Applications of Computational Chemistry Towards Combatting Challenges in Chemical Warfare and Renewable Energy

Applications of Computational Chemistry towards Combatting Challenges in Chemical Warfare and Renewable Energy

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Ryan J. Yoder

Graduate Program in Chemistry

The Ohio State University

2013

Dissertation Committee:

Professor Christopher M. Hadad, Advisor

Professor Jonathan R. Parquette

Professor James P. Stambuli

Ryan J. Yoder

2013

Abstract

Across the globe, chemists are constantly seeking to provide answers to the challenges threatening our society. Both long-term obstacles and short-term hazards are being confronted with the commitment of time, money, and intellectual acumen. The challenges faced in this thesis span both the imminent threat of the deployment of chemical warfare agents, and the long-term challenge of renewable energy. One strategy towards combatting the toxic effects of these chemical warfare agents will focus on reversing the result of exposure to organophosphorus (OP) compounds. The other strategy will examine the use of synthetic molecular baskets as a macromolecule designed to entrap OP compounds before victims can feel their adverse effects. Also in this thesis, our contribution towards the pursuit of sources for renewable energy, namely the synthesis of important small molecules from chemical feedstocks, will be presented.

OP compounds are a grave threat to military and civilian populations, especially in the context of increasing acts of terror around the globe. These nerve agents are toxic due to their inhibition of the acetylcholinesterase (AChE) active site. Although some methods of enzyme recovery are known to exist, they succumb to the need to be presented to affected victims in an extremely short amount of time. Currently, there are no known therapeutics to recover aged AChE, a permanently dead form of the enzyme upon prolonged inhibition as a result of OP binding to the enzyme. Part of this thesis will ii present efforts towards the development of a small molecule therapeutic designed as an alkylating agent to reverse the aging process and ultimately recover the active enzyme.

Our efforts have focused on the synthesis of a library of quinone methide precursors

(QMPs) that have been shown to alkylate a model phosphonate, making them promising candidates for further study with the native enzyme.

An alternate strategy to the reversal of the inhibition of the enzyme by OP compounds would be to sequester the nerve agent before it binds with AChE. In collaboration with the Badjic research group at The Ohio State University, molecular baskets have been synthesized to accomplish this goal. A rigorous computational protocol was established to examine the conformational flexibility of the molecular baskets, and further modeling studies were performed to determine the recognition of various OP compounds by molecular baskets. An imidazole-capped basket, mimicking a decarboxylated histidine residue, has been shown to bind the small OP compound, dimethoxy methylphosphonate (DMMP).

Finally, this thesis will present efforts in collaboration with the Stambuli research group at The Ohio State University towards the development of a metal catalyst to transform a saturated fatty acid into an unsaturated fatty acid. This novel chemistry would allow access to value-added chemicals as identified by the Department of Energy.

Our molecular modeling studies provided important insight to experimental results.

These insights included the inability to dehydrogenate a pendant fatty acid and reasoning for the selective dehydrogenation of an acyl phosphine ligand.

iii

Acknowledgments

The work presented in this thesis could not be possible without the contributions of several important people towards my growth as an educator, scientist, and person.

First, I must thank my advisor, Dr. Christopher Hadad, for your valuable mentorship and support over the past five years. Your continuing guidance was instrumental in helping me to realize my scientific, academic, and personal goals, and I hope our relationship will only grow in future years. I would also like to thank collaborators at The Ohio State

University who played pivotal roles in my maturation as a research scientist, namely Dr.

James Stambuli and Dr. Jovica Badjic.

To my fellow members of the Hadad Research Group, thank you for your unconditional assistance in allowing me to become a successful researcher. Each of you, whether past or present, remains a part of creating the unique atmosphere that allows our group to function so well. Special thanks go to Dr. Jeremy Beck, Dr. Shubham Vyas, Dr.

Daniel Turner, and Dr. Hashem Taha for your patience and mentorship as you passed on the wealth of knowledge you possess. Thank you also to Jason Brown for your friendship and collaboration within the Hadad Group on our nerve agent work. I would also like to thank all of the experimental researchers who assisted in advancing this work through their collaborative efforts.

Specifically, I would like to thank Katelyn Cody, an undergraduate researcher in

Dr. Christopher Callam’s research group at The Ohio State University who I have taken an active role in mentoring the past year. Thank you for your tireless efforts towards completing and presenting our research to many different audiences. Hopefully, I have been a positive influence in your development towards reaching your personal and professional goals.

I am thankful to the funding agencies that made this research possible. These agencies include the Defense Threat Reduction Agency (DTRA) and the Ohio

Supercomputer Center (OSC).

As much as I’ve grown as a scientist during my graduate career, I feel I’ve matured just as much as a teacher. Through the generosity and guidance of several influential mentors, I’ve been able to realize my true calling in life as an educator.

Certainly, I must thank Dr. Hadad for allowing me to pursue several opportunities to teach while completing my graduate research. Also, I have to thank Dr. Christopher

Callam and Dr. Noel Paul for their mentorship while serving as their Teaching Assistant at The Ohio State University. Although both of you have your own unique style and personality, you each taught me invaluable lessons in and out of the classroom. I hold the utmost respect for both of you and look forward to our friendship continuing for years to come. Thanks also to those at Capital University and Ohio Wesleyan University for allowing me the opportunity to be an adjunct instructor during my graduate career.

Thank you especially to my undergraduate advisor, Dr. Katie Hervert, for your continued friendship and support in nurturing my chosen career path.

Finally, thank you to my friends and family for your unconditional love and support over the last five years. To my wife and best friend Stefanie, thank you for helping me through every step of this experience. I know we have had significant challenges to overcome in the last five years, but through our growing love and faith, we have reached a new stage in our life’s journey together. You have undoubtedly been the best thing that has ever happened to me and I promise to continue my career knowing it is in loving devotion to you and our family. Although you may never read or understand the contents of this dissertation, it would not have been possible without the love and support from you, Boo, and Beaker.

To my mother and father, thank you for your unending love and support in cultivating my potential to allow me to reach this point in my life. Hopefully I have made you proud by following in your footsteps with a career in academia. Every day of my life I have sought to live by the principles and values you have taught me. I will continue to do so to the best of my abilities as I begin my career and family. Surely, I would not be where I am today without the sacrifices you have made. And lastly, thank you to my twin brother Matt. I would be nowhere without your love, support, and companionship for the last twenty-seven years. My sincere hope is that our relationship will continue to stay strong as we both encounter the exciting changes and challenges ahead in each of our lives, wherever that may lead us. Onward and upward.

Vita

2004 ...... Urbana High School

2008 ...... B.A. Chemistry, Ohio Wesleyan University

2008 to present ...... Graduate Research Associate, Department

of Chemistry, The Ohio State University

Publications

1) Whittemore, S. M.; Yoder, R. J.; Stambuli, J. P. “Site-Selective Alkyl

Dehydrogenation of a Coordinated Acylphosphine Ligand” Organometallics, 2012,

31 (17), 6124.

2) Hermann, K.; Sardini, S.; Ruan, Y.; Yoder, R. J.; Chakraborty, M.; Vyas, S.;

Hadad, C. M.; Badjic, J. D. “Method for the Preparation of Derivatives of

Heptiptycene: Toward Dual–Cavity Baskets” J. Org. Chem. 2013, 78 (7), 2984.

3) Ruan, Y.; Taha, H. A.; Yoder, R. J.; Maslak, V.; Hadad, C. M.; Badjic, J. D. “The

Prospect of Selective Recognition of Nerve Agents with Modular Basket-like Hosts.

A Structure–Activity Study of the Entrapment of a Series of Organophosphonates in

Aqueous Media” J. Phys. Chem. B. 2013, 117 (11), 3240.

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Fields of Study

Major Field: Chemistry

viii

Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Vita ...... vii

Table of Contents ...... ix

List of Tables ...... xiii

List of Figures ...... xvii

List of Schemes ...... xxvii

Chapter 1 : Introduction ...... 1

1.1 THE APPLICATION OF CHEMISTRY TOWARDS PROBLEMS

ENDANGERING SOCEITY ...... 1

1.2 THE CHEMICAL DEFENSE CHALLENGE ...... 2

1.3 ORGANOPHOSPHORUS COMPOUNDS (OPs) AS CHEMICAL WARFARE

AGENTS ...... 4

1.4 THE DETECTION OF ORGANOPHOSPHORUS COMPOUNDS ...... 6

1.5 THESIS SUMMARY – PART I ...... 11

1.6 THE RENEWABLE ENERGY PROBLEM ...... 12

1.7 THE PURSUIT OF VALUE-ADDED CHEMICALS ...... 15

1.8 THESIS SUMMARY – PART II ...... 18

1.9 REFERENCES FOR CHAPTER ...... 19

Chapter 2 : The use of quinone methide precursors (QMPs) as a small molecule therapeutic towards reversing the aging of AChE upon exposure to chemical warfare agents...... 23

2.1 INTRODUCTION ...... 23

2.2 SYNTHESIS OF QUINONE METHIDE PRECURSOR ...... 31

2.3 ALKYLATION STUDIES OF QUINONE METHIDE PRECURSORS WITH

MODEL NUCLEOPHILES ...... 34

2.4 UV-VIS STUDIES OF QUINONE METHIDE PRECURSORS USING MODEL

PHOSPHONATE ...... 37

2.5 FURTHER STUDY INTO POTENTIAL DUAL HYDROLYSIS PATHWAY ... 48

2.6 CONCLUSIONS ...... 54

2.7 REFERENCES FOR CHAPTER 2 ...... 56

Chapter 3 : Molecular Baskets as a Pseudo-Bioscavenger for the Entrapment and

Hydrolysis of Organophosphorus Nerve Agents ...... 58

3.1 INTRODUCTION ...... 58

3.2 COMPUTATIONAL METHODS ...... 70

3.3 MOLECULAR BASKETS APPENDED WITH VARIOUS

DECARBOXYLATED HISTIDINES SUBJECTED TO COMPUTATIONAL

PROTOCOL ...... 72 x

3.4 CONFORMATIONAL ANALYSIS OF DECARBOXYLATED IMIDAZOLE-

CAPPED BASKETS ...... 74

3.5 MOLECULAR MODELING AND EXPERIMENTAL STUDIES WITH TRIS-

PROTONATED IMIDAZOLE-CAPPED BASKET USING DIMETHYLMETHOXY

PHOSPHONATE AS A GUEST ...... 80

3.6 OTHER MOLECULAR DOCKING STUDIES WITH METHYL

PHOSPHONATES ...... 90

3.7 CONCLUSIONS ...... 95

3.8 REFERENCES FOR CHAPTER 3 ...... 97

Chapter 4 : Design of Iridium-Based Catalysts for the Selective Dehydrogenation of Fatty

Acids ...... 100

4.1 INTRODUCTION ...... 100

4.2 COMPUTATIONAL METHODS ...... 105

4.3 CALIBRATION OF COMPUTATIONAL METHODS ON CRABTREE–

DERIVED CATALYST ...... 106

4.4 USING ISODESMIC REACTIONS TO MODEL THE OPTIMUM CRABTREE–

DERIVED CATALYST ...... 119

4.5 PROBING THE PROPOSED MECHANISM OF C–H ACTIVATION ...... 124

4.6 CONCLUSIONS ...... 135

4.7 REFERENCES FOR CHAPTER 4 ...... 137

Chapter 5 : Alternative Iridium–Based Catalysts for the Selective Dehydrogenation of

Fatty Acid Derivatives ...... 138 xi

5.1 INTRODUCTION ...... 138

5.2 COMPUTATIONAL METHODS ...... 142

5.3 STUDIES OF Ir–DMSO COMPLEX TOWARDS FAVORING

INTRAMOLECULAR C–H ACTIVATION ...... 144

5.4 USE OF Ir–CP* COMPLEX APPENDED WITH FATTY ACID DERIVATIVES

...... 149

5.5 STUDY OF PROPOSED DEHYDROGENATION MECHANISM ...... 154

5.6 CONCLUSIONS ...... 163

5.7 REFERENCES FOR CHAPTER 5 ...... 164

Chapter 6 : Thesis Conclusions and Future Directions ...... 167

6.1 THESIS CONCLUSIONS ...... 167

6.2 FUTURE DIRECTIONS OF QUINONE METHIDE PROJECT ...... 168

6.3 FUTURE DIRECTIONS OF MOLECULAR BASKETS PROJECT ...... 173

6.4 REFERENCES FOR CHAPTER 6 ...... 177

List of References ...... 179

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List of Tables

Table 2.1. Results from trapping studies with neutral QMPs and different nucleophiles. 36

Table 2.2. Results from trapping studies with protonated QMPs and different

nucleophiles...... 37

Table 2.3. Percentage alkylation of neutral QMPs using a model phosphonate...... 43

Table 2.4. Percentage alkylation of protonated QMPs using a model phosphonate...... 45

Table 2.5. Results from HPLC/ESI-MS-TOF analysis of reaction between 1 and model

phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated product were

observed. The major observed products resulted from a dual hydrolysis degradation

of the intermediate alkylated product...... 50

Table 2.6. Results from HPLC/ESI-MS-TOF analysis of reaction between 1-HCl and

model phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated

product were observed. The major observed products resulted from a dual

hydrolysis degradation of the intermediate alkylated product...... 51

Table 2.7. Results from HPLC/ESI-MS-TOF analysis of reaction between 1-Acetal-HCl

and model phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated

product were observed. The major observed products resulted from a dual

hydrolysis degradation of the intermediate alkylated product...... 52

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Table 3.1. Effect of protonation state and linker size on cavity size (N–N distance) and

torsional distribution (Out:In ratio) of imidazole-capped baskets...... 77

Table 3.2. Summary of docking poses of DMMP in several MD snapshots of basket. ... 83

Table 3.3. Summary of apparent binding constants of select OP ligands showing a

decrease in binding as the size of the O-alkyl group is increased...... 95

Table 4.1. Comparison of computed bond distances to the crystal structure of Ir(H)2(P(p-

a FPh)3)2(O2C(CH2)5CH3) ...... 109

Table 4.2. Comparison of computed bond angles to crystal structure of Ir(H)2(P(p-

a FPh)3)2(O2C(CH2)5CH3) ...... 112

Table 4.3. Comparison of computed bond distances to crystal structure of Ir(H)2(P(p-

FPh)3)2(O2C(CH2)5CH3), in angstroms, using TZVP basis set for all atoms...... 114

Table 4.4. Comparison of computed bond angles to crystal structure of Ir(H)2(P(p-

FPh)3)2(O2C(CH2)5CH3), in degrees, using TZVP basis set for all atoms...... 117

Table 4.5. ∆H (kcal/mol) of isodesmic reactions studying the optimum identity and

a orientation of PR3 in Ir(H)2(PR3)2(O2C(CH2)5CH3) ...... 122

Table 4.6. Loss of H2 without a sacrificial acceptor, in kcal/mol...... 127

Table 4.7. Loss of H2 with sacrificial acceptor in kcal/mol ...... 128

Table 4.8. Thermodynamic preference of intramolecular oxidative addition in kcal/mol as

computed with bottom-of-the-well relative energies as shown in Scheme 4.7...... 130

Table 4.9. Relative thermodynamic stability to 4.7a (refer to Scheme 4.7) using different

phosphine ligands, as computed by bottom-of-the-well energies in kcal/mol at BP86

and B3LYP...... 131

xiv

Table 4.10. Relative thermodynamic preference, computed as bottom-of-the-well

energies (in kcal/mol), of fatty acid coordination being either η1 or η2...... 133

Table 4.11. Relative energies, computed with bottom-of-the-well energies (in kcal/mol),

of η1 and η2 coordination of heptanoic acid derivatives with α-disubstitution using

BP86/Basis Set 1...... 134

Table 4.12. Relative energies, computed with bottom-of-the-well energies (in kcal/mol),

of η1 and η2 coordination of heptanoic acid derivatives with α-disubstitution using

B3LYP/Basis Set 1...... 135

Table 5.1. Relative thermodynamic favorability as measured at the bottom-of-the-well of

η2 coordination of fatty acid in presence of substituted NHC ligand using BP86/Basis

Set 1, in kcal/mol...... 146

Table 5.2. Relative thermodynamic favorability as measured at the bottom-of-the-well of

η2 coordination of fatty acid in presence of substituted NHC ligand using

B3LYP/Basis Set 1, in kcal/mol...... 147

Table 5.3. Relative thermodynamic favorability as measured at the bottom-of-the-well of

η2 coordination of fatty acid in presence of substituted NHC ligand using BP86/Basis

Set 1 in kcal/mol...... 149

Table 5.4. Relative thermodynamic favorability as measured at the bottom-of-the-well of

η2 coordination of fatty acid in presence of substituted NHC ligand using

B3LYP/Basis Set 1 in kcal/mol...... 149

Table 5.5. Relative energies of all calculated reactants, transition states, and products of

four possible β–hydride elimination pathways from 5- or 6-membered xv

cyclometallated intermediates, in kcal/mol, using M06L/Basis Set 1, as computed

with bottom-of-the-well energies for the reactions in Scheme 5.10...... 160

– Table 5.6. Thermodynamics, reaction enthalpy, and free energy of first step in PF6 as

proposed by Pellinghelli as modeled using three different levels of theory in

kcal/mol...... 162

– Table 5.7. Thermodynamics, reaction enthalpy, and free energy of second step in PF6 as

proposed by Pellinghelli as modeled using three different levels of theory in

kcal/mol...... 162

xvi

List of Figures

Figure 1.1. Naphthenic acid (left) and palmitic acid (right), two of the components of

napalm...... 3

Figure 1.2. Examples of OP compounds used as chemical warfare agents along with less

toxic pesticides amiton and paraoxon...... 5

Figure 1.3. General schematic of ion mobility spectrometry used in the detection of OP

chemical warfare agents...... 8

Figure 1.4. A colorimetric (left) and fluorogenic (right) probe developed by Anslyn to

detect the presence of OP compounds via spectroscopic techniques...... 10

Figure 1.5. Fluorogenic probe developed by Han that takes advantage of a nucleophile

initiating a cyclization reaction responsible for increase in fluorescent signal in the

presence of an OP compound...... 11

Figure 1.6. A breakdown of the type of energy used across the globe as of 2010...... 13

Figure 1.7. Original projection by Hubbert predicting the world’s production of crude oil

would peak around the year 2000...... 14

Figure 1.8. A breakdown of the types of renewable energy used across the globe as of

2010...... 15

Figure 1.9. A proposed path to 7 of the 12 value-added chemicals identified by the

Department of Energy from unsaturated fatty acids...... 19 xvii

Figure 2.1. Examples of OP compounds used as chemical warfare agents along with less

toxic pesticides, amiton and paraoxon...... 24

Figure 2.2. Pyridinium oximes that have been developed for use as reactivating agents

towards recovering the activity of inhibited AChE...... 26

Figure 2.3. Library of ortho and para QMPs derived from vanillin generated through the

joint synthetic efforts of the Callam and Hadad research groups...... 32

Figure 2.4. Subset of para QMPs derived from vanillin subjected to further analytical

studies to determine their efficacy as alkylating agents...... 33

Figure 2.5. Nucleophiles used as trapping agents to determine effectiveness of alkylation

by QMPs. A thiol (p-toluenethiol; left), amine (piperidine; center), and alcohol

(benzyl alcohol; right) were examined to sample a wide range of nucleophilic

strength...... 35

Figure 2.6. Phenacyl bromo compounds synthesized by Steinberg in attempts to

realkylate aged AChE. Also shown is 2-PAM, a known reactivator of inhibited

AChE...... 38

Figure 2.7. UV-vis spectrum of control reaction where the model phosphonate (p-

– + O2NC6H4OP(CH3)(=O)O Na ) was heated in DMF for 3 h at 100°C (red). At this

initial reading, p-nitrophenol released in the initial heating was monitored in DMF at

433 nm. After initial measurement, base was added to determine the extent of

phosphonate hydrolysis releasing the p-nitrophenoxide group, monitored at 412 nm.

...... 42

xviii

Figure 2.8. UV-vis spectrum of 4 reacted with model phosphonate (p-

– + O2NC6H4OP(CH3)(=O)O Na ) at 100°C in DMF. Initial reaction in DMF (red) and

after addition of base (blue) showed significant amounts of p-nitrophenol in solution.

The signal at 412 nm (blue) did not exceed what was seen in the background

reaction, thus no quantifiable alkylation could be observed via this indirect method.

...... 42

Figure 2.9. UV-vis spectra of protonated precursors reacted with model phosphonate at

100°C in DMF. (a) 1-HCl, (b) 2-HCl, (c) 3-HCl, and (d) 4-HCl. Initial reaction in

DMF (red) showed no evidence of a background reaction (no signal at 433 nm)

indicative of release of p-nitrophenol from the phosphonate itself. However, there is

a significant signal at 412 nm after addition of base, indicative of base-induced

cleavage of p-nitrophenol from an intermediate alkylated product. The %alkylation

was calculated from an external standardization, ignoring any background

hydrolysis...... 44

Figure 2.10. UV-vis spectrum of 3-Acetal-HCl (left) reacted with the model phosphonate

– + (p-O2NC6H4OP(CH3)(=O)O Na ) at 100°C in DMF. Initial reaction in DMF (red)

showed a slight background hydrolysis of p-nitrophenol from the model

phosphonate. After addition of base (blue), a significant amount of p-nitrophenol

was observed in solution at 412 nm. The signal at 412 nm (blue) exceeded what was

observed in all other QMPs presented...... 47

Figure 2.11. 31P NMR analysis of the reaction depicted in Scheme 2.12...... 54

xix

Figure 3.1. Potential therapeutic routes to combat the inhibition and aging of OP

exposure. (1) The use of a bioscavenger to sequester the OP before binding to

AChE. (2) The use of pyridinium oximes to reverse inhibition. (3) The use of an

alkylating agent to reverse the aging process after OP inhibition...... 59

Figure 3.2. Modified β-cyclodextrin synthesized by Smith with appended guanidinium

units acting as arms over a cavity to attract aryl phosphates through a favorable

electrostatic interaction...... 61

Figure 3.3. The modified calixarene (left) synthesized by de Mendoza and the guest

molecules (right) that were determined to be recognized by the host. Only PNPCC

was found to be hydrolyzed by the guest in chloroform...... 63

Figure 3.4. The modified cavitand (left) synthesized by Dalcanale showing the in/out

orientation of the COOH group and DMMP (right)...... 64

Figure 3.5. Substituted β-cyclodextrin designed by Kubik shown to hydrolyze GF and

remove its toxicity in the presence of human AChE under physiological conditions.

...... 66

Figure 3.6. The deprotonated pyridinium oxime appended to the β-cyclodextrin scaffold

(top left), cyclosarin (GF; bottom left), and acetylthiocholine (bottom right)...... 66

Figure 3.7. A sample molecular basket developed by the Badjic research group showing

the hydrophilic region at the top of the basket and a hydrophobic region in the cavity

of the basket, which should be a match for OP chemical warfare agents...... 68

Figure 3.8. (a) The molecular basket scaffold (top) where R=H and AA=various amino

acid side chains. (b) Various amino acid side chains studied computationally by the

Hadad group (bottom)...... 69

Figure 3.9. Imidazole-capped baskets subjected to computational protocol to determine

their conformational flexibility. The protonation state was varied to keep all three

imidazole rings neutral (H0, left), protonating one imidazole ring (H1+, center) and

protonating all three imidazole rings (H3+, right). Also, the methylene unit was

altered to allow n=1, 2, or 3 for a methylene, ethylene, or propylene linker between

the imide nitrogen in the wall of the basket and the functional imidazole ring...... 73

Figure 3.10. The two parameters examined in order to investigate the conformational

flexibility of the imidazole-capped baskets, shown with the H3+, n=2 molecular

basket. (a) The N–N distances between each of the imide nitrogens was examined in

order to learn more about the size of the cavity (dotted blue lines). (b) The C–N–C–

C dihedral angle was measured to determine the orientation of the imidazole rings;

whether they were oriented in over the cavity or out away from the cavity (solid

purple line)...... 75

Figure 3.11. Both torsional angles examined in the H3+, n=2 molecular basket (a) The N–

C–C–C dihedral angle was measured to determine whether the imidazole ring was

oriented up above the top of the basket or down towards the base (solid orange line).

(b) The C–N–C–C dihedral angle was measured to determine the orientation of the

imidazole rings; whether they were oriented in over the cavity or out away from the

cavity (solid purple line)...... 79

xxi

Figure 3.12. Two-dimensional correlation between (a) N–C–C–C UP/DOWN dihedral

angle (solid orange line, Figure 3.11) and (b) C–N–C–C IN/OUT dihedral angle in

H3+, n=2 molecular basket (solid purple line, Figure 3.11)...... 80

Figure 3.13. Two groups of snapshots used in molecular docking simulations. One group

of seven snapshots has all three arms oriented away from the center of the cavity

(left). Another group of the three snapshots has one arm oriented in over the center

of the cavity (right)...... 82

Figure 3.14. Library of OP compounds docked with imidazole-capped basket (H3+, n=2).

...... 82

Figure 3.15. Representative poses generated via docking simulations showing the

interaction between the imidazole-capped basket (H3+, n=2) and DMMP,

representative of subset of snapshots where none of the arms of the basket are

oriented in over the center of the cavity. One pose shows DMMP making a

hydrogen bond between the N–H of imidazole and resting between two walls of the

basket (left, top-down view into the basket). Another pose shows DMMP sitting in

the center of the cavity of the basket, but with the P–Me group oriented up (right,

side-on view into basket)...... 85

Figure 3.16. Representative poses generated via docking simulation showing interaction

between imidazole-capped basket (H3+, n=2) and DMMP, representative of subset of

snapshots where one of the arms of the basket are oriented in over the center of the

cavity. One pose shows DMMP making a hydrogen bond between the N–H of

imidazole and resting high up in the basket (left, top-down view into the basket).

xxii

Another pose shows DMMP sitting in the center of the cavity of the basket, with a

hydrogen bond to the imidazole ring and with the P–Me group oriented up (right,

top-down view into basket). This particular pose was the lowest-energy docking

pose generated with any snapshot and matched the hypothesis that the hydrophobic

cavity would recognize the P- ...... 87

Figure 3.17. Plot of the distance between the basket base and methyl group carbon of

DMMP as a function of simulation time, as performed by Dr. Hashem Taha in the

Hadad ...... 89

Figure 3.18. 1H NMR spectra (600 MHz, 298.0 K) of basket (H3+, n=2; 1.0 mM) obtained

upon an incremental addition of DMMP to the solution of imidazole-capped basket

(10.0 mM phosphate buffer at pH = 2.5 ± 0.1); note that the water resonance at 4.76

ppm was suppressed. These spectra were obtained by the Badjic group...... 90

Figure 3.19. Subset of docked methyl phosphonates into imidazole-capped basket where

P–Me group was held constant and size of O-alkyl group was increased...... 91

Figure 3.20. Lowest energy pose (top-down) showing MeP-OMe-OEt bound to center of

basket (imidazole cap; H3+, n=2) with P-Me group oriented down into cavity...... 92

Figure 3.21. Lowest energy pose (top-down) showing MeP-OEt-OEt bound to center of

basket (imidazole cap; H3+, n=2) with P-Me group oriented down into cavity...... 92

Figure 3.22. Lowest energy pose (top-down) showing MeP-OEt-OiPr bound to center of

basket (imidazole cap; H3+, n=2) with larger P–OiPr group oriented down into the

cavity...... 93

xxiii

Figure 3.23. Lowest energy pose (top-down) showing MeP-OiPr-OiPr bound to center of

basket (imidazole cap; H3+, n=2) with larger P–OiPr group oriented down into the

cavity...... 94

Figure 3.24. Lowest energy pose (top-down) showing MeP-OMe-OCy bound to center of

basket (imidazole cap; H3+, n=2) with larger P–OCy group oriented down into the

cavity...... 94

Figure 4.1. Various iridium pincer catalyst precursors used in alkane dehydrogenations.

