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Synthetic Developments for the Treatment of Organophosphorus Poisoning

DISSERTATION

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

By

Amneh Young

Graduate Program in Chemistry

The Ohio State University

2016

Dissertation Committee:

Professor Christopher M. Hadad, Advisor

Professor Jon R. Parquette

Professor Thomas M. Magliery

Copyrighted by

Amneh Young

2016

Abstract

Acetylcholinesterase (AChE) is a hydrolase responsible for the of the . It hydrolyzes over 25,000 acetylcholine (ACh) molecules every second. Inhibition of AChE catalytic site results in muscle contractions, blurry vision, seizures, and respiratory failure. Organophosphorus (OP) compounds are potent inhibitors of the AChE. Once the OP enters the enzyme , it phosphylates a serine residue (Ser203) to give an irreversibly inhibited AChE that is incapable of hydrolyzing ACh. This leads to a buildup of ACh at receptors and constant stimulation of nerve fibers. Reactivation of AChE can occur by hydrolysis of the phosphylated enzyme which is usually accomplished by use of a nucleophilic , such as 2-PAM, often administered after OP exposure as a treatment. However, if reactivation does not occur, the phosphylated enzyme will then undergo a spontaneous dealkylation process (called aging) to give an aged enzyme which, to date, cannot be reactivated.

This dissertation covers multiple strategies to combat OP poisoning. First is the design of a pre-treatment in the form of catalytic antibodies capable of hydrolyzing OPs before they can inhibit AChE. The potential for antibodies to catalyze hydrolysis reactions inspired several reports of abzyme neutralization of OP nerve agents. To produce the catalytic antibodies, our group designed and synthesized a small library of haptens that mimic the transition state of nerve agent hydrolysis. These haptens are then ii conjugated to an immunogenic and antibodies are raised that hopefully hydrolyze nerve agents before they can inhibit AChE. Ten haptens were synthesized and preliminary results indicate that these haptens either bind or hydrolyze our target OP nerve agent.

The second approach is the use of a cyclic peptide to mimic the active site of known OP bioscavengers. A combinatorial approach was used to develop a library of peptides synthesized on a bead using common solid phase peptide chemistry. A novel screening approach will be discussed.

The final strategy is the design of quinone methide precursors (QMPs) that will enter the active site of AChE and alkylate the “aged” enzyme. Once alkylated, AChE can be reactivated using a nucleophilic oxime (2-PAM). Our goal is to design QMPs which will bind to and selectively alkylate aged AChE. We designed four different families of

QMPs that can be easily accessed via simple transformations of commercially available starting materials. An undergraduate organic chemistry lab experiment was designed to aid in the synthesis of the QMP library and produced over 100 QMPs in gram quantities.

Approximately half of the QMPs react with model under physiological conditions. The reactivity of these QMPs with model phosphonylated peptides and aged

AChE will be discussed.

iii

Dedication

To my loving husband Philip

iv

Acknowledgments

There are many people who have helped me grow and succeed, both on an academic and personal level. I must first thank the people who mentored me through my graduate career: Professor Chris Hadad for helping me achieve my goals and for the freedom to pursue my own projects, and Dr. James Stambuli for teaching me the skills I needed to succeed and giving me continued guidance to help me find my path. Dr. Will

Henderson showed me the ropes when I first started graduate school and Dr. Matt Lauer continued my training-teaching me everything I know about Wittig reactions and softball.

All the Stambuli group members made work truly enjoyable and intellectually stimulating. I would like to thank Tom Corrigan, Chi Le, Ben Garrett, Ryan McKenney,

Dr. Jeremey Erb, Qinggeng Zhuang, and our many collaborators for making the Hapten project a success. Special thanks to Ryan McKenney for helping me turn my idea of cyclic peptides as OP scavengers into a reality. Professor Dehua Pei was instrumental in the design of that project and without his guidance our success would have been unlikely.

There are so many people involved in the QMP project, many of which I haven’t worked with directly, but without whom the project would likely still be in infancy. There are countless undergraduate students, under the guidance of Dr. Chris Callam, who did the bulk of the synthesis. Qinggeng Zhuang, worked tirelessly on this project to identify hit compounds.

v

My graduate career followed an unconventional path and I would like to thank the people who helped me navigate those waters-I would not be where I am today without them. Ben Garrett and Luke Baldwin patiently counseled me over many months while I struggled to determine my best course of action. I am really glad I listened to both of you.

Professor Chris Hadad and Dr. James Stambuli always had an open door and listened to me before doing everything in their capacity to help me accomplish my goals. I am lucky to have both of you on my side. Finally, I would like to thank my wonderful husband

Philip for all the love, support, and happiness you bring to my life. I look forward to the years ahead and all the adventures we will have along the way.

vi

Vita

2011...... B.S. Chemistry and Biochemistry, The

University of Michigan-Dearborn

2014...... M.S. Chemistry, The Ohio State University

2011 to present ...... Graduate Research Associate, Department

of Chemistry and Biochemistry, The Ohio

State University

Publications

1. Palladium-Catalyzed Reactions of Enol Ethers: Access to Enals, Furans, and

Dihydrofurans. Lauer, M.G.; Henderson, W.H.; Awad, A.; Stambuli, J.P. Org. Lett. 2012,

14, 6000 – 6003.

2. Dimeric FeFe-Hydrogenase Mimics Bearing Carboxylic Acids: Synthesis and

Electrochemical Investigation. Garrett, B.R.; Awad, A.; He, M.; Click, K.; Durr, C.;

Gallucci, J.; Hadad, C.M.; Wu, Y. Polyhedron 2016, 103, 21-27.

vii

Fields of Study

Major Field: Chemistry

viii

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vii

List of Schemes ...... xii

List of Tables ...... xv

List of Figures ...... xvi

List of Abbreviations ...... xix

CHAPTER 1: INTRODUCTION TO ORGANOPHOSPHORUS NERVE AGENTS

AND AChE ...... 1

1.1: Brief History of the Development and Use of Organophosphorus Nerve Agents ... 1

1.2: Use of OP Compounds as ...... 4

1.3: Structure and Activity of ...... 6

1.4: Current Therapies for OP Inhibition of AChE ...... 10

1.5: Statement of Purpose...... 15

CHAPTER 2: SYNTHESIS OF NOVEL PHOSPHORANE HAPTENS ...... 16

ix

2.1: Introduction ...... 16

2.2: Division of Work Described in this Chapter ...... 23

2.3: Hapten Design and Synthesis ...... 24

2.4: Results and Discussion ...... 45

2.5: Conclusions ...... 54

CHAPTER 3: DEVELOPMENT OF CYCLIC PEPTIDES FOR VX HYDROLYSIS ... 56

3.1: Introduction ...... 56

3.2: Division of Work Described in this Chapter ...... 62

3.3: Cyclic Peptide Design and Synthesis ...... 62

3.4: Model Phosphonate Design and Synthesis ...... 67

3.5: Screening Protocols and Hit Identification ...... 74

3.6: Future Directions ...... 77

CHAPTER 4: DEVELOPMENT OF ALKYLATING AGENTS FOR THE

REACTIVATION OF AGED AChE...... 81

4.1: Introduction ...... 81

4.2: Quinone Methide Precursor Design ...... 90

4.3: Division of Work Described in this Chapter ...... 92

4.4: Quinone Methide Precursor Synthesis ...... 92

4.5: Reactivity Testing of Quinone Methide Precursors ...... 98

x

4.6: Re-Aging of AChE ...... 100

4.7: Future Directions ...... 104

CHAPTER 5: EXPERIMENTAL AND SYNTHETIC DETAILS ...... 105

5.1: General Methods ...... 105

5.2: Chapter 2 Experimental Details ...... 106

5.3: Chapter 3 Experimental Details ...... 145

5.4: Chapter 4 Experimental Details ...... 150

5.5: Other Relevant Experimental Details...... 164

Appendix A: Selected NMR Spectra ...... 177

References ...... 312

xi

List of Schemes

Scheme 1.1: Mechanism of Hydrolysis of Acetylcholine in AChE Active Site ...... 8

Scheme 1.2: AChE Inhibition Mechanism by an Organophosphorus Compound ...... 8

Scheme 1.3: Mechanism of Oxime Reactivation of Inhibited AChE ...... 14

Scheme 2.1: VX Hydrolysis Mechanism and Tetrahedral Intermediate ...... 21

Scheme 2.2: Predicted Phosphorane Hydrolysis Mechanism ...... 25

Scheme 2.3: Retrosynthesis of Final Phosphorane Haptens ...... 30

Scheme 2.4: Initial Ortho-Quinone Framework Synthesis ...... 31

Scheme 2.5: Alternative Ortho-Quinone Framework Synthesis Using Tert-Butyl Acrylate as the Heck Coupling Partner ...... 32

Scheme 2.6: Attempted Reactions ...... 33

Scheme 2.7: Optimized Synthesis of 2.1 ...... 34

Scheme 2.8: Synthesis of Palmitic Acid Derived Fatty Acid Linker ...... 35

Scheme 2.9: Final Optimized Ortho-Quinone Synthesis ...... 36

Scheme 2.10: Synthesis of Symmetrical Phosphorus (III) Precursor ...... 38

Scheme 2.11: Synthesis of Unsymmetrical Phosphorus (III) Precursor ...... 39

Scheme 2.12: Final Noncovalent Class Hapten Synthesis...... 41

Scheme 2.13: Cycloaddition and Click Reactions to Give Final Haptens with Covalent

Linkers ...... 43 xii

Scheme 2.14: Hapten Synthesis with Alternative Phosphorus (III) Precursor ...... 44

Scheme 2.15: Ellman Assay for Detection ...... 46

Scheme 3.1: Reactions Catalyzed by Cyclic Peptides. a Diels-Alder Reaction. b Friedel-

Crafts Reaction...... 61

Scheme 3.2: Solid-Phase Synthesis of Linear Spacer ...... 64

Scheme 3.3: Bead Surface Modification ...... 65

Scheme 3.4: Generic Split-and-Pool Procedure...... 66

Scheme 3.5: Cyclic Peptide Library Synthesis ...... 67

Scheme 3.6: Synthesis of OP Precursor ...... 68

Scheme 37: Initial Attempts to Synthesize OP Mimic ...... 69

Scheme 3.8: Modified OP Mimic Route that Avoids Volatile Intermediates ...... 70

Scheme 3.9: Alternative Route with Different Thiol ...... 71

Scheme 3.10: Synthesis of Alternative Dye ...... 72

Scheme 3.11: Alternative Dye Route Optimization ...... 72

Scheme 3.12: Final Dye Optimized Synthesis ...... 73

Scheme 3.13: Dye-Tagged OP Synthesis ...... 74

Scheme 3.14: Reaction of Dye-Tagged OP with Cyclic Peptide Library ...... 74

Scheme 3.15: Potential On-Bead Screening of Peptide Library for Hydrolysis

...... 80

Scheme 4.1: Mechanism of AChE Aging ...... 83

Scheme 4.2: Different Aged and Inhibited Forms of AChE ...... 84

Scheme 4.3: Pyridinium Sulfonate as an Alkylating Agent ...... 85

xiii

Scheme 4.4: Methoxypyridinium Methyl Transfer Agents ...... 88

Scheme 4.5: Thermal and Photochemical Generation of a Quinone Methide and

Subsequent Reaction with an Amino Acid ...... 88

Scheme 4.6: Quinone Methide Alkylating Nucleotide Bases in DNA ...... 89

Scheme 4.7: Quinone Methide Alklyating a Phosphodiester ...... 90

Scheme 4.8: Mannich Reaction Conditions Screened to Make Naphthyl Family QMPs 95

Scheme 4.9: Formation of Iminium ...... 96

Scheme 4.10: Formylation and Subsequent Reductive Amination of 1 and 2-Naphthol . 97

Scheme 4.11: Family QMP Synthesis ...... 98

Scheme 4.12: Synthesis of Model Phosphonate ...... 99

xiv

List of Tables

Table 1.1: Symptoms of Organophosphorus Poisoning ...... 9

Table 1.2: AChE Inhibition and Aging Rates ...... 10

Table 2.1: Phosphate Haptens and Rate Enhancements of Corresponding Abzymes for

Hydrolysis Reactions ...... 20

Table 2.2: Phosphorane Haptens and Time to Complete Hydrolysis in Aqueous Solution

...... 28

Table 3.1: Catalytic Activity of Potential Bioscavengers Against Various

Organophosphorus Compounds ...... 57

Table 3.2: Catalytic Efficiency of OPH, OPAA, and PON1 Against Several

Organophosphorus Compounds ...... 58

Table 4.1: Aging Half Times of Selected OP Nerve Agents ...... 83

xv

List of Figures

Figure 1.1: Organophosphorus Nerve Agents ...... 2

Figure 1.2: Timeline of the Development and Use of Organophosphorus Nerve Agents .. 3

Figure 1.3: Use of Organophosphorus Pesticides in the USA from 1975-2007 ...... 5

Figure 1.4: Organophosphorus Pesticides (Thions) ...... 6

Figure 1.5: AChE Activity in Neuromuscular Junctions ...... 7

Figure 1.6: Possible Treatment Opportunities for Organophosphorus Poisoning ...... 11

Figure 1.7: that are Reversible AChE Inhibitors...... 12

Figure 1.8: Nucleophilic as AChE Reactivators ...... 13

Figure 2.1: Reaction Coordinate Diagrams. a Transition States of Catalyzed and

Uncatalyzed Reactions. b Reaction Coordinate with an Intermediate. The Structure of the

Intermediate will Resemble the Transition State Closest to it in Energy; in this Case, TS1

...... 17

Figure 2.2: General Abzyme Generation Outline ...... 18

Figure 2.3: A Phosphate as a TSA of Carbonate Hydrolysis ...... 19

Figure 2.4: Moriarty's First and Second Generation Phosphorane Haptens ...... 22

Figure 2.5: Phosphorane Haptens Investigated by Vayron and Co-workers ...... 22

Figure 2.6: Vayron's α-Hydroxyphosphinate Haptens ...... 23

Figure 2.7: Various Phosphorane Hapten Scaffolds Explored ...... 26 xvi

Figure 2.8: Effect of Haptens on AChE Activity as Determined by an Ellman Assay .... 47

Figure 2.9: Abzyme Anti-Hapten Titers as Determined by ELISA...... 49

Figure 2.10: Effect of Abzymes on VX Inhibition of AChE ...... 51

Figure 2.11: Effect of Abzyme Concentration of VX Inhibition of AChE ...... 52

Figure 2.12: Effect of Abzyme on VX Inhibition of AChE Over Time ...... 53

Figure 2.13: Effect of Abzyme Concentration on VX Inhibition of AChE Over Time

(Negative Control Subtracted) ...... 54

Figure 3.1: Cyclodextrin OP Scavengers ...... 59

Figure 3.2: Cyclic Peptide Therapeutics ...... 60

Figure 3.3: VX and Corresponding Dye-Tagged Mimic ...... 68

Figure 3.4: On-Bead Screening Protocol ...... 75

Figure 3.5: In-Solution Screening Protocol ...... 76

Figure 3.6: X-ray Structure of OPDA [PDB code 2D2H] Bound to Trimethyl Phosphate

...... 76

Figure 3.7: Fluorogenic OP Analogs ...... 77

Figure 3.8: RP and SP Enantiomers of VX ...... 78

Figure 3.9: and Thion OP Pesticides ...... 79

Figure 4.1: Inhibition and Aging of AChE by VX and Reactivation with 2-PAM ...... 82

Figure 4.2: Phenacyl as Phosphonate Alkylating Agents ...... 86

Figure 4.3: Model Phosphonates and Potential Anchimeric Assistance in Hydrolysis

Pathway ...... 87

xvii

Figure 4.4: Design of Quinone Methide Precursor Library Containing Features Found in

Compounds Known to Enter the AChE Active Site ...... 91

Figure 4.5: The Role of Electronic Effects in the Generation and Subsequent Reaction of o-Quinone Methides...... 92

Figure 4.6: Selection of Current Quinone Methide Precursor Library ...... 94

Figure 4.7: Reaction of Model Phosphonate Salt with Benzyl and

Corresponding 31P NMR Spectra ...... 100

Figure 4.8: and Potential Re-Aging of AChE ...... 101

Figure 4.9: Model OP Compounds Used to Probe Re-Aging of AChE ...... 102

Figure 4.10: Assay Used to Determine Re-Aging Rate ...... 103

Figure 4.11: Possible QMPs Containing a Pendant Oxime for AChE Reactivation ...... 104

xviii

List of Abbreviations

°C degrees Celcius

Å angstrom

α alpha

β beta

γ gamma

δ chemical shift in parts per million

μ micro

1H NMR proton nuclear magnetic resonance

13C NMR nuclear magnetic resonance

19F NMR fluorine nuclear magnetic resonance

31P NMR phosphorus nuclear magnetic resonance

11B NMR boron nuclear magnetic resonance

2-PAM

AA amino acid

Ac acetyl

Acm acetamidomethyl

Arg arginine

xix

Asn asparagine aq aqueous atm atmosphere

ACh acetylcholine

AChE acetylcholinesterase

B β-

BChE

Boc tert-butylcarbonate

Bn benzyl br broad tBu tert-butyl

BSA bovine serum albumin

CAM ceric ammonium molybdate

CAN ceric ammonium nitrate calcd calculated

CD cyclodextrin

CWA agent

DABCO 1,4-diazabicyclo[2.2.2]octane dba dibenzylideneacetone

DCC dicyclohexylcarbodiimide

DCM dichloromethane dd doublet of doublets

xx

DDT 1,1’-(2,2,2-trichloroethane-1,1-diyl)bis(4-chlorobenzene)

DFP diisopropyl fluorophosphate

DIPEA diisopropylethylamine

DMAP N,N-dimethylaminopyridine

DMF N,N-dimethylformamide

DMMP dimethyl methylphosphonate

DMSO dimethylsulfoxide dt doublet of triplets

DTNB 5,5’-dithiobis(2-nitrobenzoic acid) ee enantiomeric excess

ELISA enzyme-linked immunosorbent assay

EPA environmental protection agency

EtOAc equiv equivalent(s)

ESI electrospray ionization

Et ethyl

F phenylalanine

FDA food and drug administration

Fmoc fluorenylmethyloxycarbonyl g gram(s)

GABA γ-aminobutyric acid

Glu glutamate

xxi h hour(s)

HATU 1-[bis(dimethylamino)methylene]-1H,-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate

Hex hexanes

His histidine

HRMS high resolution

HSA human serum albumin

Hz Hertz

IPA isopropanol

J coupling constant in Hertz k kilo kcat/KM enzyme efficiency

KLH keyhole limpet hemocyanin

L liter

LD50 , 50%

Leu leucine

LG leaving group

M molarity, mega, m milli, meter, multiplet mAChR muscarinic acetylcholine

MALDI matrix assisted laser desorption ionization

Me methyl

xxii

MeCN

MeOH min minute(s) mol mole(s)

MOPS 3-(N-morpholino)propanesulfonic acid

MS mass spectrometry; molecular sieves n nano nAChR nicotinic

NBS N-bromosuccinimide

NHS N-hydroxysuccinimide

NMR nuclear magnetic resonance

Nu

OBOC one-bead-one-compound

OP organophosphorus

Orn ornithine p pentet

PBST phosphate buffered saline triton

Pd palladium

Pd/C palladium on carbon

PED partial Edman degradation

PEG polyethylene glycol

Ph phenyl

xxiii

Phe phenylalanine

PON1 paraoxonase-1 ppm parts per million

PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate q quartet

QM quinone methide

QMP quinone methide precursor quant quantitative

R arginine rt room temperature s singlet sat saturated sep septet sext sextet

Ser serine t triplet

TBAF tetrabutylammonium fluoride

TBDPS tert-butyldiphenylsilyl

TCEP tris(2-carboxyethyl)

TEA triethylamine tert tertiary

xxiv

TFA trifluoroacetic acid

THF tetrahydrofuran

TIPS triisopropylsilyl

TLC thin layer chromatography

TMS tetramethylsilane, trimethylsilyl

TNB 2-thio-5-nitrobenzoate

TOF time of flight

TS transition state

TSA transition state analog

Tyr tyrosine

USDA United States Department of Agriculture

UV-Vis ultraviolet-visible

Val valine

WW world war

xxv

CHAPTER 1: INTRODUCTION TO ORGANOPHOSPHORUS NERVE AGENTS

AND AChE

1.1: Brief History of the Development and Use of Organophosphorus Nerve Agents

During WWII, casualties from combat in the Pacific theater were less than casualties from malaria. It was known from the First World War, that malaria could cripple armies. In Macedonia for example, French, German, and British forces were immobilized for three years and the British army lost an estimated two million man-days due to malaria.1,2 In an attempt to prevent the debilitating effects of the disease in the

Second World War, scientists in the Allied and Axis powers researched new weapons to combat malaria: to control mosquito populations and quinine alternatives to treat malaria. In 1939, Swiss chemist Paul Müller discovered the insecticidal properties of DDT, for which he was awarded the Nobel Prize in 1948. Consequently, DDT was used for years to control mosquito populations in malaria stricken areas.

DDT was not the only potent discovered at the time. While researching new insecticides for IG Farben, German chemist Gerhard Schrader discovered a toxic organophosphorus compound (OP) named in 1936.3,4 At the time, German law required that any discoveries with potential military application be reported to military officials.5 Schrader and his team were relocated to a secret military

1 research facility where they subsequently discovered the more potent OP compound . These compounds, along with and , are termed “G agents” since

German scientists discovered them.6 During WWII, the Germans produced approximately 1,000 pounds of sarin and 12,000 tons of tabun.7 These nerve agents were not used during the war, possibly due to fear of retaliation. After the war, these large stockpiles of OP nerve agents were divided up amongst the Allied forces.5 Subsequently, a new class of nerve agents called “V agents” (V for venomous) was developed by

British, then American and Russian scientists.3,8 V agents are more viscous than G agents and are absorbed through the .9 V agents are also generally 1,000 fold more toxic than sarin. With an LD50 of 20 μg/kg, VX is so toxic that a single drop of the compound on the skin is enough for a lethal dose for a human (Figure 1.1).10

Figure 1.1: Organophosphorus Nerve Agents

Although OP nerve agents were not used in WWII, they have been used in several attacks upon civilian and military populations. During the Iran-Iraq war in the mid- 2

1980’s, the Iraqi government used and tabun against the Iranians. An estimated 45,000 casualties are a result of chemical weapons used by the Iraqi government.10 In the mid 1990’s, the terrorist group Aum Shinrikyo produced 70 tons of sarin and used it in two terrorist attacks in Japan.11,12 The nerve agent was released into the Tokyo subway system and residential areas. Thousands of people were injured and

19 people were killed.11 Most recently, during the Syrian civil war, the Syrian military attacked civilians with sarin and other chemical weapons (Figure 1.2). This resulted in over 1,300 deaths and thousands of injuries.13 The of OP compounds stems from their ability to inhibit the enzyme acetylcholinesterase (AChE).

Figure 1.2: Timeline of the Development and Use of Organophosphorus Nerve Agents

3

1.2: Use of OP Compounds as Pesticides

DDT was used in the Second World War to control the spread of malaria, typhus, and dengue fever. After the war, it was used indiscriminately to control agricultural pests. The USDA alone, sprayed hundreds of thousands of hectares with pesticides in a campaign against the S. invicta fire ant.14 Spurred by widespread insecticide use and concerns over bioaccumulation, Rachel Carson published Silent Spring in 1962. The book inspired a shift in public discourse regarding use and was instrumental in the ban of DDT in the 1970’s, and the formation of the US Environmental Protection

Agency (EPA).14 After the ban of organochlorine pesticides such as DDT, OP compounds were used as pesticides due to their lack of persistence in the environment

(Figure 1.3).15 The use of OP compounds has declined since the 1980’s however, and their use is now heavily regulated as a result of their acute toxicity.16

4

Amount of OP Insecticides Used in the US 140 120 100 80 60 40

Millions of Pounds of Millions 20 0 1975 1980 1985 1990 1995 2000 2005 2010 Year

Figure 1.3: Use of Organophosphorus Pesticides in the USA from 1975-2007

Most cases of OP poisoning are a result of OP pesticide exposure. There are approximately 3,000,000 people exposed to OP compounds every year resulting in

300,000 deaths.17 Additionally, OP compounds account for about 80% of pesticide related hospitalizations.18 OP pesticides are thions, however they are oxidized by cytochrome P450 in the liver to the more toxic oxon compounds (Figure 1.4).19 Thus, the effects of long-term, low-level exposure to OP pesticides is similar to the effects of nerve agent exposure. The need to develop effective therapeutics to treat OP poisoning from accidental, agricultural, or terrorist/combat exposure is of great importance.

5

Figure 1.4: Organophosphorus Pesticides (Thions)

1.3: Structure and Activity of Acetylcholinesterase

AChE is a serine hydrolase responsible for the hydrolysis of the neurotransmitter acetylcholine. It is found throughout the body in synaptic clefts in neuromuscular junctions.20 Acetylcholine (ACh) is a neurotransmitter that is released into the synaptic cleft following a neuronal impulse. There, ACh binds to cholinergic receptors on post- synaptic cell membranes and triggers a response, such as a muscle contraction.20

Afterwards, ACh is released into the synaptic cleft where it is rapidly hydrolyzed by

AChE (Figure 1.5). AChE is an extremely efficient enzyme: its catalytic efficiency,

9 -1 -1 kcat/KM, is 1.50x10 M •s and it hydrolyzes approximately 25,000 molecules of ACh every second making the hydrolysis of ACh by AChE near diffusion limited.21,19

6

Figure 1.5: AChE Activity in Neuromuscular Junctions

The active site of AChE lies at the bottom of a 20 Ǻ gorge and consists of a

19 catalytic triad comprised of serine (Ser203), histidine (His447), and glutamate (Glu334).

Upon entering the active site, ACh is attacked by the Ser203 hydroxyl group. This tetrahedral intermediate collapses producing and an acetylated serine residue.

Next, an activated water molecule attacks the acetylated serine to give a tetrahedral intermediate which, upon collapse, produces acetate and regenerates the active AChE

(Scheme 1.1).

7

Scheme 1.1: Mechanism of Hydrolysis of Acetylcholine in AChE Active Site

OP compounds are substrate analogs to ACh and are attacked in the AChE active site in a similar fashion. The phosphylated serine residue however, is not electrophilic enough for attack by the activated water molecule. This prevents regeneration of active

AChE and thus, AChE is unable to bind to and hydrolyze ACh (Scheme 1.2).

Scheme 1.2: AChE Inhibition Mechanism by an Organophosphorus Compound

Inhibition of AChE leads to an accumulation of ACh and overstimulation of nicotinic (nAChR) and muscarinic (mAChR) receptors which results in .

The symptoms of mild OP exposure are headache, dizziness, and . 15 Moderate and severe exposure results in muscle twitching, slurred speech, respiratory failure as a result

8 of diaphragm paralysis and increased bronchial secretions, and ultimately death (Table

1.1).15

Table 1.1: Symptoms of Organophosphorus Poisoning

Treatment of inhibited AChE with strong nucleophiles such as pyridinium oximes can restore catalytic activity of AChE. These oximes can cleave the Ser-O-P bond however, after a certain period of time, they lose their efficacy.22 At this point, the enzyme is referred to as “aged” because it is recalcitrant to reactivation. Aging occurs by

O-dealkylation of the OP-AChE adduct which results in an anionic phosphylated serine residue that is resistant to nucleophilic attack. The aging rates of different OPs vary and the half-life can be anywhere from 2-6 minutes for soman to 48 hours for VX (Table

1.2).23,24 The fast aging rates of some OPs severely shorten the window in which effective therapeutics can be administered.

9

Table 1.2: AChE Inhibition and Aging Rates

Nerve Agent Inhibition Rate (M-1•min-1) Aging Rate (h-1) Tabun (GA) 7.4 x 106 3.6 x 10-2 Sarin (GB) 2.7 x 107 2.3 x 10-1 Soman (GD) 9.2 x 107 6.6 Cyclosarin (GF) 4.9 x 108 9.9 x 10-2 VX 1.2 x 108 1.9 x 10-2 VR 4.4 x 108 5.0 x 10-3

1.4: Current Therapies for OP Inhibition of AChE

Given the mechanism of OP action, there are several opportunities to treat OP poisoning: protection of AChE from OP inhibition, reactivation of the inhibited enzyme, and realkylation of aged AChE (Figure 1.6).

10

Figure 1.6: Possible Treatment Opportunities for Organophosphorus Poisoning

Protection of AChE from inhibition can be accomplished by preventing the OP from entering the AChE active site, either by blocking the active site or sequestration of the OP in the blood. Certain carbamates such as , aminostigmine, and are reversible inhibitors (Figure 1.7).25 They bind in the

AChE active site thereby preventing the OP from irreversibly binding. Carbamates can be hydrolyzed from AChE in 30-40 minutes.19 Currently, pyridosigmine bromide is the only FDA approved drug for pretreatment against nerve agent exposure.26 However, since carbamates are inhibitors of AChE, treatment with carbamates still results in symptoms of acute AChE poisoning.

11

Figure 1.7: Carbamates that are Reversible AChE Inhibitors

An alternative treatment that avoids the symptoms of AChE inhibition is the use of a bioscavenger to sequester the OP before it can cause inhibition. These bioscavengers can be administered as a pre-exposure treatment to combat troops or post-exposure to reduce OP concentrations in the blood. Butyrylcholinesterase (BChE) is a cholinesterase enzyme similar to AChE that can bind, in a stoichiometric manner, with OP compounds such a VX.26 Human BChE offers protection against OP exposure when tested on guinea pigs and non-human primates.27,28 Based on its efficacy, human BChE was granted

Investigational New Drug status in 2006 by the FDA.27 Since BChE reacts with OP in a stoichiometric manner, large doses are needed to neutralize OPs effectively.

Large-scale production of sufficient quantities of BChE is problematic. The enzyme can be purified from human plasma but one liter of plasma provides less than one milligram of BChE.29 A recombinant version of BChE can be isolated from the milk of transgenic goats, however this process is costly.30 Catalytic scavengers such as paraoxonase-1 (PON1) can catalyze hydrolysis of the OP compound and thus offer greater than 1-to-1 protection against an OP.31 PON-1 shows promising catalytic activity for the detoxification of OP compounds and can increase survival rates of guinea pigs exposed to sarin and soman when administered as a pre-treatment.32–34 Due to the low 12 catalytic efficiency of these scavengers however, large doses are still needed for the bioscavenger to be a viable prophylactic.35 The main limitation of current enzyme bioscavenger approaches is the high cost associated with the large doses required.

The second treatment option for OP exposure is reactivation of the inhibited enzyme (Figure 1.8). The current standard of care is administration of a pyridinium oxime in conjunction with an () and anticonvulsants

(). Atropine binds to the muscarinic acetylcholine receptors, but it is an inverse and does not cause a neuronal response.36 Thus, it effectively blocks ACh from binding to its receptors and delays the cholinergic crisis. Benzodiazepines work on gamma-aminobutyric acid (GABA) receptors which inhibit neurotransmission by decreasing the synaptic release of ACh.15,37

Figure 1.8: Nucleophilic Oximes as AChE Reactivators

In the early 1950’s, Wilson discovered that the inhibited AChE could regain its activity after treatment with .38 This quickly led to the discovery that

13 aldoximes such as 2-PAM are potent AChE reactivators (Figure 1.8).38–42 The first therapeutic use of 2-PAM to treat OP poisoning was in 1956 when it was used to treat

Japanese soldiers.43 The pyridinium oximate is used to reactivate inhibited AChE.44 It enters the active site and is nucleophilic enough to attack the phosphylated enzyme, thereby returning AChE to its active state (Scheme 1.3).45 The efficacy of oximes varies depending on the OP bound to AChE and there are currently no oximes capable of restoring AChE activity against all OP used.44,46 Hence, the nerve agent must be identified and the proper oxime administered before aging has occurred (within a few minutes in the case of soman).24 Moreover, some phosphylated oximes are potent inhibitors of AChE.47 The development of a non-inhibitory and broad spectrum oxime is of significant importance to the cholinesterase community.

