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Fluorinated probes of enzyme mechanisms

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Fluorinated probes of enzyme mechanisms

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Content i

i

FLUORINATED PROBES OF ENZYME MECHANISMS
by

Candy S Hwang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2016

Copyright 2016 Candy Hwang

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DEDICATION

To Quinoline,

who has been with me from the start.

N
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ACKNOWLEDGEMENTS
I entered graduate school intrigued by the realization that small chemical changes
could contribute to dramatic functional differences in a biological context. I had to know
more and my curiosity was insatiable! I became aware that small structural changes in
biomolecules could contribute to crucially important outcomes for life processes. Thus, I
came to the understanding that to study such complex interactions would require
interdisciplinary training that integrated both biology and chemistry, and decided to
pursue graduate training in chemical biology and organic synthesis. I almost cannot
believe my Ph.D. has reached its end! I most certainly could not have accomplished this
undertaking without the help of many wonderful and patient people.
First off, I would like to thank my mentor, Prof. Charles McKenna. Prof.
McKenna taught me how to write and how to freshly analyze each piece of work with a
critical eye. It has been invaluable for my colleagues and me when writing exams, articles,
grants and fellowship applications. Over the years, Prof. McKenna has trained me in
chemistry, in running a lab, and has imparted boundless motivation for pursuing research.
As promised, my graduate work was made more productive and infinitely more enjoyable
by him allowing me to bring Quinn to the office. I am on the way to start the career I’ve
always wanted and could not have done that without his help.
I would like to thank Dr. Boris Kashemirov for his daily guidance and patience. I
learned so much from our extensive discussions on chemistry and laboratory techniques.
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The McKenna group, past and present, have been instrumental in my growth as a scientist
and a person. How would I have known how to navigate the complicated waters of
graduate school without their help? Dear members in the OCW 201 office, thank you for
all the moral support, entertaining stories and endless laughter and optimism. Dr. Dana
Mustafa, Dr. Melissa Williams and Dr. Jorge Osuna, I miss you all the time. It feels like
you left only yesterday. Kim Nguyen, I’m glad you have been with me through this entire
ride and I wish you the best of luck on your journey after USC. Dr. Elena Ferri, thank
you for helping me run exhaustive nitrogenase assays and being an overall supportive
friend and colleague.
Dr. Yue Wu and Dr. Feng Ni, thank you both for exposing me to graduate level
research and mentoring me in bioorganic chemistry. Dr. Brian Chamberlain and Dr. Ivan
Krylov showed me how to be confident and speculative. Dr. Anastasia Kadina, thank you
for always providing assistance on organic chemistry problems. Carolina Amador, thank
you for being so supportive of me and inspiring me to work harder and be better. I would
like to thank my undergraduate researcher, Kimberly Hui, for assisting me for two years
and in helping me to become a better educator for undergraduate research. I hope I have
been an adequate mentor to both of you, but even without me, I know you both will do
amazing things. Dr. Corinne Minard, Alon Chapovetsky, Beatriz Renner and Marlon
Duro, I will miss you guys and wish you the best of luck.
I would like to thank the faculty and staff of the chemistry department. Thank you
Prof. G. K. Surya Prakash, Prof. Susumu Takahashi, Prof. Chao Zhang, and Prof.
Xiaojiang Chen for participating on my screening and qualifying exam. As intimidating
as those moments were, I am glad you were there to test me and help me mature as a
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scientist. Thank you to Prof. Travis Williams, Prof. Richard Brutchey, Prof. Brent Melot,
and Prof. Matthew Pratt for words of wisdom on my career aspirations. I would like to
thank Ross Lewis, Allan Kershaw, Darrell Karrfalt and Phil Sliwoski for all their help in
running the day-to-day operations for research. Michele Dea, thank you for putting out
every fire I had and knowing how to solve every problem. Dr. Jennifer Moore and Dr.
Meredith Drake Reitan, thank you for being great role models.
I would also like to thank the USC Chemistry Department, the National Science
Foundation Graduate Research Fellowship Program, and the Dana and David Dornsife
College of Letters, Arts, and Sciences for financial support.
Dear graduate school friends, thank you for taking this adventure with me. Sean
Culver, Lena Hoober-Burkhardt, Devang Dasani, Narin Tangprasertchai, Denise Femia,
and Alvin Kung. Alvin, I’m so glad we were able to publish a paper together. Thank you
for working so hard and collaborating with me. I’d like to thank my collaborators at the
University of California, San Diego, Dr. Liang Xu and Prof. Dong Wang for their great
work on RNA polymerase. Thank you to Prof. Ralf Haiges for assisting me on the
nitrogenase project and mentoring me in gas-phase chemistry.
Last, but not least, I’d like to thank my family for never doubting my abilities or
determination. I’m so lucky to be blessed and surrounded by kind family members. Dad,
thanks for believing in me and always giving me a healthy dose of perspective. To my
brothers, Paul and Eric, I’m so happy to have great brothers who always have my back.
To my dearest Grandmother and Mom for showing me the value of hard work. To Jacob
DeForest and Quinoline, my two favorite guys in the world! Jacob, I really can’t thank
you enough; I just hope I can support you in your Ph.D. as diligently as you did for me.
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TABLE OF CONTENTS
Page
DEDICATION .................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................... iii
LIST OF TABLES ............................................................................................................ xii
LIST OF FIGURES ......................................................................................................... xiii
LIST OF SCHEMES ....................................................................................................... xxi
ABSTRACT ................................................................................................................. xxii
Fluorine in Medicinal Chemistry and Molecular Probes of Enzyme Chapter 1.
Mechanisms ..................................................................................................................... 1
1.1 Part I: Fluorinated Probes of Enzyme Mechanisms ............................................... 1
1.1.1 Chapter 2: On the Observation of Discrete Fluorine NMR Spectra for β,γ-
CHF-UTP Diastereomers at Basic pH ........................................................................ 2
1.1.2 Chapter 3: 5’-β,γ-CHF-ATP Diastereomers: Synthesis and Fluorine-
Mediated Selective Binding by c-Src Protein Kinase ................................................. 3
1.1.3 Chapter 4: Functional Interplay Between NTP Leaving Group and Base Pair
Recognition During RNA Polymerase II Nucleotide Incorporation Revealed by
Methylene Substitution ............................................................................................... 5
1.1.4 Chapter 5: Fluorinated Mechanistic Probes for Nitrogenase:
Difluorocyclopropene and Difluorodiazirine .............................................................. 6
1.2 Part II: FRET-Quenched Probes as Mechanistic Reporters ................................... 7
1.2.1 Chapter 6: Determining the Onset of Osteonecrosis of the Jaw using FRET-
Quenched Bisphosphonate Probes .............................................................................. 7
1.3 Chapter References ................................................................................................ 8
On the Observation of Discrete Fluorine NMR Spectra for Uridine 5′-β,γ- Chapter 2.
Fluoromethylenetriphosphate Diastereomers at Basic pH ................................................ 10
2.1 Introduction .......................................................................................................... 10
2.2 Results and Discussion ........................................................................................ 12
2.2.1 Preparative HPLC Purification and
1
H,
19
F, and
31
P NMR Analysis ............ 12
2.2.2 Effect of pH and Counterion on
19
F NMR Signal ......................................... 14
2.2.3 Qualitative Decomposition of 1 by
19
F NMR Analysis ................................ 17
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vii
2.2.4 Effect of Reactant Ratio (CHF-BP:UMP 5’-M) and HPLC Methods on Yield
and Purity of 1 ........................................................................................................... 18
2.2.5 Discussion ..................................................................................................... 22
2.3 Conclusion ........................................................................................................... 23
2.4 Experimental ........................................................................................................ 24
2.4.1 Materials and Methods .................................................................................. 24
2.4.2 Synthesis of 1 ................................................................................................ 24
2.4.3 Effect of Countercation and pH on
19
F NMR Spectra of 1 ........................... 27
2.4.4 Effect of Reactant Ratio (CHF-BP:UMP 5’-M) and HPLC Methods on Yield
and Purity of 1 ........................................................................................................... 28
2.5 Chapter References .............................................................................................. 29
5’-β,γ-CHF-ATP Diastereomers: Synthesis and Fluorine-Mediated Selective Chapter 3.
Binding by c-Src Protein Kinase ....................................................................................... 31
3.1 Introduction .......................................................................................................... 31
3.2 Results and Discussion ........................................................................................ 33
3.2.1 Preparation of Individual CHF-ATP Diastereomers ..................................... 33
3.2.2 Synthesis of CXY-ATP Analogues .............................................................. 35
3.2.3 Inhibition Activity on c-Src Kinase by β,γ-CXY-ATP Analogues .............. 36
3.2.4 Molecular Docking Calculations to Evaluate Stereoselectivity Exhibited by
c-Src Kinase Active Site with β,γ-CXY-ATP analogues ......................................... 37
3.3 Conclusion ........................................................................................................... 39
3.4 Experimental ........................................................................................................ 40
3.4.1 Materials and Methods .................................................................................. 40
3.4.2 Synthesis of Tetramethyl (fluoromethanediyl)bis(phosphonate), 4 .............. 41
3.4.3 Synthesis of Methyl [(dimethoxyphosphoryl)(fluoro)methyl]
phosphonate, 5 ......................................................................................................... 42
3.4.4 Synthesis of Methyl (7S)-4-fluoro-3,5-dimethoxy-7-phenyl-2,6-dioxa-3,5-
diphosphaoctan-8-oate 3,5- dioxide, 6a/6b .............................................................. 43
3.4.5 Synthesis of (2S)-({[Fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)-
(phenyl)ethanoic acid, 8a/8b .................................................................................... 44
3.4.6 Synthesis of [(R)-Fluoro{hydroxy[(1S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-
ethoxy]phosphoryl}methyl]phosphonic acid, 10a and [(S)-fluoro{hydroxy[(1S)-2-
(morpholin-4-yl)-2-oxo-1-phenylethoxy]phosphoryl}
methyl]phosphonic acid, 10b .................................................................................... 45
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3.4.7 Effect of Solvents on NMR-Based Diastereomer Differentiation in 9a/9b
Mixture ...................................................................................................................... 47
3.4.8 Synthesis of 9-{5-O-[{[((S)-Fluoro{hydroxy[(S)-morpholin-4-
yl(phenyl)methoxy]phosphoryl}methyl)(hydroxy)phosphoryl]oxy}(hydroxy)phosph
oryl]pento-furanosyl}-9H-purin-6-amine, 11a ......................................................... 49
3.4.9 Synthesis of 9-{5-O-[{[((R)-Fluoro{hydroxy[(S)-morpholin-4-
yl(phenyl)methoxy]phosphoryl}methyl)(hydroxy)phosphoryl]oxy}(hydroxy)phosph
oryl]-pentofuranosyl}-9H-purin-6-amine, 11b ......................................................... 50
3.4.10 Synthesis of 9-{5-O-[({[(S)-
Fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)phosphinato]pentofuranosyl}-
9H-purin-6-amine, 12a ............................................................................................. 50
3.4.11 Synthesis of 9-{5-O-[({[(R)-
Fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)phosphinato]pentofuranosyl}-
9H-purin-6-amine, 12b ............................................................................................. 52
3.4.12 General Synthesis of Nucleoside 5’-β,γ-Methylenetriphosphate Analogues,
12, 15-16 ................................................................................................................... 52
3.4.13 NMR Studies of Synthetic (12) and Artificial Diastereomer Mixtures,
12a/12b .................................................................................................................... 53
3.4.14 Effect of Counterion on
19
F and
31
P NMR of Diastereomer Mixture 12 ..... 53
3.4.15 Isolation and Characterization of Impurity Observed in NMR, 17 ............ 54
3.4.16 Kinase Expression and Purification ............................................................ 54
3.4.17 In vitro Kinase Activity Assay .................................................................... 55
3.4.18 In silico Analysis of Ligand/Receptor to Predict Binding Interactions ...... 56
3.5 Chapter References .............................................................................................. 57
Functional Interplay Between NTP Leaving Group and Base Pair Recognition Chapter 4.
During RNA Polymerase II Nucleotide Incorporation Revealed by Methylene
Substitution ................................................................................................................... 60
4.1 Introduction .......................................................................................................... 60
4.2 Results and Discussion ........................................................................................ 62
4.2.1 Cognate Nucleotide Incorporation ................................................................ 62
4.2.2 Non-Cognate Nucleotide Incorporation ........................................................ 64
4.2.3 Trigger Loop-Dependent Substrate Recognition and Incorporation ............ 65
4.2.4 Effect of Wobble Base-Pairing on UMP Incorporation ................................ 67
4.2.5 Discussion ..................................................................................................... 72
4.3 Conclusion ........................................................................................................... 74
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4.4 Experimental ........................................................................................................ 74
4.4.1 Materials and Methods .................................................................................. 74
4.4.2 Synthesis of Tributylammonium Salt of Methylenebis(phosphonic acid), 2
and Difluoromethylene bis(phosphonic acid), 13 ..................................................... 75
4.4.3 Synthesis of Nucleoside 5’-β,γ-Methylenetriphosphates (β,γ-CH
2
-(d)NTPs),
7 and 9 ...................................................................................................................... 76
4.4.4 Characterization of 2’-Deoxyadenosine 5’-β,γ-Methylenetriphosphate (β,γ-
CH
2
-dATP), 7 ............................................................................................................ 76
4.4.5 Characterization of 2’-Deoxythymidine 5’-β,γ-Methylenetriphosphate (β,γ-
CH
2
-dTTP), 14 .......................................................................................................... 77
4.4.6 Characterization of Commercial Adenosine 5’-β,γ-Methylenetriphosphate
(β,γ-CH
2
-ATP), 8 ..................................................................................................... 77
4.4.7 Characterization of Uridine 5’-β,γ-Methylenetriphosphate
(β,γ-CH
2
-UTP), 9 ..................................................................................................... 78
4.4.8 Re-characterization of Adenosine 5’-β,γ-Methylenetriphosphate (β,γ-CH
2
-
ATP), 10 After Dual-Pass HPLC Purification .......................................................... 78
4.4.9 Synthesis of Nucleoside 5’-β,γ-Difluoromethylenetriphosphates (β,γ-CF
2
-
(d)NTPs), 15-18 ........................................................................................................ 78
4.4.10 Characterization of 2’-Deoxyadenosine 5’-β,γ-
Difluoromethylenetriphosphate (β,γ-CF
2
-dATP), 15 ............................................... 79
4.4.11 Characterization of 2’-Deoxythymidine 5’-β,γ-
Difluoromethylenetriphosphate (β,γ-CF
2
-dTTP), 16 ............................................... 79
4.4.12 Characterization of Adenosine 5’-β,γ-Difluoromethylenetriphosphate (β,γ-
CF
2
-ATP), 17 ............................................................................................................ 80
4.4.13 Characterization of Uridine 5’-β,γ-Difluoromethylenetriphosphate (β,γ-
CF
2
-UTP), 18 ............................................................................................................ 80
4.4.14 Preparation and Determination of Concentrations of (d)NTP Analogue
Solutions ................................................................................................................... 81
4.4.15 Determining Nucleotide Analogue Stability in Assay Conditions by
LC-MS .................................................................................................................... 81
4.4.16 In vitro Transcription Assays ...................................................................... 82
4.4.17 Single Turnover Nucleotide Incorporation Assays ..................................... 83
4.4.18 Kinetic Data Analysis ................................................................................. 84
4.4.19 Molecular Modeling and Energy Minimization ......................................... 84
4.5 Chapter References .............................................................................................. 86
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Fluorinated Probes of Nitrogenase: Difluorocyclopropene and Chapter 5.
Difluorodiazirine ............................................................................................................... 89
5.1 Introduction .......................................................................................................... 89
5.2 Results and Discussion ........................................................................................ 91
5.2.1 Reduction of DFCP to Propene and 2-Fluoropropene: Product Identification
by GC-MS and NMR ................................................................................................ 91
5.2.2 Reduction of DFCP to Propene and 2-Fluoropropene: Kinetic Analysis ..... 93
5.2.3 Selectivity in DFCP Reduction: Reduction of d
2
-DFCP in H
2
O .................. 95
5.2.4 Activity of DF-DZR in Enzyme Assays ....................................................... 98
5.2.5 Discussion: Mechanistic Implications of DFCP as a Substrate for N
2
ase .. 104
5.2.6 Discussion: Implications for Dehalogenase Mechanisms ........................... 109
5.3 Conclusion ......................................................................................................... 110
5.4 Experimental ...................................................................................................... 111
5.4.1 Materials and Methods ................................................................................ 111
5.4.2 Synthesis and Characterization of DFCP .................................................... 111
5.4.3 Synthesis and Characterization of d
2
-DFCP ............................................... 112
5.4.4 Chemical Stability of DFCP Under Assay Conditions ............................... 113
5.4.5 Synthesis of Difluorodiazirine .................................................................... 113
5.4.6 Stability of Difluorodiazirine Under Assay Conditions .............................. 115
5.4.7 Assay Reagents ........................................................................................... 117
5.4.8 Nitrogenase-Catalyzed Reduction Assays .................................................. 117
5.4.9 GC-MS and NMR Sample Preparation....................................................... 118
5.4.10 GC and GC-MS Analysis ......................................................................... 119
5.4.11 Fluoride Detection .................................................................................... 119
5.4.12 Fluorescence Detection of the Liquid Phase of Reaction Mixtures .......... 120
5.4.13 Inhibition of Enzymatic Acetylene Reduction with DF-DZR .................. 120
5.5 Chapter References ............................................................................................ 121
Determining the Onset of Osteonecrosis of the Jaw using FRET-Quenched Chapter 6.
Bisphosphonate Probes ................................................................................................... 124
6.1 Introduction ........................................................................................................ 124
6.1.1 Molecular design of quenched-fluorescence CatK probes ......................... 125
6.2 Results and Discussion ...................................................................................... 127
6.2.1 Preparation of N-BP Conjugates ................................................................. 127
6.2.2 Using Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) to
Prepare β-Alanine-FAM-BP, Intermediate A ......................................................... 129
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6.2.3 Retrosynthetic Analysis and Other Attempted, Unsuccessful Routes ........ 132
6.3 Conclusion ......................................................................................................... 135
6.4 Experimental ...................................................................................................... 135
6.4.1 Materials and Methods ................................................................................ 135
6.4.2 Synthesis of TiPBP-para-dRIS 3 ................................................................ 136
6.4.3 Synthesis of para-dRIS 4 ............................................................................ 137
6.4.4 Synthesis of Glycidyl Azide 7 .................................................................... 138
6.4.5 Synthesis of para-dRIS-linker 8 ................................................................. 139
6.4.6 Synthesis of RIS-linker 10 .......................................................................... 140
6.4.7 Synthesis of Linker 17 ................................................................................ 140
6.4.8 Synthesis of Boc-β-alanine-ethylene Diamine Linker 19 ........................... 143
6.4.9 Synthesis of Boc-β-alanine-ethylene Diamine-FAM Linker 21 ................. 144
6.4.10 Synthesis of Boc-Protected Intermediate 22 via CuAAC ......................... 145
6.4.11 Synthesis of Intermediate A 23 ................................................................. 146
6.4.12 Synthesis of Penultimate Intermediate 25 ................................................. 147
6.4.13 Synthesis of Final compound 27 ............................................................... 148
6.5 Chapter References ............................................................................................ 149
Bibliography ................................................................................................................. 151
APPENDIX ................................................................................................................. 167
Appendix A. Chapter 2 Supporting Data .................................................................... 168
Appendix B. Chapter 3 Supporting Data .................................................................... 180
Appendix C. Chapter 4 Supporting Data .................................................................... 220
Appendix D. Chapter 5 Supporting Data .................................................................... 247
Appendix E. Chapter 6 Supporting Data .................................................................... 273

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LIST OF TABLES

Table 2.2.1 Effect of pH and counterions on
19
F NMR of β,γ-CHF-UTP 1 .................... 15
Table 2.2.2 Calculated purity and yield of 1 obtained for different ratios of CHF-
BP:UMP 5’-M; original and modified stage 1 HPLC methods ........................................ 21
Table 3.2.1 Effect of fluorination of β,γ-CH
2
-ATP on inhibition of c-Src kinase ........... 37
Table 3.2.2 Inhibition of c-Src kinase by β,γ-CHF-ATP diastereomers .......................... 37
Table 3.4.1 Yields for synthesis of 5 ................................................................................ 43
Table 3.4.2 Yields of 9a/9b under various reaction conditions ....................................... 47
Table 3.4.3 Conditions used to diastereomer mixtures .................................................... 53
Table 4.2.1 Kinetic data of wt NTPs and β,γ-CH
2
-NTPs ................................................. 69
Table 4.4.1 Summary of LC-MS results showing NTP stability over time ..................... 83
Table 4.4.2 AEI values of wt NTPs and β,γ-CH
2
-NTPs ................................................... 84
Table 5.2.1 Nitrogenase-Catalyzed Reduction of 3,3-Difluorocyclopropene .................. 92
Table C1. Preparative HPLC conditions and elution times ........................................... 229
Table D1. Effect of difluorination on the solubility of small, unsaturated three-
membered rings ............................................................................................................... 247
Table D2. Results from computational model of gas-phase IR of DF-DZR (CF
2
N
2
) using
GAUSSIAN .................................................................................................................... 257
Table D3. Detailed experimental description of inhibition of acetylene (constant) with
DF-DZR (variable) experiment ....................................................................................... 269
Table E1. Table of HPLC conditions ............................................................................. 300

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LIST OF FIGURES

Figure 1.1.1 Fluorinated nucleotide probes ....................................................................... 1
Figure 1.1.2 Representative fluorine NMR of fluorinated nucleotide analogue
17
............. 2
Figure 1.1.3 Application of fluorinated nucleotide to Src kinase
19
................................... 4
Figure 1.1.4 Fluorinated nitrogenase probes ...................................................................... 6
Figure 2.1.1 Synthesis of 1............................................................................................... 11
Figure 2.2.1 Effect of pH and counterion on
19
F NMR spectra of β,γ-CHF-UTP 1 (~1:1
diastereomers) ................................................................................................................... 14
Figure 2.2.2 Effect of spectrometer frequency (SF) and counterion effect on
19
F NMR
spectra ............................................................................................................................... 15
Figure 2.2.3
19
F NMR of synthetic 1 (purified by our published method)
15
in D
2
O at pH
10.1.................................................................................................................................... 16
Figure 2.2.4 Determination of β,γ-CHF-UTP 1 (~1:1 diastereomer mixture) stability at
pH 10 by
19
F NMR (D
2
O, 470 MHz, referenced to CFCl
3
) .............................................. 18
Figure 2.2.5 Effect of CHF-BP:UMP 5’-M ratio on yield and purity of 1 ...................... 19
Figure 2.2.6 Purity of 1 (~1:1 diastereomers,
19
F NMR) after preparative SAX HPLC
using modified HPLC method .......................................................................................... 20
Figure 2.2.7
19
F NMR of ~1:1 diastereomer mixture of 1 ............................................... 22
Figure 2.4.1 Preparative SAX HPLC fractionation of 1 peak ......................................... 28
Figure 3.1.1 Standard synthesis of β,γ-CXY-ATPs ......................................................... 32
Figure 3.2.1 Superimposition for A) 16 and B) 12a and 12b docked into the active site of
human Src kinase using AutoDock Vina 1.1.2 and X-ray crystallographic data for the
apoprotein (2SRC) including Mg (shown as green sphere). ............................................. 38
Figure 3.4.1 SDS-PAGE gel for purity determination ..................................................... 55
Figure 4.1.1 β,γ-CH
2
-(d)NTPs can be recognized and incorporated by RNA pol II ....... 61
Figure 4.2.1 Kinetic effects of varying the β,γ-bridging atom in NTPs during RNA pol II
incorporation ..................................................................................................................... 63
Figure 4.2.2 α-Amanitin effect index reveals that β,γ-CH
2
substitution doesn’t interfere
with patterns of TL dependence for nucleotide incorporation .......................................... 66
Figure 4.2.3 Kinetic effects of alteration of β,γ-bridging atoms in NTPs during RNA pol
II incorporation in hydrogen bond deficient scaffolds ...................................................... 68
Figure 4.2.4 CH
2
substitution effect on k
pol
in different scaffolds ................................... 70
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Figure 4.2.5 Molecular modeling of UTP binding opposite dT template at pol II active
site ..................................................................................................................................... 71
Figure 5.2.1 Double reciprocal (Lineweaver-Burk) K
m
plot for the formation of propene
and 2-fluoropropene from DFCP reduction by N
2
ase ....................................................... 93
Figure 5.2.2 GC-MS spectra of products from d
2
-DFCP reduction by N
2
ase. (FeP/MoFeP
= 20:1) ............................................................................................................................... 94
Figure 5.2.3 NMR spectra (500 MHz, CDCl
3
/C
2
Cl
4
= 1:1) of products of N
2
ase-
catalyzed reduction of d
2
-DFCP (FeP/MoFeP = 20:1) ..................................................... 96
Figure 5.2.4 Results from N
2
ase inhibition of acetylene experiment with 6% DF-DZR 99
Figure 5.2.5 N
2
ase inhibition experiment with acetylene and varying concentrations of
DF-DZR .......................................................................................................................... 100
Figure 6.1.1 Chemical structures of para-dRIS (4) and RIS (9) .................................... 125
Figure 6.1.2 Molecular design and rationale for quenched-fluorescence CatK probes . 126
Figure 6.2.1 Previous strategies employed to access FRET-quenched probes using a
commercially available Boc-protected peptide, linker 17, and N-BP conjugates 8 or 10
......................................................................................................................................... 133
Figure A1. Chromatogram trace for SAX HPLC purification of β,γ-CHF-UTP (1) ..... 168
Figure A2. Chromatogram trace for RP-C
18
HPLC purification of β,γ-CHF-UTP (1) . 169
Figure A3.
19
F NMR (470 MHz, D
2
O, pH 10.4) of β,γ-CHF-UTP 1 (~1:1 diastereomers),
Na
+
counterion ................................................................................................................ 170
Figure A4. Effect of pH and counterion on δ
F
of ~1:1 β,γ-CHF-UTP diastereomers 1 in
D
2
O ................................................................................................................................. 171
Figure A5. Effect of counterion ionic radius on Δδ
F
of β,γ-CHF-UTP in D
2
O ............. 172
Figure A6. UV Analysis of β,γ-CHF-UTP 1 (~1:1 diastereomers) ............................... 173
Figure A7.
1
H NMR (500 MHz, D
2
O, pH 7.6) of 1 (~1:1 diastereomers) ..................... 173
Figure A8.
19
F NMR (470 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers) .................. 174
Figure A9.
31
P NMR (202 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers) .................. 174
Figure A10. Representative
31
P NMR spectrum (202 MHz, D
2
O, pH 7.4) of 1 (~1:1
diastereomers) after exchange on Dowex 50WX8 (200-400 mesh, prepped NH
4
+
form)
resin ................................................................................................................................. 175
Figure A11.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.1) of 1 (~1:1 diastereomers)
after exchange with NH
4
+
(Figure A10), followed by titration with NH
4
OH ................ 175
Figure A12.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.0) of 1 (~1:1 diastereomers)
after exchange with TEAH
+
, followed by titration with TEA ........................................ 176
Figure A13.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers)
after exchange with Na
+
, followed by titration with NaOH ........................................... 177
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Figure A14.
31
P NMR spectrum (202 MHz, D
2
O, pH 12.7) of 1 (~1:1 diastereomers)
after exchange with K
+
, followed by titration with KOH ............................................... 178
Figure A15. Simulation of spectrum for 1 (~1:1 diastereomers,
19
F NMR) at a
spectrometer frequency of 235 MHz .............................................................................. 178
Figure B1.
1
H NMR (500 MHz, CDCl
3
) of 5 ................................................................ 180
Figure B2.
19
F NMR (470 MHz, CDCl
3
) of 5 ................................................................ 181
Figure B3.
31
P NMR (202 MHz, CDCl
3
) of 5 ................................................................ 181
Figure B4. MS of 5 ........................................................................................................ 182
Figure B5.
31
P NMR (202 MHz, CD
3
OD) of 6a/6b ...................................................... 183
Figure B6. MS of 9a/9b ................................................................................................. 184
Figure B7. Spectrometer Frequency and Solvent Effects in
19
F NMR Spectra of 9a/9b
(~1:1) ............................................................................................................................... 185
Figure B8.
31
P NMR (202 MHz, CD
3
OD) of Product 9a/9b (~1:1) Before Purification
......................................................................................................................................... 186
Figure B9.
31
P NMR (202 MHz, D
2
O, pH 8.9) of Product 9a/9b (~1:1) Before
Purification ...................................................................................................................... 186
Figure B10.
19
F NMR (470 MHz) Spectra of Product 9a/9b (~1:1) in CD
3
OD Spiked
with Isolated 9a (fast HPLC peak) .................................................................................. 187
Figure B11.
19
F NMR (470 MHz) Spectra of Product 9a/9b (~1:1) in D
2
O at pH 8.8
Spiked with Isolated 9a (fast HPLC peak) ..................................................................... 188
Figure B12.
31
P NMR (202 MHz) Spectra of Product 9a/9b (~1:1) in CD
3
OD Spiked
with Isolated 9a (fast HPLC peak) .................................................................................. 189
Figure B13.
31
P NMR (202 MHz) Spectra of Product 9a/9b (~1:1) in D
2
O at pH 8.8
Spiked with Isolated 9a (fast HPLC peak) ..................................................................... 190
Figure B14. RP-C
18
HPLC Analysis of 9a/9b Using Varian Microsorb C
18
HPLC
Column (5 µm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System ...... 191
Figure B15. RP-C
18
HPLC Analysis of 9a/9b Using Varian Microsorb C
18
HPLC
Column (5 µm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System ...... 191
Figure B16. RP-C
18
HPLC Analysis of 9a/9b Using Phenomenex Luna C
18
HPLC
Column (5 µm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System ...... 191
Figure B17. Preparative RP-C
18
HPLC of 9a/9b Using Phenomenex Luna C
18
HPLC
Column (5 µm, 250 mm × 21 mm) and EZStart 7.4 ....................................................... 192
Figure B18.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 9a ................................................. 192
Figure B19.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 9a ................................................ 193
Figure B20.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 9a ................................................ 193
Figure B21. MS of 9a .................................................................................................... 194
xvi

xvi
Figure B22.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 9b ................................................. 195
Figure B23.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 9b ................................................ 195
Figure B24.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 9b ................................................ 196
Figure B25. MS of 9b .................................................................................................... 197
Figure B26. Preparative Ion Exchange HPLC Analysis of 11a ..................................... 198
Figure B27.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 11a ............................................... 198
Figure B28.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 11a .............................................. 199
Figure B29.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 11a .............................................. 199
Figure B30. MS of 11a .................................................................................................. 200
Figure B31.
1
H NMR (500 MHz, D
2
O, pH 10) of 11b .................................................. 201
Figure B32.
19
F NMR (470 MHz, D
2
O, pH 10) of 11b ................................................. 201
Figure B33.
31
P NMR (202 MHz, D
2
O, pH 10) of 11b ................................................. 202
Figure B34. MS of 11b .................................................................................................. 203
Figure B35.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 12a ............................................... 204
Figure B36.
19
F NMR (470 MHz, D
2
O, pH 10.2) of 12a .............................................. 204
Figure B37.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 12a .............................................. 205
Figure B38. MS of 12a .................................................................................................. 206
Figure B39.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 12b ............................................... 207
Figure B40.
19
F NMR (470 MHZ, D
2
O, pH 10.2) of 12b ............................................. 207
Figure B41.
31
P NMR (202 MHz, D
2
O, pH 9.7) of 12b ................................................ 208
Figure B42. MS of 12b .................................................................................................. 209
Figure B43.
1
H NMR (500 MHz, D
2
O, pH ~10) of 12 .................................................. 210
Figure B44.
19
F NMR (470 MHz, D
2
O, pH ~10) of 12 ................................................. 210
Figure B45.
31
P NMR (202 MHz, D
2
O, pH ~10) of 12 ................................................. 211
Figure B46.
19
F NMR (470 MHz, D
2
O, pH 9.8) of Diastereomer Mixture, 12, and
Individual Diastereomers, 12a and 12b .......................................................................... 211
Figure B47.
31
P NMR (202 MHz, D
2
O, pH 9.8) of Diastereomer Mixture, 12, and
Individual Diastereomers, 12a and 12b .......................................................................... 212
Figure B48.
19
F NMR (470 MHz, D
2
O, pH 10.4) of Impurity 17 ................................. 212
Figure B49.
31
P NMR (202 MHz, D
2
O, pH 10.4) of Impurity 17 ................................. 213
Figure B50. MS of Impurity 17 ..................................................................................... 214
Figure B51.
19
F NMR (470 MHz, D
2
O) of Diastereomer Mixture, 12, with Various Basic
Counterions ..................................................................................................................... 215
Figure B52.
31
P NMR (202 MHz, D
2
O) of Diastereomer Mixture, 12, with Various Basic
Counterions ..................................................................................................................... 215
xvii

xvii
Figure B53.
31
P NMR (D
2
O, pH 10.9) of 12 Treated with Na
2
CO
3
at Two Spectrometer
Frequencies ..................................................................................................................... 216
Figure B54.
31
P NMR (202 MHz, D
2
O, pH 12.1) of 12 Treated with NaOH ................ 216
Figure B55.
19
F NMR (470 MHz, D
2
O, pH 9.8) of Artificial Diastereomer Mixture (3:1,
75% 12a and 25% 12b) ................................................................................................... 217
Figure B56.
31
P NMR (202 MHz, D
2
O, pH 9.8) of Artificial Diastereomer Mixture (3:1,
75% 12a and 25% 12b) ................................................................................................... 217
Figure B57. NMR Simulation Studies with 12,
19
F NMR (470 MHz, D
2
O, pH 9.8) .... 218
Figure C1.
1
H NMR (500 MHz, D
2
O, pH 9.8) of 7 ....................................................... 220
Figure C2.
31
P NMR (202 MHz, D
2
O, pH 9.8) of 7 ...................................................... 221
Figure C3. MS of 7 ........................................................................................................ 222
Figure C4.
1
H NMR (600 MHz, D
2
O, pH 10.5) of 8 ..................................................... 223
Figure C5.
31
P NMR (202 MHz, D
2
O, pH 10.5) of 8 .................................................... 223
Figure C6. MS of 8 ........................................................................................................ 224
Figure C7.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 9 ..................................................... 225
Figure C8.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 9 .................................................... 225
Figure C9. MS of 9 ........................................................................................................ 226
Figure C10.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 10 ................................................. 227
Figure C11.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 10 ................................................ 227
Figure C12. MS of 10 .................................................................................................... 228
Figure C13. Representative kinetic fitting data (β,γ-CH
2
-ATP/dT) .............................. 230
Figure C14. Representative kinetic fitting data (β,γ-CH
2
-UTP/dT). ............................. 231
Figure C15. Two potential UTP:dT forms based on reported U:U pairing ................... 232
Figure C16. Energy minimized modeling of UTP binding opposite dT template at pol II
active site ........................................................................................................................ 232
Figure C17.
1
H NMR (500 MHz, D
2
O, pH 9.9) of 14 ................................................... 233
Figure C18.
31
P NMR (202 MHz, D
2
O, pH 9.9) of 14 .................................................. 233
Figure C19. MS of 14 .................................................................................................... 234
Figure C20.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 15 ................................................. 235
Figure C21.
19
F NMR (470 MHz, D
2
O, pH 10.2) of 15 ................................................ 236
Figure C22.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 15 ................................................ 236
Figure C23. MS of 15 .................................................................................................... 237
Figure C24.
1
H NMR (500 MHz, D
2
O, pH 9.6) of 16 ................................................... 238
Figure C25.
19
F NMR (470 MHz, D
2
O, pH 9.8) of 16 .................................................. 238
Figure C26.
31
P NMR (202 MHz, D
2
O, pH 9.8) of 16 .................................................. 239
Figure C27. MS of 16 .................................................................................................... 240
xviii

xviii
Figure C28.
1
H NMR (500 MHz, D
2
O, pH 9.9) of 17 ................................................... 241
Figure C29.
19
F NMR (470 MHz, D
2
O, pH 9.9) of 17 .................................................. 241
Figure C30.
31
P NMR (202 MHz, D
2
O, pH 9.9) of 17 .................................................. 242
Figure C31. MS of 17 .................................................................................................... 243
Figure C32.
1
H NMR (500 MHz, D
2
O, pH 8.1) of 18 ................................................... 244
Figure C33.
19
F NMR (470 MHz, D
2
O, pH 10.3) of 18 ................................................ 244
Figure C34.
31
P NMR (202 MHz, D
2
O, pH 10.3) of 18 ................................................ 245
Figure C35. MS of 18 .................................................................................................... 246
Figure D1.
1
H NMR spectrum (500 MHz, CDCl
3
) of DFCP ......................................... 248
Figure D2.
1
H NMR spectra (500 MHz) of DFCP ........................................................ 248
Figure D3.
19
F NMR spectra (470.4 MHz, CDCl
3
) of DFCP ........................................ 249
Figure D4.
13
C NMR spectrum (500 MHz, CDCl
3
) of DFCP ....................................... 250
Figure D5. MS Chromatogram of DFCP and d
2
-DFCP ................................................ 251
Figure D6. Gas-phase IR of DF-DZR (20 Torr) ............................................................ 252
Figure D7.
1
H NMR of DF-DZR in CDCl
3
(600 MHz, 8 scans) ................................... 252
Figure D8.
13
C NMR of DF-DZR in CDCl
3
(151 MHz, 30,000 scans) ......................... 253
Figure D9.
14
N NMR of DF-DZR in CDCl
3
(36 MHz, overnight) ................................ 253
Figure D10.
19
F NMR of DF-DZR in CDCl
3
(546 MHz, 128 scans), referenced using
CFCl
3
standard (δ 0.00 ppm) .......................................................................................... 254
Figure D11.
1
H NMR of DF-DZR in D
2
O (600 MHz, 8 scans) .................................... 254
Figure D12.
13
C NMR of DF-DZR in D
2
O (150 MHz, 30,000 scans) .......................... 255
Figure D13.
19
F NMR of DF-DZR in D
2
O (564 MHz, 2,048 scans), referenced using
CFCl
3
standard (δ 0.00 ppm) .......................................................................................... 255
Figure D14.
19
F NMR of DF-DZR in D
2
O (564 MHz, 1,024 scans), after four days.
Impurity peak at -7.7 ppm did not significantly increase ............................................... 256
Figure D15.
19
F NMR of DF-DZR in D
2
O (470 MHz), after four days ........................ 256
Figure D16. Plot from computational model of gas phase-IR of DF-DZR (CF
2
N
2
) using
GAUSSIAN .................................................................................................................... 258
Figure D17.
19
F NMR time-dependent study of DF-DZR in H
2
O ................................. 259
Figure D18. Time-dependent study of
19
F NMR of difluorodiazirine in assay buffer (470
MHz, 256 or 1024 scans), referenced using CFCl
3
standard (δ 0.00 ppm) at 30 ˚C ...... 260
Figure D19. Design of storage vessel for DF-DZR ....................................................... 261
Figure D20. GC-MS analysis of products from DFCP reduction by N
2
ase (FeP/MoFeP =
20:1) ................................................................................................................................ 262
Figure D21.
1
H NMR (500 MHz, CDCl
3
) spectra of gas phase products of DFCP
reduction by N
2
ase .......................................................................................................... 263
xix

xix
Figure D22.
19
F NMR (470.4 MHz; CDCl
3
) spectra for DFCP reduction by N
2
ase ..... 264
Figure D23. Fluoride (F
–
) release rates (4 replicates) .................................................... 264
Figure D24. Distribution of d
2
-propene and d
2
-2-fluoropropene isomers formed under
different electron flux conditions (FeP/MoFeP = 20:1 vs. 1:5) ...................................... 265
Figure D25. Schematic of inhibition experiment of acetylene with DF-DZR ............... 266
Figure D26. Fluorescence detection method using OPA/2-ME
1,2
................................. 267
Figure D27. Calibration plot for determination of ammonia ......................................... 267
Figure D28. Example of fluorescence detection using OPA/2-ME method from assay
experiment containing 6% DF-DZR in He ..................................................................... 268
Figure D29. Image of reduction experiments with 50% DF-DZR in He ....................... 270
Figure D30. Image of inhibition experiments with addition of acetylene after exposure
and removal of 50% DF-DZR for 40 minutes ................................................................ 270
Figure D31. Calibration plot of H
2
by GC-BID ............................................................. 271
Figure D32. Detection of gases by GC-FID from add-back experiment after exposure of
buffer assay mixture to DF-DZR and removal ............................................................... 271
Figure E1. ISCO Purification of 3 ................................................................................. 273
Figure E2.
1
H NMR (500 MHz, CDCl
3
) of 3 ................................................................ 274
Figure E3.
31
P NMR (202 MHz, CDCl
3
) of 3 ................................................................ 274
Figure E4.
1
H NMR (500 MHz, D
2
O) of 4 .................................................................... 275
Figure E5.
31
P NMR (202 MHz, D
2
O) of 4 ................................................................... 275
Figure E6. Predictive iMass for 4 .................................................................................. 276
Figure E7. MS (+) of 4 .................................................................................................. 277
Figure E8. MS (-) of 4 ................................................................................................... 278
Figure E9.
1
H NMR (500 MHz, D
2
O, pH 10.26) of 8 ................................................... 279
Figure E10.
31
P NMR (202 MHz, D
2
O, pH 10.26) of 8 ................................................ 279
Figure E11. Predictive iMass of 8 ................................................................................. 280
Figure E12. MS (+) of 8 ................................................................................................ 281
Figure E13. MS (-) of 8 ................................................................................................. 282
Figure E14.
1
H NMR of 10 ............................................................................................ 283
Figure E15.
31
P NMR of 10 ........................................................................................... 283
Figure E16. Predictive iMass of 10 ............................................................................... 284
Figure E17. MS (+) of 10 .............................................................................................. 285
Figure E18. MS (-) of 10 ............................................................................................... 286
Figure E19.
1
H NMR (500 MHz, CDCl
3
) of tosylated alcohol 12 ................................ 287
Figure E20.
1
H NMR (400 MHz, CDCl
3
) of monoprotected diamine 16 ..................... 287
xx

xx
Figure E21. ISCO purification of 17 with (Attempt 1) and without 1% TEA (Attempt 2)
......................................................................................................................................... 288
Figure E22.
1
H NMR (500 MHz, D
2
O) of 17 ................................................................ 289
Figure E23.
1
H NMR (400 MHz, CDCl
3
) of 19 ............................................................ 289
Figure E24. MS (+) of 19 .............................................................................................. 290
Figure E25. ISCO Purification of 21 ............................................................................. 291
Figure E26. Predictive iMass of 21 ............................................................................... 292
Figure E27. MS (+) of 21 .............................................................................................. 293
Figure E28. MS (-) of 21 ............................................................................................... 294
Figure E29. Predictive iMass of 22 ............................................................................... 295
Figure E30. MS (+) of 22 .............................................................................................. 295
Figure E31. MS (-) of 22 ............................................................................................... 296
Figure E32. Predictive iMass of 23 ............................................................................... 297
Figure E33. MS (+) of 23 .............................................................................................. 298
Figure E34. MS (-) of 23 ............................................................................................... 299
Figure E35. Predictive iMass of 25 ............................................................................... 300
Figure E36. MS (+) of 25 .............................................................................................. 301
Figure E37. MS (-) of 25 ............................................................................................... 302
Figure E38. MALDI (-) trace of final compound 27 ..................................................... 303

xxi

xxi
LIST OF SCHEMES
Scheme 3.2.1 Modified synthesis of (S)-mandelic acid morpholinamide (R)/(S)-
monoesters 9a and 9b ....................................................................................................... 33
Scheme 3.2.2 Synthesis of 12a and 12b from 9a and 9b, respectively ............................ 35
Scheme 3.4.1 Synthesis of chiral synthon ........................................................................ 41
Scheme 5.2.1 d
2
-Propene and d
2
-2-fluoropropene isomers produced by N
2
ase-catalyzed
reduction of d
2
-DFCP (FeP/MoFeP = 20:1) ..................................................................... 95
Scheme 5.2.2 Proposed mechanism for reduction of DFCP catalyzed by N
2
ase ........... 106
Scheme 5.4.1 Synthesis of DFCP ................................................................................... 111
Scheme 6.2.1 Synthesis of para-dRIS N-BP conjugate 8 .............................................. 128
Scheme 6.2.2 Access to Intermediate A (23) ................................................................. 129
Scheme 6.2.3 Conjugation reaction to prepare final compound 27 ............................... 131

xxii

xxii
ABSTRACT

Jakeman et al. recently reported the inability to distinguish the diastereomers of
uridine 5’-β,γ-fluoromethylenetriphosphate (β,γ-CHF-UTP) by
19
F NMR under
conditions we previously prescribed for the resolution of the corresponding β,γ-CHF-
dGTP spectra, stating further that the β,γ-CHF-UTP decomposed under these basic
conditions. Here we show that the
19
F NMR spectra of β,γ-CHF-UTP (~1:1 diastereomer
mixture prepared by coupling of UMP-morpholidate with
fluoromethylenebis(phosphonic acid) in D
2
O at pH 10 are indeed readily distinguishable.
β,γ-CHF-UTP in this solution was stable for 24 h at rt.
The first preparation of the individual β,γ-CHF-ATP stereoisomers 12a and 12b is
reported. Configurationally differing solely by the orientation of the C–F fluorine, 12a
and 12b have discrete
31
P (202 MHz, pH 10.9, Δδ
Pα
6 Hz, Δδ
Pβ
4 Hz) and
19
F NMR (470
MHz, pH 9.8, Δδ
F
25 Hz) spectral signatures and exhibit a 6-fold difference in IC
50

values for c-Src kinase, attributed to a unique interaction of the (S)-fluorine of bound 12b
with R388 in the active site.
RNA polymerase II (pol II) utilizes a complex interaction network to select and
incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and
fidelity. Our previous “synthetic nucleic acid substitution” strategy has been successfully
xxiii

xxiii
applied in dissecting the function of nucleic acid moieties in pol II transcription. However,
how the triphosphate moiety of substrate influences P-O bond cleavage and formation
during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-
“substituted” NTPs, we elucidate how the methylene substitution in the pyrophosphate
leaving group affects cognate and noncognate nucleotide incorporation. Intriguingly, β,γ-
CH
2
substitution in ATP causes a ~130-fold decrease in k
pol
for AMP incorporation
opposite dT DNA template; whereas the same substitution in UTP only causes a ~3-fold
decrease in k
pol
for noncognate UMP incorporation opposite dT. Removal of the wobble
hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP.
Compared to the cognate ATP/dT state, kinetic studies and molecular modelling are both
consistent with an altered transition state during noncognate UTP/dT incorporation.
These results suggest a functional interplay among base-pairing pattern, catalytic P-O
bond cleavage and formation in the triphosphate moiety.
Reduction of the first known halogen-containing substrate by nitrogenase, 3,3-
difluorocyclopropene (DFCP), was investigated. Reduction requires both nitrogenase
proteins (MoFe and Fe protein), ATP and an exogenous reductant (dithionite), as with N
2

and known alternative substrates of the enzyme. Two major products providing evidence
for reductive C-F bond cleavage were confirmed, propene (P1, requiring 6e
−
/6H
+
) and 2-
fluoropropene (P2, 4e
−
/4H
+
). Both were identified by GC-MS and NMR spectroscopy,
with the same K
m
constants (0.022 atm, 5.4 mM). Reduction of 1,2-dideuterated DFCP
(d
2
-DFCP) further revealed that: (i) in both propene and 2-fluoropropene, two deuterium
atoms are retained, one on carbon-1 and one on carbon-3, indicating that C=C bond
cleavage rather than C−C bond cleavage is involved during DFCP reduction to these
xxiv

xxiv
products; (ii) no stereospecificity was observed in formation of cis and trans isomers of
1,3-d
2
-2-fluoropropene, whereas cis-1,3-d
2
-propene is the predominant 1,3-d
2
-propene
product, indicating that one of the bound reduction intermediates on the pathway to
propene is constrained geometrically. A reduction mechanism, consistent with hydride
transfer as a key step, is discussed. Reductive C−F bond cleavage is an ability of
nitrogenase that further demonstrates the unique and remarkable scope of its catalytic
prowess.
Osteonecrosis of the jaw (ONJ) is a well-known side effect in patients treated with
nitrogen-containing bisphosphonates (N-BPs), but only when administered through IV.
However the mechanism of how N-BPs affect the onset of ONJ is currently unknown. To
determine the pathological mechanism of ONJ, we use the synthesis and application of a
novel N-BP-based probe equipped with a Förster resonance energy transfer (FRET)-
quenched reporter. The probes will generate a fluorescent signal only in response to
osteoclast-derived cathepsin K activity, which causes separation of emitter and quencher
when incubated with osteoclasts. A short CatK-cleavable peptide bridging the fluorescent
dye and quencher are connected to a biologically active or inactive N-BP scaffold
through a clickable linker. In vivo application of the molecular probes should enable a
more comprehensive understanding of how ONJ develops in patients treated with N-BPs.