...... 102

Figure 4.2. Experimental crystal structure of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) as

observed by the Stambuli research group, but unpublished...... 107

Figure 4.3. Computational modeling of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) as optimized

at the (a) BP86 and (b) B3LYP levels of theory with the SV(P){Ir(TZVP)} basis set.

Hydrogens have been omitted for clarity...... 108

Figure 4.4. Potential sacrificial acceptors: t-butylethylene, norbornene, and

norbornadiene...... 127

Figure 5.1. Crystal structure of dehydrogenated neomenthyl unit appended to Cp*–Ir

complex...... 141

Figure 5.2. Substituted NHC ligands studied via molecular modeling studies...... 145

Figure 5.3. Equilibrium between η1 or η2 coordination of pyridonate ligand to iridium. 147

Figure 5.4. Neutral and anionic 2–pyridone ligands studied by molecular modeling. ... 148

Figure 5.5. Starting catalyst species using an acylphosphine derived from heptanoic acid.

...... 150

xxiv

Figure 5.6. Crystal structure of olefin product, with hydrogens omitted for clarity, as

obtained at The Ohio State University by Whittemore and Stambuli...... 152

Figure 5.7. Crystal structure of isolated dimeric intermediate obtained at The Ohio State

University by Whittemore and Stambuli...... 153

Figure 6.1. Two possible “control” compounds to examine the extent of SN2-like

alkylation of QMPs without the presence of a phenolic oxygen...... 170

Figure 6.2. A chiral QMP that can be separated from a diastereomeric mixture of a

Mosher’s ester...... 171

Figure 6.3. Various pyridine QMPs that could be synthesized to expand the current

library of alkylating agents. Only neutral para precursors are shown. However,

cationic precursors could be synthesized through installation of a methyl group on

the pyridine nitrogen. Also, the corresponding family of ortho precursors could also

be synthesized...... 172

Figure 6.4. Two net neutral baskets that can be subjected to our computational protocol

(Chapter 3) to examine the conformational flexibility of the molecular basket and its

binding of OP compounds. One basket is overall neutral with no charge (left) and

the other has the histidine residues at the top of the basket in their zwitterionic form

(right)...... 175

Figure 6.5. Two charged baskets that can be subjected to our computational protocol

(Chapter 3) to examine the conformational flexibility of the molecular basket and its

binding of OP compounds. One basket is acidic with a net positive charge (left) and

the other is basic with a net negative charge (right)...... 176

xxv

Figure 6.6. A modified molecular basket scaffold with an added pyrazine ring, providing

taller arms that should yield a larger cavity for the recognition of larger OP

compounds...... 177

xxvi

List of Schemes

Scheme 1.1. Proposed mechanism by Choudhary for the conversion of a furan carbonyl

to succinic acid in the presence of an acid catalyst (Amberlyst-15) and hydrogen

peroxide...... 17

Scheme 2.1. (a) The hydrolysis of acetylcholine by AChE, which occurs via the catalytic

triad (Ser-His-Glu), cleaving into choline and acetate. (b) The inhibition of AChE by

an OP compound, proceeding through a covalent bond between the OP and Ser-203.

...... 25

Scheme 2.2. The reactivation of inhibited AChE by an oxime through attack of the

phosphoryl oxygen and release of the phosphonate...... 26

Scheme 2.3. The aging of inhibited AChE upon the loss of an alkyl group, leaving a

phosphonate anion trapped in the enzyme active site...... 27

Scheme 2.4. Alkylation of amino acids by quinone methide intermediate. The quinone

methide was generated by either photochemical or thermal means...... 28

Scheme 2.5. Alkylation of a phosphodiester by quinone methide intermediate. The

quinone methide was generated by a lead (II) oxidation...... 29

Scheme 2.6. Alkylation of deoxyadenosine by quinone methide intermediate The quinone

methide was generated by a fluoride-induced cleavage of a silyl ether...... 30

xxvii

Scheme 2.7. Generation of a para-quinone methide intermediate via thermal generation

from a benzyl ammonium salt...... 31

Scheme 2.8. Reductive amination protocol developed to synthesize QMPs...... 34

Scheme 2.9. Reaction conditions for trapping studies of QMPs with model nucleophiles

using neutral (top) and protonated (bottom) QMPs derived from p-vanillin...... 35

Scheme 2.10. Alkylation of a model phosphonate appended with a p-nitrophenol group in

order to indirectly monitor the appearance of p-nitrophenol upon addition of base to

the intermediate product. This scheme was adapted from previous work by

Steinberg...... 39

Scheme 2.11. Proposed dual hydrolysis pathway to explain suppressed alkylation of

model phosphonate by QMPs. Previous calculations in the indirect UV-vis screen

assumed only the presence of the pathway generating p-nitrophenol (right). Also,

the other P–O bond in the intermediate alkylated phosphonate could be cleaved to

yield a benzyl alcohol product that would not produce a signal at 412 nm...... 48

Scheme 2.12. Alkylation of 1-Acetal-HCl by model phosphonate under microwave

conditions. This reaction was then examined by 31P NMR spectroscopy...... 53

Scheme 4.1. Proposed mechanism of Crabtree’s dehydrogenation...... 103

Scheme 4.2. Path to value-added chemicals...... 104

Scheme 4.3. Isodesmic reactions studying the optimum identity of PR3 in

Ir(H)2(PR3)2(O2C(CH2)5CH3): (a) exchange of PH3 to PR3; (b) exchange of PPh3 with

P(p-FPh)3, P(m-FPh)3, P(m, p-FPh)3; (c) axial/axial vs. axial/equatorial orientation of

PPh3...... 121

xxviii

Scheme 4.4. Isodesmic reaction studying the coordination of a fatty acid to an iridium

Crabtree catalyst...... 124

Scheme 4.5. Proposed mechanism for selective dehydrogenation of a coordinated

heptanoic acid by “Crabtree–like” system...... 125

Scheme 4.6. Loss of hydrogen from the proposed starting catalyst to an η1 intermediate

for the carboxylate group...... 126

Scheme 4.7. Unsaturated Ir intermediate proceeding through intramolecular oxidative

addition to form potential 5- (a), 6- (b), and 9-membered (c) iridalactones

intermediates...... 129

Scheme 4.8. Equilibrium between η1 and η2 coordination of pendant fatty acid to iridium.

...... 132

Scheme 4.9. Equilibrium between Ir η1 and η2 coordination of pendant fatty acid

derivatives with α-disubstituted groups (R = H, F and Me)...... 134

Scheme 5.1. Proposed mechanism of cyclohexane dehydrogenation through a dimeric

intermediate...... 140

Scheme 5.2. Dehydrogenation of cyclohexane appended to NHC ligand...... 142

Scheme 5.3. Equilibrium between η1 and η2 coordination of pendant fatty acid in

presence of substituted NHC ligand using L1...... 146

Scheme 5.4. Equilibrium between η1 and η2 coordination of pendant fatty acid in

presence of anionic 2–pyridonate ligand...... 148

Scheme 5.5. Dehydrogenation of pendant cyclohexyl substituent of acylphosphine. .... 151

xxix

Scheme 5.6. Dehydrogenation from C–H insertion along the aliphatic chain of the

acylphosphine...... 151

Scheme 5.7. Isolated dimeric species leads to identical olefin product as starting catalyst,

suggesting dimer is on reaction pathway...... 153

Scheme 5.8. Potential mechanistic pathways from isolated dimeric intermediate...... 155

Scheme 5.9. Proposed mechanistic pathway for selective dehydrogenation of pendant

acylphosphine C–H chain studied via molecular modeling...... 156

Scheme 5.10. Four possible β–hydride elimination pathways from 5– or 6–membered

cyclometallated intermediates...... 159

– Scheme 5.11. First step in hydrolysis of PF6 as proposed by Pellinghelli...... 161

– Scheme 5.12. Second step in hydrolysis of PF6 as proposed by Pellinghelli...... 162

Scheme 6.1. (A) The stepwise mechanism of alkylation through formation of the quinone

methide intermediate, followed by alkylation. (B) The concerted mechanism of

alkylation consisting of direct displacement of the leaving group by the nucleophile

at the benzylic position...... 169

xxx

Chapter 1 : Introduction

1.1 THE APPLICATION OF CHEMISTRY TOWARDS PROBLEMS

ENDANGERING SOCEITY

Many chemical researchers around the globe have dedicated their scientific endeavors towards attempting to better society. There is, of course, value in scientific research for pursuing theoretical or academic interest in the ongoing quest for knowledge.

However, the truest contribution by science as a part of society comes when research alleviates the troubles that threaten the immediate and ongoing issues facing human health and sustainability. There are countless areas where chemists have provided guidance and innovation in these areas, and the following work presented in this thesis does not portend to be anything but a minimal contribution within this larger framework.

The short-term threat considered in this thesis involves the use of organophosphorus compounds (OPs) and their potential use as chemical warfare agents against military and civilian populations in the context of the emerging threat of global terrorism. The long- term challenge confronted in this thesis is the development of an efficient methodology to better access small, large-volume chemicals of great value to our chemical industry from renewable energy sources.

1.2 THE CHEMICAL DEFENSE CHALLENGE

Throughout the last century, there has been a grave need to provide appropriate measures to defend against the deployment of chemical weapons. However, the employment of chemical weapons in times of war is almost as old as warfare itself. As long ago as ancient Greece, Homer’s Iliad depicted the use of poisoned arrows by forces of Odysseus in order to inflict maximum damage on their opposition.1 In more recent times, chemical weapons have been used against military populations across the globe.

The First World War was marred by the use of sulfur mustards as vesicant chemical weapons in a widespread manner. In fact, it is estimated that 125,000 tons of chemical weapons were used during World War I.2

Chemical and biological weapons have continued to be used after their deployment in World War I. While nerve agents were being developed in German and

British labs during World War II (see below), they were not actually employed during that conflict. However, phosphorus grenades and napalm were used extensively to attack soldiers that were entrenched in position. Napalm, in particular, was one of the more deadly chemical weapons used in warfare during the 20th century. The mixture of petroleum, naphthenic acid and palmitic acid (Figure 1.1) would stick to skin and burn at an extremely high temperature upon ignition. Napalm was estimated to be responsible for over 300,000 deaths in Japan during the Second World War.3 The substance was also used with devastating effect during later conflicts in Korea and Vietnam. Ultimately, the use of napalm was banned against civilians in 1980 by the United Nations.4

O O

OH OH 13

Figure 1.1. Naphthenic acid (left) and palmitic acid (right), two of the components of napalm.

However, actions by the United Nations and other global organizations did not completely curtail the use of such chemical weapons. In the Iran-Iraq War (1980s), sulfur mustard gas (among other chemical weapons) was used by Saddam Hussein’s forces against the Iranians. According to some estimates, as many as 45,000 soldiers have continued to suffer long-term effects from exposure to sulfur mustard gas during the conflict.5 Mustard gas has been used as a chemical warfare agent since World War I as a highly toxic blistering agent.6 In fact, mustard gas alone was determined to be responsible for over 1 million casualties during the First World War. Mustard gas is highly toxic because it can act as an alkylating agent, thereby targeting DNA, RNA, and proteins. Exposure to mustard gas can also cause blindness and pulmonary edema, among other debilitating side effects.7

While the use of chemical weapons in military campaigns has occurred throughout the 20th century, biological weapons have emerged as new threats within the context of global terrorism. Of course, the terrorist attacks on the United States of

America on September 11, 2001 has crystallized the need for not just the military, but also civilian populations, to be prepared for hostile aggression. The imminent threat of chemical and biological weapons to the public became all the more clear only a month after the 9/11 attacks, in October of 2011. 3

During this time, letters contaminated with anthrax were sent through the mail to several locations across the country, including the nation’s capital, which resulted in the closing of Congress and the Supreme Court.8 Although no person or terrorist group was held responsible, the attacks confirmed the need for the public to be vigilant against the use of biological and/or chemical weapons. These events also underscored the need for the detection and detoxification of chemical and biological weapons. Should the stockpiles of chemical weapons around the globe fall into the wrong hands, the results could be tragic to large civilian populations. Thus, ongoing efforts by the scientific community to more successfully and efficiently detect chemical and biological weapons, and attempts to prevent or reverse toxicity upon exposure, have been of grave interest in the last decade.

1.3 ORGANOPHOSPHORUS COMPOUNDS (OPs) AS CHEMICAL WARFARE

AGENTS

The focus of a portion of this thesis centers specifically on combatting the effects of exposure to toxical chemical warfare agents known as organophosphorus (OPs) compounds. OPs containing a phosphoryl group have been used as pesticides and chemical warfare agents throughout the last several decades (Figure 1.2). The use of OP compounds as toxic nerve agents dates back to the accidental discovery of tabun’s toxicity in the lab of the German scientist, Dr. Gerhard Schrader.9 These compounds did not see immediate deployment in the Second World War; however, countries around the globe have continued to stockpile these nerve agents. For example, while initially

4 developed by the British government, 4400 tons of VX were produced by the United

States from 1961 to 1968 for the purposes of deployment in times of war.10 The fact that these most toxic OP compounds have been employed in various conflicts around the globe since, often with devastating effect, makes their threat very apparent. Most recently, the 2013 civil war in Syria provided an example of this chemical weapon threat with U.S., Israeli and Turkish accusations of Syria’s usage of chemical nerve agents against its own people.11

O O O O O P P P P N O P O O O CN O F F F F Tabun (GA) Sarin (GB) Soman (GD) Ethyl Sarin (GE) Cyclosarin (GF)

NO2 O O O O

P N P N P P N O S S O S O O O O O VX VR Amiton (VG) Paraoxon

Figure 1.2. Examples of OP compounds used as chemical warfare agents along with less toxic pesticides amiton and paraoxon.

In addition to sulfur mustard gas (see above), the Iraqis deployed other chemical weapons such as tabun against the Iranians in the Iran-Iraq War.12 More recently, sarin gas was used by a terroist group in Japan in the 1995 “Tokyo Subway Attack”, which killed 13 people and injured several thousands due to exposure to the toxic OP compound. Even today, it is estimated that around 3 million people each year come into contact with OPs, resulting in approximately 300,000 deaths around the world. In the

United States alone, 8,000 exposures and 15 deaths were reported during 2008.13 And 5 while most of these exposures are not the result of the deployment of the most toxic OPs as agents of chemical warfare, the ease of synthesis as well as the large stockpiles of these nerve agents around the world are a grave matter of concern. These OP compounds are highly toxic to humans due to their inhibition of the enzyme acetylcholinesterase

(AChE), which is responsible for hydrolyzing the neurotransmitter acetylcholine.

This inhibition of AChE and resultant accumulation of acetylcholine leads to many adverse side effects on the human body. Low-level symptoms could range from muscle twitching to disorientation to reduced vision. More serious symptoms involve paralysis, vomitting, and convulsions. Ultimately though, high levels of exposure and inhibition of the AChE enzyme can lead to death by asphyxiation. Exposure to OP compounds also has been suspected to increase the risk of attention deficit/hyperactivity disorder (ADHD) and Alzheimer’s disease.14,15 These side effects are why even low- level exposure to less toxic OP compounds, such as pesticides, must be taken quite seriously. When the more toxic nerve agents are then weaponized as chemical warfare agents, the danger to society becomes even more apparent.

1.4 THE DETECTION OF ORGANOPHOSPHORUS COMPOUNDS

Certainly, the ability to detect the presence of these OP nerve agents is of critical importance for military and civilian populations who may be under seige from the use of chemical weapons. As part of this introduction, the advances made in this endeavor will be briefly presented as an example of just one facet of the contribution that science has already made towards the challenges presented by the presence of these toxic nerve

6 agents. Mere detection of OP compounds would not remove or reverse the toxicity of these compounds, at least not initially. Yet, it is important to establish a scientific precedent that knowledge of these particular chemical warfare agents is expanding.

Perhaps the knowledge gained from the development of efficient, wide-ranging detection methods of OP compounds could then be applied towards ubiquitous methods aimed at removing or reversing their toxicity.

The ideal detection method to determine the presence of OP compounds would be a portable, sensitive indicator that could quickly signal the presence of a wide variety of nerve agents in an unambiguous result. Unfortunately, such an ideal method is still elusive, although great strides have been made. Current detection methods seem to fall in one of two categories. One family of detection methods rely on sophisticated chemical instrumentation to monitor trace amounts of the OP compounds. Another method relies on some type of signal produced by the presence of a chemical warfare agent to yield some type of colorimetric or fluorogenic response from a probe. Both methods certainly have their pros and cons, preventing any single method from becoming universal in its application.

One of the dominant techniques to detect OPs and their degradation products has been mass spectrometry (MS). Even within the broad category of MS techniques, several different options are available to detect chemical warfare agents in soil, water, and the air. Gas chromatography coupled with mass spectrometry (GC/MS) has been chosen because of its reliablity, and this method allows analysis of more volatile species.16 Other researchers have continued to develop liquid chromatography/mass spectrometry

(LC/MS) techniques, which avoid the need to pretreat the sample as in GC/MS.17 An

alternate MS technique is ion mobility spectrometry (IMS), which allows for more

portable detection of vapors that could contain OP nerve agents. This IMS technology

(Figure 1.3) has been employed already by civilian and military agencies.18 An

alternative to MS techniques, capillary electrophoresis has been used to separate various

degradation products of chemical warfare agents; however, a liquid sample is required for

analysis.19

Figure 1.3. General schematic of ion mobility spectrometry used in the detection of OP chemical warfare agents.18

While these mass spectrometry techniques have been developed, if not fully

optimized, there have also been advancements in the development of sensors that detect

OP compounds through some sort of colorimetric or fluorogenic signal that is produced

in their presence. This technology has the ability to be more portable and less expensive

Figure 1. (a) General set up; (b) illustration of the voltages supplied to the drift tube (left) and the timing of the voltage pulse for the electron gun and the reaction region (right). The electron gun produces free electrons and thus triggers the formation of analyte ions (top). Then a delay ∆t is introduced in which the ions recombine (middle). Finally the extraction pulse is supplied which accelerates the remaining ions into the drift region where they are separated due to their different mobilities (bottom).

G3). With certain potentials at grid G1, electrons can enter the nitride window is about 0.5 mm2.Theregularradioactivesource tube as a constant current or pulsed. With a volatge supplied in the Draeger IMS is a 3Hsource(circularshapedmetalplate to grid G2, electrons are accelerated to the focus system G3 with 10 mm diameter) having 50 MBq of activity. It has been consisting of two parallel metal plates and a corresponding replaced by a source punch with a diameter of about 10 mm focus voltage. The emission and focusing groups are on a that emits the electron beam produced by the described potential UK. For application in IMS, the potential UK can be nonradioactive source. A summary of the voltages applied can set to values between 8 and 12 kV. The kinetic electron energy be found in Table 1. is then high enough to pass through the silicon nitride foil and The silicon nitride window limits the maximum kinetic energy ionize molecules in the reaction room. The size of the silicon (and thus UK) as well as the amount of electrons (which can be

3758 Analytical Chemistry, Vol. 82, No. 9, May 1, 2010 than the MS techniques mentioned previously, although reliability issues with false positives are an ever present concern. One early example from Anslyn used a

“supernucleophile” to cause a chemical reaction which could be followed through ultraviolet/visible (UV-vis) spectroscopy.20 The detection of a chemical warfare agent simulant could be observed by a shift of 50 nm in the UV-vis spectrum. However, the probe was not developed for aqueous conditions. In fact, detection conditions required a basic pH, which could interfere with detection by performing its own nucleophilic attack on the OP before detection can occur.

A further development by Anslyn continued to seek a fluorogenic detection method, instead of a colorimetric, “naked eye” detection.21 While these colorimetric techniques yield a clearly visible signal, fluoresence techniques have often been found to be more sensitive to low concentrations of the analyte. Once again, Anslyn employed a

“supernucleophile” in the form of a deprotonated oxime, which has been shown to act itself as a therapeutic towards OP compounds that have inhibited AChE. By slightly altering the chemical structure of the probe (Figure 1.4), Anslyn allowed for more sensitive, fluorogenic detection.

H N O O N

H NO 2 N O NH2

Figure 1.4. A colorimetric (left) and fluorogenic (right) probe developed by Anslyn to detect the presence of OP compounds via spectroscopic techniques.20,21

Recently, Han has developed a rhodamine-based sensor that can detect the presence of OP compounds in the parts per million range.22 The presence of the OP compound triggers a cyclization reaction which converts the weakly fluorescent probe into a strongly fluorescent signal (Figure 1.5). However, the probe was found to be most sensitive in a DMF solution, which again is not ideally compatible with physiological conditions. A similar cyclization reaction was used by Royo to employ an azo dye probe to detect nerve agent mimics via “naked eye” detection.23 Rebek was also able to use a similar cyclization reaction brought on by nucleophilic attack of an OP by an oxime to create a fluorescent indicator that was actually successful in aqueous solution.24

Figure 1.5. Fluorogenic probe developed by Han that takes advantage of a nucleophile initiating a cyclization reaction responsible for increase in fluorescent signal in the presence of an OP compound.22

1.5 THESIS SUMMARY – PART I

One step beyond the mere detection of such chemical warfare agents though would be to prevent or reverse the effects of exposure to such compounds. There are multiple strategies researchers have taken to improve efforts towards combatting the effects of toxic chemical warfare agents due to their danger to military and civilian populations and the increase around the globe of terrorist acts of aggression. Of course, much is dependent upon the specific chemical warfare agent being used against the victim. One strategy towards preventing the toxicity of chemical warfare agents involves the design of some entity that can preferentially bind and sequester the chemical warfare agent in order to prevent its interaction with AChE. Another alternative strategy would be the development of a therapeutic that would attempt to reverse the effects on the body upon exposure to the chemical warfare agents. Both strategies have been pursued to varying effect by chemical researchers, but so far there have been no successfully ubiquitous developments that would reverse or prevent the toxicity of OP chemical warfare agents.

A portion of this thesis will focus on pursuing each strategy to deal with combatting the effects of exposure to OP nerve agents. The first strategy (Chapter 2) will report preliminary results towards the development of a small molecule therapeutic, which would attempt to reverse the inhibition and aging of AChE upon exposure to OP compounds, thus restoring activity to the enzyme. The second strategy (Chapter 3) will report the progress made towards the development of a molecular basket to act as a scavenger to selectively bind the OP nerve agent before it can interact and inhibit AChE.

Through these efforts, we hope to contribute to chemical strategies that can minimize this imminent problem in society. The latter portion of this thesis, though, will shift its focus to a different problem facing society: that of the quest for efficient sources of value-added chemicals from renewable resources.

1.6 THE RENEWABLE ENERGY PROBLEM

The long-term health of our globe and a functioning society will clearly be affected by our response to the need to find efficient sources of renewable energy in the next decade. Currently, fossil fuels dominate the consumption of energy across the globe. It has been estimated that as much as 80 percent of consumed energy comes from fossil fuels such as coal, oil, and natural gas. On the other hand, as of 2010, only 17 percent of the world’s energy consumption was derived from sources that could be defined as renewable, which includes biofuels, wind, hydropower, and solar power to name a few.25

Figure 1.6. A breakdown of the type of energy used across the globe as of 2010.25

The concern over the need to convert energy consumption from fossil fuels to renewable sources is not a new phenomenon. As early as the 1950s, M. King Hubbert of the Shell Oil Company was expressing his doubts towards the sustainability in production of fossil fuels due to their depletion from constant use by a rapidly developing planet. In fact, Hubbert’s original projection had the peak in crude oil production around the world occurring around the year 2000 (Figure 1.7).26 This “Hubbert’s Peak” theory, while undergoing some critiques as the efficiency and production of fossil fuels has also increased since its original unveiling, underscores the need for renewable energy to surpass and eventually replace the consumption of fossil fuels. More recent estimates project coal reserves lasting at current production and consumption rates until the year

2112, but all other fossil fuels being fully consumed by 2042, less than thirty years from now.27

Figure 1.7. Original projection by Hubbert predicting the world’s production of crude oil would peak around the year 2000.26

As mentioned previously, there are several different sources of renewable energy that are in use around the globe to varying degrees. As of 2010, over half of the consumed renewable energy in the world came from biomass or biofuels.24 By 2022, it has been projected that the United States alone will use 36 billion gallons of biofuels, which would account for 11 percent of fuels consumed for transportation.28 No matter the form of renewable energy though, chemical research has played a vital role in the development of such technologies and will continue to be at the forefront of research and development in this area. This thesis will only briefly mention a few narrow examples of these contributions while recognizing the vast commitment by scientists around the globe 14 towards solving the conundrum of renewable energy resources. The focus of this thesis, though, will be the development of methods to harness the energy of biomass, specifically towards the modification of chemical feedstocks into value-added chemicals.

Figure 1.8. A breakdown of the types of renewable energy used across the globe as of

2010.25

1.7 THE PURSUIT OF VALUE-ADDED CHEMICALS

In 2004, twelve value-added chemicals were identified by the Department of

Energy, believed to be “building block chemicals” that could undergo further transformation to other “high-value bio-based chemicals or materials”.29 These twelve chemicals have been targeted due to the presence of numerous functional groups that chemists can manipulate towards transforming these building blocks into other important

15 molecules. Thus, chemical researchers have been interested in (1) the transformation of renewable materials into these value-added chemicals and (2) the transformation of value-added chemicals into other important compounds for the chemical industry. There have been advances shown in the literature towards the latter of these pursuits. For example, much research has been performed on the small molecule glycerol in order to transform it via some chemical manipulation to other value-added chemicals.30

The work herein will focus on the development of a novel method to generate value-added chemicals from other potential materials. One approach has taken advantage of carbohydrates as starting materials, for example glucose and fructose, in order to convert them into the intermediate compound 5-hydroxymethylfurfural (HMF).31 Other furan-based carbonyl compounds have been obtained in a similar fashion from a variety of starting materials that can be easily obtained from biomass. These furan-based compounds can then be transformed into various other small molecules of interest. A recent example showed the conversion of 2-furaldehyde (furfural) into succinic acid, a value-added chemical, in upwards of 74 percent yield in the presence of an acid catalyst and hydrogen peroxide (Scheme 1.1).32

Scheme 1.1. Proposed mechanism by Choudhary for the conversion of a furan carbonyl to succinic acid in the presence of an acid catalyst (Amberlyst-15) and hydrogen peroxide.32

However, the conversion of carbohydrates directly to value-added chemicals presents its own challenges with regards to the depletion of crop and food supplies for consumption. An alternative approach has been used to convert small molecules into value-added chemicals in a bottom-up approach instead of breaking down larger molecules into desired products. For example, even molecules as simple as methane and carbon dioxide have been used as starting materials to access slightly more complex value-added chemicals.33 In fact, Pombeiro has used vanadium as a heterogeneous catalyst to convert methane and carbon dioxide into acetic acid, an atom neutral 17 process.34 Our research presented herein likewise attempts a similar approach using a heterogeneous catalyst to pursue novel methodology in the pursuit of value-added chemicals from biomass, specifically saturated fatty acids.

1.8 THESIS SUMMARY – PART II

Currently, the most common renewable sources for generating value-added chemicals are carbohydrates,35 lignin36 and algal biomass.37 This thesis will focus specifically on using a chemical feedstock that has previously gone relatively uninvestigated in the pursuit of value-added chemicals, saturated fatty acids. Upon the transformation of a saturated fatty acid to an unsaturated fatty acid via a selective catalytic dehydrogenation, a pathway can be envisioned towards synthesizing 7 of the 12 value-added chemicals identified by the Department of Energy (Figure 1.9). The research presented herein focuses on the key initial transformation of a saturated fatty acid to an unsaturated fatty acid via C–H activation and loss of molecular hydrogen.

There is a rich history in organometallic chemistry towards selective C–H activation; however, fatty acids represent a class of molecules that have yet to be shown to undergo such a transformation. Through our efforts, we hope to not only provide a novel organometallic C–H activation method, but also to contribute to the ongoing quest to advance the accessibility of renewable energy resources.