Scheme 1.3: Mechanism of Oxime Reactivation of Inhibited AChE

The third treatment opportunity is realkylation of the enzyme after aging has occurred. Pyridinium oxime reactivators are ineffective against aged AChE and there are no current methods to return the aged enzyme back to the inhibited state. In the 1960s and 1970s however, a family of alkylating agents was briefly investigated to determine if the O-alkyl group on the OP-enzyme complex could be replaced.48 These compounds were unsuccessful at regenerating the inhibited form of AChE, primarily due to the lack

14 of knowledge about the inhibition mechanism and AChE structure. Specific details about the AChE active site and structure were hypothesized until its crystal structure was published in 1991.49 In recent years, several groups have demonstrated interest in reinvestigating alkylating agents for realkylation of the aged OP-enzyme adduct.50,51,52

1.5: Statement of Purpose

The primary goal of this dissertation is to highlight three novel advances in the treatment of OP poisoning. Chapters 2-3 will highlight two potential opportunities to prevent OP nerve agents from inhibiting AChE. Chapter 2 will detail the design and synthesis of phosphorane haptens and their use to generate catalytic antibodies capable of hydrolyzing the nerve agent VX. Chapter 3 will cover an alternative bioscavenger approach wherein a random library of cyclic peptides is synthesized in a combinatorial manner and screened against hydrolysis of model OP compounds. Chapter 4 will highlight recent attempts to reactivate aged AChE by realkylating the enzyme with a library of alkylating agents. Finally, synthetic experimental details will be provided in

Chapter 5.

15

CHAPTER 2: SYNTHESIS OF NOVEL PHOSPHORANE HAPTENS

2.1: Introduction

Enzyme biocatalysts are highly efficient and can affect rate enhancements of specific reactions up to 1017 over background.53 Rate enhancement stems from an enzyme’s ability to stabilize the transition state (TS) of a reaction, thereby lowering the activation energy (Figure 2.1 a).54 The rational synthesis of with customizable specificity for catalyzing chemical reactions is a challenging problem. Most efforts focus on altering the specificity of natural enzymes by chemical modification or site-directed mutagenesis.55 Although these approaches are promising, achieving progress in rate enhancements is slow.

16

Figure 2.1: Reaction Coordinate Diagrams. a Transition States of Catalyzed and Uncatalyzed Reactions. b Reaction Coordinate with an Intermediate. The Structure of the Intermediate will Resemble the Transition State Closest to it in Energy; in this Case, TS1

An alternative to altering the specificity of existing enzymes is to produce a large library of enzymes and screen them against the desired chemical reaction. The principles of enzymatic can be used to generate antibodies capable of catalyzing reactions.

These catalytic antibodies or abzymes can be generated with the aid of hybridoma technology56 which takes advantage of the immune system’s diversity and specificity to generate monoclonal antibodies.55

Successful abzymes catalyze reactions by binding to and stabilizing the transition state of the desired chemical reaction.54 Transition states are transient species with high energy and descriptions of their structures can be predicted using computational chemistry.57 The Hammond postulate predicts that transition states will resemble intermediates closest to it in energy (Figure 2.1, b).58 Therefore, transition state analogs

(TSA) that mimic unstable reaction intermediates can serve as an approximation of transition state structure.54 Based on this assumption, antibodies that are elicited in 17 response to a TSA (hapten) will stabilize the TS relative to the starting materials and products, thereby catalyzing the reaction.59,60 Haptens are small molecules that elicit an immune response only when tethered to immunogenic carrier .

The process of generating abzymes is outlined below (Figure 2.2). First, a small molecule hapten is designed to resemble the transition state of the desired chemical reaction. This hapten is conjugated to an immunogenic carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).56 These proteins contain multiple surface lysines that can be used to attach the hapten covalently.61 The hapten- carrier protein adduct (now referred to as an immunogen) is capable of eliciting an immune response when it is injected into the body.62 To generate abzymes, the immunogen is injected into mice and after the antibodies are produced, they are harvested and screened for catalysis.55

Figure 2.2: General Abzyme Generation Outline

Early investigations of suitable TSA haptens took advantage of a change in geometry as a result of a change in hybridization in the reaction (e.g. formation of a sp3 hybridized tetrahedral intermediate from a planar, sp2 hybridized starting material).54 By designing haptens to mimic tetrahedral intermediates, Schultz and Lerner independently

18 reported abzymes capable of catalyzing the hydrolysis of aryl carbonates and respectively (Figure 2.3).63,64

Figure 2.3: A Phosphate Ester as a TSA of Carbonate Hydrolysis

In Schultz’s seminal example, antibodies with a high affinity for the TSA p- nitrophenyl cholyl phosphate were screened to determine if they could accelerate the hydrolysis of an aryl carbonate.64 Nucleophilic attack of the carbonate by generates a tetrahedral intermediate. Based on the Hammond postulate, the TS should resemble the tetrahedral intermediate.58 The unstable tetrahedral intermediate can be mimicked by the more stable phosphate ester analog. Antibodies that were isolated with a high affinity for the phosphate hapten proved to be effective in catalyzing the parent reaction.64 Since those initial reports, several other groups reported successful abzyme generation with rate enhancements up to 106 over background (Table 2.1 entry 2).65

Abzymes can be tailored to enantioselectively enhance the rate of reactions (Table 2.1 entry 3).65 Additionally, abzymes generated against certain haptens can be used to catalyze multiple reactions with good rate enhancement (Table 2.1 entry 4).65 Other reactions where abzymes offer significant rate enhancement over background rates are

19 enantioselective protonations, cyclizations of hydroxy expoxides, stereospecific reduction of α-ketoamindes, and ester linkage formation in deoxy sugars.66–70

Table 2.1: Phosphate Haptens and Rate Enhancements of Corresponding Abzymes for Hydrolysis Reactions

Due to the specificity and diversity of the reactions catalyzed by abzymes, several groups have investigated the possibility of using abzymes to hydrolyze nerve agents such as VX.71–77 The mechanism of VX hydrolysis is still disputed, but the predominant mechanism involves nucleophilic attack on the phosphorus atom (I) by hydroxide to generate a pentacoordinate phosphorane (II). The phosphorane decomposes to give an

88/12 mixture of nontoxic (III) and toxic (IV) products (Scheme 2.1).73

20

Scheme 2.1: VX Hydrolysis Mechanism and Tetrahedral Intermediate

Based on this mechanism, Moriarty and collaborators synthesized a trigonal bipyramidal pentacoordinate phosphorane as a TSA to be used as a hapten (Figure 2.4, first generation hapten) to generate abzymes for OP hydrolysis.76 The abzymes that were generated from the hapten showed a rate acceleration of 5480 for the hydrolysis of an OP compound compared to the hydrolysis in the absence of abzyme.71 The abzyme effect on rate acceleration is debated however because spontaneous acidic hydrolysis in the experiments could not be ruled out.75,78 Moriarty’s second generation haptens (Figure

2.4) were deemed to be more hydrolytically stable, however, no reports exist in the literature of abzymes generated against these haptens.79

21

Figure 2.4: Moriarty's First and Second Generation Phosphorane Haptens

Following Moriarty’s pioneering work, Vayron and co-workers synthesized several phosphoranes based on Moriarty’s first generation phosphoranes (Figure 2.5).

These phosphoranes however, were hydrolytically unstable and therefore were not good candidates for haptens.74 Phosphoranes V and VI decomposed in the presence of water and in the presence of trace amounts of within 24 hours whereas phosphoranes

VII and VIII hydrolyzed in a matter of minutes in the presence of water.74 Due to the difficulty obtaining water-stable phosphoranes to use as haptens, Vayron and co-workers sought an alternative route to obtaining haptens.

Figure 2.5: Phosphorane Haptens Investigated by Vayron and Co-workers

22

Based on the mechanism of VX hydrolysis, Vayron and co-workers then synthesized haptens to mimic the water molecule approaching the nerve agent, which is the first step in the hydrolysis mechanism (Figure 2.6). Two α-hydroxyphosphinate haptens (IX and X) were synthesized in six steps with 25-35% overall yield.74 These haptens were stable to aqueous conditions and were subsequently tethered to KLH for immunization into mice.75 The abzymes generated from these haptens were able to hydrolyze model VX compounds, however the catalytic efficiency was low.75 The low catalytic activity can be attributed to the fact that the haptens were designed to mimic a pre-transition state structure, rather than the transition state itself. Other attempts to generate abzymes from haptens designed to mimic other hydrolysis pathways were similarly unsuccessful.73 In this chapter, the synthesis of hydrolytically stable phosphorane hapten TSA’s will be described.

Figure 2.6: Vayron's α-Hydroxyphosphinate Haptens

2.2: Division of Work Described in this Chapter

The work described in this chapter represents a collaborative effort between the

Hadad group at The Ohio State University, MRIGlobal, and PrecisionAntibody. Hapten conjugation and abzyme generation and purification was performed by 23

PrecisionAntibody. In vivo hapten toxicity studies as well as hydrolysis assays of authentic samples of VX were performed by MRIGlobal. Hapten design and synthesis was performed at The Ohio State University. Work on phosphorus (III) precursors and most final hapten formation was conducted by Tom Corrigan. Amneh Young, the author of this dissertation, worked primarily on optimizing the synthesis of quinones and developing linkers for hapten conjugation. Ryan McKenney worked extensively on linker development and some final hapten production. Chi Le primarily worked on developing ways to make water soluble haptens. Ben Garrett was instrumental in initial quinone syntheses. Jeremy Erb worked on alternative hapten designs. In vitro hapten toxicity with AChE was performed by Qinggeng Zhuang. The work detailed in this chapter would not have been possible without all the contributions described above.

2.3: Hapten Design and Synthesis

The haptens were designed to make the synthesis as convergent as possible and to allow development of a large variety of haptens in a simple and efficient manner. The initial strategy for the design of the haptens was based on Moriarty’s second generation hapten that incorporated electron-withdrawing substituents (CF3) on the phosphorus center to increase the stability of the final phosphorane compounds.80 The decomposition of phosphoranes in aqueous media is thought to proceed through a intermediate XII (Scheme 2.2). If so, it is possible to shift the equilibrium towards the trigonal bipyramidal phosphorane XI by destabilizing the cationic intermediate XIII via the strategic incorporation of an electron withdrawing group within the phosphorane

(Scheme 2.2). 77,79

24

Scheme 2.2: Predicted Phosphorane Hydrolysis Mechanism

A key step utilized in the synthesis of these pentavalent phosphoranes is a

Kukhtin-Ramirez addition of an ortho-quinone on a phosphorus (III) center.81,82 The limited stability of the ortho-quinones required the presence of at least one bulky tert- butyl group on the aromatic ring. This prevented any undesired chemistry on the quinone intermediates, which are otherwise highly susceptible to 1,4-Michael addition reactions.83,84 Initial synthetic investigations used commercially available 3,5-di-tert- butyl- as a convenient precursor to the ortho-benzoquinone used for the cyclization reaction. The lack of remaining functionalizable positions on the aromatic ring required the phosphorus center to contain a tether that could be linked to a carrier protein for antibody recognition. The remaining substituents around phosphorus could be varied accordingly to increase the stability of the final phosphorane (Figure 2.6). Initial attempts of phosphorane synthesis utilized di-tert-butyl ortho-quinone, a trifluoromethyl group, and two alkoxy substituents on the phosphorus (XIV). The appearance of the pentavalent phosphorane peak on the 31P NMR spectrum confirmed successful synthesis of the target hapten, however, this peak typically degraded on the timescale of minutes.

25

This result is in agreement with Vayron’s initial phosphorane synthesis, where phosphoranes containing alkoxy groups proved to be hydrolytically unstable.74

Following this encouraging preliminary result, a variety of phosphoranes were synthesized to determine what features could increase the stability of the final products.

A screen of various alkoxy and amino substituents on the phosphorus center revealed that the most stable phosphoranes contained two secondary amines. Thus, a secondary that had some linking functionality for attachment to a desired carrier protein (XV) was pursued. This proved to be unsuccessful when bis(amino)trifluoromethylphosphine was reluctant to undergo the cycloaddition with the ortho-quinone. Efforts then turned to synthesizing a library of novel, stable ortho- quinones with pendant linking capabilities. Phosphorus (III) precursors with a trifluoromethyl and two secondary amine groups were subjected to a cycloaddition reaction with the ortho-quinones to give the desired phosphoranes (XVI).

Figure 2.6: Various Phosphorane Hapten Scaffolds Explored

26

Herein the syntheses of these compounds along with the results of stability testing in an aqueous acetonitrile solution are reported. The stability of the phosphoranes were examined in 9:1 MeCN:MOPS buffer (pH 7.24) solution by monitoring the 31P NMR signal as the product quartet around -30 ppm decreased and a new oxidation product grew in around 20 ppm. Once the samples were subjected to this aqueous environment, an initial scan was taken, then every hour until no product signal remained. From these stability tests, the appreciable stabilities of the phosphoranes were conclusively determined (anywhere from a few hours to days) in aqueous environments. The phosphorane stabilities are summarized in Table 2.2 with the trends for various about the phosphorus center, as well as different ortho-quinone substitutions. The 3,5-di- tert-butyl-ortho-benzoquinone provided the greatest stability in an aqueous MeCN solution, which is attributed to the added steric bulk of the second tert-butyl group. The bulky substituent effectively shields the phosphorus center from nucleophilic attack by an approaching water molecule (entry 3). Exchanging the second tert-butyl group for the terminal alkynyl ester provided a handle for linkage of the final hapten to a carrier protein, at the cost of some phosphorane stability (entries 5-6). Secondary amino ligands on the phosphorus center provided a significantly higher level of stability alkoxy ligands (entries 1-4). Among the amino ligands, bulkier groups performed the best, which again, can be attributed to the added bulk effectively protecting the phosphorus center from nucleophilic attack.

27

Table 2.2: Phosphorane Haptens and Time to Complete Hydrolysis in Aqueous Solution

28

After determining the appropriate substitution at the phosphorus center necessary for stable phosphoranes, two strategies to attach the haptens to a carrier protein were explored. The phosphoranes would utilize a covalent linkage via the use of an N- hydroxysuccinimide (NHS) ester to make amide bonds with surface lysines on the immunogenic protein. Alternatively, a non-covalent hydrophobic interaction between a long alkyl substituent on the phosphorus compound and hydrophobic pockets of carrier proteins could be exploited to tether the phosphorane to the protein. From a retrosynthetic standpoint, a cycloaddition reaction of the ortho-quinone on the phosphorus (III) precursor would give the pentavalent phosphorus center. The various linkers could be installed on the quinone via a DCC mediated coupling reaction. Finally, a click reaction would be used to attach the NHS ester on the haptens via a terminal alkyne on the ester of the ortho-quinone that would utilize a covalent link to the carrier protein.85 The ortho-quinone would be derived from commercially available 4-tert- butylcatechol whereas the phosphorus (III) precursor would be derived from (Scheme 2.3).

29

Scheme 2.3: Retrosynthesis of Final Phosphorane Haptens

30

Initial attempts to synthesize ortho-quinone frameworks did not employ any protecting groups on the catechol framework. The Heck coupling reaction with several (acrylonitrile, methyl acrylate) suffered from very low yields. A benzyl protecting group was used to improve the yield of the Heck coupling reaction. Benzyl protection of the catechol proceeds in quantitative yield and coupling the protected (XVII) with acrylonitrile gave the desired styrene (XVIII) in 70% yield (Scheme

2.4). Reduction of the with an in situ generated nickel borohydride in the presence of an amine protecting group produced the protected amine (XIX) in low yields.

Attempts to improve the yield of the reduction were unsuccessful. Hydrogenolysis of the benzyl groups unmasked the catechol (XX); however, oxidation to the quinone (XXI) with iron (III) chloride did not produce the product. Alternative Heck coupling partners were then explored, but none of these attempts were successful.

Scheme 2.4: Initial Ortho-Quinone Framework Synthesis

31

While optimizing the Heck reaction conditions, it was observed that tert-butyl acrylate could react with the aryl halide (2.2) in appreciable yields in the absence of protecting groups. Efforts then focused on optimizing the quinone synthesis using tert- butyl acrylate as a coupling partner. This route could produce the quinone in three steps from the aryl halide (2.2). Although oxidation to the quinone (XXIV) could occur using the iron (III) chloride oxidant used in earlier synthesis, it was observed that sodium periodate could produce the desired product in near quantitative yield and less reaction time (Scheme 2.5).

Scheme 2.5: Alternative Ortho-Quinone Framework Synthesis Using Tert-Butyl Acrylate as the Heck Coupling Partner

At this stage in the synthesis, a transesterification reaction with an was explored to attach various linkers to the quinone framework (Scheme 2.6). Acidic and basic conditions were explored starting from the catechol (XXIII) or quinone (XXIV).

Reactions with the catechol under acidic conditions gave a lactonization product (XXV), 32 not the intermolecular transesterification. Reactions starting from the quinone framework only gave degradation products.

Scheme 2.6: Attempted Transesterification Reactions

Deprotection of the tert-butyl ester (XXIII) to give the free acid that could be used in DCC mediated coupling reactions produced the lactone (XXV) and attempts to deprotect at the quinone (XXIV) stage only produced degradation products. Thus, it was determined that the catechol would need to be protected before the carboxylic acid was unmasked, and that quinone formation should be the last step in the synthesis due to its poor stability. A literature search revealed that ceric ammonium nitrate (CAN) could directly oxidize methyl protected to the desired quinone. The optimized synthesis is described below.

The various ortho-quinones were all derived from the carboxylic acid 3-(5-tert- butyl)-2,3-dimethoxyphenyl)propanoic acid (2.1). The synthesis of this acid was accomplished by starting from commercially available 4-tert-butylcatechol. Electrophilic 33 bromination of the ring followed by methyl protection of the catechol provided the coupling precursor in excellent yield. The aryl bromide was coupled with methyl acrylate via Heck reaction to give the α,β-unsaturated ester, which was subsequently hydrogenated and saponified to give 2.1 in 93% overall yield (Scheme 2.7). This route proved to be scalable and robust, providing the desired acid in multi-gram quantities.

The carboxylic acid provides a convenient handle to couple either covalent or non- covalent linkers to be utilized later by a carrier protein.

Scheme 2.7: Optimized Synthesis of Carboxylic Acid 2.1

A DCC mediated coupling reaction between the acid 2.1 and various alcohol linkers were used to provide three unique quinone precursors. All of the were commercially available except for the alcohol (2.5) derived from palmitic acid and pentaerythritol. To synthesize this linker, pentaerythritol was mono protected with a silyl ether. The remaining free alcohols were DCC coupled to palmitic acid to give the 34 protected tris-palmitoyl ester 2.7 which was deprotected to give the alcohol linker 2.5

(Scheme 2.8).

Scheme 2.8: Synthesis of Palmitic Acid Derived Fatty Acid Linker

With the alcohol linkers in hand, the final steps for the synthesis of the ortho- quinones proceeded smoothly in two steps. Esterification of the acid 2.1 using DCC and various alcohols (, 2.5, or 4-pentyn-1-ol) provided the quinone precursors which were oxidized using ceric ammonium nitrate to give the final quinones (Scheme

2.9).

35

Scheme 2.9: Final Optimized Ortho-Quinone Synthesis

The final ortho-quinone product could be stabilized by introducing steric bulk to the framework. To accomplish this goal, several strategies were attempted.

Cyclopropanation using diethyl and diiodomethane gave only unreacted starting material. Initially, it was thought that no reaction occurred because the zinc source was not activated enough. After activating the zinc however, the was still resistant to cyclopropanation, likely due to the deactivated nature of the olefin. Next, conjugate addition was attempted to increase the bulk. This reaction was attempted several times, however the only product observed was the 1,2-addition product (XVIII) (Scheme 2.10).

36

Scheme 2.10: Attempts to Increase Steric Bulk at Benzylic Carbon

The phosphorus (III) precursors used in the cycloaddition reaction with the quinones were either symmetrical or unsymmetrical. Symmetrical precursors were synthesized by substitution on phosphorus trichloride with two identical amines to give monochlorophosphines 2.14 and 2.15. Substitution of the remaining with a trifluoromethyl group by means of Ruppert’s reagent furnished the desired symmetrical 2.16 and 2.17 that were stable to air and silica gel column conditions (Scheme

2.11).35

37

Scheme 2.11: Synthesis of Symmetrical Phosphorus (III) Precursor

Synthesis of the unsymmetrical phosphines, with intentions of better representing the substitution found on VX, began with a single substitution of phosphorus trichloride with diethylamine to give the dichlorophosphine 2.18. Commercially available isonipecotic acid was Boc protected followed by conversion of the acid into the corresponding diisopropylamide 2.20 via an acid chloride intermediate. Removal of the protecting group was accomplished using TFA to give 2.21. Substitution of the second chlorine on phosphine 2.18 and subsequent trifluoromethylation gave the desired unsymmetrical phosphine 2.22 (Scheme 2.12).

38

Scheme 2.12: Synthesis of Unsymmetrical Phosphorus (III) Precursor

With the phosphorus (III) and ortho-quinone framework in place, each could be submitted to a cycloaddition reaction, producing the desired pentavalent phosphorus species, which was isolatable by silica gel column chromatography. For the non-covalent 39 class, the cycloaddition was the final step producing phosphoranes 2.23-2.26 (Scheme

2.13).

40

Scheme 2.13: Final Noncovalent Class Hapten Synthesis 41

For the covalent class, the different phosphines and the quinone 2.12 underwent a cycloaddition reaction to phosphoranes 2.27-2.29. These phosphoranes were then subjected to copper (I) catalyzed click conditions using azido linkers capped with an

NHS ester moiety (A, B). These conditions were gentle enough to generate the triazole without compromising the phosphorus center (Scheme 2.14).

42

Scheme 2.14: Cycloaddition and Click Reactions to Give Final Haptens with Covalent Linkers

43

The scope of the phosphorus core was explored further with alternative substituents. An attempt to stabilize the core using alkyl substituents was explored given that the presence of electron-withdrawing alkoxy substituents results in an unstable phosphorus (III) precursor, but the less electronegative amine substituents give a stable phosphorus center. Initial attempts showed this new core to be mildly stable to DCC coupling conditions yielding the non-covalently linked compounds 2.37 and 2.38

(Scheme 2.15). However, synthesis of covalently linked compounds using typical click chemistry was unsuccessful. Alternative routes to this motif will be explored in the future.

Scheme 2.15: Hapten Synthesis with Alternative Phosphorus (III) Precursor

Several novel phosphoranes were produced using the route detailed above. The route is convergent for the individual components and the entire synthesis is moderately

44 yielding. Additionally, the mild reaction conditions employed can tolerate a variety of substituents thereby allowing access to a wider assortment of scaffolds.

2.4: Results and Discussion

After the synthesis was completed, the ten final haptens were observed for signs of hydrolysis under aqueous conditions. Haptens with covalent linking capability (2.30-

2.33) were stable in aqueous conditions (9:1 MeCN:MOPS buffer) for 48 hours. The hydrophobic haptens with noncovalent linking capability (2.23-2.26, 2.37-2.38) were not soluble in with large amounts of water present. These haptens however, showed appreciable stability in solution for over 96 hours.

The Ellman assay is a well-established method to measure free thiol concentration.87 Free react stoichiometrically with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) rapidly to produce 2-thio-5-nitrobenzoate (TNB) which has a yellow color and absorbs strongly at 412 nm.28 AChE activity can be measured by using acetylthiocholine as a substrate (Scheme 2.16). Hydrolysis produces free thiocholine which can react with DTNB to produce TNB that can be measured spectrophotometrically.

45

Scheme 2.16: Ellman Assay for Thiol Detection

The haptens were tested for potential toxicity by determining their ability to inhibit AChE in vitro. Hapten solutions (1 µM, 10 µM and 100 µM) were individually incubated with electric eel AChE and the activity was measured using a modified

Ellman’s assay (Figure 2.7). While most of the haptens did not show any significant inhibition of AChE activity, haptens F and G demonstrated inhibition at all three concentrations tested.

46

Figure 2.7: Effect of Haptens on AChE Activity as Determined by an Ellman Assay

Following in vitro toxicity studies, all haptens were individually injected into groups of CD-1 mice (1 µg/mouse via tail vein injection; 5 mice per group) to measure in vivo toxicity. Mice were observed twice daily for clinical signs of toxicity and death for four days. All mice gained weight over the study period except for those injected with haptens C and K. However, none of the hapten candidates were statistically different from the negative control group L when analyzed via ANOVA.88 Therefore, while groups

C and K had a weight loss over the course of the four day study, it is not statistically significant due to the variability in the weights of the five animals within each of these groups. Since no adverse observations were observed for these animals, it is unlikely that the differences in weights of groups C and K were real. They are likely due to the variability within the data set and the animals. None of the haptens demonstrated any acute toxicity when administered at a dose of 1 µg/mouse via IV (4 days). There were no 47 deaths due to hapten injection and no definitive trend in body weight loss as compared to the negative control (vehicle only) group nor any statistically significant difference in body weight. All in vivo toxicity studies were conducted by collaborators at MRIGlobal.

Since the haptens did not display significant in vivo toxicity, they were conjugated to either KLH (A, B, L, and V) via amide formation from surface lysines and the NHS ester, or human serum albumin (HSA) or BSA via a cholesterol/fatty acid hydrophobic interaction (C, D, E, F, G, and W) to form hapten-carrier protein complexes for immunization. The immunogen complexes were mixed with AGVant adjuvant solution and sonicated to give an emulsion for vaccination. CD-1 mice (10 mice/group) were immunized with a different immunogen for every group. In order to determine the specific immune response against each test vaccine, a tail bleed screening was performed.

Titers against antigen were determined by ELISA (Figure 2.8).89 Most of the hapten immunogens induced a robust response in mice with the highest titers (300,000 and

1,000,000) achieved by haptens that were covalently linked to KLH (Haptens A and L).

Hapten conjugation, vaccination, and abzyme collection/purification was performed by collaborators at PrescionAntibody.

48

Figure 2.8: Abzyme Anti-Hapten Titers as Determined by ELISA

The ability of the antibody test samples to hydrolyze VX (generating a free thiol) was determined using an Ellman assay.90 Since this method can be used to detect any free thiol, it can be used to detect formation of thiols as a result of VX hydrolysis when the P-S bond is cleaved.29,30 VX can be hydrolyzed at several locations, but cleavage of the P-S bond is the only variant that renders the molecule inactive.31 Antibody test samples were diluted (1:10) and added to VX for 20 h at 24 °C. VX hydrolysis was determined using Ellman’s assay; however, none of the abzyme samples demonstrated any thiol generation when incubated with VX. This observation may be explained by any of the following reasons. First, the abzymes are not active against VX. Second, the

49 abzymes hydrolyze VX but not at the P-S bond (no thiols generated). Third, the abzymes bind to VX but do not hydrolyze the OP. Lastly, there could be possible interference from organic solvents with the assay (e.g. IPA).

In order to address the above scenarios, an alternative assay was explored.

Abzymes were diluted (1:2) and incubated with VX for 18 h at 24 °C. The mixture was then incubated with human AChE for 1 hour at 24 °C and AChE activity was determined using an Ellman assay. If the abzymes have no effect on VX, then AChE activity will be low in the presence or absence of abzymes. Most of the abzyme samples (9/10; all except V) demonstrated an increase in AChE activity compared to the naïve control sample (Figure 2.9). Therefore, these results suggests that these abzymes interfere with

VX inhibition of AChE.

50

Figure 2.9: Effect of Abzymes on VX Inhibition of AChE

Abzyme test samples that demonstrated potential VX inactivation (all but sample

V) were retested against VX at three concentrations (10%, 50%, and 90% sample concentrations). All nine of the abzyme samples demonstrated a clear dose-dependent effect on AChE activity compared to the naïve control sample (Figure 2.10). This effect was significantly different from the naïve control at 50% (p<0.05) and 90% (p<0.01).

These results suggest that the abzymes interfere with VX inhibition of AChE by either binding directly to VX and/or hydrolyzing VX.

51

Figure 2.10: Effect of Abzyme Concentration of VX Inhibition of AChE

In order to determine the mechanism of VX inactivation (binding versus hydrolytic activity), the abzyme test samples were incubated with VX over 139 hours.

Aliquots of the reaction were taken at 0.5, 18, and 139 hours during the incubation period and added to AChE. If the abzymes bind VX but do not hydrolyze it catalytically, then

AChE activity over the reaction time should remain unchanged. All nine of the abzyme samples demonstrated a clear time-dependent effect on AChE activity compared to the naïve control sample (Figure 2.11). At 50% abzyme concentration, this effect was significantly different from the naïve control at 0.5 h (p<0.05), 18 h (p<0.05), and 139 h

(p<0.01). The effect of the abzymes on VX continued through 139 hours which suggest the abzymes may be hydrolyzing VX.

52

Figure 2.11: Effect of Abzyme on VX Inhibition of AChE Over Time

Unfortunately, the naïve control sample demonstrated significant background activity at the 139 hour time point. Therefore, the naïve control values were subtracted from all of the data in order to remove this background activity. Again, all nine of the abzyme samples demonstrated a clear dose-dependent effect on AChE activity over the time period (Figure 2.12). This suggests that VX inactivation is specifically induced by the abzymes. The VX inactivation activity increased through the 139 hour time point for all abzyme test samples which again suggests that the abzymes may be hydrolyzing VX.

All abzyme hydrolysis studies with authentic samples of VX were performed by collaborators at MRIGlobal.

53

Figure 2.12: Effect of Abzyme Concentration on VX Inhibition of AChE Over Time (Negative Control Subtracted)

2.5: Conclusions

Ten novel phosphoranes with appreciable stabilities in aqueous environments were synthesized for the first time. The synthesis is convergent and will allow further development of different scaffolds to increase the potential library of active phosphoranes. These phosphorane haptens were conjugated to immunogenic proteins and injected into mice. Most of the hapten immunogens produced a robust immune response. The anti-serum that was produced was pooled and tested for VX hydrolysis activity in vitro. Most of the abzyme samples (9 out of 10) demonstrated significant VX inactivation. This inactivation could be a result of catalytic activity of the abzymes on

VX. This course of treatment focuses on a vaccine-like treatment to be used in addition to acute therapies. A vaccine that is able to generate catalytic antibodies within the host could, in theory, be used as a prophylactic to actively hydrolyze agent and reduce the toxicity of a nerve agent. This approach provides two key advantages over the current bioscavenger (BChE). The first key advantage is a greater than one-to-one activity

54

(antibodies produced by the host). The second key advantage is that this work would ultimately lead to a more effective prophylaxis for protection against nerve agent exposure because of the longevity of protection provided by active immunity (persistent protection). This approach would enable even relatively low concentrations of antibodies present between boosters, to effectively reduce the concentration of VX circulating in the blood and provide additional time for acute therapies to be administered

55

CHAPTER 3: DEVELOPMENT OF CYCLIC PEPTIDES FOR VX HYDROLYSIS

3.1: Introduction

Since their discovery in 1932, OP compounds have been used as both effective pesticides and chemical warfare agents (CWA). Current strategies to treat OP poisoning are to reactivate the inhibited AChE by using a nucleophilic oxime in conjunction with and anticonvulsants (e.g. atropine and benzodiazepines) or to prevent OP inhibition of AChE by blocking the active site with a reversible inhibitor (e.g. pyridostigmine bromide).36,37,44,46,47,94 Bioscavengers on the other hand (stoichiometric and catalytic), can prevent AChE inhibition by binding free OP in the blood. This protects the AChE and minimizes any cholinergic crisis. Current bioscavengers are proteins that sequester (stoichiometric) or hydrolyze (catalytic) OPs in the blood.

Stoichiometric scavengers such as butyrylcholinesterase (BChE) offer protection against

OP compounds by binding an equimolar amount of the OP.26–28 However, due to the large molecular weight of the protein, large doses are required to offer adequate protection.95 Catalytic scavengers such as paraoxonase-1 (PON1) can catalyze hydrolysis of the OP compound and thus offer greater than 1-to-1 protection against an OP.31–34 Due to the low catalytic efficiency of these scavengers however, large doses are still needed for the bioscavenger to be a viable prophylactic.

56

Bioscavengers which could be used to hydrolyze any OP compound are highly desirable because exposure to multiple nerve agents is a possibility. Table 3.1 summarizes the activity of several potential protein bioscavengers against a series of nerve agents.96 PON1, OPAA, and OPH show catalytic activity against multiple nerve agents and the bacterial OP hydrolyzing protein OPH shows the broadest substrate specificity.