1

1
Fluorine in Medicinal Chemistry and Molecular Probes of Chapter 1.
Enzyme Mechanisms
1.1 Part I: Fluorinated Probes of Enzyme Mechanisms
Expanding our understanding of enzymes by the use of small molecule probes is
critical for discovering the underlying mechanism behind biological processes and the
development of therapeutics for the treatment of diseases. My dissertation research
focuses on the study of several enzymes, including RNA polymerase II (pol II), Src
protein kinase, osteoclast-derived cathepsin K (CatK) and nitrogenase (N
2
ase). It also
incorporates the design and synthesis of fluorinated and nonfluorinated molecular probes
and their application to enzyme systems. Fluorine plays a critical role in the design and
development of drug therapies in medicinal chemistry, demonstrated by the prevalence of
fluorine in 20-25% of commercial pharmaceuticals.
1
Fluorine substitution is used to
improve metabolic stability, bioavailability, and protein-ligand interactions by
modulating the pharmacokinetic and pharmacodynamic properties of a drug.
2
Although
fluorine and hydrogen have drastically different chemical properties, their similarity in
size allows for the incorporation of fluorine with minimal steric perturbation.
Furthermore, selective introduction of fluorine can exploit stereoisomers as
conformational tools in enzyme interactions.
3

1
1
Nucleotides are important to study, as they are ubiquitous in biology as cellular
regulators and sources of energy. Nucleoside triphosphates (NTPs) are required for the
biosyntheses of DNA and RNA and therefore have values for the structural investigation
of nucleic acids of crucial interest. Modified (d)NTPs have important therapeutic and
diagnostic applications, and synthesis of these (d)NTPs are needed for biochemical and
pharmacological investigations.
4
These triphosphate analogues have proven to be
effective chemical probes to extract molecular detail from enzymatic catalysis.

Figure 1.1.1 Fluorinated nucleotide probes

The deoxynucleoside triphosphate (dNTP) analogues have demonstrated their
utility as probes in mechanistic studies with DNA polymerase β (Figure 1.1.1).
3,5,6
The
synthesis of dNTP analogues, with the bridging β,γ-oxygen replaced by a
monofluoromethylene (CHF) group, results in a pair of stereoisomers that are essentially
identical except for the orientation of the C-F fluorine (Figure 1.1.1). The newly
introduced and stable P-C-P link that comprises the bisphosphonate group confers
chemical and metabolic stability not available to natural nucleotide triphosphates.
7

Bisphosphonate analogues of natural nucleotides can provide unique information about
the structure and function of enzymes that consume a dNTP or NTP substrate. Several
monofluoromethylene (CHF) phosphonate compounds,
8-12
including β,γ-CHF NTP and
O
OH
O
P
O
OH
P
OH
P
HO
OH
X
O O O
R
Y
Base
β
γ α
Base = U, A, T
R = H (deoxyribose) or OH (ribose)
X,Y = H, F

2
2
dNTP analogues,
3,13,14
have already demonstrated utility as probes in enzyme systems
exhibiting selectivity toward one of the isomers, which makes them attractive probes,
however there are only a handful of studies exploring the effect of fluorination or the
utilization of individual CHF stereoisomers.
10

Since simple coupling of CHF-BP with an activated dNTP (or NTP) results in both
β,γ-CHF-dNTP stereoisomers, their complete chemical and biochemical utility require
the ability to resolve the diastereomer resonances by NMR.
15
In lieu of demonstrated
diastereoselectivity exhibited by a variety of enzymes
3,8,10,11,13
and the lack of fluorine in
natural biological systems,
16
creates a readily detectable NMR probe and therefore
establishes a convenient means to determine relative reaction rates of each stereoisomer
after turnover.
12
Several of my dissertation chapters involved extending this approach to
ribosylnucleotides (NTPs).

1.1.1 Chapter 2: On the Observation of Discrete Fluorine NMR Spectra for β,γ-CHF-
UTP Diastereomers at Basic pH

Figure 1.1.2 Representative fluorine NMR of fluorinated nucleotide analogue
17

19
F
NH4OH
pH 7.56
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
OH
NH
N
O
O
F
470 564
MHz
ppm
ppm
pH 10.06
1

3
3
In previous reports, uridine 5’-β,γ-fluoromethylenetriphosphate isomers (β,γ-
CHF-UTPs) were investigated as a probe of thymidylyltransferase Cps2L. These
transferases catalyze the formation of sugar nucleotides and release inorganic
pyrophosphate.
14,18
Results suggested stereoselective turnover by Cps2L, but were
deemed inconclusive due to an inability to resolve the
19
F NMR resonances of the β,γ-
CHF-UTP stereoisomers.
14
The authors claimed that at pH ~10, their β,γ-CHF-UTPs
decomposed.
14
In this chapter, β,γ-CHF-UTP diastereomers were synthesized and
characterized (Figures 1.1.1 and 1.1.2). Our results establish unequivocally that the
19
F
and
31
P NMR spectra of the individual diastereomers are resolvable and were published
in the Journal of Organic Chemistry.
17
Additionally, the compounds are completely
stable for 24 hours at room temperature at pH ~10. The broader significance of this work
demonstrates that the stereoisomers represent useful NMR probes for NTP-utilizing
enzymes. A resolved
19
F NMR spectrum can potentially reveal a) if there is a selective
interaction with one diastereomer during enzyme binding and/or b) additional
information about the active site.

1.1.2 Chapter 3: 5’-β,γ-CHF-ATP Diastereomers: Synthesis and Fluorine-Mediated
Selective Binding by c-Src Protein Kinase

β,γ-CHF-ATPs
N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
12b
IC
50
= 150 µM
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
12a
IC
50
= 23 µM (S)-F
(R)-F
R388
3.2 Å
C277

4
4
Figure 1.1.3 Application of fluorinated nucleotide to Src kinase
19

Eukaryotic protein kinases transfer a phosphoryl group from ATP to a serine,
threonine or tyrosine residue on a peptide substrate. The transfer of a phosphate catalyzed
by Src kinase regulates fundamental cellular processes. This phosphorylation event
serves as a posttranslational modification that typically alters the activity of an enzyme or
modulates protein-protein binding interactions. However, aberrant expression or
activation of this enzyme can often lead to a variety of human diseases. Understanding
the catalytic mechanism of Src and its role in oncogenesis is critical for design of
therapeutic agents to target the diseases associated with its disregulation.
To explore the mechanism underlying phosphoryl transfer of protein kinases,
structural probes can be employed. ATP is a ubiquitous nucleotide and functions as a
source of energy within the cell. The lack of availability of individual β,γ-CHF-ATPs has
been recognized as a serious impediment to their application as enzymatic mechanistic
probes. In this chapter, both stereoisomers of β,γ-CHF-ATP were synthesized and
characterized for the first time (Figures 1.1.1 and 1.1.3). Their stereochemistry was
established by the absolute configurations of the chiral bisphosphonate synthons, and
confirmed by the observation of discrete
19
F and
31
P NMR spectra and was reported in
Organic Letters.
19
Investigating the interaction of Src with these two stereoisomers
revealed a 6:1 difference in K
d
and greater IC
50
value potency with increased fluorination.
Molecular modeling calculations were performed on (R)- and (S)-β,γ-CHF-ATP vs. β,γ-
CF
2
-ATP docked into the active site of human Src and suggested that the (S)-fluorine is
within 3.0-3.2 Å from Arg388. The results provide a unique assessment of role of
noncovalent C-F fluorine interactions with proteins, which has broad significance given

5
5
the ubiquity of fluorinated pharmaceuticals, and the poorly understood nature of fluorine
interactions within enzyme active sites.

1.1.3 Chapter 4: Functional Interplay Between NTP Leaving Group and Base Pair
Recognition During RNA Polymerase II Nucleotide Incorporation Revealed by
Methylene Substitution
Nucleoside triphosphates (NTPs) are required for the biosyntheses of DNA and
RNA and therefore have value for the structural investigation of nucleic acids of crucial
interest. These can be modified at the pyrimidine or purine base, at the (deoxy)ribose
sugar, or at the triphosphate chain. Analogues with substitutions at the nucleobase or
sugar moieties of a nucleoside precursor have been the basis for structure-activity
relationships and are a classical method for developing chemotherapeutic agents targeting
enzymes that process nucleotides.
7
Moreover, modification at the triphosphate can reveal
new and intriguing insights into the mechanism because they contain the locus of
chemical transformations catalyzed in the enzyme active site.
13

RNA polymerase II (pol II) utilizes a complex interaction network among its
residues and RNA-DNA hybrid to select and incorporate correct nucleoside triphosphate
(NTP) substrates with high transcriptional efficiency and fidelity. Previous “chemical
mutation” strategy has been successfully applied in dissecting the function of nucleic acid
moieties, including the nucleobase and sugar. However, how the triphosphate moiety of
substrate is recognized and involved in P-O bond cleavage and formation during
nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-“mutated”
NTPs, we elucidate how pol II recognizes the triphosphate moiety and how the chemistry

6
6
step affects transcriptional efficiency. Results from this chapter were recently published
in an article in Nucleic Acids Research.
20

1.1.4 Chapter 5: Fluorinated Mechanistic Probes for Nitrogenase:
Difluorocyclopropene and Difluorodiazirine
Figure 1.1.4 Fluorinated nitrogenase probes

N
2
ase enzymes are essential components of biological processes necessary to
sustain life, as they are capable of fixing atmospheric N
2
to ammonia (NH
3
) for
assimilation by plants. The main industrial process available accounts for ~50% of
worldwide N
2
fixation, but requires high pressures and temperatures to reduce N
2
, and
relies on fossil fuels. In contrast, N
2
ase reduces N
2
at ambient temperature and pressure.
In the face of ever-increasing energy costs, it is critical to find more efficient alternatives
to the century-old Haber-Bosch catalyst. Establishing the chemical mechanism of N
2
ase
is expected to provide insights into the design of more effective catalysts for sustainable
production of NH
3
.
N
2
ase reduces a variety of small, unsaturated substrates apart from N
2
(Figure
1.1.4). In our 2013 JACS paper, it was demonstrated that N
2
ase unexpectedly cleaves C-F
N N N N
F F
F F
CPE F
2
CPE
F
2
DZR DZR

7
7
bonds in the reduction of fluorinated 3,3-difluorocyclopropene (F
2
CPE).
21
Building on
this reactivity, fluorinated diazirine (F
2
DZR) was investigated to provide exciting new
insights into the mechanistic pathway (Figure 1.1.4). The compound has been synthesized
and characterized by
13
C,
15
N,
19
F NMR, and IR spectroscopy. F
2
DZR has been tested as
a potential substrate for N
2
ase, including its inhibition of N
2
reduction and H
2
evolution.
The substrate reduction rates elucidated by gas chromatography has revealed that the
compound exhibits reactivity toward the exogenous reductant dithionite, resulting in
overall inactivation of the enzyme.

1.2 Part II: FRET-Quenched Probes as Mechanistic Reporters
1.2.1 Chapter 6: Determining the Onset of Osteonecrosis of the Jaw using FRET-
Quenched Bisphosphonate Probes
Osteonecrosis of the jaw (ONJ) is a well-known side effect in patients treated
with nitrogen-containing bisphosphonates (N-BPs), but only when administered through
IV. These N-BPs therapeutically target osteoclasts, however the mechanism of how N-
BPs affect the onset of ONJ is currently unknown. To determine the pathological
mechanism of ONJ, three novel N-BP-based probes equipped with a Förster resonance
energy transfer (FRET)-quenched reporters were prepared and applied in vivo in mice
ONJ models. The probes will generate a fluorescent signal only in response to osteoclast-
derived cathepsin K (CatK) activity, which causes separation of emitter and quencher
when incubated with osteoclasts. A short CatK-cleavable peptide bridging the fluorescent
dye and quencher are connected to a biologically active or inactive N-BP scaffold

8
8
through a clickable linker. In vivo application of the molecular probes should enable a
more comprehensive understanding of how ONJ develops in patients treated with BPs.

1.3 Chapter References
(1) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chem. Soc. Rev. 2008, 37,
320.
(2) Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
(3) Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard,
W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Biochemistry 2012, 51, 8491.
(4) Weinschenk, L.; Meier, C. Chemical Synthesis of Nucleoside Analogues 2013,
209.
(5) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.;
Pedersen, L. C.; Beard, W. A.; Wilson, S. H. J. Am. Chem. Soc. 2007, 129, 15412.
(6) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.;
Wilson, S. H.; McKenna, C. E. J. Am. Chem. Soc. 2012, 134, 8734.
(7) McKenna, C. E.; Kashemirov, B. A.; Peterson, L. W.; Goodman, M. F. Biochim.
Biophys. Acta 2010, 1804, 1223.
(8) Xu, Y.; Qian, L.; Prestwich, G. D. The Journal of Organic Chemistry 2003, 68,
5320.
(9) Cox, R. J.; Gibson, J. S.; Hadfield, A. T. Chembiochem 2005, 6, 2255.
(10) Berkowitz, D. B.; Bose, M.; Pfannenstiel, T. J.; Doukov, T. J. Org. Chem. 2000,
65, 4498.
(11) Forget, S. M.; Bhattasali, D.; Hart, V. C.; Cameron, T. S.; Syvitski, R. T.;
Jakeman, D. L. Chemical Science 2012, 3, 1866.
(12) Nieschalk, J.; Batsanov, A. S.; O'Hagan, D.; Howard, J. Tetrahedron 1996, 52,
165.
(13) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.;
Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617.
(14) Beaton, S. A.; Jiang, P. M.; Melong, J. C.; Loranger, M. W.; Mohamady, S.;
Veinot, T. I.; Jakeman, D. L. Org. Biomol. Chem. 2013, 11, 5473.
(15) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin Trans. 1 1984,
1119.
(16) Berkowitz, D. B.; Karukurichi, K. R.; de la Salud-Bea, R.; Nelson, D. L.; McCune,
C. D. J. Fluorine Chem. 2008, 129, 731.
(17) Hwang, C. S.; Kashemirov, B. A.; McKenna, C. E. J. Org. Chem. 2014, 79, 5315.
(18) Blankenfeldt, W.; Asuncion, M.; Lam, J. S.; Naismith, J. H. The EMBO Journal
2000, 19, 6652.
(19) Hwang, C. S.; Kung, A.; Kashemirov, B. A.; Zhang, C.; McKenna, C. E. Org.
Lett. 2015, 17, 1624.

9
9
(20) Hwang, C. S.; Xu, L.; Wang, W.; Ulrich, S.; Zhang, L.; Chong, J.; Shin, J. H.;
Huang, X.; Kool, E. T.; McKenna, C. E.; Wang, D. Nucleic Acids Res. 2016.
(21) Ni, F.; Lee, C. C.; Hwang, C. S.; Hu, Y.; Ribbe, M. W.; McKenna, C. E. J. Am.
Chem. Soc. 2013, 135, 10346.

10
10
On the Observation of Discrete Fluorine NMR Spectra for Chapter 2.
Uridine 5′-β,γ-Fluoromethylenetriphosphate Diastereomers at Basic
pH
1

2.1 Introduction
Ribonucleoside (NTP) and deoxyribonucleoside (dNTP) triphosphate analogues
can be modified at the base, sugar, or triphosphate moiety to generate nucleotide
derivatives able to function as substrate mimics or inhibitors of enzymes that utilize
nucleotides.
1-5
Replacing the bridging oxygen between the β- and γ-phosphates of the
triphosphate moiety with a methylene carbon generates a nonhydrolyzable
bisphosphonate (BP) inhibitor for enzymes that cleave or transfer the γ-phosphate, such
as protein kinases.
4,6-8
Conversely, these analogues can act as substrates for enzymes,
such as DNA polymerases, that process nucleotides with release of pyrophosphate.
7-11

Variation of carbon substitution at the CXY group when X ≠ Y generates two possible
diastereomers due to the introduction of a new chiral center, in principal resulting in
differing interactions with a binding enzyme.
12-16
We recently synthesized the first
examples of individual β,γ-CXY-stereoisomers, namely both diastereomers of β,γ-CHF-
and β,γ-CHCl-dGTP.
17
The configuration at the CHX carbon was found to affect both K
d

and k
pol
with DNA polymerase β (pol β),
16
an important base excision repair (BER)
enzyme that typically inserts a single dNTP replacing an excised damaged or mismatched

1
Reproduced with permission from Hwang, C. S.; Kashemirov, B. A.; McKenna, C. E. J.
Org. Chem. 2014, 79, 5315. Copyright 2014 American Chemical Society.

11
11
residue
18,19
(in the case of these substrate analogues, the corresponding bisphosphonate is
released instead of pyrophosphate).

Figure 2.1.1 Synthesis of 1

As simple coupling
20
of fluoromethylenebis(phosphonic acid) (CHF-BP, 2) with
an activated dNMP (or NMP) results in formation of both β,γ-CHF-dNTP stereoisomers
together (Figure 2.2.1), the ability to resolve their
19
F NMR spectra is important,
particularly because this also offers a convenient means to determine their relative
reaction rates after turnover.
16
We previously demonstrated that synthetic β,γ-CHF-dGTP
diastereomer mixtures exhibit discrete
19
F NMR (at 376 and 470 MHz) at pH 10,
14,15

confirmed by preparation and analysis of the individual stereoisomers.
17
Subsequently,
Jakeman and coworkers studied the reaction of thymidylyltransferase Cps2L with a
mixture of uridine-5’-β,γ-fluoromethylenetriphosphate (β,γ-CHF-UTP) diastereomers,
and found that ca. 50% of the substrate was rapidly consumed, suggesting
stereospecificity for one of the two isomers present.
21
However, they could not verify this
intriguing result by
19
F NMR at 235 MHz, due to inability to distinguish the individual
isomers, reporting
21
that “…the
19
F spectra of [β,γ-CHF-UTP]
22
failed to show two sets
of overlapping multiplets as observed by McKenna and co-workers for [β,γ-CHF-
dGTP]… at the basic pH of the McKenna studies compound [β,γ-CHF-UTP] broke
down.”
NH
O
O N
O
OH OH
O P N
OH
O
O
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
F
NH
O
O N
OH
P P
OH
OH
O O
F
HO
+
UMP 5'-M CHF-BP, 2 β,γ-CHF-UMP, 1

12
12
We found these observations to be surprising, and therefore have sought to
reproduce them. Here we report that a) two discrete sets of
19
F NMR multiplets are
readily observable for a ~1:1 mixture of 1 diastereomers (Figure 2.1.1) in D
2
O under the
basic conditions (pH 10) we prescribed for obtaining distinguishable
19
F NMR spectra for
a similar β,γ-CHF-dGTP diastereomer mixture; and b) the same β,γ-CHF-UTP
diastereomer mixture solution is stable at rt under these solution conditions (pH ~10) for
more than 24 h.

2.2 Results and Discussion
2.2.1 Preparative HPLC Purification and
1
H,
19
F, and
31
P NMR Analysis
Jakeman and Mohamady originally proposed a rapid synthesis for nucleoside
triphosphate bisphosphonate analogues from nucleoside monophosphates using activation
with trifluoroacetic anhydride (TFAA) and N-methylimidazole as base.
22
They
subsequently reported difficulties associated with the activation of UMP by this method.
23

As a result, we applied standard NMP-morpholidate coupling
14,15,20
to synthesize the
diastereomeric mixture of 1. After dual pass HPLC purification,
14
including modification
of the SAX HPLC purification step to remove a persistent, minor side product with
19
F
NMR δ near -216.90 ppm (see Results 2.2.4 and Experimental 2.4.4), the product was
treated with Chelex-100 to remove traces of paramagnetic metal ions. To confirm the
identity of the product, its
1
H,
19
F, and
31
P NMR (Figure A7-A9, respectively) were
determined and found generally to agree with those reported by Mohamady et al. for 1,
22

apart from discrepancy in the
19
F NMR spectral resolution as discussed below. The MS

13
13
analysis using an ESI probe operated in negative mode gave the expected m/z of 499.1
[M-H]
-
.
Our
1
H spectrum (500 MHz, D
2
O, pH 7.6, Figure A7) displays the same peaks as
reported previously
22
(the CHF signal is partially obscured by the large HDO peak at 4.79
ppm), and the reported J values for the H-5 and H-6 multiplets are consistent with ours
(7.9 vs. 8.2 Hz). Our
31
P NMR spectrum (202 MHz, D
2
O, pH 10.4, Figure A9) displays
similar J values for each phosphorus resonance, but the δ of P
β
is shifted downfield by ca.
4 ppm (a function of the pH
12
). We also observe additional P
α
peaks, attributed to
diastereomer peak resolution (Δδ 4.0 Hz), which was supported by the spectrum obtained
at 243 MHz (Δδ 4.6 Hz, predicted 4.7 Hz).

Under the conditions used for resolution of the
19
F NMR β,γ-CHF-dGTP
diastereomers,
14,15
our
19
F NMR spectrum for 1 (470 MHz, D
2
O, pH 10.4, Figure A3 and
A8) displays two sets of multiplets assigned to the diastereomers with δ
1
-216.91 and δ
2
-
216.96 (ddd, J = 67.5, 54.6, 45.0 Hz). The largest J value (67.5 Hz) is assigned to P
β

coupling, confirmed by the
31
P NMR spectrum (J
F,Pβ
= 65.3 Hz). The second J value (54.6
Hz; 55.6 Hz for P
γ
in the
31
P NMR spectrum) is in fair agreement with the Mohamady
value (59.8 Hz).
22
Although the CHF proton peak is partly obscured in our
1
H NMR, the J
value (45.0 Hz) is also in agreement with theirs (46.2 Hz).
22
In Mohamady’s study, the
19
F signal was found at δ -213.33, (reported as δ 213.33),
22
with coupling constant
assignments as follows: dd, J
F,P
β
85.6, J
F,P
α
62.1 Hz.
22
However, in their
31
P NMR
spectrum, they report
2
J
P
β
,F
62.8 Hz, suggesting that the smaller coupling constant

14
14
observed in the
19
F spectrum is not due to splitting by P
α
, but rather to splitting by P
β

consistent with our assignment.

2.2.2 Effect of pH and Counterion on
19
F NMR Signal

Figure 2.2.1 Effect of pH and counterion on
19
F NMR spectra of β,γ-CHF-UTP 1 (~1:1 diastereomers)
Compound 1 was purified by our previously described
14
dual-pass preparative HPLC method (D
2
O, 470
MHz, referenced to CFCl
3
). A) Series of spectra for samples near neutral pH (7.4, 8.3, 7.6, and 7.9 for NH
4
+
,
K
+
, TEAH
+
, and Na
+
, respectively); B) Series of spectra for samples at basic pH 10, (10.1, 12.7, 10.0, and
10.4 for NH
4
+
, K
+
, TEAH
+
, and Na
+
, respectively). The minor impurity peak (1-5% by
19
F NMR) that
appears upfield in the resolved spectra can be completely removed by adjustment of the SAX HPLC step
(see Experimental 2.4.4).

We next examined pH and counterion effects on the
19
F NMR spectra of 1. This
was of particular interest because as obtained, the synthetic product contains both
triethylammonium (TEAH
+
) and Na
+
counterions. The expected
19
F splitting pattern for a
single stereoisomer of 1 is predicted to be a doublet of doublet of doublets (ddd, 8 peaks)
at pH ≥ 10.
14,15,17
With our experimental 1:1 diastereomer mixture, we would therefore
expect up to 16 peaks depending on the degree of overlap (Figure A3). After exchange of
the TEAH
+
cation with Na
+
,

K
+
, or NH
4
+
using a preparative ion-
Counterion Effect
ppm
A B
NH4
+
K
+
TEAH
+
Na
+

15
15

Figure 2.2.2 Effect of spectrometer frequency (SF) and counterion effect on
19
F NMR spectra
A) Series of spectra at 470 MHz (D
2
O, referenced to CFCl
3
) of β,γ-CHF-UTP 1 (~1:1 diastereomers) for
NH
4
+
(J = 67.1, 56.1, and 45.4 Hz), K
+
(J = 67.2, 55.3, and 45.5 Hz), Na
+
(J = 67.5, 54.6, and 45.1 Hz); B)
Series of spectra at 564 MHz for NH
4
+
(J = 65.8, 56.0, and 45.4 Hz), K
+
(J = 67.5, 54.4, and 45.6 Hz) and
Na
+
(J = 65.8, 55.5, and 45.7 Hz). It can be seen that the appearance of the splitting pattern is sensitive to
small changes in the magnitude of Δδ.

Table 2.2.1 Effect of pH and counterions on
19
F NMR of β,γ-CHF-UTP 1

19
F NMR
Base Counterion
a
pH Δδ (Hz)
b
SF (MHz)
c
Δδ (ppm)
b
dG
d
Na
+
10.5 22.6 376 0.060
U Na
+
10.4 24.0 470 0.051
U Na
+
10.4 29.6 564 0.052
U K
+
12.7 23.5 470 0.050
U K
+
12.7 28.4 564 0.050
U NH
4
+
10.1 22.5 470 0.048
U NH
4
+
10.1 26.8 564 0.047
U
e
NH
4
+
<10
f
nd 235 nd
a
The ionic radii of for the Na
+
, K
+
, and NH
4
+
are 1.02, 1.38 and 1.50 Å, respectively.
24
Ionic radii are not
readily available for bulky alkylammonium cations, but instead rely on partial molar volume.
25
As a
representative example, the ionic radius of tetramethylammonium is 3.47 Å.
24

b
Δδ is the δ difference between the overlapping diastereomers given in Hz and ppm, respectively.

c
SF is the spectrometer frequency given in MHz.

d
Lit. value.
17

e
Lit. value.
22

f
The pH was not reported for the spectrum given but is likely below 10.
22
A simulation for SF = 235 MHz
(Figure A15) assuming a line width at half height of 8 Hz and Δδ 12 Hz at pH 10 generated a 12-peak
multiplet with substantially resolved outer peaks.

Counterion Effect
A B
NH4
+
K
+
Na
+
ppm
Δδ = 22.5 Hz
Δδ = 23.5 Hz
Δδ = 24.0 Hz
Δδ = 26.8 Hz
Δδ = 28.4 Hz
Δδ = 29.6 Hz

16
16
exchange resin column,
19
F (470 MHz) spectra of 1 in D
2
O could not be resolved into
contributions from discrete isomers at pH < 10 (Figure 2.2.1A). For each of these
countercations, titration of the NMR sample solution to a pH ≥ 10 revealed discrete
19
F
NMR spectra for the two diastereomers (14 of 16 peaks, Figure 2.2.1B and Table 2.2.1),
with the exception of TEAH
+
, which gave broad, unresolved multiplets. Conditions that
did not yield diastereomeric differentiation were omitted from Table 2.2.1.

Figure 2.2.3
19
F NMR of synthetic 1 (purified by our published method)
15
in D
2
O at pH 10.1
A) Spectrum for synthetic 1:1 diastereomer mixture. The individual stereoisomers have: δ –216.66 (red)
and –216.70 (blue), ddd,
2
J
F,Pβ
= 65.8 Hz,
2
J
F,Pγ
= 56.0 Hz,
2
J
F,H
= 45.0 Hz; B) Calculated individual
19
F
NMR (blue) for one of the two diastereomers; C) Calculated individual
19
F NMR (red) for the other
diastereomer. NMR spectra were simulated using spin simulation on MestReNova (Mnova 8.1.4)
15

ppm
A
B
C

17
17
Figure 2.2.1 depicts the dramatic transition from an unresolved, broad multiplet to
a sharp, distinct set of multiplets when the pH is raised from 7 to 10, which narrows the
line width to ~ 8 Hz. At 470 MHz, the spectrum at pH 10 exhibits 14 of the expected 16
peaks for each of the three countercations due to partial overlap (Figure 2.2.1B). To
assign Δδ and J values for the spectra, they were also measured at 564 MHz (Figure
2.2.2B). To further validate the assignment, the spin systems of the individual
diastereomers were simulated using MestReNova (Mnova 8.1.4). The calculated spectra
display satisfactory congruence with the experimental spectra (Figure 2.2.3), yielding a
Δδ of 0.05-0.06 ppm for the mixed 1 diastereomers.

2.2.3 Qualitative Decomposition of 1 by
19
F NMR Analysis
Although Jakeman et al. asserted that 1 decomposed under the basic conditions
we previously recommended to obtain resolved
19
F (and
31
P) spectra of β,γ-CHF-dGTP
diastereomers, no direct evidence or specific experimental conditions were given and the
pH of the NMR samples was not provided.
21,22
Curiously, the
19
F NMR of their purified
product does not show decomposition in their synthesis of 1, despite adjustment of the
pH to 9.5 prior to lyophilization. Even though the actual pH was not specified, it can be
inferred that the pH of their NMR sample was below pH 7.5, based on a series of
31
P
spectra we acquired over the range pH 7-8 (δ P
β
2.78; pH 7.4; NH
4
+
, Figure A10). Most
importantly, in our hands 1 was quite stable, without detectable NMR decomposition
under “basic” conditions (e.g., pH 10.1, counterion NH
4
+
, 48 h at rt, Figure 2.2.4). Even
after 72 h at rt, only a very slight decomposition to 2 (2.0%) was detected by
19
F NMR
(Figure 2.2.4).

18
18

Figure 2.2.4 Determination of β,γ-CHF-UTP 1 (~1:1 diastereomer mixture) stability at pH 10 by
19
F NMR
(D
2
O, 470 MHz, referenced to CFCl
3
)
A) Spectrum acquired prior to final preparative RP-C
18
HPLC remove 2, revealing unreacted 2 at δ -214.95,
1 at δ -216.62 in D
2
O solution, with the pH adjusted to 10.4; B) Spectrum acquired after removal of 2 by
the RP-C
18
HPLC purification step, pH adjustment to 10.1 and then 24 h at rt. No evidence of hydrolysis to
2 can be seen.

2.2.4 Effect of Reactant Ratio (CHF-BP:UMP 5’-M) and HPLC Methods on Yield and
Purity of 1
Using the standard conjugation of activated UMP 5’-M with the tri-n-
butylammonium salt of CHF-BP (2) persistently resulted in formation of unknown
byproduct. Even after subsequent dual-pass purification by HPLC, trace amounts of the
byproduct (1-5% by NMR) were detected (Table 2.2.2 and Figure A8). The stoichiometry
of the reactants was modified, using 6:1, 3:1, and 1:1 ratios of CHF-BP:UMP 5’-M, in an
attempt to minimize or eliminate formation of byproduct. These reaction mixtures were
then purified by the original preparative SAX HPLC method. In addition, another set of
A
B
ppm

19
19
experiments was employed to completely remove the byproduct during preparative SAX
HPLC by using the original purification method but fractionating the product peak (P1,
P2, and P3, see Experimental, Figure 2.4.1). The separated fractions were evaporated by
vacuum and their
19
F NMR spectra were acquired.

Figure 2.2.5 Effect of CHF-BP:UMP 5’-M ratio on yield and purity of 1
A) Preparative SAX HPLC trace of product mixture for 6:1 CHF-BP:UMP 5’-M; B) Preparative SAX
HPLC trace of product mixture for 1:1 CHF-BP:UMP 5’-M; C) Effect of CHF-BP:UMP 5’-M ratio on
product yield calculated from % peak areas in HPLC traces (preparative SAX HPLC trace for 3:1 CHF-
BP:UMP 5’-M reactant ratio is shown in Figure A1); D) Key to labels for plots in panel C.

Figure 2.2.5 and Figure A1 are chromatogram traces that reveal the effect of
reactant ratio on product formation. From a purely quantitative observation, Figure 2.2.5
panels A and B, and Figure A1 reveal that product peak 1 (referred to as d in Figure
2.2.5D) grows with increasing the stoichiometric amount of 2. This trend is reflected in

20
20
panel C of Figure 2.2.5. It appears that increasing bisphosphonate may inhibit dimer
formation of the activated morpholidate. Yields for each reaction mixture are reported in
Table 2.2.2. Using a 6:1 CHF-BP:UMP 5’M gave a 65% yield. This was in stark
comparison to 1:1 ratio, which led to a yield of 14%. Whereas 3:1 led to a 20% yield.

Figure 2.2.6 Purity of 1 (~1:1 diastereomers,
19
F NMR) after preparative SAX HPLC using modified
HPLC method
Experimental (black) and simulated individual diastereomer
19
F NMR spectra (blue and red) of the product
from 6:1 CHF-BP:UMP 5’-M. A) P3 Fraction obtained using modified preparative HPLC method (98.5%);
B) Total peak obtained using modified preparative SAX HPLC (99.5%); C) Preparative SAX HPLC trace
corresponding to A. Negative ion ESI-MS results were identical for each sample (m/z 499 [M-H]
-
).
Fractionation is defined in Figure 2.4.1.

Product peak 1 was fractionated into three parts during SAX preparative
chromatography (shown in Figure 2.4.1). The fractions were rotavapped and taken up in

21
21
D
2
O, then a
19
F NMR spectrum was acquired. Results from the fractionation are shown in
Figure 2.2.7A-C and the amounts of impurity were calculated from the
19
F NMR and
listed in Table 2.2.2. Although the NMR spectrum of fraction 1 (P1) appeared to have the
least amount of impurity, this purification technique was not sufficient to remove the
unknown impurity entirely.
Table 2.2.2 Calculated purity and yield of 1 obtained for different ratios of CHF-BP:UMP 5’-M; original
and modified stage 1 HPLC methods

Integration

Reactant Ratio Product
a
Impurity
b
Purity % Yield
3:1 CHF-BP:UMP 5’-M T 2.0 0.31 86.6% 20%
6:1 CHF-BP:UMP 5’-M T* 2.0 0.01 99.5%
6:1 CHF-BP:UMP 5’-M T
c
nd nd 96.0% 65%
6:1 CHF-BP:UMP 5’-M P1 2.0 0.04 98.0%
6:1 CHF-BP:UMP 5’-M P2 2.0 0.13 93.9%
6:1 CHF-BP:UMP 5’-M P3 2.0 0.15 93.0%
1:1 CHF-BP:UMP 5’-M T 2.0 0.22 90.1% 14%
1:1 CHF-BP:UMP 5’-M P1 2.0 0.17 92.2%

a,b
Cf. Figure 2.2.6 and 2.2.7.
c
A combined peak fraction was not acquired for 6:1 CHF-BP:UMP 5’-M, but the weighted, theoretical
purity was calculated based on the yield and purity for each of the three time-dependent fractions collected.

An alternate SAX purification technique was performed on P3 and labeled T*
(Figure 2.2.6). Washing the column with several portions of water creates a method to
separate the impurity from product peak (Figure 2.2.6). Based on the
19
F NMR of the
dried fraction taken up in D
2
O, the amount of impurity present in sample was around 0.05%
based on integration of the NMR. This indicates that the majority of the impurity can be
successfully eliminated using the new purification technique, fully elaborated in the
experimental section 2.4.4.
19
F NMR from these samples are shown in Figure 2.2.6B and
Figure 2.2.7D.

22
22

Figure 2.2.7
19
F NMR of ~1:1 diastereomer mixture of 1
Experimental (black) and simulated individual diastereomer
19
F NMR spectra (blue and red) of isolated 1
prepared using a 6:1 CHF-BP:UMP 5’-M. A) First fraction (P1; 98%); B) Second fraction (P2; 93.9%); C)
Last fraction (P3; 93%); D) Combined peak fractions (T*) using modified SAX HPLC method (99.5%).
Negative ion ESI-MS gave m/z 499 [M-H]
-
for each sample (simulation parameters are given in Figure
2.2.3).

2.2.5 Discussion
Several monofluoromethylene (CHF) phosphonate compounds,
26-30
including β,γ-
CHF-dNTP analogues,
13,15,16
have demonstrated utility as probes in enzyme systems. The
lack of fluorine in natural systems
31
creates a convenient and readily available nuclear
spin label for fluorinated analogues, which can use
19
F NMR to readily detect
stereoselective binding or consumption.
19
F and
31
P NMR spectroscopy have been
extensively used as tools to determine pH and to explore metal cation effects, due to the
sensitivity of the chemical shifts of these nuclei to the local chemical environment.
32,33

19
F
A C
B D
Δδ = 29.2 Hz
Δδ = 25.3 Hz
Δδ = 25.2 Hz
Δδ = 26.1 Hz
ppm ppm
ppm ppm

23
23
NMR is an attractive alternative to
31
P NMR, due to its greater sensitivity relative to
1
H
(83% vs. 6%), as well as the usual absence of interfering fluorine signals in natural
systems. In addition to producing a stronger signal at an equivalent concentration,
19
F
NMR typically offers a more sensitive response to the local environment compared to
31
P
NMR and the detection of subtle structural changes.
34

19
F NMR probes have been frequently utilized as indicators of local pH.
33

Between pH 7-9.5, the δ
F
of 1 shows a linear downfield-shifted pH-dependence with little
or no countercation effects (Figure S4A), suggesting that negative charge generation at
the last ionization of the “triphosphate” moiety (pK
a4
7.5)
21
contributes significantly to
this change in δ. When the pH significantly exceeds pK
a4
(pH > 10) little or no
dependence of δ on pH is observed (Figure S4B). For our present purposes of resolving
β,γ-CHF-diastereomer resonances, a similar pH effect is observed. At pH 7-8, the line
width is >> 10 Hz presumably due to exchange of the remaining γ-phosphate OH
proton,
32
in close proximity to the CHF stereocenter.
Although the individual diastereomers are not yet unequivocally assigned, it is
possible that the more downfield peak in
19
F NMR belongs to the (S)-CHF isomer by
analogy with the corresponding dGTP analogue assignment.
17

2.3 Conclusion
In conclusion, we have re-investigated the
19
F NMR and stability properties of 1,
synthesized (for the first time) by NMP-morpholidate route.
20
Our results establish
unequivocally, that under our previously reported conditions, the spectra of the individual
diastereomers of 1 are easily distinguishable. Furthermore, at the “basic” pH (~10)

24
24
required to resolve the spectra by decreasing the line width, 1 was stable for 24 h at rt
(and indeed, showed little change even after 72 h, Figure 2.2.4).

2.4 Experimental
2.4.1 Materials and Methods
Uridine 5'-monophosphate disodium salt was purchased from Sigma.
Fluoromethylenebis(phosphonic acid) (CHF-BP) (2)
35,36
and uridine 5'-monophosphate
morpholidate (UMP 5’-M) (3)
20
were prepared according to literature procedures. All
other reagents were purchased from commercial sources and used as obtained, unless
specified otherwise.
1
H,
19
F, and
31
P NMR spectra were obtained on 500 and 600 MHz (3-
Channel) NMR spectrometers. Multiplicities are quoted as singlet (s), doublet (d), triplet
(t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of doublets
(ddd), doublet of triplets (dt) or broad signal (br). All chemical shifts (δ) are given in
parts per million (ppm) relative to internal CD
3
OD (δ 3.34,
1
H NMR), D
2
O (δ 4.79,
1
H
NMR),

external 85% H
3
PO
4
(δ 0.00,
31
P NMR) and 80% CFCl
3
(δ 0.00,
19
F NMR).
31
P

NMR spectra were proton-decoupled, and
1
H,
19
F, and
31
P coupling constants (J values)
are given in Hz. Low-resolution mass spectrometry (LRMS) was performed on a mass
spectrometer equipped with an ESI source operated in the negative ion mode.