NH2 O HO OH OH O reduction OH O 3-hydroxy- O HO H tetrahydrofuran OH OH O O or Aspartic Acid dehydration OH O NH3 HO HO O OH – H2O O O O HO 3-amino- OH tetrahydrofuran O Malic Acid O H N 2 HO Fumaric Acid oxidation O 3-Hydroxybutyrolactone OH O O reduction n OH γ-butenyl lactone Succinic Acid allylic O O oxidation HO THF, NMP, HO OH or 1,4 BDO OH O 5 O O Succinic Acid oxidation n 4 OH + O epoxidation 1) hydrolysis OH – H m m = n – 4 2 2) oxidation O O O OH RECYCLE n Fatty Acid n m OH

– H O 2 epoxidation oxidation O 3 m OH O m = n – 3 n OH 4 OH + acrylic acid O O reduction 3-Hydroxy O propionic CN H OH Acid (3-HPA) HO OH acrylonitrile O

OCH3 methyl acrylate Scheme 1. Selective chemical conversion of saturated fatty acids to mono-unsaturated (Δ3,4 or Δ4,5) derivatives (red), followed by standard conversions to produce value-added chemicals. The structures shown in blue are 7 representatives compounds Figure 1.9ou.t o Af 12 c proposedhemical building blopathcks wit h t tohe "gr 7ea te ofst p ot theential " b 12ased o valuen carbohy-daddedrate "bioref in chemicalseries". identified by the Department of Energy29 from unsaturated fatty acids.

1.9 REFERENCES FOR CHAPTER

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12. Iraq’s Chemical Warfare Program – Comprehensive Report of the Special Advisor to the DCI, 2004. Retrieved online at https://www.cia.gov/library/reports/general-reports- 1/iraq_wmd_2004/chap5.html

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16. Hooijschuur, E. W. J.; Hulst, A. G.; de Jong, A. L.; de Reuver, L. P.; van Krimpen, S. H.; van Baar, B. L. M.; Wils, E. R. J.; Kientz, C. E.; Brinkman, U. A. T. TrAC-Trends Anal. Chem. 2002, 21, 116.

17. D’Agostino, P. A.; Hancock, J. R.; Provost, L. R. J. Chromatogr. A 2001, 912, 291.

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Chapter 2 : The use of quinone methide precursors (QMPs) as a small molecule therapeutic towards reversing the aging of AChE upon exposure to chemical warfare agents.

Dr. Carolyn Reid in the Hadad research group at The Ohio State University developed the experimental synthesis presented in this chapter. Leah Guerra, Tyler Secor, Katelyn

Cody, Ali Esfahani and Dr. Christopher Callam at The Ohio State University performed further experimental and analytical work. The 31P NMR and HPLC/MS-TOF data presented in this chapter were obtained by Katelyn Cody and Dr. Christopher Callam.

2.1 INTRODUCTION

Organophosphorous (OP) compounds containing a phosphoryl group have been used as pesticides and chemical warfare agents (Figure 2.1). These OP compounds are toxic due to their inhibition of the enzyme acetylcholinesterase (AChE), a serine hydrolase typically found in the central and peripheral nervous system that regulates concentrations of the neurotransmitter acetylcholine.1 Under normal function, AChE hydrolyzes acetylcholine into choline and acetate via the catalytic triad of glutamate, histidine, and serine residues (Scheme 2.1), similar to other serine hydrolases found throughout the body.2 However, upon exposure to an OP compound, the critical Ser-203 residue in AChE makes a covalent bond to the phosphoryl center, creating a complex between the AChE active site and the bound OP. The phosphonylation of the Ser oxygen by the OP results in the enzyme being in an inhibited state. Inhibition of AChE can lead 23 to death by respiratory failure due to overstimulation of acetylcholine receptors at the neuromuscular junctions.3

O O O O O P P P P N O P O O O CN O F F F F Tabun (GA) Sarin (GB) Soman (GD) Ethyl Sarin (GE) Cyclosarin (GF)

NO2 O O O O

P N P N P P N O S S O S O O O O O VX VR Amiton (VG) Paraoxon

Figure 2.1. Examples of OP compounds used as chemical warfare agents along with less toxic pesticides, amiton and paraoxon.

H447 H447 S203 H447 S203 S203 E334 E334 E334

–HO(CH2)2NMe3

O O O O H Me OH O HN N HN C O N C O N NH O O C O O O O Me Me

NMe3 ! NMe3

–CH3COOH

H447 H447 S203 S203 E334 E334

O O O HN H OH C O N N O NH C O O O HO Me Me H

H447 H447 S203 H447 S203 S203 E334 E334 E334

-X " O O O O O H OH OH N HN N RO P O N NH P O NH O P O O RO Me R O X Me Me X

Scheme 2.1. (a) The hydrolysis of acetylcholine by AChE, which occurs via the catalytic triad (Ser-His-Glu), cleaving into choline and acetate. (b) The inhibition of AChE by an OP compound, proceeding through a covalent bond between the OP and Ser-203.

There are some known therapeutics (specifically, pyridinium oximes) that are effective under certain, limited circumstances to reverse OP inhibition of AChE.4 These pyridinium oximes can reactivate AChE by facilitating a nucleophilic attack on the phosphoryl center of the OP–Ser complex, releasing the phosphonate as a leaving group

(Scheme 2.2).5 This attack can then regenerate the Ser-203 residue, which can again be involved in its native function, the hydrolysis of acetylcholine. Several pyridinium oximes have been developed, the most common of these being pralidoxime, also referred to as 2-PAM (Figure 2.2). The cationic moeity of 2-PAM, similar to the cationic amine 25 in acetylcholine, helps the oxime to bind into the inhibited AChE active site. However, the efficacy of these pyridinium oximes varies greatly on the structure of the OP,6 thus they are far from a ubiquitious solution to combatting the effects of exposure to chemical warfare agents.

S203 S203 S203

O O–N=CHR" P N O O HO Me O CHR" O R'O P O P R'O R'O Active Phosphonylated Me Me O–N=CHR" Enzyme Oxime

Scheme 2.2. The reactivation of inhibited AChE by an oxime through attack of the phosphoryl oxygen and release of the phosphonate.

NOH

N N O N NOH N O N HON NOH O

NH 2–PAM 2 HI–6 Obidoxime

NOH

N N N N N O N HON NOH HON NOH O NOH

NH2 HLö–7 MMB–4 TMB–4

Figure 2.2. Pyridinium oximes that have been developed for use as reactivating agents towards recovering the activity of inhibited AChE.

If left untreated after inhibition, the O-alkyl group of the OP can undergo a cleavage reaction, thereby leading to a phosphonylated serine anion in the AChE active

26 site in a process known as “aging”.7 Aging occurs when the already inhibited AChE–OP complex within the enzyme active site (with the OP bound to Ser-203 and leaving group expelled) undergoes dealkylation of its O-alkyl group (Scheme 2.3). This aging leaves a stable phosphonate anion in the active site. The rate of aging varies from minutes to hours between different OPs.8 This difference usually depends on the size of the OP compound that inhibits AChE. Currently there is no known therapeutic to reactivate aged

AChE, due to the presence of the unreactive phosphonate anion, bound to the critical Ser-

203 residue, in the enzyme active site. Previous investigations utilized alkyl sulfonates9 and phenacyl bromides10 to alkylate a model phosphonate, but attempts to alkylate the aged enzyme were unsuccessful. Thus, the search for a ubiquitous therapeutic to recover

AChE activity after the aging process remains elusive.

Ser(203) Ser(203) O O O P O P O R O Inhibited AChE Aged AChE

Scheme 2.3. The aging of inhibited AChE upon the loss of an alkyl group, leaving a phosphonate anion trapped in the enzyme active site.

The benefit of designing a therapeutic to realkylate aged AChE is twofold: a single alkylating therapeutic will treat the identical aged structures as each of the common OPs, except for tabun, result in the the same phosphonate structure after aging.

Therefore, the same oxime should be effective for all realkylated OPs. The preferred alkylating agent must be reactive enough to alkylate the phosphonate anion in the aged 27

AChE enzyme, but selective enough to minimize alkylation of other biological scaffolds in vivo. Thus, the alkylating agent must be designed in a way that would selectively target the aged AChE active site.

When examining potential scaffolds, our efforts quickly turned to quinone methides for use as a small molecule therapeutic. Quinone methides (QMs) have been used as alkylating agents for other biological applications in vitro, thus could be an intriguing target for a small molecule therapeutic. An example by Freccero showed the ability of a quinone methide intermediate to alkylate various amino acids under physiological conditions (Scheme 2.4).11 Several different amino acids were chosen to determine the ability of the quinone methide to alkylate different nucleophiles (nitrogen, oxygen, and sulfur-based amino acid side chains) and the alkylation was determined to be reversible. The ortho quinone methide intermediate was generated by either photochemical irradiation or thermal generation at 37°C at a pH of 7.8. The basic conditions allowed for more facile generation of the quinone methide intermediate after deprotonation of the phenolic oxygen.

Scheme 2.4. Alkylation of amino acids by quinone methide intermediate. The quinone methide was generated by either photochemical or thermal means.13

An example from Turnbull demonstrated the ability of a quinone methide intermediate to alkylate a phosphodiester (Scheme 2.5).12 In this case, the quinone methide was generated by oxidation with lead (II) oxide. Once again, the precursor in this case was a phenolic compound. The target for Turnbull’s alkylating agent was DNA modification, specifically the phosphodiester backbone. Further, because of the reversibility of quinone methide alkylation, Turnbull took advantage of a subsequent, intramolecular lactonization to prevent reversibility. Importantly, the acid catalyzed nucleophilic attack of water was found not to compete with alkylation of the phosphodiester.

O O O O O O

P OBu O HO O OBu 35˚ C

R R R H H H N O N O N O R= H, CH3 O O

O P OBu O P OBu O O OBu O OBu

Scheme 2.5. Alkylation of a phosphodiester by quinone methide intermediate. The quinone methide was generated by a lead (II) oxidation.14

Along with the targeting of the DNA phosphodiester backbone, Rokita has demonstrated the ability of these intermediates to target nucleoside bases of DNA.13 In this instance, the quinone methide intermediate was generated via the cleavage of a silyl ether in the presence of fluoride. Due to the fluoride-induced cleavage, the generation of the quinone methide intermediate was performed in DMF instead of aqueous conditions.

Interestingly, the quinone methide intermediate was shown to alkylate the less

29 nucleophilic nitrogen of the deoxyadenosine molecule (Scheme 2.6). The authors hypothesized this result was determined by the thermodynamic stability of the alkylated product through attack by the exocyclic nitrogen.

Scheme 2.6. Alkylation of deoxyadenosine by quinone methide intermediate.15 The quinone methide was generated by a fluoride-induced cleavage of a silyl ether.

In the literature, quinone methides have predominantly been generated by oxidative or photochemical means due to higher yield of the reactive intermediate, but for in vivo applications, the QM intermediate must be generated thermally. Often, these quinone methide precursors (QMPs) are benzyl ammonium salts, which expel the cationic amine leaving group upon heating to yield the reactive quinone methide intermediate (Scheme 2.7). The structure of such potential QMPs also strongly mimics edrophonium, an oxyanilinium-based inhibitor of AChE that is known to bind in the active site14 and even has an extant X-ray crystal structure bound in the active site of

AChE.15 Another potentially useful aspect of QMs is their ability to be delivered in an unreactive prodrug form. Moreover, they have been shown to possess highly tunable reactivity through modification of substituents on the aromatic ring16 or the amine leaving

30 group.17 Therefore, we seek to focus on a subset of para-QMPs as candidates for the realkylation of aged AChE.

R2N Cl heat or hv + R2NH O O OH O

Scheme 2.7. Generation of a para-quinone methide intermediate via thermal generation from a benzyl ammonium salt.

2.2 SYNTHESIS OF QUINONE METHIDE PRECURSOR

A reliable and efficient method for producing amines via reductive amination was developed due to the efforts of Dr. Carolyn Reid and the Callam research group at The

Ohio State University. This protocol aided in establishing a facile approach towards the synthesis of a large library of QMPs. For example, many different QMPs have been generated through the starting aldehyde vanillin, in either the ortho or para configuration, which showed the ability to append a wide variety of amines to function as the leaving group (Figure 2.3). The library as a whole also allowed for an examination of the electronic effects on alkylation by our QMPs by varying substituents on the aromatic ring.

O O N N N N N N N N OH OH OH OH

O O O O O O O O OH OH OH OH

O O H H H H H H H H N N N N N N N N Cl Cl OH Cl OH OH Cl OH Cl Cl Cl Cl

O O O O O O O O OH OH OH OH

O O N N N N N N N N I I OH I OH I OH OH I I OH I I

O O O O O O O O OH OH OH OH

O O N N N N N N N N I OH I OH I I I OH I OH I I

O O O O O O O O OH OH OH OH

Figure 2.3. Library of ortho and para QMPs derived from vanillin generated through the joint synthetic efforts of the Callam and Hadad research groups.

As developed by Reid and Callam, a typical reductive amination would include the reaction of an aldehyde with an amine along with sodium cyanoborohydride in acidic methanol. Typical yields for this step to generate the neutral amine products were 80%.

The quarternary ammonium compounds could then be protonated by HCl addition in

~90% yield. All compounds were characterized by Reid and Callam by NMR spectroscopy, MS and other analytical techniques.

The results presented herein will only focus on a small subset of this QMP library, specifically a selection of four neutral QMPs derived from p-vanillin and four protonated

QMPs derived from p-vanillin. In this small subset, the amine leaving group varied between dimethyl amine, pyrrolidine, piperidine, and morpholine (Figure 2.4). In the

32 established protocol (Scheme 2.8), secondary amines were reacted with an aldehyde substrate to produce a conjugated iminium ion which was reduced in situ by sodium cyanoborohydride (77–86%). The resulting amines (1–4) were isolated via column chromatography and then dissolved in methanol and toluene followed by slow addition of hydrochloric acid to yield the corresponding hydrochloride ammonium salt in 93–98% yield (1–4 HCl).

O H H H H N N N N

Cl Cl Cl Cl

O O O O OH OH OH OH 1-HCl 2-HCl 3-HCl 4-HCl

O N N N N

O O O O OH OH OH OH 1 2 3 4

Figure 2.4. Subset of para QMPs derived from vanillin subjected to further analytical studies to determine their efficacy as alkylating agents.

H R HN R2N O R NH, HCl, NaCNBH 2 2 3. Cl CH3OH, reﬂux, 2 h 5M HCl

CH3OH, toluene, rt, 5 min O O O OH OH OH

77-86% 93-98% O R NH = N NH 2 , NH, NH H ,

Scheme 2.8. Reductive amination protocol developed to synthesize QMPs.

2.3 ALKYLATION STUDIES OF QUINONE METHIDE PRECURSORS WITH

MODEL NUCLEOPHILES

In order to determine the effectiveness of these QMPs, studies with model nucleophiles were undertaken. Our efforts sought to begin to understand the ability of our QMPs to act as alkylating agents against a broader range of nucleophiles first, instead of immediately focusing on closer mimics to the target nucleophile, aged AChE. This approach would allow us to study the effect of our QMPs in a wide variety of reaction conditions to optimize the necessary conditions for alkylation. These experiments would seek to trap the thermally-generated quinone methide intermediate with a thiol (p- toluenethiol), alcohol (benzyl alcohol), or amine (piperidine) as nucleophiles (Figure 2.5).

SH OH N H

Figure 2.5. Nucleophiles used as trapping agents to determine effectiveness of alkylation by QMPs. A thiol (p-toluenethiol; left), amine (piperidine; center), and alcohol (benzyl alcohol; right) were examined to sample a wide range of nucleophilic strength.

Each of the compounds (Figure 2.4) was separately subjected to reaction with p- toluenethiol, piperdine and benzyl alcohol at 100 °C for 3 h (Scheme 2.9). All of the above reactions were performed in a 1:1 mixture of methanol:water at 100 °C for 3 h.

All reactions were monitored by GC/MS for percent conversion based on an internal standard calibration. Isolated yields were determined after column chromatography.

R2N Nuc Nuc, 100 °C

1:1 (CH3OH:H2O) O O OH OH

R2N Nuc Cl Nuc, 100 °C

1:1 (CH3OH:H2O) O O OH OH

Scheme 2.9. Reaction conditions for trapping studies of QMPs with model nucleophiles using neutral (top) and protonated (bottom) QMPs derived from p-vanillin.

Across both the neutral and protonated compounds, the results throughout each of the different QMPs were fairly consistent. In the case of the neutral precursors (Table

2.1), the highest yield of product was seen with the thiols, which was predicted due to the greater nucleophilic strength of the sulfur-based nucleophile. The nitrogen-based piperidine saw similarly high alkylation of each of the neutral precursors. However, there was very little alkylation observed with benzyl alcohol. Across each of the different leaving groups, there was a fair amount of consistency, which also makes sense when examining similar secondary amines.18

Table 2.1. Results from trapping studies with neutral QMPs and different nucleophiles.

Compound Nucleophile Conversion Yield 1 4-methyl-benzenethiol 96 84 1 piperdine 86 80 1 benzyl alcohol 0 0 2 4-methyl-benzenethiol 95 87 2 piperdine 86 77 2 benzyl alcohol 11 8 3 4-methyl-benzenethiol 96 83 3 piperdine 88 81 3 benzyl alcohol 14 11 4 4-methyl-benzenethiol 95 83 4 piperdine 93 79 4 benzyl alcohol 15 13

In the case of the protonated precursors (Table 2.2), the highest yield of product was seen with the thiols, which were found to be in similar yield to the neutral precursors.

The nitrogen-based piperidine nucleophile revealed similarly high alkylation of each of the protonated precursors. In the case of the benzyl alcohol, there was some limited alkylation observed, even though it was not comparable to the nitrogen or sulfur-based nucleophiles. However, alkylation with the oxygen-based nucleophile was more prevalent when the leaving group was protonated. This result may suggest the protonated 36 leaving group would produce a more reactive alkylating agent with less-reactive nucleophiles. Once again, there was some consistency seen across the different secondary amine leaving groups employed in the QMP. With this initial proof-of- concept completed, we felt comfortable with our efforts and ability to show the efficacy of these QMPs to act as alkylating agents against model nucleophiles.

Table 2.2. Results from trapping studies with protonated QMPs and different nucleophiles.

Compound Nucleophile Yield 1-HCl 4-methyl-benzenethiol 83 1-HCl Piperdine 78 1-HCl benzyl alcohol 8 2-HCl 4-methyl-benzenethiol 83 2-HCl Piperdine 78 2-HCl benzyl alcohol 12 3-HCl 4-methyl-benzenethiol 85 3-HCl Piperdine 81 3-HCl benzyl alcohol 10 4-HCl 4-methyl-benzenethiol 84 4-HCl Piperdine 69 4-HCl benzyl alcohol 16

2.4 UV-VIS STUDIES OF QUINONE METHIDE PRECURSORS USING MODEL

PHOSPHONATE

In order to test the efficacy of our precursors against a phosphonate as a simple model for the aged AChE active site, we sought to replicate a study from Steinberg10 to indirectly monitor alkylation by UV-vis spectroscopy. The original study by Steinberg was also focused on discovering a selective alkylating agent to target the aged AChE active site. Instead of benzyl ammonium salts (like our QMPs), Steinberg synthesized a

37 family of phenacyl bromo compounds to act as alkylating agents (Figure 2.6). Similar to acetylcholine and 2-PAM, known compounds that bind the enzyme active site, these phenacyl bromo compounds contained a cationic moeity, which should aid in the recognition by AChE. However, Steinberg was unable to observe recovery of activity by aged AChE upon exposure to these phenacyl bromo compounds.

O O O O Br H3C Br Br Br N N

VII VIII XXV XXXV O

Br CH3 N

N NOH H3C 2–PAM XXXVI

Figure 2.6. Phenacyl bromo compounds synthesized by Steinberg in attempts to realkylate aged AChE.10 Also shown is 2-PAM, a known reactivator of inhibited AChE.

Within the report of Steinberg’s efforts though, there was an intriguing study performed to indirectly monitor the ability of these phenacyl bromo compounds to alkylate a model phosphonate that more closely resembled aged AChE. By appending a model phosphonate with a p-nitrophenol leaving group, alkylation of Steinberg’s phenacyl bromo compounds was indirectly monitored after addition of base to the synthesized alkylated product. This observation could be made by following the appearance of free p-nitrophenol, after base-induced hydrolysis from the intermediate alkylated phosphonate product. By measuring the amount of free p-nitrophenol 38 generated from its UV-vis absorption, one could then calculate the amount of phosphonate alkylated by our QMPs, assuming 100% of alkylated product is hydrolyzed to only two products (Scheme 2.10). By following this protocol, we would be able to downselect which QMPs might be more likely to alkylate the aged AChE active site.

NaO O P O O P O O O 1-4 p-NO2(C6H4) P p-NO2(C6H4) ONa OH NaOH, H2O or O + DMF, 100 °C 1-4 HCl O ONa OH

NO2 412 nm

Scheme 2.10. Alkylation of a model phosphonate appended with a p-nitrophenol group in order to indirectly monitor the appearance of p-nitrophenol upon addition of base to the intermediate product. This scheme was adapted from previous work by Steinberg.10

– + For typical experiments, the model phosphonate (p-O2NC6H4OP(CH3)(=O)O Na ,

Scheme 2.10) and a chosen QMP were dissolved separately to a concentration of approximately 5.00 x 10–4 M in DMF. The two were added to a test tube (2 mL each) and heated under N2 for 3 hours at 100 °C. The UV-vis absorption was examined directly after heating by adding the reaction mixture (1 mL) to DMF (1 mL). The background hydrolysis of the p-nitrophenol leaving group was monitored at ~433 nm in DMF. A second sample of reaction mixture (1 mL) was then added to a pH=10 solution of NaOH

(1 mL) to completely hydrolyze the p-nitrophenol leaving group, which was monitored at

412 nm in a 1:1 DMF/NaOH solution. The measure of the absorbance at 412 nm was an 39 indirect measurement of alkylation of QMP by the model phosphonate. Following the reaction between our precursor and the model phosphonate, addition of base would cause scission of the P–O(PhNO2) bond to generate p-nitrophenol, which was then monitored appropriately at 412 nm in a 1:1 solution of DMF and pH 10 sodium hydroxide.

A control reaction was performed by adding 2 mL of the model phosphonate (p-

– + O2NC6H4OP(CH3)(=O)O Na ) solution to DMF (2 mL), specifically to maintain the same concentration of phosphonate as the alkylation reactions. The background hydrolysis was monitored at 412 nm upon addition of pH=10 NaOH after heating for 3 h

(A=0.919). This value was then subtracted from the absorbance of the alkylation reactions with 1-4, as these solutions were yellow after heating, suggesting a background hydrolysis of p-nitrophenoxide that was measured at 433 nm.

However, the background hydrolysis was not included in the %alkylation calculations for 1-HCL to 4-HCL because these solutions were clear after heating, suggesting no background hydrolysis of p-nitrophenol had occurred during the course of the reaction. The concentration of p-nitrophenol was determined by an external standardization method of known concentrations of the chromophore (1.65 x 10–5 M to

7.00 x 10–5 M) in a 1:1 DMF:(pH=10)NaOH solution. A linear fit was calculated to fit a

Beers Law equation (y = 18249x + 0.1541, R2 = 0.9934) to solve for the concentration of the reaction mixtures upon hydrolysis at 412 nm, where y was set equal to the absorbance at 412 nm and x was set equal to the concentration of the known stock solutions of p- nitrophenol.

Our initial experimental protocol was not ideal for mimicking physiological conditions necessary to the in vivo goal of a specific realkylating agent for aged AChE.

Certainly, the reaction conditions of DMF and 100°C will need optimization in the future.

However, our initial goal was to determine if our QMPs could be suitable under any circumstances for the alkylation of an electron-poor phosphonate. The results of our model phosphonate studies varied greatly between the neutral QMPs studied and their protonated counterparts. Presumably due to the high temperature necessary to increase the rate of alkylation, there was a large background of p-nitrophenol observed in our control experiment (Figure 2.7), evident by a yellow solution observed directly after heating at 433 nm (Figure 2.8). Of the four neutral precursors tested with the model phosphonate, only 3 showed an absorbance at 412 nm (after base-induced hydrolysis of any intermediate alkylated product) above the control reaction. Thus, for 1, 2, and 4, no quantifiable alkylation could be determined. The extent of alkylation for 3 was calculated to be 8.5% based on external standardization methods (Table 2.3), an amount of alkylation similar to the alcohol-based nucleophiles used in our previous experiments.

Phosphonate Control

1 DMF Base Absorbance 0 300 350 400 450 500 550 600 -1 Wavelength (nm)

Figure 2.7. UV-vis spectrum of control reaction where the model phosphonate (p- – + O2NC6H4OP(CH3)(=O)O Na ) was heated in DMF for 3 h at 100°C (red). At this initial reading, p-nitrophenol released in the initial heating was monitored in DMF at 433 nm. After initial measurement, base was added to determine the extent of phosphonate hydrolysis releasing the p-nitrophenoxide group, monitored at 412 nm.

Reaction of 4 with Model Phosphonate

4 3 2 DMF 1 Base Absorbance 0 300 350 400 450 500 550 600 -1 Wavelength (nm)

Figure 2.8. UV-vis spectrum of 4 reacted with model phosphonate (p- – + O2NC6H4OP(CH3)(=O)O Na ) at 100°C in DMF. Initial reaction in DMF (red) and after addition of base (blue) showed significant amounts of p-nitrophenol in solution. The signal at 412 nm (blue) did not exceed what was seen in the background reaction, thus no quantifiable alkylation could be observed via this indirect method.

Table 2.3. Percentage alkylation of neutral QMPs using a model phosphonate.

Compound Absorbance (412 nm) Percentage Alkylation 1 0.97 N/Aa 2 0.48 N/Aa 3 1.26 8 4 0.84 N/Aa a In case of 1–4, background hydrolysis of control reaction was accounted for in %alkylation calculation. Absorbance at 412 nm of reaction was less than that seen in the control reaction, thus no quantifiable alkylation by QMP could be calculated.

Interestingly, each of the four protonated para-QMPs showed no initial background reaction after 3 h of heating, presenting a clear solution with no observable absorption at 433 nm directly after heating (Figure 2.9) . Presumably, this lack of an observed background reaction is due to the presence of the proton on the amine leaving group, although the specific mechanism behind the lack of a background reaction remains unclear. The yellow color assigned to the presence of p-nitrophenol only appeared upon addition of base, presumably indicating the cleavage of the chromophore from an intermediate alkylated product. Absorbance at 412 nm in the case of the protonated

QMPs showed more substantial alkylation than any of the neutral precursors tested, ranging from 43–50% (Table 2.4).

Reaction of 1-HCl with Model Phosphonate

1 DMF Series1 Absorbance 0 300 350 400 450 500 550 600 -1 Wavelength (nm)

Reaction of 2-HCl with Model Phosphonate

5 4 3 2 DMF 1 Base Absorbance 0 -1 300 350 400 450 500 550 600 -2 Wavelength (nm)

continued Figure 2.9. UV-vis spectra of protonated precursors reacted with model phosphonate at 100°C in DMF. (a) 1-HCl, (b) 2-HCl, (c) 3-HCl, and (d) 4-HCl. Initial reaction in DMF (red) showed no evidence of a background reaction (no signal at 433 nm) indicative of release of p-nitrophenol from the phosphonate itself. However, there is a significant signal at 412 nm after addition of base, indicative of base-induced cleavage of p- nitrophenol from an intermediate alkylated product. The %alkylation was calculated from an external standardization, ignoring any background hydrolysis.

Figure 2.9 continued

Reaction of 3-HCl with Model Phosphonate

2 DMF 1 Base Absorbance 0 300 350 400 450 500 550 600 -1 Wavelength (nm)

Reaction of 4-HCl with Model Phosphonate

5 4 3 2 DMF Base

Absorbance 1 0 300 350 400 450 500 550 600 -1 Wavelength (nm)

Table 2.4. Percentage alkylation of protonated QMPs using a model phosphonate.