Table 3.1: Catalytic Activity of Potential Bioscavengers Against Various Organophosphorus Compounds

Enzyme -S DFP Tabun Sarin Soman Cyclosarin VX R-VX AChE − − − − − − − − − BChE − − − − − − − − − PON1 + − + + + + + − − OPAA + − + + + + + − − OPH + + + + + + + + +

Among the potential catalytic scavengers, OPH shows the highest catalytic efficiency overall (Table 3.2).96 Since OPH is a bacterial protein however, fast immunological responses would severely decrease the concentration of this protein in the blood, rendering it ineffective. There has been some work to lower the immunological response due to injection of bacterial proteins. Bacterial proteins could be PEGylated or encapsulated within liposomes prior to injection.97,98 Mice exposed to paraoxon who were injected with atropine, 2-PAM, and OPH encapsulated in a biodegradable enzyme carrier prior to exposure showed less symptoms of cholinergic crisis than mice who received injections lacking the catalytic scavenger.98 57

Table 3.2: Catalytic Efficiency of OPH, OPAA, and PON1 Against Several Organophosphorus Compounds

Enzyme/substrate kcat/Km (M−1s−1) OPH Paraoxon 6.46 107 DFP 1.3 106 Sarin 8 104 Soman 1 104

OPAA Paraoxon 4.8 102 DFP 2.5 105 Sarin 2.8 105 Soman 6.1 104

PON1 Paraoxon 2.4 104/ 1.1 105 DFP 6.3 102/ ndf Sarin 1.5 104/ 1.1 103 Soman 4.6 104/ 3.5 104

The main limitation of protein bioscavenger approaches is the mass production of large quantities of protein at a low cost. Alternatively, a small molecule scavenger offers several advantages over traditional bioscavengers: their molecular weight ratio relative to the OP is smaller which means that a lower dose would be required, their chemical production makes the synthesis of these molecules highly amenable to scale up, and the modularity of the synthesis simplifies the production of more efficacious and less toxic derivatives.

Research into small molecule OP scavengers that can act as artificial enzymes traditionally have focused on cyclodextrins (CDs). Cyclodextrins are relatively nontoxic 58 cyclic oligosaccharides made from glucopyranose (Figure 3.1). Their structure can be modified chemically and they are known to form host-guest complexes with small molecules in the hydrophobic cavity interior.99 Native cyclodextrins (β-cyclodextrin has the highest affinity) can degrade the nerve agents soman and sarin, but recent efforts focus on improving the efficacy of these compounds by incorporating a nucleophilic group such as an oxime.99–103 Although these results are promising, the efficacy of CDs is still low.

Figure 3.1: Cyclodextrin OP Scavengers

An alternative molecular scavenger could be derived from a cyclic peptide scaffold. Cyclic peptides benefit from a rigid structure which increases their selectivity toward their target molecule and reduces non-specific and potentially toxic binding to other targets.104 In addition, cyclic peptides lack free carboxyl and amino termini rendering them resistant to hydrolysis by exopeptidases thereby increasing the length of 59 time they can act as scavengers in the blood.104 Eventual degradation of cyclic peptides produces non-toxic amino acids.104

Several cyclic peptides are known therapeutics (Figure 3.2). Gramicidin S and tyrocidine are cyclic peptides with antibacterial activity. The mixture of the two peptides was the first commercialized antibiotic, however, it can only be used topically due to toxicity issues. Cyclosporin A is another therapeutic cyclic peptide used as an immunosuppressant to prevent graft rejection and to treat rheumatoid arthritis and psoriasis.104 Oxytocin is a naturally occurring hormone that is used as a drug to induce labor.

Gramicidin S - 1944 oxytocin

Figure 3.2: Cyclic Peptide Therapeutics

Linear peptides are known to catalyze several reactions such as epoxidations, aldol reactions, Michael additions, Morita-Baylis-Hillman reactions, and hydrocyanation of among others.105 Recently, Herrmann and coworkers demonstrated that the 60 conformational rigidity of cyclic peptides could be exploited to enantioselectively catalyze reactions (Scheme 3.1).106 They synthesized a semi-random library of 15 cyclic nonapeptides and complexed Cu2+ to a histidine anchoring group.106 The best performing peptides were able to catalyze Diels-Alder and Friedel-Crafts reactions with enantioselectivities up to 99% ee and 86% ee respectively. 106 Linearization of the peptides resulted in loss of enantioselectivity which suggests that the rigid structure of the cyclic peptide is essential for success.106,107

Scheme 3.1: Reactions Catalyzed by Cyclic Peptides. a Diels-Alder Reaction. b Friedel- Crafts Reaction

Landry and Deng reported the synthesis of a random library of linear peptides for

OP hydrolysis.108 They found one compound in their library successfully made a phosphate ester with a sarin analog.108 No testing with authentic samples of nerve agents or catalytic activity was reported. A cyclic peptide benefits from structural rigidity and that could be exploited to improve the binding and hydrolysis of OP compounds. One

61 major advantaged cyclic peptides have over other scavengers is that large libraries can be synthesized in a combinatorial manner and then screened in a high-throughput fashion.

Additionally, since the peptides are small molecules, a cocktail of different cyclic peptides could be co-administered in the event of exposure to multiple OPs. This chapter will detail current efforts to develop small, cost effective, cyclic peptides that can efficiently bind and hydrolyze OP compounds.

3.2: Division of Work Described in this Chapter

The work described in this chapter represents a collaborative effort between the

Hadad and Pei groups at The Ohio State University. Ryan McKenney, with guidance from Professor Dehua Pei and his students, synthesized the initial cyclic peptide library.

The model OP compound was synthesized by Amneh Young (the author of this chapter) and the synthesis of the model OP compound was optimized by the combined efforts and suggestions of Amneh Young and Ryan McKenney. The project described in this chapter is the invention of Amneh Young, under the guidance of Professors Christopher Hadad and Dehua Pei.

3.3: Cyclic Peptide Design and Synthesis

The cyclic peptide library is synthesized in a combinatorial fashion using one- bead-one-compound (OBOC) solid-phase peptide synthesis.109–111 The fast reaction times, high yields, and reliability of coupling reactions make them excellent candidates for potential scale up. The catalytic molecular scavengers will mimic the structure of efficient OP hydrolyzing proteins by incorporating amino acids found in the active sites of these proteins. A combinatorial approach will allow for the synthesis of a fairly large

62 library of cyclic peptides that samples a broad scope of the total reactive space.

Furthermore, OBOC synthesis of cyclic peptides simplifies purifications since impurities can be washed away, leaving desired compounds bound to the bead throughout the synthesis. The modular synthesis will allow for tunability of ring size, cyclic conformation, and binding affinity with ease.

TentaGel resin was chosen as the solid support because it displays free amine functional groups on the surface of the resin, it is nonsticky, and it is uniform in size

(Scheme 3.2).112 Each bead contains the same linear spacer (MRRFBB). Methionine

(M) is used to cleave the peptide from the bead with bromide, arginine (R) is used to help ionize the peptide under positive mode on a MALDI-TOF mass spectrometer

(MS), phenylalanine (F) is used to increase the molecular weight of the peptide to minimize interference from the MALDI matrix, and β-alanine (B) is used to identify the end of the cyclic peptide since it cannot be hydrolyzed under partial Edman degradation

(PED) conditions.104 Standard peptide coupling reactions with Fmoc protected amino acids was used. Fmoc protected amino acids were used instead of Boc amino acids due to the milder deprotection method (20% /DMF). Each coupling reaction was performed two times and the Kaiser test with ninhydrin was used to confirm greater than

99% completion of the coupling reactions.113

63

Scheme 3.2: Solid-Phase Synthesis of Linear Spacer

After the same linear sequence was installed on all beads, the surface of the beads was modified to serve three distinct functions (Scheme 3.3). Each bead contains three motifs: 50% of the bead surface contains the linear segment of the peptide which will allow for the sequence of any hits by PED, 45% of the surface contains a trap to detect any OP hydrolysis, and 5% of the bead surface contains the cyclic peptide scavenger.

Formation of the three motifs can be controlled by the molar equivalents and protecting groups of amino acids in the coupling reactions.114 with two different side-chain protecting groups was used to distinguish between the linear peptide sequence and the cyclic peptide sequence. The γ-allyl ester was used for the cyclic peptide portion of the bead and the tert-butyl ester was used for the linear portion. Selective allyl deprotection allows for peptide cyclization using the carboxylic acid side chain. S- acetamidomethyl-L- was used as a handle to install a labile disulfide trap after completion of the library synthesis.

64

Scheme 3.3: Bead Surface Modification

The cyclic peptide library contains amino acids commonly found in the most efficient OP hydrolyzing proteins and a mixture of D and L amino acids. This will allow for exploration of different conformations of the cyclic peptide for activity. The peptides were constructed using a split-and-pool (Scheme 3.4)109,115 synthesis using 11 different amino acids (Orn, His, Glu, D-Phe, Tyr, Asn, Gly, D-Val, Leu, Ser, Arg) and the length of the peptide was adjusted to be 6-8 amino acids long.

65

Scheme 3.4: Generic Split-and-Pool Procedure

66

After synthesis of the random peptide sequence, hydrogenolysis was used to remove the γ-allyl protecting group (Scheme 3.5). The free carboxylic acid was then used to cyclize the peptide sequence using PyAOP as the coupling reagent. Side-chains were then deprotected using reagent K (, water, thioanisole, ethanedithiol, anisole, and TFA). The penultimate step to library synthesis is Acm deprotection of the cysteine to give the free thiol. A disulfide exchange with 2,2’-dithiodipyridine produced the labile handle to be used to detect VX hydrolysis. One gram of tentagel resin was used which corresponds to 2.3 million beads and consequently, a library of 2.3 million unique compounds.

Scheme 3.5: Cyclic Peptide Library Synthesis

3.4: Model Phosphonate Design and Synthesis

There are several desirable features for a safe nerve agent mimic. The mimic should accurately represent the nerve agent of choice, VX in this case, it should be a non-

67 volatile solid that is easy to handle, and it should provide a functional handle to detect hydrolysis. There are several VX hydrolysis possibilities, but only P-S cleavage gives benign products. Thus, the VX mimic for this project was designed to contain a pendant visible dye that can be used to identify the hit beads in a colorimetric assay only when P-

S hydrolysis occurred (Figure 3.3).

Figure 3.3: VX and Corresponding Dye-Tagged Mimic

Synthesis of the model VX compound begins with commercially available triethylphosphite (3.1). An Arbuzov reaction with iodomethane produces the methylphosphonate (3.2) which is reacted with oxalyl chloride to give the methylphosphonochloridate (3.3) in 83% overall yield (Scheme 3.6). This precursor later reacted with a free thiol to produce the model VX compound.

Scheme 3.6: Synthesis of OP Precursor

68

Formation of the dye-tagged free thiol begins with commercially available cysteamine hydrochloride. Selective protection of the thiol is needed to ensure the dye tag will react only at the amine portion, leaving the thiol free to react with the phosphorus precursor 3.3. Various strategies were used to accomplish this goal. Initial strategies relied on in situ protection of the thiol using TMSCl followed by Fmoc protection of the amine (Scheme 3.7). Acidic workup removed the transient thiol protecting group to produce the free thiol 3.4. This thiol was reacted with 3.3 and produced the desired 31P signal (55 ppm) in the NMR spectrum, but that signal disappeared upon Fmoc deprotection and work-up.

Scheme 3.7: Initial Attempts to Synthesize OP Mimic

Loss of the desired 31P signal could be due to the volatile nature of 3.6.

Therefore, deprotection of 3.5 was attempted and commercially available DABCYL-SE was added without prior work-up. However, the reaction of the NHS ester with the free amine did not produce the desired amide. To avoid synthesis of volatile 3.6, the route was modified such that the desired amide (3.7) was formed immediately after transient

69 protection of the thiol (Scheme 3.8). Reaction of 3.7 with the phosphorus precursor 3.3 however, did not produce the desired phosphonate 3.8.

Scheme 3.8: Modified OP Mimic Route that Avoids Volatile Intermediates

An alternative thiol protecting group that would allow for isolation and purification of intermediates was then explored (Scheme 3.9). Disulfide 3.9 was formed in near quantitative yield by reaction with 2,2’-dithiopyridine. The desired amide 3.10 was successfully produced by reaction of the free amine with DABCYL-SE. Mild deprotection with produced the free thiol 3.7 while leaving the azo bridge intact. Upon reaction with the phosphorus precursor 3.3, however, the desired phosphorus signal was not observed in the 31P NMR spectrum.

70

Scheme 3.9: Alternative Route with Different Thiol Protecting Group

Amine bases can displace chlorides in phosphoryl chlorides to make phosphoramides.80,116,117 To eliminate the possibility that the dimethylaniline moiety is causing undesired phosphoramide formation, an alternative visible dye (3.15) was synthesized (Scheme 3.10). Sodium was used to generate the diazonium salt of 4- aminobenzoic acid which was the used in a typical azo coupling reaction with phenol.118

The azo dye (3.11) was methylated to prevent any side reactions between the phenol and the phosphorus precursor 3.3.118 Carboxylic acid 3.12 was converted into the amide 3.14 via an acid chloride intermediate 3.13. The final step of the synthesis is phosphine deprotection of the disulfide 3.14 to give the free thiol 3.15, however the yield for this reaction was unusually low. This was attributed to the presence of minor impurities in the reaction mixture since the acid chloride intermediate 3.13 could not be easily purified.

To circumvent this problem, the synthesis was revised to avoid acid chloride formation.

71

Scheme 3.10: Synthesis of Alternative Dye

An activated NHS ester (3.16) was used as the isolatable intermediate to make disulfide 3.14 (Scheme 3.11). As expected, the yield of the subsequent deprotection improved. Removal of the tributylphosphine oxide byproduct was accomplished by repetitive trituration with large quantities of hexanes. To scale up production of the free thiol 3.15, the synthesis was modified a final time.

Scheme 3.11: Alternative Dye Route Optimization

72

The final dye synthesis was optimized to produce free thiol 3.15 in six overall steps, in multigram quantities, and near quantitative yield (Scheme 3.12). Carboxylic acid 3.12 was converted directly to disulfide 3.14 using standard peptide coupling conditions. Tris(2-carboxyethyl)phosphine (TCEP) was used for the reduction of disulfide 3.14 instead of tributylphosphine because the resulting phosphine oxide is water soluble and can be washed away during work-up.119 This greatly improved the purity of the final free thiol 3.15.

Scheme 3.12: Final Dye Optimized Synthesis

With ample quantities of free thiol 3.15 on hand, the final step to synthesize the model OP is reaction with the phosphonate precursor 3.3 (Scheme 3.13). Initial reaction produced the desired phosphorus signal in the 31P NMR (55 ppm), however the reaction suffers from poor yield. This reaction is currently being optimized by varying , base, equivalents, and temperature.

73

Scheme 3.13: Dye-Tagged OP Synthesis

3.5: Screening Protocols and Hit Identification

Once ample quantities of the OP mimic 3.17 are on hand, screening of the 2.3 million OBOC library can commence. Initial hits will be identified from on-bead screening with the model OP 3.17.120 If the cyclic peptide hydrolyzes the P-S bond of

3.17, then a free thiol 3.15 will be generated which will quickly react with the labile disulfide trap on the bead (Scheme 3.14). The covalent attachment of the dye will enable detection of the bead under a fluorescence microscope.

Scheme 3.14: Reaction of Dye-Tagged OP with Cyclic Peptide Library

74

On-bead screening will occur by incubating the beads with the model OP 3.17 in buffered solution and mixing on a rotary shaker (Figure 3.4). After 20-24 hours, the beads will be washed to remove any unreacted OP and transferred to a petri dish.

Fluorescent beads will be removed with a micropipette and transferred to another petri dish containing TCEP. Beads that retain their fluorescence will be removed as false positives (caused by nonspecific binding of the dye to the bead) whereas beads that lose their fluorescence due to cleavage of the disulfide bond and release of the dye will be removed for sequence identification by partial Edman degradation-mass spectrometry

(PED-MS).121

Figure 3.4: On-Bead Screening Protocol

Once the peptide sequence of the hit compounds is known, the individual peptides will be resynthesized and cleaved from the bead (Figure 3.5). An Ellman assay will be performed on the individual compounds by incubating with dithionitrobenzoic acid

(DTNB) and the model OP 3.17 in a well plate. The formation of 2-nitro-5-thiobenzoate

(TNB-) will be monitored using a UV-Vis spectrometer. This assay will confirm P-S cleavage and will provide a way to measure the rate of hydrolysis. Thus, the catalytic efficiency of the cyclic peptides can be quantified. This information will be used for the rational design of other derivatives of the cyclic peptides. 75

Figure 3.5: In-Solution Screening Protocol

All of the protein bioscavengers discussed earlier contain a metal ion in or near the active site. OPH contains two metal centers: one metal is used to bind the OP and the other is used to activate the water molecule for hydrolysis (Figure 3.6).122 Therefore, it is possible that the cyclic peptides will require coordinated metals in order to efficiently hydrolyze the model OP. If so, non-toxic divalent metal cations (e.g Ca2+, Zn2+) will be incorporated into the library screening.123

Figure 3.6: X-ray Structure of OPDA [PDB code 2D2H] Bound to Trimethyl Phosphate

76

3.6: Future Directions

There are several opportunities to expand on the utility of cyclic peptides as therapeutics to remove OP compounds from the blood before the onset of cholinergic crisis. Cyclic peptides that can hydrolyze multiple OPs, that favor more toxic enantiomers in a racemic OP mixture, or that can hydrolyze OP pesticides will be explored. OP hydrolyzing proteins like OPH have a broad substrate scope. Thus, hit cyclic peptides that can hydrolyze VX mimics will be screened in-solution with fluorogenic analogs of other nerve agents to determine substrate specificity (Figure

3.7).124 Upon hydrolysis, the OP analogs release a 7-hydroxycoumarin derivative that can be monitored at 460 nm. The rate of OP hydrolysis can be measured and the best performing peptides could be modified by varying amino acids that are not essential to hydrolysis, but may play a role in the binding of OP substrates.

Figure 3.7: Fluorogenic OP Analogs

The SP enantiomers of OP nerve agents are more toxic than the RP enantiomers and bacterial OP hydrolyzing proteins favor hydrolysis of the less toxic (RP) enantiomer of OP compounds (Figure 3.8).125 Thus, a more efficacious therapeutic could be developed if the selectivity of the cyclic peptide could be tuned to favor the more toxic SP 77 enantiomers of OP compounds. Tuning of the cyclic peptides could be accomplished by replacing D amino acids with their L counterparts.

Figure 3.8: RP and SP Enantiomers of VX

Most civilian exposure to OP compounds is in the form of thion pesticides.18

Thions are acute AChE inhibitors but once they are absorbed in the body, they are oxidized to oxons which are far more potent inhibitors (Figure 3.9).17 The oxon derivatives cause the majority of the resultant cholinergic crisis and eventual death.

Treatment for OP typically targets the oxon derivatives, after oxidation by cytochrome P450 occurs.17 If a treatment could be developed that removes the thion derivative from the blood before oxidation can occur, cholinergic crisis and its symptoms can be avoided.

78

Figure 3.9: Oxon and Thion OP Pesticides

Cyclic peptide scavengers with high affinity for thions could hydrolyze the pesticide before the onset of any toxic effects. Cyclic peptide hits from nerve agent hydrolysis will be used to determine if they can effect thion hydrolysis as well.

Hydrolysis of parathion will be explored initially, because it produces p-nitrophenoxide which can be monitored at 412 nm. In-solution screening of the lead compounds with parathion will provide a simple, and quick way to determine if the peptides have any promising hydrolysis activity.

If the peptides do not display any hydrolysis, a new cyclic peptide library could be synthesized. On-bead screening of the large library would be used to isolate initial hits since in-solution screening of millions of compounds is tedious and impractical.

79

Hydrolysis of thions produces alkoxides. Therefore, the cyclic peptide library will be modified to include an alkoxide trap instead of a thiol trap (Scheme 3.15). Alkoxides released after thion cleavage will remove the silyl protecting group on a fluorescein based dye. Desilylation will trigger formation of a highly fluorescent quinone and thus enables detection of active cyclic peptides.

Scheme 3.15: Potential On-Bead Screening of Peptide Library for Parathion Hydrolysis

The main limitation of this strategy is the selectivity of the alkoxide trap. The trimethylsilyl protecting group is rapidly hydrolyzed under basic conditions and if the background hydrolysis of this group is higher than silyl transfer to the released alkoxide, an alternative alkoxide trap will need to be developed. One way to overcome this potential obstacle is to screen various silyl protecting groups to find the right balance between rapid silyl transfer and slow background hydrolysis.

80

CHAPTER 4: DEVELOPMENT OF ALKYLATING AGENTS FOR THE

REACTIVATION OF AGED AChE

4.1: Introduction

Acetylcholinesterase (AChE) is a serine hydrolase responsible for the hydrolysis of the neurotransmitter acetylcholine. It hydrolyzes over 25,000 acetylcholine (ACh) molecules every second.19 The active site of AChE is a catalytic triad composed of serine

203, histidine 447, and glutamate 334. Organophosphorus compounds (OPs) are potent inhibitors of the enzyme AChE. Inhibition of AChE results in muscle contractions, blurry vision, seizures, and respiratory failure.

Once the OP enters the enzyme active site, it phosphylates a serine residue

(Ser203) to give an irreversibly inhibited AChE that is no longer capable of hydrolyzing

ACh. This leads to a buildup of ACh at cholinergic receptors and constant stimulation of nerve fibers. Reactivation of AChE can occur by hydrolysis of the phosphylated enzyme which is usually accomplished by use of a nucleophilic oxime, such as 2-PAM, often administered after OP exposure as a treatment. However, if reactivation does not occur, the phosphylated enzyme will then undergo a spontaneous dealkylation process (called aging) to give an aged enzyme which, to date, cannot be reactivated (Figure 4.1).126,127

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Figure 4.1: Inhibition and Aging of AChE by VX and Reactivation with 2-PAM

Most studies to reverse AChE inhibition focus on developing improved oxime therapeutics however, there is still no universal oxime capable of reactivating inhibited

AChE regardless of the OP used.128 Additionally, once aging occurs, oximes are no longer able to dephosphylate the enzyme. This is because the phosphonate anion in the aged enzyme-OP adduct is stabilized by interaction with the protonated histidine 447

(Scheme 4.1).129 Furthermore, the negative charge on the aged enzyme-OP adduct prevents approach of the nucleophilic oximate anion thereby preventing any OP hydrolysis once aging occurs.129

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Scheme 4.1: Mechanism of AChE Aging

Given the rapid aging rates of some OP compounds (Table 4.1), there is considerable need to develop a method to treat aged AChE.129 The previous chapters in this dissertation focused on preventing cholinergic crisis by scavenging OP compounds in the blood before they can inhibit AChE. This chapter will detail efforts to treat the aged form of the enzyme.

Table 4.1: Aging Half Times of Selected OP Nerve Agents

Nerve Agent Aging half time

Soman 2-4 minutes

Sarin 5 hours

Tabun 46 hours

VX 48 hours

Since aged AChE is resistant to reactivation with oximes due to the presence of the phosphonate anion, reinstatement of the alkyl group to restore the enzyme to the inhibited state should then allow for traditional oxime treatment. Regardless of the OP used, the aged enzyme will have only three unique structures: a phosphonate, a 83 phosphate, or, in the case of aging with tabun, a phosphoramidate (Scheme 4.2). The benefit of this approach is that a single alkylating agent could be used to return to the inhibited state and, since the inhibited enzymes would then have a very similar structure, a single oxime could reactivate the enzyme regardless of the OP used.

Scheme 4.2: Different Aged and Inhibited Forms of AChE

There are several small studies dating back to the 1960’s that attempt to identify alkylating agents capable of returning the aged enzyme back to the inhibited state. In

1969, Blumbergs and co-workers designed and synthesized a series of water soluble pyridinium based alkylating agents.130 These compounds were then tested against various anions including a model phosphonate to represent aged AChE (Scheme 4.3).131

Although these compounds were successful in alkylating several anions, they reacted poorly with the model phosphonate and no studies with authentic aged AChE were reported.

84

Scheme 4.3: Pyridinium Sulfonate as an Alkylating Agent

Following that study, Steinberg reported a family of phenacyl bromides that reacted with a model phosphonate anion (Figure 4.2).48 Upon alkylation, the model phosphonate was subjected to basic conditions to hydrolyze the p-nitrophenoxide leaving group which could be monitored at 412 nm. Using this indirect hydrolysis method,

Steinberg could measure the amount of alkylated model phosphonate.

85

Figure 4.2: Phenacyl Bromides as Phosphonate Alkylating Agents

Steinberg hypothesized that the presence of the on his alkylating agent could accelerate the rate of subsequent hydrolysis via neighboring group participation to return the enzyme to its native, uninhibited state.48 Using this approach, there would be no need for subsequent treatment with oximes such a 2-PAM. Indeed, a comparison of the hydrolysis of two model phosphonates (VII and VIII) revealed that the phosphonate bearing a carbonyl moiety (VIII) was hydrolyzed 66 times faster than

86 the phosphonate lacking a carbonyl (VII) (Figure 4.3)48. Despite these encouraging hydrolysis results, no alkylation of aged AChE was observed.48

Figure 4.3: Model Phosphonates and Potential Anchimeric Assistance in Hydrolysis Pathway

Most recently, Quinn described a series of 2-methoxypyridinium based methyl transfer compounds to reactivate aged AChE (Scheme 4.4).50,132 Hydrolytic stabilities and rates of methyl transfer were monitored by 1H and 31P NMR spectroscopy. The best performing compound (X) was capable of realkylating 40% of a model phosphonate anion to give a 2-pyridone species as well as dimethyl methyl phosphonate within 10 minutes.50 Although this rapid alkylation is within the time frame needed to treat OP exposure, no significant realkylation was observed for authentic aged AChE.50

87

Scheme 4.4: Methoxypyridinium Methyl Transfer Agents

One class of alkylating agents hitherto unexplored for alkylating aged AChE are quinone methides (QM). Quinone methides are known to alkylate several different compounds under biological conditions. In 2001, Freccero and coworkers reported a family of ortho-quinone methides capable of reacting with amino acids under aqueous conditions (Scheme 4.5).133 The quinone methides were generated from a quinone methide precursor by elimination from a benzylammonium salt under thermal or photochemical conditions.133 The subsequent alkylation of amino acids was discovered to be reversible under photochemical or thermal conditions.133

Scheme 4.5: Thermal and Photochemical Generation of a Quinone Methide and Subsequent Reaction with an Amino Acid

88

In addition to amino acids, quinone methides are capable of alkylating nucleotide bases in DNA. Zhou and coworkers studied the ability of a quinolinium substituted ortho-quinone methide to react with nucleotide bases in DNA under aqueous conditions

(Scheme 4.6).134,135 The N-methyl quinolinium portion of their ortho-quinone methide is thought to associate with DNA and disrupt the hydrogen bond network to afford alkylation at the N1 position of deoxyguanosine which is usually disfavored.134,135

Scheme 4.6: Quinone Methide Alkylating Nucleotide Bases in DNA

Turnbull demonstrated the ability of para-quinone methides to reversibly alkylate phosphodiesters (Scheme 4.7).136 Even though phosphodiesters are poor nucleophiles, the competitive hydrolysis of the QM with water was considerably slower. This allowed the alkylated phosphate to be trapped via a subsequent lactonization for further study.136

Since the QM is electrophilic enough for attack by poor nucleophiles, it is possible the phosphonate anion in aged AChE could be alkylated with an appropriately designed QM.

89

Scheme 4.7: Quinone Methide Alklyating a Phosphodiester

These examples show that quinone methides can be generated under near physiological conditions, they can react with poor nucleophiles such as phosphodiesters, and appropriate substitution of the QM framework can allow for traditionally disfavored reactivity. Given these advantages along with the ease of synthesis of a variety of tunable

QM frameworks, quinone methides were selected as potential alkylating agents of aged

AChE. The design and synthesis of quinone methide precursors and investigations of their potential use as alkylating agents of aged AChE will be described in this chapter.

4.2: Quinone Methide Precursor Design

To increase the likelihood of phosphonate anion alkylation in aged AChE, the desired quinone methide scaffold must be selective for the AChE active site and must be tunable such that the desired QM can be generated at will from a less reactive quinone methide precursor (QMP). Ideally, the synthesis of these QMPs will be simple so that a large library of potential alkylating agents can be quickly synthesized and tested. To ensure fit into the AChE active site, the QMP library was designed to contain features of known AChE substrates and inhibitors since these compounds can enter the active site

(Figure 4.4). Four families of QMPs were pursued: phenyl, pyridyl, naphthyl, and quinolinyl. The aromatic rings in the QMPs resemble those found in , 90 pyridostigmine bromide, and 2-PAM.137,138 A quaternary ammonium group was chosen as the leaving group in the QMP library to mimic the ammonium moiety found in acetylcholine, 2-PAM, edrophonium, and other AChE inhibitors (Figure 4.4).19,132,139,140

Structural analog to 2-PAM and edrophonium

Modulate reactivity Analogous to quaternary of QMP and QM ammonium on acetylcholine and edrophonium

Figure 4.4: Design of Quinone Methide Precursor Library Containing Features Found in Compounds Known to Enter the AChE Active Site

Both ortho and para quinone methides are included in the library. The QMPs will contain various R groups to help tune the reactivity of both the QMP and QM. A study by Weinert in 2006 showed that the formation of ortho quinone methides is influenced by the nature of the substituents on the QMP framework.141 When R is an electron withdrawing substituent, formation of the QM is suppressed but when R is an electron donating group, QM formation is promoted (Figure 4.5).141 Furthermore, the subsequent nucleophilic attack on the QM is also controlled by the R group. When R is electron withdrawing, nucleophilic attack is promoted, but when R is electron donating, the attack is suppressed (Figure 4.5).141

91

Figure 4.5: The Role of Electronic Effects in the Generation and Subsequent Reaction of o-Quinone Methides

4.3: Division of Work Described in this Chapter

Initial QMP library synthesis for phenyl and pyridyl families was developed by

Dr. Chris Callam and Dr. Carolyn Reid. Ryan McKenney and Amneh Young, the author of this dissertation, worked on extending the library to include the naphthyl, quinolinyl, and isoquinolinyl frameworks and synthesized a variety of OP compounds for re-aging studies. Screening of the QMP library for aged AChE realkylation and re-aging kinetics were performed by Qinggeng Zhuang. A large portion of the QMP library was synthesized with the helping hands of various undergraduate researchers in the Hadad group, the Callam group, or as a part of the REEL program.

4.4: Quinone Methide Precursor Synthesis

One benefit of using QMs as potential alkylating agents is that the synthesis of a large library of QMPs is simple. Reid and Callam optimized the synthesis of phenyl and pyridyl QMP libraries by mild reductive amination of aromatic aldehydes or by Mannich reaction of .142 These QMPs could be further diversified by either protonation or 92 alkylation. Using these methods, a large library of QMPs (over 100 compounds) was synthesized, a subset of which is shown in Figure 4.6.

93

Figure 9: Selection of Current Quinone Methide Precursor Library

94

An extension of the above methodology was explored for the synthesis of QMP’s derived from both 1-naphthol and 2-naphthol. Several different Mannich reaction conditions were screened with five different amines (dimethylamine, diethylamine, pyrrolidine, piperidine, and morpholine) however, these reactions did not produce sufficient quantities of the desired product (Scheme 4.8). Under all Mannich reaction conditions explored, a large quantity of unreacted amine was found. It was hypothesized that the slow formation of ample quantities of the iminium electrophile was the reason for the poor reaction yields.

Scheme 4: Mannich Reaction Conditions Screened to Make Naphthyl Family QMPs

To further probe the Mannich reaction and to circumvent the issues of in situ iminium generation described above, the desired iminium salts were pre-formed.

Condensation of the desired amine with formaldehyde formed the corresponding aminals which were converted to the desired iminium ions by treatment with acetyl chloride or trifluoroacetic anhydride (Scheme 4.9).143 Due to the hygroscopic nature of these salts however, reaction with either 1-naphthol or 2-naphthol gave a mixture of products that were difficult to purify. Thus, an alternative avenue to synthesize the desired QMPs was explored.