2.4.2 Synthesis of 1
The compound as an (R/S)-CHF mixture was synthesized using the standard
conjugation of activated uridine 5’-monophosphate morpholidate (UMP 5’-M)
20
with the
tri-n-butylammonium salt of 2.
14
Uridine 5’-monophosphate (UMP) disodium salt (265

25
25
mg, 0.72 mmol) was treated with Dowex 50WX8 (200-400 mesh, H
+
form) to generate
the free acid, which was evaporated to dryness. The residue was dissolved in 10 mL of t-
butanol (t-BuOH:H
2
O (2:1). The pH was adjusted to 2 with 0.1 M HCl, a stir bar was
added to the reaction flask and morpholine (distilled, 311.5 µL, 3.60 mmol, 5 equiv) was
added dropwise using a 500 µL gas-tight syringe. After stirring for 30 min, the pH of the
mixture was measured and readjusted to 7.5 using 0.1 M HCl. The solution was brought
to reflux. N,N’-Dicyclohexylcarbodiimide (DCC, 594 mg, 2.88 mmol, 4 equiv) was
dissolved in 5 mL of t-BuOH, and the resulting solution was divided into ten aliquots of
500 µL. An aliquot was added dropwise every 10 min to the refluxing solution. After an
additional 20 min, the progress of the reaction was checked by
31
P NMR (UMP 5’-M
δ 7.25 (s); UMP δ 3.59 (s)). The mixture was cooled to rt and precipitated N,N’-
dicyclohexylurea was removed by vacuum filtration. The filtrate was evaporated to
dryness, and the residue taken up in 10 mL H
2
O. After extraction with ether (3 x 10 mL),
the aqueous layer was rotavapped and dissolved and coevaporated with dioxane (3 x 5
mL) yielding a dry white powder of the N,N’-dicyclohexyl-4-morpholinecarboxamidine
salt (341 mg, 69%). It should be noted that over time degradation of product to UMP was
observed, but was accounted for using UV and
1
H NMR analysis for stoichiometric
determination.
Compound 2 was prepared from tetraisopropyl methylenebis(phosphonic acid)
according to the literature procedure.
35,36
2 (231 mg, 0.119 mmol, 3 equiv) was dissolved
in 5 mL EtOH:H
2
O (1:1). The pH was slowly adjusted to 4.5 by addition of 10%
tributylamine (NBu
3
) in EtOH, and the solution was allowed to stir for 30 min at rt. The
solvent was evaporated under vacuum and the residue dried by co-evaporation with

26
26
anhydrous DMF (3 x 3 mL). Then a solution of UMP 5’-M (28 mg, 0.04 mmol, 1 equiv)
in anhydrous DMSO (2 mL) was added and the mixture stirred for 72 h at rt under N
2
.
The reaction mixture was then passed through a column (25 mm x 15 cm) of a strong
anion exchange (SAX) resin eluted with a gradient method (0-10 min, 0-60%; 10-15 min,
60%; 15-25 min, 60-100%) of 0.5 M triethylammonium bicarbonate (TEAB) buffer pH
7.0 with a flow rate of 8.0 mL/min (Figure A1). To eliminate the minor impurity in
19
F
NMR (δ near -216.9 ppm), a modified
14,15
gradient method (0-20 min, 0%; 20-35 min,
60-100%; 35-45 min, 100%) of 0.5 M TEAB buffer pH 7.0 with a flow rate of 8.0
mL/min was used with the SAX resin column. The desired compound eluted at 30.0 min
(UV detection at 259 nm; HPLC trace and
19
F NMR spectra, Figure 2.2.6). The fractions
containing it were evaporated to yield the product as a TEAH
+
salt.
19
F and
31
P NMR
analysis revealed traces of 2 (D
2
O; pH 8.6; at δ

10.67 (d)) in the purified product.
The product was next dissolved in 2 mL of 0.1 M TEAB (pH 7.0) and purified on
a C
18
reversed-phase (RP-C
18
) preparative column (5 µm, 250 mm x 21 mm) by isocratic
elution with 3.75% CH
3
CN in 0.1 M TEAB pH 7.0 at a flow rate of 8.0 mL/min . The
product 1 eluted at 18.7 min (Figure A2). Evaporation of the corresponding collected
fractions gave 6.3 mg (7.8 µmol by UV, 20%) of a clear film as a TEAH
+
salt. ESI-MS:
m/z 499 [M-H]
-
.
1
H NMR (500 MHz, D
2
O, pH 7.6, Figure A7) δ 7.99 (d, J = 8.2 Hz, 1H),
6.01 (t, J = 6.5 Hz, 2H), 4.53 – 4.35 (m, 2H), 4.31 (s, 1H), 4.26 (s, 2H).
19
F NMR (470
MHz, D
2
O, pH 10.4, Figure A8) δ -216.91 and -216.96 (ddd, J = 67.5, 54.6, 45.0 Hz, Δδ
24.0 Hz).
31
P NMR (202 MHz, D
2
O, pH 10.4, Figure A9) δ 7.46 (dd, J = 55.6, 14.3 Hz),
5.26 (ddd, J = 65.4, 28.3, 14.3 Hz), -10.55 (d, J = 28.7, Δδ
P
α 4.0 Hz).

27
27
2.4.3 Effect of Countercation and pH on
19
F NMR Spectra of 1
The dual pass HPLC-purified product was further treated with Chelex-100 to
remove trace metals. The
1
H,
19
F, and
31
P NMR spectra (D
2
O, pH 7.6) were acquired and
did not manifest distinguishable peaks for the individual diastereomers. Following
treatment with Chelex-100, the compound was reisolated by evaporation and then
dissolved in 2 mL of H
2
O, and the resulting solution divided into four aliquots. Dowex
50WX8 (200-400 mesh, H
+
form) resin was converted to alternate cation forms by
treatment with 1 M HCl, rinsing with H
2
O until the eluate was neutral (pH paper), and
subsequent treatment with the desired cation as a 10% (w/w) of its hydroxide salt solution
in H
2
O (i.e., NaOH for Na
+
, KOH for K
+
, and NH
4
OH for NH
4
+
), followed by additional
washing with H
2
O until the eluate was again neutral. A 500 µL aliquot of 1 as described
above was passed into each exchange-resin column, which was next washed with several
portions of H
2
O. The samples were then rotavapped to yield four samples with a
different countercation. NMR samples were prepared by adding 500 µL of D
2
O (yielding
nucleotide concentrations of ca. 3.9 mM) and the pH (NMR tube electrode) was
determined prior to acquiring the
1
H,
19
F, and
31
P NMR spectra. The spectra of 1 near
physiological pH did not result in resolved diastereo-peaks, irrespective of the counterion
used.
Immediately after NMR acquisition, the pH of the NMR sample was adjusted to
≥10 using a 10% (w/w) solution of the counterion hydroxide (or TEA) in D
2
O. Because
the nucleotide concentrations in the NMR sample were low, the pH adjustment required
minimal added solution. After determining the sample pH, spectra were reacquired for

28
28
each countercation sample. Overnight spectra were acquired on both 500 and 600 MHz
spectrometers for each counterion sample to clarify assignment of Δδ vs. J values.

2.4.4 Effect of Reactant Ratio (CHF-BP:UMP 5’-M) and HPLC Methods on Yield and
Purity of 1
The synthesis of 1 was carried out at 6:1, 3:1, and 1:1 ratios of CHF-BP:UMP 5’-
M. The stoichiometry with respect to UMP 5’-M was determined by UV-Vis analysis and
1
H NMR spectroscopy, with accounting for any UMP 5’-monophosphate present. The
stoichiometry with respect to CHF-BP was determined by
19
F NMR, using trifluoroacetic
acid (1-5 mM), together with a capillary tube containing 25 µl of 20 mM trifluoroethanol
as a standard to construct a calibration curve. Standard Moffatt coupling
20
was conducted
at the different reactant ratios as described in the manuscript. The results are summarized
in Figure 2.2.5 and Table 2.2.2.

Figure 2.4.1 Preparative SAX HPLC fractionation of 1 peak
Where T is combined peak fractions.

Product mixtures were separated by preparative SAX HPLC (Figure A1). In
addition, three time-dependent fractions of the peak corresponding to 1 were collected,
where P1 refers to the first fraction, P2 the second fraction, and P3 the last fraction

29
29
(Figure 2.4.1). The results are summarized in Table 2.2.2. A modified elution condition
was also applied to an aliquot of the 6:1 CHF-BP:UMP 5’-M reaction mixture wherein
the column was first washed with water for 20 min, followed by 60% 0.5 M TEAB buffer
(pH 7.0; 8.0 mL/min; gradient 60%-100% 0.5 M TEAB buffer for 10 min; 100% 0.5 M
TEAB buffer for 5 min).

2.5 Chapter References

(1) Engel, R. Chem. Rev. 1977, 77, 349.
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Beabealashvilli, R.; Hamilton, C.; Roberts, S. J. Chem. Soc., Perkin Trans. 1 1999, 1039.
(9) Martynov, B. I.; Shirokova, E. A.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A.
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(10) Alexandrova, L. A.; Skoblov, A. Y.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A.
A. Nucleic Acids Res. 1998, 26, 778.
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Biophys. Acta 2010, 1804, 1223.
(12) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin Trans. 1 1984,
1119.
(13) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martínek, V.;
Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E. Biochemistry
2007, 46, 461.
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Pedersen, L. C.; Beard, W. A.; Wilson, S. H. J. Am. Chem. Soc. 2007, 129, 15412.
(15) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.;
Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617.
(16) Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard,
W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Biochemistry 2012, 51, 8491.
(17) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.;
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83, 659.
(21) Beaton, S. A.; Jiang, P. M.; Melong, J. C.; Loranger, M. W.; Mohamady, S.;
Veinot, T. I.; Jakeman, D. L. Org. Biomol. Chem. 2013, 11, 5473.
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31
31
5’-β,γ-CHF-ATP Diastereomers: Synthesis and Fluorine- Chapter 3.
Mediated Selective Binding by c-Src Protein Kinase
2

3.1 Introduction
ATP is ubiquitous in biological systems where it plays a central role in energy
storage, transduction, and utilization. Modification of the triphosphate moiety by
replacing the oxygen between the β- and γ-phosphate with a methylene (CH
2
)
1
creates
nonhydrolyzable inhibitors for enzymes that cleave the γ-phosphate, such as ATPases and
protein kinases.
2,3
It was proposed many years ago that α-fluorination of the bridging
methylene affords pCHFp analogues (CXY, X, Y = HF) that are more isoacidic with
ATP than the corresponding pCH
2
p analogues.
4-6
Besides adjusting the electronegativity
of the bridging carbon to more closely resemble that of the bridging oxygen atom with
minimal steric perturbation, the general absence of the fluorine atom from natural
biological systems allows it to function as a bioorthogonal and readily detectable probe
for
19
F NMR studies. At the same time, replacement of the pOp oxygen by CHF
introduces a new chiral center into the nucleotide, resulting in two diastereomeric forms
of β,γ-CHF-ATP that offer the potential advantage of comparing biochemical properties
of two enzyme inhibitors essentially identical in structure except with respect to the
orientation of the C-F fluorine within a protein binding site. By switching a defined

2
Reproduced with permission from Hwang, C. S.; Kung, A.; Kashemirov, B. A.; Zhang,
C.; McKenna, C. E. Org. Lett. 2015, 17, 1624. Copyright 2015 American Chemical
Society.

32
32
fluorine orientation in an otherwise similarly bound ATP analogue, a unique platform for
investigating weak F bonding interactions
7-11
with proteins is thus made available.
Beginning in the late 1990s, reports began to appear
12-15
suggesting that the CHF-
stereochemistry of α-fluorinated monophosphonates can affect their binding to enzymes,
and interest in this phenomenon has continued to grow.
9,16-18
However, the preparation of
the individual diastereomers of β,γ-CHF-ATP itself has remained an unmet challenge,
preventing this unique set of enzyme probes from realizing their original potential.
Standard approaches to the synthesis of β,γ-CXY ATPs such as conjugation of the
appropriate methylenebis(phosphonic acid) salt with a 5’-activated AMP
19
(Figure 3.1.1),
result in a mixture of both diastereomers when X ≠ Y.

Figure 3.1.1 Standard synthesis of β,γ-CXY-ATPs

β,γ-CXY and α,β-CXY desoxyNTP analogues have recently demonstrated their
utility as probes of the ground and transition states of DNA polymerase β (pol β).
20-24
As
part of these studies, the individual synthesis of all four β,γ-CHF and β,γ-CHCl dGTP
diastereomers was achieved, with the absolute configurations assigned by X-ray
crystallography of their ternary complex with DNA and pol β.
25
These analogues were
found to exhibit significantly different K
d
and k
pol
values with the enzyme, modulated
only by the position of the halogen atom substituent at the CXY carbon.
24
Furthermore,
the
19
F NMR spectra of these CHF diastereomers were found to be resolvable and useful
N
N
N
N
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
NH
2
OH
N
N
N
NH
2
N
O
OH
O
P
OH
O
OH
OH
P
OH
P
HO
OH
O O
+
AG
XY XY
12: X,Y = H,F (mixture)
15: X,Y = H
16: X,Y = F
AG = activating group

33
33
to monitor their stereoselective turnover catalyzed by the enzyme.
20,24
Early work on
synthetic β,γ -CHF-ATP mixtures
26
and a recent paper on β,γ-CHF-UTPs
18
reported
inability to distinguish the ribosylnucleotide CHF-stereoisomers by
19
F NMR, although
the former observation was not confirmed when the fluorinated ATP derivative was
prepared by an alternate method.
27
These results prompted us to attempt the long-awaited
synthesis of the individual β,γ-CHF-ATPs.

3.2 Results and Discussion
3.2.1 Preparation of Individual CHF-ATP Diastereomers
Our strategy was based on the key chiral bisphosphonate synthons 9a and 9b
(separable by preparative HPLC) prepared in eight steps from tetraisopropyl
methylenebis(phosphonate) 1;
25
however, the synthesis of 9a and 9b was modified at
several steps, improving the yield significantly (Scheme 3.2.1). In the first modification,
monodemethylation of 4 (previously prepared with NaI
25
) with triethylamine
28,29
under
reflux raised the isolated yield of 5 to 90% with a substantial reduction in reaction time to
10-20 min.
Scheme 3.2.1 Modified synthesis of (S)-mandelic acid morpholinamide (R)/(S)-monoesters 9a and 9b
P P
OiPr
OiPr
O
iPr O
iPr O
O
P P
OMe
OMe
O
MeO
MeO
O
F
P P
O
-
OMe
O
MeO
MeO
O
F
P P
O
OMe
O
MeO
MeO
O
F
(S) (S) OMe
O
1. DOWEX H
+
2. DIAD, PPh
3
Dioxane, N
2
HO
(R) (R)
OMe
O
P P
O
OH
O
HO
HO
O
F
(S) (S) OH
O
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
1. DCC
Morpholine
t-BuOH, ∆
O
O
1 4 5
6a/6b 8a/8b 9a/9b
TEA
∆
Et
3
NH
+
96%
65%
90%
2. C
18
HPLC

34
34

Compound 5 was then transformed into the stereoisomer ester mixture 6a/6b,
purified on a silica gel column replacing the original preparative TLC procedure for
convenient scalability.
25
In the second important modification, after conversion of 6a/6b
to the acids 8a/8b,
25
the latter were derivatized to the chiral dimorpholidates 9a/9b using
a nonaqueous solvent system, shortening the reaction time from several hours to <1 h
after addition of DCC and also modestly increasing the yield from 87% to 96%,
additionally aided by a more performant reversed-phase HPLC column.
Prior to separation and purification of the individual isomers, reaction mixture
9a/9b taken up in D
2
O showed discrete, but overlapping multiplets in the
19
F (470 or 564
MHz, pH 8.8-9.3, Figure B7C,D and B11, Appendix B) and
31
P NMR spectra (202 MHz,
pH 8.8-8.9, Figures B9 and B13, Appendix B). Interestingly, the same sample dissolved
in CD
3
OD gave a clean separation between the isomer multiplets in
19
F NMR (470 or 564
MHz, Figure B7A-B and B10) but the relative position of the two resonances was
inverted, which was confirmed at higher spectrometer frequency (564 MHz) and by
spiking with 9a (Figure B10-11, Appendix B).
The separated individual isomers in acid form (10a, 10b)
25
were coupled
conventionally with AMP-morpholidate
19
(AMP 5’-M, 14) and then treated to remove the
protecting groups as described previously
25
(Scheme 3.2.2). Gratifyingly, coupling of 14
to the individual synthons did not require additional protection of the 2’,3’-hydroxyl
groups of the ribose sugar. The (R)/(S)-CHF chirality of the separated and purified
dimorpholidated chiral synthons (9a and 9b) was assigned by HPLC and NMR analysis
in comparison with the literature values.
25

31
P NMR spectral splitting patterns in D
2
O

(Figures B20, B24), where 9a exhibits second order splitting, additionally distinguish the

35
35
two isomers. (The original assignments of the absolute configurations of 9a and 9b were
established retrosynthetically from the X-ray structure of (R)-β,γ-CHF-dGTP complexed
to DNA pol.
25
)
Scheme 3.2.2 Synthesis of 12a and 12b from 9a and 9b, respectively

3.2.2 Synthesis of CXY-ATP Analogues
β,γ-Methylene (CH
2
)-, mixed β,γ-CHF- and β,γ-difluoromethylene (CF
2
)-ATPs
were synthesized by conjugating 14 with the appropriate bisphosphonate (Figure
3.1.1).
1,6,19
A discrete
19
F and
31
P NMR spectrum for each diastereomer 12a/12b in a
synthetic mixture (12) was observed (Figures B44-45, Appendix B), contrary to early
observations
26
but consistent with prior observations for β,γ-CHF-dGTP
20,22,25
and -UTP
analogues.
30
The discrepancy may be attributed to the crucial role of the sample pH
31
(≥
(R) (R)
P P
O
OH
O
HO
HO
F
O
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
(R) (R)
P P
O
OH
O
HO
N
O
O
F
(S) (S)
O
N O
(S) (S)
O
N O
1. DOWEX H
+
2. 1 M HCl
N
N
N
NH
2
N
O
OH
O
P
OH
HO
N
N
N
N
O
OH
O
P
N O
DCC, Morpholine
t-BuOH:H
2
O (1:1)
DMSO
30% Pd/C, H
2
0.1 M TEAB: MeOH (1:1), pH 8
OH
O
O
O O O
O O O
or (S)
or (S)
or (R)
or (R)
OH
OH
NH
2
OH
NH
2
OH
9a/9b
10a/10b
11a/11b
12a/12b
∆
78% (
31
P NMR)
26-30%
85-87%
quant
yield
13
14

36
36
10) and possibly, the nature of the countercation (e.g., NH
4
+
gives broadened line
widths).
30,32
A 3:1 mixture of the separated diastereomers was made and its
19
F and
31
P
NMR spectra were measured to confirm (Figures B55-56, Appendix B) that 12a (the (S)-
CHF-isomer) resonates more downfield in the
19
F NMR and has P
α
and P
β
, more
downfield and upfield, respectively in the
31
P NMR.
25
To further validate the assignment
of each pairwise resonance
25
to a specific diastereomer, the
19
F NMR spectra were
simulated
33
and compared to that of the experimental mixture of isomers (Figure B57).
22,30
The ability to discern each diastereomer by
19
F and
31
P NMR provides a convenient
method for determining selective substrate consumption by a selective enzyme.
18,24

3.2.3 Inhibition Activity on c-Src Kinase by β,γ-CXY-ATP Analogues
In search of an enzyme interaction that might be stereospecific for 12a vs 12b, we
examined a protein kinase. Protein kinases catalyze the transfer of a phosphoryl group
from ATP to substrate proteins. As a major mode of post-translational modification, the
phosphorylation event catalyzed by protein kinases regulates fundamental cellular
processes such as cell proliferation, differentiation and motility. Aberrant expression or
activation of these enzymes can often lead to a variety of human diseases.
34-38
For
example, overexpression of Src tyrosine kinases is frequently found in cancerous
tumors.
3,34,39-41
Understanding the catalytic mechanism of Src and its role in oncogenesis
is critical for design of therapeutic agents to target the diseases associated with its
disregulation.
3,37-40

37
37
Table 3.2.1 Effect of fluorination of β,γ-CH
2
-ATP on inhibition of c-Src kinase
IC
50

X,Y = H
2
15 (µM)
X,Y = HF
12 (µM)
X,Y = F
2

16 (µM)
mean ± SE (n)
a
145 ± 25 (3) 47 ± 7 (6) 46 ± 7 (3)
a
SE = standard error; n = number of replicates; variance test revealed no significant deviation prior to running an
ANOVA. Differences (ANOVA, F (2, 6) = 12, p = 0.008 < 0.05) are statistically significant.

Table 3.2.2 Inhibition of c-Src kinase by β,γ-CHF-ATP diastereomers
IC
50

X,Y = H,F
12a (µM)
X,Y = F,H
12b (µM)
mixture (~1:1)
12 (µM)
mean ± SE (n)
a
23 ± 4 (6) 150 ± 8 (5) 47 ± 7 (6)
a
SE = standard error; n = number of replicates; variance test revealed no significant deviation prior to ANOVA.
Differences (ANOVA, F (2, 12) = 105, p < 0.001) are statistically significant.

We tested the β,γ-CXY-ATP analogues 12, 15, and 16 (Figure 3.1.1) as inhibitors
against the kinase domain of Src (c-Src, E.C. 2.7.10.2) and determined their IC
50
values
using the radioactive disk assay.
42
The data from the inhibition assays show that
fluorination at the β,γ-position increases inhibitor potency (Table 3.2.1). β,γ-CH
2
-ATP 15
was already shown to be a competitive inhibitor of c-Src kinase with respect to ATP and
a noncompetitive inhibitor versus the peptide substrate.
2
The K
m

for ATP and c-Src kinase
is reported at around 80 μM.
43
The Src kinase inhibition assay IC
50
data (Table 3.2.2)
indicate a 6-fold selectivity for the (S)-CHF-ATP diastereomer 12a. The relative potency
of 16 appears to exclude a steric origin for the selectivity exhibited by the enzyme.

3.2.4 Molecular Docking Calculations to Evaluate Stereoselectivity Exhibited by c-Src
Kinase Active Site with β,γ-CXY-ATP analogues
We hypothesize that the specific orientation of the CHF-fluorine in 12a makes
possible a specific dipolar interaction, adding additional stabilization energy to the

38
38
complex, which is reflected in the IC
50
difference. To examine this possibility, in silico
molecular docking simulations using AutoDock Vina 1.1.2
44
were performed to explore
potential protein-ligand interactions (Figure 3.2.2). Although our kinase assays were
performed using chicken Src kinase, we chose a published X-ray crystal structure of
human Src (2SRC) for molecular docking calculations due to the higher resolution
available (1.8 Å vs 2.3 Å for chicken Src (3DQX)). The human and chicken Src proteins
share 99% sequence identity (using BLASTP 2.2.30+
45
) and are identical within the
active site.
46

Figure 3.2.1 Superimposition for A) 16 and B) 12a and 12b docked into the active site of human Src
kinase using AutoDock Vina 1.1.2 and X-ray crystallographic data for the apoprotein (2SRC) including Mg
(shown as green sphere).
The (S)-fluorine in both compounds is oriented toward Arg388. The (R)-fluorine is oriented toward the
pocket entrance, with no protein atoms closer than 3.8 Å. The figures were generated using Accelerys
Discovery Studio.
47

The calculations suggested interactions of both the (S)-fluorine of 16 and the
γ−phosphate with the active site Arg388 (the latter observation is consistent with the
crystal structure of c-Src kinase
48
): the (S)-fluorine atom of 16 is located 3.0 Å from the
(R)-F
R388
3.2 Å
C277
(S)-F
(R)-F
R388
3.0 Å
C277
A B
(S)-F

39
39
guanidinium N of Arg388. (Figure 3.2.2A) Additionally, Lys295 stabilizes two
phosphates, and Asp404 interacts with a structural water and Mg
2+
. In contrast,
interactions of the (R)-fluorine with residues in the active site were not identified: the
nearest group was the amido NH of Cys277 at 3.6 Å, too distant from the (R)-fluorine to
have a significant interaction (Figure 3.2.2B). The (R)-fluorine was found to be 3.8 Å
from the carbonyl carbon of Gly276 and 4.6 Å from the nearer N of Arg388.
Fluorine atoms proximal to Arg side chain nitrogens have been frequently
documented in the literature,
14,49
and it has been proposed that the guanidinium moiety of
Arg is fluorophilic.
50-52
We previously observed an apparent interaction of the CHF-
fluorine of β,γ-CHF-dGTP with Arg183 (PDB entry 4DO9) in the nucleotide-binding site
of pol β.
20,22,25
The IC
50
ratio of 6:1 observed for 12b vs 12a corresponds to a bonding
interaction on the order of 1.1 kcal/mol, which is consistent with literature values for
weak noncovalent fluorine bonds.
50,53

3.3 Conclusion
In summary, for the first time the individual diastereomers of β,γ-CHF-ATP have
been obtained using an improved method for preparing the separable (S)-mandelic acid
morpholinamide (R)/(S)-monoesters 9a and 9b as synthons. The two diastereomers have
discrete
19
F and
31
P NMR spectra in D
2
O and CD
3
OD and show significantly different
inhibition constants for c-Src protein kinase, associated with the presence or absence of a
noncovalent bonding interaction of C-F with R388 in the active site.

40
40
3.4 Experimental
3.4.1 Materials and Methods
Adenosine 5'-monophosphate monosodium salt monohydrate (13) and (R)-(-)-
methyl mandelate (ee: 97%) were purchased from Aldrich.
(Fluoromethylene)bis(phosphonic acid) (3)
4,54
and adenosine 5'-monophosphate
morpholidate (AMP 5’-M, 14)
19
were prepared according to literature procedures. All
other reagents were purchased from commercial sources and used as obtained.
Compounds 4-5 and 6a,b-12a,b were synthesized as described below.
1
H,
19
F, and
31
P
NMR spectra were obtained on Varian 400-MR, VNMRS-500 and VNMRS-600 NMR
spectrometers. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved
multiplet (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of
triplets (dt) or broad signal (br). All chemical shifts are given on the δ–scale in parts per
million ((ppm) relative to internal CD
3
OD (δ 3.34,
1
H NMR), CDCl
3
(δ 7.26,
1
H NMR),
D
2
O (δ 4.79,
1
H NMR),

or external 85% H
3
PO
4
(δ 0.00,
31
P NMR) and C
6
F
6
(δ -164.9,
19
F NMR).
31
P

NMR spectra were proton-decoupled, and
1
H,
19
F, and
31
P coupling
constants (J values) are given in Hz. The concentration of the NMR samples was in the
range of 2-5 mg/mL. NMR samples with D
2
O as the solvent have pH reported as pH
*
, the
direct reading of the NMR sample using an H
2
O-calibrated NMR pH meter,
32
as in our
previous publications.
20,22,25,30,55
Preparative HPLC was performed using a Varian ProStar
equipped with a Shimadzu SPD-10A UV detector (0.5 mm path length) with detection at
wavelength at 256 or 259 nm. Mass spectrometry (MS) was performed on a Finnigan
LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion

41
41
mode. The IUPAC names of compounds were assigned using ACD/Labs, Release 12.00,
Product Version 12.01.
Scheme 3.4.1 Synthesis of chiral synthon
Significant modifications to our original synthesis
25
are highlighted in red and are elaborated in the text.

3.4.2 Synthesis of Tetramethyl (fluoromethanediyl)bis(phosphonate), 4

The compound was synthesized using our literature procedure.
25
3 (145 mg, 0.75
mmol) was methylated to yield 182 mg (0.73 mmol, 97%) of compound 4, as a colorless
oil.
1
H

NMR (500 MHz; CD
3
OD): δ 5.62 (dt, J

= 44.5, 14.0 Hz, 1H), 3.88 – 3.82 (m,
12H).
19
F NMR (470 MHz; CD
3
OD): δ -232.03 (dt, J

= 63.0, 44.6 Hz).
31
P NMR (202
P P
OiPr
OiPr
O
iPr O
iPr O
O
P P
OiPr
OiPr
O
iPr O
iPr O
O
P P
OH
OH
O
HO
HO
O
F
F
Selectfluor®
NaH
DMF/THF, 0 ˚C HCl
∆
P P
OMe
OMe
O
MeO
MeO
O
F
P P
O
-
OMe
O
MeO
MeO
O
F
P P
O
OMe
O
MeO
MeO
O
F
TEA
∆
HC(OMe)
3
∆
(S) (S) OMe
O
1. DOWEX H
+
2. DIAD, PPh
3
Dioxane, N
2
HO
(R) (R) OMe
O
P P
O
OH
O
HO
HO
O
F
(S) (S) OMe
O
P P
O
OH
O
HO
HO
O
F
(S) (S) OH
O
1.BTMS
CH
3
CN
2. MeOH
Na
2
CO
3

pH 8
H
2
O
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
DCC
Morpholine
t-BuOH
O
O
1 2 3
4 5 6a/6b
7a/7b 8a/8b 9a/9b
44% (
31
P NMR) Quant. Yield
Quant. Yield
90%
65% (
31
P NMR)
Quant. Yield
96%
79%
P P
OH
OH
O
HO
HO
O
F
3
∆
HC(OMe)
3
P P
OMe
OMe
O
MeO
MeO
O
F
4

42
42
MHz; CD
3
OD): δ 13.82 (d, J = 62.7 Hz), consistent with literature values.
25

3.4.3 Synthesis of Methyl [(dimethoxyphosphoryl)(fluoro)methyl]phosphonate, 5

Our original procedure
25
for monodemethylation of 4 called for 1.1 equiv of NaI
at rt and gave a yield near 50%. Our subsequent efforts to optimize this reaction were
unsuccessful (Table 3.4.1). Accordingly, we have devised a new procedure for this step,
in which refluxing triethylamine (TEA)
28
is used to monodemethylate 4. This procedure
has the advantages of decreasing the reaction time and workup from >12 h to less than an
hour, eliminates tedious workup of the NaI reaction by slow precipitation and instead
requires simple extraction with CHCl
3
, and increases the yield from 51% to 90%. If the
reaction is monitored carefully by
31
P NMR for completion and uses a minimal amount of
acetonitrile, the reaction can avoid undesired didemethylation byproduct.
Compound 4 (182 mg, 0.73 mmol) was dissolved in 0.5 mL of acetonitrile
(CH
3
CN), followed by addition of TEA (3 mL, excess). The solution was allowed to
reflux for 20 minutes and monitored by
31
P NMR (95% by NMR). The organic phase was
concentrated under vacuum. The residue was redissolved in 10 mL of H
2
O and any
unreacted tetramethyl ester 4 was extracted with CHCl
3
(3 × 10 mL). The aqueous phase
was dried under vacuum to yield 155 mg (90%) of compound 5, obtained as a slightly
brown oil.
1
H NMR (500 MHz, CDCl
3
) δ 5.02 (dt, J = 46.7, 12.3 Hz, 1H), 3.85 (dd, J =
18.8, 10.7 Hz, 6H), 3.68 (d, J = 10.5 Hz, 3H).
19
F NMR (470 MHz, CDCl
3
) δ -223.15
(ddd, J = 65.0, 53.1, 46.4 Hz).
31
P NMR (202 MHz, CDCl
3
) δ 19.06 (dd, J = 65.6, 16.0
P P
OMe
OMe
O
MeO
MeO
O
F
P P
O
-
OMe
O
MeO
MeO
O
F
TEA
∆
4 5

43
43
Hz), 4.38 (dd, J = 52.8, 15.9 Hz),
31
P NMR is

consistent with literature values (
1
H and
19
F
NMR values were not provided).
25
ESI-MS: MS (m/z): calcd for C
4
H
10
FO
6
P
2
-
: 235.0,
found: 235.1 [M − H]
−
.
Table 3.4.1 Yields for synthesis of 5
Entry Conditions T (˚C) t (h) Yield (%)
1 1.1 equiv NaI 0 12 20
2 1.1 equiv NaI 25 12 10-51%
25

3
1.1 equiv NaI, EtOAc:Acetone
(15:5)
25 12 30
4 TEA reflux 0.3 90

Yields were determined by
31
P NMR. Reactions using NaI at both 0 ˚C and 25 ˚C
resulted in didemethylated by product. Bringing reaction to reflux temperature was not
feasible with NaI because higher temperature increased the didemethylation byproduct,
as commonly observed for literature didemethylation of phosphonates.
56
Formation of
didemethylated byproduct could be abated in synthesis of 5 using TEA under reflux, with
limited reaction time and minimal CH
3
CN.

3.4.4 Synthesis of Methyl (7S)-4-fluoro-3,5-dimethoxy-7-phenyl-2,6-dioxa-3,5-
diphosphaoctan-8-oate 3,5- dioxide, 6a/6b

P P
O
-
OMe
O
MeO
MeO
O
F
P P
O
OMe
O
MeO
MeO
O
F
(S) (S) OMe
O
1. DOWEX H
+
2. DIAD, PPh
3
Dioxane, N
2
HO
(R) (R) OMe
O
5 6a/6b

44
44
The product was synthesized from 5 (298 mg, 1.26 mmol) using our literature
procedure,
25
with a modified isolation method. To improve convenience and scalability,
preparative TLC (50% hexane/ethyl acetate) was replaced by silica gel column
chromatography with elution by CH
2
Cl
2
(10-100% in acetone), giving 314 mg (0.82
mmol, 65%) of 6a/6b as a colorless oil.
19
F NMR (376 MHz, CD
3
OD) δ -228.86 – -
233.97 (m).
31
P NMR (162 MHz, CD
3
OD) δ 14.80 – 10.02 (m),
31
P NMR

consistent with
literature values (
19
F NMR values were not reported).
25

3.4.5 Synthesis of (2S)-({[Fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)-
(phenyl)ethanoic acid, 8a/8b

The product was synthesized using our literature procedure.
25
6a/6b (205 mg,
0.53 mmol) yielded intermediate, 7a/7b (144 mg) as a clear film (0.42 mmol, 79%).
19
F
NMR (470 MHz, CD
3
OD) δ -225.51 – -229.74 (m).
31
P NMR (202 MHz, CD
3
OD) δ
11.37 – 8.43 (m). Literature values were not previously reported for intermediate for
7a/7b,
25
which was dissolved in 10 mL of H
2
O, the pH adjusted to 8 using Na
2
CO
3
and
the solution was allowed to stir overnight. The solvents were evaporated and the product
8a/8b was used in the next step as obtained.
25

P P
O
OMe
O
MeO
MeO
O
F
(S) (S) OMe
O
1. BTMS
CH
3
CN
2. MeOH
P P
O
OH
O
HO
HO
O
F
(S) (S) OMe
O
P P
O
OH
O
HO
HO
O
F
(S) (S) OH
O
Na
2
CO
3

pH 8
H
2
O
7a/7b 8a/8b 6a/6b

45
45
3.4.6 Synthesis of [(R)-Fluoro{hydroxy[(1S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-
ethoxy]phosphoryl}methyl]phosphonic acid, 10a and [(S)-fluoro{hydroxy[(1S)-2-
(morpholin-4-yl)-2-oxo-1-phenylethoxy]phosphoryl}methyl]phosphonic acid, 10b

Our previously reported conditions used large excesses of reagents (8 equiv of
morpholine and 20 equiv of DCC). By replacing the aqueous alcohol solvent with a non-
aqueous solvent system, the reaction time, after addition of DCC, was reduced from > 2 h
to less than 1 h, with a slight gain in yield (87%
25
to 96%). The modified reaction also
reduces the amount of DCC from 20 equiv to 4 equiv. This is particularly advantageous
because unreacted DCC forms the byproduct dicyclohexylurea, which complicates
purification and separation of the desired products during preparative C
18
HPLC
purification.
Our literature procedure
25
was modified by replacing the aqueous alcohol solvent
with neat t-BuOH (Table 3.4.2).Thus, the mixture of diastereomers 8a/8b (43 mg, 0.11
mmol) was dissolved in 6 mL of t-BuOH, followed by dropwise addition of morpholine
(46 µL, 5 equiv). The reaction mixture was first stirred at rt for fifteen minutes and then
set to reflux. Dicyclohexylcarbodiimide (DCC, 88 mg, 4 equiv) was dissolved in 3 mL of
t-BuOH and divided into 12 aliquots. An aliquot was added dropwise to the reaction
mixture under reflux every ten minutes. After 2 hr, DCC addition was complete and
reflux was continued for another 5 min. After reaction was completed, the mixture was
cooled to rt and volatiles were removed under vacuum. The residue was treated with 2
P P
O
OH
O
HO
HO
O
F
(S) (S) OH
O
8a/8b
(R) (R) P P
O
OH
O
HO
N
O
F
(S) (S) N
O
1. DCC
Morpholine
t-BuOH
O
O
9a/9b
(R) (R)
P P
O
OH
O
HO
HO
F
O
(S) (S)
O
N O
or (S)
10a/10b
1. DOWEX H
+
2. 1M HCl
2. C
18
HPLC
or (S)

46
46
mL of water. Solids were removed by filtration and the aqueous layer was concentrated
under vacuum to yield 50 mg (0.11 mmol, 96%, HPLC) of 9a/9b as a mixture of
diastereomers.
Purification and separation of 9a/9b were performed by preparative HPLC using a
Phenomenex Luna C
18
HPLC column (5 µm, 250 mm × 21 mm) with 15% CH
3
CN in 0.1
M triethylammonium bicarbonate (TEAB) buffer, pH 7.2 at a flow rate of 8.0 mL/min.
This column provided significant advantages over the previously described column
Varian Microsorb RP-C
18
)
25
, reducing the diastereomer separation time to two minutes
with improved peak resolution (cf. Figures B13-15). Isomer 9b consistently elutes later
than 9a under the conditions we have described herein and in reference 5. The UV
detector was operated at 256 nm. Diastereomer 9a eluted at 23 min and was obtained as a
triethylammonium salt (47%, 12 mg, 26 µmol). After evaporation of volatiles under
vacuum, a colorless film was obtained .
1
H NMR (500 MHz, D
2
O, pH 10.0) δ 7.62 – 7.44
(m, 5H), 6.17 (d, J = 8.2 Hz, 1H), 4.77 – 4.62 (m, 1H), 3.85 – 3.72 (m, 4H), 3.64 (tdd, J =
9.5, 7.1, 4.3 Hz, 8H), 3.04 (q, J = 7.2 Hz, 4H).
19
F NMR (470 MHz, D
2
O, pH 10.0) δ -
218.48 (ddd, J = 61.6, 58.9, 45.4 Hz).
31
P NMR (202 MHz, D
2
O, pH 10.0) δ 9.86 (dd, J =
62.5, 13.6 Hz), 9.56 (dd, J = 58.9, 10.0 Hz). NMR spectra were consistent with literature
values (see also discussion of solvent effects below).
25
ESI-MS: MS (m/z): calcd for
C
17
H
24
FN
2
O
8
P
2
-
: 465.1, found: 465.1 [M − H]
−
.

Diastereomer 9b was eluted at 25 min and obtained as triethylammonium salt.
After removal of volatiles a colorless film was obtained (42%, 11 mg, 23 µmol).
1
H NMR
(500 MHz, D
2
O, pH 10.3) δ 7.51 (ddd, J = 19.1, 10.7, 6.9 Hz, 5H), 6.16 (d, J = 8.6 Hz,
1H), 4.71 (d, J = 13.1 Hz, 1H), 3.76 (d, J = 13.3 Hz, 4H), 3.73 – 3.54 (m, 8H), 3.46 –

47
47
3.31 (m, 4H).
19
F NMR (470 MHz, D
2
O, pH 10.0) δ -218.32 (td, J = 61.3, 60.9, 45.7 Hz).

31
P NMR (202 MHz, D
2
O, pH 10.0) δ 10.22 (dd, J = 62.5, 12.7 Hz), 9.71 (dd, J = 59.2,
12.7 Hz).

NMR spectra were consistent with literature values (see also discussion of
solvent effects below).
25
ESI-MS: MS (m/z): calcd for C
17
H
24
FN
2
O
8
P
2
-
: 465.1, found:
465.1 [M − H]
−
.
Table 3.4.2 Yields of 9a/9b under various reaction conditions

a
Reaction time reflects total time after addition of DCC.

Conversion of 9a or 9b to the corresponding free acid 10a or 10b was effected by
passage through a column of Dowex 50WX8 (100-400 mesh, H
+
form) eluted with H
2
O.
The eluates were evaporated at reduced pressure, leaving 10a (or 10b) as a colorless film,
which was used directly in the next step.

3.4.7 Effect of Solvents on NMR-Based Diastereomer Differentiation in 9a/9b Mixture
After completion of the dimorpholidation of 9a/9b was observed by MS-ESI (-),
the solvent was evaporated and the residue was resuspended in 10 mL of H
2
O. The
precipitate was filtered and the solvent evaporated to yield a brown film. The product
was dissolved in 1.5 mL of CD
3
OD and 750 µL of the mixture was transferred to an
Entry Conditions Solvent T (˚C) t (h)
a
Yield (% by
31
P NMR)
1
8 equiv morpholine,
20 equiv DCC
t-BuOH:H
2
O
(1:1)
100 2 87
25

2
5 equiv morpholine,
5 equiv DCC
t-BuOH:H
2
O
(1:1)
100 12 30
3
5 equiv morpholine,
4 equiv DCC
t-BuOH 100 <1 96

48
48
NMR tube to acquire the
19
F and
31
P NMR spectra. The remainder was evaporated and
redissolved in 750 µL of D
2
O for NMR analysis. Both spectra are illustrated in Figure
B7, with insets that are scaled to the same 2 ppm range. In the
19
F NMR CD
3
OD spectra
(470 MHz), the two diastereomers are separated by ∆δ
F
279 Hz (in Figure B7A), which
spans a 1 ppm range, revealing two distinct sets of fluorine resonances. However, in the
19
F NMR spectra with D
2
O (470 MHz, pH 9.3), the diastereomers cluster together in a
range of 0.5 ppm, overlapping the two isomer resonances, but exhibit a significantly
narrower line width (~3 Hz vs. ~8 Hz in CD
3
OD). Diastereomeric differentiation was
confirmed in samples dissolved in CD
3
OD by comparing the outermost peaks of the two
putative diastereomers and comparison of J values acquired at 470 and 564 MHz (∆δ
F

346 Hz in Figure B7B) in the
19
F NMR spectra.

19
F NMR (470 MHz, CD
3
OD) δ -219.30 (td, J = 60.3, 46.8 Hz), -219.89 (td, J = 60.3,
46.7 Hz).
19
F NMR (470 MHz, D
2
O, pH 9.3) δ -216.90 – -217.20 (m), -217.20 – -217.49
(m).
19
F NMR (564 MHz, CD
3
OD) δ -219.22 (td, J = 60.0, 46.6 Hz), -219.83 (td, J =
60.1, 46.6 Hz).
19
F NMR (564 MHz, D
2
O, pH 8.9) δ -218.28 – -218.54 (m), -218.56 – -
218.90 (m).
31
P NMR (202 MHz, CD
3
OD) δ 10.51 – 9.18 (m).
31
P NMR (202 MHz, D
2
O,
8.9) δ 10.46 – 9.53 (m).

49
49
3.4.8 Synthesis of 9-{5-O-[{[((S)-Fluoro{hydroxy[(S)-morpholin-4-yl(phenyl)methoxy]-
phosphoryl}methyl)(hydroxy)phosphoryl]oxy}(hydroxy)phosphoryl]pento-
furanosyl}-9H-purin-6-amine, 11a

Compound 10a (25 mg, 62.9 µmol) was dissolved in 5 mL of 1:1 EtOH:H
2
O.
Tributylamine in EtOH (1:10) was slowly added to the mixture to reach pH 4.5. After
mixing for 30 min at rt, the solution was evaporated under vacuum and the residue dried
by co-evaporation with anhydrous DMF (3 × 3 mL). It was then dissolved in a 2 mL
solution of 1.5 equiv. AMP 5’-M (14) in anhydrous DMSO. The reaction mixture was
stirred under rt for 72 h under N
2
. Purification of 11a was performed on a Macherey-
Nagel Nucleogel SAX 1000-10 (25 mm × 15 cm) preparative column, using a gradient
(0-10 min, 60%; 10-16 min, 60%; 16-25 min, 100%) of 0.5 M TEAB buffer pH 7.4 at a
flow rate of 8 mL/min. Compound 11a eluted at 17 min to give 13.6 mg (30%) of a
colorless film.
1
H NMR (500 MHz, D
2
O, pH 10.0) δ 8.52 (s, 1H), 8.26 (s, 1H), 7.50 –
7.31 (m, 5H), 6.14 (d, J = 17.3 Hz, 1H), 5.09 – 4.92 (m, 1H), 4.73 (s, 1H), 4.54 (s, 1H),
4.38 (s, 1H), 4.22 (s, 1H), 3.72 (d, J = 4.7 Hz, 4H), 3.58 (d, J = 24.1 Hz, 8H), 3.36 (d, J =
10.5 Hz, 4H).
19
F NMR (470 MHz, D
2
O, pH 10.0) δ -218.73 (td, J = 61.1, 45.8 Hz).
31
P
NMR (202 MHz, D
2
O, pH 10.0) δ 8.75 (dd, J = 60.4, 17.9 Hz), -0.11 (ddd, J = 61.6, 27.8,
17.9 Hz), -11.28 (d, J = 27.7 Hz). Values are consistent with published data,
25
except the
(R) (R)
P P
O
OH
O
HO
HO
F
O
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
(S) (S)
O
N O
N
N
N
N
O
OH
O
P
N O
DMSO
OH
O
O O O
or (S)
or (R)
OH
NH
2
OH
10a/10b
11a/11b

50
50
reported
19
F quartet was resolved to a td. ESI-MS: calcd for C
23
H
29
FN
6
O
14
P
3
-
:

725.1,
found m/z 725.1 [M − H]
−
.

3.4.9 Synthesis of 9-{5-O-[{[((R)-Fluoro{hydroxy[(S)-morpholin-4-yl(phenyl)methoxy]-
phosphoryl}methyl)(hydroxy)phosphoryl]oxy}(hydroxy)phosphoryl]-
pentofuranosyl}-9H-purin-6-amine, 11b
The diastereomer 11b (33 mg, 83.1 µmol) was synthesized and purified as
described for 11a. Compound 11b eluted at 17 min and after evaporation of volatiles, was
obtained as a colorless film (15.7 mg, 26%).
1
H NMR (500 MHz, D
2
O, pH 10.0) δ 8.46 (s,
1H), 8.22 (s, 1H), 7.44 – 7.27 (m, 5H), 6.10 (dd, J = 8.4, 6.9 Hz, 1H), 4.99 (dt, J = 45.9,
13.1 Hz, 1H), 4.70 (t, J = 5.4 Hz, 1H), 4.51 (dd, J = 5.0, 3.6 Hz, 1H), 4.36 (t, J = 3.0 Hz,
1H), 4.21 (td, J = 5.0, 4.5, 2.9 Hz, 1H), 3.73 – 3.66 (m, 4H), 3.66 – 3.47 (m, 8H), 3.34 –
3.23 (m, 4H).
19
F NMR (470 MHz, D
2
O, pH 10.0) δ -218.40 (td, J = 61.0, 45.9 Hz).
31
P
NMR (202 MHz, D
2
O, pH 10.0) δ 8.88 (dd, J = 60.1, 18.4 Hz), -0.07 (d, J = 60.7 Hz), -
11.28 (d, J = 28.0 Hz). Values are consistent with published data,
25
except that the
19
F
multiplet was observed as a td. ESI-MS: calcd for C
23
H
29
FN
6
O
14
P
3
-
: 725.1, found m/z
725.1 [M − H]
−
.