Compound Absorbance (412 nm) Percentage Alkylation 1-HCl 1.26 50 2-HCl 1.21 47 3-HCl 1.19 46 4-HCl 1.13 43

After observing these results, we were able to determine that the para protonated precursors provided the most robust alkylation of the model phosphonate. In an effort to further investigate our expansive library of para QMPs, we sought to test our model phosphonate UV-vis screen against a modified QMP appended with an acetal group instead of a hydroxyl group. At first inspection, one might consider this type of QMP disadvantageous because of the apparent inability to form the quinone methide intermediate. However, this type of functional group could allow the small molecule therapeutic to be introduced in more of a pro-drug form. Our experiments found this acetal-based QMP actually yielded the largest signal at 412 nm of any QMP subjected to our model phosphonate screen (Figure 2.10). When the small, observable background hydrolysis was taken into account, the %alkylation was calculated to be 59.9% for 3-

Acetal-HCl.

Reaction of 3-Acetal-HCl with Model Phosphonate

3# 2.5# O 2# O 1.5# 1# DMF H Cl 0.5# N 0# Base Absorbance !0.5# 0# 200# 400# 600# 800# 1000# 1200# !1# !1.5# !2# Wavelength (nm)

Figure 2.10. UV-vis spectrum of 3-Acetal-HCl (left) reacted with the model phosphonate – + (p-O2NC6H4OP(CH3)(=O)O Na ) at 100°C in DMF. Initial reaction in DMF (red) showed a slight background hydrolysis of p-nitrophenol from the model phosphonate. After addition of base (blue), a significant amount of p-nitrophenol was observed in solution at 412 nm. The signal at 412 nm (blue) exceeded what was observed in all other QMPs presented.

Thus, we believed our efforts using this methodology originally developed by

Steinberg10 gave us an indirect method of monitoring the alkylation of a model phosphonate by our QMPs. However, the UV-vis protocol proved itself to be somewhat unreliable over the course of replicate trials. Further, the relatively low amounts of alkylation caused us to think more critically about our indirect method of monitoring alkylation. Namely, we reconsidered the assumption made about the base-induced hydrolysis of the intermediate alkylated phosphonate (Scheme 2.10). In our previous calculations, we assumed that only p-nitrophenol was hydrolyzed upon the addition of base. Yet, if the alkylating agent is also hydrolyzed in an equilibrium process as a benzyl alcohol (Scheme 2.11), then the percentage alkylation of the model phosphonate may

47 actually be significantly higher than the results of the indirect UV-vis screen should suggest. Thus, our efforts moved away from the indirect UV-vis screen and were refocused on determining if our QMPs were suitable alkylating agents for model phosphonates.

NaO O P O ONa O p-NO2(C6H4) P O O O O p-NO (C H ) P 2 6 4 OH + NaOH, H2O O NaOH, H2O + NaO O ONa OH

O OH NO2

Scheme 2.11. Proposed dual hydrolysis pathway to explain suppressed alkylation of model phosphonate by QMPs. Previous calculations in the indirect UV-vis screen assumed only the presence of the pathway generating p-nitrophenol (right). Also, the other P–O bond in the intermediate alkylated phosphonate could be cleaved to yield a benzyl alcohol product that would not produce a signal at 412 nm.

2.5 FURTHER STUDY INTO POTENTIAL DUAL HYDROLYSIS PATHWAY

In order to further examine our dual hydrolysis hypothesis, and to confirm alkylation of the model phosphonate by QMPs, the reaction was monitored by tandem

HPLC/ESI-MS-TOF, and subsequently by 31P NMR spectroscopy. These techniques would allow for a better opportunity to monitor the potential products generated, and perhaps other previously unconsidered pathways that may be responsible for the degradation of the alkylated product.

A representative QMP of the neutral (1), protonated (1-HCl), and acetal (1-

Acetal-HCl) form were each reacted with the model phosphonate in water at 100°C for 3 h and analyzed using the HPLC/ESI-MS-TOF to identify each of the species in solution after heating. The HPLC analysis was performed on an Agilent G6230A HPLC/TOF mass spectrometer. The components included a CTCPAL auto-injector with 1 µL sample loop, Bin Pump (G42220A) operating a a maximum pressure of 1200 bar, and a solvent ratio of 50:50 water:acetonitrile with 0.1% formic acid for ionization that was flowing at

0.3 mL/min for 3 minutes.. The HPLC column was a C-18 50 mm high pressure column without guard. The time-of-flight (TOF) was calibrated daily and data were acquired in positive ion mode with total ion counting turned on. The source parameters for the TOF were: gas temperature of 325 °C, gas flow of 10 L/min, nebulizer of 20 psi, sheath gas temperature of 400 °C and a sheath gas flow of 10 °C. The scan source parameters were run with a voltage capacity of 3000. Reference masses during the scan were obtained at

121.0508 and 922.0979 amu.

For each species, only trace amounts of the intermediate alkylated product were observed. However, the major peaks observed in each reaction were the four products yielded from a dual hydrolysis degradation of the intermediate alkylated product (Scheme

2.11). The %alkylation from this method could not be quantified though, due to the inability to separate each peak in the HPLC. Efforts are ongoing to examine different conditions to separate each individual species for quantitative analysis. In the case of 1

(Table 2.5) and 1-HCl (Table 2.6), the observed products were identical, due to the identical QMP scaffold for each precursor. The observed products in the case of 1-

Acetal-HCl (Table 2.7) also matched the four products resulting from a dual hydrolysis cleavage of the intermediate alkylated phosphonate. Thus, not only did we confirm the alkylation of our model phosphonate by these QMPs, but also our efforts uncovered a more accurate, efficient means of screening our QMP alkylation studies. The tandem

HPLC/ESI-MS-TOF showed evidence of each of the two-hydrolysis pathways when examining the alkylation of 1-HCl as both the p-nitrophenol and the QMP benzyl alcohol were present.

Table 2.5. Results from HPLC/ESI-MS-TOF analysis of reaction between 1 and model phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated product were observed. The major observed products resulted from a dual hydrolysis degradation of the intermediate alkylated product.

Major MS QMP Hydrolysis Products Peaks

139.0271

232.0496

154.0621

217.0144

Table 2.6. Results from HPLC/ESI-MS-TOF analysis of reaction between 1-HCl and model phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated product were observed. The major observed products resulted from a dual hydrolysis degradation of the intermediate alkylated product.

Major MS QMP Hydrolysis Products Peaks

139.0261

232.0510

154.0611

217.0136

Table 2.7. Results from HPLC/ESI-MS-TOF analysis of reaction between 1-Acetal-HCl and model phosphonate in water at 100°C for 3 h. Trace amounts of the alkylated product were observed. The major observed products resulted from a dual hydrolysis degradation of the intermediate alkylated product.

Major MS QMP Hydrolysis Products Peaks OH HO O P O 139.0301

O O NO 230.0333 O 2 O Exact Mass: 139.03 Exact Mass: 230.03

H N HO O N 2 O Exact Mass: 179.09 152.0431 P O OH O O 217.0156 Exact Mass: 152.05 Exact Mass: 217.01

To further examine the alkylation of the model phosphonate by our QMPs, 31P

NMR spectroscopy was used to follow the different phosphorus species generated from heating the two together in D2O for 3 h at 100°C (Scheme 2.12). Initial attempts showed that this reaction was sluggish in D2O, so interestingly, the reaction was also performed under microwave conditions. A solution of a precursor QMP and p-nitrophenoxy methylphosphonate were reacted in D2O using microwave irradiation. The reaction mixture was sealed in a pressure-tight vessel and subjected to microwave irradiation using a CEM Discover SP-D with Explorer 24/28 microwave reactor. Microwave experiments were conducted under a programmed method of heating parameters. Under continuous stirring, the reaction was heated to 180°C for 20 min at 175 W for 5 cycles. 52

O O O O O N P 2 O microwave O P O N O O 2 D2O H N O Cl O

Scheme 2.12. Alkylation of 1-Acetal-HCl by model phosphonate under microwave conditions. This reaction was then examined by 31P NMR spectroscopy.

In experiments conducted with Katelyn Cody, an undergraduate researcher, the results of our 31P NMR studies indicated the presence of four different phosphorus species after heating (Figure 2.11). The peak at 25.1 ppm is representative of the model phosphonate starting material itself. However, this species is also regenerated in the dual hydrolysis pathway along with the corresponding benzyl alcohol. The other most prominent peak at 20.6 ppm actually corresponds to a phosphonic acid species. This particular phosphorus species would be resultant from cleavage of both P–O bonds in the intermediate alkylated product. The other two signals in the 31P NMR remain unidentified. However, it is surmised that these two signals may correspond to the other phosphonate anion from the dual hydrolysis pathway and trace amounts of the intermediate alkylated product (Scheme 2.11).

O N 2 O P O O

Figure 2.11. 31P NMR analysis of the reaction depicted in Scheme 2.12.

2.6 CONCLUSIONS

There is a dire need for the development of small molecule therapeutics that can protect military and civilian populations from exposure to toxic OP compounds.

Specifically, our efforts targeted an alkylating agent that could reactivate aged AChE to restore enzyme function. A quinone methide precursor (QMP) was designed to act as a small molecule therapeutic to thermally generate a reactive quinone methide precursor that could target the phosphonate anion in the aged AChE active site. With a reductive amination protocol established by our synthetic collaborators, a library of QMPs was created.

A small subset of this library, with QMPs derived from p-vanillin, was subjected to further studies to determine their effectiveness in alkylating model nucleophiles. Both the neutral and protonated precursors were shown to alkylate amines and thiols to a robust extent, but were less effective in alkylating an alcohol nucleophile. After this initial proof-of-concept, our attention turned to an indirect UV-vis screen based on a protocol previously developed by Steinberg to determine the efficacy of our QMPs against a model phosphonate.

These UV-vis studies initially seemed to indicate the ability of the protonated

QMPs derived from p-vanillin, and an acetal-based QMP, to be effective at alkylating a

– + model phosphonate (p-O2NC6H4OP(CH3)(=O)O Na ). However, closer inspection of our data revealed a dual hydrolysis pathway that could be responsible for suppressed quantification of alkylation using the indirect UV-vis method. In order to confirm the alkylation of the model phosphonate, HPLC/ESI-MS-TOF analysis and 31P NMR spectroscopy were used to confirm the presence of the products derived from two degradation pathways of the alkylated phosphonate.

In the larger context of the project, these alkylation studies have helped to identify potential lead compounds throughout the QMP library generated in the Hadad research group. Further computational studies by other colleagues have sought to learn more about the operative mechanism of alkylation by these QMPs. Other proteomic studies using lysozyme as a model protein have further identified QMPs that are not overly reactive with the protein surface itself. Based on the criteria that a QMP (1) alkylates model nucleophiles, (2) alkylates the model phosphonate, and (3) does not alkylate the

55 surface of a model protein, certain QMPs have been identified as lead compounds that may be suitable for further study with aged AChE.

2.7 REFERENCES FOR CHAPTER 2

1. Quinn, D. M. Chem. Rev. 1987, 87, 955-979.

2. Botos, I.; Wlodawer, A. Curr. Opin. Struct. Biol. 2007, 17, 683-690.

3. Jokanovic, M.; Prostran, M. Curr. Med. Chem. 2009, 16, 2177-2188.

4. Worek, F.; Szinicz, L.; Eyer, P.; Thiermann, H. Toxicol. Appl. Pharmacol. 2005, 209, 193-202.

5. Tara, S.; Elcock, A. H.; Kirchhoff, P. D.; Briggs, J. M.; Radic, Z.; Taylor, P.; McCammon, J. A. Biopolymers 1998, 46, 465-474.

6. Worek, F.; Thiermann, H.; Szinicz, L.; Eyer, P. Biochem. Pharmacol. 2004, 68, 2237- 2248.

7. Michel, H. O.; Hackley, B. E., Jr.; Berkowitz, L.; List, G.; Hackley, E. B.; Gillilan, W.; Pankau, M. Arch. Biochem. Biophys. 1967, 121, 29-34.

8. Barak, D.; Ordentlich, A.; Segall, Y.; Velan, B.; Benschop, H. P.; De Jong, L. P. A.; Shafferman, A. J. Am. Chem. Soc. 1997, 119, 3157-3158.

9. Blumbergs, P.; Ash, A. B.; Daniher, F. A.; Stevens, C. L.; Michel, H. O.; Hackley, B. E., Jr.; Epstein, J. J. Org. Chem. 1969, 34, 4065-4070.

10. Steinberg, G. M.; Lieske, C. N.; Boldt, R. J. Med. Chem. 1970, 13, 435-446.

11. Modica, E.; Zanaletti, R.; Freccero, M.; Mella, M. J. Org. Chem. 2001, 66, 41.

12. Zhou, Q.; Turnbull, K. D. J. Org. Chem. 2001, 66, 7072.

13. Veldhuyzen, W. F.; Shallop, A. J.; Jones, R. A.; Rokita, S. E. J. Am. Chem. Soc. 2001, 123, 11126.

14. Grove, S. J.; Kaur, J.; Muir, A. W.; Pow, E.; Tarver, G. J.; Zhang, M. Q. Bioorg. Med. Chem. Lett. 2002, 12, 193-196.

15. Ravelli, R. B.; Raves, M. L.; Ren, Z.; Bourgeouis, D.; Roth, M.; Kroon, J.; Silman, I.; Sussman, J. L. Acta Crystallogr., Sect. D 1998, 54, 1359-1366.

16. Freccero, M. Mini-Rev. Org. Chem. 2004, 1, 403-415.

17. Weinert, E. E.; Dondi, R.; Colloredo-Melz, S.; Frankenfield, K. N.; Mitchell, C. H.; Freccero, M.; Rokita, S. E. J. Am. Chem. Soc. 2006, 128, 11940-11947.

18. Dewick, P. M. Essentials of Organic Chemistry: For Students of Pharmacy, Medicinal Chemistry and Biological Chemistry; Wiley: Chichester, England, 2006.

Chapter 3 : Molecular Baskets as a Pseudo-Bioscavenger for the Entrapment and Hydrolysis of Organophosphorus Nerve Agents

3.1 INTRODUCTION

The previous chapter introduced one method towards combatting the effects of toxic nerve agents by attempting to find a suitable re-alkylating agent for aged acetylcholinesterase (AChE). And, progress was shown to be made towards the use of quinone methide precursors (QMPs) as a potential small-molecule therapeutic to reverse the aging process upon OP inhibition of AChE. However, an alternate therapeutic route could be imagined that would attempt to counter the effects of organophosphorus (OP) nerve agent exposure before inhibition of the enzyme could occur. If a bioscavenger, or a similar synthetic macromolecule, could be designed that could sequester and perhaps hydrolyze the OP compound before it binds and inhibits AChE, then the toxic effects of exposure could be prevented (Figure 3.1). A catalytic bioscavenger of OP compounds would be even more beneficial towards reducing or removing their toxicity upon inhibition.

Figure 3.1. Potential therapeutic routes to combat the inhibition and aging of OP exposure. (1) The use of a bioscavenger to sequester the OP before binding to AChE. (2) The use of pyridinium oximes to reverse inhibition. (3) The use of an alkylating agent to reverse the aging process after OP inhibition.

There have been previous attempts at targeting a true bioscavenger for the purpose of binding and hydrolyzing OPs. Significant efforts have been focused on altering butyrylcholinesterase (BuChE) as a catalytic bioscavenger because of its similarities to AChE, a sister protein. BuChE has a consistent active site when compared to AChE, except for some missing aromatic amino acid residues, which make for a larger active site in BuChE.1 Therefore, BuChE can bind and hydrolyze OP compounds much like AChE binds OPs; however, BuChE is only a stoichiometric scavenger of OPs, thus requiring an extremely high concentration in order to be an effective bioscavenger.2 The

Hadad Group at The Ohio State University has studied various BuChE mutants towards understanding how mutants interact with various OPs towards finding a catalytic 59 bioscavenger using molecular modeling techniques such as molecular docking and molecular dynamics simulations.3

Another enzyme found in the human body that has been studied as a potential bioscavenger for OPs is paraoxonase-1 (PON1). The exact function of PON1 in the body remains unclear; however, the enzyme has been shown to hydrolyze various pesticides and chemical warfare agents.4 Much like BuChE, PON1 also has not shown sufficient activity to suggest it would be an efficient, catalytic bioscavenger for a wide range of OP compounds. Further complicating matters, the exact active site and mechanism of hydrolysis remains unknown, although efforts have been made in the Hadad group at the

Ohio State University to elucidate the reaction mechanism, along with continued studies of potential mutants optimized for use as a catalytic bioscavenger towards OPs.5

While these efforts to find a true catalytic bioscavenger by mutating native enzymes, another potential route pursued in the literature has been the synthesis of a synthetic macromolecule that could bind and hydrolyze OPs in much the same way as these true bioscavengers. Many different scaffolds have been studied in the literature that could act as a pseudo-bioscavenger for various OP compounds, a small portion of which will be presented here as background for the inspiration of our current research. The synthetic macromolecule could then be tuned and optimized to perform catalytic hydrolysis on the guest OP molecule.

One such system was synthesized by Smith where a β-cyclodextrin (a cyclic oligosaccharide) was appended with guanidinium units; indeed, the cationic guanidinium units were shown to aid in the binding of various aryl phosphates when compared to

60 unmodified β-cyclodextrin due to a favorable electrostatic interaction.6,7 In fact, unmodified β-cyclodextrin itself has been shown to hydrolyze sarin and soman to a very slight extent, removing some of its toxicity, although certainly not enough to be used as a therapeutic.8 The use of a pentamethylene linker between the two guanidinium units actually was shown to weaken the binding event due to the association of the guanidinium with the host cavity, not the aryl phosphates (Figure 3.2). Also, although this modified β-cyclodextrin was shown to bind certain aryl phosphates, there was no observed hydrolysis of bound OP molecules that were recognized by the guest.

NH2 H2N H N NH 2 (CH2)5 2 H2N NH2 N N H H NH HN HN NH

!-CD !-CD

Figure 3.2. Modified β-cyclodextrin synthesized by Smith6 with appended guanidinium units acting as arms over a cavity to attract aryl phosphates through a favorable electrostatic interaction.

Another macromolecule scaffold that has been investigated is a calixarene synthesized by de Mendoza that was shown to bind a novel phosphate.9 The calixarene itself was modified with a guanidinium unit along with an aromatic moeity that allowed for attraction of various guest molecules through hydrogen bonding and electrostatic

61 interactions. The phosphate DOPC (Figure 3.3) which was shown to have affinity for the calixarene was appended with a cationic ammonium moiety, which is similar to the structure of acetylcholine, the native ligand hydrolyzed in vivo by AChE. The modified calixarene was shown to bind both acetylcholine and DOPC, although in chloroform, and not under aqueous physiological conditions. Also, the hydrolysis of these molecules was not studied, although a carbonate (PNPCC) was found to be hydrolyzed by the host molecule (Figure 3.3). However, the addition of a cyclohexyl group into the calixarene scaffold showed a stronger recognition of the guest molecules studied, presumably because of a stronger preorganization of the host. Other macromolecules, including those studied by Rebek, have shown the ability to bind acetylchoine and be monitored by fluoresence.10

OH N N

O H H

O NH

Me O

N tBu tBu Me O Me OR ACh OMe MeO

NO2 Me O OMe MeO N Me OMe O O Me

tBu tBu PNPCC

Me O (CH2)6CH3 O N tBu Me O P O O Me O O R = H O (CH2)6CH3 R = CH 2 DOPC

Figure 3.3. The modified calixarene (left) synthesized by de Mendoza and the guest molecules (right) that were determined to be recognized by the host. Only PNPCC was found to be hydrolyzed by the guest in chloroform.9

While these macromolecules have shown the ability to bind phosphates, these specific examples do not target a phosphonate that more closely mimics the structure of

OP chemical warfare agents. Dalcanale showed the ability for a cavitand to detect dimethoxy methylphosphonate (DMMP, Figure 3.4) at very low concentrations (as low as ppm) when attached to a solid support by surface plasmon resonance (SPR) spectroscopy.11 The interesting structural feature of this particular cavitand is the

63 presence of the carboxylic acid moiety at the upper rim of the cavity (Figure 3.4). This carboxylic acid group can be oriented either in over the center of the cavity, or out away from the cavity. Binding was shown to be preferred when the carboxylic acid group was oriented in over the cavity of the host molecule. The carboxylic acid seems to be acting as a hydrogen-bond donor, associating with the phosphoryl oxygen of DMMP. Once the guest molecule is recognized, it can then associate with the π system of the cavitand.

Although recognition and binding of the small OP compound was observed, hydrolysis of the OP was not found to occur upon the recognition event.

O O O P P P O O O O O O

Figure 3.4. The modified cavitand (left) synthesized by DalcanaleO showing the in/out O orientation of the COOH group and DMMP (right).11 O P P P O O O O O O

There is an example in the literature, however, that has demonstrated the ability of a synthetic macromolecule to act as a host for a chemical warfare agent (cyclosarin, GF).

Not only does the modified cyclodextrin bind GF, but it has also been shown to hydrolyze it as well and remove its toxicity under physiological conditions. The macromolecule designed by Kubik was a β-cyclodextrin scaffold appended with a

64 deprotonated oxime (Figure 3.5).12 Similar oximes have been used in the literature as a nucleophile to restore AChE activity upon inhibition by a chemical warfare agent.13 In order to determine whether the macromolecule hydrolyzed the relatively small GF, Kubik developed an indirect method of quantifying the removal of toxicity upon OP exposure.

Kubik reported the reduction of GF under physiological conditions (an aqueous

TRIS-HCL buffer at a pH of 7.40) within seconds. Kubik monitored the hydrolysis of

GF by exposing the nerve agent to the AChE enzyme in the presence of the β- cyclodextrin and acetylthiocholine. If the enzyme were inhibited by GF, the acetylthiocholine would be unable to be hydrolyzed by the human enzyme. However, if the β-cyclodextrin were found to be successful, then hydrolysis of acetylthiocholine would be observed via an Ellman assay.14 In fact, when the variable R group is equal to the deprotonated pyridinium oxime shown below (Figure 3.6), no enzyme inhibition by

GF is observable upon the initial measurement.

Figure 3.5. Substituted β-cyclodextrin designed by Kubik shown to hydrolyze GF and remove its toxicity in the presence of human AChE under physiological conditions.12

Figure 3.6. The deprotonated pyridinium oxime appended to the β-cyclodextrin scaffold (top left), cyclosarin (GF; bottom left), and acetylthiocholine (bottom right).12

Thus, Kubik has shown the ability to design a specific host complex for the hydrolysis of a specific OP. However, a challenge still facing this host/guest chemistry with regards to the sequestration and hydrolysis of chemical warfare agents is the search for a ubiquitous solution that would be appropriate under physiological conditions for a 66 wide variety of OPs. This understanding of the present state of the host/guest chemistry between pseudo-bioscavengers and chemical warfare agents caused us to pursue other molecular scaffolds that have shown tunability towards the encapsulation of a wide variety of small molecules. Namely, our attention was drawn to a potential collaboration with the Badjic Research Group at The Ohio State University and their efforts towards designing molecular baskets as small molecule receptors.

There has been a history of collaboration between the Badjic and Hadad research groups in recent years. While the Badjic research group has synthesized several molecular baskets and studied their interaction with small molecule guests, the Hadad group has provided support through molecular modeling simulations to examine the conformational flexibility of the hosts themselves, and the study of the interaction between the host and guest molecules.15,16 Through the use of Monte Carlo simulations to randomly examine various torsions, to molecular dynamics simulations to examine the molecular trajectory of the host/guest complex, to molecular docking simulations to examine the interaction of host and guest, the Hadad group has been able to provide understanding to experimental results seen in the Badjic research group and has aided in the guidance of synthetic efforts.

The molecular basket scaffold designed by Badjic is an attractive host for the recognition of OPs for many reasons. First, the three arms at the top of the basket, which often serve as molecular gates, can be functionalized in various ways to attract a small molecule of choice. Second, based on the structure of the base and walls of the basket, the size of the cavity can be tuned to match various chemical warfare agents. Third, by

67 nature, the basket itself has a hydrophilic region at the rim of the basket and a hydrophobic region deep inside of the cavity (Figure 3.7). This dualistic nature of the cavity should be attractive for chemical warfare agents, which contain an alkyl group on the phosphonate that should be recognized by the hydrophobic inner cavity.

Figure 3.7. A sample molecular basket developed by the Badjic research group showing the hydrophilic region at the top of the basket and a hydrophobic region in the cavity of the basket, which should be a match for OP chemical warfare agents.

The particular molecular basket first studied by this collaborative effort between the Hadad and Badjic research groups towards a more universal pseudo-bioscavenger used the following scaffold (Figure 3.8). At the base of the scaffold is a benzene ring, which has attached a bicycloheptane ring on three sides, which begins to build the three walls of the basket. At the top of the basket, there is an imide that can be functionalized to a variety of hydrophilic groups. Since we are targeting a macromolecule that could be a potential in vivo therapeutic for OP compounds, we first sought to use decarboxylated amino acids (essentially the amino acid side chain) as the gate of the basket. Several different amino acid side chains were chosen to represent polar, non-polar, basic, and 68 acidic residues. The following research will focus on the use of the histidine side chain for each of the three arms of the molecular basket.

R AA O N O

O R N AA O

O N AA O R

O OH NH2 OH SER TYR ASN

CH3 CH3 HN CH3

ALA TRP LEU PHE

N NH O OH

HIS ASP

Figure 3.8. (a) The molecular basket scaffold (top) where R=H and AA=various amino acid side chains. (b) Various amino acid side chains studied computationally by the Hadad group (bottom).

3.2 COMPUTATIONAL METHODS

17 Each molecular basket was built with the GaussView program using a C3v symmetric core, and the basket arms were each capped with an amino acid side chain

(Figure 3.8). The resulting structures were then utilized in a Monte Carlo conformational search using the MacroModel program.18 The MCMM Torsional Sampling protocol was used as the search method. The energy window for saving structures was set to 100 kJ/mol to ensure that higher energy structures are sampled, and redundant conformers were eliminated using a root mean square deviation (RMSD) cutoff of 2.5 Å. The five lowest energy and five unique highly populated structures for each basket were chosen for our subsequent geometry optimizations and partial atomic charge calculations.

All basket structures were optimized with the Gaussian 09 program19 using the

B3LYP/6-31+G* level of theory.20,21 Partial atomic charges were computed using electrostatic potentials calculated at the same level of theory and sampled with the Merz–

Kollman scheme.22,23 For each molecular basket, the charges were averaged over ten conformations and these were used for subsequent molecular dynamics (MD) simulations.

The basket conformations were prepared for MD simulations using the antechamber and leap modules of the AMBER 10 program.24 Each basket was solvated with explicit TIP3P water or chloroform molecules within 10 Å of the basket in a periodic octahedral box. MD simulations of all baskets were carried out using the

SANDER implementation in AMBER with the general AMBER force field (FF03).25 A three-step equilibration scheme was performed on each basket with an initial

70 minimization step where the basket was held fixed and the positions of solvent molecules were relaxed. A subsequent minimization allowed for all atoms to move to minimize the system as a whole. Once sufficiently relaxed, the system underwent 20 picoseconds of a heating phase from 0 to 300 K with a small restraint on the basket to prevent drastic fluctuations in structure. The volume was kept constant, and the SHAKE algorithm26 was used to constrain bonds involving hydrogen atoms. Production dynamics were run over 5 ns using an isothermal–isobaric ensemble (NPT conditions), where temperature and pressure were held constant using a Langevin thermostat27 with a collision frequency of 1 and a constant pressure barostat with isotropic position scaling, respectively. Long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) algorithm28 with a cutoff of 10 Å.