95

Scheme 5: Formation of Iminium Salts

There are several aromatic formylation reactions that can produce salicyladehydes from phenols. The Duff reaction uses hexamethylenetetramine as a formyl source,

Skattebøl conditions rely on paraformaldehyde, and Reimer-Tiemann conditions use a carbene generated from chloroform to produce the .144–146 Both Skattebøl and

Duff conditions were attempted on 1-naphthol and 2-naphthol, but Duff conditions proved to be more reliable and scalable (Scheme 4.10). The corresponding aldehydes of

1-naphthol (4.1) and 2-naphthol (4.2) could then be used in standard reductive amination conditions developed by Callam and Reid. The corresponding QMPs were synthesized by undergraduate students in the organic teaching labs at The Ohio State University as a part of the REEL program.

96

Scheme 6: Formylation and Subsequent Reductive Amination of 1 and 2-Naphthol

A similar formylation approach was used for the 8-hydroxyquinoline scaffold, but none reliably produced the desired product. Thus, an alternative procedure to produce the desired QMPs was explored. Gaseous was bubbled through an aqueous solution of 8-hydroxyquinoline and formaldehyde to produce the

4.3 (Scheme 4.11). Initially, oxidation of the corresponding followed by reductive amination was envisioned to be the route to generate the desired QMPs.

Substitution of the chlorine with the desired amine however, proved to be a more efficient route. With access to substrates from the four different QMP families of interest, a large library of potential aged AChE alkylating agents can be developed. To expedite discovery of effective alkylating agents, a quick screening protocol of the alkylating agents with various nucleophiles can provide valuable information about the utility of the different scaffolds.

97

Scheme 7: Quinoline Family QMP Synthesis

4.5: Reactivity Testing of Quinone Methide Precursors

A quick and robust screening protocol of the QMP libraries with a model nucleophile was sought to help identify compounds capable of alkylating aged AChE.

The best performing compounds would then be screened with authentic aged AChE to identify efficacious hit compounds. The model nucleophile initially used in this study was also used in Steinberg’s original study of phenacyl bromides.48 The phosphonate salt is an easy to handle, crystalline solid. The product of realkylation however, can inhibit aged AChE. This work was conducted with undergraduate researchers in the Hadad group, thus, from a safety standpoint, an alternative model nucleophile that would not inhibit AChE was sought. From our work which will be discussed in the following section, we knew that a model OP without a good leaving group cannot inhibit AChE.

Thus a phosphonate salt made with benzyl alcohol instead of p- was synthesized (Scheme 4.12).

98

Scheme 8: Synthesis of Model Phosphonate Salt

Methyl phosphonic dichloride was stirred with two equivalents of benzyl alcohol to make the dibenzyl OP compound 4.4. This compound was then selectively hydrolyzed and deprotonated to produce the model OP salt 4.5. To assess the ability of the salt to act as a model nucleophile, the phosphonate 4.5 was incubated with a good electrophile

() at room temperature (Figure 4.7). After two days, the appearance of the product OP 4.4 and the disappearance of the salt 4.5 was observed in the 31P NMR spectrum (Figure 4.7). Although this result is promising, a quicker method to screen the large QMP library still needs to be developed. Initial attempts to accelerate the alkylation by heating the reaction to 60 °C resulted in decomposition. Further optimization of the alkylation procedure is still necessary before widespread screening of the QMP library can be attempted.

99

4.4

4.5

4.4

Figure 4.7: Reaction of Model Phosphonate Salt with Benzyl Bromide and Corresponding 31P NMR Spectra

4.6: Re-Aging of AChE

The goal of this project is the synthesis and identification of a quinone methide precursor capable of alkylating the aged form of AChE in situ and consequently returning the enzyme to its inhibited state. The inhibited enzyme can then be treated with an oxime to return to the resurrected native state. Once the enzyme returns to the inhibited state, however, it is possible that it will age once again (Figure 4.8). This phenomenon will be referred to as “re-aging” hereafter. In order for the QMP to be a viable treatment, the re- aging of the enzyme must be slow to broaden the oxime treatment window.

100

Re-Aging

Figure 4.8: Alkylation and Potential Re-Aging of AChE

In order to measure re-aging, a series of OP compounds were developed with alkyl groups corresponding to the broad QMP families. Once these compounds inhibit

AChE, the enzyme has the same structure as it would after realkylation with a QMP.

Aging of this inhibited enzyme then, is equivalent to the re-aging of AChE. Initial realkylation OP mimics (4.4, 4.6-4.7) were disubstituted with benzyl alcohols (Figure

4.9). Since these OP compounds lacked a good leaving group however, they failed to inhibit AChE. The second generation realkylation mimics therefore, contain a coumarin or p-nitrophenol leaving group.

101

Figure 4.9: Model OP Compounds Used to Probe Re-Aging of AChE

The second generation OP mimics successfully inhibit AChE. Compounds 4.8,

4.9, and 4.20 can provide valuable information about the reactivity of the different QMP families. Additionally, aging rate constants of OP compounds 4.12-4.20 can be used to establish a free energy relationship that can provide information about the mechanism of

102 aging. To determine the aging rate, AChE was individually incubated with the various

OP compounds until the AChE was completely inhibited (as determined by an Ellman assay). The inhibited AChE was allowed to age and aliquots of the sample were removed and treated with a reactivator. Upon treatment with a reactivator (oxime or fluoride), the inhibited AChE was returned to its native state. The AChE activity was measured again using an Ellman assay. The difference in the AChE activity before the reaction and after treatment with OP and reactivator provides the amount of aged enzyme at each time point

(Figure 4.10).

Figure 10: Assay Used to Determine Re-Aging Rate

Initial aging kinetics of this family of OP compounds with electric eel AChE suggest that aging is rapid, and in some cases immeasurable. Compounds with electron donating groups such as 4.10 and 4.15 generally showed slower aging rates. This study is

103 inconclusive however, and must be reproduced using human AChE. Currently, the stability of human AChE under our reaction conditions is being examined.

4.7: Future Directions

There are many features of this project that need to be explored further. Beyond synthesizing varied quinolinyl and naphthyl scaffolds and testing the QMP library with model nucleophiles, model OP compounds, and aged AChE, the best oxime for reactivation of the re-alkylated AChE still needs to be determined. This oxime could be a known AChE reactivator such as 2-PAM, or a previously undiscovered one. One possibility would be a bifunctional AChE re-alkylator with a pendant oxime (Figure

4.11). That way, a single molecule can both re-alkylate and reactivate the aged enzyme.

Figure 11: Possible QMPs Containing a Pendant Oxime for AChE Reactivation

An oxime can be attached to the alkylator like XVIII so that it would be close to the phosphonate after re-alkylation occurs. One complication of such a strategy would be formation of a large ring as the product of the reaction. Alternatively, a realkylator could be designed such that the leaving group contains the pendant oxime like XIX.

Realkylation would release the oxime into the AChE active site so that it could reactivate the enzyme.

104

CHAPTER 5: EXPERIMENTAL AND SYNTHETIC DETAILS

5.1: General Methods

All reactions were performed in flame or oven dried glassware and cooled under inert atmosphere. Unless otherwise noted, all reactions were conducted under a or argon atmosphere. Reactions were monitored by TLC and visualized by a dual short/long wave UV lamp and stained with an ethanolic solution of , ceric ammonium molybdate, p-anisaldehyde, or iodine. Column flash chromatography was conducted using normal phase Aldrich 40-63 μm 60 Å silica gel.

Deactivation of silica gel was accomplished by treatment with a 1% triethylamine solution prior to packing the column. Triethylamine and diisopropylamine were freshly distilled over calcium hydride prior to use. Dichloromethane, , , pentane, and tetrahydrofuran were obtained from a solvent purification system (activated alumina columns) and used without further drying. Chemicals were either used as received or purified according to the procedures outlined in Purification of Common

Laboratory Chemicals. NMR spectra were obtained on a 400 or 600 MHz Bruker spectrometer and reported relative to TMS. Data are presented as follows: multiplicity (s

= singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad, bm = broad multiplet, dd = doublet of doublets, dt = doublet of triplet, etc), coupling

105 constant (J/Hz) and integration. Infrared spectra were recorded on a PerkinElmer

Spectrum RX1 FTIR spectrometer. Absorptions are given in wave numbers (cm-1). High resolution mass spectra were obtained on a Bruker MicroTOF II instrument from the mass spectrometry facility at The Ohio State University. 6-azidohexanoic acid147, 6- azidohexanoic acid succinimidyl ester147, 2-(pyridin-2-yldisulfanyl)ethan-1-amine hydrochloride148, (E)-4-((4-hydroxyphenyl)diazenyl)benzoic acid118, (E)-4-((4- methoxyphenyl)diazenyl)benzoic acid118, (E)-4-((4-methoxyphenyl)diazenyl)benzoyl chloride118 were synthesized according to known literature procedures. Azido-dPEG 4-

NHS ester was purchased from Nanocs and used without further purification. DABCYL-

SE was purchased from Sigma and used without further purification.

5.2: Chapter 2 Experimental Details

General Care of Mice for Hapten Study:

CD-1 mice were obtained from Charles River Laboratories (Raleigh, NC) and were 8 weeks of age and an average 34 g at the time of dosing. All animals were identified by alphanumerical ear tags and animal care and housing were in accordance with the guide for the Care and Use of Laboratory Animals (Institute of Laboratory

Animal Research, Commission on Life Sciences, National Research Council, National

Academic Press, 1996). The animals were co-housed (up to 5 mice/cage) in environmentally controlled rooms with at least 10 air changes/hour. The rooms were maintained at a temperature between 22.6-24.5 °C with a relative humidity of 28.4-49.1% and a 12-hour light/dark cycle per day. The housing was polycarbonate Tecniplast caging with wire top (Tecniplast, Phoenixville, PA) with paper chip bedding. Prior to the

106 beginning of the study, the mice were housed at MRIGlobal for nine days. All animals were provided with Lab Diet Certified Rodent Food and offered food and water ad libitum. No contaminants were present in the water that could affect the results of the study.

In vivo Hapten Toxicity Studies:

For in vivo toxicity studies, haptens were individually administered via IV injection in the tail vein at a target dose of 1 μg in vehicle (MOPS buffer, 1% DMSO,

0.24% DCM). Five mice were injected per hapten and the mice were weighed within 24 hours prior to dose administration and prior to termination. The mice were observed twice daily for general signs of toxicity.

Hapten Conjugation and Immunogen Vaccination:

Haptens were conjugated to carrier proteins via a covalent or noncovalent linkage.

Four haptens (A, B, L, and V) were conjugated to KLH (1 mg) via amine linkage to form the desired immunogen. For these haptens, an additional 100 μg was also conjugated to

BSA for anti-hapten titer screening. The remaining haptens (C, D, E, F, G, and W) were conjugated to either fatty acid-free BSA or HSA (1 mg) for immunization and anti-hapten titer screening. The immunogens were mixed with AGVand adjuvant solution and sonicated to form an emulsion for injection into mice. Affinity Boost technology was used to generate high affinity antibodies. One hundred mice (10 mice/group) were immunized with different immunogens for each group with six subcutaneous injection sites. Each injection site was administered 20 μL emulsion (10 μg immunogen/injection) for every mouse. Mice were boosted six times and test bled for anti-hapten titer

107 screening. All haptens except A and B received additional boosts. In order to increase the immune response towards the pentavalent phosphorus core, each group was also cross immunized with lipid loaded happens with identical hapten group.

Procedure for Abzyme Titer Determination:

In order to determine the specific immune response against each vaccine, a tail bleed screening was performed and titers against antigen were determined by ELISA.

High-binding 96-well plates were coated with immunogens (500 ng in 100 μg) diluted in

PBS buffer at 37 °C for 30 minutes. The plates were then blocked with 300 μL/well of

50% milk in PBST for 30 minutes at 37 °C. Pooled test bleed serum was diluted 1:10k,

1:30k, 1:100k, 1:300k, and 1:1000k with serum dilution buffer (PBST and 1% BSA) and

100 μL of diluted serum was administered to each well, with 1:10k pre-immune serum added to the control well. Plates were incubated for one hour at room temperature, washed three times with 300 μL washing buffer (PBST) per well, then treated with 100

μL HRP-goat-anti-mouse IgG gamma chain-specific secondary antibody diluted 1:4000 in PBST + 5% milk and incubated for one hour. Plates were again washed three times with washing buffer and incubated for 15 minutes at room temperature with 100 μL of one component TMB substrate. Absorbance was measured using a 96-well plate reader

(OD605).

Abzyme Sample Preparation:

Mice were tail-bled and approximately 1 mL of blood was collected from each mouse and pooled according to group. Blood samples were spun and the sera as collected. Ammonium sulfate was slowly added and dissolved into two-thirds of the

108 collected serum to the 33% saturation level. The gamma globulin fraction was separated by centrifugation (18,000 x g for 5 minutes), then re-dissolved in PBS and added to the remaining one-third untreated serum. This mixture was then dialyzed against excess volume PBS in a 30k MWCO dialysis tube to produce 10 unique abzyme test samples.

VX Hydrolysis Assay:

VX hydrolysis was determined using a modified Ellman assay in a 96-well plate format. Samples were tested at 1% and 10% total volume against 112 μM and 337 μM

VX in assay buffer (50 mM Tris buffer, 3.3% IPA and 10% IPA respectively, pH 7.5) with 1 mM acetylthiocholine and 0.5 mM DTNB. This solution was monitored spectrophotometrically for 24 hours at 412 nm. The slopes were calculated as ΔOD412/m.

VX Inactivation Assay:

7.5 nM VX was incubated with 10%, 50% and 90% abzyme concentrations at 24

°C for 0.5, 18, and 139 hours. At each time point, 10 μL of sample-incubated VX was added to a 96 well plate containing 10 μL of 1.7 μg/mL AChE and 30 μL of 50 mM Tris buffer (pH 7.5). This mixture was allowed to incubate for one hour at 24 °c and then 50

μL of Ellman solution (1 mM acetylthiocholine and 0.5 mM DTNB) was added. The mixture was monitored spectrophotometrically for 30 minutes at 412 nm. The slopes were calculated as ΔOD412/m.

109

3-bromo-5-(tert-butyl)-1,2-diol (2.2): A solution of NBS (17.70 g, 99.45 mmol, 1.1 equiv) in DMF (60 mL) was added slowly, via an addition funnel, to a solution of 4-tert-butyl-catechol (15.15 g, 91.15 mmol, 1 equiv) in DMF, which was cooled to 0

°C. After the addition was complete the mixture was slowly warmed to 23 °C and stirred for 18 h. This yielded an orange-red oil that solidified in the freezer overnight. This solid was then rinsed with cold hexanes to remove any color, yielding pure, white crystals

(22.30 g, 90.98 mmol, 99%). Spectroscopic data corresponded with that in the literature.

149 1 H NMR (400 MHz, CDCl3) 1.26 (s, 9 H), 6.92 (d, J=2.2 Hz, 1 H) 6.99 (d, J=2.2 Hz,

1 H).

1-bromo-5-(tert-butyl)-2,3-dimethoxybenzene (2.3): Potassium carbonate

(27.50 g, 197.60 mmol, 4 equiv) was added to a stirring solution of 3-bromo-5-tert-butyl- catechol (12.11 g, 49.40 mmol, 1 equiv) in DMF (120 mL). This mixture was allowed to stir for a few minutes, slowly turning the solution a deep blue. Iodomethane (12.30 mL,

197.40 mmol, 4 equiv) was added, turning the solution greenish. This mixture was allowed to stir for 20 h at 23 °C. The mixture was diluted with EtOAc (300 mL), washed with water (6 X 150 mL), and brine (3 X 100 mL). The organic layer was dried over

110 sodium sulfate and the solvent was removed under reduced pressure to yield an off white

1 solid (13.36 g, 48.90 mmol, 99%). H NMR (400 MHz, CDCl3) 1.29 (s, 9 H), 3.83 (s, 3

H), 3.87 (s, 3 H), 6.86 (d, J=2.2 Hz, 1 H), 7.12 (d, J=2.2 Hz, 1 H). 13C NMR (100 MHz,

CDCl3) 31.30, 34.77, 56.14, 60.52, 109.55, 117.16, 121.80, 144.23, 148.48, 153.08;

+ HRMS (ESI) calcd for [C12H17BrO2 + Na] 295.0304, found 294.0299.

Methyl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)acrylate (2.4): This reaction was set up in a flame dried round bottom flask in a dry glove box under a nitrogen atmosphere. Methyl acrylate (6.63 mL, 73.21 mmol, 2 equiv), Pd2(dba)3 (0.23 g, 0.25 mmol, 0.007 equiv), tri-tert-butylphosphine (0.10 g, 0.51 mmol, 0.014 equiv) and N,N- dicyclohexylmethylamine (8.62 mL, 40.27 mmol, 1.1 equiv) were added to a solution of

1-bromo-5-(t-butyl)-2,3-dimethoxybenzne (10.0 g, 36.61 mmol, 1 equiv) in dioxane (100 mL). The flask was sealed, and heated to 80 °C for 18 h (solid started precipitating after

1 h). The solution was then filtered through a silica plug with EtOAc and the solvent was removed under reduced pressure. This yielded an oil (10.19 g, 36.61 mmol, quant) and

1 was used without further purification. H NMR (400 MHz, CDCl3) 1.3 (s, 9 H), 3.81 (s, 3

H), 3.84 (s, 3 H), 3.88 (s, 3 H) 6.50 (d, J=16.2 Hz, 1 H), 6.96 (d, J=2.2 Hz, 2 H), 7.13 (d,

111

J=2.2 Hz, 2 H), 7.98 (d, J=16.2 Hz, 1 H). *Singlet at 3.70 ppm in the proton NMR spectrum is dioxane.

Methyl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoate (2.4a): A solution of methyl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)acrylate (10.19 g, 36.63 mmol, 1 equiv) in (150 mL, 200 proof) was added slowly to a slurry of Pd/C (1.58 g, 10 wt %) in water (1 mL). The flask was purged 4 times with hydrogen and then the mixture was heated to 70 °C, for 24 h under a balloon of hydrogen. The mixture was filtered through a plug of celite and then the solvent was removed under reduced pressure. This yielded a

1 light yellow/orange oil (10.26 g, 36.61 mmol, quant). H NMR (400 MHz, CDCl3) 1.29

(s, 9 H), 2.59-2.63 (m, 2 H), 2.91-2.96 (m, 2 H) 3.68 (s, 3 H), 3.82 (s, 3 H), 3.86 (s, 3 H),

13 6.77 (d, J=2.3 Hz, 1 H), 6.81 (d, J=2.3 Hz, 1 H); C NMR (100 MHz, CDCl3) 26.07,

31.47, 34.60, 35.05, 51.52, 55.75, 60.55, 108.42, 118.61, 133.23, 144.88, 146.95, 152.01,

+ 173.70; HRMS (ESI) calcd for [C16H24O4 + Na] 303.1567, found 303.1560.

112

3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoic acid (2.1): Powedered KOH

(16.00 g, 285.35 mmol, 8 equiv) was added to a solution of methyl 3-(5-(tert-butyl)-2,3- dimethoxyphenyl)propanoate (10.00 g, 35.67 mmol, 1 equiv) in THF (300 mL). The mixture was stirred under reflux for 20 h. The mixture was cooled and diluted with water

(200 mL). The aqueous layer was washed with diethyl ether and then acidified with aqueous HCl (6 M) until pH of 1. The aqueous layer was then extracted with EtOAc (3 X

250 mL), dried over sodium sulfate and then removed under reduced pressure. This

1 yielded a pale yellow solid (9.00 g, 33.79 mmol, 95%). H NMR (400 MHz, CDCl3) 1.29

(s, 9 H), 2.65-2.69 (m, 2 H), 2.92-2.97 (m, 2 H), 3.82 (s, 3 H), 3.87 (s, 3 H), 6.79 (d,

13 J=2.2, 1 H), 6.82 (d, J=2.2 Hz, 1 H); C NMR (100 MHz, CDCl3) 25.80, 31,46, 34.61,

34.79, 55.77, 60.56, 108.55, 118.64, 132.90, 144.87, 147.03, 152.02, 178.58; HRMS

+ (ESI) calcd for [C15H22O4 + Na] 289.1410, found 289.1406.

113

Pent-4-yn-1-yl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoate (2.9): A solution of DCC (1.55 g, 7.51 mmol, 1 equiv) in DCM (10 mL) was added to a solution of 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoic acid (2.00g, 7.51 mmol, 1 equiv), 4- pentyl-1-ol (0.69 g, 8.26 mmol, 1.1 equiv) and DMAP (0.09 g, 0.751 mmol, 0.1 equiv) in

DCM (70 mL) at 0 °C. The mixture was slowly warmed to 23 °C and stirred for 18 h.

The mixture was filtered through a plug of celite, washed with water (3 X 50 mL) and brine (2 X 30 mL), dried over sodium sulfate and removed under reduced pressure. This

1 yielded a pale, light yellow oil (2.47 g, 7.43 mmol, 99%). H NMR (400 MHz, CDCl3),

1.29 (s, 9 H), 1.83 (quin, J=6.9 Hz, 2 H), 1.95 (t, J=2.7 Hz, 1 H), 2.24(dt, J=2.7, 7.1 Hz, 2

H), 2.60-1.64 (m, 2 H), 2.92-2.96 (m, 2 H), 3.82 (s, 3 H), 3.86 (s, 3 H), 4.17 (t, J=6.3 Hz,

13 2 H), 6.77 (d, J=2.2 Hz, 1 H), 6.81 (d, J=2.2 Hz, 1 H); C NMR (100 MHz, CDCl3),

15.19, 26.05, 27.60, 31.48, 34.60, 35.13, 55.76, 60.55, 62.88, 68.93, 83.10, 108.46,

+ 118.58, 133.19, 144.90, 146.95, 152.02, 173.20; HRMS (ESI) calcd for [C20H28O4 + Na]

355.1880, found 355.1872.

Pent-4-yn-1-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1-yl)propanoate

(2.12): A solution of CAN (3.28 g, 6 mmol, 2 equiv) in water (10 mL) was added to a solution of pent-4-yn-1-yl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoate (1.0 g, 3

114 mmol, 1 equiv) in acetonitrile (10 mL), stirring at 23 °C. The solution quickly turned a deep red color and was done by TLC (25% EtOAc/Hex) after 2 hours. The mixture was diluted with water (20 mL) and EtOAc (20 mL). The aqueous layer was extracted with

EtOAc (3 X 25 mL). The organic layer was then washed with water (2 X 25 mL) and brine (2 X 25 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a dark red oil (0.65 g, 2.16 mmol, 72%). This was purified by flash

1 chromatography using 25% EtOAc/Hex and 1% triethylamine, isolating the top spot. H

NMR (400 MHz, CDCl3) 1.22 (s, 9 H), 1.84 (quin, J=6.8 Hz, 2 H) 1.96 (t, J=2.6 Hz, 1H),

2.27 (dt, J=2.7, 7.0, 2H), 2.55-2.58 (m, 2H), 2.70-2.73 (m, 2H), 4.18 (t, J=6.3, 2H), 6.20

13 (d, J=2.3, 1 H), 7.00-7.01 (m, 1 H); C NMR (100 MHz, CDCl3) 15.20, 25.20, 27.47,

27.77, 32.60, 35.79, 63.19, 69.10, 82.84, 122.27, 136.81, 140.15, 162.85, 172.29, 180.12,

+ 180.66; HRMS (ESI) calcd for [C18H22O4 + Na] 325.1410, found 325.1399.

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-

115 yl 3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoate (2.8): A solution of DCC (0.77 g,

3.75 mmol, 1 equiv) in DCM (5 mL) was added to a solution of 3-(5-(tert-butyl)-2,3- dimethoxyphenyl)propanoic acid (1.00 g, 3.75 mmol, 1 equiv), cholesterol (1.60 g, 4.13 mmol, 1.1 equiv) and DMAP (0.05 g, 0.375 mmol, 0.1 equiv) in DCM (35 mL) at 0 °C.

The mixture was slowly warmed to 23 °C and stirred for 18 h. The mixture was filtered through a plug of celite, washed with water (3 X 25 mL) and brine (2 X 20 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a pale, light yellow oil (2.01 g, 3.17 mmol, 84%). This was purified by flash chromatography using

1 10% EtOAc/Hex. H NMR (400 MHz, CDCl3) 0.68 (s, 3 H), 0.86 (dd, J=1.76, 6.6 Hz, 6

H), 0.91 (d, J=6.5 Hz, 3 H), .94-.99 (m, 2 H), 1.01 (s, 3 H), 1.04-1.22 (m, 8 H) 1.29 (s, 9

H), 1.33-1.61 (m, 12 H), 1.81-1.86 (m, 3 H), 1.94-1.96 (m, 2 H) 2.28-2.30 (m, 2 H), 2.56-

2.60 (m, 2 H), 2.91-2.95 (m, 2 H), 3.82 (s, 3 H), 3.86 (s, 3 H), 4.57-4.65 (m, 1 H), 6.77

13 (d, J=2.2 Hz, 1 H), 6.80 (d, J=2.2 Hz, 1 H); C NMR (100 MHz, CDCl3) 11.88, 18.76,

19.34, 21.06, 22.60, 22.85, 23.87, 24.31, 26.09, 27.81, 28.02, 28.26, 31.52, 31.68, 31.92,

34.58, 35.46, 35.82, 36.22, 36.60, 37.03, 38.17, 39.55, 39.77, 42.33, 50.06, 55.73, 56.17,

56.72, 60.50, 73.84, 76.79, 77.11, 77.43, 108.40, 118.64, 122.53, 133.33, 139.72, 144.96,

+ 146.78, 152.00, 172.60; HRMS (ESI) calcd for [C42H66O4 + Na] 657.4853, found

657.4820. *The alkyl region in the 1H NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values.

116

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1-yl)propanoate (2.11): A solution of

CAN (0.86 g, 1.57 mmol, 2 equiv) in water (10 mL) was added to a solution of

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-

(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoate (0.50 g, 0.79 mmol, 1 equiv) in tetrahydrofuran, stirring at 23 °C for 24 h. The solution quickly turned a dark brown color. The mixture was diluted with water (20 mL) and EtOAc (20 mL). The aqueous layer was extracted with EtOAc (3 X 25 mL). The organic layer was then washed with water (2 X 25 mL) and brine (2 X 25 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a dark red/brown oil (0.26 g, 0.43 mmol, 54%). This was purified by deactivated flash chromatography, run with hexanes (200 mL), 2.5%

EtOAc/Hex (200 mL) and 5% EtOAc/Hex (500 mL). The desired product was the

1 bottom spot that was visibly yellow. H NMR (400 MHz, CDCl3) 0.67 (s, 3 H), 0.86 (d,

J=1.8 Hz, 3 H), 0.87 (d, J=1.8 Hz, 3 H), 0.91 (d, J=6.5 Hz, 4 H), 1.00-1.58 (m, 42 H),

117

1.80-1.88 (m, 3 H), 1.93-2.03 (m, 3 H), 2.28 (d, J=7.6 Hz, 2 H), 2.53 (t, J=7.0 Hz, 2 H),

2.71 (t, J=7.0 Hz, 2 H), 3.63 (d, J=2.8 Hz, 1 H), 4.56-4.64 (m, 1 H), 5.34-5.35 (m, 1 H),

13 6.20(d, J=2.4 Hz, 1 H), 6.98-6.99 (m, 1 H); C NMR (100 MHz, CDCl3) 11.85, 14.19,

18.71, 19.29, 21.04, 22.55, 22.81, 23.82, 24.27, 25.24, 27.79, 28.01, 28.21, 31.85, 31.90,

32.95, 35.78, 35.80, 36.18, 36.59, 36.94, 38.16, 39.51, 39.72, 42.31, 50.03, 56.14, 56.70,

60.38, 74.33, 122.20, 122.74, 136.62, 139.53, 140.33, 162.91, 171.13, 171.77, 180.14,

+ 180.73; HRMS (ESI) calcd for [C40H60O4 + Na] 604.4384, found 604.4376. *The alkyl region in the 1H NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values.

2-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol

(2.6): A solution of pentaerythritol (5.00 g, 36.72 equiv) was dissolved in DMF (300 mL) and cooled to 0 °C. (2.47 g, 36.28 mmol) was added and the solution was stirred for 10 min, then TBDPSCl (5.095 g, 18.54 mmol) was added dropwise over 10 min. The reaction was allowed to slowly warm to 23 °C and stir for 24 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (3 X 150 mL). The organic layer was washed with brine (2 X 100 mL), dried over sodium sulfate and the solvent was removed under reduced pressure. This yielded a clear oil (3.66 g, 9.76 mmol, 53%). This was purified via flash chromatography using 70% EtOAc/Hex, isolating the bottom spot.

118

1 H NMR (400 MHz, CDCl3) 1.07 (s, 9 H), 2.34 (bs, 3 H), 3.67 (s, 2 H), 3.73 (d, J=4.7

Hz, 6 H), 7.38-7.48 (m, 6 H), 7.64-7.66 (m, 4 H).

2-(((tert-butyldiphenylsilyl)oxy)methyl)-2-((palmitoyloxy)methyl)propane-

1,3-diyl dipalmitate (2.7): A solution of DCC (6.04 g, 29.28 mmol, 3 equiv) in DCM

(20 mL) was added to a solution of palmitic acid (8.26 g, 32.21 mmol, 3.3 equiv), 2-

(((tert-butyldiphenylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol (3.66 g, 9.76 mmol, 1 equiv) and DMAP (0.35 g, 2.93 mmol, 0.3 equiv) in DCM (80 mL) at 0 °C. The mixture was slowly warmed to 23 °C and stirred for 18 h. The mixture was filtered through a plug of celite, washed with water (3 X 100 mL) and brine (2 X 100 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a clear oil (8.88 g,

1 8.15 mmol, 83%). H NMR (400 MHz, CDCl3) 0.86-0.90 (m, 12 H), 1.04 (s, 9 H), 1.25

(bs, 99 H), 1.53-1.67 (m, 8 H), 2.23 (t, J=7.4 Hz, 6 H), 2.28 (apparent quint, J=7.4 Hz, 1

H), 2.44 (t, J=7.4 Hz, 1 H), 3.62 (s, 2 H), 4.12 (s, 7 H), 7.35-7.46 (m, 6 H), 7.60-7.62 (m,

13 + 4 H); C NMR (100 MHz, CDCl3); HRMS (ESI) calcd for [C69H120O7Si + Na]

1111.8696, found 1111.8630.

119

2-(hydroxymethyl)-2-((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate

(2.5): A solution of TBAF (24.45 mL, 1 M, 3 equiv) was added to a solution of 2-(((tert- butyldiphenylsilyl)oxy)methyl)-2-((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate

(8.88 g, 8.15 mmol, 1 equiv) in THF (100 mL). This mixture was stirred at 23 °C for 18 h. The mixture was diluted with EtOAc, washed with water (100 mL), dried over sodium sulfate and the solvent was removed under reduced pressure. This yielded a white solid

(4.57 g, 5.37 mmol, 66%) which was purified by flash chromatography using 10%

1 EtOAc/Hex (Rf 0.10). H NMR (400 MHz, CDCl3) 0.86-0.90 (m, 12 H), 1.26 (s, 97 H),

1.59-1.65 (m, 8 H), 2.3-2.37 (m, 8 H), 3.57 (s, 2 H), 4.11-4.15 (m, 6 H); 13C NMR (100

+ MHz, CDCl3); HRMS (ESI) calcd for [C53H102O7 + Na] 873.7518, found 873.7479.

*Peak at 3.49 (s, 1.3 H) in the proton NMR spectrum corresponds to tetrabutylammonium salt impurity

2-(((3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoyl)oxy)methyl)-2-

((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate (2.10): A solution of DCC (0.39

120 g, 1.88 mmol, 1 equiv) in DCM (5 mL) was added to a solution of 3-(5-(tert-butyl)-2,3- dimethoxyphenyl)propanoic acid (0.50 g, 1.88 mmol, 1 equiv), 2-(hydroxymethyl)-2-

((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate (1.76 g, 2.07 mmol, 1.1 equiv) and

DMAP (0.023 g, 0.19 mmol, 0.1 equiv) in DCM (15 mL) at 0 °C. The mixture was slowly warmed to 23 °C and stirred for 18 h. The mixture was filtered through a plug of celite, washed with water (3 X 20 mL) and brine (2 X 15 mL), dried over sodium sulfate and the solvent was removed under reduced pressure. This yielded a white solid (0.73 g,

1 0.66 mmol, 37%). H NMR (400 MHz, CDCl3) 0.88 (t, J=6.6 Hz, 20 H), 1.25 (s, 170 H),

1.57-1.61 (m, 13 H), 2.27-2.32 (m, 12 H), 2.59-2.63 (m, 2 H), 2.89-2.93 (m, 2 H), 3.81 (s,

3 H), 3.86 (s, 3 H), 4.11-4.11 (m, 8 H), 6.75 (d, J=2.2 Hz, 1 H), 6.81 (d, J=2.2 Hz, 1 H);

13 + C NMR (100 MHz, CDCl3); HRMS (ESI) calcd for [C68H122O10 + Na] 1121.8930, found 1121.8904.