3.4.10 Synthesis of 9-{5-O-[({[(S)-Fluoro(phosphono)methyl](hydroxy)phosphoryl}-
oxy)phosphinato]pentofuranosyl}-9H-purin-6-amine, 12a

N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
or (R)
OH
11a/11b
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
30% Pd/C, H
2
0.1 TEAB: MeOH (1:1),
pH 8
O O O
or (R)
NH
2
OH
12a/12b

51
51
10% wt. % of Pd/C
25
used in the synthesis of the corresponding dGTP derivative
25

did not effect deprotection of the adenosine nucleotide 11a, which however was achieved
by increasing the amount of catalyst and the reaction time to 6 h. The triethylammonium
salt of 11a (13.6 mg, 18.8 µmol, determined by UV) was dissolved in 5 ml of 0.1 M
TEAB:MeOH (1:1, pH 7.8), followed by addition of 30 wt. % Pd/C (18.0 mg) and a stir
bar. The pH of the solution was adjusted to 8 by bubbling in CO
2
. The reaction mixture
was first frozen (dry ice in acetone), and then degassed under vacuum with flushing N
2

gas over 3 cycles. The vessel was then filled with H
2
gas several times. After the final fill
of H
2
, the solvent was thawed. The reaction was stirred under rt under 1 atm of H
2
for 6 h.
The completion of reaction was confirmed by mass spectrometry by monitoring the ratio
of peaks at: m/z: 522 and 725 (ESI-MS). Purification was performed on a Macherey-
Nagel Nucleogel SAX 1000-10 25 mm × 15 cm preparative column, using a gradient (0-
10 min, 55%; 10-16 min, 55%; 16-25 min, 100%) of 0.5 M TEAB buffer pH 7.4 at a flow
rate of 8 mL/min. Compound 12a eluted at 22 min (8 mg, 85%) as a triethylammonium
salt after evaporation, a colorless film was obtained.
1
H NMR (500 MHz, D
2
O, pH 10.2)
δ 8.88 (s, 1H), 8.59 (s, 1H), 6.48 (d, J = 6.0 Hz, 1H), 4.94 (t, J = 4.3 Hz, 1H), 4.81 – 4.70
(m, 1H), 4.66 – 4.49 (m, 2H).
19
F NMR (470 MHz, D
2
O, pH 10.2) δ -216.53 (ddd, J =
65.3, 55.3, 45.2 Hz).
31
P NMR (202 MHz, D
2
O, pH 10.2) δ 7.02 (dd, J = 56.0, 14.9 Hz),
4.54 (ddd, J = 65.5, 28.9, 15.8 Hz), -11.00 (d, J = 28.6 Hz). ESI-MS: calcd for
C
11
H
16
FN
5
O
12
P
3
-
: 522.0, found m/z 522.1 [M − H]
−
.

52
52
3.4.11 Synthesis of 9-{5-O-[({[(R)-Fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)-
phosphinato]pentofuranosyl}-9H-purin-6-amine, 12b
A triethylammonium salt of compound 11b (15.7 mg, 21.6 µmol, determined by
UV) was prepared and purified as described for the synthesis of 12a. The product 12b
eluted on the HPLC column at 22 min and was obtained as a triethylammonium salt (10
mg, 87%) after evaporation to a colorless film.
1
H NMR (500 MHz, D
2
O, pH 10.2) δ 8.55
(s, 1H), 8.25 (s, 1H), 6.13 (d, J = 6.1 Hz, 1H), 4.92 – 4.84 (m, 1H), 4.60 (dd, J = 5.1, 3.4
Hz, 1H), 4.39 (t, J = 2.9 Hz, 1H), 4.33 – 4.14 (m, 2H).
19
F NMR (470 MHz, D
2
O, pH
10.2) δ -216.53 (ddd, J = 65.9, 55.6, 45.2 Hz).
31
P NMR (202 MHz, D
2
O, pH 9.7) δ 6.81
(dd, J = 55.7, 14.7 Hz), 4.60 (ddd, J = 65.8, 29.0, 14.6 Hz), -11.13 (d, J = 29.0 Hz). ESI-
MS: calcd for C
11
H
16
FN
5
O
12
P
3
-
: 522.0, found m/z 522.1 [M − H]
−
.

3.4.12 General Synthesis of Nucleoside 5’-β,γ-Methylenetriphosphate Analogues, 12, 15-
16

The following β,γ-methylene-nucleoside triphosphate analogues: methylene (CH
2
;
X,Y=H, 15), mixed monofluoromethylenes (CHF; X=H, Y=F, 12), and
difluoromethylene (CF
2
; X,Y=F, 16) were synthesized according to literature
procedures,
19
by conjugation of activated AMP 5′-M, 14 with the tri-n-butyl-ammonium
salt of the appropriate bisphosphonic acid, followed by dual-pass HPLC purification.
P P
OH
O
-
O
-
O
-
O
O
N
N
N
N
O
OH
O
P
N O
OH
O
OH
NH
2
+
DMSO
3d
X Y
N
N
N
N
O
OH
O
P
O
OH
P
OH
P
HO
OH
X
O O O
NH
2
OH
Y
X=H,F
Y=H,F
Bu
3
NH
+
Bu
3
NH
+
Bu
3
NH
+
12: X,Y=H,F
15: X,Y=H
16: X,Y=F
14

53
53
3.4.13 NMR Studies of Synthetic (12) and Artificial Diastereomer Mixtures, 12a/12b
Compound 12 was dissolved in 2 mL of D
2
O and 500 µL aliquots were added to
four NMR tubes and treated with Chelex. The pH of the sample was adjusted using KOH,
NH
4
OH, triethylamine, or Na
2
CO
3
to determine if there was a counterion effect on the
spectra. Conditions used are listed in Table 3.4.3. Then
19
F and
31
P NMR spectra were
acquired for each counterion. Addition of Na
2
CO
3
results in adequate diastereomeric
differentiation. To assess if the sodium cation was responsible for the differentiation,
another sample was prepared and adjusted using NaOH, which resulted in sharp
resolution and satisfactory diastereomeric differentiation.
3.4.14 Effect of Counterion on
19
F and
31
P NMR of Diastereomer Mixture 12
Table reflects the pH and relative amount of solvent that was added to each NMR
tube to adjust the pH. Each NMR tube originally contained 500 µL of D
2
O, therefore the
relative concentrations were not exactly the same. However, it appears the counterion
effect is independent of the concentration of 12 (NaOH sample has a lower concentration
but greater resolution).
30

Table 3.4.3 Conditions used to diastereomer mixtures
Base pH
Solvent Depth in
NMR Tube (mm)
a

KOH 11.1 34
NH
4
OH 10.8 59
Na
2
CO
3
11.0 39
Et
3
N 10.9 34
NaOH 12.1 53
a
Solvent after adjusting pH with base in D
2
O.

54
54
3.4.15 Isolation and Characterization of Impurity Observed in NMR, 17
A minor impurity peak (1−20% by NMR) that appears upfield of the resolved
19
F
NMR peaks of 12 (downfield of P
α
in the
31
P NMR) was occasionally observed in the
reaction mixture. This impurity (17) could be completely removed by adjustment of the
SAX HPLC conditions.
30
Based on the
19
F,
31
P NMR and MS analysis of isolated
impurity (HPLC), it is speculated that the impurity may be a tetraphosphate dimer, based
on the symmetry of the
31
P NMR peaks. Dimerization has been previously noted to occur
under these coupling conditions,
25,57
but can be avoided by increasing the bisphosphonate
to activated nucleoside ratio or addressed by HPLC separation as demonstrated in our
previous work.
30

31
P NMR (202 MHz, D
2
O, pH 10.4) δ 0.42 (dd, J = 61.2, 24.4 Hz), -
10.89 (d, J = 21.3 Hz).
19
F NMR (470 MHz, D
2
O, pH 10.4) δ -218.22 (td, J = 59.5, 46.3
Hz). ESI-MS: calcd for C
21
H
28
FN
10
O
18
P
4
-
: 851.1, found m/z 851.1 [M − H]
−
.
IC
50
values obtained from bioassays (next section) with 12 (>99% pure) were consistent
with values acquired from 12 (95%, with 5% impurity), 45 µM (± 8 µM, n = 2) vs. 47 µM
(± 7 µM, n = 6), respectively.
3.4.16 Kinase Expression and Purification
The kinase domain of chicken c-Src (residues 260-533) in modified pET-28a
vector with tobacco etch virus (TEV) protease cleavable site was co-expressed with full-
length YopH phosphatase from Yersinia as previously decribed.
58
Briefly, E. coli BL21
(DE3) co-transformed with Src (pET28a) and YopH (pCDFDuet) was grown at 37 °C,
induced with IPTG (0.5 mM) between OD
600
of 0.6 - 0.8 units, and then grown at 18 °C
for 12-16 hours. The cells were harvested by centrifugation at 4,300 rpm, re-suspended

55
55
in lysis buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 7.5), and lysed by
sonication (Q500 Sonicator, Qsonica). After the cell lysate was centrifuged at 10,200 × g
for 1 hour at 4 °C, the supernatant was collected and filtered through at 0.45 µm syringe
filter (VWR) and purified through a Ni-affinity column (HisTrap, GE Lifescience) via
FPLC (BioRad BioLogic System). The wash buffer contains 20 mM Tris, 300 mM NaCl,
and 20 mM imidazole at pH 7.5 while the elution buffer is identical except a higher
concentration (250 mM) of imidazole. The elution peak was collected and dialyzed
against dialysis buffer (20 mM Tris, 300 mM NaCl, pH 7.5) along with the addition of
TEV protease overnight at 4 °C. The cleaved Src was reapplied to the Ni-affinity column
and the flow-through was collected and analyzed by SDS-PAGE gel for purity
determination (Figure 1.4.1). The purified Src kinase was used for in vitro kinase assays.

Figure 3.4.1 SDS-PAGE gel for purity determination

3.4.17 In vitro Kinase Activity Assay

YopH
WT Src
Cleaved WT Src
Pre-TEV digest
Post-TEV digest
OH
peptide
kinase
ATP
32
ADP
O
peptide
P
32
O
-
O
OH
IYEFGKKK
membrane
disk
wash
radiation detected
via scintillation
counter (cpm)
Inhibitor (µM)
CPM (count)
IC
50
Inhibitor competes with
ATP
32
and reduces
cpm count
AT
32
P
AT
32
P
IYGEFKKK
O
32
P
O
OH
O
-

56
56
The kinase activity was measured using [γ-
32
P] –ATP as the phosphoryl donor to
the peptide substrate. The assay was carried out in 50 mM Tris (pH 8.0), 10 mM MgCl
2
,
5.5 nM [γ-
32
P] –ATP (PerkinElmer), 1 mg/mL BSA, 0.5 mM peptide substrate
(IYGEFKKK) along with different concentrations (from 0.3 µM to 3 mM) of ATP
analogues (12, 12a, 12b, 15-16) at 25°C in 30 µL reaction. The kinase reaction was
initiated by the addition of [γ-
32
P]–ATP and was allowed to proceed for 30 minutes. The
reaction was transferred onto phosphocellulose filter disc paper (Whatman P81/GE
Healthcare), quenched with 10% acetic acid, washed with 0.5% phosphoric acid (3 times),
and rinsed with acetone. The dried phosphocellulose filter was added scintillation fluid
(BetaMax-ES, MP Biomedicals), and counted on a scintillation counter (Beckman
LC6500). The data was plotted using Prism and analyzed using a nonlinear regression fit
to calculate the IC
50
.

3.4.18 In silico Analysis of Ligand/Receptor to Predict Binding Interactions
Molecular docking calculations were performed using human Src kinase X-ray
crystallographic data (PDB entry 2SRC) and AutoDock Vina 1.1.2,
44
to explore potential
ligand-receptor interactions within the active site of the protein. The geometries for each
ligand structure (12, 16) were optimized using SPARTAN ’14 Quantum Mechanics
software suite. The solvation parameters and charges were applied by Autodock Tools
(MGL). Rotatable and non-rotatable bonds were identified and all non-polar hydrogens
and water molecules were removed, except for structural waters. A grid map for the space
to be docked within the protein set at a maximum of 26 × 26 × 26 points.

57
57
Although enzyme assays were performed using chicken kinase, a Src kinase X-ray
crystal structure with the higher resolution was found more optimal for molecular
docking calculations. The available chicken kinase structure has a resolution of 2.3 Å
(PDB entry 3DQX), vs. 1.8 Å for human Src kinase. To verify active site homology,
protein sequences were aligned using BLASTP 2.2.30+
45
for the wild-type c-Src kinase
domain of the two species: human (length: 452) and chicken (length: 286). BLASTP
alignment revealed a 99% sequence overlap of 283 amino acids. Alignment of the
nucleotide-binding site using PyMol was similar.

3.5 Chapter References

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Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461.
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Biochemistry 2008, 47, 870.
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Wilson, S. H.; McKenna, C. E. J. Am. Chem. Soc. 2012, 134, 8734.
(26) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc. Perkin 1 1984, 1119.
(27) McKenna, C. E.; Harutunian, V. FASEB J. 1988, 2, 6148.
(28) Vepsäläinen, J. J.; Kivikoski, J.; Ahlgrén, M.; Nupponen, H. E.; Pohjala, E. K.
Tetrahedron 1995, 51, 6805.
(29) Ahlmark, M. J.; Vepsäläinen, J. J. Tetrahedron 1997, 53, 16153.
(30) Hwang, C. S.; Kashemirov, B. A.; McKenna, C. E. J. Org. Chem. 2014, 79, 5315.
(31) The pH values for deuterated water solutions as reported here and by us
previously (ref. 20, 22, 25, 30) are given as measured with a glass electrode, without
correction to pD (ref. 32).
(32) Krȩżel, A.; Bal, W. J. Inorg. Biochem. 2004, 98, 161.
(33) Fluorine NMR were simulated using spin simulation on MestReNova 8.1.2
(34) Schwartz, P. A.; Murray, B. W. Bioorg. Chem. 2011, 39, 192.
(35) Kalesh, K. A.; Liu, K.; Yao, S. Q. Org. Biomol. Chem. 2009, 7, 5129.
(36) Xie, J.; Corneau, A. B.; Seto, C. T. Org. Lett. 2003, 6, 83.
(37) Moffat, D.; Nichols, C. J.; Riley, D. A.; Simpkins, N. S. Org. Biomol. Chem.
2005, 3, 2953.
(38) Zhang, C.; Lopez, M. S.; Dar, A. C.; Ladow, E.; Finkbeiner, S.; Yun, C. H.; Eck,
M. J.; Shokat, K. M. ACS Chem. Biol. 2013, 8, 1931.
(39) Kwarcinski, F. E.; Fox, C. C.; Steffey, M. E.; Soellner, M. B. ACS Chem. Biol.
2012, 7, 1910.
(40) Brandvold, K. R.; Steffey, M. E.; Fox, C. C.; Soellner, M. B. ACS Chem. Biol.
2012, 7, 1393.
(41) Ulrich, S. M.; Kenski, D. M.; Shokat, K. M. Biochemistry 2003, 42, 7915.
(42) Witt, J. J.; Roskoski Jr, R. Anal. Biochem. 1975, 66, 253.
(43) Knight, Z. A.; Shokat, K. M. Chem. Biol. 2005, 12, 621.
(44) Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
(45) Altschul, S. F.; Madden, T. L.; Schäffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.;
Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389.

59
59
(46) Via visual inspection of the active site using PyMol, The PyMOL Molecular
Graphics System, Version 1.2r3pre, Schrödinger, LLC.
(47) Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.1,
San Diego: Accelrys Software Inc., 2007
(48) Xu, W.; Harrison, S. C.; Eck, M. J. Nature 1997, 385, 595.
(49) Morgenthaler, M.; Aebi, J. D.; Grüninger, F.; Mona, D.; Wagner, B.; Kansy, M.;
Diederich, F. J. Fluorine Chem. 2008, 129, 852.
(50) Zhou, P.; Zou, J.; Tian, F.; Shang, Z. J. Chem. Inf. Model. 2009, 49, 2344.
(51) Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
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(53) Dalvit, C.; Invernizzi, C.; Vulpetti, A. Chem. Eur. J. 2014, 20, 11058.
(54) Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E.
J. Fluorine Chem. 2005, 126, 1467.
(55) Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E. J. Org.
Chem. 2011, 76, 5132.
(56) McKenna, C. E.; Kashemirov, B. A.; Roze, C. N. Bioorg. Chem. 2002, 30, 383.
(57) Mohamady, S.; Taylor, S. D. J. Org. Chem. 2011, 76, 6344.
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1991, 88, 1187.

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60
Functional Interplay Between NTP Leaving Group and Chapter 4.
Base Pair Recognition During RNA Polymerase II Nucleotide
Incorporation Revealed by Methylene Substitution
3

4.1 Introduction
RNA pol II is the central enzyme responsible for the synthesis of mRNA during
eukaryotic gene transcription.
1,2
It is essential for pol II to catalyze nucleotide addition
with high efficiency and fidelity. Previous studies revealed that a key conserved motif,
the trigger loop (TL), plays important roles in substrate selection, catalysis, and
translocation during each nucleotide addition cycle in the transcription elongation
phase.
3-10
Upon correct substrate binding, the TL undergoes important conformational
changes, switching from inactive open conformations to an active closed conformation.
7,8

This closure of the TL stabilizes the correctly bound substrate and facilitates subsequent
nucleotide addition reaction.
3-10
The pol II TL, bridge helix, along with other active site
residues, the nucleoside triphosphate (NTP) substrates, and the RNA-DNA hybrid form a
well-organized substrate recognition network to ensure high transcriptional fidelity
(Figure 4.1.1A).
11-13
Multiple types of non-covalent interactions, such as base stacking,
hydrogen-bonding, hydrophobic interaction, and salt bridge interactions, are involved in
substrate recognition (Figure 4.1.1A).
14
Dissecting individual contributions from

3
Reproduced with permission from Hwang, C. S.; Xu, L.; Wang, W.; Ulrich, S.; Zhang,
L.; Chong, J.; Shin, J. H.; Huang, X.; Kool, E. T.; McKenna, C. E.; Wang, D. Nucleic
Acids Res. 2016. Copyright 2016 Oxford University Press.

61
61
these interactions within this sophisticated network is pivotal to understanding the overall
molecular mechanism of pol II transcription and the molecular basis of how DNA
modifications and lesions affect transcription fidelity.
15-18

Figure 4.1.1 β,γ-CH
2
-(d)NTPs can be recognized and incorporated by RNA pol II
A) Interaction network in pol II catalytic site; B) Chemical structures of β,γ-CH
2
(d)NTPs; C) Gel analysis
of nucleotide incorporation of wt ATP and its analogue (2.5 µM). Time points were 15 s, 30 s, 1 min, 2
min, 4 min, 8 min, 15 min and 30 min.

Synthetic nucleic acid analogues are powerful tools to probe interactions between
nucleic acids and pol II by “substituting” individual nucleic acid moieties or ablating
specific interactions.
19-21
These nucleic acid analogues enabled us to investigate the
individual contributions of the nucleobase and sugar moieties to pol II transcription.
These studies have greatly advanced our understanding of how the intrinsic structural

62
62
features of nucleic acid moieties are recognized and how specific interactions are
involved in substrate selection and incorporation during pol II transcription.
22-25

However, the interactions involved at the triphosphate moiety of NTPs during nucleotide
selection and incorporation have not yet been extensively explored. Investigations
targeting the triphosphate moiety can provide further mechanistic insights into how pol II
catalyzes the chemical bond cleavage and formation during nucleotide addition.
Deoxynucleoside triphosphate (dNTP) analogues were applied to probe the
mechanisms of several nucleic acid enzymes.
26-28
The synthesis of these dNTP analogues
involves replacing the bridging β,γ-oxygen with a methylene group,
29
resulting in a non-
hydrolysable analogue for enzymes that cleave the terminal phosphate (Figure
4.1.1B).
30,31
For enzymes such as DNA pol β that release pyrophosphate ion (PP
i
) after
nucleotidyl transfer,
26-28
these β,γ-CXY-dNTP analogues function as substrate analogues
with leaving group properties that can be tuned by substituents on the methylene
carbon.
32-34
The bridging oxygen substituted by the less electronegative methylene group
increases the leaving group's basicity relative to pyrophosphate and thereby provides a
method to examine the rate-determining step (RDS) of DNA pol β.
32-34
Here, by
employing β,γ-CH
2
modified (d)NTPs (Figure 4.1.1B), we explore how RNA pol II
recognizes modified triphosphate groups and how the chemical step affects nucleotide
incorporation efficiency.

4.2 Results and Discussion
4.2.1 Cognate Nucleotide Incorporation
During transcription, pol II catalyzes bond cleavage at the P
α
-O-P
β
site of an NTP
substrate and bond formation of NMP with the 3’-OH of RNA terminus. As a result, the

63
63
RNA transcript is extended by 1 nt at its 3’-end and a PP
i
, the leaving group, is
subsequently released from the pol II active site. Substitution of the β,γ-bridging oxygen
in the triphosphate with methylene (Figure 4.1.1B)
29
leads to a poorer leaving group than
PP
i
. Such nucleotide analogues allow us to determine the incorporation rates of the wt
and β,γ-CH
2
modified nucleotide triphosphates (β,γ-CH
2
-ATP (10), β,γ-CH
2
-dATP (7),
and β,γ-CH
2
-UTP (9)) and to probe the mechanisms of pol II catalysis and transcriptional
fidelity control.

Figure 4.2.1 Kinetic effects of varying the β,γ-bridging atom in NTPs during RNA pol II incorporation
A) Scaffold used for studying incorporation efficiency of cognate nucleotide (AMP/dT) and noncognate
nucleotides (dAMP/dT and UMP/dT). Catalytic rates (k
pol
); B) and specificity constants (k
pol
/K
d
); C) of wt
and β,γ-CH
2
-(d)NTPs.

In vitro transcription assays were first performed to evaluate substrate
incorporation efficiencies for wt ATP and β,γ-CH
2
-ATP (Figure 4.1.1B and C). As shown
in Figure 4.1.1C, β,γ-CH
2
-ATP can be recognized by pol II as a substrate and becomes
incorporated into the RNA primer via a Watson-Crick (W-C) base pair with dT.
However, β,γ-CH
2
substitution in ATP greatly reduces the nucleotide incorporation rate
by pol II in comparison to wt ATP (Figure 4.1.1C and 4.2.1B).

64
64
To quantitatively identify the effect of the bridging β,γ-CH
2
substitution, single-
turnover pre-steady-state kinetic assays were performed to measure the kinetic
parameters of pol II nucleotide incorporation, specifically k
pol
and K
d,app
, and the ratio of
k
pol
/K
d,app
to calculate the substrate specificity constant of pol II (Table 4.2.1). The
kinetic analysis revealed that the incorporation of β,γ-CH
2
-ATP was significantly slower
than wt ATP, with a ~130-fold decrease in k
pol
. Interestingly, the binding affinity (K
d,app
)
of β,γ-CH
2
-ATP in the dT template did not vary substantially from wt ATP, with both
values in the moderately low micromolar range. As a result, nucleotide incorporation
specificity k
pol
/K
d,app
was greatly reduced (~50-fold). Thus, in the cognate ATP/dT
scaffold, the CH
2
substitution of the bridging oxygen atom results in decreased catalytic
efficiency, indicating that the CH
2
substitution substantially slows down the “chemistry
step” (P-O bond cleavage of the triphosphate moiety between P
α
-O-P
β
and formation of
NMP with the 3’-OH of RNA terminus) and the “chemistry step” becomes the rate-
determining step of the overall mechanism.
32,33

4.2.2 Non-Cognate Nucleotide Incorporation
Since the cognate (matched) nucleotide incorporation was negatively affected by
the β,γ-CH
2
substitution, it is of interest to determine whether this substitution also
altered noncognate nucleotide incorporation, where the nucleobase or sugar moiety of the
incoming NTP is incorrect.
28
We first investigated the CH
2
substitution effect on
noncognate (mismatched) substrate incorporation using UTP as an example (Figure
4.1.1B). Intriguingly, β,γ-CH
2
-UTP exhibits only a 3-fold decrease in k
pol
and a 9-fold
decrease in specificity constant (k
pol
/K
d,app
) in comparison with wt UTP (Figure 4.2.1 and

65
65
Table 4.2.1). This pattern is strikingly distinct from that of the cognate ATP/dT
nucleotide incorporation.
We then investigated the CH
2
substitution effect on noncognate nucleotide dATP
incorporation. Interestingly, β,γ-CH
2
-dATP exhibits a ~100-fold decrease in k
pol
and ~20-
fold in specificity constant (k
pol
/K
d,app
) in comparison with wt dATP (Figure 4.2.1 and
Table 4.2.1). This pattern is comparable with the results of β,γ-CH
2
-ATP for the cognate
ATP/dT nucleotide incorporation.
These results indicate that both wt and β,γ-CH
2
modified analogues can be
recognized by pol II as substrates. However, replacement of the natural pyrophosphate
leaving group in ATP (or UTP) with the more basic methylenebisphosphonate group has
a profound effect on nucleotide addition efficiency. Specifically, the methylene
modification effects on nucleotide incorporation are sensitive to the nature of substrate
base-pairing interactions but not sugar identity.

4.2.3 Trigger Loop-Dependent Substrate Recognition and Incorporation
The trigger loop (TL) plays key roles in controlling efficient pol II nucleotide
selection and catalysis.
3,4,7,8
The TL undergoes a conformational change from an inactive
open state to a closed active state in the presence of correct W-C base pairs with the DNA
template strand, allowing efficient and accurate nucleotide addition.
7,8
To further
understand the effect of methylene substitution of (d)NTPs on pol II nucleotide-binding
induced TL conformational changes and the extent of TL-dependent nucleotide
incorporation, we measured the α-amanitin effect index (AEI). AEI is the ratio of
nucleotide incorporation efficiencies in the absence and presence of α-amanitin. This was

66
66
determined for ATP/dT, dATP/dT and UTP/dT and their CH
2
modified analogues to
assess the involvement of active TL during substrate incorporation (Table 4.4.2).
24
α-
Amanitin is a potent specific inhibitor for pol II transcription that binds nearby bridge
helix (BH) and TL, and traps TL in an open inactive conformation, preventing it from
closing to an active closed conformation.
4,35
As a result, AEI-sensitivity in NTP
incorporation indicates the extent of TL involvement (requires the closed active TL
conformation) during nucleotide incorporation. The TL is actively involved in substrate
recognition for cognate nucleotide incorporation and reaches fully closed state upon
cognate substrate binding, and hence, it results in high AEI values (~80-100). In contrast,
the TL fails to reach its fully closed state effectively in the noncognate nucleobase or
sugar scenario, and thus smaller AEI values are observed (~1 or ~10, respectively).
24

Figure 4.2.2 α-Amanitin effect index reveals that β,γ-CH
2
substitution doesn’t interfere with patterns of TL
dependence for nucleotide incorporation

Indeed, as shown in Figure 4.2.2 and Table 4.4.2, the wt ATP revealed a high AEI
value (~80) for the cognate AMP incorporation, indicating that the TL recognizes the wt
substrate. It is important to note that a high AEI value was also observed with β,γ-CH
2
-
ATP (~80). These results establish that β,γ-methylene substitution in ATP does not

67
67
interfere with TL closure during matched AMP incorporation. In the base-noncognate
incorporation (UTP/dT) scenario, similar AEI values (<5) were observed for both UTP
and β,γ-CH
2
-UTP incorporation, indicating that the TL remains in an inactive
conformation for both wt UTP and β,γ-CH
2
-UTP analogue incorporation (Figure 4.2.2).
Similarly, we observed comparable AEI values (~5-10) for both wt dATP and β,γ-CH
2
-
dATP for dATP/dT incorporation (noncognate sugar). Taken together, methylene
substitution of the β,γ-bridge atom of the triphosphate moiety does not modify TL open-
close conformational profile and its role in the selection of cognate substrate (ATP) over
noncognate substrate (UTP and dATP).

4.2.4 Effect of Wobble Base-Pairing on UMP Incorporation
The observation that TL recognizes β,γ-CH
2
-NTPs and NTPs in a similar manner
indicates that they are appropriate probes to investigate the mechanism of pol II catalysis
by modulating the leaving group and the chemistry step. One intriguing finding is that the
methylene substitution generates a significant difference in k
pol
for cognate AMP
incorporation but a minor difference for UMP incorporation. Since UTP forms a wobble-
base pair with dT that potentially positions its triphosphate moiety in a different binding
environment from that of ATP, we were interested in investigating whether these distinct
methylene substitution effects rely on the patterns of hydrogen bonding to the incipient
pair. To this end, we introduced the hydrogen-bonding deficient mutant (dF), a nonpolar
isostere of dT,
36
to ablate hydrogen-bonding interactions between base pairs
22,37
and
measured the β,γ-CH
2
substitution effects for both AMP and UMP incorporation on the
dF template (Figure 4.2.3 and Table 4.2.1).

68
68

Figure 4.2.3 Kinetic effects of alteration of β,γ-bridging atoms in NTPs during RNA pol II incorporation in
hydrogen bond deficient scaffolds
A) Scaffold used for studying incorporation efficiency in the hydrogen-bonding deficient template (dF).
Catalytic rates (k
pol
); B) and specificity constants (k
pol
/K
d
) (c) of wt NTPs, and β,γ-CH
2
-NTPs with the dF
template.

For the incipient ATP/dF pair, methylene substitution also resulted in a ~70-fold
decrease in k
pol
, which is comparable to the result observed for the ATP/dT system. This
indicates that the elimination of Watson-Crick base pair hydrogen bonding in the ATP/dF
scaffold did not significantly alter the strong CH
2
substitution effect relative to the
ATP/dT scaffold. It is also very interesting to note that we observe the similar methylene
substitution effects from dT and dF scaffold, even though the initial wt ATP
incorporation rate for dF scaffold is substantially (~10
4
) lower than that for dT scaffold,
indicating that the effect of methylene substitution does not rely on the initial rate values.
In sharp contrast with the UTP/dT case (3-fold decrease in k
pol
), the CH
2
substitution
greatly reduced incorporation rate (80-fold decrease in k
pol
) in the UTP/dF setup.
Notably, enhancement of the methylene substitution effect in the UTP/dF pair compared
to the UTP/dT pair was due to the strong increase in the incorporation rate for the natural
UTP/dF case, while the incorporation rate for methylene substituted UTP stayed in a
similar level in these two pairs. These intriguing findings indicate that the removal of

69
69
wobble pair hydrogen bonding between UTP/dT pairing abolishes its unique response to
methylene substitution in the triphosphate moiety.
Table 4.2.1 Kinetic data of wt NTPs and β,γ-CH
2
-NTPs
Scaffold
O CH
2
CH
2
effect
a

k
pol

(min
-1
)
K
d

(µM)
k
pol
/K
d

(µM
-1

min
-1
)
k
pol
(min
-1
)
K
d

(µM)
k
pol
/K
d
(µM
-1

min
-1
)
k
pol
effect
k
pol
/K
d
effect
ATP/dT
750 ±
210
90 ± 20 8.3 ± 3 5.7 ± 0.1 37 ± 2
0.16 ±
0.01
0.0076 ±
0.0021
0.019 ±
0.007
dATP/dT
0.93 ±
0.03
650 ± 50
0.0014 ±
0.0001
0.0087 ±
0.0003
120 ± 20
(7.3 ±
0.9) ×
10
-5

0.0094 ±
0.0005
0.051±
0.008
UTP/dT
0.015 ±
0.03
800 ± 60
(1.9 ±
0.4) ×
10
-5

0.0043 ±
0.0003
2000 ±
300
(2.1 ±
0.4) ×
10
-6

0.29 ±
0.06
0.11 ±
0.03
ATP/dF
0.13 ±
0.02
540 ± 30
(2.4 ±
0.4) ×
10
-4

0.0018 ±
0.0001
550 ± 80
(3.3 ±
0.5) ×
10
-6

0.014 ±
0.002
0.014 ±
0.003
UTP/dF
0.19 ±
0.04
760 ± 60
(2.5 ±
0.6) ×
10
-4

0.0025±
0.0001
710 ± 70
(3.5 ±
0.4) ×
10
-6

0.013 ±
0.001
0.014 ±
0.004
a
k
pol
effect = k
pol
(CH
2
)/ k
pol
(wt); k
pol
/K
d
effect = k
pol
/K
d
(CH
2
)/ k
pol
/K
d
(wt)

The methylene substitution effects on NTP incorporation were examined in a
systematic manner and summarized in Figure 4.2.4 and Table 4.2.1. Methylene
substitution greatly reduced incorporation of ATP for both the dT and dF templates,
indicating that the strong CH
2
substitution effect does not rely on W-C hydrogen bonding
between ATP and the matched DNA template. In sharp contrast, CH
2
substitution of
noncognate UTP incorporation leads to a ~3-fold decrease for UTP/dT system, whereas it
causes a ~80-fold reduction for UTP/dF incorporation. These results suggest that wobble
hydrogen bonding between UTP:dT may be critical for counteracting the strong
methylene substitution effect for noncognate UTP incorporation. We found that the CH
2

substitution effect on the UTP/dT pair is distinct from the rest of the groups (Figure 4.2.4
and Table 4.2.1).

70
70

Figure 4.2.4 CH
2
substitution effect on k
pol
in different scaffolds

To gain further structural insight into how UTP binds in the pol II active site, we
performed molecular modeling based on a previously published high resolution wobble
base-pairing crystal structure.
38
Based on previously reported crystal structures of U:U
pairing in RNA duplex, two possible wobble-base pairing patterns were obtained for
UTP:dT in the context of the RNA:DNA hybrid duplex (Figures C15).
38
When we
superimposed these two states in the pol II active site,
3
we found that only one UTP:dT
wobble-pair pairing was energetically preferred (Figure 4.2.5A), whereas the other
wobble-pair causes strong steric clash with neighboring pol II residues (Figure 4.2.5B).
Further energy minimization of the favorable wobble-base pairing form in Figure 4.2.5A
reveals that UTP is well tolerated at pol II catalytic site (Figure C16A).
Interestingly the uracil base of incoming UTP may likely form additional
electrostatic interactions with the main chain of nearby pol II residues in fork loop 3,
which we previously reported to play a recognition role towards modified cytosine in the
template.
39
In parallel, we also applied the same energy minimization procedure for the

71
71

Figure 4.2.5 Molecular modeling of UTP binding opposite dT template at pol II active site
A) UTP forms a wobble bonding pair with dT template, resulting in the misalignment of its triphosphate
group compared to cognate ATP recognition; B) An alternative form of UTP:dT pairing In this form, the
UTP shifts towards the minor groove of the RNA/DNA hybrid, leading to steric clash between pol II
residues and the incoming nucleotide. The two potential base pairing behaviors were modeled based on the
previously reported U:U pair.
38

“sterically clashed” wobble-base pairing form in Figure 4.2.5B. Although the strong
steric clash was partially resolved by pushing both the protein residue and the uracil base
of UTP away from each other, it is not energetically favorable for closed contact between
the hydrophobic residue (P448) and the hydrophilic uracil base (Figure C16B), Hence,
we believe that wobble base paring of UTP:dT in Figure 4.2.5A (and Figure C15A) may
represent the major form during UTP/dT incorporation. In this energy-favored
conformation, the UTP:dT wobble geometry causes UTP to shift away from the
canonical position (nucleotide addition site). Such a positional shift also causes the
triphosphate moiety of UTP to move away from its canonical binding site, which contains
several positively charged interacting residues. Thus, this positional shift of the
triphosphate may prevent nucleotidyl transfer reaction, disfavor the stabilization of the

72
72
transition state,
3,40
and ultimately results in the unique CH
2
substitution pattern observed
for the UTP/dT pair.

4.2.5 Discussion
RNA pol II possesses high nucleotide incorporation efficiency and fidelity
during transcriptional elongation to ensure accurate and timely genetic information
transfer. Application of a “synthetic nucleic acid substitution” strategy has revealed
several unique contributions from the nucleic acid nucleobase and sugar backbone
moieties.
22,23,37
In this study, we utilized β,γ-CH
2
-NTP “analogues” as probes to dissect
the relationship between “triphosphate” moiety and the mechanism of nucleotide
incorporation. These probes provide unique information pertaining to the roles of base-
pairing and how the leaving group affects cognate (matched) and noncognate nucleotide
addition by pol II.
It was reported that the chemistry step is not the rate-limiting step for pol II
nucleotide incorporation. Rather, non-chemistry steps, such as trigger loop
conformational changes upon substrate binding and translocation steps, are likely to be
rate-limiting steps.
41-44
On the other hand, direct evidence for dissecting and comparing
the relative time-scales of the chemistry step with those non-chemistry rate-limiting steps
is lacking. Here we reported a group of β,γ-CH
2
-NTP analogues that act as “functional
mutants” and fit into the pol II active site without disrupting the key recognition process
of TL closure. Thus, these β,γ-CH
2
-NTP “analogues” allow us to specifically modulate
the chemistry step without interfering in non-chemistry rate-limiting steps (e.g., trigger
loop conformational changes).

73
73
To our surprise, we found that CH
2
substitution resulted in a ~130-fold
decrease in k
pol
in the cognate ATP/dT scaffold. The effect on the rate of chemical bond
cleavage and formation is significantly higher than previously observed for DNA pol
β.
32,33
Indeed, comparison of methylene substitution on RNA pol II and DNA polymerase
catalyzed DNA-dependent substrate incorporation reveals both similarities and
differences. In terms of similarities, we find that methylene substitution causes a decrease
in incorporation efficiency in both enzymes. However, RNA pol II is more sensitive to
methylene substitution with around ~100-fold rate decrease in cognate nucleotide
incorporation, whereas DNA polymerase β is only affected by ~10-fold. This result may
suggest that pol II active site is highly sensitive to electrostatic effect, in which variations
in the basicity of pyrophosphate leaving group can greatly affect the interaction network,
leading to a more significant response in the chemistry step than that of DNA polymerase
β during nucleotide addition. The weaker leaving group resulting from CH
2
substitution
is expected to slow down P-O bond cleavage of the triphosphate and incorporation into
the mRNA. These results in a shift of the overall rate-limiting step to the “chemistry” step.
Another striking result is that the effect of the β,γ-CH
2
substitution on the
noncognate UTP/dT scaffold is significantly different from that of the matched ATP/dT
scaffold. We found that methylene substitution leads to ~130-fold less incorporation
efficiency for the ATP/dT scaffold, but only causes a ~3-fold decrease in incorporation
efficiency for the UTP/dT scaffold. This striking distinction likely unveils a unique
altered transition state for bond formation and cleavage for UTP/dT incorporation
compared with matched ATP/dT incorporation. Our structural modeling suggests that
UTP likely adopts a distinct position (via wobble pair with dT template) in the active site

74
74
with a different set of interacting pol II residues. Indeed, ablation of the wobble hydrogen
bonds in the UTP/dF scaffold leads to a strong CH
2
substitution effect that resembles the
matched ATP/dT pair (Figure 4.2.4).

4.3 Conclusion
Collectively, our data from comparative studies of wt NTPs and β,γ-CH
2
-bridging
NTP analogues reveals a pol II catalytic site that is highly sensitive to variation in the
basicity of the leaving group. RNA pol II transcriptional efficiency is not only determined
by effective substrate recognition but also influenced by leaving group properties.
Notably, we found two distinct states probed by these NTP analogues. One is the base
cognate state, in which the interaction network is built on the canonical W-C base pair
and the complementary shape for high incorporation efficiency. The other one is the
mismatched UTP/dT form, in which a different interaction network is constructed via the
geometry induced by wobble UTP:dT hydrogen bonds. The uniqueness of UTP/dT
incorporation may reveal how pol II takes advantage of UTP:dT wobble pairing to
discriminate against UTP misincorporation. Future studies will seek to obtain the direct
structural evidence for this alternate UTP:dT binding and bond formation state and
systematically investigate additional noncognate incorporations using β,γ-CXY

NTP
analogues.

4.4 Experimental
4.4.1 Materials and Methods
Tetraisopropyl methylenebis(phosphonate) 1 was a gift from Rhodia. Nucleoside

75
75
5’-monophosphates were purchased from Sigma-Aldrich (UMP 4, dTMP 11) and Chem-
Impex International (dAMP, 3). Nucleoside 5'-monophosphate morpholidates ((d)NMP-
morpholidate, (d)NMP 5’-M, 5-6, 12) were prepared according to published
procedure.
27,45,46
All other reagents were purchased from commercial sources and used as
obtained, unless specified otherwise. β,γ-CH
2
-ATP (10) is commercially available from
Sigma-Aldrich (Figure 1B).
1
H and
31
P NMR spectra were obtained on VNMRS-500 and
VNMRS-600 3-Channel NMR spectrometers. Multiplicities are quoted as singlet (s),
doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd) or broad signal
(br). All chemical shifts are given on the δ –scale in parts per million ((ppm) relative to
internal D
2
O (δ 4.79,
1
H NMR),

or external 85% H
3
PO
4
(δ 0.00,
31
P NMR).
31
P

NMR
spectra were proton-decoupled, and
1
H and
31
P coupling constants (J values) are given in
Hz. The concentration of the NMR samples was in the range of 2-5 mg/mL. Preparative
HPLC was performed using a Phenomenex C
18
equipped with a Shimadzu SPD-10A UV
detector (0.5 mm path length) with detection at wavelength at 260 nm. Low-resolution
mass spectrometry (MS) was performed on a Finnigan LCQ Deca XP Max mass
spectrometer equipped with an ESI source in the negative ion mode. All exact masses
were computed for the following isotopic compositions:
1
H,
12
C,
14
N,
16
O, and
31
P. The
pH values reported for NMR samples with D
2
O as solvent are a direct reading of the
NMR sample using an H
2
O-calibrated NMR pH meter (pH
*
).
47

4.4.2 Synthesis of Tributylammonium Salt of Methylenebis(phosphonic acid), 2 and
Difluoromethylene bis(phosphonic acid), 13
This compound was prepared from 1 according to the literature.
26,27,48

76
76

4.4.3 Synthesis of Nucleoside 5’-β,γ-Methylenetriphosphates (β,γ-CH
2
-(d)NTPs), 7 and
9
In a flask with dried tributylammonium salt of methylenebis(phosphonic acid) 2
was added a stir bar and a solution of (d)NMP 5’-M in a 4:1 ratio under N
2(g)
. The
reaction was allowed to stir for 3 d at rt. The compounds were purified through dual-pass
purification (Table C1) using the modified strong anion exchange (SAX) gradient method
according to the literature.
46
Compounds were characterized by UV,
31
P and
1
H NMR and
MS (for spectra and HPLC data refer to Figures C1-12 and Table C1 in the supplemental
information). Compounds 7, 9, 10 were used for bioassays with RNA pol II (Figure
4.1.1B). The stability of these analogues was also confirmed by LC-MS during our
experimental time scale under assay conditions (Table 4.1.1).

4.4.4 Characterization of 2’-Deoxyadenosine 5’-β,γ-Methylenetriphosphate (β,γ-CH
2
-
dATP), 7
1
H NMR (500 MHz; D
2
O; pH 9.8, Figure C1): δ 8.5 (s, 1H), 8.2 (s, 1H), 6.5 (t, J
= 6.8 Hz, 1 Hz), 4.3 (s, 1H), 4.2 (m, 2H), 2.8 (dt, J = 13.7, 6.9 Hz, 1H), 2.6 (m, 1H), 2.2
(t, 20.3 Hz, 2H).
31
P NMR (202 MHz; D
2
O; pH 9.8, Figure C2): δ 12.4 (d, J = 28.6 Hz,
1P), 11.4 (d, J = 7.9 Hz, 1P), -8.2 (d, J = 27.6 Hz, 1P). ESI-MS (Figure C3): MS (m/z):
calcd for C
11
H
17
N
5
O
11
P
3
-
: 488.0, found: 488.0 [M − H]
−
.

77
77
4.4.5 Characterization of 2’-Deoxythymidine 5’-β,γ-Methylenetriphosphate (β,γ-CH
2
-
dTTP), 14
1
H NMR (500 MHz; D
2
O; pH 9.8, Figure C17): δ 8.5 (s, 1H), 8.2 (s, 1H), 6.4 (t, J
= 6.9 Hz, 1 Hz), 4.7 (s, 1H), 4.2 (s, 3H), 2.4 (m, 2H), 2.2 (t, J = 20.1 Hz, 2H), 1.9 (s, 3H).
31
P NMR (202 MHz; D
2
O; pH 9.8, Figure C18): δ 12.9 (dd, J
PP
= 27.5 Hz, J = 8.3 Hz,
1P), 11.5 (d, J = 7.7 Hz, 1P), -11.2 (d, J = 27.1 Hz, 1P). Values are consistent with
literature,
34
except the beta and gamma phosphate values are inverted in the
31
P NMR,
which may be due to pH differences of the NMR samples.
49
The relative J values for
each phosphorus peak are consistent. ESI-MS (Figure C19): MS (m/z): calcd for
C
11
H
18
N
2
O
13
P
3
-
: 479.0, found: 479.0 [M − H]
−
.