Molecular docking simulations were performed using Autodock 4.0.29 For each basket, a total of ten ‘snapshots’ were taken from the MD trajectories that represent clusters of conformations obtained using the ptraj module of AMBER based on structural

RMS deviations. These ten snapshots were used for the docking simulations. Automated receptor preparation was performed, including merging of non-polar hydrogen atoms into adjacent heavy atoms, and solvent molecules were removed. The basket snapshots were kept rigid in the docking simulations, and a library of OP compounds were docked into the cavity of the basket. The applied scoring grid had an 0.375 Å grid spacing, with dimensions of 18.4 x 16.7 x 18.0 Å, centered on the basket cavity, to ensure the treatment of all possible binding poses. To generate charges for the OP ligands, each structure was

71 optimized at the B3LYP/6-31+G* level of theory, and Merz–Kollman nuclear-centered atomic charges were obtained using Gaussian09.

3.3 MOLECULAR BASKETS APPENDED WITH VARIOUS DECARBOXYLATED

HISTIDINES SUBJECTED TO COMPUTATIONAL PROTOCOL

Appending the molecular basket scaffold with the basic amino acid side chain histidine presented many opportunities for examining the effect of protonation state on the conformations of the basket and potential host/guest chemistry. Ideally, the basket would recognize OP chemical warfare agents at physiological pH in water. However, initial studies may be performed at any pH range, which would affect the protonation state of the imidazole rings acting as the gates of the basket. Thus, when subjecting these baskets with decarboxylated histidines acting as the caps, varying the protonation state would be necessary in order to subject different baskets to our rigorous computational protocol.

Further, another variable that was examined was the size of linker that attaches, in effect, the wall of the basket (linking from the imide nitrogen) to the functional imidazole unit. Changing the number of methylene units would certainly affect the interaction of the three arms of the basket, which could form a hydrogen-bonding network over the center of the cavity. Thus, this linker size would also affect the size of the cavity and the interaction between the host and any guest molecules.

Thus, when subjecting these varied imidazole-capped baskets, we subjected nine different molecular basket scaffolds to our computational protocol (Figure 3.9). First, we

72 looked at changing the linker size from one, two, or three methylene units. Also, we examined leaving all three imidazole rings neutral, protonating one imidazole ring

(monoprotonated), and protonating all three imidazole rings (tris-protonated). With examining these variable characteristics of the basket, a total of nine different imidazole- capped baskets were subjected to our Monte Carlo and initial molecular dynamics simulations to determine their computational flexibility.

H N N N

NH NH NH n n O O n O N N N O O O H N N N O O O NH NH NH N n N n N n

O O O

O O O N N N O O n n O n HN HN N NH HN NH

1+ 3+ H0 H H

Figure 3.9. Imidazole-capped baskets subjected to computational protocol to determine their conformational flexibility. The protonation state was varied to keep all three 0 1+ imidazole rings neutral (H , left), protonating one imidazole ring (H , center) and protonating all three imidazole rings (H3+, right). Also, the methylene unit was altered to allow n=1, 2, or 3 for a methylene, ethylene, or propylene linker between the imide nitrogen in the wall of the basket and the functional imidazole ring.

3.4 CONFORMATIONAL ANALYSIS OF DECARBOXYLATED IMIDAZOLE-

CAPPED BASKETS

Each of the nine imidazole-capped baskets with varying protonation state and linker size was subjected to the initial Monte Carlo simulation. After the initial simulation was complete, ten unique conformations were chosen to carry forward in a subsequent DFT optimization and charge calculation. Of these ten unique conformations, five were chosen as the lowest energy structures that were unique. Another five were chosen to ensure the conformational space of the basket, specifically with respect to the orientation of the histidine side chain arms being oriented in over the cavity or out away from the cavity.

Once the charge calculations were complete, each of the ten conformations for the nine different imidazole-capped baskets were subjected to the molecular dynamics protocol mentioned previously. Once the simulations were finished for each unique basket, we chose to examine different parameters to learn more about how the conformational flexibility of the basket was explored over the 5 nanosecond simulation.

First, we examined the distance between the imide nitrogen located at the top of the wall of the basket. Measuring each of the three different nitrogen-nitrogen distances throughout the simulation and then combining and averaging them (using MATLAB) gave an indication of the width of the cavity that could be compared for each of the nine baskets. Second, we examined the appropriate C–N–C–C dihedral angle to determine the orientation of the imidazole rings. This measurement would allow us to determine the

74 availability of the cavity towards OP guests and to look at the potential hydrogen- bonding network amongst the three imidazole rings (Figure 3.10).

H N

O N O

H O N

N N H O

O N O

NH H N

Figure 3.10. The two parameters examined in order to investigate the conformational flexibility of the imidazole-capped baskets, shown with the H3+, n=2 molecular basket. (a) The N–N distances between each of the imide nitrogens was examined in order to learn more about the size of the cavity (dotted blue lines). (b) The C–N–C–C dihedral angle was measured to determine the orientation of the imidazole rings; whether they were oriented in over the cavity or out away from the cavity (solid purple line).

The addition of methylene units to the linker seemed to reduce the size of the cavity, based on the N–N distance of each basket in the case of the neutral imidazole- capped baskets and the monoprotonated molecular baskets. In the neutral basket, there is a decrease from 8.6 to 7.9 Å from the methano to the propano linker. In the monoprotonated basket, there is a decrease from 8.5 to 8.0 Å from the methano to the propano linker. It seems as the size of the linker increases, it allows the arms more flexibility to orient themselves in over the cavity and interact with one another, which shrinks the N-N distance. The effect is not present in the tris-protonated basket though. 75

In fact, the largest N–N distance observed was for the tris-protonated basket with an ethano linker, matching a decarboxylated histidine residue functioning as the gate of the basket.

The analysis of our molecular dynamics simulations suggest that protonation state has a much more pronounced effect on the orientation (in over the cavity or out away from the cavity) of the imidazole caps than the number of methylene units linking the cap to the wall of the basket (Table 3.1). In the neutral and monoprotonated states, the imidazole caps have the opportunity to establish a hydrogen-bonding network. This result is evident in the preference for the imidazole caps to be “In”, oriented over the cavity of the basket. However, when the baskets are fully protonated, the hydrogen- bonding network is disrupted because there are no more nitrogen atoms that can act as a hydrogen bond acceptor. Thus, for each of the methano (n=1), ethano (n=2), and propano (n=3) linkers, the preference of the imidazole caps switches to “Out”, extending away from the cavity of the basket in the tris-protonated molecular basket.

Table 3.1. Effect of protonation state and linker size on cavity size (N–N distance) and torsional distribution (Out:In ratio) of imidazole-capped baskets.

N–N Distance (Å) Out:In Distribution

H0, n=1 8.6 36:64

H0, n=2 8.3 27:73

H0, n=3 7.9 29:35a

H1+, n=1 8.5 31:69

H1+, n=2 8.4 36:64

H1+, n=3 8.0 27:41a

H3+, n=1 8.7 52:48

H3+, n=2 8.9 56:44

H3+, n=3 8.7 41:28a ain/out torsional distribution does not add to 100% because a third mode was found where the imidazole ring extended through the side of the basket

Based on these results, it appeared the tris-protonated imidazole-capped baskets with an ethano linker (identical to decarboxylated histidine) would be most fruitful for initial study with OP compounds, both experimentally and computationally. This is because the H3+, n=2 basket has the largest N-N distance, suggesting the widest cavity, and the largest percentage of the arms oriented outside the cavity, suggesting the most openness for recognition between host and guest. Before proceeding to our molecular docking simulations with a library of OP compounds and other ligands of interest though, another degree of freedom was examined with this particular molecular basket. 77

Similarly to the C–N–C–C dihedral angle that gave an indication of the orientation of the decarboxylated histidine cap with regards to being in over the cavity or out from the cavity, a different dihedral angle could give an indication of the imidazole rings being up above the basket, or down towards the base of the basket. By measuring the N–C–C–C dihedral angle extending from the imide nitrogen through the ethano linker and the imidazole ring, this up/down distribution can be understood in much the same way as the in/out preference of the arms (Figure 3.11). Furthermore, this up/down dihedral angle can be correlated via a two-dimensional plot to the in/out torsion discussed earlier to give a more precise idea of the preference in orientation of the arms of the basket throughout the molecular dynamics simulation (Figure 3.12).

H N

O N O

H O N

N N H O

O N O

NH H N

Figure 3.11. Both torsional angles examined in the H3+, n=2 molecular basket (a) The N– C–C–C dihedral angle was measured to determine whether the imidazole ring was oriented up above the top of the basket or down towards the base (solid orange line). (b) The C–N–C–C dihedral angle was measured to determine the orientation of the imidazole rings; whether they were oriented in over the cavity or out away from the cavity (solid purple line).

In fact, when the 2-D correlation between the two torsions is examined, it is observed that the imidazole rings are most often in the UP and OUT orientation (Figure

3.12). This suggests the imidazole rings are not blocking the cavity of the basket from being able to recognize an incoming guest molecule. Also, with the arms oriented up, perhaps they could play a role in attract the OP guest to the hydrophobic cavity in order to create the recognition event. According to the 2-D plot, the imidazole arms also spend a significant amount of time in the UP and IN orientation. As the 2-D plot shows, the imidazole arms spend the least amount of time in the IN and DOWN position. This would be the most problematic orientation of the imidazole rings because they would be blocking the hydrophobic cavity, which should recognize the alkyl group of most OPs. If

79 the hydrophobic cavity preferentially interacted with the arms of the basket instead of an

OP guest, recognition of chemical warfare agents would be very difficult.

Figure 3.12. Two-dimensional correlation between (a) N–C–C–C UP/DOWN dihedral angle (solid orange line, Figure 3.11) and (b) C–N–C–C IN/OUT dihedral angle in H3+, n=2 molecular basket (solid purple line, Figure 3.11).

3.5 MOLECULAR MODELING AND EXPERIMENTAL STUDIES WITH TRIS-

PROTONATED IMIDAZOLE-CAPPED BASKET USING DIMETHYLMETHOXY

PHOSPHONATE AS A GUEST

In carrying forward our computational protocol with this particular molecular basket (H3+, n=2), the next step was to perform a clustering analysis after the ten different molecular dynamics trajectories were combined. This clustering analysis then produced ten new structures (snapshots) that were representative of all of the MD simulations. It 80 was these ten structures that were used for docking simulations with our library of OP compounds and other ligands of interest. Each docking simulation produced 200 docking poses between the ligand and snapshot of interest that measured the electrostatic interaction between the host and guest.

Upon studying the ten snapshots produced from the clustering analysis, it became evident that the structures could be placed in one of two distinct groups (Figure 3.13).

One group of seven snapshots had each of the three imidazole-capped arms oriented out away from the central cavity of the basket. This would create a completely open cavity in order to recognize OP guest molecules. Another group of three snapshots had two of the imidazole-capped arms out away from the cavity, but one arm was oriented in over the center of the basket. These snapshots were then subjected to a library of OP compounds where the effect of the size of the alkyl and O-alkyl groups could be established (Figure 3.14).

Figure 3.13. Two groups of snapshots used in molecular docking simulations. One group of seven snapshots has all three arms oriented away from the center of the cavity (left). Another group of the three snapshots has one arm oriented in over the center of the cavity (right).

O O O P O P O P O O O O

O O O O P O P O P O P O O O O O

O O O O P O P O P O P O OH OH O OH

Figure 3.14. Library of OP compounds docked with imidazole-capped basket (H3+, n=2).

The first ligand of interest was dimethoxy methylphosphonate (DMMP). This particular OP compound was the smallest in our library, so it would be an appropriate lower limit of study to determine if any OP ligands would be a suitable guest for the particular imidazole-capped basket (H3+, n=2) subjected to the molecular docking 82 simulations. The results from our molecular docking simulation generated a variety of poses for the DMMP ligand to orient inside the basket’s cavity. Our results were analyzed taking into account the two different groups of snapshots mentioned earlier: those snapshots where the cavity was completely open, and snapshots where one of the protonated imidazole rings was oriented in over the center of the cavity. The results of the docking simulations are presented below (Table 3.2).

Table 3.2. Summary of docking poses of DMMP in several MD snapshots of basket.

Absolute Energy Overall Relative Snapshot # # of Conformations (kcal/mol) Energy (kcal/mol)

5 -0.82 0.92

355 180 -0.75 0.99

15 -0.71 1.03

45 -0.65 1.09 1867 155 -0.61 1.13

486 200 -0.70 1.04

191 -0.82 0.92 855 9 -0.74 1.00

continued

Table 3.2 continued

194 -0.53 1.21 1021 6 -0.49 1.25

116 -0.87 0.87 1807 84 -0.82 0.92

196 -0.90 0.84

1099 3 -0.82 0.92

1 -0.77 0.97

199 -0.91 0.83 1233 1 -0.81 0.93

185 -0.91 0.83

1356 14 -0.88 0.86

1 -0.83 0.91

197 -1.74 0.00 1556 3 -1.64 0.10

In the case of the cavity being completely open (snapshots #355-1099), DMMP is free to be recognized by the cavity in a variety of orientations (Figure 3.15). In the majority of the docking orientations generated (~53%), there is a degree of hydrogen bonding between the phosphoryl oxygen of DMMP with one of the N–H bonds of the imidazole ring of the basket arm. With this hydrogen bond intact; however, the DMMP ligand often finds itself oriented between two of the walls of the basket, and not in the 84 center of the cavity. The second most frequent orientation generated by the simulation did not have this hydrogen bond present between the basket and DMMP. In these snapshots (~34%), the DMMP ligand orients itself with the P-Me group oriented facing up towards the top of the basket. This result is contrary to the hypothesis that the hydrophobic cavity of the basket would spur the recognition of the P-alkyl group of the

OP. These two poses were very close in energy, between 0.1 and 0.2 kcal/mol.

Figure 3.15. Representative poses generated via docking simulations showing the interaction between the imidazole-capped basket (H3+, n=2) and DMMP, representative of subset of snapshots where none of the arms of the basket are oriented in over the center of the cavity. One pose shows DMMP making a hydrogen bond between the N–H of imidazole and resting between two walls of the basket (left, top-down view into the basket). Another pose shows DMMP sitting in the center of the cavity of the basket, but with the P–Me group oriented up (right, side-on view into basket).

In the case of the other subset of snapshots, with one of the imidazole rings oriented over the center of the cavity, there was an even higher prevalence of hydrogen bonding between DMMP and the N–H bond of the imidazole ring that was oriented in over the cavity (Figure 3.16). The majority of docking poses in this subset of snapshots

(~66%) oriented DMMP higher in the cavity with a hydrogen bond between the phosphoryl oxygen of DMMP and the protonated imidazole. The other docking pose generated from these snapshots (~33%) saw not only the presence of a hydrogen bond, but the DMMP ligand oriented in the center of the cavity with the P–Me group oriented into the hydrophobic base of the basket. This particular pose generated was the lowest energy pose generated by the docking simulation by almost 1 kcal/mol when compared with other poses. In this result, the imidazole ring being oriented over the center of the cavity does not obstruct the cavity and prevent the recognition of DMMP by the molecular basket. Instead, it seems that having an imidazole ring oriented over the center of the cavity helps to attract the ligand to the cavity.

Figure 3.16. Representative poses generated via docking simulation showing interaction between imidazole-capped basket (H3+, n=2) and DMMP, representative of subset of snapshots where one of the arms of the basket are oriented in over the center of the cavity. One pose shows DMMP making a hydrogen bond between the N–H of imidazole and resting high up in the basket (left, top-down view into the basket). Another pose shows DMMP sitting in the center of the cavity of the basket, with a hydrogen bond to the imidazole ring and with the P–Me group oriented up (right, top-down view into basket). This particular pose was the lowest-energy docking pose generated with any snapshot and matched the hypothesis that the hydrophobic cavity would recognize the P-

In order to further investigate the poses generated by the molecular docking simulations, molecular dynamics simulations of the lowest energy pose and/or most populated pose of each guest from each of the 10 docking simulations (with the snapshots generated from the clustering analysis) were performed for 8 nanoseconds in a box of

TIP3P water molecules. We monitored the dynamics of the guest–basket complex as a function of time. Initially, we looked at the dynamics of DMMP guest within each of the different basket snapshots, and found that regardless of the starting orientation, the guest eventually equilibrates to have P–Me group pointing towards the base of the basket for 11 of the 12 docking poses examined by this further molecular dynamics simulation.

This result can be seen clearly by monitoring the distance between the center of the basket base and the alkyl group carbon (Figure 3.17). This crucial result suggests that any recognition by the molecular basket would necessarily occur between the hydrophobic cavity of the basket with the P–Me group of the OP compound, no matter the initial orientation of the ligand. Also, these results suggest the particular scaffold studied, with protonated imidazole rings and the ethylene linker, also aid in orienting the

DMMP ligand into the host.

Figure 3.17. Plot of the distance between the basket base and methyl group carbon of DMMP as a function of simulation time, as performed by Dr. Hashem Taha in the Hadad Group.

Experimental evidence from the Badjic research group has validated the results of our computational protocol using this particular imidazole-capped basket (H3+, n=2) and

DMMP as the OP guest.30 The basket was allowed to interact with the guest DMMP at varying concentrations in a phosphate buffer. The pH was held acidic (pH=2.5) in order to ensure each of the imidazole rings was protonated, and to aid in the solubility of the basket in aqueous solution. A downfield shift was seen in the aromatic residues of both the wall of the basket and the protons on the imidazole ring (Figure 3.18). An analysis of

NMR data was able to yield an association constant of ~321 M–1. A larger change in

89 chemical shift was seen in the P–Me group as opposed to the P–OMe group, suggesting the P–Me group was indeed binding to the hydrophobic cavity of the basket.

Figure 3.18. 1H NMR spectra (600 MHz, 298.0 K) of basket (H3+, n=2; 1.0 mM) obtained upon an incremental addition of DMMP to the solution of imidazole-capped basket (10.0 mM phosphate buffer at pH = 2.5 ± 0.1); note that the water resonance at 4.76 ppm was suppressed. These spectra were obtained by the Badjic group.

3.6 OTHER MOLECULAR DOCKING STUDIES WITH METHYL PHOSPHONATES

In order to further examine the preference of this particular imidazole-capped basket for the P–Me group, other OPs in our docking library were examined where the O- alkyl groups were increased in size. This subset of docked ligands is shown below

(Figure 3.19). The size of the O-alkyl groups increase from O-ethyl to O-cyclohexyl, allowing an opportunity to determine if the P-Me group is recognized even in the presence of larger, more hydrophobic alkyl groups. Due to the results of our previous molecular docking simulation with DMMP, our attention would be most focused on the

90 lowest-energy docking pose generated by each simulation with these other methyl phosphonates.

O O O P P P O O O O O O

MeP-OMe-OEt MeP-OEt-OEt MeP-OEt-OiPr

O O P P O O O O

i i MeP-O Pr-O Pr MeP-OMe-OCy

Figure 3.19. Subset of docked methyl phosphonates into imidazole-capped basket where P–Me group was held constant and size of O-alkyl group was increased.

As could be imagined, there was great similarity when comparing the results of our docking simulation with DMMP to the methyl phosphonate MeP-OMe-OEt. The lowest energy pose of this methoxyethoxy methylphosphonate also gave the most favorable pose with the P–Me group oriented down into the cavity of the basket (Figure

3.20). This particular pose was only about 0.2 kcal/mol more stable than a similar pose with the O-Et group oriented down in the cavity.

Interestingly, similar to the recognition of DMMP, this lowest energy pose was observed when one of the imidazole arms was oriented over the center of the cavity, able to make a hydrogen bond with the phosphoryl oxygen of the OP. Similarly, the methyl phosphonate MeP-OEt-OEt (diethoxy methylphosphonate) also has the lowest energy pose orienting the P–Me group down into the cavity of the basket (Figure 3.21). Once

91 again, this pose was generated when one of the imidazole rings was oriented in over the center of the cavity, but was still only about 0.2 kcal/mol more stable than a similar pose with the O-Et group oriented down in the cavity.

Figure 3.20. Lowest energy pose (top-down) showing MeP-OMe-OEt bound to center of basket (imidazole cap; H3+, n=2) with P-Me group oriented down into cavity.

Figure 3.21. Lowest energy pose (top-down) showing MeP-OEt-OEt bound to center of basket (imidazole cap; H3+, n=2) with P-Me group oriented down into cavity.

However, when a more bulky isopropyl group is installed on the methyl phosphonate, the lowest-energy docking pose generated oriented the O-isopropyl group down into the hydrophobic cavity instead of the P-Me group. This suggests the larger van der Waals surface of the isopropyl group is favored to bind in the hydrophobic cavity more than the P–Me group. In the case of both MeP-OEt-OiPr (Figure 3.22) and MeP-

OiPr-OiPr (Figure 3.23), the O–iPr was oriented down in the center of the cavity. Once again, each of these poses were generated with one of the imidazole rings oriented over the center of the cavity, aiding the recognition of the OP by making a hydrogen bond with the phosphoryl oxygen. Finally, consistent with this observed trend in the molecular docking simulations, the largest O-alkyl group studied (MeP-OMe-OCy) oriented the O– cyclohexyl group down into the cavity of the basket (Figure 3.24).

Figure 3.22. Lowest energy pose (top-down) showing MeP-OEt-OiPr bound to center of basket (imidazole cap; H3+, n=2) with larger P–OiPr group oriented down into the cavity.

Figure 3.23. Lowest energy pose (top-down) showing MeP-OiPr-OiPr bound to center of basket (imidazole cap; H3+, n=2) with larger P–OiPr group oriented down into the cavity.

Figure 3.24. Lowest energy pose (top-down) showing MeP-OMe-OCy bound to center of basket (imidazole cap; H3+, n=2) with larger P–OCy group oriented down into the cavity.

Preliminary experimental results somewhat confirmed the observations from our molecular docking simulations on these other methyl phosphonates. As the size of the

OP compound increased, the apparent binding constant decreased (Table 3.3). This result suggests larger, more flexible O-alkyl groups hinder the ability of the P–Me group to bind the hydrophobic cavity of the basket. This result was seen in several 1H NMR experiments where the chemical shift of the P–Me group was not shifted as far downfield as the size of the O-alkyl group increased. However, even though the binding of these larger methyl phosphonates is decreased, there is clearly still a recognition event between the molecular basket and the OP compound.

Table 3.3. Summary of apparent binding constants of select OP ligands showing a decrease in binding as the size of the O-alkyl group is increased.

3 -1 Volume (Å ) Experimental Kapp (M )

MeP-OMe-OMe

(DMMP) 118 321

MeP-OMe-OEt 137 242

MeP-OEt-OEt 155 154

MeP-OEt-OiPr 173 98

MeP-OiPr-OiPr 192 87

3.7 CONCLUSIONS

A computational protocol was established to explore the conformational flexibility of molecular baskets capped with imidazole rings for the purpose of binding

OPs in order to act as a pseudo-bioscavenger. The size of the linker between the wall of the basket and the functional imidazole ring and the protonation state of the three 95 imidazole arms were varied. The decarboxylated histidine-capped basket (tris-protonated with an ethylene linker) was found to have the largest N–N distance and the highest frequency of the arms oriented away from the cavity, suggesting it had the most open cavity. The results of the Monte Carlo, DFT optimizations, and molecular dynamics simulations were then carried forward into a molecular docking procedure with various

OP compounds.

The snapshots chosen for docking from the clustering analysis of the particular basket of interest were organized into two representative conformations. One set of snapshots contained conformations where none of the imidazole arms are oriented over the center of the basket, leaving an open cavity for recognition. With the cavity open, each of the ligands was able to bind to the basket receptor. However, when one arm was oriented over the cavity, the lowest-energy docking pose included a hydrogen bond between the phosphoryl oxygen of the OP and one of the His side chains. This oriented the P–Me group down into the cavity of the basket for smaller OPs such as DMMP, which was confirmed by experimental 1H NMR studies in the Badjic group. Guests with larger alkyl groups also preferred to have these groups pointed toward the hydrophobic base of the basket, and as the size of the alkyl group increased, the propensity for guest to be in this orientation also increased. This preference lowered the binding affinity for the

P–Me group for other methyl phosphonates studied. These observations suggest that molecular baskets are suitable to further OP binding studies, having shown the ability to recognize small OPs in aqueous buffer; but these scaffolds may require further optimization to be efficient against a wide range of potential guests.

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Chapter 4 : Design of Iridium-Based Catalysts for the Selective Dehydrogenation of Fatty Acids

4.1 INTRODUCTION

Alkane dehydrogenation, also known as C–H activation, of unfunctionalized alkanes has long been an area of importance in organometallic chemistry,1 largely due to the significance of alkene synthesis in industrial applications. Until recently, the many challenges associated with alkane dehydrogenation have resulted in few examples appearing in the chemical literature. Due to the relative stability of the C–H bond, catalytic methods often require high temperatures or photochemical excitation.2

However, high temperatures often promote unwanted side products through isomerization of terminal alkenes to internal alkenes. These isomerization side products have prevented a truly selective C–H activation catalyst from being discovered. Despite these challenges, metal–catalyzed alkane dehydrogenation holds great promise as a reaction that can convert renewable resources to important value–added chemicals.

To the best of our knowledge, the investigation of metal-catalyzed dehydrogenations of fatty acids has not been undertaken, yet fatty acids comprise an under-utilized component of biomass. While there have been many efforts towards using carbohydrates from renewable sources,3 there have been less efforts at extracting value

100 from fatty acids and lignins. This work focuses on creating value-added chemicals from the fatty acids available in renewable biomass.

One set of the most successful alkane dehydrogenation catalysts currently in the literature are the so-called pincer catalysts (Figure 4.1) discovered by Jensen and

Goldman.4 These pincer catalysts are very thermodynamically stable compared to other iridium catalysts currently employed for alkane dehydrogenation. The pincer catalysts rely on phosphorus ligands connected to an aromatic ring through various meta– substituted linkers. Although alkane isomerization is still present as an unwanted side reaction, the pincer catalysts have been shown to accomplish C–H activation in the absence of a sacrificial acceptor alkene. Usually, alkane dehydrogenation often involves a sacrificial acceptor (most commonly t-butylethylene), to receive a mole of molecular hydrogen that is reductively eliminated from many starting catalysts. The addition of another molecule to the system often leads to unwanted side products and makes isolation of the target compound more difficult. Therefore, the demonstrated ability of the pincer catalysts to perform the reaction without the presence of a sacrificial acceptor greatly simplifies alkane dehydrogenation.5

101

OMe OMe OMe OMe

O O HN NH R P Ir PR R2P Ir PR2 R2P Ir PR2 2 2 R2P Ir PR2 H H H H Cl Cl Cl Cl

Figure 4.1. Various iridium pincer catalyst precursors used in alkane dehydrogenations.5

One of the original dehydrogenation catalysts to gain prominence was developed by Crabtree in the late 1970s.6 Crabtree first employed an iridium hydride catalyst with two coordinating acetone ligands, [Ir(H)2(acetone)2(PPh3)2]BF4, and suggested an operative mechanism. The alkane adds to the catalyst and is dehydrogenated through β- hydride elimination and released in an intermolecular catalytic cycle. Further work by

2 Crabtree replaced the acetone ligands with trifluoroacetic acid (TFA), IrH2(η -

O2CCF3)(PAr3), where the aryl substituent is a p-fluorinated triphenylphosphine. The catalyst also participates in alkane dehydrogenation, either photochemically or under thermal conditions in the presence of a sacrificial olefin.7 The proposed mechanism of alkane dehydrogenation using Crabtree’s TFA-based catalyst (Scheme 4.1) involves removal of hydrogen gas at high temperature or the presence of a sacrificial acceptor, followed by intermolecular C–H activation and β-hydride elimination to yield the desired alkene.

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Scheme 4. Crabtree's Ir-Carboxylate Dehydrogenation Catalyst high temperature L O H O or sacrificial acceptor L Ir CF Ir O H O 3 L L CF3 L = P(p-FC6H4)3 -H2 intermolecular H C-H activation R R O R L β-H elimination L O H O CF3 Ir O Ir H H CF L L 3 R

Scheme 4.1. Proposed mechanism of Crabtree’s dehydrogenation.

The research presented herein focuses on developing iridium catalyst systems inspired by the catalysts that were first introduced by Crabtree. However, instead of using a pendant TFA ligand, a fatty acid is directly substituted onto the iridium center to promote intramolecular C–H activation, ideally resulting in selective dehydrogenation to unsaturated fatty acids. In previous mechanistic studies by Crabtree,6 the TFA ligand has been shown to stay on the catalyst during dehydrogenation, suggesting that intramolecular C–H activation of a fatty acid could ultimately be a favorable process.

These unsaturated fatty acids, if isolated, would serve as a “gateway” molecule to several of the twelve value-added chemicals identified by the Department of Energy8 (Scheme

4.2).

103

– H O 2 epoxidation oxidation O 3 m OH O m = n – 3 n OH 4 OH + acrylic acid O O reduction 3-Hydroxy O propionic CN H OH Acid (3-HPA) HO OH acrylonitrile O

OCH3 methyl acrylate Scheme 1. Selective chemical conversion of saturated fatty acids to mono-unsaturated (Δ3,4 or Δ4,5) derivatives (red), followed by standard conversions to produce value-added chemicals. The structures shown in blue are 7 representatives compounds Scheme 4.o2ut .o fPath 12 chem toical bvalueuilding blo-caddedks with the "chemicals.greatest potential" b ased on carbohydrate "biorefineries".

A new pathway to these important compounds, many of them currently generated on the metric ton scale from petroleum feedstocks, could revolutionize their production and provide a renewable alternative to crude oil. As shown in Scheme 4.2 (red box), the selective dehydrogenation of a fatty acid would provide access to unsaturated fatty acids that can be converted to 6 of the 12 identified value-added chemicals using well-known oxidative reactions. To accomplish this feat, however, an efficient and selective dehydrogenation procedure for the initial conversion of saturated fatty acids to unsaturated fatty acids must be developed. 104

4.2 COMPUTATIONAL METHODS

Computational modeling of the proposed catalyst systems provided support via several in silico experiments to our experimental collaborators (Dr. James Stambuli and

Dr. Sean Whittemore, The Ohio State University) in the design of a selective C–H activation catalyst. Both Crabtree and Hall9 have reported computational studies of similar iridium catalysts previously used in catalytic dehydrogenation reactions.

Previously, Crabtree has performed computational studies of his TFA-bound catalysts using density functional theory (DFT) methods. Also, Hall has shown that DFT methods such as BP8610 (Becke’s 1988 exchange and Perdew’s 1986 correlation) and B3LYP11

(Becke’s 3–parameter exchange and Lee-Yang-Parr’s correlation) are suitable for studying iridium pincer catalysts, and Hall has claimed that the BP86 functional performs better than the more popular B3LYP method for such inorganic complexes. Although

Hall concluded that many mechanisms could be operative at high temperatures, his computational studies did show that DFT methods were appropriate to probe the mechanisms of similarly catalyzed dehydrogenation reactions.9

As DFT methods have been shown to be accurate for modeling similar iridium– based dehydrogenation catalysts, it is likely that, by extension, they will also be sufficient for the proposed catalysts based on derivatives of Crabtree’s original catalysts.

Therefore, both BP86 and B3LYP methods were examined against proposed starting catalysts and possible intermediates to determine which level of theory can better model the coordination environment around the iridium center.

105

Initial calculations were performed using the Turbomole program.12 A single basis set (referred to as Basis Set 1) was used that modeled all atoms, except iridium, with an SV(P) basis set (similar to 6–31G*)7 while a TZVP (similar to 6–311G**)7 basis set was used for iridium. Moreover, iridium was also treated with an effective core potential (ecp) in Turbomole. Available crystal structures were also studied using a

TZVP basis set on every atom. The resolution–of–the–identity (RI) approximation13 was also employed for all Turbomole calculations to increase the computational efficiency.

Each structure was optimized to a stationary point, using the default optimization criteria in Turbomole. In many cases, a vibrational frequency analysis was also performed to characterize the stationary point as a minimum or maximum on the potential energy surface.

4.3 CALIBRATION OF COMPUTATIONAL METHODS ON CRABTREE–DERIVED

CATALYST

Before probing the possible mechanism of selective fatty acid dehydrogenation, it was important to calibrate our computational methods to ensure they were well suited to model the coordination environment of these iridium catalysts. Therefore, our first goal was to compare the experimental crystal structure of a Crabree-derived metal catalyst with heptanoic acid attached, Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3), to a geometry optimization at the BP86 and B3LYP levels of theory. By comparing measured bond distances and bond angles between the crystal structure (Figure 4.2) and our

106 computational models (Figure 4.3), we could ensure these levels of theory were sufficient for further study of the proposed intramolecular C–H activation of fatty acid substituents.

Figure 4.2. Experimental crystal structure of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) as observed by the Stambuli research group, but unpublished.

107

Figure 4.3. Computational modeling of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) as optimized at the (a) BP86 and (b) B3LYP levels of theory with the SV(P){Ir(TZVP)} basis set. Hydrogens have been omitted for clarity.

The first measure of the accuracy of our computational methods was to compare a large subset of the experimental bond measurements from the crystal structure to the results of the BP86 and B3LYP geometry optimizations (Table 4.1). From the mean average deviation between experiment and the absolute average deviation from experiment, there seems to be no quantitative difference between the two DFT functionals. The B3LYP functional is only 0.002 Å more accurate in terms of absolute average deviation than the BP86 functional. Thus, at least when establishing the bond distances around the iridium Crabtree catalyst, the less computationally expensive

BP86/SV(P){Ir(TZVP)} seems to be a sufficient functional.

108

Table 4.1. Comparison of computed bond distances to the crystal structure of Ir(H)2(P(p- a FPh)3)2(O2C(CH2)5CH3) BP86 B3LYP Experimentb

Ir–O(1) 2.335 2.343 2.335

Ir–O(2) 2.322 2.334 2.272

Ir–C(37) 2.692 2.701 2.606

C(37)–O(1) 1.279 1.267 1.269

C(37)–O(2) 1.278 1.268 1.270

Ir–H(1A) 1.590 1.583 1.453

Ir–H(2A) 1.593 1.584 1.424

Ir–P(1) 2.336 2.357 2.292

Ir–P(2) 2.335 2.356 2.285

C(1)–P(1) 1.849 1.847 1.826

C(7)–P(1) 1.852 1.849 1.826

C(13)–P(1) 1.853 1.849 1.826

C(2)–H(2) 1.101 1.092 0.950

C(1)–C(2) 1.414 1.406 1.392

C(2)–C(3) 1.401 1.399 1.389

C(4)–F(1) 1.346 1.339 1.365

continued

109

Table 4.1 continued

C(19)–P(2) 1.854 1.849 1.826

C(25)–P(2) 1.849 1.847 1.830

C(31)–P(2) 1.851 1.848 1.826

C(20)–H(20) 1.101 1.092 0.950

C(19)–C(20) 1.414 1.408 1.409

C(20)–C(21) 1.402 1.394 1.387

C(22)–F(4) 1.346 1.339 1.354

C(37)–C(38) 1.519 1.518 1.501

C(38)–C(39) 1.538 1.532 1.495

C(39)–C(40) 1.504 1.533 1.525

C(40)–C(41) 1.539 1.534 1.494

C(41)–C(42) 1.514 1.534 1.552

C(42)–C(43) 1.536 1.532 1.401

C(38)–H(38A) 1.12 1.11 0.99

C(39)–H(39A) 1.11 1.11 0.99

C(40)–H(40A) 1.11 1.11 0.99

C(41)–H(41A) 1.12 1.11 0.99

C(42)–H(42A) 1.12 1.11 0.99

C(43)–H(43A) 1.11 1.11 0.99

continued

110

Table 4.1 continued

Mean Average

Deviation 0.056 0.055

Mean Absolute

Deviation 0.061 0.059 a Using the SV(P) basis set for all atoms, except for iridium, which used the TZVP basis set. All values are listed in Angstroms. b As obtained at The Ohio State University by Dr. James Stambuli, but unpublished.

The next measure of our ability to properly model the coordination environment of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) was to compare a subset of various bond angles between our computational optimizations and the experimental crystal structure (Table

4.2). Even though the mean average deviation is less than one degree for each level of theory, a more realistic depiction of the accuracy of the DFT methods comes when examining the absolute average deviation. When this analysis is performed, the less expensive BP86 is once again the more accurate functional to compare the computational model of Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) to its crystal structure by about 0.5 degrees.

Based on these results, it can be concluded that the BP86 functional is entirely appropriate to model the coordination environment around the metal center in Ir(H)2(P(p-

FPh)3)2 (O2C(CH2)5CH3) and other Crabtree–type catalysts for the purpose of our fatty acid study. This is consistent with prior reports by Hall and co–workers.9

111

Table 4.2. Comparison of computed bond angles to crystal structure of Ir(H)2(P(p- a FPh)3)2(O2C(CH2)5CH3) BP86 B3LYP Experimentb

C(1)–P(1)–Ir 115.56 115.68 114.33

C(19)–P(2)–Ir 114.19 113.77 116.58

C(37)–O(1)–Ir 91.56 91.97 91.90

C(37)–O(2)–Ir 92.16 92.36 90.20

O(1)–Ir–P(1) 90.44 90.04 92.43

O(1)–Ir–P(2) 96.02 96.28 95.80

O(2)–Ir–P(1) 95.48 95.89 89.42

O(2)–Ir–P(2) 92.26 91.23 93.01

P(2)–Ir–P(1) 171.83 172.35 171.48

H(1A)–Ir–H(2A) 81.20 81.34 81

P(1)–Ir–H(1A) 88.73 88.99 84

P(2)–Ir–H(1A) 86.08 85.84 87.5

P(1)–Ir–H(2A) 85.38 85.51 91.4

P(2)–Ir–H(2A) 87.59 81.14 87.9

continued

112

Table 4.2 continued

C(37)–Ir–H(1A) 138.89 138.88 140

C(37)–Ir–H(2A) 139.91 139.77 139

O(1)–Ir–H(1A) 167.11 166.66 168.9

P(1)–Ir–C(37) 92.93 93.05 89.37

P(2)–Ir–C(37) 95.13 94.56 96.73

C(7)–P(1)–C(1) 103.77 104.24 103.83

C(19)–P(2)–C(31) 103.35 103.23 102.96

O(1)–C(37)–O(2) 119.59 119.75 119.2

C(1)–C(2)–C(3) 120.79 120.84 121

F(1)–C(4)–C(3) 119.20 119.25 118.4

C(37)–C(38)–C(39) 114.43 114.92 116.1

C(37)–C(38)–H(38A) 108.65 106.53 108.3

Mean Average Deviation 0.06 –0.26

Mean Absolute Deviation 1.57 2.05 a Using the SV(P) basis set for all atoms, except for iridium, which used the TZVP basis set. All values are listed in degrees. b As obtained at The Ohio State University by Dr. James Stambuli, but unpublished.

Other basis sets could also be employed in further studies to come even closer to reproducing experimental values. Basis Set 1 is fairly inexpensive computationally, except for the TZVP basis function, which was only placed on iridium. In studying the data from Basis Set 1, some of the largest error comes from the modeling of the angles

113 between the P and H atoms bonded to iridium. Basis sets could be imagined that also include polarization functions on other atoms in the catalyst, such as P and the H bonded directly to iridium. In order to test the accuracy of a bigger basis set, a full TZVP basis set was assigned to every atom in Ir(H)2(P(p-FPh)3)2(O2C(CH2)5CH3) and its geometry optimized using the BP86 and B3LYP functionals. The same bond lengths (Table 4.1) and bond angles (Table 4.2) were examined again (Tables 4.3 and 4.4) to determine if this bigger basis set can ensure a more accurate optimized geometry than Basis Set 1.

Table 4.3. Comparison of computed bond distances to crystal structure of Ir(H)2(P(p- FPh)3)2(O2C(CH2)5CH3), in angstroms, using TZVP basis set for all atoms. BP86/AllTZVP B3LYP/AllTZVP Experimenta

Ir–O(1) 2.341 2.352 2.335

Ir–O(2) 2.347 2.359 2.272

Ir–C(37) 2.707 2.718 2.606

C(37)–O(1) 1.280 1.268 1.269

C(37)–O(2) 1.279 1.267 1.270

Ir–H(1A) 1.575 1.568 1.453

Ir–H(2A) 1.574 1.566 1.424

Ir–P(1) 2.338 2.359 2.292

Ir–P(2) 2.338 2.359 2.285

continued

114

Table 4.3 continued

C(1)–P(1) 1.856 1.852 1.826

C(7)–P(1) 1.855 1.852 1.826

C(13)–P(1) 1.858 1.855 1.826

C(2)–H(2) 1.091 1.082 0.950

C(1)–C(2) 1.404 1.398 1.392

C(2)–C(3) 1.398 1.391 1.389

C(4)–F(1) 1.360 1.353 1.365

C(19)–P(2) 1.859 1.856 1.826

C(25)–P(2) 1.855 1.852 1.830

C(31)–P(2) 1.858 1.854 1.826

C(20)–H(20) 1.090 1.082 0.950

C(19)–C(20) 1.406 1.399 1.409

C(20)–C(21) 1.395 1.389 1.387

C(22)–F(4) 1.360 1.351 1.354

C(37)–C(38) 1.517 1.515 1.501

C(38)–C(39) 1.532 1.530 1.495

C(39)–C(40) 1.534 1.530 1.525

continued

115

Table 4.3 continued

C(40)–C(41) 1.533 1.530 1.494

C(41)–C(42) 1.535 1.531 1.552

C(42)–C(43) 1.532 1.529 1.401

C(38)–H(38A) 1.10 1.09 0.99

C(39)–H(39A) 1.10 1.09 0.99

C(40)–H(40A) 1.10 1.10 0.99

C(41)–H(41A) 1.11 1.10 0.99

C(42)–H(42A) 1.10 1.10 0.99

C(43)–H(43A) 1.10 1.09 0.99

Mean Ave Deviation 0.057 0.053

Mean Abs Deviation 0.057 0.056 a As obtained at The Ohio State University by Dr. James Stambuli, but unpublished.

116

Table 4.4. Comparison of computed bond angles to crystal structure of Ir(H)2(P(p- FPh)3)2(O2C(CH2)5CH3), in degrees, using TZVP basis set for all atoms. BP86/AllTZVP B3LYP/AllTZVP Experimenta

C(1)–P(1)–Ir 115.36 114.93 114.33

C(19)–P(2)–Ir 114.98 114.71 116.58

C(37)–O(1)–Ir 91.99 92.42 91.90

C(37)–O(2)–Ir 91.78 92.11 90.20

O(1)–Ir–P(1) 92.20 91.71 92.43

O(1)–Ir–P(2) 96.66 96.34 95.80

O(2)–Ir–P(1) 96.45 96.30 89.42

O(2)–Ir–P(2) 91.04 90.52 93.01

P(2)–Ir–P(1) 170.62 171.50 171.48

H(1A)–Ir–H(2A) 80.6 80.7 81.0

P(1)–Ir–H(1A) 87.1 87.6 84.0

P(2)–Ir–H(1A) 84.84 85.11 87.5

P(1)–Ir–H(2A) 85.53 85.73 91.4

P(2)–Ir–H(2A) 88.39 88.77 87.9

C(37)–Ir–H(1A) 140.1 139.9 140.0

C(37)–Ir–H(2A) 139.4 139.4 139.0

O(1)–Ir–H(1A) 168.2 167.6 168.9

continued

117

Table 4.4 continued

P(1)–Ir–C(37) 87.12 94.61 89.37

P(2)–Ir–C(37) 94.24 93.78 96.73

C(7)–P(1)–C(1) 103.86 104.22 103.83

C(19)–P(2)–C(31) 102.80 103.18 102.96

O(1)–C(37)–O(2) 119.9 119.9 119.2

C(1)–C(2)–C(3) 120.7 120.8 121.0

F(1)–C(4)–C(3) 118.69 118.8 118.4

C(37)–C(38)–C(39) 114.9 115.0 116.1

C(37)–C(38)–H(38A) 107.7 107.7 108.3

Mean Average Deviation –0.22 0.10

Mean Absolute Deviation 1.42 1.61 a As obtained at The Ohio State University by Dr. James Stambuli, but unpublished

Surprisingly, adding the extra polarization functions of the triple–zeta basis set does not remarkably decrease the mean absolute deviation of the calculated bond lengths or bond angles when compared to the crystal structure of Ir(H)2(P(p-FPh)3)2

(O2C(CH2)5CH3). When comparing the calculated bond lengths of Basis Set 1 and the complete TZVP Basis Set, there is only an increase of accuracy of less than 0.005 Å

(mean absolute deviation) when using the bigger basis set. Such an insignificant increase in accuracy surely does not justify the expanded computational expense of the expanded basis set. In a similar fashion, there is not much of an advantage when examining the

118 calculated bond angles using the TZVP Basis Set instead of the less computationally expensive Basis Set 1, only increasing the accuracy of the calculated bond angle of the optimized structure by less than half a degree, again when examining the mean absolute deviation. Thus, at this point in our study, we were confident that Basis Set 1 was sufficiently accurate to employ for optimization studies of these Crabtree-like catalysts.

4.4 USING ISODESMIC REACTIONS TO MODEL THE OPTIMUM CRABTREE–

DERIVED CATALYST

The use of isodesmic reactions is a powerful tool in chemistry, especially when applied to computational chemistry. Electronic energies garnered from geometry optimizations tell nothing about the stability of one compound compared to another because the stoichiometry may be different between each molecule. However, if an isodesmic reaction is used, with equal stoichiometry between products and reactants, a

∆H can be derived that can yield useful chemical information about the stability of one compound versus another. For our purposes, these isodesmic reactions could prove to be quite useful in discovering the optimum catalyst for the selective C–H activation of saturated fatty acids.

The first set of isodesmic reactions calculated focused on discovering the optimum phosphine ligand for Ir(H)2(PR3)2(O2C(CH2)5CH3). The crystal structure examined for the catalyst used a para-fluorinated triphenylphosphine ligand to increase the solubility of the catalyst. But, is it possible that the p–substituted fluorine does more than increase the solubility, but also aids in the stability of the catalyst? Would the

119 addition of a second fluoride in the meta position give even greater stability? Another question to be studied is the energy difference between exchanging various phosphine ligands. Would alkyl ligands such as ethyl, isopropyl, or t-butyl phosphines bring more stability than phenyl–substituted phosphines?

Often times in computational chemistry, PH3 is substituted as a ligand for bulkier phosphines because of the reduction in computational time. Using isodesmic reactions, we can tell if the difference between PH3 and other phosphines is significant enough to consistently use the more convenient PH3 or if the actual ligand in use should be modeled instead. If there is a substantial difference in energy between catalysts with PH3 as ligand or other phosphines, the catalyst systems should be modeled exactly as they are being studied in the laboratory. Finally, the position of phosphine ligands was also studied around the metal center to discover whether an axial/axial or axial/equatorial orientation is more energetically favorable. A number of isodesmic reactions were studied (Scheme

4.3), and the resulting ∆H values (Table 4.5) indeed provided interesting results.

120

(a)

PH3 PR3 H O H O Ir 2PR3 Ir 2PH3 H O 5 H O 5 PH PR 3 3

(b)

PPh3 P(x-FPh)3 H O H O Ir 2P(x-FPh)3 Ir 2PPh3 H O 5 H O 5 PPh P(x-FPh) 3 3

(c)

PR3 PR3 H O H O Ir 5 Ir H O R3P O 5 PR 3 H

Scheme 4.3. Isodesmic reactions studying the optimum identity of PR3 in Ir(H)2(PR3)2(O2C(CH2)5CH3): (a) exchange of PH3 to PR3; (b) exchange of PPh3 with P(p-FPh)3, P(m-FPh)3, P(m, p-FPh)3; (c) axial/axial vs. axial/equatorial orientation of PPh3.

121

Table 4.5. ∆H (kcal/mol) of isodesmic reactions studying the optimum identity and a orientation of PR3 in Ir(H)2(PR3)2(O2C(CH2)5CH3) BP86/Basis Set 1 B3LYP/Basis Set 1 Cone Angle 14 Relative to PH3 o (a) PH3 to PEt3 –25.85 –26.81 45

i o PH3 to P Pr3 –14.43 –14.93 73

t o PH3 to P Bu3 –5.17 –4.88 87

o PH3 to PPh3 –14.17 –14.85 58

(b) PPh3 to P(p-FPh)3 –0.95 –0.80

PPh3 to P(m-FPh)3 0.17 0.32

PPh3 to P(m, p-FPh)3 –0.40 –0.27

Ax/Ax to Ax/Eq P(p-FPh)3 9.63 10.53 a Refer to Scheme 4.3 for the specific reactions being computed.

The results of these isodesmic reactions showed that there is no precise, positive correlation between the increase in cone angle and added stabilization in energy. The

t largest increase in cone angle from PH3 to P Bu3 only gave an added stabilization of approximately 5 kcal/mol. Interestingly, less bulky ligands such as PEt3 gave the greatest stabilization, in upwards of over 25 kcal/mol. The most common ligand in the Crabtree catalyst system, PPh3 provided an added stabilization of approximately 14 kcal/mol, still a significant gain in energy. Based on these results, it can be concluded that there is a significant stabilization from exchanging PH3 with other phosphines that the actual ligand

122 being studied in the laboratory should be studied computationally to provide the most accurate results when examining the thermodynamics of any proposed catalytic cycle.

From the isodesmic reaction exchanging PPh3 to P(p-FPh)3, a slight gain in energetic favorability from placing a fluoride in the para position was observed. Thus, these results show the addition of fluorides in the phosphine not only aids in solvation, but also slightly aids in the stability of the catalyst. When a single fluoride was placed in the meta position, however, there was a slight increase in energy, indicating less stabilization. When two fluorides were placed on the phenyl ring, there was only approximately 0.5 kcal/mol stabilization, less than a single fluoride in the para position.

Thus, the most effective fluoride substitution on the phenyl ring providing the most stabilization was a single fluoride in the para position.

The final question answered from Table 4.5 showed there was a definite thermodynamic cost in converting between axial/axial PPh3 and P(p-FPh)3 and the axial/equatorial orientation of the phosphine ligands. The rise in energy of approximately

10 kcal/mol helps explain why bulky phosphine ligands are more stable in the axial/axial orientation, even in the presence of hydride ligands, which have a similarly high trans

15 effect. The bulky nature of P(p-FPh)3 and PPh3 also must play a role in the energetic preference for these ligands to be trans in the iridium coordination environment. Finally, of the two levels of theory studied, both BP86 and B3LYP results were in agreement, within 1 kcal/mol of their energies.

Another isodesmic reaction can be employed to calculate the energetic difference in coordinating a fatty acid to an iridium-based Crabtree catalyst instead of other popular

123 carboxylate ligands, such as trifluoroacetate (TFA). The first critical step in the proposed dehydrogenation of a saturated fatty acid is, of course, the coordination of the fatty acid to the iridium catalyst. To calculate the energy difference between the coordination of a

C7 fatty acid (heptanoic acid) and TFA to the iridium catalyst center, the ∆H of the reaction below (Scheme 4.4) was calculated.

L L H O O H O O Ir CF3 Ir 5 H O OH H O F3C OH L L

Scheme 4.4. Isodesmic reaction studying the coordination of a fatty acid to an iridium Crabtree catalyst.

The calculation for Scheme 4.4 showed a small energetic cost in replacing the coordinating TFA with a C7 fatty acid. The BP86 functional calculated the iridium coordinated to TFA to be more energetically favorable by 2.91 kcal/mol, while the

B3LYP functional calculates the TFA coordinated catalyst to be more energetically favorable by 3.69 kcal/mol. While the iridium catalyst bound to TFA is slightly more stable, the ability to overcome this energy difference in coordination of a different ligand has been proven through the crystal structure shown in Figure 4.2.

4.5 PROBING THE PROPOSED MECHANISM OF C–H ACTIVATION

With the study of these isodesmic reactions completed, the study of key intermediates in the proposed mechanism of the dehydrogenation of saturated fatty acids while focusing on the most useful catalysts was undertaken. Mechanistic studies focused 124 on examining Crabtree catalysts using various phosphines as ligands. A proposed mechanism was developed through a collaboration with the Stambuli research group

(Scheme 4.5). The first step, starting with the initial catalyst E (Scheme 4.5), would be the loss of hydrogen to decrease the valence of iridium and allow for a subsequent intramolecular oxidative addition. Depending on which C–H bond inserts into the metal, either a 5, or 6–membered iridalactone could be formed. Upon β-hydride elimination, two different unsaturated fatty acids could be isolated depending upon the selectivity of the catalyst system.

Scheme 4. Proposed Mechanism for the Dehydrogenation of Palmitic Acid O intramolecular intramolecular O L O O H H L H C-H activation L H H C-H activation O H Ir Ir O H Ir H H L β H I L H F H H 10 L β H β G β Hβ H H Hβ H 10 β 10 Hβ H H heat or H2 β β sacrificial acceptor β-Hydride L β-Hydride Elimination H O Elimination Ir H O L E 10 O O H L O O H L O H H H Ir Ir HO H H H 10 L H O or O L H O J H H 10 HO OH H 10 HO 10 10 10 controlled by ancillary ligands

Scheme 4.5. Proposed mechanism for selective dehydrogenation of a coordinated heptanoic acid by “Crabtree–like” system.

125

The first step in the proposed mechanism of selective C–H activation of saturated fatty acids involves the reductive elimination of H2 from the initial Crabtree catalyst

(Scheme 4.6) to form an iridium (I) species. Once a mole of hydrogen has been lost, then the pendant fatty acid can oxidatively add back into the metal center. The thermal preference of this initial step of the proposed mechanism was computed using PEt3, PPh3, and P(p-FPh)3 as ligands. These ligands were chosen based on earlier computational results that suggested these phosphine ligands provided the most stable catalyst species.

Results showed though that a significant thermal energy penalty existed to remove hydrogen from the starting catalyst species (Table 4.6). This highly endothermic step could be mitigated one of two ways: (1) removal of hydrogen throughout the course of the reaction or (2) the introduction of a sacrificial acceptor to bond with a molecule of hydrogen.

PR 3 O H O Ir H O L -H2 Ir O 5 PR3 4 L Scheme 4.6. Loss of hydrogen from the proposed starting catalyst to an η1 intermediate for the carboxylate group.

126

Table 4.6. Loss of H2 without a sacrificial acceptor, in kcal/mol.

BP86/Basis Set 1 B3LYP/Basis Set 1

L=P(p-FPh)3 60.47 57.24

L=PPh3 60.40 57.12

L=PEt3 65.41 61.19

Due to its high endothermicity, the thermal preference of this reaction was then also computed using one of three sacrificial acceptors for the mole of hydrogen to be lost from the metal: t-butylethylene, norbornene, and norbornadiene (Figure 4.4). An isodesmic reaction was calculated, taking into account both the sacrificial acceptor as a reactant, and the resulting alkane as a product. As expected, this loss of H2 was more thermodynamically favorable in the presence of one of these sacrificial acceptors, with the thermal energy penalty being reduced by upwards of 40 kcal/mol. Overall, the loss of hydrogen is still endothermic; however, the computed value is much less so than without the presence of the sacrificial acceptor (Table 4.7).

Figure 4.4. Potential sacrificial acceptors: t-butylethylene, norbornene, and norbornadiene.

127

Table 4.7. Loss of H2 with sacrificial acceptor in kcal/mol t-butylethylene BP86 (kcal/mol) B3LYP (kcal/mol)

L=P(p-FPh)3 22.05 18.57

L=PPh3 21.98 18.46

L=PEt3 27.00 22.52

Norbornene

L=P(p-FPh)3 21.18 16.80

L=PPh3 21.11 16.68

L=PEt3 26.12 20.75

Norbornadiene

L=P(p-FPh)3 15.61 11.30

L=PPh3 15.54 11.19

L=PEt3 20.55 15.25

Following this initial step of the proposed mechanism is the key step that would allow for selective synthesis of unsaturated fatty acids directly from saturated fatty acids.

In this step, the coordinated fatty acid would oxidatively add back to the iridium center in an intramolecular fashion to form either a five or six–membered ring. After this intramolecular addition, β–H elimination can occur to convert the saturated fatty acid into a ∆3,4–fatty acid or ∆4,5–fatty acid, or a terminal saturated fatty acid depending on the

128 cyclic intermediate produced. A second equivalent of the initial saturated fatty acid could then displace the newly formed unsaturated fatty acid and return the initial catalyst, continuing the catalytic cycle.

The study of the thermodynamic and kinetic preference of these intermediates is critical in the determination of which unsaturated fatty acid will be formed (Scheme 4.7).

If either the 5-, 6- or 9-membered intermediate is heavily favored energetically, then the eventual product should proceed with a high degree of selectivity. However, if there is not a large energetic difference between these intermediates, there may be no selectivity in the synthesis of a particular unsaturated fatty acid.

O O O L O O L O H Ir L O L or H Ir or O H Ir Ir 5 L L L L 4 C4H9 C3H7 4.7a 4.7b 4.7c Scheme 4.7. Unsaturated Ir intermediate proceeding through intramolecular oxidative addition to form potential 5- (a), 6- (b), and 9-membered (c) iridalactones intermediates.

Because the stoichiometry of these compounds are identical, their electronic energies can be directly compared to yield useful information about the exothermic or endothermic nature of the intramolecular oxidative addition and the thermodynamic preference between the three proposed intermediates (Table 4.8). The thermodynamic energy difference between the three different ring–sized intermediates was also calculated using both the BP86 and B3LYP functionals (Table 4.9).

129

Table 4.8. Thermodynamic preference of intramolecular oxidative addition in kcal/mol as computed with bottom-of-the-well relative energies as shown in Scheme 4.7.

Formation of 4.7a BP86 B3LYP

L=P(p-FPh)3 –12.58 –5.02

L=PPh3 –13.26 –5.93

L=PEt3 –17.00 –6.55

Formation of 4.7b BP86 B3LYP

L=P(p-FPh)3 –11.18 –1.37

L=PPh3 –11.85 –4.25

L=PEt3 –14.74 –6.63

Formation of 4.7c BP86 B3LYP

L=P(p-FPh)3 –10.25 –2.49

L=PPh3 –10.27 –4.09

L=PEt3 –14.89 –8.40

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Table 4.9. Relative thermodynamic stability to 4.7a (refer to Scheme 4.7) using different phosphine ligands, as computed by bottom-of-the-well energies in kcal/mol at BP86 and B3LYP.

4.7a

BP86 B3LYP

L=P(p–FPh)3 0.00 0.00

L=PPh3 0.00 0.00

L=PEt3 0.00 0.00

4.7b

BP86 B3LYP

L=P(p–FPh)3 1.40 3.64

L=PPh3 1.41 1.67

L=PEt3 2.26 –0.08

4.7c

BP86 B3LYP

L=P(p–FPh)3 2.33 2.33

L=PPh3 2.99 1.84

L=PEt3 2.11 –1.84

Both functionals suggested the intramolecular oxidative addition should be exothermic, which was an encouraging sign for our proposed mechanism. However, these results suggested the potential selectivity of the proposed mechanism of C–H activation may be quite low based on the thermodynamic data. First, the BP86 functional 131 estimates the change in energy after intramolecular oxidative addition to be almost twice as high as the B3LYP functional, a discrepancy between the two theories not seen until this point. Second, when comparing between the ring closings, there was a very small thermodynamic difference in energy between the three proposed pathways. In all but one case (B3LYP with L=PEt3), the 5-membered ring is the most thermodynamically preferred.

However, this step in the proposed mechanism may not even be possible if the initial loss of hydrogen leads to an iridium species that is getting “stuck” in an η2 conformation instead of the desired η1 conformation (Scheme 4.8). In fact, if the catalyst is stuck in this conformation, then it may not be possible for the pendant fatty acid to oxidatively add back into the metal center. Calculations showed the possible square planar coordination is significantly more stable thermodynamically than the η1 conformation, by around 30 kcal/mol (Table 4.10). Further exploration of this possible alternate mechanism was performed to understand more fully the true catalytic cycle in the proposed mechanism.

O L O 4 L Ir Ir O 5 L O L

Scheme 4.8. Equilibrium between η1 and η2 coordination of pendant fatty acid to iridium.

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Table 4.10. Relative thermodynamic preference, computed as bottom-of-the-well energies (in kcal/mol), of fatty acid coordination being either η1 or η2.

η1 η2

BP86 B3LYP BP86 B3LYP

L=P(p-FPh)3 0.00 0.00 –30.75 –26.20

L=PPh3 0.00 0.00 –30.39 –23.57

L=PEt3 0.00 0.00 –34.30 –29.92

In order to determine a way to favor the desired η1 conformation, instead of the inactive η2 conformation of the pendant fatty acid, several fatty acid derivatives were examined that might shift the calculated equilibrium. Three gem-disubstituted derivatives of heptanoic acid were examined: dimethyl, difluoro, and diphenyl (Scheme

4.9). The hypothesis being that more steric bulk around iridium could favor the η1 coordination of the fatty acid derivative. Results showed that, even with disubstituted fatty acids, the η2 coordination of the pendant fatty acid was thermodynamically favored by a significant margin (Table 4.11 and 4.12). The difference in thermal stability (~20–

40 kcal/mol) seems too significant to overcome using feasible reaction variables.

133

O 4 L O 4 R Ir L R L O R Ir O R L

Scheme 4.9. Equilibrium between Ir η1 and η2 coordination of pendant fatty acid derivatives with α-disubstituted groups (R = H, F and Me).

Table 4.11. Relative energies, computed with bottom-of-the-well energies (in kcal/mol), of η1 and η2 coordination of heptanoic acid derivatives with α-disubstitution using BP86/Basis Set 1.

α-difluoro η1 η2

L=P(p-FPh)3 0.00 –34.30

L=PPh3 0.00 –30.39

L=PEt3 0.00 –30.75

α-dimethyl

L=P(p-FPh)3 0.00 –37.51

L=PPh3 0.00 –32.30

L=PEt3 0.00 –32.69

α-diphenyl

L=P(p-FPh)3 0.00 –30.05

L=PPh3 0.00 –20.77

L=PEt3 0.00 –32.91

134

Table 4.12. Relative energies, computed with bottom-of-the-well energies (in kcal/mol), of η1 and η2 coordination of heptanoic acid derivatives with α-disubstitution using B3LYP/Basis Set 1.

α-difluoro η1 η2

L=P(p-FPh)3 0.00 –29.92

L=PPh3 0.00 –23.57

L=PEt3 0.00 –26.20

α-dimethyl

L=P(p-FPh)3 0.00 –33.67

L=PPh3 0.00 –28.56

L=PEt3 0.00 –29.24

α-diphenyl –33.78

L=P(p-FPh)3 0.00 –28.58

L=PPh3 0.00 –29.04

L=PEt3 0.00 –32.91

4.6 CONCLUSIONS

From these initial DFT studies, we have come closer to discovering the optimum iridium–based catalysts to employ in the selective dehydrogenation of saturated fatty acids. Using our Basis Set 1, it was determined that the more common, computationally expensive B3LYP functional is no more accurate in modeling the coordination 135 environment of iridium than the computationally less expensive BP86 functional when studying these Crabtree–like catalysts. Further, the use of bigger basis sets with added polarization functions does not increase the accuracy of computed bond lengths and bond angles. Using isodesmic reactions, we found Crabtree catalysts gain added stabilization from phosphine ligands such as PPh3 and P(p-FPh)3 while also confirming the preference for axial/axial orientation. When modeling Crabtree catalysts, it is also necessary to model the precise catalyst, instead of substituting PH3 for other phosphine ligands.

Meanwhile, studies of the proposed mechanism of selective C–H activation using

Crabtree catalysts have thus far shown little thermodynamic preference between synthesis of a ∆3,4-fatty acid, ∆4,5-fatty acid or a terminal fatty acid from the formation of a 5-, 6-, or 9–membered intermediate via intramolecular oxidative addition of an unsaturated fatty acid. However, ultimately this particular catalyst system was found to be experimentally unviable because the catalyst was likely getting stuck in an η2 square planar conformation after an initial loss of hydrogen. The addition of substituents in the α-position of heptanoic acid were computed to have no effect in favoring the η1 conformation of the pendant fatty acid. Because of these significant issues with the proposed mechanism, and a lack of successful synthetic results, this particular catalyst system was quickly abandoned. Hence, no kinetic barriers were examined in the proposed mechanism by optimizing transition states. However, the significant contribution of the computational studies presented herein allowed the collaborative effort to continue forward onto a new approach towards the selective dehydrogenation of pendant fatty acids.

136

4.7 REFERENCES FOR CHAPTER 4

1. Crabtree, R. H. Chem. Rev. 1985, 85, 245.

2. Jensen, C. M. Chem. Commun. 1999, 2443.

3. Filho, M.V.; Araujo, C.; Bonfa, A.; Porto, W. Enzyme Research 2011, 2011, 1.

4. Liu, F.; Pak, E. B.; Singh, B.; Jensen, C. M.; Goldman, A. S. J. Am. Chem. Soc. 1999, 121, 4086.

5. Krogh–Jespersen, K.; Czerw, M.; Summa, N.; B., R. K.; Achord, P. D.; Goldman, A. S. J. Am. Chem. Soc. 2002, 124, 11404.

6. Crabtree, R. H.; Mihelcic, J. M.; Quirk, J. M.. J. Am. Chem. Soc. 1979, 101, 7738.

7. Gérard, H.; Eisenstein, O.; Lee, D.–H.; Chen, J.; Crabtree, R. H. New J. Chem. 2001, 25, 1121.

8. Werpy, T.; Petersen, G., Top Value Added Chemicals from Biomass. Volume 1: Results of Screening for Potential Candidates from Sugars and Synthesis Gas. U.S. Department of Energy: Oak Ridge, 2004, Vol 1.

9. Fan, H.–J.; Hall, M. B. J. Mol. Catal. A–Chem. 2002, 189, 111.

10. (a) Becke, A. D. Phys Rev. A. 1988, 38, 3098. (b) Perdew, J. P. Phys. Rev. B. 1986, 33, 8822.

11. (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785.

12. (a) Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Chem. Phys. Lett. 1989, 62, 165. (b) Häser, M.; Ahlrichs, R. J. Comput. Chem. 1989, 10, 104. (c) Treutler, O.; Ahlrichs, R. J. Chem. Phys. 1995, 102, 346. (d) Arnim, M. v.; Ahlrichs, R. J. Comput. Chem. 1998, 19, 1746.

13. Whitten, J. L. J. Chem. Phys. 1973, 58, 4496.

14. Tolman, C. Chem. Rev. 1977, 77, 313.

15. Hegedus, L. Transition Metals in the Synthesis of Complex Organic Molecules, University Science Books, 1999.

137

Chapter 5 : Alternative Iridium–Based Catalysts for the Selective Dehydrogenation of Fatty Acid Derivatives

This chapter is partially adapted and reproduced from a manuscript published in

Society. Experimental studies were performed by Dr. Sean Whittemore in the Stambuli

Research Group at The Ohio State University. Experimental crystal structures were obtained by Dr. Judith Gallucci.

5.1 INTRODUCTION

As mentioned in the previous chapter, iridium has been used in the literature as a metal catalyst center to attempt C–H activation of saturated alkanes. However, several other iridium catalysts have been developed since the early efforts of Crabtree to affect a wide spectrum of organometallic transformations. This brief introduction seeks to highlight some of the notable efforts in the development of organometallic reactions that were influential towards inspiring the work presented herein. Through the use of different ancillary ligands as directing groups, iridium has been shown to be incredibly versatile when applied to novel organometallic reactions, including further attempts at C–

H activation. Specifically, this includes the ability for iridium to perform intramolecular

138 oxidative addition to form a cyclometallated product, the type of intermediate that would be crucial towards selective dehydrogenation if a C–H bond was inserted into the metal.

One of the earliest examples of intramolecular C–H activation of aliphatic hydrocarbons to form a cyclometallated intermediate was found by Vrieze, where a pendant tricyclohexyl phosphine group was transformed into an olefin.1 The tricyclohexylphosphine ligand was originally employed because of its high basicity. The steric crowding of the cyclohexyl groups also brought the C–H bond that was inserted closer to the iridium metal center, defined as the “proximity effect”. Vrieze proposed the catalytic cycle proceeded first through a coordinatively unsaturated iridium complex, achieved after loss of a phosphine ligand.

Perhaps even more interestingly, the mechanism proposed by Vrieze was subsequently surmised to proceed through a dimeric intermediate (Scheme 5.1). This would imply one iridium metal center would insert into the C–H bond from the ancillary cyclohexane ring of another iridium metal center. However, the dehydrogenation was not selective because the product mixture contained different olefin products as observed by

NMR. When the resulting metal–olefin complex was reacted with CO, the olefin remained intact, but was replaced in the coordination sphere of iridium.

139

solv. H

R3P Cl solv. R3P Cl solv. Ir Ir Ir Ir solv. PR3 Ir H Cl PR2 H Cl PR2

R3P Cl

Scheme 5.1. Proposed mechanism of cyclohexane dehydrogenation through a dimeric intermediate.1

Another example of cyclometallation by insertion of an aliphatic C–H bond came through efforts from Bergman.2 Once again, it was an ancillary ligand that was ultimately dehydrogenated, this time though, not through a phosphine. Instead, a neomenthyl group that was appended to a cyclopentadiene (Cp) ligand was found to be dehydrogenated by an unsaturated iridium center (Figure 5.1). Once again though, several isomers were produced, which suggests the dehydrogenation lacked selectivity.

Interestingly though, Bergman performed limited molecular modeling studies using low– level molecular mechanics to examine the competing intramolecular C–H activation to intermolecular C–H activation.

140

Figure 5.1. Crystal structure of dehydrogenated neomenthyl unit appended to Cp*–Ir complex.2

Bergman found the addition of the bulky neomenthyl group increased the activation energy of intermolecular C–H activation, while decreasing the activation energy of the corresponding intramolecular insertion. The role of the ancillary methyl group was also determined to favor intramolecular C–H insertion at iridium due to added steric hindrance of the cationic metal center. Without the presence of the methyl group, intermolecular C–H activation was observed exclusively. Silver hexafluoroantimonate was also employed as an additive to increase the rate of the dehydrogenation.

Herrmann showed another example of cyclometallation by C–H insertion into iridium using an N–heterocyclic carbene (NHC) ligand appended with cyclohexyl groups.3 A permethylated cyclopentyldiene ligand (Cp*) was employed as well as ancillary methyl groups, similar to Bergman’s previous study.4 The addition of trifluoromethylsulfonic acid leads to the elimination of methane, which results in an open

141 coordination site on iridium to insert the aliphatic C–H bond of the cyclohexane ring

(Scheme 5.2). Due to the nature of the ligand, four possible isomers were possible.

However, Herrmann suggested, on the basis of spectral and crystal structure evidence, that the syn double bond was the favored product formed.

OSO CF OSO2CF3 2 3 OSO2CF3 H Cp* CH3 Ir Cp* Ir Cp* Ir C6H11 N N N C H C H N 6 11 N N 6 11

Scheme 5.2. Dehydrogenation of cyclohexane appended to NHC ligand.3

5.2 COMPUTATIONAL METHODS

Calculations on the Ir–DMSO complexes presented herein were performed using the Turbomole program5. The previously referred Basis Set 1 was used, which modeled all atoms except iridium with an SV (P) basis set and a TZVP basis set on iridium.

Iridium was also treated by default with an effective core potential (ECP) in Turbomole.

Once again, the RI (resolution–of–the–identity) approximation6 was also employed for all

Turbomole calculations to increase computational efficiency. Each structure was optimized to a stationary point, using the default optimization criteria in Turbomole. In many cases, a vibrational frequency analysis was also performed to characterize the stationary point as a minimum or maximum on the potential energy surface.

Further calculations presented herein on the Ir–Cp* complexes appended with acylphosphines were performed using Gaussian 09.7 In terms of basis sets, iridium was

142 treated with an ECP to reduce the large number of electrons calculated while accurately representing relativistic effects of the core electrons.8 Often, iridium has been treated with the LANL2DZ5 basis set; however, the SDD9 pseudopotential was used in these calculations, which has previously been used to treat organometallic systems in the

Hadad group. When using Gaussian, the aforementioned Basis Set 1 previously used in

Turbomole–based calculations was maintained, except for the alterations made to treat the iridium center with a different pseudopotential. In addition to DFT functionals mentioned in previous chapters, a slightly different functional was examined, M06L, developed by Cramer and Truhlar to more accurately treat long–range correlations in organometallic systems.10 Each intermediate in the mechanistic studies was confirmed to be a minimum on the potential energy surface with no imaginary frequencies resulting from a vibrational frequency analysis.

When calculating the thermodynamics of PF6 hydrolysis, multiple computational methods were employed. Two DFT methods (M06L and B3LYP11) along with one semiempirical method (PM312) were used to examine the isodesmic reactions necessary to compute the thermodynamics of the hydrolysis. At the two DFT levels of theory, an

SV(P) basis set was used on all atoms. A vibrational frequency analysis was performed to find the enthalpic (ΔH) and free energy (ΔG) corrections to the bottom-of-the-well energies. A scaling factor was not applied for the vibrational frequencies or the zero– point vibrational energy correction. Also, the M06L calculations were repeated using the

Polarizable Continuum Model13 (PCM) in Gaussian 09 to model the effect of solvation around the species studied, using dichloromethane as the solvent.

143

5.3 STUDIES OF Ir–DMSO COMPLEX TOWARDS FAVORING

INTRAMOLECULAR C–H ACTIVATION

As mentioned in Chapter 4, the previously studied iridium catalyst system derived from Crabtree’s dehydrogenation catalyst could not participate in intramolecular C–H activation in order to selectively dehydrogenate a pendant fatty acid. Our computational efforts discovered the overwhelming thermal stability of an η2 coordination of the pendant heptanoic acid upon loss of hydrogen from the starting catalyst, which would prevent intramolecular C–H activation that would necessarily need to occur from an η1 coordination to form an intermediate iridalactone. Thus, our experimental collaborators,

Dr. Sean Whittemore and Dr. James Stambuli of The Ohio State University, and ourselves necessarily explored alternate iridium catalyst systems.

Our new goal was to adapt our iridium catalyst to favor the necessary η1 coordination of the fatty acid, while maintaining necessary unsaturation to allow for an intramolecular C–H activation of the long chain of the same fatty acid. A particular system of interest was an Ir–DMSO complex. Studies have shown such Ir–DMSO complexes to add water oxidatively under room temperature conditions,14 along with performing other C–H activation chemistry, which would suggest it to be a promising scaffold for our desired purpose. The Ir–DMSO complex as mentioned above would provide a significantly different catalytic skeleton than the Crabtree–inspired ligands that were previously studied that employed phosphine ligands instead of sulfoxides.

In order to provide guidance to our experimental collaborators, a small library of ligands were chosen that could provide flexibility in terms of their coordination to the

144 iridium center. These ligands would be used to examine the equilibrium between the η1 or η2 coordination of the pendant fatty acid, where the flexible ligand would occupy the extra coordination site in the η1 coordinated fatty acid. The overall goal would be to discover a ligand that could ultimately favor that η1 coordination of the fatty acid, unlike the previous catalytic systems that were studied computationally (see Chapter 4). Thus, this equilibrium was studied for several ligands at BP86 and B3LYP levels of theory using Basis Set 1.

Several modified N–heterocyclic carbene (NHC) ligands were studied to provide an alternate ligand that could simultaneously occupy more than one coordination site on iridium. An NHC scaffold was modified to include an oxygen–based substituent that could coordinate to iridium. These carbene ligands have found great use in organometallic chemistry to carry out a variety of transformations.15 Four different carbene ligands were used (Figure 5.2), changing the oxy–based substituents to examine the coordination of an alcohol, acyclic ether, cyclic ether, and lactone to iridium. A

DMSO ligand was also used to coordinate to the iridium center.

O N O N N N O N N OH N O N

L1 L2 L3 L4

Figure 5.2. Substituted NHC ligands studied via molecular modeling studies.

145

Each of the NHC ligands studied computationally in the Ir–DMSO complexes still ultimately favored the η2 coordination of the pendant fatty acid, heptanoic acid

(Scheme 5.3). Although, each NHC ligand showed less thermodynamic favorability for the η2 coordination of the fatty acid than the previously studied Crabtree–like iridium catalysts. Where the previously studied system proved a dead end to the potential catalytic dehydrogenation, favoring the η2 coordination of the fatty acid by around 30 kcal/mol, the NHC ligands favored η2 coordination by as little as 5 kcal/mol when THF was substituted on the NHC (Tables 5.1 and 5.2). The prearrangement of the cyclic ether

(L3) seems to orient the oxygen of THF to occupy one of the coordination sites on iridium. However, even this preorganization is not enough to favor the necessary η1 coordination of the fatty acid.

O O O O S S O C6H13 Ir Ir C H O 6 13 N N OH N N OH

Scheme 5.3. Equilibrium between η1 and η2 coordination of pendant fatty acid in presence of substituted NHC ligand using L1.

Table 5.1. Relative thermodynamic favorability as measured at the bottom-of-the-well of η2 coordination of fatty acid in presence of substituted NHC ligand using BP86/Basis Set 1, in kcal/mol.

Ligand η1 Fatty Acid η2 Fatty Acid L1 0.00 –23.07 L2 0.00 –15.94 L3 0.00 –7.49 L4 0.00 –16.83 146

Table 5.2. Relative thermodynamic favorability as measured at the bottom-of-the-well of η2 coordination of fatty acid in presence of substituted NHC ligand using B3LYP/Basis Set 1, in kcal/mol.

Ligand η1 Fatty Acid η2 Fatty Acid L1 0.00 –23.40 L2 0.00 –16.05 L3 0.00 –5.90 L4 0.00 –16.48

Another particular ligand of interest that could conceivably occupy one or more coordination sites on the iridium metal center was the pyridonate ligand developed by

Fujita.16 These ligands have the ability to coordinate in an η1 or η2 fashion themselves

(Figure 5.3), much like the pendant fatty acids that would be coordinated to the iridium center. Thus, if the coordination of the pyridonate (or other) ligand could be controlled such that it would have a more favorable η2 coordination than the pendant fatty acid, intramolecular C–H activation could be enhanced in our catalyst system by favoring η1 coordination of the pendant fatty acid.

Cp* Cl Cp* Cl Ir Ir N Cl N O OH

Figure 5.3. Equilibrium between η1 or η2 coordination of pyridonate ligand to iridium.

The 2–pyridone ligand of Fujita was also studied in two forms, both neutral and anionic (Figure 5.4). Perhaps unsurprisingly, the neutral form of the ligand still favored the η2 coordination of the fatty acid by around 16 kcal/mol. However, the anionic 2–

147 pyridonate ligand actually does favor the necessary η1 coordination of the fatty acid

(Tables 5.3 and 5.4) in order to promote intramolecular C–H activation in a subsequent step. Certainly, the preorganization of the substituted pyridine ring and the strong binding of the anionic oxygen favor the coordination of the anionic ligand instead of the weaker η2 coordination of the fatty acid (Scheme 5.4). However, despite this potential breakthrough towards an active iridium catalyst that could promote intramolecular C–H activation, there were no successful results observed from this ligand/catalyst system by our experimental collaborators in the Stambuli group. Instead, another alternate system was experimentally pursued that would use a derivatized heptanoic acid to create a directing group effect with a slightly different iridium scaffold.

N OH N O

L5 L6

Figure 5.4. Neutral and anionic 2–pyridone ligands studied by molecular modeling.

O O O O S S O C6H13 Ir Ir C H O N O 6 13 N O

Scheme 5.4. Equilibrium between η1 and η2 coordination of pendant fatty acid in presence of anionic 2–pyridonate ligand.

148

Table 5.3. Relative thermodynamic favorability as measured at the bottom-of-the-well of η2 coordination of fatty acid in presence of substituted NHC ligand using BP86/Basis Set 1 in kcal/mol.

Ligand η1 Fatty Acid η2 Fatty Acid L5 0.00 –20.62 L6 0.00 13.28

Table 5.4. Relative thermodynamic favorability as measured at the bottom-of-the-well of η2 coordination of fatty acid in presence of substituted NHC ligand using B3LYP/Basis Set 1 in kcal/mol.

Ligand η1 Fatty Acid η2 Fatty Acid L5 0.00 –19.87 L6 0.00 12.74

5.4 USE OF Ir–CP* COMPLEX APPENDED WITH FATTY ACID DERIVATIVES

With limited success using phosphines or DMSO ligands with appended fatty acids, our experimental collaborators in the Stambuli group explored another iridium catalyst system to promote the selective dehydrogenation transformation. Instead of looking at saturated fatty acids as ligands, which our computational studies had shown were unsuitable to intramolecular, catalytic dehydrogenation, a fatty acid derivative was used to coordinate to iridium. Specifically, via a simple synthetic transformation, the fatty acids were converted to acylphosphines and other derivatives. Indeed, acylphosphines have been used much less often in organometallic chemistry than unmodified fatty acids.17 However, the acylphosphine ligand is not a very basic ligand,

149 so instead of using other phosphines or DMSO ligands, a Cp* ligand was introduced

(Figure 5.5) to add electron density to the starting catalyst.

Ir Cl Cl R2P

()4 O

Figure 5.5. Starting catalyst species using an acylphosphine derived from heptanoic acid.

At first, two cyclohexyl units were appended by Whittemore and Stambuli to the acylphosphine derived from heptanoic acid. However, this starting catalyst produced C–

H activation on the cyclohexyl ring instead of the extended fatty acid chain (Scheme 5.5) when reacted with silver hexafluorophosphate in dichloromethane at 37°C. This observation had actually been reported previously in the literature for similar catalytic systems.18 This experimental observation was confirmed by using a deuterated acylphosphine, which did not yield a C–D insertion product. Substituting t–butyl groups instead of cyclohexyl groups also failed to yield the desired unsaturated fatty acid derivative.

150

PF Cp* Cp* 6 Cl H Ir Ir Cy P CyP 2 Cl AgPF6 (2.2 equiv) O O DCM, 45°C

4 4

Scheme 5.5. Dehydrogenation of pendant cyclohexyl substituent of acylphosphine.

However, once the acylphosphine was substituted with isopropyl groups at the same reaction conditions mentioned above, there was evidence of selective dehydrogenation along the chain of the heptanoic acid derivative (Scheme 5.6). The presence of the singular C–H activation product was confirmed by X–ray crystallography

(Figure 5.6) at the Δ3,4 position with a cis orientation about the double bond. This selectivity is an important advancement in C–H activation chemistry when considering similar results was shown for either symmetric alkanes or was unselective, generating multiple isomeric products.

PF6

Cl AgPF6 (2.2 equiv) Ir Ir Cl P DCM, 45°C P H ()4 O O

Scheme 5.6. Dehydrogenation from C–H insertion along the aliphatic chain of the acylphosphine.

151

Ir P H

Figure 5.6. Crystal structure of olefin product, with hydrogens omitted for clarity, as obtained at The Ohio State University by Whittemore and Stambuli.

The dehydrogenation of the heptanoic acid derivative was not found to be catalytic; however, the overall yield of the reaction was found to be over 80%. The working mechanism was studied further via several spectroscopic techniques, which showed several different signals in 31P NMR (suggesting multiple intermediates along the pathway) and the appearance of an Ir–hydride signal in the 1H NMR. The hydrolysis of the non–coordinating hexafluorophosphate anion was also observed as well as other peaks in the 31P NMR that could not immediately be assigned.

One additional intermediate was isolated though, which was shown to be an unusual diiridium complex where the carbonyl oxygen of the acylphosphine coordinated to another iridium metal center. The structure of this intermediate was proven by X–ray crystallography (Figure 5.7) and was shown to be on the same reaction pathway as the ultimate unsaturated product (Scheme 5.7). With these experimental observations in mind, our goals were to use knowledge gained from similar calculations on other iridium

152 catalyst systems to model potential mechanistic pathways for the selective dehydrogenation of the pendant acylphosphine.

Cl1b

Ir1b Ir2b

Cl2b

O1b P1b

Figure 5.7. Crystal structure of isolated dimeric intermediate obtained at The Ohio State University by Whittemore and Stambuli.

(PF6)2 PF6

Cl Ir Ir DCM, 45°C, 4h Ir Cl P i H ( Pr)2P O

() O 5

Scheme 5.7. Isolated dimeric species leads to identical olefin product as starting catalyst, suggesting dimer is on reaction pathway.

153

5.5 STUDY OF PROPOSED DEHYDROGENATION MECHANISM

On the basis of our past experience in calculating the thermodynamics of proposed transformations mediated by an iridium catalyst, possible mechanisms were studied using Density Functional Theory. However, instead of using more standard

Becke–based functionals (BP86 and B3LYP), the M06L functional was chosen instead due to its more accurate treatment for organometallic systems.10 Our initial focus in attempting to gain a better understanding of the experimental observations from our collaborators focused not on the final olefin product, but instead on the isolated diiridium species.

In order to examine the importance of the unique diiridium intermediate that was isolated, a plausible computational mechanism had to be established to determine the degradation of the dimeric complex. Since the dimeric species was shown to exist on the reaction pathway, determining its fate would allow us to surmise potential mechanisms from the resultant degradation products. To that extent, two pathways were imagined that could plausibly yield the dehydrogenated olefin product based upon the degradation of the diiridium complex. Depending upon the results of this initial computational study, a reasonable mechanism for the entire dehydrogenation could then be explored in more detail, since the diridium species was shown to exist on the reaction pathway towards the unsaturated acylphosphine product.

Thus, two pathways considering the possible degradation of the diiridium species were calculated to determine their thermodynamic favorability (Scheme 5.8). The first pathway, dependent upon an additional equivalent of phosphine, could see the isolated

154 dimer decompose into the tetravalent starting material and a highly unsaturated dicoordinate iridium complex (Pathway A). The second pathway would involve the decomposition of the dimer into two equal tricoordinate iridium species upon the addition of one equivalent of phosphine (Pathway B). Also, the effects of counter ions were ignored to reduce the computational expense.

*Cp Cl (PF6)2 (PF ) Pathway A Ir i 6 2 i () 5 Cl ( Pr)2P Ir Cp* ( Pr)2P (iPr) P *Cp Cl Cp* 2 ()5 ()5 Ir Ir Cl O O O i ( Pr)2P O () *Cp Cl PF6 5 Ir Pathway B 2 i () 5 ( Pr)2P O

Scheme 5.8. Potential mechanistic pathways from isolated dimeric intermediate.

The thermodynamic preference of each of these two pathways was calculated, once again using isodesmic reactions, which ensured the electronic energies could be directly compared. In each potential pathway, the equivalent of phosphine was added to the diiridium complex to balance the reaction stoichiometry. Using bottom-of-the-well energies, Pathway A was found to be endothermic by 52.22 kcal/mol while Pathway B was found to be exothermic by –48.4 kcal/mol. Thus, our results show Pathway B is favored by around 100 kcal/mol. Due to the extremely large difference in thermodynamic stability, kinetic barriers were not calculated. The instability of the highly unsaturated, dicoordinate 14–electron iridium complex formed in Pathway A may be responsible for the high endothermicity of this proposed decomposition of the dimer. 155

Any dicoordinate iridium complex, though highly reactive, seems to be too high in energy to play a role in the reaction mechanism.

These results indicated the next important intermediate in the proposed mechanism should be the tricoordinate iridium species. Based on the isolated crystal structures and previously mentioned computational data, the tricoordinate iridium species must be the critical intermediate that allows dehydrogenation to occur. From that tricoordinate iridium, the next step of the proposed mechanism was hypothesized to be the intramolecular C–H activation, which would then yield a proposed 5–membered cyclometallated intermediate. Further elimination of HCl followed by β–hydride elimination would yield the isolated final product (Scheme 5.9).

(PF6)2 *Cp Cl PF6 Cl Ir H Ir ()3 2i () 5 i ( Pr)2P ( Pr)2P

O O

PF6 (PF6) H Ir Ir ()3 i i ( Pr)2P ( Pr)2P

O HCl O HCl

Scheme 5.9. Proposed mechanistic pathway for selective dehydrogenation of pendant acylphosphine C–H chain studied via molecular modeling.

156

The proposed C–H activation to make the initial cyclometallated species from the tricoordinate iridium intermediate was found to be endothermic by 38.41 kcal/mol. The kinetic barrier of this step of the mechanism was not found through our computational efforts. The subsequent elimination of HCl was found to be relatively thermoneutral, with a gain in thermodynamic stability of less than 0.5 kcal/mol as measured at the computed bottom-of-the-well energy. The final β–hydride elimination step was then determined to be exothermic by 11.5 kcal/mol relative to the previous cyclometallated intermediate as measured at the bottom-of-the-well. A molecule of HCl was used in the last two steps to balance each step of the mechanism stoichiometrically. Overall though, from the initial tricoordinate iridium species, the overall transformation was found to be endothermic by around 26 kcal/mol.

While the cis Δ3,4 unsaturated product was the only alkene product isolated experimentally, there are 4 plausible products that could be formed based on the formation of a 5–membered or 6–membered cyclometallated intermediate, depending upon the particular C–H bond inserted into the metallic center. From a 5–membered cyclometallated intermediate, the trans Δ3,4 alkene can be synthesized in addition to the cis Δ3,4 alkene.

From a 6–membered cyclometallated species (if a different C–H bond would

4,5 insert along the alkyl chain of the pendant acylphosphine), either a cis or trans Δ alkene can be produced. However, experimentally only the singular isomer was formed, suggesting some selectivity in the operating mechanism. Thus, our computational efforts sought to provide further insight into the final β–hydride elimination step to determine if 157 it was the source of the selectivity, or if the final product’s stereochemistry is determined earlier in the reaction mechanism.

Each of the four possible β–hydride elimination reactions from a 5– or 6– membered intermediate, the final step in the proposed mechanism, was found to be exothermic (Table 5.5). Also, both 5–membered cyclometallated species were found to be more stable than either of the corresponding 6–membered cyclometallated intermediates. The most stable cyclometallated intermediate, leading to the trans Δ3,4 alkene product, does not yield the most thermodynamically alkene stable product

(Scheme 5.10, Reaction 2). DFT calculations have shown the ultimately isolated cis Δ3,4 product to be the most thermodynamically stable product by around 3 kcal/mol compared to any of the other three possible alkene products (Scheme 5.10, Reaction 1). The stabilization of the cis product seems to be a result of the Cp* ligand disfavoring trans formation due to steric interactions with the long alkyl chain. Kinetic favorability in the actual β–hydride elimination also doesn’t seem to play much of a factor in this step as each of the activation barriers were between 1 and 2 kcal/mol for each of the four β– hydride elimination reactions studied (Scheme 5.10). This suggests the final olefin product is determined by its overall thermal stability compared to other possible alkene isomers.

158

Ir Ir (1) P P H

O O

Ir Ir (2) P P H

O O

Ir (3) Ir P P H

O O

Ir Ir (4) P P H

O O

Scheme 5.10. Four possible β–hydride elimination pathways from 5– or 6–membered cyclometallated intermediates.

159

Table 5.5. Relative energies of all calculated reactants, transition states, and products of four possible β–hydride elimination pathways from 5- or 6-membered cyclometallated intermediates, in kcal/mol, using M06L/Basis Set 1, as computed with bottom-of-the-well energies for the reactions in Scheme 5.10.

Reactant Transition State Alkene Product

Reaction 1 0.0 1.5 –11.5

Reaction 2 –1.7 –0.1 –8.4

Reaction 3 0.7 2.0 –6.9

Reaction 4 4.5 5.6 –5.1

Another aspect of the operating mechanism we wished to explore was the observed hydrolysis of the non–coordinating counterion: hexafluorophosphate. A series of reactions explaining the hydrolysis of hexafluorophosphate was originally proposed by

Pellinghelli, which consisted of a stepwise hydrolysis through the intermediate species

– 19 POF4 to ultimately yield PO2F2 . To determine if this hydrolysis of the counterion played any role in the operating mechanism, the proposed reactions of Pellinghelli were examined by several different computational theories. If the hydrolysis were found to be highly exothermic, perhaps it could be a thermodynamic driving force for the dehydrogenation. In order to balance each of the reactions of the stepwise hydrolysis, so that an accurate enthalpy could be calculated, HF and H2O were used to ensure the same stoichiometry on either side of the equation to balance each reaction .

The hydrolysis reactions were examined using the M06L DFT functional in the

13 gas phase and in CH2Cl2, using a Polarized Continuum Model (PCM). We also examined the hydrolysis using a different DFT functional (B3LYP) and a semiempirical 160 method (PM3) to compare the different treatments of the calculations. Overall, the

– hydrolysis of PF6 to PO2F2 was found to be highly endothermic by every computational

– method examined (Table 5.6 and 5.7). Once the intermediate POF4 is formed (Scheme

– 5.11), the second hydrolysis to yield PO2F2 was found to be much less endothermic than

– – the initial hydrolysis from PF6 to PO2F2 (Scheme 5.12). The normally less accurate semiempirical method was found to be less endothermic in the first step, but came close to matching the values found by DFT methods in the second step. A stabilization of about 10–15 kcal/mol was found after the free energy correction was made for each of the two steps of hydrolysis.

However, the large endothermicity of the initial step of hydrolysis suggests the

– proposed transformation of PF6 is not a thermodynamic driving force for the overall dehydrogenation. In fact, the uphill nature of the hydrolysis suggests a Lewis Acid may be mediating the observed hydrolysis by trace amounts of water.

F O F F F H O P 2 P 2 HF F F F F F F

– Scheme 5.11. First step in hydrolysis of PF6 as proposed by Pellinghelli.

161

– Table 5.6. Thermodynamics, reaction enthalpy, and free energy of first step in PF6 as proposed by Pellinghelli as modeled using three different levels of theory in kcal/mol.

Method Bottom-of-the-Well ∆H ∆G

M06L 57.77 55.16 42.92

M06L (DCM) 53.42 50.85 38.76

B3LYP 59.44 56.76 44.52

PM3 29.43 28.35 17.87

O O F

F P O P H2O F F 2 HF F F

– Scheme 5.12. Second step in hydrolysis of PF6 as proposed by Pellinghelli.

– Table 5.7. Thermodynamics, reaction enthalpy, and free energy of second step in PF6 as proposed by Pellinghelli as modeled using three different levels of theory in kcal/mol.

Method Bottom-of-the-Well ∆H ∆G

M06L 16.90 14.72 4.59

M06L (DCM) 13.13 10.89 0.74

B3LYP 18.97 16.83 6.72

PM3 20.39 18.47 8.31

162

5.6 CONCLUSIONS

Though not an example of catalytic dehydrogenation, the efforts towards the selective C–H activation of an extended fatty acid chain have proven beneficial to ongoing scientific efforts in this field. Further computational study confirmed the difficulty of performing C–H insertion on an unmodified fatty acid due to the thermodynamic favorability of the η2 coordination of the carboxyl group. This particular coordination seemed to prohibit C–H insertion that would form the necessary cyclometallated product to favor β–hydride elimination and an olefin product.

Thus, other ligands were explored that could block the η2 coordination of the pendant fatty acid by having a similar ability to occupy one or two coordination sites on iridium. Modified NHC ligands, with an oxy–based substituent, failed to overcome the

η2 coordination of the pendant fatty acid. However, the use of Fujita’s 2–pyridonate did ultimately favor the η1 coordination of the fatty acid, presumably due to the strong binding of the anionic oxygen and its proximity to the iridium center. Still, because sufficient synthetic experiments were not carried out using this catalyst/ligand system, it is currently unknown whether this ligand would allow the desired intramolecular C–H insertion of a long–chain fatty acid. Perhaps the η2 coordination of the 2–pyridonate ligand would leave the intermediate catalyst species stuck in an unreactive state, much like the η2 coordination of the fatty acid itself.

Thus, instead of altering the ancillary ligands around the iridium center, the fatty acid itself was converted to an acylphosphine that would instead be coordinated to the metal center. When further substituting the acylphosphine with two isopropyl groups, a 163

3,4 single olefin product was observed experimentally with a cis orientation at the Δ position. An intriguing dimeric intermediate, which was shown to exist on the reaction pathway, was also isolated and its structure confirmed through X–ray crystallography.

Molecular modeling studies were able to study a proposed mechanism for this novel transformation, specifically examining the fate of the diiridium intermediate and the final, crucial β–hydride elimination step.

3,4 It was determined the thermodynamic stability of the cis Δ alkene was the determining factor in the selectivity shown in this particular organometallic system. The experimentally observed hydrolysis of the PF6 anion was found to be far too endothermic to be considered a thermodynamic driving force, suggesting the use of the acylphosphine as a directing group was influential in the observed, selective dehydrogenation.

However, even though the C–H activation proceeded in sufficient yield, the transformation was not catalytic. The next advancement in this particular chemistry then must be to optimize the dehydrogenation so that it may become catalytic. Once the acylphosphine dehydrogenation becomes catalytic, then the challenge of catalyzing the

C–H activation of an aliphatic fatty acid chain can once again be explored.

5.7 REFERENCES FOR CHAPTER 5

1. Hietkamp, S.; Stufkens, D. J.; Vrieze, K. J. Organomet. Chem. 1978, 152, 347.

2. Ma, Y.; Bergman, R.G. Organometallics. 1994, 13, 2548.

3. Prinz, M.; Grosche, M.; Herdtweck, E.; Herrmann, W. Organometallics. 2000, 19, 1692.

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4. Arndtsen, B. A.; Bergman, R. G. Science. 1995, 270, 1970.

5. (a) Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Chem. Phys. Lett. 1989, 62, 165. (b) Häser, M.; Ahlrichs, R. J. Comput. Chem. 1989, 10, 104. (c) Treutler, O.; Ahlrichs, R. J. Chem. Phys. 1995, 102, 346. (d) Arnim, M. v.; Ahlrichs, R. J. Comput. Chem. 1998, 19, 1746.

6. Whitten, J. L. J. Chem. Phys. 1973, 58, 4496.

7. Gaussian 09, Revision A.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2009.

8. Niu, S.; Hall, M. B. J. Am. Chem. Soc. 1999, 121, 3992–3999.

9. Andrae, D.; Haubermann, U.; Dolg, M.; Stoll, H.; Preub, H. Theor Chim Acta 1990, 77, 123.

10. Cramer, C. J.; Truhlar, D. G. Phys. Chem. Chem. Phys. 2009, 11, 10757.

11. (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785.

12. Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209.

13. Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Rev. 2005, 105, 2999–3093.

14. Dorta, R.; Rozenberg, H.; Shimon, L. J. W.; Milstein, D. J. Am. Chem. Soc. 2002, 124, 188.

15. Hitchcock, P. B.; Lappert, M. F.; Terreros, P. J. Organomet. Chem. 1982, 239, C26.

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16. Yamaguchi, R.; Ikeda, C.; Takahashi, Y.; Fujita, K. J. Am. Chem. Soc. 2009, 131, 8410.

17. Baber, R. A.; Clarke, M. L.; Orpen, A. G.; Ratcliffe, D. A. J. Organomet. Chem. 2003, 667, 112.

18. Grelier, M.; Vendier, L.; Sabo–Etienne, S. Angew. Chem., Int. Ed. 2007, 46, 2613.

19. Fernandez–Galan, R.; Manzano, B. R., Otero, A.; Lanfranchi, M.; Pellinghelli, M. A. Inorg. Chem. 1994, 33, 2309.

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Chapter 6 : Thesis Conclusions and Future Directions

6.1 THESIS CONCLUSIONS

This thesis has shown contributions made to both objectives set out in the introduction. First, progress has been made on multiple fronts towards combatting the effects of exposure to organophosphorus (OP) chemical nerve agents. On one hand, quinone methide precursors (QMPs) have been developed and shown to alkylate a model phosphonate, similar to the phosphylated serine residue in the aged acetylcholinesterase

(AChE) active site. Also, a family of molecular baskets has been developed, with an imidazole-capped basket shown to bind dimethoxy methylphosphonate (DMMP) in aqueous solution. Both of these efforts are ongoing in the Hadad research group, and the majority of this chapter will focus on future directions that these research projects may pursue.

Also, this thesis presented a novel application of organometallic chemistry towards the selective C–H activation of a pendant acyl phosphine unit. However, this area of research still leaves much to accomplish in order for the goal of catalytic dehydrogenation of a fatty acid to be realized. Ideally, if a saturated fatty acid can be selectively converted to an unsaturated fatty acid, many of the Department of Energy’s value-added chemicals (Chapter 1) could be easily obtained. Thus far though, the chemistry has not been developed to a point where the dehydrogenation is catalytic or 167 performed on an unmodified fatty acid. Further ligand systems could be studied in a joint computational and experimental approach in the future, although efforts continue to meet the ultimate goal of the original proposal. Despite the remaining challenges, a significant achievement was made in the novel dehydrogenation of a pendant acyl phosphine.

6.2 FUTURE DIRECTIONS OF QUINONE METHIDE PROJECT

There are several different directions to continue to explore the use of quinone methide precursors as a small molecule therapeutic to recover aged AChE after exposure to an OP nerve agent. While our efforts have isolated certain para-QMPs as potential alkylating agents, much work remains to truly determine if one of our alkylating agents would be selective for the aged AChE active site. Furthermore, there are several experiments that can be performed to optimize the ability of our QMPs to alkylate by examining slightly different molecular frameworks based on our quinone methide scaffold. Also, there are various mechanistic studies that can be performed, as the operative mechanism of alkylation is still elusive.

There have been some preliminary computational studies within the Hadad group to further examine the possible reaction mechanism of the alkylation of our QMPs. Both

Jason Brown1 and Dr. Jeremy Beck2 have performed density functional theory (DFT) calculations to examine the effect of electronic substituents on the two different conceivable mechanisms of alkylation. One possible operative mechanism would involve a stepwise process where the actual quinone methide intermediate is generated in the first step. Then, the nucleophile would attack at the electrophilic and exocyclic

168 carbon to restore aromaticity, while alkylating the quinone methide (Scheme 6.1, A).

The alternative mechanism would involve a direct nucleophilic attack on the benzylic position of the QMP in an SN2-like mechanism (Scheme 6.1, B). The computational work performed in the Hadad group has suggested that the operating mechanism is subject to the starting structure of the QMP. Specifically, differences in the electron- donating or electron-withdrawing nature of substituents on the aromatic ring impacts the kinetic favorability of the two possible mechanisms.

Base H—Base

H H O O O

R R R R R R

NR3 Nu

H H O O

R R R R

NR3 Nu Nu

Scheme 6.1. (A) The stepwise mechanism of alkylation through formation of the quinone methide intermediate, followed by alkylation. (B) The concerted mechanism of alkylation consisting of direct displacement of the leaving group by the nucleophile at the benzylic position.

In addition to these preliminary computational studies, there are several experimental studies that could be performed to further examine the operating mechanism of alkylation. One could imagine the synthesis of compounds very similar to 169 our QMPs; however, without the presence of a phenolic oxygen that would be critical for the base-catalyzed generation of the quinone methide intermediate. The replacement of a phenolic oxygen by either a hydrogen or a methyl group (Figure 6.1) would only allow alkylation through an SN2-like mechanism. Comparing the extent of alkylation with these compounds to other QMPs already tested would help to determine the preferred mechanism of alkylation.

R R R R

NR2 NR2

Figure 6.1. Two possible “control” compounds to examine the extent of SN2-like alkylation of QMPs without the presence of a phenolic oxygen.

A similar strategy would be the synthesis of a stereogenic center at the benzylic position of the QMP, thereby providing some information as to the roles of both mechanisms (Scheme 6.1) for alkylation. By making the benzylic position chiral, one could observe if an inversion of stereochemistry occurs through direct attack by a nucleophile. If an SN2-like mechanism is predominant, there will be an inversion of configuration at the site of attack, the benzylic carbon. If instead the mechanism is proceeding through a planar intermediate (the quinone methide), then a racemic mixture should be observed in the alkylated product, indicative of nucleophilic attack from both faces of the alkene.

170

There are different methods to achieve a chiral QMP with one specific stereocenter at the benzylic position, modified to incorporate a deuterium atom. One path to a chiral QMP would be the synthesis of a Mosher’s ester from the original phenolic compound.3 By reacting the deuterated QMP with Mosher’s acid (MTPA), a mixture of diastereomers can be yielded, with both possible configurations at the benzylic position.

Then, as long as the mixture of diastereomers can be separated, the Mosher’s acid can be removed, yielding the chiral QMP of a single enantiomer (Figure 6.2). A similar approach could be taken by installing some type of chiral amine as the leaving group.

F C 3 O O O O N O D

Figure 6.2. A chiral QMP that can be separated from a diastereomeric mixture of a Mosher’s ester.

Aside from further study of the operative reaction mechanism, similar scaffolds to our QMPs can be examined to further enhance reactivity of alkylation and selectivity for aged AChE. By modifying the benzene ring in our QMPs to a pyridine ring, the electronics of the QMP should be dramatically altered, certainly affecting its reactivity as an alkylating agent. Also, the addition of the nitrogen atom to the ring leads to an even

171 wider variety of precursors that can be synthesized. The nitrogen can be placed in the two, three, or five position relative to the phenolic oxygen (Figure 6.3). Further, the nitrogen atom could also be methylated to make a cationic QMP that could better bind in the AChE active site due to its structural similarity to the known AChE reactivator 2-

PAM. These pyridine QMPs could be synthesized via a similar reductive amination protocol from the corresponding aldehydes.4

HO N HO HO N NR NR2 NR 2 N 2

Figure 6.3. Various pyridine QMPs that could be synthesized to expand the current library of alkylating agents. Only neutral para precursors are shown. However, cationic precursors could be synthesized through installation of a methyl group on the pyridine nitrogen. Also, the corresponding family of ortho precursors could also be synthesized.

Finally, in addition to computational and experimental studies performed within the Hadad research group, a third component to these efforts involves proteomic studies on lead QMPs identified through the results of the experimental and theoretical observations. As was mentioned briefly in Chapter 2, preliminary proteomic studies have been undertaken with lysozyme as a model protein. MS-TOF studies have been performed to determine how many times our QMPs alkylate a model protein. Ideally, when subjecting the alkylating agent to the aged AChE sample, the QMP would selectively alkylate once at the phosphonate anion in the aged active site. However, if the

QMP is alkylating multiple residues on the surface of a protein, that outcome would be evident via the results of the mass spectrometric investigations. 172

Our preliminary studies have identified several QMPs that do not alkylate the surface of a model protein. Of course, the results of our studies with lysozyme are not a perfect substitute for studies with the target enzyme, AChE. Still, the benefit of the model enzyme studies is two-fold. First, the protein surfaces should be similar enough that if a QMP does not overalkylate the surface of lysozyme, it likewise should not overalkylate the surface of AChE. Second, the trials with lysozyme will allow for optimization of analytical techniques. As our development of the QMPs and other precursors continues to unfold, the proteomic studies with AChE and aged AChE will ultimately determine how successful our alkylating agents are at reversing the toxic effects of exposure to OP nerve agents.

6.3 FUTURE DIRECTIONS OF MOLECULAR BASKETS PROJECT

Similarly to the quinone methide project discussed previously, there are multiple directions to carry forward the gains already made in the entrapment of OP compounds by molecular baskets. Already, our efforts have shown a molecular basket with the ability to bind the OP compound, dimethoxy methylphosphonate (DMMP). Yet, ongoing efforts must be made to show this particular macromolecular scaffold is suitable for the entrapment, and possible hydrolysis of a wide range of chemical warfare agents. By further modifying the amino acid residues at the gate of the basket and altering the molecular framework of the basket itself, different OP compounds could be targeted for preferential binding. And ultimately, the goal of the research would be the synthesis of a

173 basket that could not only bind the OP compound, but also hydrolyze it in order to eliminate the OP’s toxic effects.

As presented in Chapter 3, the molecular basket that was successful in the entrapment of DMMP was modified with decarboxylated histidine residues acting as the arms of the basket. Each of the three imidazole rings on the decarboxylated histidine residues were also protonated. This protonation state allowed for a hydrogen-bonding interaction, which helped to orient the DMMP ligand into the cavity of the basket.

Naturally, the next iteration of this particular basket may be to install the native amino acid residue, instead of the decarboxylated analog, at the rim of the basket. The addition of the carboxylate unit at the α-position of the gate may increase water solubility and also keep the imidazole rings over the center of the cavity more frequently.

As with the previously presented imidazole-capped basket, in using the native histidine residue, several different protonation states can be subjected to our full computational protocol. There are two different neutral molecular baskets that could be studied. One molecular basket would have a neutral imidazole ring and carboxylic acid, thus giving the basket an overall net neutral charge (Figure 6.4). Also, one could imagine a basket where the individual amino acid residues are in their zwitterionic form. In this case, there would be a carboxylate at the α-position and a protonated imidazole. This form of the basket would be particularly interesting, as this zwitterionic form is how histidine exists at physiological pH. There are other baskets as well that can be studied containing an overall charge of +3 or –3 (Figure 6.5). The +3, or acidic basket, would have a carboxylic acid at the alpha position and each of the imidazole rings protonated.

174

The –3, or basic basket, would have carboxylates at the α-position and would have the imidazole rings being neutral. Examining these protonation states will allow us to predict the behavior of the histidine-capped baskets at several different experimental conditions.

N NH HN NH

HO O O O O N O N O O

O O N NH N N N N H H OH O O O O O

O O HO N O N O O O O HN HN

N N H

Figure 6.4. Two net neutral baskets that can be subjected to our computational protocol (Chapter 3) to examine the conformational flexibility of the molecular basket and its binding of OP compounds. One basket is overall neutral with no charge (left) and the other has the histidine residues at the top of the basket in their zwitterionic form (right).

175

HN NH N NH

HO O O O O N O N O O

O O NH N N N N N H H OH O O O O O

O O HO N O N O O O O HN HN

N N H

Figure 6.5. Two charged baskets that can be subjected to our computational protocol (Chapter 3) to examine the conformational flexibility of the molecular basket and its binding of OP compounds. One basket is acidic with a net positive charge (left) and the other is basic with a net negative charge (right).

Another possible extension of this work would involve the synthesis of other molecular basket scaffolds. The particular scaffold used to recognize DMMP could be expanded so that it may be more suitable to bind larger OP compounds. For example, another molecular basket scaffold that has begun to be subjected to preliminary experimental and theoretical studies is shown below (Figure 6.6). This molecular basket has been modified to contain an added pyrazine ring into the wall of the basket. The addition of this heterocycle makes the basket taller and gives it a larger cavity. Similar modifications could also be performed to the benzene base and other parts of the basket to continue to optimize this class of macromolecules towards the entrapment of OP compounds. A recently developed program could aid in this effort by allowing our

176 researchers to calculate the volume of the interior of the cavity. This Voss Volume

Voxelator could aid in understanding how modifications to the basket scaffold can impact the size of the cavity.5 Once the understanding and optimization of these molecular baskets has become better understood, the hydrolysis of OP compounds can be examined, in addition to their entrapment.

O R N O

N N O N R N N O N N

O N O R

Figure 6.6. A modified molecular basket scaffold with an added pyrazine ring, providing taller arms that should yield a larger cavity for the recognition of larger OP compounds.

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