2-(((3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1- yl)propanoyl)oxy)methyl)-2-((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate

(2.13): A solution of CAN (1.51 g, 2.76 mmol, 4 equiv) in water (10 mL) was added to a solution of 2-(((3-(5-(tert-butyl)-2,3-dimethoxyphenyl)propanoyl)oxy)methyl)-2-

((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate (0.76 g, 0.69 mmol, 1 equiv) in

121 tetrahydrofuran, stirring at 23 °C for 24 h. The solution slowly turned a dark brown color. The mixture was diluted with water (20 mL) and EtOAc (20 mL). The aqueous layer was extracted with EtOAc (3 X 25 mL). The organic layer was then washed with water (2 X 25 mL) and brine (2 X 25 mL), dried over sodium sulfate and the solvent was removed under reduced pressure. This yielded a dark red/brown oil (0.44 g, 0.41 mmol,

60%). This was purified by deactivated flash chromatography, run with hexanes (200 mL), 1% EtOAc/Hex (250 mL), 2% EtOAc/Hex (250 mL) and 5% EtOAc/Hex (1000 mL) and 10% EtOAc/Hex (1000 mL). The desired product was the bottom spot that was visibly yellow, UV active and CAM stain was used to see the spot. 1H NMR (400 MHz,

CDCl3) 0.86-0.90 (m, 12 H), 1.23 (s, 8 H), 1.25 (s, 97 H), 1.57-1.61 (m, 8 H), 2.28-2.31

(m, 8 H), 2.54-2.58 (m, 2 H), 2.67-2.71 (m, 2 H), 4.10-4.11 (m, 8 H), 6.21 (d, J=2.3 Hz, 1

13 H), 6.99 (d, J=2.3 Hz, 1 H); C NMR (100 MHz, CDCl3) 11.85, 18.71, 19.29, 21.03,

22.55, 22.81, 23.82, 24.28, 25.24, 27.79, 28.01, 28.22, 29.24, 31.85, 31.90, 32.95, 35.80,

36.18, 36.59, 36.95, 38.16, 39.52, 39.72, 42.32, 50.03, 56.14, 56.70, 74.33, 122.20,

122.75, 136.62, 139.53, 140.34, 162.91, 180.17, 180.73; HRMS (ESI) calcd for

+ [C66H116O10 + Na] 1091.8461, found 1091.8411.

122

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl propionate (2.11a): A solution of DCC (55 mg, 0.27 mmol, 1 equiv) in DCM (1 mL) was added to a solution of propanoic acid (20 mg, 0.27 mmol, 1 equiv), cholesterol (116 mg, 0.30 mmol, 1.1 equiv) and DMAP (12 mg, 0.027 mmol, 0.1 equiv) in DCM (2 mL) at 0 °C. The mixture was slowly warmed to 23 °C and stirred for 18 h. The mixture was filtered through a plug of celite, washed with water (3 X 10 mL) and brine (2 X 10 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a pure white

1 solid, (120 mg, 0.27 mmol, Quant). H NMR (400 MHz, CDCl3) 0.86 (dd, J=1.8, 6.6 Hz,

6 H), 0.91-1.61 (34 H), 1.78-1.88 (m, 3 H), 1.94-2.03 (m, 2 H), 2.27-2.32 (m, 4 H), 4.57-

13 4.66 (m, 1 H), 5.36-5.38 (m, 1 H); C NMR (100 MHz, CDCl3) 9.18, 11.85, 18.72,

19.32, 21.04, 22.56, 22.81, 23.83, 24.29, 27.80, 27.93, 28.01, 28.23, 31.87, 31.91, 35.79,

36.19, 36.60, 37.00, 38.16, 39.52, 39.74, 42.32, 50.04, 56.14, 56.70, 73.75, 122.58,

+ 139.75, 173.94; HRMS (ESI) calcd for [C30H50O2 + Na] 465.3703, found 465.3682.

*The alkyl region in the 1H NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values.

123

2-((palmitoyloxy)methyl)-2-((propionyloxy)methyl)propane-1,3-diyl dipalmitate (2.13a): A solution of DCC (55 mg, 0.27 mmol, 1 equiv) in DCM (1 mL) was added to a solution of propanoic acid (20 mg, 0.27 mmol, 1 equiv), 2-

(hydroxymethyl)-2-((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate (256 mg, 0.30 mmol, 1.1 equiv) and DMAP (12 mg, 0.027 mmol, 0.1 equiv) in DCM (2 mL) at 0 °C.

The mixture was slowly warmed to 23 °C and stirred for 18 h. The mixture was filtered through a plug of celite, washed with water (3 X 10 mL) and brine (2 X 10 mL), dried over sodium sulfate and removed under reduced pressure. This yielded a pure white solid,

1 (120 mg, 0.27 mmol, Quant). H NMR (400 MHz, CDCl3) 0.86-0.90 (m, 12 H), 1.25 (s,

94 H), 1.58-1.61 (m, 8 H), 2.28-2.36 (m, 10 H), 4.11 (s, 10 H); 13C NMR (100 MHz,

CDCl3) 1.01, 9.02, 14.11, 22.69, 24.87, 24.92, 26.56, 27.39, 29.15, 29.26, 29.36, 29.48,

29.62, 29.66, 29.70, 31.93, 34.10, 34.17, 62.00, 62.14, 127.72, 134.79, 173.27, 173.83;

+ HRMS (ESI) calcd for [C56H106O8 + Na] 929.7780, found 929.7719.

124

General procedure for the synthesis of bis(amino)chlorophosphines: A stirred solution of phosphorus trichloride (1 equiv) in 200 mL of dry pentane was cooled to 0 °C under a nitrogen atmosphere and the amine (4 equiv) was slowly added over 20 minutes.

After the addition was complete, the reaction was stirred at 0 °C for an additional 10 minutes after which it was warmed to 23 °C and stirred for 3 hours. The precipitated white solid was filtered under a nitrogen atmosphere and the filtrate was condensed to give the pure compound as a pale white oil.

Bis(N,N-diethylamino)chlorophosphine (2.14): This compound was prepared using the above general procedure using 6 mL of phosphorus trichloride (68.7 mmol) and

28.4 mL of piperidine (63 mmol). The crude compound was purified by distillation, 75

°C at 1.0 Torr to give 10.573 g, 50.1 mmol, 72%. Spectroscopic data corresponded to that

150 31 reported in the literature. P NMR (162 MHz, CDCl3), δ: 160.0 (s).

125

Bis(N,N-diethylamino)trifluoromethylphosphine (2.16): Bis(N,N- diethylamino)chlorophosphine (1.4 g, 5.96 mmol) was dissolved in 25 mL THF under a nitrogen atmosphere. To this solution was added potassium fluoride (415 mg, 7.15 mmol) followed by trifluoromethyltrimethyl silane (0.88 mL, 5.96 mmol). 18-crown-6 was added (315 mg, 1.19 mmol) and reaction was stirred 24 h. Reaction was filtered through celite and the filtrate was concentrated under reduced pressure to give a clear oil. This oil was purified by chromatography on silica gel (10% EtOAc in Hexanes). Spectroscopic

151 1 data corresponded to that reported in the literature. H NMR (400 MHz, CDCl3), δ:

13 1.08 (t, J=7.0 Hz, 12 H); 3.08-3.21 (m, 8 H); C NMR (100 MHz, CDCl3), δ: 14.4 (d,

J=2.9 Hz); 43.7 (d, J=19.4 Hz); 129.5 (dq, J=26.9, 324.5 Hz); 31P NMR (162 MHz,

31 19 CDCl3), δ: 74.2 (q, J= 87.9 Hz); P NMR (162 MHz, CDCl3), δ: 74.1 (q, J=92.3 Hz); F

+ NMR (376 MHz, CDCl3), δ: -62.1 (d, J=90.4 Hz); HRMS (ESI) calcd for [C9H21F3N2P]

245.1389, found 245.1401.

Pent-4-yn-1-yl 3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate (2.27): A solution of Bis(N,N-

126 diethylamino)trifluoromethylphosphine (151 mg, 0.62 mmol) in DCM (5 mL) was added to a stirred solution of pent-4-yn-1-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1- yl)propanoate (188 mg, 0.62 mmol) in DCM (5 mL). The resulting solution was stirred at

23 °C for 24 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel with 10% ethyl acetate:hexanes and 1% triethylamine (92 mg, 0.17 mmol, 27%). 1H NMR (400 MHz,

CDCl3), δ: 6.82 (s, 1 H); 6.60 (m, 1 H); 4.17 (t, J= 6.0 Hz, 2 H); 3.17 (m, 8 H); 2.95-2.89

(m, 2 H); 2.69-2.64 (m, 2 H); 2.22 (dt, J= 2.7, 7.0 Hz, 2 H); 1.95 (t, J=2.4 Hz, 1 H); 1.82

(quint, J= 6.8 Hz, 2 H); 1.27 (s, 9 H); 1.06 (t, J= 6.9 Hz, 12 H); 31P NMR (162 MHz,

1 CDCl3), δ: -31.27 (q, J=62.9 Hz). * H NMR spectrum contains excess starting material peaks overlapping with the product diethylamino peaks.

2,5-dioxopyrrolidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-

1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (2.30): A solution of Azido- dPEG 4-NHS ester (158 mg, 0.41 mmol) in DMSO (4 mL) was added to 223 mg (0.41 mmol) of pent-4-yn-1-yl 3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-(trifluoromethyl)-2λ5- 127 benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate followed by addition of CuSO4•5H2O (20 mg, 0.082 mmol) and sodium (L)-ascorbate (64 mg, 0.33 mmol). The resulting solution was stirred for 24 hours at 23 °C. Ether was added and the mixture was filtered through a pad of celite. The filtrate was concentrated to give the product which was used without

1 further purification (100 mg, 0.107 mmol, 26%). H NMR (400 MHz, CDCl3), δ: 7.48 (s,

1 H); 6.81 (s, 1 H); 6.66 (s, 1 H); 4.50 (t, J=5.0 Hz, 2 H); 4.14 (t, J=6.4 Hz, 2 H); 3.87-

3.82 (m, 5 H); 3.69-3.61 (bm, 20 H); 3.39 (m, 1 H); 3.16-3.01 (m, 9 H); 2.94-2.87 (m, 5

H); 2.83 (s, 5 H); 2.77-2.74 (m, 3 H); 2.69-2.55 (m, 5 H); 2.04-1.97 (m, 2 H); 1.27-1.24

31 (bm, 14 H); 1.07-1.03 (bm, 14 H); P NMR (162 MHz, CDCl3), δ: -31.63 (q, J=60.9

+ Hz); HRMS (ESI) calcd for [C42H66F3N6O12P + Na] 957.4321, found 957.4311. *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

3-(1-(3-ethyl-5-oxo-4,8,11,14,17-pentaoxa-3-azanonadecan-19-yl)-1H-1,2,3- triazol-4-yl)propyl 3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate (2.30a): A solution of 2,5- 128 dioxopyrrolidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-

1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (30 mg, 0.032 mmol), diethylamine (3.3 μL, 0.032 mmol), and DMAP (1.1 mg, 0.0096 mmol) in DCM (1 mL) was stirred at room temperature for 4 hours. The solvent was removed and the product

1 was used without further purification. H NMR (400 MHz, CDCl3), δ: 7.47 (s, 1 H); 6.81

(s, 1 h); 6.66 (m, 1 H); 4.51-4.49 (m, 2 H); 4.13 (t, J=6.4 Hz, 2 H); 3.87-3.84 (m, 2 H);

3.81-3.77 (m, 2 H); 3.66-3.61 (bm, 19 H); 3.39-3.28 (bm, 5 H); 3.13-2.90 (bm, 22 H);

2.77-2.59 (bm, 17 H); 2.04-1.97 (m, 2 H); 1.26 (bs, 29 H); 1.18-1.08 (m, 8 H); 1.05 (t,

31 J=7.0 Hz, 13 H); P NMR (162 MHz, CDCl3), δ: -31.33 (q, J=63.0 Hz). *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl 3-(7-(tert-butyl)-2,2-bis(diethylamino)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-5-yl)propanoate (2.24): A solution of Bis(N,N-

129 diethylamino)trifluoromethylphosphine (48.8 mg, 0.2 mmol) in DCM (0.5 mL) was added to a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6- methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H- cyclopenta[a]phenanthren-3-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1- yl)propanoate (133 mg, 0.2 mmol) in DCM (0.5 mL). The resulting solution was stirred at 23 °C for 48 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel with 15% ethyl acetate:hexanes and 1% triethylamine (20 mg, 0.024 mmol, 12% yield). 1H NMR (400

MHz, CDCl3), δ: 6.81 (s, 1 H); 6.65 (m, 1 H); 5.34 (m, 1 H); 4.58 (m, 1 H); 3.19-3.02 (m,

8 H); 2.91 (t, J=7.24 Hz, 2 H); 2.63 (t, J=8.12 Hz, 2 H); 2.31-2.21 (m, 2 H); 2.02-1.37

31 (bm, 33 H); 1.37-0.83 (bm, 217 H); P NMR (162 MHz, CDCl3), δ: -31.30 (q, J=62.9

+ Hz); HRMS (ESI) calcd for [C49H81F3N2O4P] 849.5881, found 849.5863. *The alkyl region in the 1H NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values.

2-(((3-(6-(tert-butyl)-2,2-bis(diethylamino)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)methyl)-2- 130

((palmitoyloxy)methyl)propane-1,3-diyl dipalmitate (2.23): A solution of Bis(N,N- diethylamino)trifluoromethylphosphine (12 mg, 0.05 mmol) in chloroform (0.5 mL) was added to a stirred solution of pent-4-yn-1-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3- dien-1-yl)propanoate (60 mg, 0.05 mmol) in chloroform (0.5 mL). The resulting solution was stirred at 23 °C for 56 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel (56 mg, 0.02

1 mmol, 39%). H NMR (400 MHz, CDCl3), δ: 0.86-0.89 (m, 13 H); 1.05 (t, J=7.0 Hz, 12

H); 1.25-1.27 (m, 92 H); 1.54 (s, 12 H); 1.57-1.61 (m, 6 H); 2.28-2.31 (m, 6 H); 2.64-

2.68 (m, 2 H); 2.88-2.92 (m, 2 H); 3.01-3.15 (m, 8 H); 4.10 (s, 6 H); 4.14 (s, 2 H); 6.63-

31 6.64 (m, 1 H); 6.82 (m, 1 H); P NMR (162 MHz, CDCl3), δ: -31.36 (q, J=63.9 Hz);

+ HRMS (ESI) calcd for [C75H137F3N2O10P] 1313.9957, found 1313.9982.

Bis(piperidinyl)chlorophosphine (2.15): This compound was prepared using the above general procedure using 5.5 mL of phosphorus trichloride (63 mmol) and 2.5 mL of piperidine (63 mmol). The crude oil was purified by distillation; 83 °C at 1.0 Torr to give 11.09 g, 47.25 mmol, 75%. Spectroscopic data corresponded to that reported in the

150 31 literature. P NMR (162 MHz, CDCl3), δ: 155.9 (s).

131

Bis(piperidinyl)trifluoromethylphosphine (2.17):

Bis(piperidinyl)chlorophosphine was (500 mg, 2.1 mmol) was dissolved in 15 mL THF.

To this solution was added potassium fluoride (145 mg, 2.5 mmol), followed by trifluoromethyl silane (0.31 mL, 2.1 mmol). 18-crown-6 (111 mg, 0.42 mmol) was added and reaction was stirred 24 h. Reaction was filtered through celite and the filtrate was concentrated under reduced pressure to give pale white oil. The oil was purified by

1 chromatography on silica (10% EtOAc in Hexanes). H NMR (400 MHz, CDCl3), δ:

1.47-1.52 (m, 8 H); 1.54-1.58 (m, 4 H); 2.98-3.21 (bm, 8 H); 13C NMR (100 MHz,

CDCl3), δ: 24.6; 27.2 (d, J=4.8 Hz); 50.8 (d, J=18.2 Hz); 129.5 (dq, J=27.6, 325.3 Hz);

31 19 P NMR (162 MHz, CDCl3), δ: 74.2 (q, J= 87.9 Hz); F NMR (376 MHz, CDCl3), δ: -

+ 61.34 (d, J=87.2 Hz); [C11H21F3N2P] 269.1389, found 269.1393.

132

Pent-4-yn-1-yl 3-(6-(tert-butyl)-2,2-di(piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate (2.28): A solution of

Bis(piperidinyl)trifluoromethylphosphine (80 mg, 0.33 mmol) in DCM (0.5 mL) was added to a stirred solution of pent-4-yn-1-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3- dien-1-yl)propanoate (100 mg, 0.33 mmol) in DCM (0.5 mL). The resulting solution was stirred at 23 °C for 24 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel with 10% ethyl acetate:hexanes and 1% triethylamine (78 mg, 0.14 mmol, 41%). 1H NMR (400 MHz,

CDCl3), δ: 6.86 (m, 1 H); 6.67 (m, 1 H); 4.17 (t, J=6.9 Hz, 2 H); 3.16-2.98 (m, 8 H);

2.94-2.9 (m, 2 H); 2.69-2.65 (m, 2 H); 2.22 (dt, J= 2.7, 7.2 Hz, 2 H); 1.95 (t, J=2.9 Hz, 1

H); 1.82 (quint, J=6.5 Hz, 2 H); 1.59-1.47 (m, 12 H); 1.27 (s, 9 H); 31P NMR (162 MHz,

CDCl3), δ: -35.30 (q, J=62.3 Hz).

2,5-dioxopyrrolidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2,2-di(piperidin-1-yl)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-

1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (2.31): A solution of Azido- dPEG 4-NHS ester (53 mg, 0.137 mmol) in DMSO (2 mL) was added to 78 mg (0.137 133 mmol) of pent-4-yn-1-yl 3-(6-(tert-butyl)-2,2-di(piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate followed by addition of CuSO4•5H2O (6.8 mg, 0.027 mmol) and sodium (L)-ascorbate (21 mg, 0.11 mmol). The resulting solution was stirred for 36 hours at 23 °C. Ether was added and the mixture was filtered through a pad of celite. The filtrate was concentrated to give the product which was used without

1 further purification (135 mg, 0.137 mmol, 100% crude). H NMR (400 MHz, CDCl3), δ:

1.14-1.18 (m, 4 H); 1.20 (s, 9 H); 1.36 (s, 2 H); 1.44-1.50 (bm, 14 H); 1.44-1.59 (bm, 20

H); 1.90-1.97 (m, 2 H); 2.16-2.24 (m, 1 H); 2.41-2.45 (m, 1 H); 2.53-2.70 (bm, 14 H);

2.77 (s, 3 H); 2.80-2.87 (m, 4 H); 2.90-3.10 (bm, 10 H); 3.31-3.36 (bm, 21 H); 3.69-3.72

(m, 2 H); 3.76-3.81 (m, 4 H); 4.06 (t, J=6.5 Hz, 2 H); 4.29 (t, J=7.0 Hz, 1 H); 4.43-4.45

31 (m, 2 H); 6.60-6.61 (m, 1 H); 6.77 (m, 1 H); P NMR (162 MHz, CDCl3), δ: -35.43 (q,

J=61.2 Hz). *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

Piperidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2,2-di(piperidin-1-yl)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-

1,2,3-triazol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (2.31a): A solution 2,5- 134 dioxopyrrolidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2,2-di(piperidin-1-yl)-2-(trifluoromethyl)-

2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-1,2,3-triazol-1-yl)-

3,6,9,12-tetraoxapentadecan-15-oate (30 mg, 0.031 mmol), diethylamine (3.3 μL, 0.031 mmol), and DMAP (1.1 mg, 0.0096 mmol) in DCM (1 mL) was stirred at room temperature for 4 hours. The solvent was removed and the product was used without

1 further purification. H NMR (400 MHz, CDCl3), δ: 7.47-7.41 (m, 1 H); 6.93-6.58 (m, 2

H); 4.47-4.45 (m, 2 H); 4.10-4.05 (m, 2 H); 3.83-3.79 (m, 2 H); 3.76-3.71 (m, 2 H); 3.65-

3.56 (bm, 17 H); 3.50-3.45 (m, 3 H); 3.38-3.34 (m, 3 H); 3.12-2.85 (bm, 18 H); 2.72-2.56

(bm, 14 H); 2.00-1.91 (m, 2 H); 1.74 (bs, 8 H); 1.59-1.48 (bm, 25 H); 1.39 (s, 1 H); 1.22-

1.19 (m, 11 H); 31P NMR (162 MHz, DMSO), δ: -35.30 (q, J=62.4 Hz). *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl 3-(7-(tert-butyl)-2,2-di(piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-5-yl)propanoate (2.25): A solution of 135

Bis(piperidinyl)trifluoromethylphosphine (40.2 mg, 0.15 mmol) in DCM (0.5 mL) was added to a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6- methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H- cyclopenta[a]phenanthren-3-yl 3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1- yl)propanoate (94 mg, 0.15 mmol) in DCM (0.5 mL). The resulting solution was stirred at 23 °C for 48 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel with 15% ethyl acetate:hexanes and 1% triethylamine (30 mg, 0.034 mmol, 23% yield). 1H NMR (400

MHz, CDCl3), δ: 6.85 (s, 1 H); 6.67 (m, 1 H); 5.34 (m, 1 H); 4.56 (m, 1 H); 3.21-2.97 (m,

12 H); 2.91 (t, J=7.44 Hz, 2 H); 2.63 (t, J=8.12 Hz, 2 H); 2.31-2.20 (m, 2 H); 2.02-1.93

(bm, 2 H); 1.87-1.75 (bm, 3 H); 1.62-0.9 (bm, 60 H); 0.86 (dd, J= 1.7, 6.6 Hz, 6 H); 0.67

31 + (s, 3 H); P NMR (162 MHz, CDCl3), δ: -35.34 (q, J=61.2 Hz); C51H81F3N2O4P]

873.5881, found 873.5863. *The alkyl region in the 1H NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values. Peak at 74 ppm in

31P NMR spectrum is residual starting material.

N,N-diethylamino bischlorophosphine (2.18): Trichlorophosphine (3 mL, 34.38 mmol) was dissolved in dry pentane (75 mL) and chilled to 0 °C. To this solution diethylamine (7.1 mL, 68.76 mmol) was added dropwise over 20 min. Reaction was

136 allowed to reach room temperature and stirred 4 h. The reaction was filtered through celite under a nitrogen atmosphere, and filtrate was concentrated under reduced pressure to give a pale white oil. This oil was purified by distillation (62 °C at 1.0 Torr) to give 5.3 g, 30.9 mmol, 90% yield of product. Spectroscopic data corresponded to that reported in

152,153 31 the literature. P NMR (162 MHz, CDCl3), δ: 162.0 (s).

N-boc-isonipecotic acid (2.19): A stirred solution of isonipecotic acid (1.457 g,

11.3 mmol) and potassium carbonate (3.118 g, 22.6 mmol) in water was cooled to 0 °C.

A solution of Boc anhydride (2.462 g, 11.3 mmol) in THF was added dropwise over 30 minutes. The reaction was then allowed to warm to 23 °C and stirred for 12 hours. The solvent was removed and the residue was diluted with water and washed with DCM (15 mL). The aqueous layer was acidified with 6 M HCl and the solid was extracted with dichloromethane (3x30 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 2.036 g (8.98 mmol,

80%) of a white solid which was used without further purification. Spectroscopic data

154 1 corresponded to that reported in the literature. H NMR (400 MHz, CDCl3), δ: 4.08-

3.94 (m, 2 H); 2.89-2.83 (m, 2 H); 2.53-2.46 (m, 1 H); 1.70-1.60 (m, 3 H); 1.46 (s, 9 H).

137

Tert-butyl 4-(diisopropylcarbamoyl)piperidine-1-carboxylate (2.20): N-boc- isonipecotic acid (230 mg, 1 mmol) was dissolved in DCM (8 ml) under a nitrogen atmosphere. To this solution was added oxalyl chloride (0.09 mL, 1 mmol), followed by

DMF (20 L). Reaction was stirred for 30 min, and the solvent was removed under reduced pressure to give crude material as a yellow oil. The crude oil was dissolved in

DCM (8 mL), and chilled to 0 °C. Diisopropylamine (0.56 mL, 4 mmol) was added drop- wise over 10 min. Reaction was stirred for 30 min, then warmed to 25 °C and stirred an additional 1 h. The reaction was then concentrated under reduced pressure to give crude oil, which was suspended in . The precipitate was removed by filtration and washed with acetone. The filtrate was concentrated and purified by column chromatography on silica (50% EtOAc in Hexanes) to yield 202 mg, 65% product as

1 white solid. H NMR (400 MHz, CDCl3), δ: 4.27-4.05 (m, 2 H); 3.99-3.84 (m, 1 H);

2.82-2.69 (m, 2 H); 2.56-2.48 (m, 1 H); 1.79-1.68 (m, 2 H); 1.64-1.62 (m, 1 H); 1.45 (s, 9

H); 1.34 (d, J=6.7 Hz, 6 H); 1.23 (d, J=6.7 Hz, 6 H).

138

N,N-diisopropylpiperidine-4-carboxamide (2.21): Tert-butyl-4-

(diisopropylcarbamoyl)piperidine-1-carboxylate (548 mg, 2.58 mmol) was dissolved in 3 mL of DCM and cooled to 0 °C. TFA was added dropwise and the resulting solution was stirred for 5 minutes. The solvent was removed under reduced pressure and the crude oil was dissolved in 5 mL of water. The pH was adJusted to 11 using 2.5 M NaOH and the aqueous layer was extracted with DCM (5x15 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give the product (395 mg, 1.86 mmol, 76%) as an oil which was used without further purification. 1H NMR

(400 MHz, CDCl3), δ: 4.09-3.93 (m, 1 H); 3.19-3.11 (m, 2 H); 2.69-2.62 (m, 2 H); 2.57-

2.50 (m, 1 H); 1.93-1.62 (m, 6 H); 1.34 (d, J=6.6 Hz, 6 H); 1.22 (d, J=6.7 Hz, 6 H);

+ HRMS (ESI) calcd for [C12H25N2O] 213.1961, found 213.1975.

1-((diethylamino)(trifluoromethyl)phosphanyl)-N,N-diisopropylpiperidine-4- carboxamide (2.22): Unless otherwise noted, all reactions and work-ups were conducted under a nitrogen atmosphere. A stirred solution of N,N-diethylamino bischlorophosphine

(26 mg, 0.15 mmol) in dry dichloromethane (1 mL) was cooled to 0 °C and a solution of triethylamine (0.25 mL, 0.18 mmol) in dichloromethane (1 mL) was added drop-wise over 5 minutes. The resulting solution was stirred for 18 hours at 23 °C. The solvent was

139 removed under reduced pressure. Ether was added and the solution was filtered through a pad of celite and the pad was washed with additional small portions of ether. The combined filtrate and washings were concentrated and the resulting oil was used without

31 further purification in the next step. P NMR (162 MHz, CDCl3), δ: 156.4 (s). To a solution of 1-(chloro(diethylamino)phosphanyl)-N,N-diisopropylpiperidine-4- carboxamide (52 mg, 0.15 mmol) in dry THF (2 mL) under an argon atmosphere was added TMSCF3 (21 mg, 0.15 mmol), potassium fluoride (10 mg, 0.18 mmol), and 18- crown-6 (8 mg, 0.03 mmol). The resulting solution was stirred at 23 °C for 24 hours. The solvent was removed under reduced pressure and the residue was purified via flash column chromatography on silica gel with 5% ethyl acetate:hexanes and 1% triethylamine (the product was visualized with an I2 stain, 9 mg, 0.02 mmol, 6% yield).

31 19 P NMR (162 MHz, CDCl3), δ: 74.15 (q, J=90.5 Hz); F NMR (376 MHz, CDCl3), δ: -

+ 61.96 (d, J=91.8 Hz); HRMS (ESI) calcd for [C17H34N3F3OP] 384.2386, found

384.2412.

140

Pent-4-yn-1-yl 3-(6-(tert-butyl)-2-(diethylamino)-2-(4-

(diisopropylcarbamoyl)piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate (2.29): A solution of 1-

((diethylamino)(trifluoromethyl)phosphanyl)-N,N-diisopropylpiperidine-4-carboxamide

(60 mg, 0.15 mmol) in DCM (0.5 mL) was added to a stirred solution of pent-4-yn-1-yl

3-(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1-yl)propanoate (47 mg, 0.15 mmol) in

DCM (0.5 mL). The resulting solution was stirred at 23 °C for 48 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column chromatography on silica gel with 15% ethyl acetate:hexanes and 1% triethylamine (the

1 product was visualized with an I2 stain, 35 mg, 0.05 mmol, 34%). H NMR (400 MHz,

CDCl3), δ: 6.82 (s, 1 H); 6.66 (m, 1 H); 4.16 (t, J=6.3 Hz, 2 H); 3.26-3.05 (m, 4 H); 2.96-

2.87 (m, 2H); 2.80-2.63 (m, 2 H); 2.22 (dt, J=2.8, 7.2 Hz, 2 H); 1.94 (t, J=2.8 Hz, 1 H);

1.88-1.76 (m, 4 H); 1.35-1.34 (m, 6 H); 1.26 (s, 9 H); 1.26-1.20 (m, 6 H); 1.05 (t, J=6.8

13 Hz, 6 H); C NMR (100 MHz, CDCl3), δ: 173.0; 121.1; 117.9; 105.7; 105.6; 83.0; 68.9;

62.8; 45.8; 45.6; 39.4; 34.5; 34.0; 31.6; 29.7; 27.6; 25.9; 21.4; 20.7; 15.1; 13.6; 13.5; 31P

19 NMR (162 MHz, CDCl3), δ: -31.27 (q, J=62.9 Hz); F NMR (376 MHz, CDCl3), δ: -

+ 58.10 (d, J=63.8 Hz). HRMS (ESI) calcd for [C35H55N3F3O5P + Na] 708.3724, found

708.3702.

141

2,5-dioxopyrrolidin-1-yl 6-(4-(3-((3-(6-(tert-butyl)-2-(diethylamino)-2-(4-

(diisopropylcarbamoyl)piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-1,2,3-triazol-1- yl)hexanoate (2.33): A solution of 6-azidohexanoic acid succinimidyl ester (13.2 mg,

0.052 mmol) in DMSO (0.6 mL) was added to 35.9 mg (0.052 mmol) of pent-4-yn-1-yl

3-(6-(tert-butyl)-2-(diethylamino)-2-(4-(diisopropylcarbamoyl)piperidin-1-yl)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate followed by addition of CuSO4•5H2O (2.6 mg, 0.01 mmol) and sodium (L)-ascorbate (8.24 mg, 0.042 mmol). The resulting solution was stirred for 18 hours at 23 °C. Ether was added and the mixture was filtered through a pad of celite. The filtrate was concentrated to give the

1 product which was used without further purification. H NMR (400 MHz, CDCl3), δ:

7.32 (s, 1 H); 6.81 (s, 1 H), 6.66 (m, 1 H); 4.33 (m, 3 H); 4.12 (m, 3 H); 3.32-3.28 (m, 5

H); 3.17-3.03 (m, 10 H); 2.99 (s, 4 H); 2.88-2.71 (bm, 16 H); 2.36-2.25 (bm, 3 H); 2.07-

1.87 (bm, 9 H); 1.85-1.58 (bm, 15 H); 1.56-1.39 (bm, 10 H); 1.30-1.22 (bm, 20 H); 1.12-

13 1.00 (bm, 17 H); C NMR (100 MHz, CDCl3), δ: 169.1; 168.3; 76.7; 51.1; 42.6; 40.9;

39.7; 31.6; 30.7; 30.6; 29.7; 28.4; 25.8; 25.6; 25.4; 24.1; 23.9; 13.5; 13.5; 31P NMR (162

142

19 MHz, CDCl3), δ: -31.33 (q, J=62.9 Hz); F NMR (376 MHz, CDCl3), δ: -58.01 (d,

+ J=63.0 Hz). HRMS (ESI) calcd for [C37H57N6F3O8P] 801.3922, found 801.3869.