4.4.6 Characterization of Commercial Adenosine 5’-β,γ-Methylenetriphosphate (β,γ-
CH
2
-ATP), 8
1
H NMR (600 MHz, D
2
O, pH 10.5, Figure C4) δ 8.53 (s, 1H), 8.26 (s, 1H), 6.14
(d, J = 5.8 Hz, 1H), 4.58 (dd, J = 5.1, 3.8 Hz, 1H), 4.40 (t, J = 3.1 Hz, 1H), 4.29 – 4.16
(m, 2H), 2.18 (dd, J = 21.2, 18.8 Hz, 2H).
31
P NMR (202 MHz, D
2
O, pH 10.5, Figure C5)
δ 13.48 (dd, J = 27.2, 7.3 Hz), 11.43 (d, J = 7.3 Hz), -10.86 (d, J = 26.9 Hz). ESI-MS:
m/z 504 [M − H]
−
. Values are consistent with literature except for the beta- and gamma-
phosphate, which are shifted down four and up three ppm units, respectively.
50
These
differences are likely due to pH differences in the NMR sample.
49
ESI-MS (Figure C6):
MS (m/z): calcd for C
11
H
17
N
5
O
12
P
3
-
: 504.0, found: 504.0 [M − H]
−
.

78
78
4.4.7 Characterization of Uridine 5’-β,γ-Methylenetriphosphate (β,γ-CH
2
-UTP), 9
1
H NMR (500 MHz, D
2
O, pH 10.2, Figure C7) δ 7.86 (d, J = 8.6 Hz, 1H), 6.04
(dd, J = 5.2, 2.2 Hz, 1H), 5.91 (d, J = 7.7 Hz, 1H), 4.44 – 4.39 (m, 1H), 4.36 (td, J = 5.2,
1.8 Hz, 1H), 4.24 (ddd, J = 20.1, 4.9, 2.3 Hz, 2H), 2.18 (dd, J = 21.3, 18.9 Hz, 2H).
31
P
NMR (202 MHz, D
2
O, pH 10.2, Figure C8) δ 13.17 (dd, J = 27.2, 7.3 Hz), 11.33 (d, J =
7.3 Hz), -11.05 (d, J = 27.2 Hz). ESI-MS: m/z 481 [M − H]
−
. Values are consistent with
the literature except for the beta- and gamma-phosphate, which are shifted four units.
50

Differences are a result of pH differences in the NMR samples.
49
ESI-MS (Figure C9):
MS (m/z): calcd for C
10
H
16
N
2
O
14
P
3
-
: 481.0, found: 481.1 [M − H]
−
.

4.4.8 Re-characterization of Adenosine 5’-β,γ-Methylenetriphosphate (β,γ-CH
2
-ATP),
10 After Dual-Pass HPLC Purification
1
H NMR (500 MHz, D
2
O, pH 10.2, Figure C10) δ 8.42 (s, 1H), 8.13 (s, 1H), 6.01
(d, J = 5.8 Hz, 1H), 4.46 (t, J = 4.4 Hz, 1H), 4.29 – 4.24 (m, 1H), 4.17 – 4.02 (m, 2H),
2.06 (t, J = 20.0 Hz, 2H).
31
P NMR (202 MHz, D
2
O, pH 10.2, Figure C11) δ 13.09 (d, J
= 25.0 Hz), 11.32 (d, J = 7.5 Hz), -11.00 (d, J = 27.2 Hz). ESI-MS (Figure C12): MS
(m/z): calcd for C
11
H
17
N
5
O
12
P
3
-
: 504.0, found: 504.1 [M − H]
−
.

4.4.9 Synthesis of Nucleoside 5’-β,γ-Difluoromethylenetriphosphates (β,γ-CF
2
-
(d)NTPs), 15-18
To a flask containing the dried tributylammonium salt of
difluoromethylenebis(phosphonic acid) 13 was added a stir bar and a solution of (d)NMP
5’-M in a 4:1 ratio under N
2(g)
. The reaction was allowed to stir for 3 d at rt. The

79
79
compounds were purified by dual-pass HPLC on RP-strong anion exchange (SAX)
columns.
46

4.4.10 Characterization of 2’-Deoxyadenosine 5’-β,γ-Difluoromethylenetriphosphate
(β,γ-CF
2
-dATP), 15
1
H NMR (500 MHz, D
2
O, pH 10.2, Figure C20) δ 8.49 (s, 1H), 8.22 (s, 1H), 6.50
(t, J = 6.8 Hz, 1H), 4.30 (s, 1H), 4.27 – 4.12 (m, 2H), 2.83 (dt, J = 13.7, 6.8 Hz, 1H), 2.60
(ddd, J = 14.0, 6.3, 3.6 Hz, 1H).
19
F NMR (470 MHz, D
2
O, pH 10.2, Figure C21) δ -
117.81 (dd, J = 90.3, 72.3 Hz).
31
P NMR (202 MHz, D
2
O, pH 10.2, Figure C22) δ 3.77
(td, J = 70.7, 58.3 Hz), -2.77 (tdd, J = 90.0, 56.5, 32.0 Hz), -10.88 (d, J = 32.1 Hz). ESI-
MS (Figure C23): MS (m/z): calcd for C
11
H
15
F
2
N
5
O
11
P
3
-
: 524.0, found: 524.1 [M − H]
−
.

4.4.11 Characterization of 2’-Deoxythymidine 5’-β,γ-Difluoromethylenetriphosphate
(β,γ-CF
2
-dTTP), 16
1
H NMR (500 MHz, D
2
O, pH 9.6, Figure C24) δ 7.75 (d, J = 1.1 Hz, 1H), 6.37 (t, J = 7.0
Hz, 1H), 4.68 (dt, J = 6.4, 3.4 Hz, 1H), 4.26 – 4.18 (m, 3H), 2.44 – 2.32 (m, 2H), 1.94 (d,
J = 1.2 Hz, 3H).
19
F NMR (470 MHz, D
2
O, pH 9.8, Figure C25) δ -117.89 (dd, J = 90.2,
72.2 Hz).
31
P NMR (202 MHz, D
2
O, pH 9.8, Figure C26) δ 3.75 (td, J = 72.2, 56.5 Hz), -
2.85 (tdd, J = 89.4, 56.5, 32.2 Hz), -11.14 (d, J = 32.3 Hz). Values are consistent with
literature, except
19
F NMR values which were reported as multiplet for the beta
phosphate and a dt for the gamma phosphate, versus the tdd and td reported here,
respectively.
34
ESI-MS (Figure C27): MS (m/z): calcd for C
11
H
16
F
2
N
2
O
13
P
3
-
: 515.0,
found: 515.0 [M − H]
−
.

80
80

4.4.12 Characterization of Adenosine 5’-β,γ-Difluoromethylenetriphosphate (β,γ-CF
2
-
ATP), 17
1
H NMR (500 MHz, D
2
O, pH 9.9, Figure C28) δ 8.53 (s, 1H), 8.22 (s, 1H), 6.12
(d, J = 6.0 Hz, 1H), 4.60 (dd, J = 5.1, 3.5 Hz, 1H), 4.40 (t, J = 3.0 Hz, 1H), 4.32 – 4.18
(m, 2H).
19
F NMR (470 MHz, D
2
O, pH 9.9, Figure C29) δ -117.78 (dd, J = 90.5, 72.2
Hz).
31
P NMR (202 MHz, D
2
O, pH 9.9, Figure C30) δ 3.71 (td, J = 72.4, 56.5 Hz), -2.82
(tdd, J = 89.3, 56.3, 31.8 Hz), -10.91 (d, J = 32.3 Hz). Values are consistent with
literature, except for the coupling constant in
31
P NMR between the beta- and gamma
phosphates. Literature reports a J value of 22.4 Hz for 25, but 58.2 Hz for 26,
50
which is
consistent with the J values found in this report for compounds 23-26.
19
F NMR values
were not reported.
50
ESI-MS (Figure C31): MS (m/z): calcd for C
11
H
15
F
2
N
5
O
12
P
3
-
: 540.0,
found: 540.1 [M − H]
−
.

4.4.13 Characterization of Uridine 5’-β,γ-Difluoromethylenetriphosphate (β,γ-CF
2
-
UTP), 18
1
H NMR (500 MHz, D
2
O, pH 8.1, Figure C32) δ 8.01 (d, J = 8.1 Hz, 1H), 6.21 –
5.85 (m, 1H), 4.51 – 4.44 (m, 1H), 4.42 (t, J = 5.3 Hz, 1H), 4.35 – 4.17 (m, 2H).
19
F NMR
(470 MHz, D
2
O, pH 10.3, Figure C33) δ -117.09 (dd, J = 91.7, 72.8 Hz).
31
P NMR (202
MHz, D
2
O, pH 10.3, Figure C34) δ 3.49 (td, J = 72.7, 56.4 Hz), -3.17 (tdd, J = 90.8, 56.3,
32.8 Hz), -11.22 (d, J = 32.9 Hz). Values are consistent with literature.
50
ESI-MS (Figure
C35): MS (m/z): calcd for C
10
H
14
F
2
N
2
O
14
P
3
-
: 517.0, found: 516.9 [M − H]
−
.

81
81
4.4.14 Preparation and Determination of Concentrations of (d)NTP Analogue Solutions
Compound was dissolved in 1 mL of H
2
O and then 30 µL of this stock solution
was added to 300 µL of 100 mM phosphate buffer. The UV sample was prepared by
diluting 100 µL of second stock solution with 0.9 mL of 100 mM phosphate buffer at pH
7.0. Three samples were made and analyzed. Samples were further diluted with 100 mM
phosphate buffer if the absorbance exceeded 1.0 during sample analysis.

4.4.15 Determining Nucleotide Analogue Stability in Assay Conditions by LC-MS
As control standards, AMP and PP
i
(expected degradation products for ATP) or
BP (CH
2
-BP for β,γ-CH
2
-ATP), were made at a concentration of 5 mM in 1xEB buffer
(20 mM Tris-HCl, 40 mM KCl, 5 mM MgCl
2
, pH 7.5). BP and PP
i
are not UV active and
could not be detected by MS; AMP can be observed by UV at 260 nm (ε = 15,400 M
-1

cm
-1
, pH 7.5) and can be detected by MS. The data are summarized in the table below
(Table 4.4.1). β,γ-CH
2
-ATP analogues are very stable and soluble in physiological
conditions, as revealed by LC-MS analysis. No detectable AMP or other degradation
products were found in the β,γ-CH
2
-ATP sample after 4 day incubation at room
temperature (i.e., the longest time point in kinetic assays).
Sample preparation: A solution of ATP or β,γ-CH
2
-ATP (5 mM) was made in
1xEB Buffer (20 mM Tris-HCl, 40 mM KCl, 5 mM MgCl
2
), and 10 uL of sample were
taken at specific time points and run on LC -MS. Samples were kept at room temperature.
Retention times of standards:
AMP: R
t
= 3.3 min by UV at 260 nm
ATP: R
t
= 2.8 min by UV at 260 nm

82
82
CH
2
-ATP: R
t
= 2.7 min by UV at 260 nm
LC-MS Conditions for representative spectra of LC-MS stability experiment:
Method: Isocratic/50 mM NH
4
OAc, 7.5% CH
3
CN, pH = 6.0
Column: Phenomenex Luna 5u C
18
(2) 100A, 250 x 4.6 mm
ESI-MS: (-) Mode
Expected m/z(-): ATP—505.99; CH
2
ATP—504.01
Exact Mass: ATP—507.00 g/mol; CH
2
ATP—505.02 g/mol

4.4.16 In vitro Transcription Assays
RNA pol II was purified from Saccharomyces cerevisiae as previously
described.
3,51
The DNA template and non-template oligonucleotides were purchased from
IDT. RNA primers were purchased from TriLink Biotechnologies and radiolabelled using
[γ-
32
P] ATP and T4 Polynucleotide Kinase (NEB). The ultrapure NTPs were purchased
from Affymetrix.
The pol II elongation complexes for transcription assays were assembled using
established methods.
22,23
Briefly, an aliquot of 5’-
32
P-labeled RNA (10 µM) was annealed
with a 1.5-fold amount of template DNA (15 µM) and 2-fold amount of non-template
DNA (20 µM) to form the RNA/DNA scaffold (final stock concentration: 1 µM, defined
by RNA concentration) in elongation buffer (20 mM Tris-HCl (pH 7.5), 40 mM KCl, 5
mM MgCl
2
). An aliquot of the annealed scaffold of RNA/DNA (50 nM) was then
incubated with a 4-fold amount of pol II (200 nM) at room temperature for 10 min to
ensure the formation of a pol II elongation complex. The pol II elongation complex is
ready for in vitro transcription upon mixing with equal volumes of NTP solution of

83
83
various concentrations. Final reaction concentrations after mixing were 25 nM scaffold,
100 nM pol II, 5 mM DTT, 5 mM MgCl
2
, 40 mM KCl, 20 mM Tris-HCl (pH = 7.5), and
NTP. The quenched products were analyzed by 16% denaturing urea/TBE PAGE and
visualized using a storage phosphor screen and Pharos FX imager (Bio-Rad). All in vitro
transcription assays described in this manuscript were repeated two to four times.
Table 4.4.1 Summary of LC-MS results showing NTP stability over time

ATP Series β,γ-CH
2
-ATP Series
Time ATP
a
AMP
b
CH
2
ATP
c
AMP
0 506.07 n.d.
d
504.05 n.d.
1 h 506.08 n.d. 504.06 n.d.
4 h 506.06 n.d. 504.01 n.d.
1 d 505.99 n.d. 504.00 n.d.
2 d 506.03 n.d. 504.00 n.d.
4 d 506.02 n.d. 504.03 n.d.
Control N/A 346.11 N/A 346.11
a
Peak was detected in negative mode with expected m/z of 505.99
b
Peak was detected in negative mode with expected m/z of 346.06

c
Peak was detected in negative mode with expected m/z of 504.01

d
n.d. stands for not detected

4.4.17 Single Turnover Nucleotide Incorporation Assays
The assay was carried out as previously described.
22,23
Briefly, nucleotide
incorporation assays were conducted by pre-incubating 50 nM scaffold with 200 nM pol
II for 10 min in elongation buffer at 22 °C. The pre-incubated enzyme:scaffold complex
was then mixed with an equal volume of solution containing 40 mM KCl, 20 mM Tris-
HCl (pH 7.5), 10 mM DTT, 10 mM MgCl
2
, and 2-fold concentrations of various
nucleotides. Final reaction concentrations after mixing were 25 nM scaffold, 100 nM pol
II, 5 mM MgCl
2
, and various nucleotide concentrations in elongation buffer. Reactions

84
84
were quenched at various times by the addition of one volume of 0.5 M EDTA (pH 8.0),
and analyzed as described above.

4.4.18 Kinetic Data Analysis
Nonlinear-regression data fitting was performed using Prism 6. The time
dependence of product formation was fit to a one-phase association Equation (1) to
determine the observed rate (k
obs
). The substrate concentration dependence was fit to a
hyperbolic Equation (2) to obtain values for the maximum rate of NTP incorporation (k
pol
)
and apparent K
d
(K
d,app
) governing NTP binding.
[Product] = Ae
(-kobs t)
+ C (1)
k
obs
= k
pol
[Substrate] / (K
d,app
+ [Substrate]) (2)
Representative data and kinetic fittings are shown in Figure C13 and C14. The specificity
constant was determined by k
pol
/K
d,app
. Discrimination was calculated as the ratio of
specificity constants governing incorporation of cognate substrate over noncognate
substrate as described.
22,23

Table 4.4.2 AEI values of wt NTPs and β,γ-CH
2
-NTPs
Scaffold O CH
2

ATP/dT 81 ± 23 84 ± 10
dATP/dT 6.4 ± 1.3 7.5 ± 1.5
UTP/dT 2.6 ± 0.5 1.4 ± 0.3

4.4.19 Molecular Modeling and Energy Minimization
The pol II elongation complex model was built based on the crystal structures of
the pol II complex
52
with bound cognate ATP (PDB ID: 4BY1). The model of pol II
elongation complex with a noncognate UTP opposite dT was obtained in the following
procedure: First, the crystal coordinates of two stable U:U pairs were obtained from the

85
85
crystal structure of an RNA duplex (PDB ID: 4U38).
38
These U:U pairs form specific
wobble hydrogen bonding patterns. One of the UMPs was then substituted by UTP (from
the PDB ID: 2NVZ) to generate a UTP:U pair.
3
The nucleobase of the UMP
(corresponding to the template strand) in UTP:U pairs was superimposed with the dT at
the i+1 site of template strand in pol II complex using COOT.
53
This superposition
allowed us to define the potential positions of UTP in the pair of UTP:dT at pol II active
site. All structure figures were rendered in PyMOL (http://www.pymol.org).
Energy minimization of described model above was then performed. Protein
subunits Rpb4 (chain D) and Rpb7 (chain G) were removed. Missing residues were built
using Modeller.
54,55
The missing side chains of amino acids were fixed by the “what if”
suite.
56
Protonation states of the titratable residues were predicted using Propka
57,58
in
Pdb2pqr package.
59
The Amber99sb force field parameters
60
were chosen to simulate
protein, nucleic acids and ions. Amber99sb force field parameters for uridine were
employed for the base and sugar ring of UTP. Amber-compatible polyphosphate
parameters were developed from Meagher et al.
61
and were used for the triphosphate tail
of UTP.
Each system was solvated in a triclinic box with the box edges at least 10 Å away
from the Pol II surface. Then 85 sodium ions were added to make the system electrically
neutral. The whole simulation model contained ~450,000 atoms, including ~129,000
TIP3P water molecules.
62
Afterwards, the package Gromacs 5.0
63,64
was used for two-
phase energy minimizations: a 5000-step energy minimization with the steepest descent
algorithm was performed first with positional restraint (i.e., a force constant of 10 kJ mol
-
1
Å
-2
) on the heavy atoms of the nucleotides and UTP; followed by another 10,000-step

86
86
energy minimization performed for the whole system. The long-range electrostatic
interactions beyond the cut-off of 12 Å were treated with the Particle-Mesh Ewald
method.
65
The van der Waals interactions were smoothly switched off between 10 Å and
12 Å.

4.5 Chapter References

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89
89
Fluorinated Probes of Nitrogenase: Difluorocyclopropene Chapter 5.
and Difluorodiazirine
4

5.1 Introduction
Nitrogenase (N
2
ase) reduction of N
2
to ammonia is coupled to the hydrolysis of 16
equivalents of ATP to ADP/P
i
and is accompanied by the formation of one molecule of
H
2
.
1-4
Thus, in contrast to the familiar biochemical process of oxidative phosphorylation,
this enzyme mediates a reductive dephosphorylation, consuming energy to promote
catalysis despite the favorable thermodynamics of the reaction at ambient temperature.
There are at least three major known classes of N
2
ases. Each type is encoded by a unique
set of genes, and each has a different combination of bound metals (i.e., Fe/Mo, Fe/V and
Fe/Fe). The most widely studied N
2
ase is the molybdenum-containing enzyme, which
consists of two component metalloproteins, the MoFe-protein (MoFeP) and the Fe-
protein (FeP). The Fe protein transfers an electron to the MoFe protein coupled with the
hydrolysis of two MgATP. The MoFe-protein contains two different types of unusual
metallocenters, the FeMo-cofactor and the 8Fe7S P-cluster. Each electron-transfer step
between Fe protein and MoFe protein involves an obligatory cycle of association and
dissociation of the protein complex, with dissociation proposed to be the rate-determining
step for the overall reaction,

4
Reproduced with permission from Ni, F.; Lee, C. C.; Hwang, C. S.; Hu, Y.; Ribbe, M.
W.; McKenna, C. E. J. Am. Chem. Soc. 2013, 135, 10346. Copyright 2013 American
Chemical Society.

90
90
and the FeMo-cofactor identified as the site of substrate binding and reduction.
3,5,6

Besides reduction of the natural substrates, N
2
and H
+
, wild-type N
2
ase can reduce
a wide range of small molecules containing a triple bond (HCN, CH
3
NC, HN
3
/N
3
−
,
C
2
H
2
).
4
Very recently, our appreciation of the catalytic versatility of N
2
ase has expanded
to include the reduction of CO, long recognized as a non-competitive inhibitor of N
2
ase.
7-
9
However, even slightly larger homologues of terminal alkynes are very poor substrates
(e.g., propyne) and non-terminal alkynes are not reduced at all (e.g., 2-butyne).
10
Simple
alkenes (e.g., C
2
H
4
) are also virtually unreactive with wild-type N
2
ase,
10
but the strained-
ring C=C hydrocarbon cyclopropene (K
m
= 0.1 mM) is a relatively good substrate (N
2
:
K
m
= 0.1 mM) that undergoes both reductive ring cleavage (to propene) and reduction to
an alkane (cyclopropane).
11-14
Diazirine (K
m
= 0.05−0.09 mM), containing the azo
(−N=N−) group in a strained, three-membered ring, is a slightly better substrate than N
2

itself and is reduced by N
2
ase to methane, ammonia and methylamine, consistent with
reductive cleavage of both the N=N and C−N bonds.
15
Cis- and trans-dimethyldiazene
are reduced to the same products, with decreasing effectiveness (K
m
= 60 mM and
500~600 mM, respectively).
15,16
Simeonov and McKenna also showed that
monomethyldiazene inhibits H
2
evolution and C
2
H
2
reduction and detected at least one
reduction product, methylamine. Monomethyldiazene and also diazene, both of which are
unstable under assay conditions, were subsequently shown to give rise to
spectroscopically detectable enzyme-bound intermediates.
17-19

Thus cyclopropene is the only alkene known that is an effective substrate of wild-
type N
2
ase. To examine the effect of electron-deficiency on the C=C bond reactivity, 3,3-
difluorocyclopropene (DFCP) is of interest as a potential N
2
ase substrate. In addition, we

91
91
also propose to create a fluorinated diazirine (DF-DZR) probe that will provide exciting
new insights into the mechanistic pathway. This new substrate combines the properties of
DFCP and diazirine, as a unique marker for the redox chemistry of the enzyme. The C−F
bond is typically unreactive chemically due to its large bond strength, and fluorine has a
minimal steric impact as a substituent.
20,21
N
2
ase-catalyzed reduction of fluorinated
substrates, is also of interest in relation to biological degradation of halogenated
compounds
22,23
and organometallic activation of C−F bonds.
24-26
Apart from representing
a novel class of substrate if reduced, the
19
F nucleus-containing substrate will be unique
when bound into the protein complex, and thus might be useful for nuclear spin
resonance-dependent spectroscopies such as EPR/ENDOR and ESEEM, which are
important tools
1,2,27
to elucidate N
2
ase structure and function.
Here, we report a mechanistic investigation of DFCP and DF-DZR, and establish
DFCP as the first halogen-containing substrate of N
2
ase, and confirm
28
that the enzyme
catalyzes a remarkable reductive C−F bond cleavage to give the 6e
−
/6H
+
and 4e
−
/4H
+

reduction products propene (P1) and 2-fluoropropene (P2), respectively, consistent with a
hydride-transfer step and C=C cleavage, suggested by analysis of the reduction products
from 1,2-d
2
-DFCP. We also describe an improved synthesis of DFCP that conveniently
provides the pure compound in gram quantities and the first reported sets of NMR for
DF-DZR.

5.2 Results and Discussion
5.2.1 Reduction of DFCP to Propene and 2-Fluoropropene: Product Identification by
GC-MS and NMR
The exposure of DFCP (0.05 atm in Ar) to 1 mL of a N
2
ase assay mixture

92
92
containing ATP (5 µM), MgCl
2
(5 µM), CP (50 µM), CPK (50 units), HEPES (50 µM,
pH 7.5), and DT (50 µM) results in the formation of substantial quantities of propene and
2-fluoropropene, as detected by GC analysis (Porapak N column) of the gas phase.
Further analysis of the gas phase by GC-MS and
1
H,
19
F NMR confirmed the products as
propene and 2-fluoropropene, by comparison with spectra of authentic samples. As
shown in Figure D20, product peaks were detected at t
retention
= 1.66 min (m/z = 41 [M-
H]
+
, propene) and at t
retention
= 2.56 min m/z = 59 [M-H]
+
, consistent with a
fluoropropene. The
1
H NMR spectrum (Figure D21) of the product mixture is consistent
with a mixture (~1:1) of authentic propene and 2-fluoropropene. The
19
F NMR spectrum
of the reduction product eluting at 2.56 min (Figure D22) is identical to that of authentic
2-fluoropropene.
Table 5.2.1 Nitrogenase-Catalyzed Reduction of 3,3-Difluorocyclopropene
Expt
No.
Assay
mixture
a, b

FeP
(mg/vial)
MoFeP
(mg/vial)
FeP/MoFeP
Products formed
(nmol/min/mg MoFeP)
c
Ratio
P1/P2
Propene
(P1)
2-fluoropropene
(P2)
1 Complete 0.2 4 1:5 6.6 2.9 2.3
2 Complete 0.2 0.8 1:1 11.9 4.3 2.7
3 Complete 0.4 0.8 2:1 15.4 4.9 3.1
4 Complete 1.0 0.8 5:1 19.1 5.9 3.3
5 Complete 4.0 0.8 20:1 15.8 4.5 3.5
6 Complete 8.0 0.8 30:1 17.3 5.1 3.4
7 -ATP 1.5 0.2 30:1 nd nd /
8 -DT 1.5 0.2 30:1 trace trace /
9 -FeP 1.5 0.2 30:1 nd nd /
10 -MoFeP 1.5 0.2 30:1 nd nd /
a
Initial partial pressure of DFCP 0.03 atm. in 1 atm. Ar, determined manometrically
b
Reaction quenched by 0.1 mL 100 mM EDTA (pH = 7.5) after 15 min
c
Determined by GC

Pertinent data concerning the reduction are presented in Table 5.2.1. Neither
product was generated by control mixtures lacking FeP, MoFeP, ATP or dithionite (DT).

93
93
The reduction requirements for DFCP are therefore the same as those of other N
2
ase
substrates, including N
2
, acetylene and cyclopropene. H
2
(0.1 atm) could not be
substituted for DT as a reductant. It is important to note that the propene/2-fluoropropene
ratio varies when the ratio of FeP/MoFeP (electron flux) is changed. Higher electron flux
favors the formation of propene, the 6e
−
/6H
+
reduction product.
The release rate of the implied third reduction product, fluoride, from DFCP
under assay conditions was also determined. The fluoride release rate corresponding to
addition of enzyme to the assay mixture correlates with propene and 2-fluoropropene
formed by N
2
ase-catalyzed DFCP reduction (Figure D23).

5.2.2 Reduction of DFCP to Propene and 2-Fluoropropene: Kinetic Analysis

Figure 5.2.1 Double reciprocal (Lineweaver-Burk) K
m
plot for the formation of propene and 2-
fluoropropene from DFCP reduction by N
2
ase
K
m
for both products is the same (0.022 atm, 5.4 mM).

Gas phase studies with commercially obtained 2-fluoropropene (0.1 atm)
established its stability under assay conditions. No propene was observed when 2-
fluoropropene (0.1 atm) was reacted with the N
2
ase assay mixture, verifying that propene

94
94
produced during DFCP reduction did not arise from the reduction of free 2-fluoropropene
by N
2
ase. In addition, 2-fluoropropene was not detectably a product from propene and F
−

(released during the reduction). This was confirmed by incubating propene with N
2
ase
under varying concentrations of F
−
(as NaF). No 2-fluoropropene was observed, ruling
out the possibility that the F
−
ion participated in the reaction at the active site to form the
observed organo-fluorine product, although F
−
(133 pm in NaF crystal) has a similar
radius to hydride (146 pm in NaH crystal).
29
Incubation of C
2
H
2
or C
2
H
4
with N
2
ase under
varying concentrations of F
−
also did not give detectable vinyl fluoride, which further
supports this conclusion.

Figure 5.2.2 GC-MS spectra of products from d
2
-DFCP reduction by N
2
ase. (FeP/MoFeP = 20:1)
A) GC chromatogram; B) MS of P1, identified as d
2
-propene ([M-H]
+
, m/z = 43); C) MS of P2, identified
as d
2
-2-fluoropropene ([M-H]
+
, m/z = 61).

Double-reciprocal Lineweaver-Burk plots of the product-forming reaction were
constructed for each product, P1 and P2 (Figure 5.2.1). Both plots were linear over the

95
95
range examined and extrapolated to the same K
m
value, 0.022 atm, giving V
m
values of
16.4 and 5.1 nmol/min/mg MoFeP, respectively (Figure 5.2.1). Using a value of 0.25
M/atm for the solubility of DFCP in assay buffer at 30 °C, the estimated K
m
for DFCP is
5.4 mM, 50-100 times larger than those of its analogues cyclopropene and diazirine.

5.2.3 Selectivity in DFCP Reduction: Reduction of d
2
-DFCP in H
2
O
Scheme 5.2.1 d
2
-Propene and d
2
-2-fluoropropene isomers produced by N
2
ase-catalyzed reduction of d
2
-
DFCP (FeP/MoFeP = 20:1)

In order to explore regioselectivity in DFCP reductions, 1,2-dideutero-DFCP (d
2
-
DFCP) was synthesized (see characterization in Appendix D). d
2
-DFCP was incubated
with N
2
ase assay mixtures having FeP/MoFeP ratios of 20:1 and 1:5, and the gas phase
products were subjected to GC-MS analysis. As shown in the MS spectrum (Figure 5.2.2,
FeP/MoFeP = 20:1), the propene peak (t
retention
= 1.67 min) gives a m/z = 43 (d
2
-propene,
[M-H]
+
) and the corresponding 2-fluoropropene peak (t
retention
= 2.55 min) also shows a 2
Da increase (m/z = 61, [M-H]
+
), consistent with retention of both deuterium atoms
present in the substrate.
The gas phase product mixture was further characterized by
1
H NMR,
2
H-
decoupled
1
H NMR and
1
H-decoupled
2
H NMR (Figures 5.2.3A, B, and C, respectively,
FeP/MoFeP = 20:1). The two major products were identified as mixtures of d
2
-propenes
and d
2
-2-fluoropropenes. In the
1
H NMR spectrum (Figure 5.2.3A), a doublet at 4.463
F F
D D
DH
2
C
H H
D DH
2
C
H D
H
DH
2
C
F H
D DH
2
C
F D
H
~ 72% ~ 5%
~ 12% ~ 11%
N2ase
Fe/MoFe = 20:1
3.3
1
d
+
+

96
96
ppm (J
H-F
= 16.5 Hz) was assigned to the cis-CHD= proton in 1,3-d
2
-2-fluoropropene. A
0.016 ppm upfield shift was observed for this doublet (geminal deuterium isotope
effect)
30
compared to the corresponding chemical shift of the cis-CH
2
= protons in 2-
fluoropropene. Similarly, the doublet at 4.175 ppm (J
H-F
= 48.5 Hz) was assigned to the
trans-CHD= proton in 1,3-d
2
-2-fluoropropene (0.03 ppm upfield shift relative to the trans
proton in the CH
2
= of 2-fluoropropene). The doublet of triplets at ~1.9 ppm (J
H-F
= 16 Hz,
J
H-D
= 2.5 Hz, 0.016 ppm upfield shift related to undeuterated 2-fluoropropene) is
assigned to CH
2
D− in cis- or trans-1,3-d
2
-2-fluoropropene. This signal appears as a
doublet (J
H-F
= 16 Hz) in the
2
H-decoupled
1
H NMR spectrum, confirming the presence

Figure 5.2.3 NMR spectra (500 MHz, CDCl
3
/C
2
Cl
4
= 1:1) of products of N
2
ase-catalyzed reduction of d
2
-
DFCP (FeP/MoFeP = 20:1)
A)
1
H NMR spectrum; B)
2
H-decoupled
1
H NMR spectrum; C)
1
H-decoupled
2
H NMR spectrum

97
97
of one deuterium coupled to the protons. The
2
H NMR (
1
H-decoupled) spectrum shows a
doublet (J
D-F
= 2.0 Hz) at 1.926 ppm, consistent with the presence of fluorine in a CH
2
D-
C(F)= moiety (Figure 5.2.3B). By integration of the CHD= proton peaks in the
1
H NMR
spectrum, the ratio of d
2
-2-fluoropropenes was determined as 1:1 cis-1,3-d
2
-2-
fluoropropene:trans-1,3-d
2
-2-fluoropropene.
The remaining peaks in the
1
H NMR spectrum were assigned to d
2
-propene as
follows (Figure 5.2.3). The doublet of triplets at 1.70~1.75 ppm (J
H-H
= 6.0 Hz, J
H-D
= 2.0
Hz, 0.016 ppm upfield shift relative to undeuterated propene) belongs to a CH
2
D− group.
This signal appears as a doublet (J
H-H
= 6.0 Hz) in the
2
H-decoupled
1
H NMR spectrum.
The
2
H NMR (
1
H-decoupled) spectrum shows a

singlet at 1.744 ppm, consistent with the
assignment to a CH
2
D- group in d
2
-propene (Figure 5.2.3C). The doublet (J
H-H
= 10.0 Hz)
signal at δ = 4.919 ppm is assigned to the CHD= proton in cis-1,3-d
2
-propene. A 0.018
ppm upfield shift is observed for this doublet (geminal deuterium isotope effect) relative
to the corresponding peak in propene. The
2
H NMR (
1
H-decoupled) spectrum shows a

singlet at 5.075 ppm, supporting the presence of a CHD= group in cis-1,3-d
2
-propene
(Figure 5.2.3C). The weak doublet (J
H-H
= 17.0 Hz) signal at 5.012 ppm, 0.022 ppm
upfield shift relative to propene, is assigned to the CHD= moiety in trans-1,3-d
2
-2-
fluoropropene. Resonances at 5.75−5.85 ppm are contributed by the H on carbon-2 in
both cis-1,3-d
2
-propene and trans-1,3-d
2
-propene. The ratio of cis-1,3-d
2
-propene:trans-
1,3-d
2
-propene was determined as 14:1 by integration of the CHD= proton peaks in the
1
H NMR spectrum. It should be noted that signals from the geminal protons of
CH
2
=CH(CH
3-x
D
x
) were observed as two doublets at 4.936 ppm (merged with the proton
signal of CHD= in cis-1,3-d
2
-propene) and 5.022 ppm, respectively. This result indicates

98
98
the presence of a minor amount of d
1
-propene as a mixture of propene isomer consistent
with the observation of characteristic mass peak (m/z = 41, [M-D]
+
ion) for d
1
-propene in
Figure 5.2.2B. Combined with the GC data analysis, the constitution of the identified
product mixture from d
2
-DFCP reduction is summarized in Scheme 5.2.1. Figure D24
shows the dependence of the d
2
-DFCP reduction product distribution under different
electron flux conditions: higher electron flux favors a relatively greater amount of cis-
1,3-d
2
-propene.

5.2.4 Activity of DF-DZR in Enzyme Assays
DF-DZR was synthesized and characterized by
13
C,
14
N, and
19
F NMR and gas-IR
(Figure D6, D8-10). Computational modeling of gas phase IR simulation at three
different levels using GAUSSIAN show strong agreement with literature values,
indicating identity and purity of DF-DZR (Table D2 and Figure D16).
31
Based on time-
dependent
19
F NMR studies, DF-DZR demonstrates stability in water and in assay buffer
over time (Figure D17-18, respectively). To determine if DF-DZR is a substrate of N
2
ase,
acetylene inhibition experiments were performed with concentrations of 6% DF-DZR in
He. A schematic of the inhibition experiment is shown in Figure D25. Briefly, the
amount of acetylene with vary in five to six airtight assay bottles, with one series in the
absence of DF-DZR and the other in the presence of the gas. Enzyme was added to each
vial, along with all other assay buffer components. After completion of the reaction (i.e.,
10 minutes), detection of potential products in the gas phase was monitored by GC-FID
and the liquid phase was analyzed for ammonia using an OPA/2-ME method to measure
fluorescence.
32,33

99
99

Figure 5.2.4 Results from N
2
ase inhibition of acetylene experiment with 6% DF-DZR
A) Fluorescence detection at 490 nm for all samples with standard error (SE, n=3) bars shown; B)
fluorescence detection at 472 nm with standard error (SE, n=3) bars shown; C) amount of ethylene detected
by GC-FID for vials; D) schematic of experimental setup with varying amounts of acetylene and a constant
amount of DF-DZR (6%). Vessels depicted in images represent airtight assay vials used in experiments.

Preliminary inhibition experiment with varying amount of acetylene and a
constant amount of DF-DZR (Figure 5.2.4D) revealed presence of a putative ammonia
product in the liquid phase of assay mixtures. According to Figure 5.2.4A and B, vials in
the presence of DF-DZR and enzyme produce greater fluorescence than samples without
DF-DZR or without enzyme. Although the standard sample with DF-DZR – Enzyme
showed a substantially decreased fluorescence at both 490 and 472 nm, after two weeks
(which was the overall storage time for these samples), the fluorescence grew, indicating
that DF-DZR may interact with either the assay buffer or OPA/2-ME. In addition,

100
100
reduction of acetylene to ethylene was inhibited by the addition of DF-DZR at low
concentration of acetylene. This finding indicates that DF-DZR may be a potent inhibitor
of acetylene reduction and may be a substrate of N
2
ase.

Figure 5.2.5 N
2
ase inhibition experiment with acetylene and varying concentrations of DF-DZR
A) Fluorescence measurements using OPA/2-ME buffer of all samples; B) schematic of experimental setup
with varying amounts of DF-DZR and constant amount of acetylene. Vessels depicted in images represent
airtight assay vials used in experiments; C) Fluorescence detection at 472 nm for all samples with standard
error (SE, n=3) bars shown; D) fluorescence detection at 490 nm with standard error (SE, n=3) bars shown

To further elucidate the inhibitory capability of DF-DZR, a set of experiments
were performed and shown in Table D3. Five assay vials were prepared with the same
amount of acetylene and varying amounts of DF-DZR (decreasing by 10-fold). In
addition, several controls were also used. In vials with all components including an
atmosphere of 6% DF-DZR in He, one component was systematically removed (e.g.,

101
101
ATP stock or dithionite, etc.). A positive control was made with all buffer components
and N
2(g)
with enzyme. An EDTA control was also used to monitor if EDTA was
adequately quenching the enzyme reaction.
After allowing the enzyme assays to react at 30 ˚C for ten minutes, the reactions
were quenched with EDTA. The EDTA control revealed substantially more ethylene by
GC-FID when EDTA was not added, indicating that EDTA effectively quenches the
reaction. Most control vials with DF-DZR exhibited a strong fluorescence measurement,
except for controls lacking either CPK buffer mixture or DT (Figure 5.2.5C and D). In
addition, the fluorescence measurement decreases in the series with decreasing DF-DZR,
as expected. These experiments show that some byproduct of DF-DZR is interacting with
the assay mixture to produce an intermediate that can react with the OPA/2-ME buffer to
form a fluorescent product, as is the case in the control reactions that contain DF-DZR.
Furthermore, it shows that the OPA/2-ME cannot differentiate ammonia production from
N
2
ase reduction and DF-DZR byproduct reactions.
Although byproducts interfere with some of the fluorescence analysis, it is still
possible that DF-DZR is a substrate of N
2
ase. To explore this question, a 20:1 Fe: MoFe
protein ratio was employed with a high concentration of protein (375 units of MoFe),
with either acetylene or DF-DZR and allowed to react in a shaker bath at 30 ˚C for 60
minutes. A control with all buffer components and DF-DZR, excluding enzyme was also
employed. Experiments with acetylene revealed that the protein is active for over 60
minutes, continuously producing ethylene (real-time monitoring by GC-FID). However,
no reduction products were detected in assay vials with DF-DZR (concentration 50% DF-
DZR in He). Instead, a cloudy precipitate was observed only in vials with DF-DZR with

102
102
enzyme (Figure D29). There is also a slight decrease in concentration of DF-DZR in
assay vials with enzyme compared to vials with DF-DZR lacking enzyme (GC-FID). GC-
BID measurement of gas-phase of vial with DF-DZR showed four distinct peaks, with N
2

as the only identified peak.
19
F NMR of the reaction mixture revealed that the major
compound in solution corresponds to DF-DZR.
The experiment was repeated, except the enzyme concentration was doubled and
the ratio of enzymes was adjusted to 10:1 Fe:MoFe. Analysis of the assays vials revealed
similar findings to those in the previous experiment. A calibration plot of H
2
was
constructed to determine the amount produced from N
2
ase (Figure D31). H
2
was detected
in vials with acetylene but not for vials containing DF-DZR, with and without enzyme
(GC-BID). Enzyme assays without any substrate added, also resulted in H
2
production. In
a subsequent experiment, the reaction was repeated, except after 40 minutes, DF-DZR
was removed from the gas phase and argon and acetylene were added to the assay vial
(Figure D30A). Twenty minutes after the addition of acetylene, the gas-phase was
monitored by GC-FID. However, no ethylene production was detected, indicating that
DF-DZR interacts with the assay mixture and somehow irreversibly inhibits the enzyme.
The same cloudy solutions were observed in samples of DF-DZR in the presence of
enzyme as well. Assay mixtures with acetylene gave a pH reading of 7.2 and assay vials
with DF-DZR and enzyme measured at a pH 4.0. A
19
F NMR stability experiment was
performed again, and DF-DZR appeared to be stable, indicating that
19
F NMR might not
be sensitive enough to detect side reactions, especially due to the poor solubility of DF-
DZR in D
2
O.

103
103
The precipitate in the liquid phase of the assay mixture was analyzed using two
different protein concentration methods. A control was also prepared using a freshly
prepared assay mixture and treating it with TFA to precipitate the proteins. The mixtures
were centrifuged at 5,000 rpm for ten minutes. A Pierce Bicinchoninic acid (BCA) assay
kit and the Bradford reagent were used to independently determine protein concentrations.
A calibration plot was constructed for each method, using albumin as the standard. Based
on the calibration curve, both methods gave similar concentrations for the samples,
indicating that precipitated material from assays with DF-DZR in the presence enzyme is
a protein in nature.
Precipitation of the protein may be from DF-DZR interacting with buffer
components and subsequently lowering the pH, or from DF-DZR directly reacting with
protein through carbene or addition chemistry. To test whether DF-DZR is interacting
with buffer components, vials were prepared all containing DF-DZR for twenty minutes
without enzyme (Figure D32). Controls were made without any exposure to DF-DZR,
and only containing acetylene and enzyme. Then, DF-DZR was removed from the gas
phase, and in vials 1 and 2, only enzyme and acetylene were added. In vials 3 and 4, an
additional portion of ATP buffer was added along with enzyme and acetylene. To vials 5
and 6 were added an additional portion of dithionite solution, followed by enzyme and
acetylene. Vials 7 and 8 were controls with no exposure to DF-DZR.
Based on detection by GC-FID, the enzyme was active in reduction of acetylene
for the control vials (Figure D32, vials 7 and 8). In vials 1 and 2, no ethylene production
was observed. In vials 3 and 4, ethylene production was observed but not as much as the
controls. Addition of dithionite solution to vial 5 also recovered some ethylene

104
104
production, however, curiously there was no ethylene detected in vial 6. The findings
from this experiment unequivocally demonstrate that enzyme inactivation occurs in the
absence of DF-DZR, and only requires pretreated DF-DZR assay buffer to deactivate
acetylene reduction. In addition, add-back of ATP stock solution or dithionite solution
recovers some enzyme activity, but does not fully restore activity. Results from these
experiments suggest reduction assays with DF-DZR requires a completely different
reductant (dithionite) or assay mixture altogether.

5.2.5 Discussion: Mechanistic Implications of DFCP as a Substrate for N
2
ase
N
2
ase cleaves the strained cyclopropene ring of DFCP, adding reductively two H,
mirroring its reduction of cyclopropene itself to propene (2e
−
). However with DFCP,
additional electrons are transferred before product release, cleaving one (2e
−
) or both (4e
−
)
C−F bonds, giving 2-fluoropropene and propene, respectively. The choice of 3,3-
difluorinated cyclopropene (DFCP) rather than a 1- or 2-fluorinated cyclopropene was
based on the known fact that in alkyne substrates, extension of the carbon chain length
along the –C≡C– axis decreases effectiveness as a N
2
ase substrate (e.g., propyne, butyne).
Nevertheless, the K
m
(5.4 mM) and V
m
(16.4 and 5.1 nmol/min/mg MoFeP) of DFCP
implies lower affinity and lower N
2
ase activity than cyclopropene, suggesting that a
possible topological restriction is imposed on the substituents of carbon-3 of the
cyclopropene ring. Alternatively, the low affinity and low activity of DFCP might also be
due to the effect of the electronegative fluorine substituents on alkene reactivity.
Although reduction chemistry of DFCP has not been previously reported, it is recognized
that fluorine substitution at the methylene carbon of cyclopropene results in significant

105
105
lengthening of the C=C bond and shortening of the C–C single bonds,
34
where fluorine
substitution at C-3 in cyclopropene appears to actually stabilize the three-membered
ring.
35
This will decrease the “alkyne-like” property of the strained C=C bond, making it
more “alkene-like” and thus less effective as a N
2
ase substrate.
With cyclopropene, only 2e
−
reduction products were found (cyclopropane and
propene). With fluorine substitution, the reduction product pattern is changed to 4e
−
/6e
−

product, 2-fluoropropene/propene. These products require an unprecedented reductive
C−F bond cleavage by N
2
ase. The “missing” 1,1-difluorocyclopropane product (b.p.
−16 °C)
36
might be explained by a destabilizing effect on the three-membered ring of
cyclopropane by fluorine substitution, unlike the stabilizing effect of fluorine on
unsaturated cyclopropene.
35
Such an effect could make the proposed cyclopropane
intermediate 5 in Scheme 5.2.2 prone to release strain energy through ring-opening or by
stabilizing the ring through fluoride release, without formation of 1,1-
difluorocyclopropane. Assuming that no fluorine migration takes place during reduction,
the fluorine atom on the 2-fluoro product provides a mechanistic marker indicating that
DFCP is cleaved symmetrically with respect to its C=C bond.
In the d
2
-DFCP reduction experiment, both MS (Figure 5.2.2B) and NMR (Figure
5.2.3) data indicate that a d
1
-propene is an apparently minor product of the reduction,
most likely 3-d-propene (Figure 5.2.3A, B). Chemical exchange of d
2
-DFCP with H
2
O is
unlikely under the assay conditions. An enzyme-catalyzed exchange of d
2
-DFCP with a
protiated species at the MoFe protein active site, e.g. addition of metal-hydride to the d
2
-
DFCP C=C bond, could be followed by elimination of a deuterium atom to generate d
1
-
DFCP, which could then be reduced to the d
1
-propene.