Note: No molecular ion peak was observed. *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl 3-(7-(tert-butyl)-2-(diethylamino)-2-(4-(diisopropylcarbamoyl)piperidin-1-yl)-2-

(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-5-yl)propanoate (2.26): A solution of 1-((diethylamino)(trifluoromethyl)phosphanyl)-N,N-diisopropylpiperidine-4- carboxamide (60 mg, 0.15 mmol) in DCM (0.5 mL) was added to a stirred solution of

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-

(3-(tert-butyl)-5,6-dioxocyclohexa-1,3-dien-1-yl)propanoate (94 mg, 0.15 mmol) in

DCM (0.5 mL). The resulting solution was stirred at 23 °C for 48 hours. The solvent was removed under reduced pressure and the crude oil was purified via flash column

143 chromatography on silica gel with 15% ethyl acetate:hexanes and 1% triethylamine (the

1 product was visualized with an I2 stain, 53 mg, 0.05 mmol, 35%). H NMR (400 MHz,

CDCl3), δ: 6.81 (s, 1 H); 6.65 (m, 1 H); 5.34 (m, 1 H); 4.56 (m, 1 H); 3.17-3.05 (m, 10

H); 2.91 (t, J=7.5 Hz); 2.62 (t, J=8.1 Hz, 2 H); 2.30-2.19 (m, 2 H); 2.06-0.86 (bm, 144

13 H); 0.67 (s, 5 H); C NMR (100 MHz, CDCl3), δ:76.7; 73.8; 56.7; 56.1; 50.0; 42.3; 39.6;

39.5; 38.1; 36.9; 36.5; 36.2; 35.8; 34.5; 34.4; 31.8; 31.6; 31.5; 29.7; 28.2; 28.0; 24.2;

31 23.8; 22.8; 22.6; 22.5; 21.0; 19.3; 18.7; 14.1; 13.5; 11.8; P NMR (162 MHz, CDCl3), δ:

19 -31.29 (q, J=62.9 Hz); F NMR (376 MHz, CDCl3), δ: -58.07 (d, J=63.2 Hz). HRMS

+ 1 (ESI) calcd for [C57H94N3F3O5P] 988.6878, found 988.6889. *The alkyl region in the H

NMR spectrum matches that of cholesterol, however, the broad peaks give elevated integration values.

2,5-dioxopyrrolidin-1-yl 1-(4-(3-((3-(6-(tert-butyl)-2-(diethylamino)-2-(4-

(diisopropylcarbamoyl)piperidin-1-yl)-2-(trifluoromethyl)-2λ5- benzo[d][1,3,2]dioxaphosphol-4-yl)propanoyl)oxy)propyl)-1H-1,2,3-triazol-1-yl)-

3,6,9,12-tetraoxapentadecan-15-oate (2.32): A solution of Azido-dPEG 4-NHS ester

144

(9.32 mg, 0.024 mmol) in DMSO (0.3 mL) was added to 16.3 mg (0.024 mmol) of pent-

4-yn-1-yl 3-(6-(tert-butyl)-2-(diethylamino)-2-(4-(diisopropylcarbamoyl)piperidin-1-yl)-

2-(trifluoromethyl)-2λ5-benzo[d][1,3,2]dioxaphosphol-4-yl)propanoate followed by addition of CuSO4•5H2O (1.2 mg, 0.0048 mmol) and sodium (L)-ascorbate (3.8 mg,

0.019 mmol). The resulting solution was stirred for 24 hours at 23 °C. Ether was added and the mixture was filtered through a pad of celite. The filtrate was concentrated to give the product which was used without further purification (19.3 mg, 0.018 mmol, 73%). 1H

NMR (400 MHz, DMSO), δ: 7.79 (s, 1 H); 6.87 (s, 1 H); 6.73 (s, 1 H); 4.46 (t, J= 4.0 Hz,

2 H); 4.37-4.33 (m, 2 H); 4.03 (t, J= 6.0 Hz, 3 H); 3.79 (t, J= 5.2 Hz, 2 H); 3.71 (t, J= 6.4

Hz, 2 H); 3.54-3.47 (m, 16 H); 3.34 (bs, 21 H); 3.13-3.01 (m, 4 H); 2.84-2.79 (bs, 6 H);

2.59 (s, 3 H); 2.52-2.49 (m, 3 H); 1.25-1.21 (m, 18 H); 1.14-1.11 (m, 6 H); 13C NMR

(100 MHz, CDCl3), δ: 172.1; 168.0; 167.9; 165.8; 145.7; 121.1; 120.1; 116.8; 104.6;

104.5; 69.7; 69.6; 69.5; 69.4; 69.3; 68.5; 64.7; 62.5; 49.1; 44.8; 44.7; 38.4; 33.5; 33.0;

31.1; 30.6; 24.5; 21.0; 20.3; 19.7; 13.1; 12.6; 12.5; 12.4; 9.9; 31P NMR (162 MHz,

DMSO), δ: -31.79 (q, J=60.4 Hz); 19F NMR (376 MHz, DMSO), δ: -56.84 (d, J=60.5

+ Hz). HRMS (ESI) calcd for [C50H79N7F3O13P + Na] 1096.5318, found 1096.5324. *Due to the sensitive nature of the NHS ester moiety, this product could not be further purified and the NMR spectra indicate the presence of some impurities.

5.3: Chapter 3 Experimental Details

145

Diethyl methylphosphonate (3.2): A solution of triethylphosphite (3.40 mL, 20 mmol) and methyl (1.40 mL, 22 mmol) was refluxed for 12 h. The ethyl iodide produced was removed in vacuo and the resulting clear liquid was used without further purification (2.92 g, 96 % yield). Spectroscopic data corresponds to that reported in the

1 literature. H NMR (400 MHz, CDCl3), δ: 4.17-4.01 (m, 4 H); 1.47 (dd, J=17.4, 1.84 Hz,

31 3 H); 1.35-1.31 (dt, J=7.04, 1.92 Hz, 6 H); P NMR (162 MHz, CDCl3), δ: 30.3.

Ethyl methylphosphonochloridate (3.3): Oxalyl chloride (2.34 mL, 27.3 mmol) was added dropwise to a stirred solution of diethyl methylphosphonate (3.19 g, 21 mmol) in chloroform (25 mL) at room temperature and the resulting solution stirred at room temperature for 20 hours. The solvent was removed and the product distilled under reduced pressure to give a clear liquid (2.48 g, 87% yield). Spectroscopic data

1 corresponds to that reported in the literature. H NMR (400 MHz, CDCl3), δ: 4.31-4.08

(m, 2 H); 1.91(d, J=17.2 Hz, 3 H); 1.33 (t, J=7.08 Hz, 3 H); 31P NMR (162 MHz,

CDCl3), δ: 30.0.

146

(9H-fluoren-9-yl)methyl (2-mercaptoethyl) (3.4): Cysteamine hydrochloride (1.1 g, 10 mmol) was dissolved in acetonitrile (7.5 mL) under a nitrogen atmosphere and cooled to 0 °C. DIPEA (4.0 mL, 23 mmol) was added slowly and the resulting solution was stirred for five minutes. TMSCl (1.6 mL, 13 mmol) was added dropwise and the solution was allowed to stand for ten minutes. A solution of FmocCl

(2.5 g, 10 mmol) in MeCN (2.5 mL) was added slowly with stirring followed by DIPEA

(1.7 mL, 10 mmol). The reaction was stirred for 30 minutes at 0 °C then for two hours at

23 °C. The mixture was dumped into ice-water (25 mL) then extracted with DCM (2 x

50 mL) followed by washing with water (100 mL), 1 M HCl (100 mL), 0.1 M NaHCO3

(100 mL), and brine (100 mL). This was used in the next step without further

1 purification. H NMR (400 MHz, CDCl3), δ: 7.77 (m, 2 H); 7.59 (m, 2 H); 7.40 (t, J=7.4

Hz, 2 H); 7.32 (m, 2 H); 5.12 (s, 0.6 H), 4.43 (m, 2 H); 4.422 (m, 1 H); 3.38 (m, 1.6 H);

2.65 (m, 1.6 H); 1.33 (m, 0.8 H).

S-(2-(2-(9H-fluoren-9-yl)acetamido)ethyl) O-ethyl methylphosphonothioate

(3.5): A solution of the thiol 3.5 (1.14 g, 5 mmol) in DCM (25 mL) was added to a stirred solution of the chloridate 3.3 (0.71 g, 5 mmol) and trimethylamine (0.84 mL, 6 mmol) in

DCM (25 mL) at 0 °C. The resulting solution was allowed to warm to room temperature

147 and stirred overnight. The solvent was removed under vacuum and the resulting oil was diluted with DCM (100 mL) and washed with excess water (3 x 150 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The product was used

1 without further purification (0.70 g, 34 % yield). H NMR (400 MHz, CDCl3), δ: 7.68

(m, 2 H); 7.52 (m, 2 H); 7.32 (t, J=7.4 Hz, 2 H); 7.23 (t, J=7.4 Hz, 2 H); 5.63 (s, 1 H);

4.32 (m, 2 H); 4.12 (m, 2 H); 3.41 (m, 2 H); 2.94 (m, 2 H); 1.72 (d, J=15.7 Hz, 3 H); 1.26

31 (t, J=7.1 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 54.1.

(E)-4-((4-(dimethylamino)phenyl)diazenyl)-N-(2-mercaptoethyl)benzamide

(3.7): A solution of DABCYL-SE (0.1 g, 0.3 mmol) and trimethylamine (84 μL, 0.6 mmol) in DMF (1 mL) was stirred at 0 °C for 1 hour, then allowed to warm to 23 °c and stirred overnight. The solid that precipitated was filtered, washed with ether, and dried.

The solid was then dissolved in a 2:1 solution of MeCN:H2O (3 mL) and tributylphosphine (43 mg, 0.2 mmol) was added. The reaction was stirred for 30 minutes after which the solvent was removed under vacuum. Ether was added and the solid was filtered, washed with ether, and used without further purification (63 mg, 63 % yield over

1 two steps). ). H NMR (400 MHz, CDCl3), δ: 7.93 (m, 6 H); 6.78 (m, 6 H); 6.61 (m, 1

148

H); 3.67 (q, J= 6.2 Hz, 2 H); 3.12 (s, 6 H); 2.82 (m, 2 H); 1.43 (t, J=8.5 Hz, 1 H). HRMS

+ (ESI) calcd for [C17H20N4SO + H] 329.1431, found 329.1427.

(E)-N-(2-mercaptoethyl)-4-((4-methoxyphenyl)diazenyl)benzamide (3.15):

HATU (0.38 g, 1 mmol) was added to a solution the carboxylic acid 3.12 (0.26 g, 1 mmol) in DMF (5 mL) and the solution was stirred for 30 minutes at room temperature.

A solution of the disulfide 3.9 (0.24 g, 1.1 mmol) and DIPEA (0.52 mL, 3 mmol) in DMF

(5 mL) was added slowly and the reaction was stirred for 1 hour at 23 °C. The reaction was diluted with excess EtOAc (50 mL) and washed successively with water (5 x 50 mL) and brine (3 x 50 mL). The product (3.14) was used in the following step without further purification. Solid TCEP hydrochloride (54 mg, 0.19 mmol) was added to a solution of

3.14 in 2:1 THF:H2O (3 mL) and this was stirred for 30 minutes at 23 °C. THF was removed under reduced pressure and the resulting solid was filtered and washed with

1 excess water (68 mg, 65% yield over two steps). H NMR (400 MHz, CDCl3), δ: 7.86 (m,

6 H); 6.96 (m, 2 H); 3.84 (s, 3 H); 3.61 (q, J=6 Hz, 2 H); 2.76 (m, 2 H). HRMS (ESI)

+ calcd for [C16H17N3SO2 + Na] 338.0920, found 338.0934.

149

(E)-O-ethyl S-(2-(4-((4-methoxyphenyl)diazenyl)benzamido)ethyl) methylphosphonothioate (3.17): A solution of the thiol 3.15 (0.31 g, 1 mmol) in THF

(2.5 mL) was added dropwise to a stirred solution of the chloridate 3.3 (0.21 g, 1.5 mmol) in THF (2.5 mL) under an argon atmosphere. The reaction was stirred for 48 hours at 23

°C. The solvent was removed and the residue was dissolved in DCM and washed successively with saturated sodium bicarbonate solution, water, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. A small amount of the product was purified on prep TLC (DCM to remove top band, product eluted with EtOAc

1 containing 2% trimethylamine, Rf 0.3). H NMR (600 MHz, CDCl3), δ: 8.03 (m, 2 H);

7.86 (m, 5 H); 2.96 (m, 2H); 4.06-3.97 (m, 2 H); 3.86 (s, 3 H); 3.79-3.65 (m, 2 H); 3.23-

3.04 (m, 2 H); 2.28 (t, J=4.8 Hz, 1 H); 1.76 (d, J= 15.72 Hz, 3 H); 31P NMR (243 MHz,

+ CDCl3), δ: 55.8. HRMS (ESI) calcd for [C19H24N3SPO4 + Na] 444.1105, found

444.1117.

5.4: Chapter 4 Experimental Details

General Procedure for the Formylation of Naphthols: A solution of HMTA

(9.8 g, 70 mmol) and the naphthol (10 g, 70 mmol) in TFA (100 mL) was refluxed for 6 hours. 10% HCl solution (100 mL) was added and the reaction was refluxed for another hour. After cooling to room temperature, the reaction was allowed to sit overnight then

150 diluted with water (200 mL) and extracted with DCM (3 x 150 mL). The organic layer was washed with water (400 mL) and brine (400 mL), dried over sodium sulfate, filtered and concentrated. Hexanes (250 mL) was added to the solid which was heated until the solid dissolved, at which point a red oil remained in the bottom of the flask. The yellow hexanes solution was decanted and concentrated to give the pure aldehyde (78-80% yield).

1 1-hydroxy-2-naphthaldehyde (4.1): H NMR (400 MHz, CDCl3), δ: 13.25 (s, 1

H); 10.24 (s, 1 H); 10.08 (s, 1 H); 9.31 (m, 1 H); 8.54 (m, 1 H); 8.07 (s, 1 H); 7.88 (m, 1

H); 7.68 (m, 1 H).

1 2-hydroxy-1-naphthaldehyde (4.2): H NMR (400 MHz, CDCl3), δ:13.16 (s, 1

H); 10.83 (s, 1 H); 8.36 (d, J= 8.52 Hz, 1 H); 7.99 (d, J= 9.08 Hz, 1 H); 7.81 (d, J= 8.08

Hz, 1 H); 7.62 (m, 1 H); 7.44 (m, 1 H); 7.14 (d, J= 9.08 Hz, 1 H).

151

5-(chloromethyl)quinolin-8-ol hydrochloride (4.3): HCl gas was bubbled slowly through a stirred mixture of 8-hyrdoxyquinoline (14.6 g, 100 mmol), 37% formaldehyde solution (16 mL, 100 mmol), and concentrated hydrochloric acid (16 mL,

194 mmol) at 0 °C for five hours. The reaction was then allowed to sit without stirring for

12 hours. The yellow solid was filtered and recrystallized from 95% EtOH (14.9 g, 65 % yield).155 1H NMR (400 MHz, DMSO-d6), δ: 9.06 (m, 2 H); 8.01 (m, 1 H); 7.80 (m, 1 H);

+ 7.34 (m, 1 H); 5.30 (s, 2 H). HRMS (ESI) calcd for [C10H8NO + CH3OH] 191.0941, found 191.0934.

General Procedure for the : Potassium carbonate (2 equiv) was added to a stirred solution of 4.3 (1 equiv) in acetonitrile (0.2 M) at 0 °C. The mixture was stirred for 10 minutes then the amine (1 equiv) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred for 12 hours. The

152 mixture was diluted with DCM, washed with sat. sodium bicarbonate solution, washed with brine, dried over sodium sulfate, filtered, and concentrated.

1 5-((dimethylamino)methyl)quinolin-8-ol (4.3a): H NMR (600 MHz, CDCl3),

δ: 8.77 (dd, J=1.8, 4.2 Hz, 1 H); 8.62 (dd, J= 1.5, 8.5 Hz, 1 H); 7.46 (dd, J=8.5, 6.3 Hz, 1

H); 7.68 (d, J= 11.5 Hz, 1 H); 7.07 (d, J=11.5 Hz, 1 H); 3.70 (s, 2 H); 2.24 (s, 6 H).

+ HRMS (ESI) calcd for [C12H14N2O + H] 203.1179, found 203.1174.

1 5-((diethylamino)methyl)quinolin-8-ol (4.3b): H NMR (600 MHz, CDCl3), δ:

8.77-8.74 (m, 2 H); 7.45-7.35 (m, 1 H); 7.36-7.35 (m, 1 H); 7.07-7.06 (m, 1 H); 3.86 (s, 2

H); 2.53 (q, J=7.1 Hz, 4 H); 1.04 (t, J=7.1 Hz, 6 H). HRMS (ESI) calcd for [C14H18N2O

+ H]+ 231.1492, found 231.1486.

153

1 5-(pyrrolidin-1-ylmethyl)quinolin-8-ol (4.3c): H NMR (600 MHz, CDCl3), δ:

8.77 (dd, J=1.6, 4.2 Hz, 1 H); 8.66 (dd, J=1.6, 8.5 Hz, 1 H); 7.46 (dd, J=4.2, 8.5 Hz, 1

H); 7.36 (d, J=7.7 Hz, 1 H); 7.07 (d, J=7.7 Hz, 2 H); 3.91 (s, 2 H); 2.5 (m, 4 H); 1.75 (m,

+ 4 H). HRMS (ESI) calcd for [C14H16N2O + H] 229.1335, found 229.1338.

1 5-(piperidin-1-ylmethyl)quinolin-8-ol (4.3d): H NMR (600 MHz, CDCl3), δ:

8.77-8.76 (m, 1 H); 8.72-8.70 (m, 1 H); 7.45-7.43 (m, 1 H); 7.32-7.30 (m, 1 H); 3.73 (s, 2

H); 2.37 (m, 2 H); 1.51 (m, 6 H); 1.43 (m, 2 H). HRMS (ESI) calcd for [C15H18N2O +

H]+ 243.1492, found 243.1495.

154

1 5-(morpholinomethyl)quinolin-8-ol (4.3e): H NMR (600 MHz, CDCl3), δ:

8.79-8.77 (m, 1 H); 8.68-8.66 (m, 1 H); 7.47-7.45 (m, 1 H); 7.33-7.32 (m, 1 H); 7.07-7.06

(m, 1 H); 3.79 (s, 2 H); 3.66-3.65 (m, 4 H); 2.45 (m, 4 H). HRMS (ESI) calcd for

+ [C14H16N2O2 + H] 245.1285, found 245.1288.

General Procedure for the Synthesis of Dibenzyl OP compounds: A solution of trimethylamine (2 equiv) and the benzyl alcohol (2 equiv) in DCM (20 mL) was added dropwise over 30 minutes into a solution of methylphosphonic dichloride (1 equiv) in

DCM (30 mL) at 0 °C under an argon atmosphere. The resulting solution was allowed to warm to room temperature and stirred for 48 hours. Ether was added and the solution was filtered through a pad of celite. The filtrate was concentrated under reduced pressure. The residue was diluted with DCM (50 mL) and washed with water (3 x 30 mL), dried over sodium sulfate, filtered, and concentrated. The residue was purified via flash column chromatography.

155

Dibenzyl methylphosphonate (4.4): Column conditions: 80% EtOAc/hexanes.

1 H NMR (400 MHz, CDCl3), δ: 7.36-7.31 (m, 10 H); 5.09-4.95 (m, 4 H); 1.48 (d, J= 17.6

31 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 31.67. HRMS (ESI) calcd for [C15H17PO3 +

Na]+ 299.0808, found 299.0799.

Bis(benzo[d][1,3]dioxol-5-ylmethyl) methylphosphonate (4.6): Column

1 conditions: 80 % EtOAc/hexanes. H NMR (400 MHz, CDCl3), δ: 6.84-676 (m, 6 H);

5.96 (s, 4 H); 4.96-4.84 (m, 4 H); 1.44 (d, J= 17.6 Hz, 3 H); 31P NMR (162 MHz,

+ CDCl3), δ: 31.54. HRMS (ESI) calcd for [C17H17PO7 + Na] 387.0604, found 387.0598.

bis(pyridin-2-ylmethyl) methylphosphonate (4.7): Column conditions: 5 % MeOH/DCM

1 with 1% triethylamine. H NMR (400 MHz, CDCl3), δ: 8.56-8.55 (m, 2 H); 7.72-7.67 (m,

2 H); 7.44-7.42 (m, 2 H); 7.23-7.19 (m, 2 H); 5.24-5.12 (m, 4 H); 1.65 (d, J= 17.7 Hz, 3

31 H); P NMR (162 MHz, CDCl3), δ: 32.09.

156

General Procedure for the Synthesis of Second Generation OP Mimics A: A solution of the coumarin (1 equiv) and trimethylamine (2.3 equiv) in DCM (15 mL) was added dropwise over 30 minutes to a stirred solution of methyl phosphonic dichloride (1 equiv) in DCM (10 mL) at 0 °C under an argon atmosphere. The resulting solution was allowed to warm to room temperature and stirred for 24 hours. The reaction was then cooled to 0 °C and a solution of the benzyl alcohol (1 equiv) and trimethylamine (2.3 equiv) in DCM (15 mL) was added dropwise over 30 minutes. The resulting solution was allowed to warm to room temperature and stirred for 24 hours. Ether was added and the solution was filtered through a pad of celite. The filtrate was concentrated under reduced pressure. The residue was purified via flash column chromatography.

General Procedure for the Synthesis of Second Generation OP Mimics B: A solution of p-nitrophenol (1 equiv) and trimethylamine (2.3 equiv) in DCM (15 mL) was added dropwise over 30 minutes to a stirred solution of methyl phosphonic dichloride (1 equiv) in DCM (10 mL) at 0 °C under an argon atmosphere. The resulting solution was allowed to warm to room temperature and stirred for 24 hours. The reaction was then cooled to 0 °C and a solution of the benzyl alcohol (1 equiv) and trimethylamine (2.3 equiv) in DCM (15 mL) was added dropwise over 30 minutes. The resulting solution was allowed to warm to room temperature and stirred for 24 hours. Ether was added and the solution was filtered through a pad of celite. The filtrate was concentrated under reduced pressure. The residue was purified via flash column chromatography.

157

4-nitrophenyl (pyridin-2-ylmethyl) methylphosphonate (4.8B): General procedure B was followed. The produce was purified by washing as column

1 chromatography resulted in decomposition. H NMR (400 MHz, CDCl3), δ: 8.16-8.15

(m, 4 H); 7.46-7.44 (m, 2 H); 7.27-7.25 (m, 2 H); 5.29-5.14 (m, 2 H); 1.77 (d, J=17.3 Hz,

31 3 H); P NMR (162 MHz, CDCl3), δ: 29.76.

Naphthalen-2-ylmethyl (4-nitrophenyl) methylphosphonate (4.9B): General procedure B was followed. Purified on Prep TLC using 100% EtOAc. 1H NMR (400

MHz, CDCl3), δ: 7.94-7.92 (m, 2 H); 7.75-7.71 (m, 2 H); 7.68-7.65 (m, 2 H); 7.43-7.74

(m, 2 H); 7.36-7.34 (m, 1 H); 714-7.12 (m, 2 H); 5.23-5.20 (m, 2 H); 1.65 (d, J=17.9 Hz,

31 3 H); P NMR (162 MHz, CDCl3), δ: 29.24.

158

4-nitrophenyl (3,4,5-trimethoxybenzyl) methylphosphonate (4.10B): General procedure B was followed. Purified on Prep TLC using 100% EtOAc. 1H NMR (400

MHz, CDCl3), δ: 8.21-8.19 (m, 2 H); 7.33-7.30 (m, 2 H); 6.60-6.57 (m, 2 H); 5.14-5.00

31 (m, 2 H); 3.84-3.82 (m, 9 H); 1.71 (d, J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ:

28.97.

Benzo[d][1,3]dioxol-5-ylmethyl (4-methyl-2-oxo-2H-chromen-7-yl) methylphosphonate (4.11A): General Procedure A was followed. 1H NMR (600 MHz,

CDCl3), δ: 7.46-7.44 (m, 1 H); 7.71-7.08 (m, 1 H); 6.97-6.96 (m, 1 H); 6.63-6.62 (m, 1

H); 6.17 (s, 1 H); 5.89-5.87 (m, 2 H); 4.99-4.92 (m, 2 H); 2.35 (s, 3 H); 1.60 (d, J=17.8

31 Hz, 3 H); P NMR (243 MHz, CDCl3), δ: 28.87. HRMS (ESI) calcd for [C19H17PO7 +

Na]+ 411.0604, found 411.0594.

159

4-nitrobenzyl (4-nitrophenyl) methylphosphonate (4.12B): General Procedure

1 B was followed. Purified on Prep TLC using 100% EtOAc. H NMR (400 MHz, CDCl3),

δ: 8.2-8.21 (m, 4 H); 7.53-7.51 (m, 2 H); 7.37-7.34 (m, 2 H); 5.33-5.16 (m, 2 H); 1.77 (d,

31 J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 29.29.

4-nitrophenyl (4-(trifluoromethyl)benzyl) methylphosphonate (4.13B):

General Procedure B was followed. Column conditions: 50% EtOAc/hexanes. 1H NMR

(400 MHz, CDCl3), δ: 8.20-8.18 (m, 2 H); 7.62-7.60 (m, 2 H); 7.47-7.45 (m, 2 H); 7.33-

7.312 (m, 2 H); 7.30 (m, 2 H): 5.27-5.13 (m, 2 H); 1.74 (d, J=17.8 Hz, 3 H); 19F NMR

31 (376 Hz, CDCl3), 62.76; P NMR (162 MHz, CDCl3), δ: 29.21.

4-cyanobenzyl (4-nitrophenyl) methylphosphonate (4.14B): General Procedure

1 B was followed. Column conditions: 100% EtOAc. H NMR (400 MHz, CDCl3), δ: 8.23-

8.21 (m, 2 H); 7.67-7.65 (m, 2 H); 7.47-7.45 (m, 2 H); 7.36-7.33 (m, 2 H); 5.29-5.11 (m,

31 2 H); 1.75 (d, J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 29.24. 160

4-methoxybenzyl (4-nitrophenyl) methylphosphonate (4.15B): General

Procedure B was followed. Column conditions: 100% EtOAc on Florisil. 1H NMR (400

MHz, CDCl3), δ: 8.17-8.13 (m, 2 H); 7.30-7.24 (m, 4 H); 6.90-6.82 (m, 2 H); 5.13-5.02

31 (m, 2 H); 3.80 (s, 3 H); 1.66 (d, J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 28.92.

4-fluorobenzyl (4-nitrophenyl) methylphosphonate (4.16B): General Procedure B was followed. Column Conditions: 50% EtOAc/hexanes. 1H NMR (400 MHz, acetone-d6), δ:

8.26-8.25 (m, 2 H); 7.50-7.47 (m, 4 H); 7.16-7.12 (m, 2 H); 5.23-5.17 (m, 2 H); 1.76 (d,

J=17.8 Hz, 3 H); 19F NMR (376 Hz, acetone-d6), -115.06; 31P NMR (162 MHz, acetone- d6), δ: 28.73 (hept, J =9.0 Hz).

161

4-chlorobenzyl (4-nitrophenyl) methylphosphonate (4.17B): General

Procedure B was followed. Column Conditions: 50% EtOAc/hexanes. 1H NMR (400

MHz, CDCl3), δ: 8.20-8.18 (m, 2 H); 7.32-7.28 (m, 5 H); 5.17-5.04 (m, 2 H); 1.70 (d,

31 J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ: 29.07.

4-bromobenzyl (4-nitrophenyl) methylphosphonate (4.18B): General

Procedure B was followed. Column Conditions: 50% EtOAc/hexanes. 1H NMR (400

MHz, CDCl3), δ: 8.20-8.18 (m, 2 H); 7.47-7.45 (m, 2 H); 7.30-7.28 (m, 2 H); 7.22-7.20

31 (m, 2 H); 5.16-5.02 (m, 2 H); 1.71 (d, J=17.8 Hz, 3 H); P NMR (162 MHz, CDCl3), δ:

29.11.

4-(tert-butyl)benzyl (4-nitrophenyl) methylphosphonate (4.19B): General

Procedure B was followed. Column Conditions: 50% EtOAc/hexanes. 1H NMR (400

162

MHz, CDCl3), δ: 8.18-8.16 (m, 2 H); 7.37-7.35 (m, 2 H); 7.29-7.25 (m, 4 H); 5.18-5.06

31 (m, 2 H); 1.69 (d, J=17.8 Hz, 3 H); 1.31 (s, 9 H); P NMR (162 MHz, CDCl3), δ: 28.82.

Benzyl (4-methyl-2-oxo-2H-chromen-7-yl) methylphosphonate (4.20A):

General procedure A was followed. Column conditions: 80-100% EtOAc/hexanes. 1H

NMR (600 MHz, CDCl3), δ: 7.46-7.44 (m, 1 H); 7.29-7.25 (m, 5 H); 7.11-7.09 (m, 1 H);

7.03-7.02 (m, 1 H); 6.17 (m, 1 H); 5.14-5.02 (m, 2 H); 2.34 (s, 3 H); 1.61 (d, J=17.8 Hz,

31 + 3 H); P NMR (243 MHz, CDCl3), δ: 28.78. HRMS (ESI) calcd for [C18H17PO5 + Na]

367.0706, found 367.0697.

Benzyl (4-nitrophenyl) methylphosphonate (4.20B): General procedure B was followed. Column conditions: 50% EtOAc/hexanes. 1H NMR (600 MHz, DMSO-d6),

δ:8.26-8.24 (m, 2 H); 7.46-7.34 (m, 7 H); 5.21-511 (m, 2 H); 1.79 (d, J=17.6 Hz, 3 H);

31P NMR (243 MHz, DMSO-d6), δ: 29.54. 163

5.5: Other Relevant Experimental Details

3-cyano-4-methyl-2-oxo-2H-chromen-7-yl ethyl methylphosphonate (EMP):

To a stirred solution of 3-cyano-7-hydroxy-4-methylcoumarin (1.92 g, 9.5 mmol) and triethylamine (3.9 mL, 28 mmol) in dichloromethane (60 mL) at 0 °C a solution of ethyl methylphosphonochloridate (1.5 g, 10.5 mmol) in dichloromethane (40 mL) was added dropwise over 20 minutes. The resulting solution was allowed to warm to room temperature and stirred for 20 hours. Ether was added to the reaction and the resulting precipitate was filtered through celite and washed with ether. The combined filtrate and washings were run through a plug of silica and concentrated. The residue was purified by recrystallization from ethyl acetate:hexanes (0.73 g, 25% yield). FTIR (film, cm-1) 2988,

2928, 2230, 1737, 1613, 1555, 1507, 1425, 1385, 1382, 1316, 1236, 1140, 1033, 974,

1 906, 764; H NMR (400 MHz, CDCl3), δ: 7.71 (d, J= 8.9 Hz, 1 H); 7.34 (ddd, J= 5.44,

2.44, 1.04 Hz, 1 H); 7.25 (d, J= 1.2 Hz, 1 H); 4.32-4.14 (m, 2 H); 2.75 (s, 3 H); 1.71 (d,

13 J= 17.7 Hz, 3 H); 1.35 (t, J= 7.0 Hz, 3 H); C NMR (100 MHz, CDCl3), δ:161.6; 156.4;

155.8 (d, J= 7.9 Hz); 154.5; 127.8; 118.2 (d, J= 4.5 Hz); 115.2; 113.3; 109.3 (d, J= 5.2

Hz); 101.4; 63.1 (d, J= 6.6 Hz); 18.2; 16.3 (d, J= 6.2 Hz); 11.8 (d, J= 144.4 Hz); 31P

164

+ NMR (162 MHz, CDCl3), δ: 28.4; HRMS (ESI) calcd for [C14H14NO5P + Na] 330.0502, found 330.0501.

3-cyano-4-methyl-2-oxo-2H-chromen-7-yl cyclohexyl methylphosphonate

(CMP): To a stirred solution of methylphosphonic dichloride (1 g, 7.52 mmol) in dichloromethane (40 mL) at 0 °C, a solution of 3-cyano-7-hydroxy-4-methylcoumarin

(1.51 g, 7.52 mmol) and triethylamine (1.05 mL, 7.52 mmol) in dichloromethane (60 mL) was added dropwise over 20 minutes. The resulting solution was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was then cooled to 0 °C and a mixture of (0.78 mL, 7.52 mmol) and triethylamine (1.05 mL, 7.52 mmol) was added dropwise over 20 minutes The resulting solution was allowed to warm to room temperature and stirred for 16 hours. Ether was added to the reaction and the resulting precipitate was filtered through celite and washed with ether. The combined filtrate and washings were run through a plug of silica and concentrated. The residue was purified by recrystallization from ethyl acetate:hexanes (0.52 g, 19% yield). FTIR (film, cm-1) 3086, 2939, 2860, 2663, 2472, 2230, 1746, 1620, 1562, 1504, 1453, 1427, 1384,

165

1361, 1314, 1237, 1158, 1085, 1014, 973, 919, 871, 766, 730; 1H NMR (400 MHz,

CDCl3), δ: 7.72 (d, J= 8.9 Hz, 1 H); 7.35 (m, 1 H); 7.27 (m, 1 H); 4.56(m, 1 H); 2.76 (s, 3

13 H); 2.02-1.21 (m, 10 H); 1.71 (d, J= 17.7 Hz, 3 H); C NMR (100 MHz, CDCl3),

δ:161.8; 156.5; 155.9 (d, J= 7.7 Hz); 154.4; 127.5; 118.3 (d, J= 4.6 Hz); 114.2 (d, J=

165.4 Hz); 109.3 (d, J= 5.1 Hz); 101.2; 77.2; 33.6; (d, J= 4.2 Hz); 33.5 (d, J= 3.8 Hz);

31 24.9; 23.5; 18.3; 12.5 (d, J=144.7 Hz); P NMR (162 MHz, CDCl3), δ: 27.3; HRMS

+ (ESI) calcd for [C18H20NO5P + Na] 384.0971, found 384.0971.

4-(trifluoromethyl)-7-((triisopropylsilyl)oxy)-2H-chromen-2-one: A stirred solution of 7-hydroxy-4-trifluoromethyl coumarin (50 mg, 0.22 mmol) in THF (10mL) was cooled to 0 °C and sodium hydride (6.5 mg, 0.26 mmol) was added. The resulting solution was stirred for 5 minutes then warmed to room temperature. TIPS-Cl (55 μL,

0.26 mmol) was added and the resulting mixture was stirred at room temperature for 12 hours. The solvent was removed and the residue was dissolved in DCM, washed with water, brine, dried over sodium sulfate, and concentrated. The crude product was purified by flash column choromatography on silica gel with 95:5 hexanes:ethyl acetate (33 mg,

65% yield). Spectroscopic data corresponds to that reported in the literature. 1H NMR

(400 M0Hz, CDCl3), δ: 7.67-7.64 (m, 1 H); 7.00-6.95 (m, 2 H); 6.70 (m, 1 H); 1.41-1.32

(m, 3 H); 1.15-1.13 (s, 18 H).

166

4-methyl-2-oxo-7-((triisopropylsilyl)oxy)-2H-chromene-2-carbonitrile: The above procedure was used with 7-hydroxy-4-methyl-2-oxo-2H-chromene-3-carbonitrile

(43 mg, 0.22 mmol). The crude product was purified by flash column chromatography

1 using silica gel and 1:1 hexanes:ethyl acetate. H NMR (400 MHz, CDCl3), δ: 7.61-7.58

(m, 1 H); 6.93-9.90 (m, 1 H); 6.80-6.82 (m, 1 H); 2.72 (s, 3 H) 1.37-1.25 (m, 3 H); 1.12-

+ 1.10 (s, 18 H). HRMS (ESI) calcd for [C20H27NO3Si + Na] 380.1652, found 380.1642.

2-(ethylthio)ethanethiol: A solution of ethyl 2-hydroxyethyl sulfide (2.12 mL,

20 mmol), thiourea (1.52 g, 20 mmol) in 48% hydrobromic acid was refluxed for 12 hours. After cooling to room temperature, a solution of (1.6 g, 40 mmol) in water (8 mL) was added slowly. The resulting solution was refluxed for 12 h, cooled to room temperature, and quenched with concentrated sulfuric acid. The reaction was diluted with dichloromethane (20 mL), the layers were separated, and the aqueous layer was extracted with dichloromethane (3 X 20 mL). The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The pungent oil was used without further purification (2.25 g, 92% yield).

167

1 H NMR (400 MHz, CDCl3), δ: 2.80-2.69 (m, 4 H); 2.56 (q, J=7.4 Hz, 2 H); 1.74 (m, 1

H); 1.26 (t, J=7.4 Hz, 3 H).

Disulfoton oxon: A solution of 2-(ethylthio)ethanethiol (1.42 g. 11.2 mmol) in ethyl ether (50 mL) was cooled to -78 °C and a solution n-butyllithium in hexane was added dropwise (12 mmol). Into the resulting suspension was added a solution of dimethyl chlorophosphate in ethyl ether (50 mL), which was cooled to -78 °C prior to addition. The mixture was then stirred at room temperature for 3 hours. The reaction was filtered through a plug of celite and washed with ether. The combined filtrate and washings were concentrated and the residue was purified by flash column chromatography on silica gel using 25-30 % ethyl acetate: hexanes as the eluent (1.87 g,

65% yield). FTIR (film, cm-1): 2980, 2360, 1443, 1391, 1250, 1162, 1014, 970, 748, 605,

1 570; H NMR (400 MHz, CDCl3), δ: 2.80-2.69 (m, 4 H); 2.56 (q, J=7.4 Hz, 2 H); 1.74

13 (m, 1 H); 1.26 (t, J=7.4 Hz, 3 H); C NMR (100 MHz, CDCl3), δ: 63.7 (d, J= 6.1 Hz);

32.5 (d, J=4.2 Hz); 30.7 (d, J=3.8 Hz); 25.9; 16.0 (d, J=7.2 Hz), 14.8; 31P NMR (162

+ MHz, CDCl3), δ: 27.3; HRMS (ESI) calcd for [C8H19O3PS2 + Na] 281.0405, found

281.0411.

168

Disulfoton: A solution of O,O’-diethyldithiophosphate (3.5 mL, 20 mmol), 2- chloroethyl ethyl sulfide( 2.35 mL, 20.2 mmol), and pyridine (1.6 mL, 20 mmol) in benzene (100 mL) was refluxed for 14 hours. After cooling to room temperature, the reaction mixture was filtered through a plug of celite and the filtrate was concentrated.

The residue was diluted with dichloromethane, washed with water, dried over sodium sulfate and concentrated. The product was purified through a short plug of silica using

30% ethyl acetate: hexanes as the eluent. Any residual starting material was removed under vacuum overnight (3.18 g, 70% yield). FTIR (film, cm-1):2978, 2928, 2869, 1473,

1442, 1388, 1260, 1204, 1160, 1097, 1013, 958, 825, 769, 658, 536; 1H NMR (400 MHz,

CDCl3), δ: 4.26-4.09 (m, 4 H); 3.11-2.78 (m, 4 H); 2.60 (q, J= 7.4 Hz, 2 H); 1.37-1.35

13 (m, 6H); 1.28 (t, J=7.4 Hz, 3H); C NMR (100 MHz, CDCl3), δ: 64.0 (d, J= 6.3 Hz);

33.5 (d, J=3.6 Hz); 32.1 (d, J=4.0 Hz); 25.8; 15.9 (d, J=8.2 Hz), 14.9; 31P NMR (162

+ MHz, CDCl3), δ: 94.8; HRMS (ESI) calcd for [C8H19O2PS3 + Na] 297.0177, found

297.0181.

169

Disulfoton oxon sulfoxide: A stirred solution of disulfoton oxon (0.4882 g. 1.9 mmol) in acetic acid (1 mL) was cooled to 0 °C and 35% hydrogen peroxide solution in water (170 µL, 2.01 mmol) was added dropwise. The resulting solution was allowed to warm to room temperature and stirred for 12 h. The solution was cooled to 0 °C and a saturated sodium bicarbonate solution was added dropwise until the pH was neutral. The solution was diluted with dicholormethane (20 mL) and the layers separated. The aqueous layer was extracted with dichloromethane (3 x 20 mL) and the combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by flash column chromatography on silica gel using 30 % ethyl acetate: hexanes and 10% methanol: dichloromethane as the eluent (0.3336 g, 64% yield). FTIR (film, cm-1): 2982, 2935, 1644, 1444, 1391, 1245, 1161, 1097, 1014, 973,

1 791, 755, 602, 568, 520; H NMR (400 MHz, CDCl3), δ: 4.26-4.11 (m, 4 H); 3.28-3.21

(m, 2 H); 3.19-3.09 (m, 1 H), 3.03-2.98 (m, 1 H); 2.80-2.74 (m, 2 H); 1.39-1.34 (m, 9 H);

13 C NMR (100 MHz, CDCl3), δ: 64.0 (d, J= 6.3 Hz); 52.2; 45.8; 23.7 (d, J= 4.0 Hz); 16.0

31 (d, J= 7.0 Hz); 6.8; P NMR (162 MHz, CDCl3), δ: 26.0; HRMS (ESI) calcd for

+ [C8H19O4PS2 + Na] 297.0355, found 297.0342.

Disulfoton oxon sulfone: A stirred solution of disulfoton oxon sulfoxide (1 g. 3.8 mmol) in acetic acid (2 mL) was cooled to 0 °C and 35% hydrogen peroxide solution in water (0.36 mL, 4.05 mmol) was added dropwise. The resulting solution was stirred at 170 room temperature for 20 h. The solution was diluted with dichloromethane (50 mL), cooled to 0 °C, and a saturated sodium bicarbonate solution was added dropwise until the pH was neutral. The layers were separated and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by flash column chromatography on silica gel using 30 % ethyl acetate: hexanes and 10% methanol: dichloromethane as the eluent (0.69 g, 63% yield). FTIR (film, cm-1): 2983,

2938, 1636, 1477, 1444, 1392, 1316, 1249, 1135, 1118, 1014, 975, 793, 755, 603, 568;

1 H NMR (400 MHz, CDCl3), δ: 4.17-4.05 (m, 4 H); 3.38-3.32 (m, 2 H); 3.17-3.07 (m, 2

13 H); 3.00 (q, J= 7.5 Hz, 2 H); 1.36-1.27 (m, 9 H); C NMR (100 MHz, CDCl3), δ: 64.0

(d, J= 6.5 Hz); 52.5 (d, J= 2.2 Hz); 47.6; 22.8 (d, J= 4.1 Hz); 16.0 (d, J= 6.9 Hz); 6.4; 31P

+ NMR (162 MHz, CDCl3), δ: 25.4; HRMS (ESI) calcd for [C8H19O5PS2 + Na] 313.0304, found 313.0308.

Ethyl phenylphosphinate: A solution of ethanol (11 mL, 270 mmol) and pyridine (13.1 mL, 162.5 mmol) in toluene (20 mL) was added dropwise to a stirred solution of phenyldichlorophosphine in toluene (80 mL) over 30 minutes. The resulting suspension was stirred for 1.5 h then allowed to sit without stirring for 3 days. The suspension was diluted with saturated sodium bicarbonate solution and the layers were

171 separated. The aqueous layer was extracted with dichloromethane (3 x 40 mL) and the combined organic layers were dried over sulfate, filtered, and concentrated to give an oil which was used without further purification (20.3 g, 95% yield). 1H NMR

(400 MHz, CDCl3), δ: 7.80-7.68 (m, 2 H); 7.62-7.58 (m, 1 H); 7.59 (d, JPH= 562 Hz, 1 H)

7.54-7.49 (m, 2 H); 4.24-4.11 (m, 2 H); 1.39 (t, J=7.08, 3 H) 31P NMR (162 MHz,

CDCl3), δ: 24.5 (d, J=562 Hz).

O,S-diethyl phenylphosphontioate (DEPP): Elemental (4.12 g, 128.5 mmol) was slowly added over 20 minutes to a stirred solution of ethyl phenylphosphinate

(20.3 g, 119 mmol) and dicyclohexylamine (25.5 mL, 128.5 mmol) in ether (200 mL).

The yellow reaction mixture was stirred at room temperature for 4 hours. The resulting solid was filtered, washed with ether, and dried to yield the dicyclohexylammonium salt of the O-ethyl phenylphosphonothioate (1) which was used without further purification.

The salt 1 (10g, 26 mmol) was added slowly to a stirred solution of ethyl iodide (4.8 mL,

60 mmol) in toluene (200 mL). The reaction mixture was stirred at room temperature for

3 days, filtered, washed with hexanes, and filtered again. The filtrate was concentrated and the oil was purified by flash column chromatography on silica gel using hexanes→20

% ethyl acetate:hexanes to yield the pure O,S-diethyl phenylphosphontioate (4.2 g, 62% yield over two steps). FTIR (film, cm-1): 2982, 1645, 1433, 1389, 1262, 1225, 1120,1023,

1 949, 904, 740, 696, 561; H NMR (400 MHz, CDCl3), δ: 7.90-7.85 (m, 2 H); 7.55-7.52 172

(m, 1 H); 7.50-7.52 (m,2 H); 4.31-4.21 (m,2 H); 2.80-2.71 (m, 2 H); 1.39 (t, J= 7.08 Hz,

13 2 H); 1.25 (t, J=7.4 Hz, 3 H); C NMR (100 MHz, CDCl3), δ:132.9 (d, J=149 Hz); 132.4

(d, J=3.1 Hz); 131.1 (d, J=10.9 Hz); 128.5 (d, J=14.6 Hz); 62.1 (d, J=11.6 Hz); 24.8 (d,

31 J=2.76 Hz); 16.3 (d, J=6.9 Hz); 16.1 (d, J=5.43 Hz); P NMR (162 MHz, CDCl3), δ:

+ 44.5; HRMS (ESI) calcd for [C10H15O2PS + Na] 253.0423, found 253.0427.

Methyl pent-4-enoate: A solution of 4-pentenoic acid (0.975 g, 9.7 mmol) and a catalytic amount of concentrated sulfuric acid (1 mL) in methanol (50 mL) was refluxed with stirring and monitored by TLC until all the acid was consumed (20 h). The solution was allowed to cool to room temperature and quenched with a saturated sodium bicarbonate solution. The reaction was diluted with ether (50 mL), the layers were separated, and the aqueous layer was extracted with ether (5 x 30 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated to give a light yellow liquid which was used without further purification (0.8 g, 80% yield). 1H NMR (400

MHz, CDCl3), δ: 5.87-5.77 (m, 1 H); 5.08-5.03 (m, 1 H); 5.02-4.99 (m, 1 H); 3.68 (s, 3

H), 2.44-2.34 (m, 4 H).

173

Methyl 4-oxobutanoate: Ozone was bubbled into a stirred solution of methyl pent-4-enoate(0.8 g, 7.8 mmol) in 1:1 DCM:methanol (25 mL) at -78 °C. Once the reaction was complete (the solution turned blue), dimethyl sulfide (0.72 g, 11.7 mmol) was added and the resulting solution was allowed to slowly warm to room temperature and stirred overnight. The reaction was washed with a 50 % brine solution (3 x 25 mL), and the aquoues phase was extracted with DCM ( 3 x 50 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated to give an oil which was used

1 without further purification (0.8 g, 99% yield). H NMR (400 MHz, CDCl3), δ: 9.82 (t,

J= 0.64, 1 H); 3.69 (s, 3 H); 2.82-2.78 (m, 2 H); 2.66-2.62 (m, 2 H).

5-(thiobutyl) butyrolactone (TBBL): Butanethiol (0.78 g, 8.6 mmol) was added to a stirred solution of methyl 4-oxobutanoate ( 0.9 g, 8.6 mmol) and trifluoroacetic acid

(0.98 g, 8.6 mmol) in THF (20 mL) and the resulting solution was refluxed for 24 h. The solution was cooled to room temperature, quenched with saturated sodium bicarbonate solution and diluted with ether (20 mL). The layers were separated and the aqueous layer was extracted with ether (3 x 20 mL). The combined organic phases were dried over

MgSO4, filtered, and concentrated. The product was purified by flash column chromatography on silica gel using 5-20% ethyl acetate/ hexanes to give a clear oil (350 mg, 24% yield). FTIR (film, cm-1): 2956, 2930, 1781, 1644, 1458, 1419, 1330, 1282, 174

1 1173, 1035, 973, 925, 744; H NMR (400 MHz, CDCl3), δ: 5.69-5.66 (m, 1 H); 2.79-

2.46 (m, 4 H); 2.14-2.02 (m, 2 H); 1.67-1.55 (m, 2 H); 1.45-1.46 (m, 2 H); 0.93-0.89 (t,

13 J=7.28 Hz, 3 H); C NMR (100 MHz, CDCl3), δ: 176.2, 84.7, 31.6, 31.4, 28.6, 28.3,

+ 21.9, 13.6; HRMS (ESI) calcd for [C8H14O2S + Na] 197.0607, found 197.0605.

4-(5,5-difluoro-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10- yl): A solution of 4-formylbenzoic acid (0.7000 g, 4.6 mmol), pyrrole (0.72 mL, 10.5 mmol), and TFA (100 μL) in DCM (350 mL) and dioxane (10 mL) was stirred for 20 hours in the dark under an argon atmosphere. A solution of DDQ (1.0578, 4.6 mmol) in DCM (70 mL) was added and the resulting dark red-purple solution was stirred for one hour. Triethylamine (9.3 mL) and BF3OEt2 (9.3 mL) were added and the resulting solution stirred for a further 2 hours. The reaction mixture was filtered and the remaining solid was washed with DCM (100 mL). The combined organic layers were concentrated and the crude product was filtered through a plug of silica with 10% MeOH/DCM. The solvent was removed and the crude oil was subjected to column choromatography three times (10%MeOH/DCM) to give the product as a deep purple solid. 1H NMR (400 MHz,

CDCl3), δ: 8.29-8.27 (d, J = 8.40 Hz, 2 H); 7.98 (s, 2 H); 7.71-7.68 (d, J = 8.40 Hz, 2 H); 175

13 6.91-6.89 (m, 2 H); 6.58-6.57 (m, 2 H); C (100 MHz, CDCl3), δ: 169.94, 144.98,

11 138.83, 134.72, 131.37, 130.51, 130.18, 119.02, 29.69; B NMR (128 Hz, CDCl3), δ:

19 0.26 (t, J = 28.60 Hz); F NMR (376 Hz, CDCl3), δ: 145.06 (q, J= 28.62 Hz); HRMS

- - (ESI) (M-H) calcd for [C16H10BF2N2O2] 311.0809, found 311.0803.

176

Appendix A: Selected NMR Spectra

177

2.3

178

2.3

179

2.4

180

2.4a

181

2.4a

182

2.1

183

2.1

184

2.9

185

2.9

186

2.12

187

2.12

188

2.8

189

2.8

190

2.11

191

2.11

192

2.6

193

2.7

194

2.5

195

2.10

196

2.13

197

2.13

198

2.11a

199

2.11a

200

2.13a

201

2.13a

202

ppm -100

-50 -0.09

0

16.53

20.28

24.26

27.50

31.30 32.57 50 ppm

100

121.11 122.37

160

156.34

150

159.88 162.41 200 P31CPD CDCl3 P31CPD

203

ppm -100

-50

2.82

5.27

5.43 0

5.55

5.65

5.85

20.29 52.07

50

73.28

73.85

74.42 74.98 ppm 100

73

127.19

127.39

147.88 154.18 150 74 75 200 P31CPD CDCl3 P31CPD

204

ppm ppm -200 -180 -62.2 -160 -62.0 -140 -120

-61.8 -97.33

-100 -94.61 -61.6

-80

-62.16 -61.91 -60 -40 -20 0 F19 CDCl3

205

ppm -0.000

0.5

1.061

1.079 6.00

1.0 1.097 1.5

2.0

3.094 3.112

2.5 3.123

3.130

3.140

3.0 4.00 3.148

3.155 3.165

3.5 3.172 3.190 4.0 4.5 5.0 5.5 6.0 6.5

7.0 7.260 7.5 8.0 8.5 9.0

PROTON_OSU CDCl3 PROTON_OSU

206

ppm

-0.07

0

14.39

10

14.42

14.82

20

30 38.57

38.59

38.77

38.79

40

43.65

43.84

50

60

76.67 70

76.99

77.19

77.30

80

90

ppm

100

124 110

124.50

124.76

120

127.74

128.01 126

130.99

131.25 130

134.23

134.50

140

128

150

130

160

170

132

180

134

190

200

210 C13CPD CDCl3 C13CPD

207

1.041

1.059 ppm

1.078

1.267

1.800 1.816

0.5 1.833

1.849

1.938 16.13

1.0

1.944 9.91

1.951 2.197

1.5

2.204

2.08

2.215

0.95 2.222

2.0

2.05 2.233

2.240

2.647

2.5 2.04

2.666

2.670 2.10

2.687 10.82

3.0

2.908

2.929

2.947

3.5

3.057

3.072

3.090

4.0 2.05

3.107

3.125

3.141

4.5

3.147

3.155 4.166 5.0 5.5

6.0

6.650

6.5 1.02 6.654

1.00 6.818

7.0 7.259 7.5 8.0 8.5 PROTON_OSU CDCl3 PROTON_OSU 9.0

208

ppm -100

-50

-31.86

-31.47

-31.08 -30.69 0 ppm 50 -32.0 -31.5 100 -31.0 150 -30.5 200 P31CPD CDCl3 P31CPD

209

ppm

0.5 12.06

1.0 10.02 1.5

2.0 10.38

2.5

3.11

3.83 8.45

3.0 16.20

3.5

3.31 2.15

4.0 2.13 4.5 5.0 5.5

6.0 0.95

6.5 0.96

7.0 0.95 7.5 8.0 8.5 PROTON_OSU CDCl3 PROTON_OSU 9.0

210

ppm -80 -70 -60 -50

-40

-31.97

-31.58

-31.19 -30.80 -30 -20 ppm -10 0 -32.5 10 -32.0 20 30 -31.5 40 50 -31.0 60 -30.5 70 80 P31CPD CDCl3 P31CPD

90

211

1.037

1.055 ppm

1.072

1.084

1.102 1.120

0.5 1.147

13.61 1.165

3.88 1.182

1.0

4.62 1.261

29.01

1.432

0.88 2.590

1.5

2.608

2.621

2.59

2.625

2.0

2.648

2.667

2.688

2.5 17.43

2.702

2.712

22.71

2.738 3.0

2.757

4.99

2.776

19.19

2.908 3.5

2.24 2.929

2.51 2.947

2.38

2.979 4.0

2.998

3.016 2.40

3.034 4.5

3.052

3.068

3.085 5.0

3.103

3.121

3.303 5.5

3.321

3.357

3.375 6.0

3.613

3.625

6.5 1.00 3.658

0.96 3.662 3.665

7.0 3.778

3.795

1.10 3.850

7.5 3.862

3.875 4.113

8.0 4.129

4.491 4.504

8.5 4.517

6.659 6.662

9.0

6.812 7.474 PROTON_OSU CDCl3 CDCl3 PROTON_OSU 212

ppm -100

-50

-31.91

-31.52

-31.13 -30.74 0 ppm 50 -32.5 -32.0 100 -31.5 150 -31.0 -30.5 200 P31CPD CDCl3 CDCl3 P31CPD

213

0.836

0.839 ppm

0.852

0.858

0.867 0.871

0.5

0.876

13.04 0.884 0.901

1.0

0.908 16.72

0.925 0.958

1.5

32.72 0.975

0.997 1.040

2.0

1.058 1.95

1.075

1.079

2.5 2.03

1.090

1.097 2.06

1.105

8.44

3.0

1.108

1.118 1.123

3.5

1.126

1.141

1.159

4.0

1.238

1.247

1.256 1.04

4.5

1.262

1.267

1.274

5.0

1.286

1.00

1.305

1.319

5.5

1.321

1.323

1.337

6.0

1.428

1.487

1.491

6.5 1.01

1.497

1.00

1.499

1.506

7.0

1.510

1.518

1.527

7.5

1.530

1.536

1.555

8.0

1.613

1.847

2.038

8.5

2.628

3.074

3.091

9.0 3.109

3.126 CDCl3 PROTON_OSU 214

ppm

-100

-50

-31.89

-31.50

-31.11

-30.71

0

ppm

50

-32.0

100

-31.5

-31.0

150

200 P31CPD CDCl3 CDCl3 P31CPD

215

0.879

0.895 ppm

1.035

1.052

1.070 1.253

0.5

1.269

11.84 1.540

12.49 1.574

1.0

1.592 92.23

1.610

11.52

2.276

1.5 5.73

2.295

2.314

2.643

2.0

2.662 6.32

2.668

2.674

2.5 2.05

2.684

2.18 2.881

8.60 2.903

3.0

2.922

3.011

3.029 3.5

3.047

3.064

6.32

3.081 4.0 1.97

3.099

3.118

3.136 4.5

3.154

4.104 4.138 5.0 5.5

6.0

6.639

6.5 1.00 6.644

1.00 6.817 7.0 7.5 8.0 8.5 9.0 PROTON_OSU CDCl3 PROTON_OSU

216

ppm -100

-50

-31.47 -31.07

0

ppm 50

-31.94 -32

100

-31.56

-31.16

-31 -30.77 150 200

P31CPD CDCl3 P31CPD 217

ppm -100 -50

0

5.93

15.38

18.84

20.03

25.86

28.29

29.55 35.71 ppm 50 156

100 155.90

150 157.32 200 P31CPD CDCl3

218

ppm ppm -200 -180 -63 -160 -62 -140 -120 -61 -100 -80 -60 -40 -20 0 F19 CDCl3 F19

219

ppm ppm -100 74 -50 75

0

18.58

42.11 45.22

50

73.46

74.02

74.56 75.10

100

115.64 148.65 150 200 P31CPD CDCl3 P31CPD

220

ppm 0 10

20 24.58

27.13 27.18 30

40

50.70 50.88 50 60

70

76.68

77.00 77.31 80 90 ppm 100

110

124.46

126

124.73

120

127.71

127.99

130.96

131.24 130 134.22

128 134.49 140 150 130 160 132 170 180 134 190 200 210 C13CPD CDCl3 CDCl3 C13CPD

221

ppm

1.469

1.482

1.496

1.509

0.5

1.522

1.540

1.543

1.0

1.552

1.562 8.13

1.569 3.99

1.5

1.581

1.583

2.975

2.0

2.986

2.991

3.001 2.5

3.011

3.017

8.00

3.026 3.0

3.070

3.083

3.101 3.5

3.115

3.132

3.148 4.0

3.162

3.176

3.194 4.5 3.207 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 PROTON_OSU CDCl3 PROTON_OSU