106
106
Scheme 5.2.2 Proposed mechanism for reduction of DFCP catalyzed by N
2
ase

d
2
-DFCP reduction exhibits two important features. First, the two deuteriums are
retained in the major products (propene and 2-fluoropropene), and are found about
equally on carbon-1 and carbon-3, in both, indicating that C=C bond cleavage is the main
reaction pathway rather than initial ring C−C bond cleavage, but via a 4e
−
process
involving the elimination of F
−
. N
2
ase-catalyzed reduction of cyclopropene in D
2
O to
cyclopropane gave a cis-1,2-dideuterated product, also consistent with symmetrical
addition of hydrogen across the C=C bond.
14
Reduction of cyclopropene gave 1,3-d
2
-
propene as the major propene product.
14

The second notable result is that the reduction product 1,3-d
2
-2-fluoropropene is
formed as equal amounts of the cis and trans isomers, while cis-1,3-d
2
-propene is the
major isomer in the 1,3-d
2
-propene product. This suggests that the reduction intermediate
on the pathway to propene is effectively constrained and that the formation of P1 and P2
M
F F
D D
+
DH
2
C
H H
D
DH
2
C
H D
H
F H
D
D
M
H H
H
H F
D
D
M
H H
H
H F
D D
M
H H
F H
D D
M
H H
e-
H
+
e-
H
+
H F
D D
M
F H
D D
M
F F
D D
M
H H
DH
2
C
F H
D
DH
2
C
F D
H
F F
D
D
M
H H
H
e-
2H
+
2e
-
e-
H
+
H
-
~ 72%
~ 5%
~ 12%
~ 11%
FeMoco
e-
e-
F
-
F
-
F
-
F
-
2H
+
2e
-
2H
+
2e
-
1
2
5
6a 7a 8a 9a
6b 7b 8b 9b
3
4a
4b
F F
D D
M
F
-
F
-
unstable
Path A):
3-membered
ring opening
Path B): Fluoride release

107
107
may involve distinct mechanisms. It has been proposed that both electron flux and steric
constraints around the active site affect the stereochemistry of N
2
ase reductions.
14,37

Comparing the product distributions at high electron flux (FeP/MoFeP = 20:1) and low
(FeP/MoFeP = 1:5), more cis isomer was observed for d
2
-propene at higher electron flux
(Figure D24). This trend was also observed in d
2
-acetylene reduction catalyzed by N
2
ase,
where higher electron flux favors formation of cis-d
2
-ethylene.
14

A DFCP reduction mechanism catalyzed by N
2
ase that accounts for the observed
results and other available information is proposed in Scheme 5.2.2. In this mechanism,
we focus on substrate transformation leading to product, but do not speculate on how
cluster metals are involved in the reduction, or the source of protons. The symbol M thus
refers to either Fe or Mo. Based on an intermediate trapped during propargyl alcohol
reduction by a mutant, an intermediate alkene bound side-on to a single Fe ion
38
was
proposed. Initial binding of DFCP to FeMo-cofactor is analogously proposed to give
intermediate 5 (Scheme 5.2.2). Hydrometalation and dimetalation of the electron-rich
C=C bond in olefins are well-established chemistry.
39
Oxidative addition of the C-F bond
by a transition metal, especially Ni
0
, Pd
0
and Pt
0
complexes,
26
is also well known, and
this reaction requires the metal to be in a low oxidation state. In a recent report, when
low-coordinate Fe(II) complexes were reacted with fluoroolefins having both C=C and
C-F moieties, initial addition by the Fe(II)-hydride of the C=C groups, rather than
insertion into C-F bond, was observed.
40

In Path A, reduction of 5 by two electrons and two protons (plausibly as hydride
species) results in the C−C bond cleavage and intermediate 2. After further reduction of 2
by one electron and one proton along with cleavage of the M−C bond, intermediate 3 is

108
108
proposed as the precursor to the final products 4a and 4b. Since departure of each
fluorine on C-3 is equally likely upon β-elimination, the final step entails formation of a
C=C bond with no stereoselectivity, resulting in the observed equivalent amounts of the
isomeric 1,3-d
2
-2-fluoropropenes. (It is also interesting to consider the observed
selectivity in terms of theoretically postulated non-equivalent H-transfer pathways.
5
)
The other pathway (Path B), leading to propene, is proposed to involve initial
loss of fluoride from intermediate 5, facilitated by hydride attack at the CF
2
carbon via an
S
N
2 mechanism. The intermediate 5 is proposed to undergo a facial-selective attack by
hydride on the front or back face of the constrained three-membered ring. Since 5 covers
the M site, a hydride transferred from the FeMo cofactor would preferentially attack 5 on
the “back-face” (blue arrow), instead of the “front-face”(red arrow), of the 3-membered
carbon ring in 5 (Scheme 5.2.2), thus predominantly producing 6a over 6b. 6a and 6b are
further reduced to intermediates 8a and 8b, respectively. 8a and 8b then undergo “anti-
elimination” of fluoride ion to give 9a and 9b, respectively. The proposed pathway to
propene formation involves attack of hydride on intermediate 5, implying a metal hydride
species available to the bound DFCP. This is supported by recently suggested mechanism
proposing a FeMo-cofactor hydride species.
41,42
As depicted in Scheme 5.2.2, formation
of intermediates 2 and 5 should be competitive. Propene as the dominant product over 2-
fluoropropene might be explained on the basis that the fluoride release step (Path B)
might be a more energetically favorable than the 3-membered ring-opening step (Path A).
In summary, not only does ring-strain imposed on the −CH
2
=CH
2
− group
transform it from a non-substrate (by wild-type N
2
ase) to a fairly efficient 2e
−
substrate
(cyclopropene), but the latter is further converted into a 6e
−
substrate (DFCP, mimicking

109
109
N
2
in this respect) by incorporation of two F atoms. This is not a binding effect because
DFCP is more poorly bound than cyclopropene itself, but rather a novel “electron sink”
effect from fluorine substitution, in which 2e
−
(4) or 4e
−
(9) are consumed to cleave C−F
bonds rather than C−C bonds. The results support the earliest indications
16,28
that hydride
transfer precedes protonation.
41,42

5.2.6 Discussion: Implications for Dehalogenase Mechanisms
It has long been known that transition-metal nucleophiles readily displace fluoride
from highly fluorinated arenes and alkenes to afford metal-arene and metal-vinyl
complexes, respectively. These reactions are commonly viewed as simple nucleophilic
substitution reactions.
24
Our results document a novel C−F cleavage mediated by N
2
ase
that may offer new insights into biological C−F bond degradation.
Under aerobic conditions, compounds such as fluorobenzoate, fluorophenol and
fluorobenzene can be catabolized by microbial enzymes via established aromatic
hydrocarbon pathways. However, under anaerobic conditions, little is known about
degradation of fluoroaromatic compounds.
22
Interestingly, benzoyl-CoA reductase from
Thauera aromatica can reduce 2-, 3- or 4-fluorobenzoyl-CoA.
43
This enzyme is an
oxygen-sensitive iron-sulfur protein with a molecular mass of 160 kDa containing two
separate [2Fe-2S] clusters and two interacting [4Fe-4S] clusters in its four subunits.
Benzoyl-CoA reductase can also catalyze the ATP-dependent reduction of
hydroxylamine (K
m
= 0.15 mM) and azide. It has been suggested that some of its
properties resemble those of N
2
ase, which similarly overcomes the high activation energy
for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP.
43

110
110

5.3 Conclusion
DFCP is the first known halogen-containing substrate of N
2
ase, which reduces
DFCP to two detected reduction products, propene and 2-fluoropropene, identified by
GC-MS and NMR spectroscopy. Both propene and 2-fluoropropene have the same K
m

constants (0.022 atm, 5.4 mM), indicating that they are the reduction products of the
same substrate. The fluorine atoms create an “electron sink” that converts the strained-
ring cyclo-alkene, normally a 2 e
–
substrate, into a 4 e
–
or 6 e
–
substrate with reductive
cleavage of one or both C−F bonds to eliminate F
−
. Formation of a product that retains
one fluorine atom (2-fluoropropene) implies a bound reduction intermediate that retains a
C−F group, thus making available a potential new (
19
F) NMR or EPR/ENDOR/ESEEM
probe
27,44
for detecting active site-bound species.
DF-DZR was successfully synthesized and fully characterized. Based on initial
assessment, it appeared that DF-DZR was a strong inhibitor of N
2
and acetylene.
However, upon further inspection, it was revealed that DF-DZR interacts with the assay
medium that lowers the pH of the mixture and precipitates the nitrogenase protein. It is
possible that DF-DZR is interacting with either dithionite or ATP, drastically reducing
the pH and interfering with reduction, even after add back experiments of ATP or DT,
respectively.
Analysis of reduction products from 1,2-d
2
-DFCP indicates that one deuterium
atom is found on carbon-1 and one on carbon-3 of both propene and 2-fluoropropene,
indicating that initial C=C bond cleavage rather than ring C−C bond cleavage is the
major reaction path during DFCP reduction, consistent with C=C side-on binding of

111
111
substrate to the metal cluster in the active site. The reduction product 1,3-d
2
-2-
fluoropropene consisted of equivalent amounts of cis and trans isomers, whereas cis-1,3-
d
2
-propene was the major isomer in 1,3-d
2
-propene product, suggesting that the reduction
intermediate on the propene formation pathway is constrained.
Reductive cleavage of the C−F bond in DFCP by N
2
ase demonstrates the
extraordinary catalytic versatility of this enzyme beyond its natural role in biological N
2

fixation,
7,8
and may be compared to ATP-dependent metalloenzyme reductions in
biological C–F bond degradation.
22,23

5.4 Experimental
5.4.1 Materials and Methods
Nitrogenase. N
2
ase components were purified from continuously cultured
Azotobacter vinelandii OP. The MoFe protein (Av1, 24.5 mg/mL) had a specific activity
of 2000 (specific activity is defined as nmol of C
2
H
2
reduced per mg of protein per min)
and the Fe protein (Av2, 17 mg/mL) had a specific activity of 1900 nmol/mg min.
45

5.4.2 Synthesis and Characterization of DFCP
Scheme 5.4.1 Synthesis of DFCP
Br
F
3
C CF
O
CF
2
+
F F
Br
F
3
C
O
F
F F
190
o
C
~10 h in sealed
glass vessel
DFCP
BDFCP
1) Trap to trap vacuum distillation from
-80 to -196
o
C
2) Distillation ~ 0.1mm Hg, -80 to -50
o
C )
3) Ascarite
yield = 95%
yield = 37%
+

112
112

DFCP was synthesized on gram-scale by a modification of the method of Craig et
al.
46
as shown in Scheme 5.4.1. Specifically a low-pressure (~100 microns), low
temperature (−80 to −50
o
C) distillation of the intermediate 1-bromo, 3,3’-difluoro-
cyclopropane (BDFCP) replaced the tedious and scale limiting preparative GC
purification used in the original method. Purified BDFCP was then passed back and forth
through an Ascarite column to yield DFCP quantitatively, characterized by
1
H,
19
F,
13
C
NMR and mass spectral analysis (Figure D1, D3B, D4, and D5A, respectively).

5.4.3 Synthesis and Characterization of d
2
-DFCP

d
2
-DFCP was prepared by exposure of DFCP to D
2
O containing ~3 M NaOD. In
a typical experiment, 2 mmol of the DFCP were condensed into a 50 mL flask containing
10 mL of frozen D
2
O solution.
46
The D
2
O was then thawed as rapidly as possible while
the flask was shaken and the d
2
-DFCP quickly drawn off and trapped by liquid N
2

cooling without refreezing the D
2
O. Residual D
2
O was removed by passing the product
gas through a −70 ˚C (acetone/dry ice) trap. d
2
-DFCP was collected at −117 ˚C
(ethanol/liquid N
2
) trap.
19
F NMR and mass spectra confirmed the identity of d
2
-DFCP
(Figure D3A and D5B).

F F
D D
F F
H H
3M NaOD
0 ~ 4
o
C
yield = 85%

113
113
5.4.4 Chemical Stability of DFCP Under Assay Conditions
In a typical assay (DFCP 0.03 atm, 0.2 mg MoFeP), the total amount of detected
P1 and P2 account for ~1% of initial DFCP. During the assay excess of DFCP decrease
occurred. Control experiments with varied concentrations of DFCP incubated over 1 h
with assay mixture (without N
2
ase) were performed. DFCP decreased at rate of ~50% per
hour exposed to the assay mixture (without N
2
ase). When N
2
ase is present, no substantial
(>1%) difference was detected. DFCP in aqueous solution slowly hydrolyzes to fluoride
ion and cyclopropenone (verified to not a substrate of nitrogenase). DFCP in deuterated
chloroform (with a trace of water) was completely transformed to cyclopropenone
overnight at RT (see Figure D2A). The proton NMR spectrum of the D
2
O solution after
incubation with DFCP showed a strong cyclopropenone peak (see Figure D2B), though
no such signal was observed under corresponding complete nitrogenase assay conditions.
Similar hydrolysis behavior was previously observed with 3,3-dichlorocyclopropene, the
chlorine analogue of DFCP.
47,48
Therefore, the uncatalyzed decrease in DFCP is likely
due to reaction of DFCP with water to form cyclopropenone and fluoride.
Cyclopropenone was confirmed not to be a product of nitrogenase reduction of DFCP. No
products were detected in the gas phase by GC when cyclopropenone was exposed to
nitrogenase assay solutions.

5.4.5 Synthesis of Difluorodiazirine

difluorocyanamide
b.p. 1 ˚C
N
N
F
F
cyanamide
b.p. 260 ˚C
NH
2
N
F
(g)
in He
(g)
CsF
0˚C
3,3-difluorodiazirine
b.p. -84 ˚C
N N
F F
RT,
overnight

114
114
DF-DZR was prepared according to literature procedure.
49,50
To prepare
difluorocyanamide, a solution of 13 g Na
2
HPO
4
·7 H
2
O, 20 g NaH
2
PO
4
·H
2
O, 70 mL of 50%
aq. cyanamide solution and 20 mL water was loaded into 500 mL FEP gas washing flask
and the solution cooled to -5 °C using an ice/salt bath.
51
A slow stream of 5% fluorine in
helium was bubbled through the solution and passed through two cold traps cooled at -
78 °C (dry-ice/acetone), two traps at -196 °C (liquid nitrogen) and a bubbler filled with
perfluorinated oil to prevent back diffusion of air and moisture. After about 30 minutes,
the cyanamide solution had turned noticeably yellow-orange. The flow of 5% fluorine
gas was stopped and the replaced by a stream of pure helium. After about 30 minutes, the
gas stream was stopped. The content of the two -196 °C was combined on a grease-less
Pyrex glass vacuum line and purified by fractional condensation through a -126 °C trap
and collected at -196 °C. The contents of both traps were inspected by gas-phase IR
spectroscopy and identified as H
2
O/HCN (-126 °C trap) and F
2
NCN/CO
2
/CF
4
(-196 °C).
Various pressure, volume and temperature measurements at ambient temperature by the
expansion method determined the content of the -196 °C trap to 13.5 mmol.
Inside the glove-box, a flamed-out glass ampule (V = 200 mL) was loaded with 943 mg
(6.2 mmol) finely powdered cesium fluoride. The ampule was connected to the glass
vacuum line, evacuated and a portion of the crude difluorocyanamide (9.0 mmol) was
condensed in at -196 °C. After 72 hours at room temperature and in darkness, the content
of the ampule was pumped through traps at -126 °C (NO
2
), -156 °C (DF-DZR, some CO
2
)
and -196 °C (CF
4
, CO
2
). Various pressure, volume and temperature measurements at
ambient temperature by the expansion method determined the content of the -156 °C trap
to 5.5 mmol.

115
115
To remove the impurity, the reaction mixture was purified twice using an
isopentane/N
2
slush bath (-156˚ C), which resulted in desired product with only trace CO
2
by IR (Figure D6).
31,52
The calculations were performed at three different levels (Table
D2), which gave similar results. The calculated data are very consistent with
experimental IR frequencies with correlation coefficients >0.98 (Figure D16). The
amount was determined to be 2.2 mmols of final product was stored at rt in a specially
designed vessel (236 Torr), wrapped in Al foil (Figure D19). The product was further
characterized using 0.1 mmol in ~0.7 mL CDCl
3
, a
1
H,
13
C,
14
N, and
19
F NMR spectra
were acquired (Figure D7-10).

5.4.6 Stability of Difluorodiazirine Under Assay Conditions
DF-DZR as a pure gas was allowed to sit at rt, wrapped in Al foil (at pressures
~100-200 Torr). After several months, there does not appear to be any degradation
product according to IR. A 20 mL glass ampule was loaded with 1 mL of water. The
ampule was connected to the glass vacuum line and the water degassed. A sample of 0.2
mmol DF-DZR was condensed in at -196 °C and the ampule allowed to warm to ambient
temperature. After 24 hours in darkness, the content of the ampule was pumped through
traps at -64 °C and -196 °C. The gas-phase IR spectra and pressure, volume and
temperature measurements revealed the content of the -196 °C trap to 0.19 mmol DF-
DZR.
Solubility of DF-DZR in water was explored by NMR. A 5 mm JYoung NMR
tube was loaded with 0.8 mL D
2
O. The tube was cooled to -196 C and 0.02 mmol of DF-
DZR condensed in. The mixture was allowed to warm to room temperature and was

116
116
inspected by
1
H,
19
F and
13
C NMR spectroscopy.
1
H NMR spectrum of DF-DZR did not
indicate any degradation from compound exposed to water (Figure D11). A
13
C NMR
spectrum was acquired with 30,000 scans, but did not show much signal from DF-DZR,
indicating poor solubility of DF-DZR in D
2
O (Figure D12). Additional
19
F

NMR
experiments were performed to assess DF-DZR in H
2
O over a period of four days,
showing general stability of DF-DZR (Figure D13-15). The integration of DF-DZR by
19
F NMR was monitored by spiking samples with standard miscible in water,
trifluoroethanol, which has a chemical shift of -77.03.
53

Following stability experiments of DF-DZR in water, a time-dependent
19
F NMR
experiment was performed on the stability of DF-DZR in assay buffer (Figure D18). The
sample was prepared from 0.5 mL of ATP stock (10 mM ATP, 10 mM MgCl
2
, 100 mM
creatine phosphate, 100 mM HEPES, 1 U/mL CPK, pH 7.5), 0.2 mL dithionite (100 mM),
0.298 mL H
2
O, and 0.002 mL trifluoroethanol stock (1 mM). After the solution was
prepared, it was frozen in liquid nitrogen and DF-DZR (60 Torr) and He (700 Torr) were
added to a J. Young NMR tube. The sample was carefully monitored while it defrosted.
The NMR spectrometer was variable temperature (VT) regulated to 30 ˚C, and calibrated
using ethylene glycol. A series of
19
F NMR spectra were acquired (with 256 or 1024
scans) at 10-minute intervals (Figure D18). Based on the set of spectra, it appeared that
DF-DZR was not degrading over time and maintained a constant integration ratio with
standard trifluoroethanol. Based on the set of NMR stability experiments, DF-DZR was
used in substrate inhibition experiments with nitrogenase.

117
117
5.4.7 Assay Reagents
ATP stock solution: ATP, MgCl
2
•6H
2
O, creatine phosphate and HEPES were
dissolved in H
2
O and adjusted to pH 7.5 with 1 M NaOH and creatine phosphokinase was
added. The final solution contained 10 mM ATP, 10 mM MgCl
2
, 100 mM creatine
phosphate, 100 mM HEPES and 100 µ/mL creatine phosphokinase. DT stock solution:
solid sodium dithionite was placed in a septum-stoppered vial, which was pumped and
filled with argon repeatedly. Argon-flushed H
2
O was added with swirling, to give a 100
mM DT solution.

5.4.8 Nitrogenase-Catalyzed Reduction Assays
(i) Kinetic assays: several 9.3 ml septum-stoppered glass vaccine bottles were
evacuated to < 20 µHg, then filled to 1 atm with a DFCP (partial pressure 0.0036 to 0.05
atm)/argon gas mixture using a vacuum line manifold. C
2
H
6
(20 µl) (Matheson, CP grade)
was injected into each bottle as an internal gas chromatography (GC) standard. GC
analysis aliquots (20 µl) were removed from each bottle (and replaced immediately by
equivalent volumes of argon). To each bottle mounted in a 30 °C shaker bath was added
in rapid sequence 0.5 mL of degassed (argon) ATP stock solution (5 µmol, ATP; 5 µmol
MgCl
2
; 50 µmol creatine phosphate, 50 µmol HEPES and 50 unit creatine
phosphokinase), 0.2 mL DT stock solution (20 µmol, DT), 0.233 mL degassed (argon)
H
2
O, 0.059 mL Fe protein and 0.008 mL MoFe protein (Fe:MoFe is 20:1) to a final assay
solution volume of 1.0 mL. After 20 min, 0.1 mL of 100 mM EDTA (pH = 7.5) was
injected into the assay vial to quench the reduction. Gas aliquots (20 µl) were removed
for GC analysis.

118
118
(ii) Reduction time course assay: a series of assay bottles containing 1 atm of a
DFCP (0.03 atm)/argon gas mixture were prepared and reductions were initiated as
described in (i). The reactions were quenched by 0.1 mL 100 mM EDTA (pH = 7.5) at
time intervals of 0, 6, 20, 30, 45, 60 min. Gas aliquots (20 µl) were removed for GC
analysis.
(iii) Reductions with varied electron flux: a series of assay bottles containing 1 atm
of a DFCP (0.03 atm)/argon gas mixture were prepared and reductions were initiated as
described in (i), except that Fe protein:MoFe protein ratios were 1:5, 1:1, 2:1, 5:1, 20:1
and 30:1 (quantities of FeP and MoFeP are shown in Table 5.2.1). The reactions were
quenched after 15 min and gas aliquots (20 µl) were removed for GC analysis.

5.4.9 GC-MS and NMR Sample Preparation
Reduction products for GC-MS and NMR analysis were produced using the
kinetic assay conditions except that bigger bottles (27.7 mL), 1 atm of a DFCP (0.05
atm)/argon gas mixture and three-fold more stock solution were used. Total volume of
assay solution is 3.0 mL. The appropriate amount of Fe protein and MoFe protein for the
derived ratio (20:1 or 1:5) was sequentially injected into the assay bottles to initiate
reaction. After 30 min, the reaction was quenched with 0.3 mL of 100 mM EDTA (pH =
7.5). The following process was utilized for product enrichment: the gas phase of the
assay bottle was removed into a syringe by gas-liquid displacement, the adding of
saturated aqueous Na
2
SO
4
. The collected gas was slowly passed through a long, "U"
shaped metal needle immersed in isopentane/liquid N
2
cooling bath (−160 °C). The
products propene (b.p. −47 °C)
54
and 2-F-propene (b.p. −24 °C)
55
collected in the "U"

119
119
shape needle were then released in 1.5 mL pre-pumped rubber-stoppered vials. GC
analysis indicated a recovery yield is > 90%. The vials were vented to the atmosphere and
the gas phase analyzed by GC-MS. After analysis, CDCl
3
or CDCl
3
/C
2
Cl
4
(1:1) was
added to the vials containing the gas mixture. Then the solution was transferred into a
pre-pumped, rubber stoppered NMR tube using a syringe.
1
H,
19
F, and
2
H NMR spectra
were recorded on Varian 500 MHz or Varian 600 MHz spectrometers.

5.4.10 GC and GC-MS Analysis
Quantitative GC analysis of reduction products were performed on a Porapak N
column (Analabs) [12 in (30.5 cm) x 3/16 in glass column, 120 ˚C, helium pressure 200
kPa], detector FID (temperature 200 ˚C, air pressure 75 kPa, Hydrogen pressure 50 kPa)
installed on Shimadzu GC-14A system. Qualitative GC-MS analysis was performed on a
Focus GC-DSQ II system (Thermo Scientific) equipped with a capillary HP-AL/S
column. Running conditions: 5 µL gas injection, column temperature 140 ˚C (0-10 min),
Helium flow rate 1.5 mL/min, split ratio 7, MS scan range 40-100 m/e (+), ion source
temperature 200 ˚C.

5.4.11 Fluoride Detection
Assay aliquots (0.1 mL) were withdrawn at 0, 5, 10 min from the assay vials (4
duplicates) and immediately frozen by liquid N
2
. The resulting samples were lyophilized
and diluted to 1.0 mL with ionic strength-adjusted buffer (ISA, pH = 5.25). The solutions
were then analyzed using a fluoride ion-selective electrode (Beckman, P/N 511141)
coupled to a pH/mv meter. The concentration of fluoride ion was determined by fitting

120
120
the readings to a standard working curve generated by testing standard fluoride solutions
under the same conditions.

5.4.12 Fluorescence Detection of the Liquid Phase of Reaction Mixtures
Ammonia can be rapidly detected using a fluorescence method well-established
from the literature.
32,33
Briefly, a reagent buffer referred to as OPA/2-ME containing 20
mM o-phthaldialdehyde, 3.5 mM 2-mercaptoethanol, 200 mM potassium phosphate and
5% v/v ethanol at pH 7.3 was prepared (Figure D26). The method requires 75 uL of
sample to be mixed with 3 mL of the OPA/2-ME buffer and allowed to react for 30
minutes, at rt in the dark. Then the fluorescence was measured and recorded at 472 and
490 nm. A calibration plot was constructed using stock concentration of ammonium
chloride for both wavelengths (Figure D27). This method was also used to analyze the
liquid phase of enzyme assay experiments. An example of the spectral characteristics of
the fluorescent isoindole can be seen in Figure D28, along with the natural fluorescence
exhibited by the buffer in the absence of ammonia (Figure D28A, H
2
O).

5.4.13 Inhibition of Enzymatic Acetylene Reduction with DF-DZR
Several inhibitions experiments were performed to determine if DF-DZR was a
substrate of N
2
ase. The first set of experiments varied the amount of acetylene and kept
the concentration of DF-DZR constant (6% DF-DZR in He, Figure ). The second set of
experiments held the amount of acetylene constant and varied the concentration of DF-
DZR in by factors of 10 (Table D3).
Additional experiments were performed with a 20:1 Fe:MoFe protein ratio, using

121
121
375 units of enzyme over 60 minutes to observe production of any putative produces
from DF-DZR reduction (Figure D29). A assay vial with only acetylene was prepared as
a control. Reactions were analyzed using GC-BID and
19
F NMR. The experiment was
repeated, doubling the enzyme concentration. No discernable product was detected, so
“add-back” experiments were performed. DF-DZR was added to complete assay mixtures
including enzyme, then after twenty minutes, DF-DZR was removed from the gas phase
and replaced with argon. Acetylene was added to the reaction mixture, however, no
ethylene was detected by GC-FID, indicating that exposure to DF-DZR killed the enzyme
activity. From these interesting findings, addition add-back experiments were performed
for each individual assay buffer component. H
2
generated from N
2
ase reduction were
detected using GC-BID, eluting within five minutes. Other gases detected by GC-BID,
such was acetylene, required a gradient temperature program (30 ˚C for 4 minutes, 30 ˚ -
180 ˚C from 4-30 minutes).

5.5 Chapter References

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124
Determining the Onset of Osteonecrosis of the Jaw using Chapter 6.
FRET-Quenched Bisphosphonate Probes
6.1 Introduction
Osteonecrosis of the jaw (ONJ) is clinically defined as an unhealed oral ulceration
or wound exposing the alveolar bone for eight weeks or longer.
1
It is a rare oral
complication experienced by cancer patients treated with nitrogen-containing
bisphosphonates (N-BPs), but only when administered through IV.
2-5
Tooth extraction
disrupts the oral mucosa continuity and results in osteoclastogenesis on the surface of the
alveolar bone, as well as in the extraction socket. N-BP has a high affinity to crystalline
calcium phosphate, and it is largely absorbed to bone, targeting osteoclasts.
6-8

Therapeutically, N-BP induces osteoclast dysfunction, which is linked to decreased
osteoblastic activity. However the mechanism of how N-BPs affect the onset of ONJ is
currently unknown.
2

We hypothesize that N-BP pre-adsorbed on the jawbone can be removed by
osteoclasts and transiently released to the oral mucosa tissue. The amount of released N-
BP may depend on the degree of osteoclastogenesis localized at the surface of alveolar
bone interfacing oral mucosa, and upon reaching a critical concentration, the released N-
BP may directly affect oral mucosa resident cells and adversely influence the healing of
oral mucosa wound. To determine the pathological mechanism of ONJ, we have prepared
a novel proof-of-principle N-BP-based probe equipped with a Förster resonance energy

125
125
transfer (FRET)-quenched reporter. The probes will generate a fluorescent signal only in
response to osteoclast-derived cathepsin K (CatK) activity, which causes separation of
emitter and quencher when incubated with osteoclasts. A short CatK-cleavable peptide
bridging the fluorescent dye and quencher are connected to a biologically active or
inactive N-BP scaffold through a clickable linker. In vivo application of the molecular
probes should enable a more comprehensive understanding of how ONJ develops in
patients treated with N-BPs.

6.1.1 Molecular design of quenched-fluorescence CatK probes

Figure 6.1.1 Chemical structures of para-dRIS (4) and RIS (9)

The novel N-BP-based fluorescent probes are designed to only generate a
fluorescent signal when osteoclast activity is present. Several fluorescent-tagged N-BPs
have been previously investigated for their adsorption to hydroxyapatite (HAP) in
vitro.
9,10
The avid bone affinity is due primarily to the two phosphonate groups.
6
Here,
we propose to use N-BPs, RIS (9) and corresponding para-dRIS (4) (which will retain
bone affinity, but lack anti-osteoclastic activity, Figure 6.1.1),
6-8
linked to a CatK peptide
substrate
11
that is tethered to a fluorescent dye at one end, and a quenching moiety at the
other (Figure 6.1.2A). Unlike RIS, the proposed inactive N-BP scaffold will lack an α-
hydroxy group (which contributes slightly to bone affinity but significantly to anti-
resorptive activity)
12
and its pyridyl side chain will be para-substituted, which will
N
OH P O
OH
P
OH
O
OH
9
HO
N
OH P O
OH
P
OH
O
OH
4

126
126
dramatically decrease anti-resorptive activity with little effect on the bone mineral
affinity (Figure 6.1.1).
6
The design allows the probes to be strongly adsorbed on the
surface of HAP (bone mineral), while selectively exhibiting or not exhibiting the
biological activities of the parent drugs. Moreover, both may be present simultaneously
and detected individually, as well as used separately.
7,8,13

Figure 6.1.2 Molecular design and rationale for quenched-fluorescence CatK probes
A) General design of molecular probes. As the peptide is cleaved by CatK, separation of the quencher and
fluorescence dye results in a fluorescent signal; B) Demonstrates the corresponding expected fluorescent
signal.

The N-BP scaffold (RIS and inactive N-BP probe, para-dRIS) will be synthesized
by direct application or a modification
14
of standard methods (Scheme 6.2.1).
6,14
The N-
BP will then be conjugated with a linker containing azide group (Scheme 6.2.1) and
modified by 1) a suitably chosen fluorescent dye (water-soluble fluorescein); and 2) an
oligopeptide designed to be a specific CatK substrate (e.g., the amino acid sequence:
GHPG●GPQG, cleavage site between GG). This sequence provides an F-Q distance that
emission
GHPG GPQG
peptide
quencher
λ = 534 nm
ε = 34,000
triazole
bisphosphonate (BP)
drug
fluorescent dye
λ = 494 nm
ε = 75,000
quencher
GPQG
triazole
bisphosphonate (BP)
drug
fluorescent dye
GHPG
energy transfer excitation
✂ ︎ enzyme
associated with
osteoclasts
excitation
energy transfer
X
0
10
20
30
40
50
60
70
80
90
100
400 450 500 550 600 650
fluorescence emission
Wavelength (nm)
A B

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127
is well within the calculated requirement for FRET (< 100 Å; the proposed 8-mer gives
~30 Å). Due to the proximity of the incorporated quencher (i.e., QC-1 or BHQ-1), the
fluorescent dye signal will be silent (due to FRET phenomenon, however, the transferred
light energy is dissipated by the absorbing quencher instead of re-emitted) (Figure 6.1.2).
When osteoclasts adhere to the bone surface and secrete CatK, the peptide substrate will
be cleaved, releasing the quencher. As a result, under external illumination immediate
emission from the fluorescent dye will occur, signaling the presence of the CatK and thus
osteoclastic activity (Figure 6.1.2). The overall synthetic route is depicted in Schemes
6.2.1-6.2.3.

6.2 Results and Discussion
6.2.1 Preparation of N-BP Conjugates
Our lab has developed a “magic linker” synthesis (e.g., strategy utilizing
intermediates 5 – 7, Scheme 6.2.1) to create a series of novel fluorescent N-BPs derived
from the modern generation of heterocyclic nitrogen-containing N-BPs and a wide range
of fluorophores within the visible to near-infrared region.
7-9
The synthesis is directly
based on attachment of a functionalized linker to the N-BP drug under exceptionally mild
reaction conditions. The proposed new bisphosphonic acid conjugates will be modified
by linkage to a short oligopeptide-specific CatK substrate, that has been derivatized by a
fluorescent dye, and a quenching molecule, such that the fluorescent signal is 'off' in the
intact peptide but turned 'on' in the event of cleavage (Figure 6.1.2).
The para-dRIS N-BP conjugate 8 was prepared using tetraisopropyl
methylenebis(phosphonate) and treatment with NaH in a mixture of DMF and THF,

128
128
followed by addition of 4-(chloromethyl)pyridine (Scheme 5.2.1). After column
chromatography purification, 3 was dissolved in freshly distilled dioxane and hydrolyzed
with concentrated BTMS under microwave (MW) conditions for 20 minutes. The
reaction was worked up with several portions of methanol and water and subsequent
removal of solvent to provide 4, quantitatively.
Scheme 6.2.1 Synthesis of para-dRIS N-BP conjugate 8

To prepare the final conjugate, the glycidyl azide linker 7 was quickly prepared
according to literature procedure with minor modifications.
15
Commercial
epichlorohydrin 5 was dissolved in an aqueous solution with sodium azide, followed by
addition of glacial acetic acid. The reaction was allowed to stir for 5 hours at 30 ˚C to
form 6. Then the reaction mixture was extracted with diethyl ether and the organic phase
was washed with sodium phosphate buffer (50 mM, pH 6.5). The organic phase was
dried sodium sulfate, filtered and solvents were removed (although not completely, due to
the low molecular weight and volatility of 6). Glycidyl azide 7 was prepared by addition
of 6 with water and 0.1 M NaOH to pH 12 for several minutes, followed by pH adjusting
to 7 with 0.1 M HCl.
Then 4 was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100
mM, pH 6.0) and a portion of 7 was added (Scheme 6.2.1). The reaction was allowed to
N
OH P O
OH
P
OH
O
OH
iPr-O
P
O
O-iPr
P
O-iPr
O
O-iPr
N
Cl
NaH, DMF/THF
N
O-iPr P O
O-iPr
P
O-iPr
O
O-iPr
2. MeOH
1. BTMS, 80 ˚C
Dioxane, MW, 20 min
O
Cl
OH
N
3
Cl
O
N
3 NaN
3
in H
2
O/AcOH
30 ˚C, 5 h
diluted NaOH
5-12 min
1 3 4
5 6 7
+
NaOH, RT, 12h
MES buffer (pH = 6.5)
N
OH P O
OH
P
OH
O
OH
OH
N
3
8

129
129
stir at rt overnight and then purified by SAX HPLC purification (Table E1). The RIS
conjugate was prepared similarly, by using commercial RIS 9 to afford RIS conjugate 10
(Scheme 6.2.2).

6.2.2 Using Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) to Prepare β-
Alanine-FAM-BP, Intermediate A
CuAAC is a 1,3-cycloaddition between an azide and an alkyne to access 1,2,3-
triazoles. This reaction has been applied successfully to BP esters,
16,17
but applications to
bisphosphonic acids have been limited
18
due to its strong ability to chelate the copper
catalyst.
19,20
Chelation of copper by BP impedes detection of the N-BP conjugate starting
material and triazole product by MS, LC-MS and MS, even after HPLC purification. To
facilitate detection, the amount of copper was limited to 0.2 stoichiometric equivalents
and water-soluble ligand THPTA was added to chelate copper during CuAAC and
prevent undesired copper-catalyzed side reactions. Addition of EDTA or Chelex for
several hours enables detection of N-BP derivatives by MS, after removal of sodium from
sodium ascorbate by either LC-MS or HPLC.
Scheme 6.2.2 Access to Intermediate A (23)

O
Cl
OH
N 3
Cl
O
N 3 NaN 3 in H 2 O/AcOH
30 ˚C, 5 h
diluted NaOH
5-12 min
5 6 7
+
NaOH, RT, 12h
MES buffer (pH = 6.5)
O O HO
COOH
O
O
1. HOBt, EDC
DMF
N
O
O
CuSO 4 (20 mol%),
THPTA (1 eq)
sodium ascorbate
3:1 H 2 O:DMSO
+
H
N OH
O
O
O
N
H
H
N
Mtt
2.
17
N
O
H
N O
O
HN O
O HO O
COOH
18 19
N
O
H
N O
O
HN
Mtt
1. 1%TFA in DCM
2.
N
OH P O
OH
P
OH
O
OH
9
HO
N
+
OH P O
OH
P
OH
O
OH
OH
N 3
10
HO
20
21
N
O
H
N O
O
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
N
O
H2N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
50:50
TFA:H 2 O
22
23

130
130

Before performing the click reaction, linker 17 and intermediate 21 needed to be
prepared. Linker 17 was proposed from original literature conditions, beginning with 3-
butynol (11).
21
Tosylation of 3-butynol proceeded quantitatively. However, alkylation by
diamine and subsequent purification by vacuum distillation according to literature
procedure proved to be challenging and ineffective. An alternate route was proposed for
the linker based on literature search, modifying the order of monoprotection by Mtt.
21

Nine equivalents of commercially available ethylene diamine (13) were used with one
equivalent of Mtt to afford a monoprotected diamine 16, purified by column
chromatography, and verified by
1
H NMR (Figure E20). ESI-MS analysis was
unsuccessful based on the stability of the Mtt carbocation (m/z (+) 257). This
monoprotected diamine was then used to alkylate tosylate 12 using two separate
conditions, one at RT and one at 50 ˚C in CH
2
Cl
2
. Reaction mixtures were purified by
column chromatography (Figure E21) and
1
H NMR confirmed formation and purity of
linker 17 (Figure E22).
The CuAAC product 22 was prepared using intermediates 10 and 21 (Scheme
6.2.2). Briefly, 21 was synthetized from commercially available Boc-β-alanine, activating
the carboxylic acid group with HOBt and EDC as catalyst with the secondary amine on
17. After column chromatography purification, 19 was selectively deprotected using 1%
TFA. The free amine was then conjugated with commercial FAM-NHS ester 20 in
aqueous DMF at pH 8 to give 21. The click reaction between 10 and 21 was performed
by first dissolving 21 in DMSO, followed by addition of 10 in water. The vessel was
capped and purged with N
2(g)
. A premixed solution of THPTA and copper was added,
followed by addition sodium ascorbate to initiate the reaction. The reaction was allowed

131
131
to stir at rt for several hours and quenched with the addition of Chelex prepped in the
sodium form. Purification using semipreparative C
18
HPLC gave pure 22 by MS.
Compound 22 was then partially deprotected using 50:50 TFA:H
2
O for three
hours. Stronger conditions were applied using 1 mL of 95:2.5:2.5 TFA:H
2
O:
triisopropylsilane (TIS), for two hours and 22 was deprotected quantitatively. The
mixture was then purified on a semipreparative C
18

column by HPLC to give desired
Intermediate A, 23.
Scheme 6.2.3 Conjugation reaction to prepare final compound 27

O
N N
N N
N
OCH
3
NO
2
HN O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
O
O
24
N
O
H
2
N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
23
+
1. TSTU, TEA, DMF
2. 23, H
2
O (pH 7.7)
Na
2
CO
3
25
1. 50:50
TFA:H
2
O
2. BHQ-NHS (26)
H
2
O (pH 8.0),
Na
2
CO
3
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
N
O
NH
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
27
OH
O
N N
N N
N
OCH
3
NO
2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
OH
+
O
N N
N N
N
OCH
3
NO
2
O
N
O
O
26

132
132
Commercially ordered peptide 24 was prepared with a Boc group at the N-
terminus. The Boc group was deprotected quantitatively using 50:50 TFA:H
2
O. The
reaction was washed with several portions of methanol and water. Then the deprotected
peptide was dissolved in aqueous DMF and BHQ-1 (26) was added and the pH was
adjusted to 8.3. The reaction was allowed to stir at rt overnight and monitored by TLC.
Compound 25 was purified by HPLC and characterized by MS (Figure E35-37).
Subsequent activation was carried out using TSTU and the intermediate was monitored
by MS. The solvents were removed and then Intermediate A 23 was added (first
dissolved in water, followed by pH adjustment with sodium carbonate to 8 to dissolve the
compound). The reaction was allowed to stir for an hour. MALDI preparation of reaction
mixture using 2′,4′,6′-Trihydroxyacetophenone monohydrate in negative mode revealed
final compound 27 (Figure E38). Preparation of final compound 27, though in small
quantities, demonstrates a proof-of-principle that these FRET-quenched N-BP based
peptide probes can be prepared to study the onset of ONJ.

6.2.3 Retrosynthetic Analysis and Other Attempted, Unsuccessful Routes
The rationale for our proposed method has some notable advantages (Figure
6.2.1). For one, the synthesis can be applied to several diverse bisphosphonates to readily
prepare N-BP conjugates. Secondly, the N-BP conjugates with an azide tag have already
been prepared in our lab and preliminary studies show that the compound readily
undergoes click reactions. Lastly, it is important to note that the azide in our new
strategy is primary; by strategically modifying the sterics of this azide, we hypothesize
that the reactivity during the final click reaction will show a lowered reaction time and a

133
133
higher yield. Additional considerations include stitching the fluorescent and quencher
elements together within the same probe. It is critical to find a linker that is flexible
enough to accommodate these bulky modalities. Our proposed linker 17 has literature
precedent for the reaction of the alkyne with the protected ethylenediamine, and has
shown to react selectively with the carboxylic acid group of a peptide.
21,22
Lastly, the
final reaction between the two intermediates is expected to go smoothly in the click
reaction. Sterically bulky groups have been historically shown to react in polymers,
bioconjugates and in several other reactions under CuAAC conditions.

Figure 6.2.1 Previous strategies employed to access FRET-quenched probes using a commercially
available Boc-protected peptide, linker 17, and N-BP conjugates 8 or 10
A) Initial strategy attempted first by Boc-deprotection of peptide, amide condensation with NHS-
commercial quencher, QC-1, with unsuccessful amide condensation with HBTU and 17; B) Second
attempted synthesis, beginning with amide condensation with HBTU and 17, Mtt-deprotection and amide
condensation with FAM-NHS, but failed CuAAC reaction; C) Third strategy employed HBTU-catalyzed
amide condensation with 17, CuAAC, Mtt-deprotection, but failed FAM-NHS amide condensation; D)
HBTU-catalyzed amide condensation with 17, Mtt-deprotection, FAM-NHS amide condensation, Boc-
deprotection, quencher-NHS amide condensation, and potentially successful CuAAC. Green circles imply a
successful reaction, red means failed reactions; and dashed lines mean undetermined or uncertain.

Figure 6.2.1 shows the previously attempted strategies to access the final
FRET-quenched probes. All four attempts utilize the same conjugation methods but are
HN CHC
H
O
H
N CHC
CH 2
O
N
NH
N
C
O
H
N CHC
H
O
H
N CHC
H
O
N
C
O
H
N CHC
CH 2
O
CH 2
C
NH 2
O
H
N CHC
H
N
O
Q
O
N
N
N
HO OH
O
OH
OH
O
P
P
N
+
O
HO
O
COOH
NH O
HO
Q = IRDye QC-1
1-2
3
4-5
6
A
HN CHC
H
O
H
N CHC
CH 2
O
N
NH
N
C
O
H
N CHC
H
O
H
N CHC
H
O
N
C
O
H
N CHC
CH 2
O
CH 2
C
NH 2
O
H
N CHC
H
N
O
Q
O
N
N
N
HO OH
O
OH
OH
O
P
P
N
+
O
HO
O
COOH
NH O
HO
Q = IRDye QC-1
2-3
1
4
5-6
B
5-6
1
2
3
4
HN CHC
H
O
H
N CHC
CH 2
O
N
NH
N
C
O
H
N CHC
H
O
H
N CHC
H
O
N
C
O
H
N CHC
CH 2
O
CH 2
C
NH 2
O
H
N CHC
H
N
O
Q
O
N
N
N
N
+
O
HO
O
COOH
NH O
HO
Q = BHQ-1
HO
HO
O
OH
HO
O P
P
OH
C
4-5
1
2
3
6
HN CHC
H
O
H
N CHC
CH 2
O
N
NH
N
C
O
H
N CHC
H
O
H
N CHC
H
O
N
C
O
H
N CHC
CH 2
O
CH 2
C
NH 2
O
H
N CHC
H
N
O
Q
O
N
N
N
N
+
O
HO
O
COOH
NH O
HO
Q = BHQ-1
HO
HO
O
OH
HO
O P
P
OH
D

134
134
performed in different permutations. In Figure 6.2.1A, the strategy was first a Boc-
deprotection of peptide, followed by an amide condensation with activated NHS-
commercial quencher, QC-1. Both reactions proceeded quantitatively, giving an overall
yield of 87% after C
18
HPLC purification and determination by UV. Although promising,
amide condensation with 17 was unsuccessful after several attempts with HBTU. Figure
6.2.1B shows the subsequent strategy that begins the synthesis with HBTU-catalyzed
amide condensation with linker 17 and Boc-protected peptide. Then, selective Mtt-
deprotection using 1% TFA in dichloromethane (DCM), without deprotecting the Boc-
group on the peptide. The free amine on the linker was then conjugated with
commercially available FAM-NHS and monitored by MS, and characterized by LC-MS
and HPLC. Following HPLC purification, the intermediate was subjected to a CuAAC
reaction in aqueous DMSO. After stirring overnight and treatment with EDTA, there was
no product or intermediate starting material detected, except for N-BP conjugate by MS
or LC-MS.
To verify that CuAAC reactions are successful, an alternative strategy was
attempted (Figure 6.2.1C). In this synthesis, the linker was attached to Boc-protected
peptide through HBTU-catalyzed amide condensation. A CuAAC reaction was
performed with THPTA and N-BP conjugate, and was monitored by LC-MS. After the
product was purified, the Mtt-group was selectively deprotected and an amide
condensation reaction was attempted with FAM-NHS ester and failed. The final synthetic
route that was attempted is shown in Figure 6.2.1.D. Here, we proceeded with an amide
condensation reaction catalyzed by HBTU to attach the linker to the peptide. The Mtt-
group was then selectively deprotected, and the free amine was condensed with FAM-

135
135
NHS. The intermediate was then stirred with 50:50 TFA:H
2
O to remove the Boc-group.
The peptide free amine was coupled with quencher, BHQ-1-NHS to form an amide. This
intermediate was then subjected to a CuAAC reaction, and was detected as a minor peak
by MALDI. However, after HPLC purification, final product could not be unequivocally
characterized. Based on these failed attempts, we altered our strategy to extend the linker
and protect the peptide from undesired side reactions with the imidazole in histidine.

6.3 Conclusion
We have demonstrated that this class of compounds, with a multitude of diverse
functionalities, including chelating bisphosphonates, two light-sensitive dyes and an 8-
mer peptide with unprotected histidine, can be prepared using simple conjugation
techniques. Once fully developed and characterized, these probes will provide a novel
molecular reagent to elucidate, in more detail, the pathological mechanisms of N-BP-
associated ONJ. The effect of N-BPs in patients who are now prescribed, or soon to be
prescribed, with other anti-resorptive agents still presents a significant challenge in
prevention and treatment of ONJ. The new probes will be critical molecular tools for
future investigations addressing the role of legacy N-BP.

6.4 Experimental
6.4.1 Materials and Methods
Tetraisopropyl methylenebisphosphonate (1, TiPBP) was received as a gift from
Rhodia. Compounds 3-4, 6-8, 10, 12, 16-17, 19, 21-25, and 27 were synthesized as
described below. 4-(Chloromethyl)pyridine- HCl (2), epichlorohydrin (5), 3-butynol (11),

136
136
ethylene diamine (13), Boc-β-alanine (18), were purchased from Sigma-Aldrich.
Risedronate (9) was a gift. 5(6)-Carboxyfluorescein (20) was purchased from Life
Technologies. Peptide 24 with Boc-protected amine was custom ordered from CHI
Scientific. BHQ-1 quencher (26) was purchased on LGC Biosearch Technologies. All
other reagents were purchased from commercial sources and used as obtained, unless
specified otherwise.
1
H,
19
F, and
31
P NMR spectra were obtained on Varian 400-MR,
VNMRS-500 and VNMRS-600 3-Channel NMR spectrometers. Multiplicities are quoted
as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd),
doublet of doublet of doublets (ddd), doublet of triplets (dt) or broad signal (br). All
chemical shifts are given on the δ –scale in parts per million ((ppm) relative to internal
CD
3
OD (δ 3.34,
1
H NMR), CDCl
3
(δ 7.26,
1
H NMR), D
2
O (δ 4.79,
1
H NMR),

or external
85% H
3
PO
4
(δ 0.00,
31
P NMR) and C
6
F
6
(δ -164.9,
19
F NMR).
31
P

NMR spectra were
proton-decoupled, and
1
H,
19
F, and
31
P coupling constants (J values) are given in Hz. The
concentration of the NMR samples was in the range of 2-5 mg/mL. Preparative HPLC
was performed using a Phenomenex C
18
or a SAX-resin column equipped with a
Shimadzu SPD-10A UV detector (0.5 mm path length) with detection at wavelength at
260 nm. Mass spectrometry (MS) was performed on a Finnigan LCQ Deca XP Max mass
spectrometer equipped with an ESI source in the negative or positive ion mode.

6.4.2 Synthesis of TiPBP-para-dRIS 3

iPr-O
P
O
O-iPr
P
O-iPr
O
O-iPr
N
Cl
NaH, DMF/THF
N
O-iPr P O
O-iPr
P
O-iPr
O
O-iPr
1 3

137
137
3 was prepared according to literature procedure from tetraisopropyl
methylenebisphosphonate 1 and 4-(chloromethyl)pyridine-HCl (2).
23
Mixture was
purified using column chromatography using dichloromethane and acetone (conditions
on Figure E1).
1
H NMR (500 MHz, CDCl
3
, Figure E2) δ 8.50 (t, J = 5.1 Hz, 2H), 7.34 –
7.18 (m, 2H), 4.76 (p, J = 6.7 Hz, 4H), 3.20 (ddd, J = 22.4, 14.2, 6.3 Hz, 2H), 2.51 (td, J
= 24.0, 6.1 Hz, 1H), 2.10 – 1.77 (m, 4H), 1.29 (dd, J = 20.3, 6.3 Hz, 26H).
31
P NMR (202
MHz, CDCl
3
, Figure E3) δ 20.20.

6.4.3 Synthesis of para-dRIS 4

The tetraisopropyl esters of the methylene bisphosphonate 3 were hydrolyzed
with concentrated BTMS under MW conditions. The solvent was then evaporated under
vacuum, and then 10 mL of methanol was added to the flask and allowed to stir for 20
minutes. The solvents were removed under vacuum and treated with several portions of
methanol and water and rotavapped, then used in the subsequent reaction without further
purification.
1
H and
31
P NMR revealed pure product, and was further corroborated by
MS-ESI. After characterizing the compound, 4 was dissolved in 2.5 mL MES buffer (pH
6.0).
1
H NMR (500 MHz, D
2
O, Figure E4) δ 8.53 (dt, J = 5.7, 1.0 Hz, 2H), 7.96 – 7.84
(m, 2H), 3.45 – 3.27 (m, 2H), 2.78 – 2.58 (m, 1H).
31
P NMR (202 MHz, D
2
O, Figure E5)
δ 19.01. Predictive iMass was performed in both positive and negative mode for
compound 4 (Figure E6). ESI-MS (Figure E7): MS (m/z): calcd for C
7
H
11
NO
6
P
2
+
: 268.0,
N
OH P O
OH
P
OH
O
OH
N
O-iPr P O
O-iPr
P
O-iPr
O
O-iPr
2. MeOH
1. BTMS, 80 ˚C
Dioxane, MW, 20 min
3 4

138
138
found: 268.3 [M − H]
+
. ESI-MS (Figure E8): MS (m/z): calcd for C
7
H
10
NO
6
P
2
-
: 266.0,
found: 266.1 [M − H]
−
.

6.4.4 Synthesis of Glycidyl Azide 7

Glycidyl azide linker 7 was prepared according to literature procedure.
15
The
epichlorohydrin 5 (5 mmol) was dissolved in an aqueous solution of sodium azide (26.0
mmol in 8.0 mL), 4.6 mL acetic acid was then added and the solution was stirred for 5 h
at 30 ˚C. The solution was extracted with diethyl ether (3 x 8 mL). The combined organic
phase was washed five times with 10 mL portions of sodium phosphate buffer (50 mM,
pH 6.5). The organic phase was dried with sodium sulfate. However, it is important to
note that not all the diethyl ether was removed from on a rotary evaporator due to the low
boiling point of the product. After removal of most of the solvent, 623 mg of 1-azido-3-
chloro-2-propanol 6 was obtained.
Glycidyl azide 7 was prepared as an aqueous solution from 1-azido-3-chloro-2-
propanol 6. A 210 mg portion of 6 was added with 0.2 mL water and stirred, 1 M NaOH
was then slowly added until the solution pH was stabilized at 12.5 for 5 min, 1 M HCl
was then added to adjust the pH to 7.0, more water was added to make the volume 3.2 ml
containing 0.5 M of glycidyl azide 7.

O
Cl
OH
N
3
Cl
O
N
3 NaN
3
in H
2
O/AcOH
30 ˚C, 5 h
diluted NaOH
5-12 min
5 6 7

139
139
6.4.5 Synthesis of para-dRIS-linker 8

Para-dRIS 4 (0.15 mmol) in 0.5 mL MES buffer (100 mM, pH 6.0) was added
with 0.08 mL of 1M NaOH and 0.6 ml of glycidyl azide 7 (500 mM), sequentially.
Solution was kept at RT overnight and then subjected to SAX-column HPLC purification,
giving 53 mg para-dRIS-linker 8 as a film.
1
H NMR (500 MHz, D
2
O, Figure E9) δ 8.46
(d, J = 6.5 Hz, 2H), 7.90 (d, J = 6.4 Hz, 2H), 3.62 (t, J = 4.8 Hz, 1H), 3.46 (dd, J = 13.2,
4.0 Hz, 1H), 3.43 – 3.33 (m, 1H), 3.29 – 3.16 (m, 2H), 2.74 – 2.63 (m, 1H), 2.46 (d, J =
4.9 Hz, 1H), 2.11 (tt, J = 20.7, 6.9 Hz, 1H).
31
P NMR (202 MHz, D
2
O, Figure E10) δ
17.70. Predictive iMass was performed in both positive and negative mode for compound
8 (Figure E11). ESI-MS (Figure E12): MS (m/z): calcd for C
10
H
17
N
4
O
7
P
2
+
: 367.1, found:
367.3 [M − H]
+
. ESI-MS (Figure E13): MS (m/z): calcd for C
10
H
15
N
4
O
7
P
2
-
: 365.0, found:
365.2 [M − H]
−
.

N
OH P O
OH
P
OH
O
OH
O
N
3
4
7
+
NaOH, RT, 12h
MES buffer (pH = 6.5)
N
OH P O
OH
P
OH
O
OH
OH
N
3
8

140
140
6.4.6 Synthesis of RIS-linker 10

Commercial RIS 9 (0.15 mmol) in 0.5 mL MES buffer (100 mM, pH 6.0) was
added with 0.08 mL of 1M NaOH and 0.6 ml of glycidyl azide 7 (500 mM), sequentially.
Solution was stirred at RT overnight and then subjected to SAX-column HPLC
purification 10 as a film.
1
H NMR (400 MHz, D
2
O, Figure E14) δ 8.69 (s, 1H), 8.44 (dd,
J = 11.3, 7.3 Hz, 2H), 7.79 (t, J = 7.1 Hz, 1H), 4.37 (dd, J = 13.5, 9.4 Hz, 1H), 4.20 (s,
1H), 3.48 (dd, J = 13.2, 4.0 Hz, 1H), 3.39 (dd, J = 13.2, 6.1 Hz, 1H), 3.34 – 3.18 (m, 6H),
3.02 (qd, J = 7.3, 1.2 Hz, 8H), 1.17 – 1.08 (m, 12H).
31
P NMR (243 MHz, D
2
O) Figure
E15) δ 16.74 – 16.35 (m). Predictive iMass was performed in both positive and negative
mode for compound 10 (Figure E16). ESI-MS (Figure E17): MS (m/z): calcd for
C
10
H
17
N
4
O
8
P
2
+
: 383.1, found: 383.4 [M − H]
+
. ESI-MS (Figure E18): MS (m/z): calcd for
C
10
H
15
N
4
O
8
P
2
-
: 381.0, found: 381.1 [M − H]
−
.

6.4.7 Synthesis of Linker 17

N
OH P O
OH
P
OH
O
OH
O
N
3
9
7
+
NaOH, RT, 12h
MES buffer (pH = 6.5)
N
+
OH P O
OH
P
OH
O
OH
OH
N
3
10
HO
HO
OTs OH
DMAP, TEA, TsCl
DCM
12 11

141
141
Tosylated alcohol was prepared according to literature procedure.
24
To a solution
of 3-butynol (11, 2.35 mL, 31 mmol, 1.0 equiv), DMAP (0.01 equiv, 36.9 mg, 0.3 mmol)
and triethylamine (1.3 equiv, 5.5 mL, 40 mmol) in CH
2
Cl
2
(15 ml) at 0 °C was added a
solution of p-toluenesulfonyl chloride (TsCl, 1.1 equiv, 6.46 g, 34 mmol) in CH
2
Cl
2
(15
mL) and the reaction mixture was stirred for 4 hours at rt. The reaction was quenched
with water (30 mL), stirred for 20 min, and extracted with CH
2
Cl
2
(3 x 40 mL), the
combined organic layers were washed with brine (60 mL), dried over Na
2
SO
4
, and
solvents were evaporated to afford product 12 as a brown oil in 97% yield (6.69 g).
1
H
NMR (500 MHz, CDCl
3
, Figure E19) δ 7.67 (t, J = 4.2 Hz, 2H), 7.22 (t, J = 4.5 Hz, 2H),
3.97 (dd, J = 7.0, 2.5 Hz, 2H), 2.42 (dt, J = 7.2, 2.7 Hz, 2H), 2.37-2.21 (m, 3H), 1.93-1.75
(m, 1H).

Monoprotected diamine was prepared according to literature procedure.
21
To a
solution of tosylated alcohol (12, 1.0 equiv, 3.63 g, 16.2 mmol) and DBU (1.0 equiv, 2.45
mL, mmol) in toluene (5 mL) was added a solution of 13 (10 equiv, 10.8 mL, 162 mmol)
in toluene (5 mL) at rt and stirred for 4 hours. The reaction mixture was then
concentrated in vaccuo and unsuccessfully purified by distillation (between 70 °C and
120 °C at 2 mbar). Reaction and subsequent distillation were attempted twice and failed.
Product polymerized during distillation.
OTs
H
2
N
NH
2
DBU, toluene
H
N
NH
2
12 14
13

142
142

An alternative route was devised based on literature procedures.
22
To a solution of
13 (9.0 equiv, 9.25 g, 153.9 mmol) was added pyridine (25 mL) and CH
2
Cl
2
(35 mL) and
stirred at 0 ˚C. Mtt (15, 1.0 equiv, 5 g, 17.2 mmol) was added in four portions. The
reaction was allowed to stir at rt and quenched with 5 mL MeOH. The solvent was
evaporated and the crude was redissolved in 1:1 (CH
2
Cl
2
:H
2
O with 1% TEA, 25 mL)
solution. The product was extracted with CH
2
Cl
2
(3 x 10 mL) and purified by column
chromatography using hexane and EtOAc to yield 4.25 g of 16 (78% yield). A
1
H NMR
indicates clean product.
1
H NMR (400 MHz, CDCl
3
, Figure E20) δ 7.56 – 7.44 (m, 4H),
7.35 (d, J = 8.3 Hz, 2H), 7.33 – 7.22 (m, 4H), 7.23 – 7.14 (m, 2H), 7.08 (dd, J = 8.0, 0.6
Hz, 2H), 2.80 (t, J = 6.0 Hz, 1H), 2.30 (s, 3H), 2.21 (t, J = 5.9 Hz, 2H).
Monoprotected diamine was then taken on to the next step for alkylation with
tosylated alcohol. To a solution of monoprotected Mtt-diamine (1.58 g, 5 mmol, 1 equiv)
was added TEA (5 mmol, 0.72 mL, 1 equiv), CH
2
Cl
2

(20 mL), followed by tosylated
alkyne (1.23 g, 5.5 mmol, 1,1 equiv) at 0 ˚C. The reaction was stirred at RT and allowed
to react for 48 h. TLC of 2:1 Et
2
O:EtOAc did not show change after 4 h. Then 15 mL of
10% NaHCO
3
was added and extracted with EtOAc (3 x 30 mL). Combined organic
layers were washed with 30 mL brine and the solvents were evaporated. The mixture was
purified using ISCO and 20-70% EtOAc in Hexanes with 1% TEA (Figure E21) to give
214 mg of product (12% yield). This reaction was repeated but modified with heating at
50 ˚C for four hours.
1
H NMR of the crude mixture indicated approximately 50% yield.
H
2
N
NH
2
H
2
N
H
N
Mtt-Cl (15), py
DCM
Mtt
OTs
TEA, DCM
N
H
H
N
Mtt
17 13 16
12

143
143
Reaction was purified using ISCO and 20-70% EtOAc in Hexanes and came out much
cleaner without use of TEA (Figure E21) and gave 291 mg of product (16% yield).
1
H
NMR (500 MHz, CDCl
3
, Figure E22) δ 7.55 (ddd, J = 8.2, 3.2, 1.7 Hz, 4H), 7.45 – 7.40
(m, 2H), 7.30 (t, J = 7.8 Hz, 4H), 7.23 – 7.16 (m, 2H), 7.12 (d, J = 8.0 Hz, 2H), 2.75 (dt,
J = 25.3, 6.0 Hz, 4H), 2.38 (tt, J = 6.6, 3.3 Hz, 2H), 2.34 (s, 3H).

6.4.8 Synthesis of Boc-β-alanine-ethylene Diamine Linker 19

Commercially available Boc-β-alanine 18 (13 mg, 71 µmol, 1.3 equiv) and
ethylene diamine linker 17 (20 mg, 54 µmol, 1 equiv) was dissolved in DMF and cooled
to 0 ˚C. 1-Hydroxybenzotriazole (HOBt, 4 mg, 27 µmol, 0.5 equiv) and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 14 mg, 71 µmol, 1.3 equiv) was
added while the reaction mixture was at 0 ˚C. After 10 minutes, the ice bath was removed
and the reaction was allowed to stir overnight. TLC indicated completion of the reaction.
The reaction mixture was then dissolved in EtOAc and washed with sat. NaHCO
3
(3 x 5
mL) and then with brine. The organic phase was dried over MgSO
4
and purified using
column chromatography with hexane and ethyl acetate to give 24 mg of purified 19 (81%
yield).
1
H NMR (400 MHz, CDCl
3
, Figure E23) δ 7.49 – 7.37 (m, 4H), 7.34 – 7.22 (m,
5H), 7.19 (t, J = 7.5 Hz, 2H), 7.13 – 7.03 (m, 2H), 3.43 (q, J = 6.7 Hz, 5H), 2.60 (dd, J =
8.5, 5.1 Hz, 2H), 2.43 – 2.35 (m, 2H), 2.31 (d, J = 3.8 Hz, 4H), 2.00 (d, J = 2.5 Hz, 1H),
1. HOBt, EDC
DMF
H
N OH
O
O
O
N
H
H
N
Mtt
2.
17
18
19
N
O
H
N O
O
HN
Mtt

144
144
1.91 (t, J = 2.6 Hz, 1H), 1.42 (d, J = 6.2 Hz, 8H). ESI-MS (Figure E24): MS (m/z): calcd
for C
34
H
42
N
3
O
3
+
: 540.3, found: 540.4 [M − H]
+
.

6.4.9 Synthesis of Boc-β-alanine-ethylene Diamine-FAM Linker 21

To 19 (24 mg, 44 µmol) was added 4 mL of DCM and 40 µL of TFA. The
reaction was stirred at rt and monitored by TLC (1:1 hexane:EtOAc). After one and a half
hours, the reaction was complete. Remaining TFA was removed and the reaction was
then used in the subsequent reaction. Product was dissolved in 1 mL of DMF and 0.5 mL
of water and pH adjusted with sat. Na
2
CO
3
to pH 7, followed by addition of 5(6)-FAM-
NHS (25 mg, 182 mg/mL in DMF, 1.2 equiv, 137 µL). The reaction was allowed to stir
overnight at rt. The reaction was monitored by TLC (100% EtOAc) and MS, and was
then diluted with 5 mL EtOAc and extracted with water. The product was extracted from
the organic phase using sat. NaHCO
3
(3 x 5 mL). Then the aqueous phase was acidified
with 10% citric acid to give a precipitate. The product was extracted with EtOAc (3 x 5
mL). The organic phase was dried with MgSO
4
and the solvents were evaporated. The
mixture was purified by column chromatography (Figure E25). Predictive iMass was
performed in both positive and negative mode for compound 21 (Figure E26). ESI-MS
O O HO
COOH
O
O
N
O
O
N
O
H
N O
O
HN O
O HO O
COOH
19
N
O
H
N O
O
HN
Mtt
1. 1%TFA in DCM
2.
20
21

145
145
(Figure E27): MS (m/z): calcd for C
35
H
36
N
3
O
9
+
: 642.2, found: 642.2 [M − H]
+
. ESI-MS
(Figure E28): MS (m/z): calcd for C
35
H
34
N
3
O
9
-
:

640.2, found: 640.2 [M − H]
−
.

6.4.10 Synthesis of Boc-Protected Intermediate 22 via CuAAC

Alkyne 21 (2 mg, 3 µmol, 1 equiv) was dissolved in 260 µL of DMSO and RIS
conjugate 10 (6 µmol, 60 µL of 100 mM solution, 2 equiv) was added mixture. The
solution was purged with N
2(g)
.

A premixed solution of CuSO
4
and Tris(3-
hydroxypropyltriazolylmethyl)amine (THPTA) in water (10 mM and 50 mM,
respectively) was added (0.6 µmol, 60 uL, 0.2 equiv Cu) to the reaction mixture.
Addition of freshly made sodium ascorbate (30 µmol, 250 mM, 10 equiv, 120 µL)
initiates the reaction. The solution was allowed to stir for several hours and monitored by
TLC (100 % MeOH or 100% EtOAc).
To selectively remove starting material and residual carboxyfluorescein, the
mixture was rotavapped then dissolved in water and acidified with acetic acid to pH 5.
Extraction with EtOAc removes the byproducts, which can be detected by TLC in EtOAc.
CuSO
4
(20 mol%),
THPTA (1 eq)
sodium ascorbate
3:1 H
2
O:DMSO
+
N
O
H
N O
O
HN O
O HO O
COOH
N
+
OH P O
OH
P
OH
O
OH
OH
N
3
10
HO
21
N
O
H
N O
O
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
22

146
146
Purification of the product is performed using C
18
semiprep HPLC using 0.1 M TEAB
(Table E1). UV analysis indicated 0.9 mg of purified product (1 µmol, 30% yield). The
reaction was repeated to obtain more material. Predictive iMass was performed in both
positive and negative mode for compound 22 (Figure E29). ESI-MS (Figure E30): MS
(m/z): calcd for C
45
H
52
N
7
O
17
P
2
+
: 1024.3, found: 1024.3 [M − H]
+
. ESI-MS (Figure E31):
MS (m/z): calcd for C
45
H
50
N
7
O
17
P
2
-
: 1022.3, found: 1022.3 [M − H]
−
.

6.4.11 Synthesis of Intermediate A 23

To 22 (1.9 mg, 2 µmol) was added 5 mL TFA and 5 mL water, and allowed to
react at rt for 4 hours. Reaction progress was monitored by MS-ESI. The reaction mixture
was then purified by HPLC using 0.1 M TEAB (pH 7.5) gradient from 0% to 50 %
CH
3
CN on a semiprep C
18
column. After purification, the UV measurement was acquired
to quantify the amount product. The analysis was carried out in 1x phosphate saline
buffer (PBS) buffer, pH 7.4 and it was determined that there was 1.5 mg of 23 (2 µmol).
Predictive iMass was performed in both positive and negative mode for compound 23
N
O
H
N O
O
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
N
O
H
2
N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
50:50
TFA:H
2
O
22
23

147
147
(Figure E32). ESI-MS (Figure E33): MS (m/z): calcd for C
40
H
44
N
7
O
15
P
2
+
: 924.2, found:
924.1 [M − H]
+
. ESI-MS (Figure E34): MS (m/z): calcd for C
40
H
42
N
7
O
15
P
2
-
: 922.2, found:
922.1 [M − H]
−
.

6.4.12 Synthesis of Penultimate Intermediate 25

Commercially designed peptide 24 (3 mg, 2 µmol, 1 equiv) was deprotected using
50% TFA in water for four hours. Afterwards, the reaction was worked up by evaporating
the solvents, followed by washes with methanol and water. The mixture was then tried
and was dissolved in aqueous DMF. Quencher 26 was added to the mixture and dissolved.
The pH was adjusted to 8.3 using a solution of saturated sodium carbonate and the
reaction was allowed to stir at rt overnight. TLC (1:1 DCM:EtOAc) indicated completion
of the reaction and the reaction was purified by HPLC. Predictive iMass was performed
in both positive and negative mode for compound 25 (Figure E35). ESI-MS (Figure E36):
HN O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
O
O
24
+
25
1. 50:50
TFA:H
2
O
2. BHQ-NHS (26)
H
2
O (pH 8.0),
Na
2
CO
3
OH
O
N N
N N
N
OCH
3
NO
2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
OH
O
N N
N N
N
OCH
3
NO
2
O
N
O
O
26

148
148
MS (m/z): calcd for C
55
H
70
N
17
O
14
+
: 1192.5, found: 1192.5 [M − H]
+
. ESI-MS (Figure
E37): MS (m/z): calcd for C
55
H
68
N
17
O
14
-
: 1190.5, found: 1190.7 [M − H]
−
.

6.4.13 Synthesis of final compound 27

DMF and TEA (7 µmol, 0.25 M in DMF, 29 µL, 3 equiv) was added to the vial to
completely dissolve the peptide. N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium
tetrafluoroborate (TSTU, 3 µmol, 0.25 M in DMF, 10 µL, 1.1 equiv) was then added to
start the reaction and allowed to react at rt for 30 minutes. The reaction mixture was
monitored using MS and revealed that all peptide had completely reacted. The solvent
was then removed and a mixture of 23 in water at pH 8.3 was added to the vial. A
MALDI sample, run in negative mode, was prepared using 2′,4′,6′-
Trihydroxyacetophenone monohydrate (THAP) and revealed presence of final compound
27 (Figure E38).

O
N N
N N
N
OCH
3
NO
2
N
O
H
2
N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
23
1. TSTU, TEA, DMF
2. 23, H
2
O (pH 7.7)
Na
2
CO
3
25
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
N
O
NH
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
27
O
N N
N N
N
OCH
3
NO
2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
OH
+

149
149
6.5 Chapter References

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M. D.; Mohla, S.; Pendrys, D. G.; Raisz, L. G.; Ruggiero, S. L.; Shafer, D. M.; Shum, L.;
Silverman, S. L.; Van Poznak, C. H.; Watts, N.; Woo, S. B.; Shane, E.; American Society
for, B.; Mineral, R. J. Bone Miner. Res. 2007, 22, 1479.
(2) Reid, I. R.; Cornish, J. Nat Rev Rheumatol 2012, 8, 90.
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Scagliotti, G. V.; Sleeboom, H.; Spencer, A.; Vadhan-Raj, S.; von Moos, R.;
Willenbacher, W.; Woll, P. J.; Wang, J.; Jiang, Q.; Jun, S.; Dansey, R.; Yeh, H. J. Clin.
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Troeltzsch, M. J. Can. Dent. Assoc. 2012, 78, 1.
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A.; Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. G. Bone 2011, 49, 20.
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B.; McKenna, C. E.; Rogers, M. J. J. Bone Miner. Res. 2010, 25, 606.
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McKenna, C. E.; Russell, R. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon, F.
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APPENDIX

168
168
Appendix A. Chapter 2 Supporting Data
5

Figure A1. Chromatogram trace for SAX HPLC purification of β,γ-CHF-UTP (1)
Purification of 1 by preparative HPLC was performed in two stages. The first stage used
on a strong anion exchange (SAX) column (1000-10 25 mm × 15 cm) using a gradient (0-
10 min, 55%; 10-16 min, 55%; 16-25 min, 100%) of 0.5 M triethylammonium
bicarbonate (TEAB) buffer (pH 7.0) at a flow rate of 8 mL/min. The elution trace (UV
detection at 259 nm) is shown below. Suggested assignments are denoted with red
arrows; confirmed assignment is marked with a green arrow; the desired product is
indicated by a blue arrow.

5
Reproduced with permission from Hwang, C. S.; Kashemirov, B. A.; McKenna, C. E. J.
Org. Chem. 2014, 79, 5315. Copyright 2016 American Chemical Society.

169
169

Figure A2. Chromatogram trace for RP-C
18
HPLC purification of β,γ-CHF-UTP (1)
A second purification pass was performed on sample 1 obtained from the first HPLC
stage using a reverse phase-C
18
(RP-C
18
) column (5 mm, 250 mm x 21 mm) by isocratic
elution with 3.75% CH
3
CN in 0.1 M TEAB pH 7.0 at a flow rate of 8.0 mL/min. The
eluted 1 was detected by UV (0.5 mm path length) at 259 nm (18.7 min).

!
Chrom. 1 0.0 mins. 30.2 mins.
1
# Filename Chan Type Created Run Duration
1 SHIII-55 C18 250ul-008 A C Wed, Oct 16, 2013 4:34 PM 30.2
! Peak No.!! Time (min)!! Area
! 1!! ! ! 13.0!! ! ! 98461
2!! ! ! 18.7!! ! ! 9296809

170
170

Figure A3.
19
F NMR (470 MHz, D
2
O, pH 10.4) of β,γ-CHF-UTP 1 (~1:1 diastereomers),
Na
+
counterion
Assigned J values were corroborated by J values obtained from the
31
P NMR spectrum.
The diastereomers’ Δδ was verified by obtaining the spectrum at two different
spectrometer frequencies (470 and 564 MHz) and by simulation. The J values are very
consistent with those previously determined for the β,γ-CHF-dGTP diastereomers (J =
44.7, 55.9, and 66.9 Hz).
1
The small impurity near δ -217.3 can be removed by the
modified stage 1 preparative HPLC method (see Experimental).

ppm
JF,Pβ
JF,Pɣ
JF,H
Δδ

171
171

Figure A4. Effect of pH and counterion on δ
F
of ~1:1 β,γ-CHF-UTP diastereomers 1 in
D
2
O
A) Correlation between pH and δ
F
below pH 10; B) Correlation between pH and δ
F
above
pH 10

y = 0.0779x - 217.58!
R² = 0.44211!
-220!
-219!
-218!
-217!
-216!
9.00! 10.00! 11.00! 12.00! 13.00!
Chemical Shift (ppm)!
pH!
y = 1.4193x - 229.09!
R² = 0.99838!
-220!
-219!
-218!
-217!
-216!
-215!
6.00! 7.00! 8.00! 9.00! 10.00!
Chemical Shift (ppm)!
pH!
Na
+
K
+
TEAH
+ NH4
+
Na
+
K
+
TEAH
+
NH4
+
A
B

172
172

Figure A5. Effect of counterion ionic radius on Δδ
F
of β,γ-CHF-UTP in D
2
O
The distance between the two phosphate oxygens (5.5 Å)
2,3
readily accommodates a
Mg
2+
with ionic radii of 0.65 Å.
4
Interestingly, the magnitude of Δδ is slightly perturbed
among the ions and may be a result of the cation complexing with the triphosphate
moiety. To observe a possible effect, the Δδ for each counterion was plotted against its
ionic radius, producing a linear correlation (Figure S6), suggesting that these subtle
differences may arise from counterion complexation by the phosphates. However, this
may be misleading, as there is some variation in colinearity with respect to the SF, which
should not influence the relationship. In this connection it is notable that among the
counterions examined (NH
4
+
, Na
+
or K
+
), only TEAH
+
, which has a substantially larger
ionic radius, was alone in producing a wide line width at pH 10 resulting in unresolvable
diastereomer spectra of 1 (Figure S6). Since the pK
a
of TEAH
+
is 10.8, rapid proton
exchange contributes to this undesirable line broadening.

173
173

Figure A6. UV Analysis of β,γ-CHF-UTP 1 (~1:1 diastereomers)
Compound 1 was detected at the λ
max
= 262 nm based on the UV spectrum of UTP (ε =
10.0 x 10
3
M
-1
cm
-1
, pH 7.0) with a calculated concentration of 3.07 x 10
-5
M (± 0.04 x
10
-5
M, SE, n = 3).

Figure A7.
1
H NMR (500 MHz, D
2
O, pH 7.6) of 1 (~1:1 diastereomers)
X = unidentified impurities; Y = triethylamine (TEA); HDO = δ 4.8
0!
0.05!
0.1!
0.15!
0.2!
0.25!
0.3!
0.35!
210! 230! 250! 270! 290! 310! 330! 350!
Absorbance!
Wavelength (nm)!
Replicate 1!
Replicate 2!
Replicate 3!

174
174

Figure A8.
19
F NMR (470 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers)
X = impurity peak, can be removed by modified stage 1 HPLC (see p. S4).

Figure A9.
31
P NMR (202 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers)
X = impurity, can be removed by modified stage 1 HPLC (see Experimental)

175
175

Figure A10. Representative
31
P NMR spectrum (202 MHz, D
2
O, pH 7.4) of 1 (~1:1
diastereomers) after exchange on Dowex 50WX8 (200-400 mesh, prepped NH
4
+
form)
resin

Figure A11.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.1) of 1 (~1:1 diastereomers)
after exchange with NH
4
+
(Figure A10), followed by titration with NH
4
OH
X = impurity, can be removed by modified stage 1 HPLC (see Experimental)
α
β
ɣ
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
OH
NH
N
O
O
F
α β γ
ppm
α
β ɣ
X
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
OH
NH
N
O
O
F
α β γ
ppm
X

176
176

Figure A12.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.0) of 1 (~1:1 diastereomers)
after exchange with TEAH
+
, followed by titration with TEA

177
177

Figure A13.
31
P NMR spectrum (202 MHz, D
2
O, pH 10.4) of 1 (~1:1 diastereomers)
after exchange with Na
+
, followed by titration with NaOH
X = impurity, can be removed by modified stage 1 HPLC (see Experimental).
The
31
P NMR spectra of 1 were also re-examined in this study. Although the individual
diastereomers of β,γ-CHF-dGTP can be distinguished by their Pα and Pβ signals,
5
the limit
of the
31
P spectral line width (0.5 Hz) obtained at pH 10 in D
2
O, a Δδ value was difficult
to assign for 1. This was attributed to the relatively small S/N for the sample, which was
limited by a low concentration (16,000 transients, overnight NMR acquisition).

178
178

Figure A14.
31
P NMR spectrum (202 MHz, D
2
O, pH 12.7) of 1 (~1:1 diastereomers)
after exchange with K
+
, followed by titration with KOH

Figure A15. Simulation of spectrum for 1 (~1:1 diastereomers,
19
F NMR) at a
spectrometer frequency of 235 MHz
ppm

179
179
Chapter 2 Supporting Data References

(1) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.;
Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617.
(2) Taylor, J. S.; Deutsch, C. Biophys. J. 1983, 43, 261.
(3) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin Trans. 1 1984,
1119.
(4) Maguire, M. E.; Cowan, J. A. BioMetals 2002, 15, 203.
(5) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.;
Wilson, S. H.; McKenna, C. E. J. Am. Chem. Soc. 2012, 134, 8734.

180
180
Appendix B. Chapter 3 Supporting Data
6

Figure B1.
1
H NMR (500 MHz, CDCl
3
) of 5
X = minor impurities; Y = residual solvent

6
Reproduced with permission from Hwang, C. S.; Kung, A.; Kashemirov, B. A.; Zhang,
C.; McKenna, C. E. Org. Lett. 2015, 17, 1624. Copyright 2016 American Chemical
Society.
Y
Y
CDCl3
MeOH
P P
OH
OMe
O
MeO
MeO
O
F
5
X
X

181
181

Figure B2.
19
F NMR (470 MHz, CDCl
3
) of 5

Figure B3.
31
P NMR (202 MHz, CDCl
3
) of 5
P P
OH
OMe
O
MeO
MeO
O
F
5
P P
O
-
OMe
O
MeO
-
O
O
F
β
α
P P
OH
OMe
O
MeO
MeO
O
F
α β
5

182
182

Figure B4. MS of 5

!
P P
O
-
OMe
O
MeO
MeO
O
F
Chemical Formula: C
4
H
10
FO
6
P
2
-
Exact Mass: 235.0
5

183
183

Figure B5.
31
P NMR (202 MHz, CD
3
OD) of 6a/6b

P P
O
OMe
O
MeO
MeO
O
F
(S) (S) OMe
O
6a/b
PPh3O

184
184

Figure B6. MS of 9a/9b

P P
O
OH
O
-
O
N
O
F
(S) (S) N
O
O
O
Chemical Formula: C
17
H
24
FN
2
O
8
P
2
-
Exact Mass: 465.1
9a/b

185
185

Figure B7. Spectrometer Frequency and Solvent Effects in
19
F NMR Spectra of 9a/9b
(~1:1)
A sample of product 9a/9b (~1:1) before purification was separated into two aliquots.
From each, the solvent was evaporated, the residue was taken up in either CD
3
OD or D
2
O
and
19
F NMR spectra were acquired. Sample dissolved in A) CD
3
OD, 470 MHz; B)
CD
3
OD, 564 MHz; C) D
2
O, 470 MHz, pH 9.3; D) D
2
O, 564 MHz, pH 8.9.

C
B
A
D

186
186

Figure B8.
31
P NMR (202 MHz, CD
3
OD) of Product 9a/9b (~1:1) Before Purification
X = minor impurities

Figure B9.
31
P NMR (202 MHz, D
2
O, pH 8.9) of Product 9a/9b (~1:1) Before
Purification
X = minor impurities
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a/9b
X
X
X
X
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a/9b
X
X
X
X
X
X

187
187

Figure B10.
19
F NMR (470 MHz) Spectra of Product 9a/9b (~1:1) in CD
3
OD Spiked
with Isolated 9a (fast HPLC peak)
A)
19
F NMR Spectra for product 9a/9b (~1:1) spiked with 9a. The individual
stereoisomers were assigned based on the average of the differences between peaks ab
and cd, respectively: δ –219.050 (green, 9a) and –219.454 (orange, 9b); B) Calculated
individual
19
F NMR spectrum (green, 9a) using the following parameters: ddd,
2
J
F,P
=
60.5 Hz,
2
J
F,P
= 60.3 Hz,
2
J
F,H
= 46.3 Hz; C) Calculated individual spectrum for the other
diastereomer (orange, 9b) using the following parameters: ddd,
2
J
F,P
= 60.5 Hz,
2
J
F,P
=
60.3 Hz,
2
J
F,H
= 46.3 Hz. NMR spectra were simulated on MestReNova (Mnova 8.1.4).
C
B
A
a b
c d

188
188

Figure B11.
19
F NMR (470 MHz) Spectra of Product 9a/9b (~1:1) in D
2
O at pH 8.8
Spiked with Isolated 9a (fast HPLC peak)
A)
19
F NMR Spectra for product 9a/9b (~1:1) spiked with 9a. The individual
stereoisomers have: δ –218.495 (green, 9a) and –218.307 (orange, 9b); B) Calculated
individual
19
F NMR spectrum (green, 9a) using the following parameters: ddd,
2
J
F,P
=
61.6 Hz,
2
J
F,P
= 58.9 Hz,
2
J
F,H
= 45.7 Hz; C) Calculated individual spectrum for the other
diastereomer (orange, 9b) using the following parameters: ddd,
2
J
F,P
= 61.3 Hz,
2
J
F,P
=
60.9 Hz,
2
J
F,H
= 45.7 Hz. NMR spectra were simulated on MestReNova (Mnova 8.1.4)
C
B
A

189
189

Figure B12.
31
P NMR (202 MHz) Spectra of Product 9a/9b (~1:1) in CD
3
OD Spiked
with Isolated 9a (fast HPLC peak)
A)
31
P NMR

Spectra for product 9a/9b (~1:1) spiked with 9a. The individual
stereoisomers have: δ 8.831 and 7.926 (green, 9a) and δ 8.850 and 7.958 (orange, 9b); B)
Calculated individual
31
P NMR spectrum (green, 9a) using the following parameters: δ
8.831 (dd,
2
J
P,F
= 61.6 Hz,
2
J
P,P
= 12.7 Hz) and 7.926 (dd,
2
J
P,F
= 58.9 Hz,
2
J
P,P
= 12.7 Hz);
C) Calculated individual spectrum for the other diastereomer (green, 9a) using the
following parameters: δ 8.850 (dd,
2
J
P,F
= 61.6 Hz,
2
J
P,P
= 12.7 Hz) and 7.958 (dd,
2
J
P,F
=
58.9 Hz,
2
J
P,P
= 12.7 Hz). NMR spectra were simulated on MestReNova (Mnova 8.1.4)
C
B
A

190
190

Figure B13.
31
P NMR (202 MHz) Spectra of Product 9a/9b (~1:1) in D
2
O at pH 8.8
Spiked with Isolated 9a (fast HPLC peak)
A)
31
P NMR

Spectra for product 9a/9b (~1:1) spiked with 9a. The individual
stereoisomers have: δ 9.775 and 9.670 (green, 9a) and δ 10.239 and 9.720 (orange, 9b);
B) Calculated individual
31
P NMR spectrum (green, 9a) using the following parameters:
δ 9.775 (dd,
2
J
P,F
= 62.4 Hz,
2
J
P,P
= 12.7 Hz) and 9.670 (dd,
2
J
P,F
= 58.9 Hz,
2
J
P,P
= 12.7
Hz); C) Calculated individual
31
P NMR for the other diastereomer (green, 9a) using the
following parameters: δ 10.239 (dd,
2
J
P,F
= 62.4 Hz,
2
J
P,P
= 12.7 Hz) and 9.720 (dd,
2
J
P,F

= 58.9 Hz,
2
J
P,P
= 12.7 Hz). NMR spectra were simulated using spin simulation on
MestReNova (Mnova 8.1.4)
C
B
A

191
191

Figure B14. RP-C
18
HPLC Analysis of 9a/9b Using Varian Microsorb C
18
HPLC
Column (5 μm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System
For a 25 µL injection, 9a eluted at 15.2 min and 9b eluted at 15.7 min. The small peak
before 9a/9b is the monomorpholidate.

Figure B15. RP-C
18
HPLC Analysis of 9a/9b Using Varian Microsorb C
18
HPLC
Column (5 μm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System
For a 100 µL injection, 9a eluted at 15.5 min and 9b eluted at 16.2 min.