222

ppm 0.5

1.0

1.273 9.37

1.952

12.61

2.218

1.5

2.224 2.09

2.656 0.94

2.675

2.0

2.03 2.679

2.696

2.903

2.5 2.02

2.924

2.08

2.943

4.11

3.017

3.0 4.01

3.030

3.042

3.049 3.5

3.089

3.110

3.123 4.0 2.05 4.169 4.5 5.0 5.5

6.0

6.670

6.675

6.5 1.01

6.855

1.00

6.859

6.863 7.0 7.261 7.5 8.0 8.5 9.0 PROTON_OSU CDCl3 PROTON_OSU

223

ppm -100

-50 -35.88

-35.49

-35.12 -34.73 0 ppm 50 -36.0 100 -35.5 -35.0 150 -34.5 200 P31CPD CDCl3 P31CPD

224

1.137

1.155

ppm

1.172

1.176

1.195

1.363

1.443 0.5

1.453

1.467

3.66

1.491 1.0 9.29

1.504 1.72

1.938 19.58

2.201 1.5

2.429

2.53

2.550

2.584 2.0 1.21

2.603

0.91

2.616 14.25

2.5 2.660 2.58

2.679 4.22

2.771 10.81

3.0 2.821

2.01 2.829

2.834 21.45

3.5 2.837 1.58

3.91 2.850

2.954 2.52

4.0

3.031

0.84

3.068

2.16 3.073

4.5 3.083

3.548 3.553

5.0 3.558

3.563 3.565

5.5 3.568

3.574 3.587

6.0 3.592

3.597

3.600

1.01

6.5

3.607 1.02

3.625 3.708

7.0 3.772

3.785 1.03 3.788

7.5 3.798

3.811 4.041

8.0

4.057

4.073 4.287

8.5

4.438

6.607

6.611

9.0 6.773 PROTON_OSU CDCl3 PROTON_OSU

7.426 225

ppm -100 -80

-60

-36.00

-35.62 -40

-35.24 -34.86 -20 ppm

0

10.46 -36.0

11.05

11.65 12.24 20 -35.5 40 -35.0 60 P31CPD CDCl3 P31CPD 80

226

1.198 1.223

ppm

1.392

1.481

1.495

1.502

1.507 0.5

1.518 1.14

1.532

1.543 1.0 11.22

1.573

0.96

1.582

25.16

1.593 1.5

7.94

1.748

2.15 1.966

2.561 2.0

2.578

2.594

13.82 2.612 2.5

2.632

18.28 2.644

2.654 3.0

3.01

2.663

2.80

2.684

17.44

2.869 3.5

2.40

2.881

1.95

3.004 2.00

4.0 3.060

3.086

1.94 3.103

4.5 3.113

3.130 3.349

5.0 3.356

3.369 3.482

5.5 3.496

3.509 3.569

6.0 3.575

3.583

3.595 0.81

6.5 0.84 3.609

0.09 3.618

0.17 3.621

7.0 3.625

3.638

1.11 3.717

7.5 3.733

3.741 3.748

8.0 3.810

3.823 4.071

8.5

4.087

4.451

4.464

9.0 4.472 PROTON_OSU CDCl3 CDCl3 PROTON_OSU

7.433

227

ppm

-100

-36.47 -50

-36.10

-35.73

-35.36

9.31 0

9.89

10.46

11.03

ppm

50

-36.5

100

-36.0

150

-35.5

200 P31CPD DMSO DMSO P31CPD

228

0.677

0.856

ppm

0.861

0.873

0.877

0.908

0.924 0.5 2.84

0.977

6.30

0.985

0.998 1.0

60.34

1.026

1.058

1.081 1.5

3.07 1.106

2.03 1.117

1.130 2.0

1.91

1.140

1.153

1.97 1.168 2.5

1.266

2.03

1.331

12.35

1.343 3.0

1.350

1.422

1.465 3.5

1.472

1.486

4.0 1.499

1.512

1.524

1.00

4.5 1.537

1.551

1.563

5.0 1.810

1.01 1.817

1.849

5.5 1.992

2.248

2.257

6.0 2.616

2.635

2.655

6.5 0.97 2.891

0.98 2.911

2.930

7.0 3.017

3.029

3.041

7.5 3.048

3.086

3.104

8.0 3.118

3.134

3.150

8.5

3.164

5.355

6.669

9.0 6.673 CDCl3 PROTON_OSU

6.854

229

ppm

-100

-50 -35.92

-35.53

-35.15

-34.77

0

ppm

50

-36.0 73.62

74.18

74.72

75.28

100

-35.5

150

-35.0

200 P31CPD CDCl3 CDCl3 P31CPD

230

-0.000

1.195

ppm

1.228

1.244

1.259 1.277

0.5 1.293

1.335

1.352

1.0 5.89 1.382

6.00 1.403

10.35 1.407

1.5 5.83 1.415

1.430 1.453

2.0 1.457

1.488

1.11 1.495

2.5

1.86 1.504

1.507

1.527

3.0

1.553

1.609

1.639

3.5

1.661

1.673 1.33

1.682

4.0 1.97

1.693

1.712

1.722 4.5

1.743

1.752

1.775 5.0

1.785

1.882

1.889 5.5

2.044

2.485

2.494 6.0

2.504

2.513

2.522 6.5

2.532 2.540

7.0 2.550

2.559 2.717

7.5 2.746

2.861 3.960

8.0 3.976

3.992 4.008

8.5 4.095

4.113 4.131

9.0 4.149 CDCl3 PROTON_OSU

7.264

231

-0.000

1.063 ppm

1.079

1.174

1.222 1.239

0.5

1.259

1.277 1.282

1.0 6.36

1.293

5.82

1.299

1.334

1.5 4.21

1.351

2.83

1.394

1.453

2.0

1.489

1.508

1.01

1.596

2.5 1.98

1.620

1.646

1.673

3.0 1.97

1.692

1.701

1.722 3.5

1.730

1.754 1.00

1.764 4.0

1.865

2.044

2.500 4.5

2.510

2.527

2.537 5.0

2.547

2.555

2.565 5.5

2.574

2.621

2.628 6.0

2.652 2.658

6.5 2.682

2.689 3.137

7.0 3.168

3.580 3.588

7.5 3.676

3.692 3.963

8.0 3.979

3.995 4.095

8.5 4.113

4.131 4.149

9.0 5.301 PROTON_OSU CDCl3 CDCl3 PROTON_OSU

7.269

232

0.857

0.866 ppm

0.875

0.884

0.896 0.900

0.5 1.061

1.068

1.078 6.00

1.0 1.086 6.34

5.86 1.096

1.87 1.103

1.5 1.215

2.42

1.231 1.256

2.0

1.263

1.334

0.97

1.351

2.5

1.453

1.75 1.558

1.586

3.0 4.37

1.615

1.728

2.62

1.734 3.5

1.748

1.758 0.95

1.781 4.0

1.787

2.044

2.170 4.5

2.480

2.865

2.886 5.0

2.894

3.093

3.111 5.5

3.123

3.129

3.140 6.0

3.147

3.154

3.164 6.5

3.171 3.182

7.0 3.189

3.502 3.508

7.5 3.518

3.524 3.534

8.0 3.540

3.545 3.555

8.5 3.566

3.573 3.587

9.0 3.596

3.605 PROTON_OSU CDCl3

233

ppm -80 ppm -60 73 -40 -20 74 0 75 20 40

60

73.30

73.87

74.44 75.00 80 100 120 140 160 180 P31CPD CDCl3 CDCl3 P31CPD

234

ppm ppm -200 -180 -62.2 -160 -62.0 -140 -61.8 -120 -100 -61.6

-80

-67.96

-67.70

-62.09

-61.84

-59.37 -59.14 -60 -40 -20 0 F19 CDCl3 CDCl3 F19

235

ppm

1.048

1.200 1.205

0.5 1.217

1.221

6.21

1.256

1.0 6.04

1.263

9.68

1.343

6.03

1.360

1.5 3.08

1.799

4.26

1.815

0.93 1.832

2.0 1.945 1.91

2.213

1.07 2.220

2.5 3.90 2.661

2.666 1.87

2.683

3.0 4.01

2.915

1.05 3.103

3.120

1.45

3.5

3.138

1.02 4.144

4.160 4.0 2.00 4.175 4.5 5.0 5.5

6.0

6.656

6.5 0.96 6.661

0.95 6.824

7.0 7.261 7.5 8.0 8.5 9.0

PROTON_OSU CDCl3 PROTON_OSU 236

ppm

0

13.58

13.61 10

15.15

20.72

20.74

21.41 20

25.95

27.58

29.69

31.66 30

34.06

34.56

39.45

40

45.68

45.86

50

62.86 60

68.92

70

83.07 80

90

100 105.63

105.76

110

117.92

121.09

120

130

140

150

160

173.03 170

180

190

200

210 C13CPD CDCl3 C13CPD

237

ppm

-100

-50

-33.87

-33.48

-33.09

-32.69

0

ppm

-34.0

50

-33.5

100

-33.0

150

-32.5

200 P31CPD CDCl3 CDCl3 P31CPD

238

ppm ppm -200 -57.9 -180 -57.8 -160 -57.7 -140 -57.6 -120 -57.5 -100

-80

-59.27

-59.04

-57.77 -60 -57.60 -40 -20 0 F19CPD CDCl3 CDCl3 F19CPD

239

1.037 1.054

ppm

1.072

1.244

1.247

1.260

1.432 0.5

1.461

1.487

17.11

1.510 1.0

20.79

1.528

9.82 1.610

8.47 1.627 1.5

6.47 1.639

8.91 1.644

1.647 2.0

3.16 1.654

2.62 1.663

1.772 2.5 5.69

9.87 1.791

4.11 1.811

10.26 1.815 3.0

4.78 1.925

1.944

1.988 3.5

2.006

2.025

4.0 2.95 2.445

2.87 2.570 2.651

4.5 2.670

2.683 2.692

5.0 2.719

2.735 2.754

5.5 2.773

2.790 2.842

6.0 2.897

2.909

2.930

6.5 0.93

2.935

0.86

2.946 2.989

7.0

3.007

0.80

3.051 3.068

7.5 3.085

3.102 3.120

8.0 3.138

3.283 3.300

8.5

3.317

4.103

4.119

9.0

4.334 PROTON_OSU CDCl3 CDCl3 PROTON_OSU 7.319

240

ppm

-0.02

0

13.52 10

23.96

24.13

25.41

25.58 20

25.85

28.36

29.67

30 30.67

30.77

31.64

39.67

40 40.98

42.66

51.09

50

60

76.74 70

77.06

77.26

77.38

80

90

100

110

120

130

140

150

160

168.35

169.11

170

180

190

200

210 C13CPD CDCl3 CDCl3 C13CPD

241

ppm -200 ppm -32.0 -150 -31.5 -100 -31.0

-50

-31.92

-31.53

-31.14 -30.75

0

19.35

20.34 23.15 50 100 P31CPD CDCl3 P31CPD

242

ppm -200 -180 ppm -160 -140 -58.0 -120 -100

-80

-67.98

-67.72

-58.10 -57.93 -60 -40 -20 0 F19 CDCl3 CDCl3 F19

243

ppm

-200

ppm

-180

-160

-58.0

-140

-120

-100

-80

-62.08

-61.84

-58.15

-60

-57.99

-40

-20 0

F19 CDCl3 CDCl3 F19

244

-0.01

ppm

11.85

13.50

14.11

18.71

19.31 0

21.03

22.56

22.65

22.81 10

23.83

24.28

28.02

28.22 20

29.70

31.59 31.67

30 31.86

34.47

34.55

34.67

40

35.79

36.19

36.58

36.97 50

38.10

39.52

39.65

42.32 60

50.02

56.14 56.70

70 73.82

76.69

77.01

77.21

80 77.32 90 100 110 120 130 140 150 160 170 180 190 200 210

C13CPD CDCl3 CDCl3 C13CPD

245

0.673 0.835

ppm

0.838

0.854

0.858

0.866

0.870

0.5 5.14

0.875

0.884

0.901 1.0

136.57

0.904

0.921

0.958 1.5

0.975 5.02

0.994

3.01

1.005 2.0

2.47 1.038

1.056

1.073

2.5 2.16

1.078

2.13 1.261

9.98

1.427 3.0

1.533

1.550

1.561 3.5

1.584

1.594

1.612 4.0

1.782

1.807

1.10

1.820 4.5

1.830

1.837

1.845

5.0

1.853

1.24

1.980

1.988

5.5

2.020

2.247

2.259

6.0

2.607

2.626

2.646

6.5 0.96

2.895

0.94

2.916

2.934

7.0

3.037

3.055

3.069

7.5

3.087

3.105

3.122

8.0

3.139

3.146

3.156

8.5

5.340

5.352

6.651

9.0 6.655 PROTON_OSU CDCl3 CDCl3 PROTON_OSU

6.815

246

ppm -200 -150 -100

-50

-31.88

-31.49

-31.10 -30.71 ppm 0 -32 50 -31 100 P31CPD CDCl3 CDCl3 P31CPD

247

ppm

-1.02

-0.00

0

9.97

12.50

12.58

12.61 10

13.09

19.69 20.38

20 21.07

24.57

30.65

31.16

30

33.09

33.55 38.46

40 44.69

44.86 49.16

50

62.55 64.71

60 68.54

69.48

69.54

69.56

70

69.64 69.71 80

90

104.55 100 104.67

110

116.86

120.17 121.09 120 130

140 145.75 150

160 165.71

167.93

167.96 172.11 170 180 190 200 210

C13CPD CDCl3 CDCl3 C13CPD

248

ppm

-74

-72

-70

-68

-66

-64

-62

-60

-58

-56.92

-56.76

-56 -54

F19 DMSO DMSO F19

249

ppm -120 -100 -80

-60

-34.36

-33.98 -40

-33.61 -33.24 ppm -20 0 -34.5 20 40 -34.0 60 -33.5 80 100 -33.0 120 P31CPD DMSO

140

250

0.991

1.009 ppm

1.026

1.044

1.061 1.066

0.5 1.079

1.094

6.42 1.112

1.0 11.56

1.129

6.65

1.133

1.215

1.5

1.231

1.248

2.583

2.0

2.598

2.621 3.32

2.648

2.5 3.02

2.654 6.19

2.807

4.64

2.828 3.0

2.894

20.93

2.909 15.85

2.924

3.5 2.18

3.048 2.16

3.065

3.11

3.340 4.0

3.361 2.39

3.378 2.09

3.396 4.5

3.413

3.433

3.444 5.0

3.450

3.462

3.483 5.5

3.494

3.505

3.510 6.0

3.514 3.519

6.5 3.524 1.02

3.533 1.05 3.696

7.0 3.711

3.726 3.774

7.5

3.788

1.00

3.801 4.012

8.0 4.028

4.044 4.349

8.5 4.445

4.458 6.726

9.0 6.730

7.792 251

0.679

0.854

ppm 0.858

0.871

0.875

0.907

0.923

0.5 3.05

1.009

6.26

1.247

16.86

1.256 1.0

1.313 5.12

1.328

11.37

1.340 1.5

1.347 3.04

1.362 2.07

1.379 2.0

2.05 1.435

1.446

1.460 2.5

ppm

1.467

1.477

1.485 3.0

0.8

1.489

1.496 0.99

1.501 3.5 1.0

1.517

1.525

1.2 1.531 4.0

1.534

1.542 1.4

1.550 4.5

1.560

1.6

1.576

5.0 1.584

1.00

1.588

1.8

1.806

5.5 1.810

1.821 2.0

1.830

6.0

1.833

2.2 1.843

1.857

6.5

1.865 2.4

1.955

1.986

7.0 1.996

2.005

2.027

7.5 2.231

2.264

2.271

8.0 2.277

2.285

2.290

8.5

3.523

3.525

5.345

9.0 5.353

5.358 91 awad.25 {C:\Bruker\TopSpin3.0} CDCl3 PROTON_OSU

252

ppm

1.0 1.317

0.85 1.339 1.359

1.5

2.0

2.638

2.655 2.5 1.57 2.671

3.0

3.371

1.63 3.385 3.4

3.5

4.208

4.224 4.0 1.00

4.240

1.87 4.428

4.444 4.5

0.66 5.125 5.0

5.5

6.0

7.301

7.303 6.5

7.320

7.322

7.338 7.0

2.34 7.341

2.06 7.389

2.03

7.407 7.5

2.01 7.426

7.584

7.603

8.0

7.762 7.780

8.5

9.0

253

1.180

ppm 1.198 1.240

0.5 1.258

1.276 1.697

1.0 1.706

2.79

1.736 1.746

1.5 1.966 3.00

1.982 2.890

2.0 2.906

2.923 2.940

2.5 2.955

2.971

1.93 2.991

3.0 3.008

3.395 3.5 1.86 3.408

3.5 3.423

4.017

4.035

2.18

4.053 4.0

1.89 4.071

4.080

4.100 4.5

4.120

4.139

4.156 5.0

4.303

4.309

0.78 4.318 5.5

4.327 5.637

6.0 7.186

6.5 7.213

7.231 7.250

7.0 2.29 7.301

2.28 7.320

2.13 7.338

7.5 2.18 7.516

7.535 7.674

8.0 7.693

8.5

9.0

9.5

254

ppm

1.412

1.433 1.25 1.454 1.5

2.0

2.796

2.812 2.5

2.817

2.00

2.828

2.833

5.84 2.849 3.0

3.115

3.649

3.664 3.5

1.95

3.680 3.695 4.0 4.5 3.7 5.0 5.5

6.0

6.621

6.5 0.84

6.760

1.95 6.782 7.0

7.5

7.890

7.898 5.88 7.922 8.0

255

ppm 0.5 1.0 1.5 2.0

2.5 2.00 3.15

3.0

2.10

3.5 3.55 4.0 4.5 5.0 5.5 6.0

6.5 2.48 7.0

7.5 7.95 8.0 8.5 9.0

256

-0.000

0.799 ppm

0.811

0.822

1.178

1.185 0.5

1.190

1.202 1.205

1.0

1.208

1.216 1.220

1.5 1.228

3.00

1.254

1.264

1.270 2.0

1.481 0.97

1.545 1.558

2.5

1.570

1.723

1.743

3.0 2.35 1.749

0.81 1.769

1.975

1.84

2.101 3.5

1.26 2.268

3.04 2.281

3.30

3.282

4.0

3.703

3.830 3.836

4.5 3.841

4.011

4.032

4.035 5.0

4.047

4.059

5.048

5.5

6.827

6.842 6.949

6.0 6.952

6.960

6.964

7.191 6.5

7.845

7.848 2.18

7.851

7.0

7.860

7.866 7.869

7.5 7.877

7.881 5.06

8.020

2.15

8.023 8.0 8.032

8.035

257

ppm -150 -100 -50

0

55.83 50 56.04 100

258

ppm 1.0 1.5

2.0

2.247 5.95 2.5 3.0

3.5 1.94 3.703 4.0 4.5 5.0 5.5

6.0

7.068 6.5

7.080

7.260

0.92

7.308 7.0

7.320 1.00

1.09 7.454

7.461 7.5

7.468

7.475

8.611 8.0

8.613

8.625

0.94

8.627 8.5

1.00 8.769

8.771

8.776 9.0 8.778 9.5

259

ppm 0.5

1.0

1.741

1.747

1.752 1.5

4.07

1.759

1.764

2.495 2.0

2.497

4.02 2.506

2.510 2.5

2.515 2.517 3.0

3.5

2.01 3.915 4.0 4.5 5.0 5.5 6.0

6.5

7.066

7.079

0.96

7.354

7.0

7.366

1.07

7.446

1.14

7.453

7.5

7.460

7.467

8.655 8.0

8.657

8.669

8.672

8.5 0.96

1.00 8.765

8.767

8.772 9.0 8.774 9.5

260

ppm 10 15 20 25

30

31.22 31.67 35 40 45 50 55

P31CPD CDCl3 CDCl3 P31CPD

261

ppm

1.456

3.00 1.500 1.5 2.0 2.5 3.0 3.5

4.0

4.945

4.5

4.966

4.975

4.996

3.98

5.039 5.0

5.061

5.068 5.090 5.5

6.0 7.317

6.5 7.320

7.324

7.330 7.331

7.0 7.333

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

7.336

9.62

7.342 7.344

7.5 7.351

7.358

7.362 7.366

8.0 7.368

262

ppm

1.0

1.422

3.00 1.466 1.5 2.0 2.5 3.0 3.5

4.0

4.840

4.862

4.869 4.5

4.891

4.04

4.913

4.935 5.0

4.942 4.964

5.5

3.94 5.956

6.0

6.755

6.775 6.803

6.5

6.807

5.81

6.822 6.827

7.0 6.838 6.841 7.5 8.0 8.5 9.0

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

263

ppm -150 -100 -50

0

31.09

31.54 31.98 50 100

P31CPD CDCl3 CDCl3 P31CPD

264

ppm

1.0

1.625

1.5 3.08

1.669

2.0

2.5

3.0

3.5

4.0

5.127

4.5

5.148

5.160

5.181

5.0 4.00

5.190

5.212

5.223 5.5

5.245

7.199

6.0 7.200

7.211

7.213

6.5 7.218

7.219

7.230

7.0 1.93 7.231

7.424 1.89

7.444

7.5 1.95 7.674

7.678

7.693

8.0

7.698

7.713

1.89

7.717

8.5

8.551

8.553

8.561

9.0

8.563

9.5 PROTON_OSU CDCl3 PROTON_OSU

265

ppm

-150

-100

-50

0

32.09

50 100

P31CPD CDCl3

266

ppm

1.0

1.5 1.749

3.00

1.794

2.0

2.5

3.0

3.5

4.0

5.141 4.5

5.162

5.173

5.194 5.0

2.02

5.231

5.255

5.263 5.5

5.287

6.0

6.5

7.251

5.10 7.254

7.274

7.0

7.277 2.39

7.443 2.58

7.448

7.5

7.460

7.465

4.97

8.134

8.0 4.17

8.154

8.158

8.5

9.0 9.5

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

267

ppm

-150

-100

-50

0

29.31

29.76

30.21

34.54

34.71

50 100

P31CPD CDCl3

268

ppm 0.5

1.0

1.625

1.5 3.00 1.670 2.0 2.5 3.0 3.5 4.0

4.5

5.206 5.0 1.89 5.232

5.5 7.120

7.124

7.144

7.147 6.0

7.341

7.345

7.362 6.5

7.366

7.406

7.412

7.0 1.88

7.421

0.95

7.430

1.94

7.436

7.5 1.88

7.659

1.95

7.666

1.88

7.682

7.716 8.0

7.731

7.736

7.747 8.5

7.754

7.923

7.928

9.0

7.941 7.945 PROTON_OSU CDCl3

269

ppm -150 -100 -50

0 29.24 50 100

P31CPD CDCl3 P31CPD

270

ppm 0.5

1.0

1.684 1.5 3.14 1.729 2.0 2.5

3.0

3.829 3.5

8.66 3.841 3.874

4.0

4.643

5.001

4.5

5.025

5.030

2.00

5.054

5.0

5.090

5.117 5.145 5.5

6.0

6.567

1.83

6.610 6.5 7.309

7.0

7.312

1.84

7.332 7.335

7.5

8.191 8.0 1.85 8.213 8.5 9.0 9.5 PROTON_OSU CDCl3 CDCl3 PROTON_OSU

271

ppm -200 -150 -100 -50

0 28.97 50 100 P31CPD CDCl3 CDCl3 P31CPD

272

ppm 0.5

1.0

1.589

1.5 3.00 1.618

2.0

2.349

3.28 2.352 2.5 3.0 3.5

4.0 4.923

4.939

4.942

4.958 4.5

4.960

4.979 1.92

4.998

5.0

5.873

5.876

5.884

5.887 5.5

5.893

2.51

6.172

6.174 6.0 1.05

6.626

6.640

6.968

6.5 0.90 6.970

6.971

0.88 6.974

1.09

7.089 7.0

7.091

7.093 0.97 7.095

7.5 7.103

7.105

7.107

7.109 8.0

7.442 7.457 8.5

273

ppm -200 -150 -100 -50

0 28.87 50 100

274

ppm

0.5

1.0

1.5 1.746

3.35

1.790

2.0

2.5

3.0

3.5

4.0

5.166

4.5

5.187

5.198

5.219

5.0 1.04

5.282

1.64

5.300

5.306

5.5

5.314

5.338

6.0

6.5

7.346

7.349

7.369 7.0

7.372

2.04

7.513

2.00

7.518 7.5

7.530

7.535

8.215 8.0

3.98

8.219

8.232

8.237 8.5 9.0

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

275

ppm

-200

-150

-100

-50

0

29.30

50 100

P31CPD CDCl3 CDCl3 P31CPD

276

ppm 0.5

1.0

1.723 1.5 3.00 1.768 2.0 2.5 3.0 3.5

4.0

5.133 4.5

5.155 5.163

5.0 5.185 1.99

5.220 5.245

5.5 5.276

6.0 7.301

6.5 7.305

7.310

7.319

7.0

7.325 1.92

7.328 1.89

7.450

7.5 1.88

7.471

7.601

7.621 8.0 1.89

8.186 8.208 8.5 9.0 9.5

PROTON_OSU CDCl3 PROTON_OSU

277

ppm

-200

-180

-160

-140

-120

-100

-80

-62.76

-60

-40

-20 0

F19CPD CDCl3 F19CPD

278

ppm -200 -150 -100 -50

0 29.22 50 100

P31CPD CDCl3

279

ppm

0.5

1.0

1.729 1.5

3.00

1.774

2.0

2.5

3.0

3.5

4.0

5.116 4.5

5.137

5.148

5.169 5.0

2.03

5.236

5.260

5.268 5.5

5.292

6.0

7.336 6.5

7.339

7.359

7.362 7.0

1.89 7.452

1.92 7.453

1.89 7.472 7.5

7.473

7.656

7.676 8.0 1.86

8.214

8.236

8.5

9.0 9.5

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

280

ppm

-200

-150

-100

-50

0

29.24

50 100

P31CPD CDCl3 CDCl3 P31CPD

281

ppm

1.0

1.643

1.5 3.02 1.688 2.0 2.5 3.0

3.5 3.795 2.74

3.38 3.810

4.0

4.626

2.36

5.027 4.5

5.051

5.055

2.00

5.080 5.0

5.109 5.137

5.5

6.827

6.849

6.883 6.0

6.889

6.900

6.904 6.5

2.04

7.245

3.95

7.253

7.257 7.0 4.71

7.262 3.64

7.267

7.276 7.5

7.279

7.284

2.88 7.306 8.0

8.136

8.152

8.159 8.5 8.174 9.0 9.5 PROTON_OSU CDCl3 PROTON_OSU

282

ppm

-200

-150

-100

-50

0

28.93

50 100

P31CPD CDCl3 P31CPD

283

ppm 1.0

1.5 1.742

3.00 1.787 2.0 2.5 3.0 3.5 4.0

4.5

5.174

5.182

5.205 5.0

1.92

5.207

5.232 5.237 5.5

6.0 7.121

7.143 7.165

6.5 7.471

7.473

7.476

1.80 7.484 7.0

7.492

7.493 3.59

7.496 7.5

7.499

7.506

8.252

8.0

8.259 1.79

8.260 8.265

8.5 8.266 8.277

PROTON_OSU Acetone PROTON_OSU 8.278 9.0

8.283

284

ppm

-200

-180

-160

-140

-115.06 -120

-100

-80

-60

-40

-20 0

F19CPD Acetone Acetone F19CPD

285

ppm ppm -200 -150 28.5 -100 29.0 -50

0

28.51

28.56

28.62

28.67

28.73

28.79

28.84

28.90 28.95 50 100

P31 Acetone

286

ppm 0.5

1.0

1.680 1.5 3.32 1.724 2.0 2.5 3.0 3.5

4.0

5.042 4.5

5.065

5.072

5.094

5.0 2.00

5.119

5.145 5.175 5.5 6.0

6.5

7.283

7.287

7.290 7.0

7.305 5.07

7.310

7.313 7.5

7.326

8.183 8.0 1.86 8.205 8.5 PROTON_OSU CDCl3 PROTON_OSU

9.0

287

ppm

-200

-150

-100

-50

0

29.07

50 100

P31CPD CDCl3 CDCl3 P31CPD

288

ppm

1.0

1.684 1.5 3.31

1.728

2.0

2.5

3.0

3.5

4.0

5.028 4.5

5.051

5.058

2.00

5.081

5.0

5.102

5.129

5.158

5.5

6.0

6.5

7.200

7.221

7.282

7.0 1.90

7.285

1.93

7.305

1.99

7.308

7.5 7.457

7.478

8.181 8.0 1.95

8.204

8.5

9.0 PROTON_OSU CDCl3 CDCl3 PROTON_OSU

289

ppm

-200

-150

-100

-50

0

29.12

50 100

P31CPD CDCl3 CDCl3 P31CPD

290

ppm

0.5

1.0

9.09 1.310

1.673 1.5 3.11

1.718

2.0

2.5

3.0

3.5

4.0

5.064 4.5

5.086

5.092

5.115

5.0 2.00

5.129

5.154

5.183 5.5

6.0

7.258

6.5 7.262

7.272

7.275

7.0 7.278

4.06

7.279 2.01

7.295

7.5 7.298

7.356

7.377

8.161 8.0 1.93

8.183

8.5 9.0

PROTON_OSU CDCl3 CDCl3 PROTON_OSU

291

ppm

-200

-150

-100

-50

0

28.83

50 100

P31CPD CDCl3 P31CPD

292

ppm 1.0

1.5 1.766

2.94 1.810 2.0 2.5 3.0 3.5

4.0 5.113

4.5 5.134

5.143

5.162

5.0 2.00 5.164

5.184 5.192

5.5 5.214

7.338 7.350

6.0

7.354

7.357 7.360

6.5

7.373

7.375 7.380

7.0

7.385

4.84

7.387

2.04 7.391

7.5

7.400

7.433 7.436

8.0

7.442 1.90

7.451 7.456

8.5

7.459

8.245 8.268 9.0 9.5

PROTON_OSU DMSO DMSO PROTON_OSU

293

ppm

-200

-150

-100

-50

0

29.54

50 100

P31CPD DMSO P31CPD

294

PROTON_OSU CDCl3 TBBL

9.0

8.5

8.0

7.5

7.0

6.5 6.0

1.00 5.5

5.0

4.5

4.0

3.5 3.0

5.39 2.5

1.06 2.0

2.13 1.5

2.18 1.0

3.21

0.5 ppm

295

CARBON_OSU CDCl3 TBBL

210

200

190 180

176.21

170

160

150

140

130

120

110

100 90

84.70 80 77.33 77.01

76.69

70

60

50 40

31.65 30 31.40 28.61 28.30

20 21.89

13.57 10

0 -0.02 ppm

296

PROTON_OSU CDCl3 disulfoton 9.0 8.5 8.0 7.5 4.245 4.238 7.0 4.227 4.220 4.213 6.5 4.202 4.195 4.192 6.0 4.185 4.178 4.174 5.5 4.167 4.160 4.156 5.0 4.149 4.139

4.5 4.132 4.124 4.114

4.00 4.0 4.107 3.113 3.099

3.5 3.094 3.087 3.073

3.0 3.068 3.93 3.053 3.049

1.80 2.5 3.041 3.028 2.917

2.0 2.898 2.877 2.858

1.5 2.822 6.28 2.809 2.76 2.801 1.0 2.797 2.782 2.633

0.5 2.615 2.596 2.578 1.387 1.385 ppm 1.369

297

CARBON_OSU CDCl3 disulfoton 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70

64.08

60 64.02 50 40

33.53 33.49 30 32.13 32.09 25.86 20 15.92 15.84

10 14.86 0 ppm

298

P31 CDCl3 disulfoton

95.19 95.13 95.09

100 95.03 94.97 94.92 94.86 94.80 94.75 94.69 94.63 94.57 50 0 -50 -100 -150 -200 ppm

299

PROTON_OSU CDCl3 disulfonton oxon 9.0 8.5 8.0 7.5 7.0 6.5 6.0

4.23

5.5 4.23 4.22 4.21 4.21 4.20 5.0 4.20 4.19 4.19 4.19

4.5 4.18 4.18 4.17 4.17 4.17 4.00 4.0 4.16 4.15 4.15 4.14 3.5 4.14 4.13 4.12 3.07

3.0 3.05 2.04 3.05 3.04 2.06 3.03 3.01 2.04 2.5 3.00 2.99 2.85 2.84

2.0 2.83 2.81 2.62 2.60 2.58 1.5 2.57 1.39 6.14 1.37 3.10 1.35 1.0 1.29 1.28 1.26 0.5 ppm

300

CARBON_OSU CDCl3 disulfonton oxon 210 200 190 180 170 160 150 140 130 120 110 100 90 80 77.33 77.01 76.69 70

63.71

60 63.65 50 40

32.58

30 32.53 30.76 30.72 25.92 20 16.10 16.03

10 14.78

0 -0.02 ppm

301

P31 CDCl3 disulfonton oxon

100 50 27.51 27.45 27.40 27.35 27.30 27.25 27.20 27.15 27.09

27.03

0

-50

-100

-150

-200 ppm

302

4.220 4.217 9.0 4.213 4.203 4.199 8.5 4.195 4.191 4.182 8.0 4.177 4.173 4.160 7.5 4.159 4.155 4.154 7.0 3.287 3.276 3.273 6.5 3.264 3.258 3.256 6.0 3.250 3.246 3.238 5.5 3.233 3.231 3.223 5.0 3.213 3.210 3.193 4.5 3.163 3.147 4.00 3.132 4.0 3.114 3.109 3.091 3.5 3.039 2.14 3.026

1.28 3.0 3.020 1.12 3.008 3.006 2.09

2.5 2.995 2.988 2.808

2.0 2.805 2.789 2.786

1.5 2.770 2.767 9.09 2.751

1.0 2.749 1.396 1.394

0.5 1.392 1.378 1.377 1.374 1.359 ppm 1.357 1.341

303

210 200 190 180 170 160 150 140 130 120 110 100 90 80 77.35 77.03 76.71 70

64.10

60 64.04

50 52.22

45.87 40 30

23.78

20 23.74 16.10 16.03 10

6.81 0 ppm

304

100 50

26.21 26.16 26.10 26.05 26.00 25.94 25.89 25.84 25.62 0 -50 -100 -150 -200 ppm

305

PROTON_OSU CDCl3 disulfoton oxon sulfone 9.0 8.5 8.0

7.5 4.242 4.235 4.225 7.0 4.221 4.217 4.214 6.5 4.203 4.199 4.196 6.0 4.192 4.181 4.179 5.5 4.175 4.172 4.161 5.0 4.157 4.154 4.149 4.5 4.139 4.132 3.446

4.00 4.0 3.440 3.426 3.421 3.5 3.413 1.92 3.401 2.11 3.246 2.03 3.0 3.245 3.234 3.226 2.5 3.224 3.207 3.200 2.0 3.188 3.181 3.179 1.5 3.175 2.91 5.97 3.161 3.160 1.0 3.090 3.071 3.053 0.5 3.034 1.451 1.433 1.414

ppm 1.402 1.400

306

CARBON_OSU CDCl3 disulfoton oxon sulfone

210

200

190

180

170

160

150

140

130

120

110

100

90 80 77.33 77.02

76.70 70

64.29

60 64.23

52.85 50 52.83

47.85

40 30

23.00 20 22.96 16.10

16.03 10

6.57

0 -0.01 ppm

307

P31 CDCl3 disulfoton oxon sulfone 100 50

25.74 25.68 25.63 25.57 25.52 25.46 25.40 0 -50 -100 -150 -200 ppm

308

ppm

-3

1.238

1.257 -2

1.276

1.382

1.399 -1

1.417

2.713

2.732 0

2.745

2.750

1 3.12

2.751

2.89

2.764

2.768

2 2.770

2.782 1.91

2.801

3

4.238

4.244

4.255

4 1.96

4.260

4.261

4.265 5

4.277

4.283

7.473 6

7.477

7.484

7.488 7 2.01

7.492 1.00

7.494 1.93

7.503 8

7.505

7.532

9 7.536

7.550

7.555

7.850 10

7.854

7.871

11 7.884

7.888

7.905

12

13

14

15 16

PROTON_OSU CDCl3 DEPP CDCl3 PROTON_OSU

309

ppm

-0.02

0

16.16 10

16.22

16.32

16.38

20 24.84

24.86

30

40

50

62.06

60

62.13

70

76.69

77.01

77.33

80

90

100

110

128.43

120 128.57

131.11

131.22

132.17

130

132.40

132.43

133.66

140

150

160

170

180

190

200 210

CARBON_OSU CDCl3 DEPP CDCl3 CARBON_OSU

310

ppm

-200

-150

-100

-50

0

44.42

44.45

44.47

44.55

50 100

P31 CDCl3 DEPP CDCl3 P31

311

References

(1) Drugs, C. on the E. of A.; Health, B. on G.; Medicine, I. of. Saving Lives, Buying

Time:: Economics of Malaria Drugs in an Age of Resistance; National Academies

Press, 2004.

(2) Melville, C. H. In The Prevention of Malaria; London, 1910; pp. 577–579.

(3) Hersh, S. M. Chemical and Biological Warfare; The Bobbs-Merrill Company:

Indianapolis, IN, 1968.

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