Figure B16. RP-C
18
HPLC Analysis of 9a/9b Using Phenomenex Luna C
18
HPLC
Column (5 μm, 250 mm × 21 mm)
1
and Rainin Dynamax MacIntegrator I System
For a 100 µL injection, 9a eluted at 23.4 min and 9b eluted at 25.4 min.
Chrom. 1 0.0 mins. 18.1 mins.
1
# Filename Chan Type Created Run Duration
1 SHIV-dimorph-001 A C Tue, Nov 25, 2014 7:51 PM 18.1
Chrom. 1 0.0 mins. 22.0 mins.
1
# Filename Chan Type Created Run Duration
1 SHIV-dimorph 2-001 A C Tue, Nov 25, 2014 8:26 PM 22.0
Chrom. 1 0.0 mins. 28.7 mins.
1
# Filename Chan Type Created Run Duration
1 dimorph 2 A C Wed, Nov 26, 2014 12:22 PM 28.7

192
192

Figure B17. Preparative RP-C
18
HPLC of 9a/9b Using Phenomenex Luna C
18
HPLC
Column (5 μm, 250 mm × 21 mm) and EZStart 7.4
X = minor impurities; Y = residual solvents

Figure B18.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 9a
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a/9b
9b 9a
(R) (R)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a
X
X
X
Y Y
Y Y

193
193

Figure B19.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 9a

Figure B20.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 9a
(R) (R)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a
(R) (R)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9a

194
194

Figure B21. MS of 9a

(R) (R)
P P
O
OH
O
-
O
N
O
F
(S) (S) N
O
O
O
Chemical Formula: C
17
H
24
FN
2
O
8
P
2
-
Exact Mass: 465.1
9a

195
195

Figure B22.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 9b
X = minor impurities; Y = residual solvents

Figure B23.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 9b
(S) (S)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9b
Y Y
Y
X
X
(S) (S)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9b

196
196

Figure B24.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 9b

(S) (S)
P P
O
OH
O
HO
N
O
F
(S) (S) N
O
O
O
9b

197
197

Figure B25. MS of 9b

(S) (S)
P P
O
OH
O
-
O
N
O
F
(S) (S) N
O
O
O
Chemical Formula: C
17
H
24
FN
2
O
8
P
2
-
Exact Mass: 465.1
9b

198
198

Figure B26. Preparative Ion Exchange HPLC Analysis of 11a

Figure B27.
1
H NMR (500 MHz, D
2
O, pH 10.0) of 11a
X = minor impurities; Y = residual solvents

!
Chrom. 1 0.0 mins. 30.0 mins.
3
2
1
# Filename Chan Type Created Run Duration
1 SAX 0.5M LiCl 25MAR11-001 A C Sun, Mar 25, 2012 5:28 PM 30.0
2 SAX 0.5M LiCl 25MAR11-002 A C Sun, Mar 25, 2012 5:58 PM 30.0
3 SAX 0.5M LiCl 25MAR11-003 A C Sun, Mar 25, 2012 6:31 PM 30.0
11a-1
AMP
AMP-M
AppA
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
OH
11a
X
X
X
X
X
Y
Y Y

199
199

Figure B28.
19
F NMR (470 MHz, D
2
O, pH 10.0) of 11a
X = minor impurities

Figure B29.
31
P NMR (202 MHz, D
2
O, pH 10.0) of 11a
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
OH
11a
X
X X
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
OH
11a

200
200

Figure B30. MS of 11a

N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
O
O
-
F
(S) (S)
N
O
O
O O O
OH
11a
Chemical Formula: C
23
H
29
FN
6
O
14
P
3
-
Exact Mass: 725.1

201
201

Figure B31.
1
H NMR (500 MHz, D
2
O, pH 10) of 11b
X = minor impurities; Y = residual solvents

Figure B32.
19
F NMR (470 MHz, D
2
O, pH 10) of 11b
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
O
O
-
F
(S) (S)
N
O
O
O O O
OH
11a
Chemical Formula: C
23
H
29
FN
6
O
14
P
3
-
Exact Mass: 725.1
X
X
X
Y Y Y Y
N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
OH
11b

202
202

Figure B33.
31
P NMR (202 MHz, D
2
O, pH 10) of 11b

N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
O
OH
F
(S) (S)
N
O
O
O O O
OH
11b

203
203

Figure B34. MS of 11b

N
N
N
NH
2
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
O
O
-
F
(S) (S)
N
O
O
O O O
OH
11b
Chemical Formula: C
23
H
29
FN
6
O
14
P
3
-
Exact Mass: 725.1
11b
A2 dimer
14
10b
Reaction Mixture

204
204

Figure B35.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 12a
X = minor impurities; Y = residual solvents

Figure B36.
19
F NMR (470 MHz, D
2
O, pH 10.2) of 12a

N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
12a
X
X
Y Y Y
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
12a

205
205

Figure B37.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 12a

N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
12a

206
206

Figure B38. MS of 12a

LCQ lnstrument Control 13 Jun 2012 01:44 PM
S#: 15910 lT: 18.95 ST: 0.91 #A: 10
522.1
264.4 361.1
I
250.3
azS.g
,i",,,*il ,,r.r,l llril[]n,,,t,lJ,ur,r,[r,,-,J,,r,,,. ,.dlJ,, In, .r.,r, ,,,.,r,
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
NL: 2.93e+006
723.1
6e5.1 I
834.8
952.6
931 21
805.5 I
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
-
O
OH
F
O O O
NH
2
OH
12a
Chemical Formula: C
11
H
16
FN
5
O
12
P
3
-
Exact Mass: 522.00

207
207

Figure B39.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 12b
X = minor impurities; Y = residual solvents

Figure B40.
19
F NMR (470 MHZ, D
2
O, pH 10.2) of 12b

N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
12b
X
X
X
X
X
X
X
Y Y Y
X
N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
12b

208
208

Figure B41.
31
P NMR (202 MHz, D
2
O, pH 9.7) of 12b

N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
12b

209
209

Figure B42. MS of 12b

N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
-
O
OH
F
O O O
NH
2
OH
12b
Chemical Formula: C
11
H
16
FN
5
O
12
P
3
-
Exact Mass: 522.00

210
210

Figure B43.
1
H NMR (500 MHz, D
2
O, pH ~10) of 12
X = minor impurities; Y = residual solvents

Figure B44.
19
F NMR (470 MHz, D
2
O, pH ~10) of 12

N
N
N
N
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
NH
2
OH
F
12
X
X
X
Y Y Y
N
N
N
N
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
NH2
OH
F
12

211
211

Figure B45.
31
P NMR (202 MHz, D
2
O, pH ~10) of 12

Figure B46.
19
F NMR (470 MHz, D
2
O, pH 9.8) of Diastereomer Mixture, 12, and
Individual Diastereomers, 12a and 12b

N
N
N
N
O
OH
O
P
O
OH
P
OH
P
HO
OH
O O O
NH2
OH
F
12
ppm
12a
12b
12a/12b
1:1
Upfield
Downfield
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
12a
N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
12b

212
212

Figure B47.
31
P NMR (202 MHz, D
2
O, pH 9.8) of Diastereomer Mixture, 12, and
Individual Diastereomers, 12a and 12b

Figure B48.
19
F NMR (470 MHz, D
2
O, pH 10.4) of Impurity 17
N
N
N
N
O
OH
O
P
O
OH
P
(S) (S)
OH
P
HO
OH
F
O O O
NH
2
OH
α β γ
12a
N
N
N
N
O
OH
O
P
O
OH
P
(R) (R)
OH
P
HO
OH
F
O O O
NH
2
OH
α β γ
12b
γ
β
α
12a-1
12a-2
12a-1/12a-2
1:1
12
12a
12b
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH OH
F
NH
2
N
N
N
N
O
OH OH
O
P
O
O
OH
NH
2
17

213
213

Figure B49.
31
P NMR (202 MHz, D
2
O, pH 10.4) of Impurity 17

N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH OH
F
NH
2
N
N
N
N
O
OH OH
O
P
O
O
OH
NH
2
17

214
214

Figure B50. MS of Impurity 17

N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH OH
F
NH
2
N
N
N
N
O
OH OH
O
P
O
O
O
-
NH
2
17
Chemical Formula: C
21
H
28
FN
10
O
18
P
4
-
Exact Mass: 851.1

215
215

Figure B51.
19
F NMR (470 MHz, D
2
O) of Diastereomer Mixture, 12, with Various Basic
Counterions

Figure B52.
31
P NMR (202 MHz, D
2
O) of Diastereomer Mixture, 12, with Various Basic
Counterions
KOH (470 MHz, pH 11.1, 16 scans)
NH4OH (470 MHz, pH 10.8, 256 scans)
Na2CO3 (470 MHz, pH 10.9, 256 scans)
Et3N (470 MHz, pH 10.9, 64 scans)
Δδ = 26.8 Hz
Δδ = 24.9 Hz
Δδ = 29.1 Hz
Δδ = 31.0 Hz
JFH = 45.6 Hz
JFPγ = 56.6 Hz
JFPβ = 64.6 Hz
JFH = 45.6 Hz
JFPγ = 56.4 Hz
JFPβ = 65.3 Hz
JFH = 45.1 Hz
JFPγ = 55.7 Hz
JFPβ = 66.3 Hz
KOH (202 MHz, pH 11.1, 512 scans)
NH4OH (202 MHz, pH 10.8, 512 scans)
Na2CO3 (202 MHz, pH 10.9, 512 scans)
Et3N (202 MHz, pH 11.0, 512 scans)
JPF = 64.6 Hz
JPP = 28.3 Hz
JPP = 28.3 Hz
JPP = 14.1 Hz
JPP = 14.1 Hz
JPF = 56.6 Hz J = 2.8 Hz ?
JPP = 28.3 Hz
Δδ = 6.1 Hz
JPP = 28.3 Hz
JPP = 28.3 Hz
JPF = 64.6 Hz
JPP = 28.3 Hz
JPP = 14.1 Hz
JPP = 14.1 Hz
JPF = 56.6 Hz
JPF = 56.6 Hz
JPP = 14.1 Hz

216
216

Figure B53.
31
P NMR (D
2
O, pH 10.9) of 12 Treated with Na
2
CO
3
at Two Spectrometer
Frequencies
X = minor impurities

Figure B54.
31
P NMR (202 MHz, D
2
O, pH 12.1) of 12 Treated with NaOH
X = minor impurities
ppm
202 MHz
162 MHz
Δδ = 4.0 Hz
Δδ = 6.1 Hz
Δδ = 3.2 Hz
Δδ = 4.9 Hz
JPF = 65.1 Hz
JPP = 27.9 Hz
JPP = 14.0 Hz
JPF = 65.0 Hz
JPP = 27.9 Hz
JPP = 14.1 Hz
JPP = 14.1 Hz
JPF = 55.2 Hz
JPP = 14.5 Hz
JPF = 55.1 Hz
JPP = 27.9 Hz
JPP = 27.9 Hz
γ β α
γ β α
X
X X
JPP = 14.5 Hz
JPF = 55.5 Hz JPF= 65.4 Hz
JPP = 28.3 Hz
JPP = 14.1 Hz
JPP = 28.4 Hz
X X

217
217

Figure B55.
19
F NMR (470 MHz, D
2
O, pH 9.8) of Artificial Diastereomer Mixture (3:1,
75% 12a and 25% 12b)
X = minor impurities

Figure B56.
31
P NMR (202 MHz, D
2
O, pH 9.8) of Artificial Diastereomer Mixture (3:1,
75% 12a and 25% 12b)
X = minor impurities
X X
X
ppm
JPP = 14.1 Hz
JPF = 55.7 Hz JPF = 65.1 Hz
JPP = 28.3 Hz
JPP = 14.1 Hz
JPP = 28.4 Hz
Δδ = 6.1 Hz
β α
X X
X X

218
218

Figure B57. NMR Simulation Studies with 12,
19
F NMR (470 MHz, D
2
O, pH 9.8)
Spectrum for synthetic 1:1 diastereomer mixture (12, purple). The individual
stereoisomers have: δ –218.06 (12a, green) and –218.12 (12b, red), ddd,
2
J
F,Pβ = 65.4 Hz,
2
J
F,Pγ = 55.5 Hz,
2
J
F,H
= 45.4 Hz; A) Calculated individual
19
F NMR (12a, green) for one
of the two diastereomers; B) Calculated individual
19
F NMR (12b, red) for the other
diastereomer. NMR spectra were simulated using spin simulation on MestReNova
(Mnova 8.1.4)
2,3

A
B

219
219
Chapter 3 Supporting Data References

(1) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.;
Wilson, S. H.; McKenna, C. E. J. Am. Chem. Soc. 2012, 134, 8734.
(2) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.;
Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617.
(3) Hwang, C. S.; Kashemirov, B. A.; McKenna, C. E. J. Org. Chem. 2014, 79, 5315.

220
220
Appendix C. Chapter 4 Supporting Data
7

Figure C1.
1
H NMR (500 MHz, D
2
O, pH 9.8) of 7
X = unidentified impurities; Y = triethylamine (TEA); Z = HDO

7
Reproduced with permission from Hwang, C. S.; Xu, L.; Wang, W.; Ulrich, S.; Zhang,
L.; Chong, J.; Shin, J. H.; Huang, X.; Kool, E. T.; McKenna, C. E.; Wang, D. Nucleic
Acids Res. 2016. Copyright 2016 Oxford University Press.
N
N
N
N
O
OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2
X
X X X X X
Y Y Z

221
221

Figure C2.
31
P NMR (202 MHz, D
2
O, pH 9.8) of 7

N
N
N
N
O
OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2
α
β
γ
α β γ

222
222

Figure C3. MS of 7
ESI-MS: m/z 488 [M - H]
-

223
223

Figure C4.
1
H NMR (600 MHz, D
2
O, pH 10.5) of 8
X = unidentified impurities; Z = HDO

Figure C5.
31
P NMR (202 MHz, D
2
O, pH 10.5) of 8

Z
X X X X
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2
α
β
γ
α β γ
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2

224
224

Figure C6. MS of 8
ESI-MS: m/z 504 [M - H]
-

225
225

Figure C7.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 9
X = unidentified impurities; Y = triethylamine (TEA); Z = HDO

Figure C8.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 9

X
X X X
X
X
X
Y Y Z
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
O
O N
α
β
γ
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
O
O N
α β γ

226
226

Figure C9. MS of 9
ESI-MS: m/z 481 [M - H]
-

227
227

Figure C10.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 10
X = unidentified impurities; Y = triethylamine (TEA); Z = HDO

Figure C11.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 10

X X X X
X X X
X
Y Y Z
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
2

228
228

Figure C12. MS of 10
ESI-MS: m/z 504 [M - H]
-

229
229
Table C1. Preparative HPLC conditions and elution times
Experiment Column Mobile Phase Retention Time
Purification of 7,
9-10, 14
Macherey-Nagel
SAX 1000-10
(150 mm × 25
mm)
0.5 M Triethylammonium
bicarbonate, pH 7.4.
Gradient: (0-10 min, 60%; 10-
15 min, 60%; 15-25 min,
100%)
7
14.1 min
14
16.7 min
0.5 M Triethylammonium
bicarbonate, pH 7.4.
Gradient (0−20 min, 0%;
20−35 min, 60−100%; 35−45
min, 100%)
9
18.8 min
10
20.5 min
Purification of 7,
9-10, 14
Phenomenex
Luna
®
C
18
HPLC
Column (5 μm,
250 mm × 21 mm)
0.1 M Triethylammonium
bicarbonate, 4.75% CH
3
CN,
pH 7.4
7
6 min *eluted off with
CH
3
CN:H
2
O (1:1)
0.1 M Triethylammonium
bicarbonate, 7.5% CH
3
CN, pH
7.4
14
9.9 min
0.1 M Triethylammonium
bicarbonate, 4.75% CH
3
CN,
pH 7.4
9
15.2 min
0.1 M Triethylammonium
bicarbonate, 7.5% CH
3
CN, pH
7.4
10
10.9 min
Purification of 15-
18
Macherey-Nagel
SAX 1000-10
(150 mm × 25
mm)
0.5 M Triethylammonium
bicarbonate, pH 7.4.
Gradient (0−20 min, 0%;
20−35 min, 60−100%; 35−45
min, 100%)
15
28.7 min
16
28.7 min
17
28.9 min
18
28.1 min
Purification of 15-
18
Phenomenex
Luna
®
C
18
HPLC
Column (5 μm,
250 mm × 21 mm)
0.1 M Triethylammonium
bicarbonate, 7.5% CH
3
CN, pH
7.4
15
13.8 min
16
11.4 min
17
12.9 min
18
9.9 min

230
230

Figure C13. Representative kinetic fitting data (β,γ-CH
2
-ATP/dT)
A) Representative gel data; B) Rate fitting based on the gel analysis; C) Specificity fitting
curve.

231
231

Figure C14. Representative kinetic fitting data (β,γ-CH
2
-UTP/dT).
A) Representative gel data. (b) Rate fitting based on the gel analysis; C) Specificity
fitting curve

232
232

Figure C15. Two potential UTP:dT forms based on reported U:U pairing

Figure C16. Energy minimized modeling of UTP binding opposite dT template at pol II
active site

233
233

Figure C17.
1
H NMR (500 MHz, D
2
O, pH 9.9) of 14

Figure C18.
31
P NMR (202 MHz, D
2
O, pH 9.9) of 14
O
OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
O
O N
X X X
Y Y Z
α β γ
α
β
γ
O
OH
O
P
O
O
OH
P P
O O
OH
HO
OH
NH
O
O N

234
234

Figure C19. MS of 14
ESI-MS: m/z 488 [M ‒ H]
‒

235
235

Figure C20.
1
H NMR (500 MHz, D
2
O, pH 10.2) of 15

X X
X
X X
X X X
X
Y Y Z
N
N
N
N
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F
X
N
N
N
N
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F

236
236
Figure C21.
19
F NMR (470 MHz, D
2
O, pH 10.2) of 15

Figure C22.
31
P NMR (202 MHz, D
2
O, pH 10.2) of 15

α
β γ
N
N
N
N
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F
α β γ

237
237

Figure C23. MS of 15
ESI-MS: m/z 524 [M ‒ H]
‒

238
238

Figure C24.
1
H NMR (500 MHz, D
2
O, pH 9.6) of 16

Figure C25.
19
F NMR (470 MHz, D
2
O, pH 9.8) of 16
X X X
Y
Y
Z
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F
X
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F

239
239

Figure C26.
31
P NMR (202 MHz, D
2
O, pH 9.8) of 16

α
β
γ
α β γ
O
OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F

240
240

Figure C27. MS of 16
ESI-MS: m/z 515 [M ‒ H]
‒

241
241

Figure C28.
1
H NMR (500 MHz, D
2
O, pH 9.9) of 17

Figure C29.
19
F NMR (470 MHz, D
2
O, pH 9.9) of 17
X X
Y Y Z
X X X X
X
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F
X
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F

242
242

Figure C30.
31
P NMR (202 MHz, D
2
O, pH 9.9) of 17

α
β γ
α β γ
N
N
N
N
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
2
F F

243
243

Figure C31. MS of 17
ESI-MS: m/z 540 [M ‒ H]
‒

244
244

Figure C32.
1
H NMR (500 MHz, D
2
O, pH 8.1) of 18

Figure C33.
19
F NMR (470 MHz, D
2
O, pH 10.3) of 18
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F
X X X X X
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F
X

245
245

Figure C34.
31
P NMR (202 MHz, D
2
O, pH 10.3) of 18

α
β γ
α β γ
O
OH OH
O
P
O
O
OH
P P
O O
OH HO
HO
NH
O
O N
F F

246
246

Figure C35. MS of 18
ESI-MS: m/z 517 [M ‒ H]
‒

247
247
Appendix D. Chapter 5 Supporting Data
8

Table D1. Effect of difluorination on the solubility of small, unsaturated three-
membered rings

8
Reproduced with permission from Ni, F.; Lee, C. C.; Hwang, C. S.; Hu, Y.; Ribbe, M.
W.; McKenna, C. E. J. Am. Chem. Soc. 2013, 135, 10346. Copyright 2016 American
Chemical Society.
Compound MW
Structural
Formula
Water
Solubility
a
Water
Solubility
Henry's Law
Constant
b
Dipole
Moment
b.p.
e
g/mol at 25˚C mg/L at 25˚C mM
at 25˚C atm/M
(x10
-3
)
D ˚C
cyclopropene
40.0648 1995 49.79 0.028 0.36
c
-27.30
difluoro-
cyclopropene
76.0457 11,100 145.96 0.199 2.98
d
-53.28
diazirine
42.0403 5811 138.22 0.025 2.34
c
-21.80
difluoro-
diazirine
78.0212 9183 117.70 0.097 0.08
c
-83.98
Table 2. Effect of difluorination on solubility of small, unsaturated three-membered rings .
a
US$EPA$EPISuite
TM
$WSKOW$v1.41
b
US$EPA$EPISuite
TM
$HENRYWIN$v3.10
c
Robin$et$al.$1969.$J"Chem"Phys.
d
Ramaprasad$et$al.$1976.$J"Chem"Phys.
e
Calculated$using$ACD/PhysChem$Suite$at$760$mmHg
F F
N N
N N
F F

248
248

Figure D1.
1
H NMR spectrum (500 MHz, CDCl
3
) of DFCP

Figure D2.
1
H NMR spectra (500 MHz) of DFCP
A) Incubated in D
2
O after overnight at RT; B) Incubated in CDCl
3
solution (with a trace
of water) after overnight at RT.

249
249

Figure D3.
19
F NMR spectra (470.4 MHz, CDCl
3
) of DFCP
A) d
2
-DFCP; B) DFCP

250
250

Figure D4.
13
C NMR spectrum (500 MHz, CDCl
3
) of DFCP

251
251

Figure D5. MS Chromatogram of DFCP and d
2
-DFCP
A) Mass spectrum of DFCP ([M–H]
+
, m/z = 75); B) Mass spectrum of d
2
-DFCP ([M–D]
+
,
m/z = 76)

252
252
Figure D6. Gas-phase IR of DF-DZR (20 Torr)
It should be noted that CO
2
absorbs at 2358 cm
-1
(asymmetric stretch), 667 cm
-1
and 541
cm
-1
(bending mode).

Figure D7.
1
H NMR of DF-DZR in CDCl
3
(600 MHz, 8 scans)

253
253

Figure D8.
13
C NMR of DF-DZR in CDCl
3
(151 MHz, 30,000 scans)

Figure D9.
14
N NMR of DF-DZR in CDCl
3
(36 MHz, overnight)

254
254

Figure D10.
19
F NMR of DF-DZR in CDCl
3
(546 MHz, 128 scans), referenced using
CFCl
3
standard (δ 0.00 ppm)

Figure D11.
1
H NMR of DF-DZR in D
2
O (600 MHz, 8 scans)

255
255

Figure D12.
13
C NMR of DF-DZR in D
2
O (150 MHz, 30,000 scans)

Figure D13.
19
F NMR of DF-DZR in D
2
O (564 MHz, 2,048 scans), referenced using
CFCl
3
standard (δ 0.00 ppm)

256
256

Figure D14.
19
F NMR of DF-DZR in D
2
O (564 MHz, 1,024 scans), after four days.
Impurity peak at -7.7 ppm did not significantly increase

Figure D15.
19
F NMR of DF-DZR in D
2
O (470 MHz), after four days

257
257
Table D2. Results from computational model of gas-phase IR of DF-DZR (CF
2
N
2
) using
GAUSSIAN

CF
2
N
2

EXP
(cm
-1
)
Calculated (cm
-1
)
m062x/aug-cc-pvdz m062x/aug-cc-pvtz B3LYP/aug-cc-pvtz
1562 1743 1731 1641
1292 1402 1397 1310
1244 1289 1299 1224
1089 1169 1171 1099
1028 - - -
898 843 849 811
804 - - -
520 556 558 539
500 498 507 496
- 485 492 482
- 457 463 450

258
258

Figure D16. Plot from computational model of gas phase-IR of DF-DZR (CF
2
N
2
) using
GAUSSIAN

259
259

Figure D17.
19
F NMR time-dependent study of DF-DZR in H
2
O
A solution of trifluoroethanol (δ -77) was added to the sample as a standard for
integration with DF-DZR (δ -121). Time points were taken every ten minutes, for a total
of sixty minutes.

260
260

Figure D18. Time-dependent study of
19
F NMR of difluorodiazirine in assay buffer (470
MHz, 256 or 1024 scans), referenced using CFCl
3
standard (δ 0.00 ppm) at 30 ˚C
Signal of the trifluoroethanol standard exhibits a δ -77, and DF-DZR has a δ -121.

t = 0
t = 10
t = 20
t = 50
Time (min)

261
261

Figure D19. Design of storage vessel for DF-DZR
A) Saturated sodium sulfate; B) Kontes® HI-VAC® straight stopcock valve with CTFE
plugs and tip O-ring; C) space where DF-DZR is stored; D) 7 mm O-ring joint; E) septum.
Opening of valve connecting A and C will allow the pressure in A to equilibrate to an
atmosphere of pressure.
B
B
B
A
C
E D

262
262

Figure D20. GC-MS analysis of products from DFCP reduction by N
2
ase (FeP/MoFeP =
20:1)
A) GC chromatogram; B) MS of P1, identified as propene ([M–H]
+
, m/z = 41); C) MS of
P2, identified as 2-fluoropropene ([M–H]
+
, m/z = 59).

263
263

Figure D21.
1
H NMR (500 MHz, CDCl
3
) spectra of gas phase products of DFCP
reduction by N
2
ase
A) Reduction products for FeP/MoFeP = 20:1; B) Reduction products with FeP/MoFeP =
1:5; C) Authentic propene/2-fluoropropene mixture (~1:1). Hydrogen atoms are labeled
by circles, which refer to the corresponding peaks adjacent to the structures.

264
264

Figure D22.
19
F NMR (470.4 MHz; CDCl
3
) spectra for DFCP reduction by N
2
ase
A) Reduction products for FeP/MoFeP = 20:1; B) Authentic propene/2-fluoropropene
mixture (~1:1).

Figure D23. Fluoride (F
–
) release rates (4 replicates)
A) Complete assay conditions, 20 ˚C, 0.03 atm. DFCP, 1.0 mg/ml MoFe protein and 1.2
mg/ml Fe protein, background-corrected; B) Substrate reduction (calculation based on the
amount of released products propene, giving 2 F
–
/molecule, and 2-F-propene, giving 1 F
–
/molecule).

265
265

Figure D24. Distribution of d
2
-propene and d
2
-2-fluoropropene isomers formed under
different electron flux conditions (FeP/MoFeP = 20:1 vs. 1:5)

266
266

Figure D25. Schematic of inhibition experiment of acetylene with DF-DZR
A) Experimental design for measuring acetylene inhibition by varying amounts of
acetylene and constant amount of DF-DZR gas; B) Technique utilized to introduce DF-
DZR into airtight assay vials. Vessels depicted in images represent airtight assay vials
used in experiments. GC-FID stands for gas-chromatography flame ionization detector,
CPK stands for creatine phosphate kinase solution (containing ATP, CP, CPK, HEPES
and MgCl
2
), DT stands for dithionite solution.

267
267

Figure D26. Fluorescence detection method using OPA/2-ME
1,2

A) Table of components needed for reagent buffer; B) Reaction that occurs with
ammonia that results in fluorescent product; C) Example of fluorescent curve generated
with water standard.

Figure D27. Calibration plot for determination of ammonia
A) Example of spectra acquired for ammonia stock and wavelength detection at 474 and
490 nm; B) Calibration curve using values obtained at 474 nm or 490 nm.

268
268

Figure D28. Example of fluorescence detection using OPA/2-ME method from assay
experiment containing 6% DF-DZR in He
A) Fluorescent measurement of three samples; B) Image of fluorescence from native
buffer reagent in water and enhanced fluorescence from putative presence of ammonia or
amine.

269
269

Table D3. Detailed experimental description of inhibition of acetylene (constant) with DF-DZR (variable) experiment

270
270

Figure D29. Image of reduction experiments with 50% DF-DZR in He
Vial 1 contains DF-DZR without enzyme; Vial 3 contains DF-DZR with enzyme; Vial 5
contains acetylene with enzyme

Figure D30. Image of inhibition experiments with addition of acetylene after exposure
and removal of 50% DF-DZR for 40 minutes
A) Vials 1 & 2 contains no DF-DZR/+ enzyme; Vials 3 & 4 contains + DF-DZR/+
enzyme; Vial 5 contains + DF-DZR/+ enzyme for 40 min then removal of DF-DZR +
acetylene (for 20 min); Vial 6 contains acetylene/+ enzyme for 20 min; B) After 24 h at rt
A
B

271
271

Figure D31. Calibration plot of H
2
by GC-BID
GC-BID stands for gas-chromatography barrier discharge ionization detector

Figure D32. Detection of gases by GC-FID from add-back experiment after exposure of
buffer assay mixture to DF-DZR and removal
All vials are exposed to DF-DZR for 20 min (except Vials 7 and 8). Then the DF-DZR
was removed and acetylene/enzyme were added. Vials 1 & 2: only enzyme and acetylene
were added back; Vials 3 & 4: an additional portion of ATP Buffer was added back;
Vials 5 & 6: an addition portion of dithionite solution was added back; Vials 7 & 8:
control, only acetylene and enzyme were added back
y"="139585x"+"224076"
R²"="0.9812"
0"
500000"
1000000"
1500000"
2000000"
2500000"
3000000"
3500000"
4000000"
4500000"
0" 5" 10" 15" 20" 25" 30"
GC#Area#
umols#H2#

272
272
Chapter 5 Supporting Data References

(1) Barney, B. M.; McClead, J.; Lukoyanov, D.; Laryukhin, M.; Yang, T. C.; Dean, D.
R.; Hoffman, B. M.; Seefeldt, L. C. Biochemistry 2007, 46, 6784.
(2) Corbin, J. L. Appl. Environ. Microbiol. 1984, 47, 1027.

273

273
Appendix E. Chapter 6 Supporting Data

Figure E1. ISCO Purification of 3
Sample: SHIV-150_400mg Rf+ Wednesday 08 July 2015 12:58PM
Page 1 of 1
RediSep Column: Silica 24g
SN: E0415056C8D3FB Lot: 251132101X
Flow Rate: 35 ml/min
Equilibration Volume: 168.0 ml
Initial Waste: 0.0 ml
Air Purge: 1.0 min
Solvent A: dichloromethane
Solvent B: ethyl acetate
Peak Tube Volume: Max.
Non-Peak Tube Volume: Max.
Loading Type: Liquid
Wavelength 1 (red): 254nm
Peak Width: 1 min
Threshold: 0.20 AU
Wavelength 2 (purple): 280nm
Evaporative Light Scattering (green)
Peak Width: 1 min
Threshold: 0.05 v
Spray Temperature: 30C
Drift Temperature: 60C
Run Notes:
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
0
10
20
30
40
50
60
70
80
90
100
0.00 0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
0.00 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
ELS
Absorbance Percent B
1
2
3
7
11
15
19
25
26
30
34
38
42
46
50
54
58
62
66
73
74
78
84
85
89
93
97
101
105
1
5
9
13
17
Run Length 42.9 Min
Duration %B Solvent A Solvent B
0.0 0.0 dichloromethane ethyl acetate
1.0 0.0 dichloromethane ethyl acetate
1.2 10.0 dichloromethane ethyl acetate
2.8 10.0 dichloromethane ethyl acetate
3.2 10.0 dichloromethane ethyl acetate
0.0 10.0 dichloromethane ethyl acetate
0.0 10.0 dichloromethane ethyl acetate
2.5 40.1 dichloromethane ethyl acetate
10.8 40.1 dichloromethane ethyl acetate
0.0 40.1 dichloromethane ethyl acetate
... ... ... ...
Peak # Start Tube End Tube
1 A:1 A:2
2 A:3 A:25
3 A:26 A:73
4 A:74 A:84
5 A:85 B:10
6 B:11 B:22
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
Rack A
13 mm x 100 mm Tubes
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22
Rack B
13 mm x 100 mm Tubes
274

274

Figure E2.
1
H NMR (500 MHz, CDCl
3
) of 3

Figure E3.
31
P NMR (202 MHz, CDCl
3
) of 3
N
O-iPr P O
O-iPr
P
O-iPr
O
O-iPr
3
N
O-iPr P O
O-iPr
P
O-iPr
O
O-iPr
3
275

275

Figure E4.
1
H NMR (500 MHz, D
2
O) of 4

Figure E5.
31
P NMR (202 MHz, D
2
O) of 4
N
OH P O
OH
P
OH
O
OH
4
N
OH P O
OH
P
OH
O
OH
4
276

276

Figure E6. Predictive iMass for 4
A) ESI-MS m/z (+); B) ESI-MS m/z (-)

A B
277

277

Figure E7. MS (+) of 4

N
H
+
OH P O
OH
P
OH
O
OH
4
Chemical Formula: C
7
H
12
NO
6
P
2
+
Molecular Weight: 268.1
278

278

Figure E8. MS (-) of 4

N
OH P O
OH
P
O
-
O
OH
4
Chemical Formula: C
7
H
10
NO
6
P
2
-
Molecular Weight: 266.1
279

279

Figure E9.
1
H NMR (500 MHz, D
2
O, pH 10.26) of 8

Figure E10.
31
P NMR (202 MHz, D
2
O, pH 10.26) of 8

N
OH P O
OH
P
OH
O
OH
OH
N
3
8
N
OH P O
OH
P
OH
O
OH
OH
N
3
8
280

280

Figure E11. Predictive iMass of 8
A) ESI-MS m/z (+); B) ESI-MS m/z (-)

A B
281

281

Figure E12. MS (+) of 8

N
OH P O
OH
P
OH
O
OH
OH
N
3
8
Chemical Formula: C
10
H
17
N
4
O
7
P
2
+
Molecular Weight: 367.2
282

282

Figure E13. MS (-) of 8

N
OH P O
O
-
P
O
-
O
OH
OH
N
3
8
Chemical Formula: C
10
H
15
N
4
O
7
P
2
-
Molecular Weight: 365.2
283

283

Figure E14.
1
H NMR of 10

Figure E15.
31
P NMR of 10

N
+
OH P O
OH
P
OH
O
OH
OH
N
3
HO
N
+
OH P O
OH
P
OH
O
OH
OH
N
3
HO
284

284

Figure E16. Predictive iMass of 10
A) ESI-MS m/z (+); B) ESI-MS m/z (-)

A B
285

285

Figure E17. MS (+) of 10

N
+
OH P O
OH
P
OH
O
OH
OH
N
3
10
HO
Chemical Formula: C
10
H
17
N
4
O
8
P
2
+
Exact Mass: 383.05
286

286

Figure E18. MS (-) of 10

N
+
OH P O
O
-
P
O
-
O
OH
OH
N
3
10
HO
Chemical Formula: C
10
H
15
N
4
O
8
P
2
-
Exact Mass: 381.04
287

287

Figure E19.
1
H NMR (500 MHz, CDCl
3
) of tosylated alcohol 12

Figure E20.
1
H NMR (400 MHz, CDCl
3
) of monoprotected diamine 16
CDCl3
H2O
S
O
O
O
H
2
N
H
N
Mtt
DCM
288

288

Figure E21. ISCO purification of 17 with (Attempt 1) and without 1% TEA (Attempt 2)

Sample: SHIV-177 Rf+ Tuesday 01 September 2015 01:05PM
Page 1 of 1
RediSep Column: Silica 40g
SN: E0415056C8A34F Lot: 251132103Y
Flow Rate: 40 ml/min
Equilibration Volume: 240.0 ml
Initial Waste: 0.0 ml
Air Purge: 1.0 min
Solvent A: hexane
Solvent B: ethyl acetate
Peak Tube Volume: Max.
Non-Peak Tube Volume: Max.
Loading Type: Liquid
Wavelength 1 (red): 254nm
Peak Width: 2 min
Threshold: 0.20 AU
Wavelength 2 (purple): 280nm
Run Notes:
0.0 5.0 10.0 15.0 20.0 25.0
0
10
20
30
40
50
60
70
80
90
100
0.00 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Absorbance Percent B
1
5
6
9
12
13
16
20
21
24
27
30
35
36
Run Length 28.1 Min
Duration %B Solvent A Solvent B
0.0 0.0 hexane ethyl acetate
1.0 20.0 hexane ethyl acetate
0.4 21.4 hexane ethyl acetate
3.4 21.4 hexane ethyl acetate
0.0 21.4 hexane ethyl acetate
13.6 70.0 hexane ethyl acetate
0.3 70.0 hexane ethyl acetate
0.1 70.0 hexane ethyl acetate
5.6 70.0 hexane ethyl acetate
0.0 70.0 hexane ethyl acetate
... ... ... ...
Peak # Start Tube End Tube
1 A:1 A:5
2 A:6 A:12
3 A:13 A:20
4 A:21 A:35
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35
Rack A
13 mm x 100 mm Tubes
Sample: SHIV-174_50C Rf+ Thursday 03 September 2015 01:51PM
Page 1 of 1
RediSep Column: Silica 40g
SN: E041505C5CB712 Lot: 251717304X
Flow Rate: 40 ml/min
Equilibration Volume: 240.0 ml
Initial Waste: 0.0 ml
Air Purge: 1.0 min
Solvent A: hexane
Solvent B: ethyl acetate
Peak Tube Volume: Max.
Non-Peak Tube Volume: Max.
Loading Type: Liquid
Wavelength 1 (red): 254nm
Peak Width: 2 min
Threshold: 0.20 AU
Wavelength 2 (purple): 280nm
Run Notes:
0.0 5.0 10.0 15.0 20.0
0
10
20
30
40
50
60
70
80
90
100
0.00 0.00
0.25
0.50
0.75
1.00
Absorbance Percent B
1
3
4
7
12
13
16
19
22
25
28
31
34
Run Length 23.8 Min
Duration %B Solvent A Solvent B
0.0 0.0 hexane ethyl acetate
1.0 0.0 hexane ethyl acetate
4.0 30.0 hexane ethyl acetate
2.1 30.0 hexane ethyl acetate
1.7 30.0 hexane ethyl acetate
0.0 30.0 hexane ethyl acetate
0.9 30.0 hexane ethyl acetate
4.0 40.0 hexane ethyl acetate
0.6 40.0 hexane ethyl acetate
3.1 40.0 hexane ethyl acetate
... ... ... ...
Peak # Start Tube End Tube
1 A:1 A:3
2 A:4 A:12
3 A:13 A:34
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34
Rack A
13 mm x 100 mm Tubes
Attempt 1:
1.! Hexane/EtOAc (1% NEt3)
2.!
1
H NMR-overlapping
peaks
Attempt 2:
1.! Hexane/EtOAc
2.!
1
H NMR-clean!
N
H
H
N
Ph Ph
289

289

Figure E22.
1
H NMR (500 MHz, D
2
O) of 17
Quartet at 4.16 is ethyl acetate.

Figure E23.
1
H NMR (400 MHz, CDCl
3
) of 19

290

290

Figure E24. MS (+) of 19

19
NH
+
O
H
N O
O
HN
Mtt
Chemical Formula: C
34
H
42
N
3
O
3
+
Exact Mass: 540.32
291

291

Figure E25. ISCO Purification of 21

Sample: ER103 Rf+ Monday 11 April 2016 05:11PM
Page 1 of 1
RediSep Column: Silica 4g
Flow Rate: 18 ml/min
Equilibration Volume: 33.6 ml
Initial Waste: 0.0 ml
Air Purge: 0.5 min
Solvent A: dichloromethane
Solvent B: ethyl acetate
Peak Tube Volume: Max.
Non-Peak Tube Volume: Max.
Loading Type: Solid
Wavelength 1 (red): 254nm
Peak Width: 30 sec
Threshold: 0.20 AU
Wavelength 2 (purple): 470nm
Evaporative Light Scattering (green)
Peak Width: 30 sec
Threshold: 0.05 v
Spray Temperature: 30C
Drift Temperature: 60C
Run Notes:
0.0 5.0 10.0 15.0 20.0 25.0
0
10
20
30
40
50
60
70
80
90
100
0.00 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.00 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
ELS
Absorbance Percent B
1
2
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
Run Length 27.8 Min
Duration %B Solvent A Solvent B
0.0 0.0 dichloromethane ethyl acetate
1.4 0.0 dichloromethane ethyl acetate
4.7 31.0 dichloromethane ethyl acetate
4.2 31.0 dichloromethane ethyl acetate
10.5 100.0 dichloromethane ethyl acetate
1.7 100.0 dichloromethane ethyl acetate
0.2 100.0 dichloromethane ethyl acetate
5.0 100.0 dichloromethane ethyl acetate
Peak # Start Tube End Tube
1 A:2 A:2
2 A:3 A:5
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 30
31 32 33 34 35 36
37 38 39 40 41 42
43 44 45 46 47 48
49 50 51 52 53 54
55 56 57 58 59 60
61 62 63 64 65 66
67 68 69 70 71 72
73 74 75 76 77 78
79 80 81 82 83 84
85 86 87 88 89 90
91 92 93 94 95 96
97 98 99 100 101 102
103 104 105 106 107 108
2 3 4 5
Rack A
13 mm x 100 mm Tubes
292

292

Figure E26. Predictive iMass of 21
A) MS (+); B) MS (-)

A B
293

293

Figure E27. MS (+) of 21

NH
+
O
H
N O
O
HN O
O HO O
COOH
21
Chemical Formula: C
35
H
36
N
3
O
9
+
Exact Mass: 642.24
294

294

Figure E28. MS (-) of 21

N
O
H
N O
O
HN O
O
-
O O
COOH
21
Chemical Formula: C
35
H
34
N
3
O
9
-
Exact Mass: 640.23
295

295

Figure E29. Predictive iMass of 22
A) MS (+); B) MS (-)

Figure E30. MS (+) of 22

A B
N
O
H
N O
O
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
Chemical Formula: C
45
H
52
N
7
O
17
P
2
+
Exact Mass: 1024.29
296

296

Figure E31. MS (-) of 22

N
O
H
N O
O
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
O
-
O
O
-
HO
Chemical Formula: C
45
H
50
N
7
O
17
P
2
-
Exact Mass: 1022.27
297

297

Figure E32. Predictive iMass of 23
A) MS (+); B) MS (-)
A B
298

298

Figure E33. MS (+) of 23

N
O
H
2
N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
23
Chemical Formula: C
40
H
44
N
7
O
15
P
2
+
Exact Mass: 924.24
299

299

Figure E34. MS (-) of 23
N
O
H
2
N
HN O
O HO O
COOH
N
N
N
OH
N
+
P
P
OH
O HO
O
-
O
O
-
HO
23
Chemical Formula: C
40
H
42
N
7
O
15
P
2
-
Exact Mass: 922.22
300

300

Experiment Column Mobile Phase Retention Time
Purification of 8,
10
Macherey-Nagel
SAX 1000-10
(150 mm × 25
mm)
0.5 M Triethylammonium
bicarbonate, pH 7.4.
Gradient: (0-5 min, 0%; 5-10
min, 0-100%; 10-15 min,
100%)
8
13 min
0.5 M Triethylammonium
bicarbonate, pH 7.4.
Gradient (0-5 min, 0-5%; 5-10
min, 5%; 10-20 min, 5%-100%)
10
13.5 min
Purification of 22-
23
Phenomenex
Luna
®
C
18
HPLC
Column (5 μm,
250 mm × 10 mm)
0.1 M Triethylammonium
bicarbonate, 5-50% CH
3
CN, pH
7.4
Gradient (0-8 min, 5% CH
3
CN,
8-13 min 5%-50% CH
3
CN, 13-
20 min, 50% CH
3
CN)
22
16.3 min
0.1 M Triethylammonium
bicarbonate, 5-50% CH
3
CN, pH
7.4
Gradient (0-5 min, 5% CH
3
CN,
5-10 min 5%-50% CH
3
CN, 10-
15 min, 50% CH
3
CN)
23
13.2 min
Table E1. Table of HPLC conditions

Figure E35. Predictive iMass of 25
A) MS (+); B) MS (-)
A B
301

301

Figure E36. MS (+) of 25
25
O
N N
N N
N
OCH 3
NO 2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH 3
+
O
H
N
O
OH
Chemical Formula: C 55 H 70 N 17 O 14
+
Exact Mass: 1192.53
302

302

Figure E37. MS (-) of 25
25
O
N N
N N
N
OCH
3
NO
2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH
2
O
H
N
O
O-
Chemical Formula: C
55
H
68
N
17
O
14
-
Exact Mass: 1190.51
303

303

Figure E38. MALDI (-) trace of final compound 27

O
N N
N N
N
OCH3
NO2
H
N O
HN
O
N
NH
N
O
H
N
O
NH
O
N
O
NH
O
NH2
O
H
N
O
N
O
NH
HN O
O HO O
COOH
N N
N
OH
N
+
P
P
OH
O HO
OH
O
OH
HO
27

Abstract (if available)

Abstract Jakeman et al. recently reported the inability to distinguish the diastereomers of uridine 5’‐β,γ‐fluoromethylenetriphosphate (β,γ‐CHF‐UTP) by ¹⁹F NMR under conditions we previously prescribed for the resolution of the corresponding β,γ‐CHF‐dGTP spectra, stating further that the β,γ‐CHF‐UTP decomposed under these basic conditions. Here we show that the ¹⁹F NMR spectra of β,γ‐CHF‐UTP (~1:1 diastereomer mixture prepared by coupling of UMP‐morpholidate with fluoromethylenebis(phosphonic acid) in D₂O at pH 10 are indeed readily distinguishable. β,γ‐CHF‐UTP in this solution was stable for 24 h at rt. ❧ The first preparation of the individual β,γ‐CHF‐ATP stereoisomers 12a and 12b is reported. Configurationally differing solely by the orientation of the C–F fluorine, 12a and 12b have discrete ³¹P (202 MHz, pH 10.9, ΔδPα 6 Hz, ΔδPβ 4 Hz) and ¹⁹F NMR (470 MHz, pH 9.8, ΔδF 25 Hz) spectral signatures and exhibit a 6‐fold difference in IC₅₀ values for c‐Src kinase, attributed to a unique interaction of the (S)‐fluorine of bound 12b with R388 in the active site. ❧ RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous “synthetic nucleic acid substitution” strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences P‐O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ‐bridging atom‐“substituted” NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and noncognate nucleotide incorporation. Intriguingly, β,γ‐CH₂ substitution in ATP causes a ~130‐fold decrease in kpol for AMP incorporation opposite dT DNA template

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DNA polymerase mechanism and fidelity: using β,γ-bridging oxygen substituted dNTPs to study the mechanism of DNA polymerase β

Asset Metadata

Creator Hwang, Candy Sonhe (author)

Core Title Fluorinated probes of enzyme mechanisms

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 06/20/2016

Defense Date 05/23/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag diastereomers,fluorine,nitrogenase,NMR,nucleotide triphosphates,OAI-PMH Harvest,osteonecrosis of the jaw,RNA polymerase II,Src kinase,stereoprobes

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Chen, Xiaojiang (committee member), Prakash, G. K. Surya (committee member)

Creator Email candyhwa@usc.edu,candyyy@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-255448

Unique identifier UC11281169

Identifier etd-HwangCandy-4453.pdf (filename),usctheses-c40-255448 (legacy record id)

Legacy Identifier etd-HwangCandy-4453.pdf

Dmrecord 255448

Document Type Dissertation

Format application/pdf (imt)

Rights Hwang, Candy Sonhe

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA