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Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function

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Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function

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Content DESIGN AND SYNTHESIS OF A SERIES OF METHYLENEBISPHOSPHONATES:
A NUCLEOTIDE ANALOGUE TOOLKIT TO PROBE NUCLEIC ACID
POLYMERASE STRUCTURE AND FUNCTION
by
Thomas George Upton
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2008
Copyright 2008 Thomas George Upton

ii
DEDICATION

The work presented here is dedicated to those individuals who supported me in
everything that I strive to be. It is because of those people that I continue to do what I do.
I also dedicate this work to those important people that so influenced my education and
growth in becoming the scientist that I am today.

In Loving Memory of:
George W. Lewis
Timothy Michael Upton

iii
ACKNOWLEDGMENTS

First, I would like to thank God for blessing me with the opportunities I am
presented with everyday as well as the blessing to be such an artistic and creative person -
it is a necessary trait to continue to do what I do. Second, I would like to thank my one
true love, Goldi. You have been the constant support that I have needed in these tough
times. I’m not sure where I would be today if we hadn’t met ... I don’t think it would be
anything close to what we have accomplished together. I love you so much and just know
that I do all things with you on my mind. Yes, I do love working in the lab but I love you
more and don’t you forget it.
I would like to thank Professor Charles McKenna for giving me the opportunity to
do research with the McKenna group. It has been an essential part of my academic
endeavors. Your advice is always welcomed and appreciated. Especially when times
were tough, your kindness and understanding has been so important and essential to my
sanity and keeping my eye on the prize.
I would like to sincerely thank my undergrad advisor, Bruce Gibb and his wife
Corinne. The Gibb group was definitely the driving force behind me choosing to continue
my education into graduate school to pursue my Ph.D. It was a fun environment to learn
and work in ... thank you for that, Bruce and Corinne.
My grandfather initially exposed me to the wonderful world of chemistry. As a
chemical engineer, he developed several cleaning solvents and household products that
were simply amazing to me. His career took him all over the world and I wanted that life.

iv
He is by far the biggest influence in shaping me into the scientist that I am today. I would
also like to thank my parents, Patric and Debbie for their constant love and support in the
many twists and turns in my journey through this wondrous life. I want to thank the
Gutierrez family for being such an inspiration to me. True “Americans” in every since of
the word. To my brother, Phil, I love you man. I would also like to extend sincere
gratitude towards my grandmother, Lois - thank you so much for the great advice and her
unique perspective on the happenings of this world. Her faith and perseverance is a
testimony to the way one should live his life.
I would especially like to thank my extended family in the University of Southern
California Chemistry Department for taking a chance on me and allowing me to continue
my education here en route to my Ph.D. Dr. Wittig, thank you so much for your
generosity and helpful words of guidance and providing a place for Goldi, the babies, and
I to live in after our bold move to the West Coast. Dr. Prakash, your advice over the past
years has been greatly appreciated. Thank you for always having your door open (your
lab as well as your office). To the Petasis lab, thanks for always having you door open for
a simple question or when I need that special chemical or device to get something to
work. Michele and Heather, thank you guys so much for always being there when I had a
strange request or just needed some guidance. Dr. Reisler, thank you so much for being
that understanding voice of reassurance that we all need in graduate school. Dr. Haw,
thank you for being an understanding and genuinely kind person.
I would especially like to thank the McKenna Lab (past and present) for all of the
helpful words of advice and encouragement throughout my time in the lab as well as

v
dealing with assisting with my duties as group meeting coordinator. I know group
meeting wasn’t everybody’s favorite but it was an essential part to making our group a
better one. And to Boris, I appreciate every bit of knowledge you have instilled in me
over the course of my time with the McKenna Group. Thank you for the giving me the
opportunity to experience chemistry at a completely new level than what I was exposed
to. I would especially like to thank Greg; thank you so much for all of the great advice
you have given me over the past four and a half years. Ulrika, your wonderful outlook on
research has been very inspiring and thanks for being there for me when you were an
acting member of the group. James, thanks for all of those times when I needed to be
reminded of things to get my mind back on track or when I just need a break from all of
that stuff - thanks for being a real friend to me. Mong, thanks for the kind words of
advice and your NMR expertise when I just needed some help assigning peaks. Larryn
and Joy, China wouldn’t have been the same without you both. Thanks for the good times
especially the last year or so in OCW 205. To those that I have yet to mention, thanks for
being the support group I’ve needed to get to where I’m going in my life. Each person
has touched me in some way.
Lastly, I would like to thank my Ph.D. dissertation committee for giving me the
opportunity to continue my education here at USC. Dr. Goodman, thank you for serving
on my dissertation committee as well as allowing me the opportunity to participate in our
ongoing collaboration. Working with you along with your group members has really been
a wonderful and exciting experience. Thank you for all the words of advice and the
mentorship you have provided to me in my time here at USC. Dr. Haworth, I really
enjoyed being allowed to participate in iPIDD. It gave me the opportunity to be exposed

vi
to many facets of pharmaceutical sciences that I have been yearning to learn about.
Thank you for your constant guidance. Dr. Qin and Dr. Wang, thank you both for the
many words of advice that you have to offer to a young scientist like myself. Your
kindness has certainly been helpful in the development of the way I approach a problem.
Dr. Qin, thanks so much for opening up your lab and equipment to my colleagues in
addition to myself. This kind gesture has been detrimental to the convenient and efficient
manner in which I have performed.
In closing, I would just like to thank everyone for the opportunity to get to know
each and every one of you. I’m a better person and more importantly, a better scientist for
it.

vii
TABLE OF CONTENTS

Dedication ......................................................................................................................... ii
Acknowledgments ............................................................................................................ iii
List of Tables .................................................................................................................... x
List of Figures .................................................................................................................. xii
List of Schemes ............................................................................................................... xvi
Abstract .......................................................................................................................... xvii
Chapter 1 - Synthesis of Nucleoside Triphosphate Derivatives of HIV I Reverse
Transcriptase Inhibitors …........................................................................ 1
Introduction 1
Synthesis of Carbovir Triphosphate 4
Synthesis of Tenofovir Diphosphate 8
Synthesis of Lamivudine Triphosphate 7
Conclusion 9
Experimental Section 9
Synthesis of Lamivudine Triphosphate 7
Chapter 1 References 14

Chapter 2 - Synthesis of a Series of Methylenebisphosphonic Acids ........................... 15
Introduction 15
Materials and Methods 16
Results and Discussion 18
Experimental Section 20
Chapter 2 References 33

Chapter 3 - Determination of pK
a
s of a Series of Methylenebisphosphonic Acids by
Potentiometric Titration ........................................................................... 34
Introduction 35
Materials and Methods 37
Results and Discussion 39
Experimental Section 41
Chapter 3 References 42

Chapter 4 - Synthesis of , -dNTPs as Inhibitors of DNA Polymerases ........................ 43
Introduction 43
Materials and Methods 47
Synthesis of , -dNTPs 47

viii
Purification of , -dNTPs 50
Results and Discussion 51
Inhibition Assays 52
Conclusion 56
Experimental Section 57
Chapter 4 References 68

Chapter 5 - Synthesis of , -Methylenebisphosphonate Nucleoside Triphosphate
Analogs as Probes For Kinetics and Fidelity of DNA Polyermase ...... 71
Introduction 71
Results and Discussion 78
Experimental Section 80
Chapter 3 References 90

Chapter 6 - , -Methylenebisphosphonate Nucleoside Triphosphate Analogs as
Probes for Kinetics and Fidelity of DNA Polymerase ..........................93
Introduction 93
Materials and Methods 97
Synthesis of , -Methylene-dGTP Analogues 97
DNA Synthesis/Purification, Radiolabeling, and Annealing 97
Buffer Preparation 98
Single-Turnover Gap-Filling Assays 98
Data Analysis 100
Results and Discussion 100
Conclusion 116
Chapter 6 References 118

Chapter 7 - , -Methylenebisphosphonate NTP Analogs - DNA in Ternary Complex
with DNA Polymerase : Evidence of Stereoselective Binding Due to
an Interesting C-F···H-N Interaction with ARG183 .............................. 121
Introduction 121
Materials and Methods 123
Results and Discussion 139
Conclusion 131
Experimental Section 132
Chapter 7 References 136

Chapter 8 - Synthesis and Characterization of CF
3
-BPs ............................................... 140
Introduction 140
Materials and Methods 142
Electrophilic Trifluoromethylation 142
Synthesis of CF
3
-Etidronate 143

ix
Results and Discussion 144
Conclusion 146
Experimental Section 148
Chapter 8 References 153

Chapter 9 - Synthesis, Analysis, and Purification of Other NTPs ................................. 145
Introduction 155
Synthesis of Known and Novel Nucleoside Triphosphate Analogues 155
Analysis and Purification of New NTP Analogues 157
Results and Discussion 165
Conclusion 167
Chapter 8 References 175

Bibliography .............................................................................................................. 177
Appendix A .................................................................................................................191
Appendix B .................................................................................................................194
Appendix C ................................................................................................................ 213
Appendix D ................................................................................................................ 257
Appendix E ................................................................................................................ 272
Appendix F .............................................................................................................. 313
Appendix G .............................................................................................................. 333
Appendix H .............................................................................................................. 339

x
LIST OF TABLES

Table 3.1 List of pK
a
s reported in literature ............................................................ 36

Table 3.2 Optimization of our methods with MBP as compared to Kabachnik
et al ..........................................................................................................38

Table 3.3 Stability constants for a series of BPs ...................................................... 40

Table 4.1 Crystallographic statistics ........................................................................ 67

Table 5.1 Summary of known , -methylene-NTP analogs ................................... 74

Table 5.2 Summary of synthesized , -dNTP analogs ............................................ 78

Table 6.1 Kinetic and thermodynamic parameters for incorporation of , -
bridging dGTP analogs in pol -catalyzed single-turnover assays .......... 99

Table 7.1 Crystallographic Statistics for 3 ............................................................. 135

Table 8.1 Summary of attempts at electrophilic trfluoromethylation using
Umemoto’s reagent ................................................................................ 145

Table A.1 Analytical HPLC of Carbovir derivatives …………………………... 192

Table A.2 Analytical HPLC of Tenofovir derivatives ………………………...… 193

Table C.1 Initial analysis of MBP for calibration of electrode............................... 213

Table D.1 HPLC summary of , -CF
2
-dNTP analogues ....................................... 257

Table E.1 HPLC summary of , -methylenebisphosphonate dNTP analogues … 272

Table F.1 Docking Results for 2 ………………................................................. 313

Table F.2 RMSD Table for 2 ………………...................................................... 313

Table F.3 Docking Results for 3 ………………................................................. 316

Table F.4 RMSD Table for 3 ………………...................................................... 318

Table F.5 Docking Results for 4 ………………................................................. 321

xi
Table F.6 RMSD Table for 4 ………………...................................................... 322

Table F.7 Docking Results for 1 ………………................................................. 325

Table F.8 RMSD Table for 1 ………………...................................................... 326

Table F.9 Docking Results for dGTP ……………….......................................... 328

Table F.10 RMSD Table for dGTP ………………............................................... 329

xii
LIST OF FIGURES

Figure 1.1 List of selected reverse transcriptase inhibitors and their active
metabolites ……………………………………………………………..... 2

Figure 1.2 Monitoring by HPLC (Carbovir) ............................................................... 5

Figure 1.3 Monitoring by HPLC (Tenofovir) ............................................................. 8

Figure 2.1 Methylenebisphosphonates and , -dNTP analogs ................................. 15

Figure 2.2 A series of MBPs .................................................................................... 17

Figure 3.1 Calculation of pK
a
s from log s .............................................................. 38

Figure 3.2 Sample of Hyperquad2006 analysis ........................................................ 40

Figure 4.1 HPLC analysis of the conversion of , -CF
2
-dCDP to , -CF
2
-dCTP ... 49

Figure 4.2 Gapped DNA insertion assay with DNA pol for dNTPs (dATP, dTTP)
and , -CXY analogs ............................................................................... 51

Figure 4.3 Analog Binding Affinity .......................................................................... 53

Figure 4.4 Superposition of the active site of the ternary complex of DNA
Polymerase with incoming , -CF
2
-dTTP and the ternary complex with
, -NH-dUTP bound ............................................................................... 54

Figure 4.5 X-ray crystallographic study of , -CF 2-dCTP 6 ……………...……… 55

Figure 5.1 Analytical HPLC ( , -dNTP analogs) ..................................................... 76

Figure 6.1 Schematic diagram of the pol active site and structures of dGTP
analogs ……………................................................................................. 96

Figure 6.2 Brønsted correlations of log(k
pol
) versus leaving-group pK
a4
................ 102

Figure 6.3 Single-turnover assay of CHBr-analog .................................................. 104

Figure 6.4 Effect of O

modification on fidelity .................................................... 110

Figure 6.5 An illustration of possible interactions between bulky halogen atoms
at the , -bridging position and other elements of the active site .......... 113

Figure 7.1 , -methylene analogs of dGTP ............................................................. 122

xiii
Figure 7.2 Comparison of DNA pol active site complex structures for dGTP and
analogs obtained from crystallographic data and computational docking
studies .................................................................................................... 125

Figure 7.3 Comparison of DNA pol active site binding and docking calculations
of the dGTP analogs 3 and 4 ................................................................ 126

Figure 7.4 X-ray crystal structures solved for DNA pol :DNA complex soaked with
, -dGTPs .............................................................................................. 127

Figure 7.5 X-ray crystallographic structures ........................................................... 128

Figure 7.6 X-ray crystallographic structures ........................................................... 129

Figure 7.7 X-ray crystallographic structures ........................................................... 130

Figure 8.1 Proposed target BPs ............................................................................... 141

Figure 9.1 HPLC analysis of the , -CF
3
-Etidronate-dGTP reaction mixture ........157

Figure 9.2 Analytical HPLC analysis of , -CF
2
-dTTP ......................................... 158

Figure 9.3 HPLC analysis (on SAX) - -CF
2
H-dTMP .......................................... 160

Figure 9.4 HPLC analysis (on SAX) ....................................................................... 162

Figure 9.5 HPLC analysis (on SAX) of the CF
2
H-dTTP reaction mixture 5 hours
after the addition of more ADK and ATP .............................................. 163

Figure 9.6 , , , -bis-(CF
2
)-dNTPs ........................................................................ 162

Figure 9.7 HPLC analysis (on SAX) - , , , -bis-(CF
2
)-dNTPs ............................ 164

Figure 9.8 HPLC analysis (on SAX) ....................................................................... 164

Figure C.1 Titration curve for MBP to calibrate electrode ..................................... 215

Figure C.2 Titration curve for titration of MBP with NaOH ................................. 216

Figure C.3 Titration curve for titration of MBP with KOH .................................. 220

Figure C.4 Titration curve for titration of MFBP with KOH ................................ 224

Figure C.5 Titration curve for titration of DFBP with KOH ................................. 228

xiv
Figure C.6 Titration curve for titration of DCBP with KOH ................................. 231

Figure C.7 Titration curve for titration of DBBP with KOH ................................. 234

Figure C.8 Titration curve for titration of FClBP with KOH ................................ 237

Figure C.9 Titration curve for titration of MBBP with KOH ................................ 240

Figure C.10 Titration curve for titration of MCBP with KOH ................................ 243

Figure C.11 Titration curve for titration of EBP with KOH .................................... 246

Figure C.12 Titration curve for titration of DMBP with KOH ............................... 250

Figure C.13 Titration curve for titration of FMBP with KOH ................................ 254

Figure D.1 HPLC analysis (on SAX) of conversion of 1 to 2 ................................. 257

Figure D.2 HPLC analysis (on SAX) of conversion of 2 to 3 ................................. 258

Figure D.3 HPLC analysis (SAX) of 3 .................................................................... 259

Figure D.4 HPLC analysis (on SAX) of conversion of 4 to 5 ................................. 259

Figure D.5 HPLC analysis (on SAX) of conversion of 5 to 8 ................................. 260

Figure D.6 HPLC analysis (on SAX) of conversion of 8 to 6 ................................. 261

Figure D.7 HPLC analysis (SAX) of 6 .................................................................... 261

Figure D.8
19
F NMR analysis of the 3 .................................................................... 269

Figure E.1 HPLC analysis (on SAX) of conversion of 1 to 4 ................................. 273

Figure E.2 HPLC analysis (SAX) of 4 .................................................................... 273

Figure E.3 HPLC analysis (on SAX) of conversion of 1 to 3 ................................. 274

Figure E.4 HPLC analysis (SAX) of 3 .................................................................... 274

Figure E.5 HPLC analysis (on SAX) of conversion of 1 to 5/6 ............................. 275

Figure E.6 HPLC analysis (SAX) of 5/6 ................................................................ 275

xv
Figure E.7 HPLC analysis (on SAX) of conversion of 1 to 9 and 10/11 ................ 276

Figure E.8 HPLC analysis (SAX) of 9 and 10/11 .................................................. 276

Figure E.9 HPLC analysis (on SAX) of conversion of 1 to 12/13 ......................... 277

Figure E.10 HPLC analysis (SAX) of 12/13 ............................................................ 277

Figure E.11 HPLC analysis (on SAX) of conversion of 1 to 14 ............................... 278

Figure E.12 HPLC analysis (SAX) of 14 .................................................................. 278

Figure E.13 HPLC analysis (on SAX) of conversion of 1 to 15/16 ......................... 279

Figure E.14 HPLC analysis (SAX) of 15/16 ............................................................ 279

Figure E.15 HPLC analysis (on SAX) of conversion of 2 to 8 ................................. 280

Figure E.16 HPLC analysis (SAX) of 8 .................................................................... 280

Figure E.17 HPLC analysis (SAX) of 7 .................................................................... 281

Figure E.18 HPLC analysis (on SAX) of conversion of 1 to 21 ............................... 281

Figure E.19 HPLC analysis (on SAX) of conversion of 1 to 17/18 ......................... 282

Figure H.1 Prep. HPLC (SAX) trace of AMP-DFBP reaction mixture ................ 348

Figure H.2 Analytical HPLC (SAX) of AMP-DFBP ............................................ 348

Figure H.3 Prep. HPLC (SAX) trace of bis-CF
2
-dTTP reaction mixture ............. 349

Figure H.4 Prep. HPLC (C18) trace of bis-CF
2
-dTTP mixture ............................. 349

Figure H.5 Analytical HPLC (SAX) trace of , -Etidronate-GMP and
, -Etidronate-dGMP ......................................................................... 349

Figure H.6 HPLC analysis (SAX) of , -Risedronate-dGMP ............................. 350

Figure H.7 Analytical HPLC (SAX) trace of , , , -bis-CF
2
-dTTP and , , , -bis-
CF 2-dATP ........................................................................................... 350

Figure H.8 Analytical HPLC (SAX) of dGMP-CF
3
Etidronate reaction mixture . 353

Figure H.9 Analytical HPLC (SAX) of dGMP-CF
3
Etidronate ............................ 353

xvi
LIST OF SCHEMES
Scheme 1.1 Synthesis of Carbovir Triphosphate ...........................................................4

Scheme 1.2 Synthesis of Tenofovir Diphosphate ..........................................................7

Scheme 1.3 Proposed Synthesis of Lamividine Triphosphate ...................................... 8

Scheme 2.1 Synthesis of various substituted BPs ....................................................... 18

Scheme 2.2 Alternative synthesis of EBP ................................................................... 19

Scheme 4.1 Synthesis of , -methylene dNTP analogs .............................................. 48

Scheme 5.1 Synthesis of , -methylene bisphosphonate dNTP analogs ..................... 75

Scheme 8.1 Synthesis of CF
3
-BPs by electrophilic trifluoromethylation using
Umemoto’s reagent ............................................................................ 142

Scheme 8.2 Synthesis of CF
3
-etidronate from trifluoroacetic acid ........................... 143

Scheme 8.3 Synthesis of CF
3
-etidronate from trifluoroacetic anhydride .................. 144

Scheme 9.1 Synthesis of , -NTP analogs ................................................................ 156

Scheme 9.2 Proposed synthesis of -CF
2
H-dTMP to -CF
2
H-dTTP using ADK, PEP,
PK, ATP
cat
.............................................................................................. 159

xvii
ABSTRACT
A stereoelectronically varied series of -substituted methylenebisphosphonic acids (X,Y
= H, F, Cl, Br, CH
3
) was synthesized and used to prepare corresponding dNTP , -CXY
and also certain , -analogues. Improved analytical and preparative HPLC methods are
described for the NTP analogues. The pK
a4
values of the bisphosphonic acids were
determined under self-consistent conditions, enabling a “toolkit” of pyrophosphate
analogues for kinetic and structural studies of DNA polymerase mechanisms and fidelity.
The dNTP analogues are substrates for DNA pol and the k
pol
values of the , -CXY
analogues were compared to the pK
a4
of the relevant bisphosphonate (BP) leaving group.
For BPs with a pK
a4
less than or equal to that of pyrophosphoric acid, log k
pol
values
were similar or decreased moderately with increasing pK
a4
. For BPs with a pK
a4
greater
than that of pyrophosphate a significant trend of decreasing log k
pol
was apparent. This
observation, and the absence of an analogous effect on ground state analog binding (K
d

values, except for the CBr
2
analogue), points to an influence of the leaving group aptitude
on the energy of the transition state. Reduced catalysis rates were observed with the
dihalo-substituted substrates particularly for the T-G mispair incorporation. X-ray
crystallographic studies with several of P
-Z-P
(Z = NH, CH
2
, CF
2
) and P
-CXY-P

dNTP analogues in ternary complex with DNA pol and DNA primer-template strand
have been carried out. Evidence consistent with a docking study was obtained for a C-
F···H-N-Arg interaction in the , -CHF-dGTP analogue – active site complex, in which
only the (R)-diastereomer of the analogue is observed. The synthesis of new

xviii
bisphosphonates incorporating a CF
3
-group was also investigated. The dNTP analogue
“toolkit” should prove useful to probe structure and function in other DNA polymerases
and to refine theoretical studies of the enzymes mechanisms.
CHAPTER 1
SYNTHESIS OF NUCLEOSIDE TRIPHOSPHATE DERIV ATIVES OF
HIV I REVERSE TRANSCRIPTASE INHIBITORS
INTRODUCTION
With the increasingly pandemic nature of the HIV virus, there has been extensive
research to try and slow down or stop the progress of the virus. The inhibition of three
different enzymes, important for HIV-1 to replicate itself, can provide the coveted results
researchers seek; inhibition of integrase, protease, and reverse transcriptase.
1
The synthe-
sis of some of the active compounds that are known to inhibit HIV-1 reverse transcriptase
is discussed herein. These triphosphate derivatives are the active metabolites of the com-
mercially available prodrugs: carbovir
2
CBV , lamivudine
3
3TC, and tenofovir
4,5
TNF -
see FIGURE 1.1. The synthesis of these triphosphates (CBV-TP, 3TC-TP, TNF-DP) has
not been thoroughly covered in the literature and only a small amount of spectroscopic
data exists for these compounds. They are, however, known to exist as the active inhibi-
tory metabolites of the aforementioned drugs.
1-4
There are several procedures for the synthesis of triphosphates of nucleosides (and
their analogues) but few with a yield higher than 20%;
2,5,6
however, synthesis through the
morpholidate intermediate
7
provides sufficient product in good yields. If performed under
close observation, using a RP-HPLC and ion-exchange HPLC, a more favorable yield of
1
Figure 1.1 List of selected reverse transcriptase inhibitors and their active metabolites
triphosphates can be achieved. The unique feature of the ion-exchange HPLC analysis
method employed is that we can discern morpholidates from monophosphates, diphos-
phate analogues, and triphosphate analogues as well as observe tetraphosphates (impuri-
ties or side-products from different synthetic approaches). The general method of analysis
for these reactions is by
31
P NMR.
2,5,7
This analytical method [by NMR] is suitable for
large-scale reactions because the product concentrations are well above the sensitivity
levels for
31
P nuclei, but in our case, for small-scale reactions, longer acquisition times or
larger concentrations are generally required for appropriate NMR analysis. As witnessed
throughout the course of these syntheses, this presents an undesired opportunity for de-
creased yields and unpractical analysis for this reaction. This HPLC method is relatively
2
quick (≤ 15 min.) and very sensitive (only requiring 2-3 µL of reaction mixture to suc-
cessfully determine product concentrations).
The conversion of monophosphate to morpholidate is usually monitored by
31
P
NMR but can also be monitored by RP-HPLC using 0.1 N triethylammonium bicarbonate
(TEAB) buffer containing 4% CH3CN. The monophosphate peak elutes first with a reten-
tion time of around 3 min. and the morpholidate elutes second with a retention time
around 5 min. The reaction of morpholidate to the triphosphate analogues is monitored by
a separate method using ion-exchange HPLC on a PUREGEL SAX (strong anion ex-
change) column. This method separates the analytes by charge and hydrophilicity and
requires a gradient of 0-100% 0.5 M TEAB buffer. The morpholidate elutes first (~4
min.) as it is the most hydrophilic compound in the reaction mixture, followed by mono-
phosphate (~7 min), then diphosphate (~9 min) and triphosphate (~15 min.) and finally if
any tetraphosphate is present, it elutes at (~19 min.).
The triphosphate analogues are isolated from the product mixture by ion-
exchange chromatography. This is generally accomplished on DEAE Sephadex.
2,7,8
How-
ever, use of these resins requires tedious swelling and column packing procedures as well
as lengthy purification trials. Here, we adapted our useful analytical HPLC method to a
semipreparative scale using two of the PUREGEL SAX columns in tandem. The peak
corresponding to the triphosphate analogue [retention time (RT) = 15 - 30 min, depending
on the nucleobase] was isolated as the triethylammonium salt via preparative anion-
exchange HPLC and lyophilized.
3
SYNTHESIS OF CARBOVIR TRIPHOSPHATE
Scheme 1.1 a) POCl3, OP(OCH3)3, r.t.; b) morpholine, DCC, 50% t-BuOH/H2O; c) (bis)tributylam-
monium pyrophosphate, DMSO
Following SCHEME 1.1, Carbovir monophosphate (CBV-MP) 2, can be synthe-
sized from Carbovir (CBV) 1 by phosphorylation POCl3 in trimethylphosphate.
2
CBV-
MP was achieved with a yield greater than 90% as the triethylammonium (TEA) salt after
purification with reverse phase HPLC on C-18 column. Carbovir morpholidate (CBV-
MORPH) 3, can then be obtained by coupling 2 with morpholine using DCC. The yield,
by RP-HPLC, was ~90% and the product is purified by washing with H2O and then di-
ethylether and dried under reduced pressure. Then, 3 is dissolved in dry DMSO and then
reacted with tributylammonium pyrophosphate to give Carbovir triphosphate (CBV-TP) 4
in a yield of ~50%. CBV-TP is then purified using ion-exchange HPLC using a PURE-
GEL SAX column and is obtained as the TEA salt. It is suspected that the increase in
overall yield is due to the close monitoring of these reactions using HPLC (see FIGURE
1.2 a & b).
4
Figure 1.2 Monitoring by HPLC: a) comparison of CBV 1 to a nucleoside monophosphate [GMP]
on C18; b) CBV-MP 2 reaction mixture after 5 hrs on C18; c) comparison of 2 with CBV-Morph 3 on C18;
d) analysis of purified 3 on C18; e) analysis of CBV-TP 4 reaction mixture after 2 days.
a)
b)
c)
d)
e)
5
Figure 1.2 (continued) Monitoring by HPLC: f) CBV-TP reaction mixture analyzed on PUREGEL
(SAX); g) 4 on PUREGEL (SAX) after semi-prep. separation on SAX; h) LC-MS of TEA salt of 4 [nega-
tive scan mode]
CBV-MP
g)
f)
CBV-DP
CBV-TP
h)*
*LC-MS spectra courtesy of Luc Bi from Stan Louie’s Lab at USC School of Pharmacy
6
SYNTHESIS OF TENOFOVIR DIPHOSPHATE
Scheme 1.2 Conversion of Tenofovir Diisoproxil Fumarate 5 to Tenofovir 6 and then to Tenofovir
Diphosphate 8
The same morpholidate methodology that was used to synthesize CBV-TP is fol-
lowed in this scheme. Following SCHEME 2, Tenofovir (TNF) 6 can be obtained by
simply deprotecting the ester Tenofovir Diisoproxil Fumarate (TNF-DF) 5, under acidic
conditions.
8
6 is then purified by washing with ether and can then be converted to the
Tenofovir-morpholidate (TNF-Morph) 7, and then upon coupling with tributylammonium
pyrophosphate, Tenofovir diphosphate (TNF-DP) 8 can be obtained. Again, HPLC has
proven to be an effective analytical tool. Following the reaction using RP-HPLC (see
FIGURE 1.2), one can determine how much TNF, TNF-Morph, TNF-MP, and TNF-DP
is in the reaction mixture. The work-up was the same as with CBV-TP.
7
Figure 1.3 Monitoring by HPLC: a) on C-18: bottom (light blue) = TNF-DF to TNF after 1 hr,
middle (green) = TNF-DF to TNF after 48 hrs, top (red) = TNF to TNF-Morph after 6 hrs; b) on PURE-
GEL: bottom (beige) = reaction mixture after 60 hrs, top (blue) = after purification and freeze-drying.
a) b)
SYNTHESIS OF LAMIVUDINE TRIPHOSPHATE
Scheme 1.3 Proposed conversion of Lamivudine (3TC) 9 to Lamivudine Triphosphate 12
8
Attempts at the synthesis of Lamivudine triphosphate (3TC-TP) 12 using the
morpholidate method (see SCHEME 3) were unsuccessful due to the inability to obtain
3TC-MP 10. Lamivudine (3TC) 9 was extracted from the tablet using MeOH. However,
attempts at phosphorylation of 9 with POCl3 proved unsuccessful presumably due to the
highly acidic conditions causing decomposition of the thio-sugar ring.
CONCLUSION
In summary, a synthesis of nucleoside triphosphate analogues has been proposed
and explored with the synthesis of CBV-MP, TNF-DP, and 3TC-TP. All yields for each
are acceptable and in the case of the triphosphates (4 and 8), an improvement in the over-
all yield has been achieved with the aid of careful monitoring by HPLC. These triphos-
phates are known to exist yet there has not been any literature that pertains to the synthe-
sis of these compounds. They are, however, suspected to be the active metabolites of the
drugs carbovir, tenofovir, and lamivudine. Bioassays, performed by our collaborator Dr.
Stan Louie, have shown that our synthesized standards 4 and 8 are in fact the active de-
rivatives in inhibiting HIV-1 reverse transcriptase and the same is expected for 12. (un-
published results)
EXPERIMENTAL SECTION
The HIV-1 nucleotide prodrugs were kindly provided as listed: Carbovir - C. L. Zim-
mermann, University of Minnesota; Tenofovir and 3TC - Stan Louie, University of
Southern California, School of Pharmacy. All other reagents were purchased from Sigma
9
Aldrich. HPLC analytical and preparative separations were carried out using a Varian
ProStar 210 pump and injector system equipped with a Shimadzu SPD-10A VP UV Vis
detector, on a) a Varian C-18 (ODS) Microsorb-MV 4.6 mm x 25 cm, 5 µm analytical
column; b) a Dynamax C-18 21.4 mm x 25 cm, 5 µm preparative column; c) a Varian
PureGel SAX 10 mm x 10 cm, 7 µm analytical column; and a Macherey-Nagel Nucleo-
gel SAX 1000-10 25 mm x 15 cm preparative column. C-18 columns were eluted iso-
cratically with 0.1 M TEAB containing 2% acetonitrile. SAX column elution conditions
are given below. NMR spectra were recorded on Bruker AM-360 or AC-250 instruments.
Detailed experimental data such as NMR and MS data are presented in APPENDIX A.
Synthesis of Carbovir monophosphate (2)
50 mg (0.201 mmol) of Carbovir is weighed out in a dried 25 mL round bottom flask. In
a separate dried 10 mL flask, 112 µL (1.206 mmol) of POCl3 is dissolved in 2 mL of ice-
chilled trimethylphosphate. The POCl3/trimethylphosphate mixture is added to Carbovir
dropwise and the mixture is set to react at 0
o
C. After 5 hours, the mixture is poured into
ice-chilled H2O (10 mL). The pH was adjusted to 2.5 with 2 M NaOH and washed with
CHCl3 (3 x 10 mL). Carbovir monophosphate was purified on C-18 and isolated as the
triethylammonium salt. Analytical HPLC (C18): RT = 3 min, (SAX): 4 min;
1
H (CD3OD:
7.61 (s), 6.10 (m), 5.80 (m), 5.43 (m), 3.51 (t), 2.91 (m), 2.64 (dt), 1.60 (dt);
31
P: 3.75 (s).
10
Synthesis of Carbovir morpholidate (3)
In a dried 5 mL flask, 15 mg (0.046 mmol) of 2 is weighed out and dissolved in 1.5 mL
of 50% tBuOH:H2O and the pH is adjusted to 2. 12.4 µL (0.143 mmol) of distilled mor-
pholine is added dropwise and set to reflux. When at reflux, 30 mg (0.145 mmol) of DCC
in 0.5 mL of tBuOH is added dropwise over 2 hrs and the reaction is monitored by HPLC
(C-18). Once the reaction has reached completion, the solvent is removed by vacuum and
the resulting product mixture is washed with diethyl ether. The milky white crude product
mixture is dissolved in a small amount of water and filtered to remove the dicyclohexy-
lurea. The water is removed by reduced pressure and the resulting yellowish foam is dried
under vacuum yielding 3 (53 mg, 93% yield). Analytical HPLC (C18): RT = 5 min,
(SAX): 3.5 min.
Synthesis of Carbovir triphosphosphate (4)
The dried morpholidate 3 (0.022 mmol) is dissolved in 0.5 mL of dried and distilled
DMSO. In a separate flask, 52 mg (0.095 mmol) of tributylammonium pyrophosphate is
dissolved in 0.5 mL of dried and distilled DMSO. The morpholidate solution is added to
the pyrophosphate solution under N2 and the reaction is set at room temperature and
monitored by HPLC (SAX). After two days, reaction has reached completion. The sol-
vent is removed by reduced pressure and the resulting product mixture is purified by
semi-preparative HPLC (SAX). Carbovir triphosphate 4 (8 mg, 74% yield) is isolated as
the triethylammonium salt. Analytical HPLC (SAX): RT = 11.7 min; LC-MS (m/z) = 486
[M-H
-
].
11
Conversion of Tenofovir diisoproxil fumarate (5) to Tenofovir (6)
25.6 mg (0.39 mmol) of 5 is dissolved in 200 µL of MeOH. 2 mL of 5M HCl is added
and is set to react at 45
o
C - 60
o
C for 48 hrs. Solvent is removed under reduced pressure
and then washed with ether and dried under reduced pressure giving a white foam 6 (10.7
mg, 90% yield). Analytical HPLC (C18): RT = 3 min, (SAX): 4 min;
1
H (D2O): 8.40 (s),
8.38 (s), 6.81 (s), 4.50 (m), 4.30 (m), 3.75 (m), 1.2 (d);
31
P: 12.5 (s).
Synthesis of Tenofovir morpholidate (7)
60 mg (0.199 mmol) of 6 is dissolved in 5 mL of 50% tBuOH:H2O. Reaction is set to
reflux and then 52 µL (0.657 mmol) of morpholine is added dropwise. 123 mg (0.696
mmol) of DCC in 0.5 mL of tBuOH is added dropwise over 2 hrs. Reaction is monitored
by RP-HPLC (0.1N TEAB 7% CH3CN) and at completion, the solvent is removed by re-
duced pressure and and producted mixture is washed with ether and then dissolved in
H2O and the dicyclohexylurea is removed by filtration. The solvent is removed under re-
duced pressure and the yellowish foam 7 (65.5 mg, 89% yield) is dried under vacuum.
Analytical HPLC (C18): RT = 5 min, (SAX): 3.5 min.
Synthesis of Tenofovir diphosphosphate (8)
The morpholidate 7 (0.075 mmol) is dissolved in 2 mL of dried and distilled DMSO and
in a separate flask dissolve 164 mg (0.299 mmol) of tributylammonium pyrophosphate in
2 mL in dried and distilled DMSO. The morpholidate solution is added to the pyrophos-
phate solution under N2 and the reaction is set at room temperature and monitored by
12
HPLC (SAX). After two days, reaction has reached completion. The solvent is removed
by reduced pressure and the resulting product mixture is purified by preparative HPLC
(SAX). Tenofovir diphosphate 8 (11 mg, 32% yield) is isolated as the triethylammonium
salt. Analytical HPLC (SAX): RT = 11.2 min.
13
CHAPTER 1 REFERENCES
1. Witvrouw, M.; Van Maele, B.; Vercammen, J.; Hantson, A.; Engelborghs, Y .; De
Clercq, E.; Pannecouque, C.; Debyser, Z. Current Drug Metabolism 2004, 5, 291-
304.
2. Exall, A. M.; Jones, M. F.; Mo, C. L.; Myers, P. L.; Paternoster, I. L.; Singh, H.;
Storer, R.; Weingarten, G. G.; Williamson, C.; Brodie, A. C.; Cook, J.; Lake, D. E.;
Meerholz, C. A.; Turnbull, P. J.; Highcock, R. M. Journal of the Chemical Society
Perkin Transactions 1 1991, 10, 2467-2477.
3. Soudeyns, H.; Yao, X. J.; Gao, Q.; Belleau, B.; Kraus, J. L.; Nghe, N. B.; Spira, B.;
Wainberg, M. A. Antimicrob. Agents Chemother. 1991, 35, 1386-1390.
4. Holy, A. Current Pharmaceutical Design 2003, 9, 2567-2592.
5. Otmar, M.; Masojidkova, M.; V otruba, I.; Holy, A. Collect. Czech. Chem. Commun.
2001, 66, 500-506.
6. Fung, E. N.; Cai, Z. W.; Burnette, T. C.; Sinhababu, A. K. Journal of Chromatogra-
phy B 2001, 754, 285-295.
7. Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649-658.
8. Jennings, L. J.; Macchia, M.; Parkin, A. Journal of the Chemical Society Perkin
Transactions 1 1992, 17, 2197-2202.
14
CHAPTER 2
SYNTHESIS OF A SERIES OF METHYLENEBISPHOSPHONIC ACIDS
INTRODUCTION
Pyrophosphoric acid (or diphosphoric acid) is important in several biochemical
reactions. Several researchers have examined its physical and biochemical properties and
over the past few decades several derivatives have been studied as analogues of
pyrophosphate.
1-6
One of the most attractive and well-studied analogues is methylene-
bisphosphonic acid (MBP).
4,7-10
Geometrically, MBP is analogous to pyrophosphate with
the angle between P-C-P being slightly smaller than the analogous P-O-P of
pyrophosphate.
2,6,11
The most important and useful aspect of MBP is that it contains the
non-hydrolyzable P-C-P bond which is certainly important in analogue (and inhibitor)
design. The replacement of -O- with -C- in pyrophosphate not only provides stability
against hydrolysis but also presents the possibility of incorporating various substituents at
the bridging C.
2,3,6,11
Several of these compounds have been proposed and synthesized by
various researchers.
1-4,6,10,12,13

Figure 2.1 Methylenebisphosphonates (R = H) and β,γ-dNTP analogues (R = dNMP)
R O P P
O
HO
OH
O
OH
Y
X
X,Y = Br, Cl, F, H, CH3
In an ongoing study with DNA polymerases and deoxynucleoside triphosphos-
phate (dNTP) analogues,
14
we have employed the versatility or tunable aspect of MBP to
15
create a series of bisphosphonates (BPs) P-CXY-P (X,Y = Br, Cl, F, H, CH3), as well as
the respective β,γ-dNTP analogues, with varying acidity compared to the “natural” pyro-
phosphate (see FIGURE 2.1). In these particular studies, kinetics and fidelity of DNA
polymerase β (Pol β) are being examined for the incorporation of a single dNTP by the
enzyme.
14
Pyrophosphate or BP is the leaving group in the chemistry step of Pol β and
the rate at which the BP leaves can be adjusted by the introduction of various electron
withdrawing groups at the bridging C. This property allows us to examine BPs that are
either higher, lower, or similar in acidity to pyrophosphate. The pKas for some of these
compounds are known
2-4,7-10,15-17
and in some cases, there are several different pKas for
the same compound.
3,4,7-10,15,16,18
This disparaging finding has compelled us to perform a
study to determine the stability constants of this series under consistent conditions which
will be discussed in the next chapter. These two studies (enzyme kinetic analysis and pKa
determination) required the synthesis of the complete series of BPs with varying pKas
(this chapter) and β,γ-substituents (see CHAPTER 5), in large quantities.
MATERIALS AND METHODS
The series of halogenated BPs (see FIGURE 2.2) can be completed following
procedures previously published by our group and others. 1-8 can be synthesized from
TiPMBP.
6
The fluorinated BPs were originally obtained by fluorination using FClO3 or
condensation of perfluoroalkylphosphonates. Unfortunately, FClO3 is no longer commer-
cially available; therefore, a new convenient approach was required to obtain 14-19.
These BPs can be prepared from TiPMBP (or 3 in the case of 18) using the fluorinating
16
agent Selectfluor. Marma et al. recently presented a procedure for the preparation of
fluorinated phosphonocarboxylates.
12
The same procedure can be modified to reflect the
necessary equivalents required to synthesize predominantly 14, 16, or 18. The free acid
forms of BPs can then be obtained by either dealkyllation by BTMS
13
or reflux in HCl or
HBr (in the case of 6 and 8).
6
Figure 2.2 A series of MBPs
9 and 11 can be synthesized by alkylation of TIPMBP with NaH and MeI in
DMF/THF. 20 can be obtained by fluorination of 11 with Selectfluor as with 16 and 18.
The free acids can be prepared as with the previously described BPs; either by dealkyla-
tion in BTMS or hydrolysis by refluxing in HCl.
17
RESULTS AND DISCUSSION
All BPs were synthesized according to SCHEME 2.1. The halogenated BPs 1-8
were obtained with acceptable yields (90-92%). There was a misprint in the original pub-
lication
6
that has a detrimental affect on preparation of 5. The original publication men-
tions the use 20 mL of NaOH; however, these conditions are too concentrated for suc-
cessful synthesis of 5. When the reaction mixture is diluted with 200 mL of NaOH, 5 is
readily obtained in a 92% overall yield. Depending on the amount of starting material,
dealkylation of the isopropyl esters in neat BTMS in most cases was achieved after 2-3
days. In order to increase the rate of the reaction, dried and distilled CH3CN was added
and the reaction can be completed overnight if heated to 45
o
C without a decrease in
yield. This method proved to be acceptable for small quantities of BPs but for larger scale
reactions, refluxing in either concentrated HCl or HBr (in the case of 6 and 8) was a more
convenient option.
Scheme 2.1 Synthesis of various substituted BPs.
a) b)
R = (CH3)2CH
X,Y = H, F, Cl, Br, CH3
P P
O O
RO
RO
OR
OR
P P
O O
RO
RO
OR
OR
X Y
P P
O O
HO
HO
OH
OH
X Y
For 2 (X,Y = Cl): a) NaOCl, 0
o
C (giving 1 - 90%); b) HCl, reflux; 6 (X,Y = Br): a) Br2, NaOH, 0
o
C (giving
5 - 90%); b) HBr, reflux; 4 (X = Cl, Y = H): 1, NaHSO3, EtOH @ 0
o
C (giving 3 - 92%) followed by hy-
drolysis in HCl (reflux); 8 (X = Br, Y = H): 5, SnCl2, EtOH @ 0
o
C (giving 7 - 90%) followed by hydrolysis
in HBr (reflux); 10 (X,Y = CH3): a) NaH, MeI, 50% THF:DMF, 0
o
C (giving 9 - 97%); b) BTMS, CH3CN,
45
o
C or HCl, reflux; 13 (X = CH3, Y = H): a) NaH, MeI, 50% THF:DMF, 0
o
C (giving 11 - 25-50%); b)
BTMS, CH3CN, 45
o
C or HCl, reflux; 15 (X,Y = F): a) NaH, Selectfluor
TM
, 50% THF:DMF, 0
o
C (giving 14
- 96%); b) BTMS, CH3CN, 45
o
C or HCl, reflux; 17 (X = F, Y = H): a) NaH, Selectfluor
TM
, 50%
THF:DMF, 0
o
C (giving 16 - 65%); b) BTMS, CH3CN, 45
o
C or HCl, reflux; 19 (X = F, Y = Cl): a) 1C,
NaH, Selectfluor
TM
, 50% THF:DMF, 0
o
C (giving 18 - 96%); b) BTMS, CH3CN, 45
o
C or HCl, reflux; 22 (X
= F, Y = CH3): a) 11, NaH, Selectfluor
TM
, 50% THF:DMF, 0
o
C (giving 20 - 96%) or 12, NaH, Select-
fluor
TM
, 50% THF:DMF, 0
o
C (giving 21 - 97%), ; b) HCl, reflux.
18
The methyl-substituted methylenebisphosphonates 9 and 11 were prepared by al-
kylation of TIPMBP with NaH and MeI by appropriate adjustment of equivalents of NaH
and MeI; however, in the case of 11, attempts at purification by column chromatography
or extraction proved troublesome (< 24% isolated yield). Therefore, we investigated al-
ternative routes. Phosphonylation of the carbanion
19
of diethylethylphosphonate with
LDA and diethylchlorophosphate provides 12 (see SCHEME 2.2) in acceptable yields
(80 - 86% yield). There was no apparent decrease in yield when scaling up to obtain gram
quantities of 12. Hydrolysis of the isopropyl and ethyl esters was achieved by refluxing in
concentrated HCl giving 10 and 13, quantitatively.
Scheme 2.2 Alternative synthesis of EBP
P P
O O
EtO OEt
OEt OEt
CH
3
P
Et
O
EtO
OEt
1) BuLi, THF
-78
o
C
P
Cl
O
EtO
OEt
2)
THF, -78
o
C
P P
O O
HO OH
OH OH
CH
3
HCl
conc.
, reflux
12 13

The fluorinated BPs 14-22 were successfully synthesized from either TiPMBP, 3,
11, or 12 using Selectfluor in good yields (65-97%). This method provided a convenient
route to fluoro-containing BPs including the previously unknown 20.
The BP free acids were obtained quantitatively after either dealkylation in BTMS
or hydrolysis in HCl or HBr and dried under vacuum at 50
o
C and analyzed by combus-
tion analysis to define absolute purity.
19
EXPERIMENTAL SECTION
All chemicals were obtained through Sigma Aldrich except tetraisopropyl methylene-
bisphosphonate (TiPMBP) which was generously provided by Albright and Wilson
Americas, Inc. The BPs were analyzed using either a Varian 400 MHz, Bruker AM-360,
or Bruker AMX-500 MHz NMR. Elemental analysis was performed by Galbraith Labora-
tories. Any relevant experimental data including NMR and elemental analysis is pre-
sented in APPENDIX B.
Synthesis of tetraisopropyl (dichloromethylene)bis(phosphonate), 1
To a stirring, ice-chilled solution of 166 g of 5.25% NaOCl, 5 g (14.5 mmol) of TiPMBP
is added dropwise. The reaction mixture is reacted at room temperature and monitored by
31
P NMR for 30 min. After completion, a white precipitate is observed and the reaction
mixture is extracted with hexane (4 x 50 mL). The organic layers are combined and dried
over MgSO4 and the solvent is removed by reduced pressure. The resulting white solid
(TiPDCBP 1) is dried. (yield = 5.6 g, 82%).
31
P (CDCl3): 7.9 (s) compared to the lit.
6

value of 7.3.
Synthesis of (dichloromethylene)bis(phosphonic acid), 2
1 g (2.42 mmol) of 1 is dissolved in 2 mL of dried and distilled CH3CN. ~2 mL of BTMS
is added and the small flask sealed under N2. The reaction is carried out at room tempera-
ture for 2 days. The reaction is monitored by
31
P NMR. After the reaction has reached
completion, the solvent is removed under reduced pressure and the excess HBr is coe-
20
vaporated with CH2Cl2. The brownish residue is then dissolved in 2 mL of H2O and al-
lowed to sit at room temperature for 10 minutes and then the solution is washed with di-
ethyl ether twice. The solvent is removed and DCBP is dried to a brownish white foam
(yield = 592 mg - 100% conversion).
31
P (D2O): 7.5 (s), compared to lit.
4
value of 7.9.
Anal. calc. for CH4Cl2O6P2: C, 4.90; H, 1.65. Found: C, 5.12; H, 2.04.
Synthesis of tetraisopropyl (chloromethylene)bis(phosphonate), 3
3.3 g (mol) of Na2SO3 is dissolved in 96 mL of H2O which is then added over 15 min. to
an ice-chilled, stirring solution of 3.0 g (8.70 mmol) of 1 dissolved in 24 mL of EtOH.
After reacting at room temperature for 60 min. The reaction mixture was extracted with
CHCl3 (4 x 50 mL). The organic layers are combined and dried over MgSO4 and the sol-
vent is removed by reduced pressure. The resulting colorless oil (TiPMCBP) is dried.
(yield = 2.5 g, 90%).
31
P (CDCl3): 12.6 (s) compared to lit.
6
value of 12.2.
Synthesis of chloromethylenebis(phosphonic acid), 4
1 g (2.64 mmol) of 3 is dissolved in 2 mL of dried and distilled CH3CN. ~2 mL of BTMS
is added and the small flask sealed under N2. The reaction is carried out at room tempera-
ture for 2 days. The reaction is monitored by
31
P NMR. After the reaction has reached
completion, the solvent is removed under reduced pressure and the excess HBr is coe-
vaporated with CH2Cl2. The brownish residue is then dissolved in 2 mL of H2O and al-
lowed to sit at room temperature for 10 minutes and then the solution is washed with di-
ethyl ether twice. The solvent is removed and MCBP is dried to a brownish white foam
21
(yield = 555 mg - 100% conversion).
1
H (D2O): 3.8 (dd);
31
P NMR: 11.8 (s). Anal. calc.
for CH5ClO6P2: C, 4.71; H, 2.39. Found: C, 5.91; H, 2.71.
Synthesis of tetraisopropyl (dibromomethylene)bis(phosphonate), 5
5.43 g (136 mmol) of NaOH in 200 mL of H2O was prepared and cooled to 0
o
C. 3.5 mL
of Br2 (67.9 mmol) was added slowly with vigorous stirring. 5.0 g (14.5 mmol) of
TIPMBP was then added. The reaction mixture was stirred at 0
o
C for 10 min. and at room
temperature for 5 min. The clear yellow solution is extracted with CHCl3 (4 x 50 mL) and
the organic layers are collected and dried over MgSO4 and the solvent is removed under
reduced pressure leaving a colorless oil, TiPDBBP (6.9 g, yield = 95%).
31
P: 8.1 (s)

com-
pared to lit.
6
value of 7.5.
Synthesis of (dibromomethylene)bis(phosphonic acid), 6
3 g (5.98 mmol) of 5 is dissolved in 60 mL of HBr. The reaction is carried out at reflux
for 4 hrs. The reaction is monitored by
31
P NMR. After the reaction has reached comple-
tion, the solvent is removed under reduced pressure and the excess HBr is coevaporated
with CH2Cl2. The brownish residue, DBBP, is dried with acetone, EtOH, and then ace-
tone washes and then dried to a brownish crystalline material (2 g, yield = 100%).
31
P:
7.4 (s). Anal. calc. for CH4Br2O6P2: C, 3.60; H, 1.21. Found: C, 3.76; H, 1.31.
22
Synthesis of tetraisopropyl (bromomethylene)bis(phosphonate), 7
1.92 g (8.5 mmol) of SnCl2 • 2H2O was dissolved in 20 mL H2O. Dissolve 4.4 g (8.5
mmol) 5 in 10 mL of EtOH and cool to 0
o
C. Add the SnCl2 solution to the TiPDBBP so-
lution dropwise (w/cooling). After reacting for 5 min at 0
o
C, the mixture is reacted at
room temperature for 5min. The reaction mixture is extracted with CHCl3 (4 x 25mL). 7
is isolated as a colorless oil after drying; 3.5 g (95% yield).
31
P: 12.1 (s)

compared to lit.
6

value of 12.3.
Synthesis of (bromomethylene)bis(phosphonic acid), 8
2.2 g (5.20 mmol) of 7 is dissolved in 60 mL of HBr. The reaction is carried out at reflux
for 4 hrs. The reaction is monitored by
31
P NMR. After the reaction has reached comple-
tion, the solvent is removed under reduced pressure and the excess HBr is coevaporated
with CH2Cl2. The white residue 8 is dried with acetone, EtOH, and then crystallized with
acetone giving white crystals (1.3 g, yield = 98%).
1
H (D2O): 3.5 (dd);
31
P: 11.4 (s). Anal.
calc. for CH5BrO6P2: C, 4.71; H, 1.21. Found: C, 4.82; H, 1.99.
Synthesis of tetraisopropyl 2,2-propanediylbis(phosphonate), 9
286 mg (11.9 mmol) of NaH (95%) is weighed out in a dried three-necked flask. While
under N2, the flask is cooled down to 0
o
C and then 7.5 mL of dried and distilled THF and
7.5 mL of anhydrous DMF is added with a dried 20 mL air-tight glass syringe. 2.7 mL
(8.72 mmol) of TiPMBP is then added dropwise with a dried 1 mL gas-tight syringe. The
reaction is carried out under N2 and allowed to react at 0
o
C. After 20 minutes, 697 µL
23
(10.1 mmol) of MeI is added dropwise. After 30 minutes, the reaction is brought back to
room temperature. The reaction is then monitored by
31
P NMR until it is determined that
the reaction is complete (roughly 1 hr). After completion, the reaction is quenched with a
saturated NH4Cl solution (until a transplant solution is achieved). The reaction mixture is
washed with ice-cold CH2Cl2 and the organic layer is extracted. The solvent is removed
via reduced pressure and the oily residue is dissolved in minimum amount of hexane and
wet-loaded onto a column of hexane and silica gel. The 9 is eluted with a 50% mixture of
EtOAc/hexanes. Fractions containing the desired bisphosphonate are combined and the
solvent is removed under reduced pressure and then the yellowish oil is dried under vac-
uum (yield = 2.0 g - 62%).
31
P: 26 (s).
Synthesis of 2,2-propanediylbis(phosphonic acid), 10
1.5 g (4.02 mmol) of 9 is dissolved in 15 mL of conc. HCl. The reaction is carried out at
reflux for 24 hrs. The reaction is monitored by
31
P NMR. After the reaction has reached
completion, the solvent is removed under reduced pressure and redissolved in water and
then extracted with diethyl ether twice. The solvent is removed and 10 is dried to a
brownish white foam (yield = 815 mg - 99%).
31
P: 26.9 (s) compared to lit.
4
value of
26.9.
1
H: 1.8 (td). Anal. calc. for C3H10O6P2 ⋅H2O: C, 17.50; H, 4.99. Found: C, 17.53; H,
4.71.
24
Synthesis of tetraisopropyl 1,1-ethanediylbis(phosphonate), 11
286 mg (11.9 mmol) of NaH (95%) is weighed out in a dried three-necked flask. While
under N2, the flask is cooled down to 0
o
C and then 7.5 mL of dried and distilled THF and
7.5 mL of anhydrous DMF is added with a dried 20 mL air-tight glass syringe. 2.7 mL
(8.72 mmol) of TiPMBP is then added dropwise with a dried 1 mL gas-tight syringe. The
reaction is carried out under N2 and allowed to react at 0
o
C. After 20 minutes, 697 µL
(10.1 mmol) of MeI is added dropwise. After 30 minutes, the reaction is brought back to
room temperature. The reaction is then monitored by
31
P NMR until it is determined that
the reaction is complete (roughly 1 hr). After completion, the reaction is quenched with a
saturated NH4Cl solution (until a transplant solution is achieved). The reaction mixture is
washed with ice-cold CH2Cl2 and the organic layer is extracted. The solvent is removed
via reduced pressure and the oily residue is dissolved in minimum amount of hexane and
wet-loaded onto a column of hexane and silica gel. The 11 is eluted with a 50% mixture
of EtOAc/hexanes. Fractions containing the desired bisphosphonate are combined and the
solvent is removed under reduced pressure and then the yellowish oil is dried under vac-
uum (yield = 1.7 g - 55%).
31
P: 22.8 (s).
Synthesis of tetraethyl 1,1-ethanediylbis(phosphonate), 12
25 mL of freshly distilled THF is added to a dried 100 mL three-necked flask and cooled
down to -78
o
C. 7.5 mL of lithium diisopropylamide (LDA) in THF is added. A solution
of 1.22 mL (7.51 mmol) of diethylethylphosphonate in 15 mL of freshly distilled THF is
added to the LDA solution. The reaction is stirred at -78
o
C for 30 minutes. Then, a solu-
25
tion of 1.08 mL (7.51 mmol) of diethylchlorophosphate in 25 mL of THF is added drop-
wise. The reaction is stirred at -78
o
C for 1 hour then allowed to warm to room tempera-
ture. The reaction mixture is then poured into a 10 mL 5% HClaq solution. The organic
layer is collected and the aqueous layer is extracted with CH2Cl2 (3 x 15 mL). The or-
ganic fractions are combined and dried over MgSO4 and the solvent is removed under
reduced pressure. The resulting 12 can be further purified on silica (50% EtOAc/Hexane)
giving 1.73 g (79% yield).
31
P: 24 (s).
Synthesis of 1,1-ethanediylbis(phosphonic acid), 13
500 mg (1.65 mmol) of 11 or 12 is dissolved in 15 mL of conc. HCl. The reaction is car-
ried out at reflux for 24 hrs. The reaction is monitored by
31
P NMR. After the reaction has
reached completion, the solvent is removed under reduced pressure and redissolved in
water and then extracted with diethyl ether twice. The solvent is removed and 13 is dried
to a brownish white foam (yield = 387 mg - quantitative).
1
H (D2O): 1.1 (td), 2.1 (dq);
31
P: 22.5 (s) compared to the lit.
4
value of 22.6. Anal. calc. for C2H8O6P2 ⋅(H2O)0.1: C,
12.52; H, 4.31. Found: C, 12.54; H, 4.13.
Synthesis of tetraisopropyl (difluoromethylene)bis(phosphonate), 14
A total of 4.53 g (12.8 mmol) of Selectfluor is weighed out and separated into two test
tubes. 502 mg (13.1 mmol) of NaH (60% oil immersion) is weighed out in a dried three-
necked flask. While under N2, the flask is cooled down to 0
o
C and then 7.5 mL of dried
and distilled THF and 7.5 mL of anhydrous DMF is added with a dried 20 mL air-tight
26
glass syringe. 0.927 mL (2.91 mmol) of TiPMBP is then added dropwise with a dried 1
mL gas-tight syringe. The reaction is carried out under N2 and allowed to react at 0
o
C.
After 20 minutes, the first portion of Selectfluor is added. After 10 minutes, the second
portion of Selectfluor is added. After 30 minutes, the reaction is brought back to room
temperature. The reaction is then monitored by
31
P NMR until it is determined that the
reaction is complete (roughly 1 hr). After completion, the reaction is quenched with a
saturated NH4Cl solution (until a transplant solution is achieved). The reaction mixture is
washed with ice-cold CH2Cl2 and the organic layer is extracted. The solvent is removed
via reduced pressure and the oily residue is dissolved in minimum amount of NaHCO3
(0.1 M) and then an excess of n-hexane. The organic layer is extracted and analyzed by
31
P NMR. The organic solution is washed with NaHCO3 until only 14 remains. The sol-
vent is removed under reduced pressure and then the yellowish oil is dried (yield = 0.936
g - 85%).
31
P: 3.5 (t);
19
F: -123.3 (t) compared to the respective lit.
13
values of 2.8 and
-121.
Synthesis of (difluoromethylene)bis(phosphonic acid), 15
0.936 g (2.46 mmol) of 14 is dissolved in ~1 mL of BTMS (neat) and placed in a small
flask and sealed. The reaction is carried out at room temperature for 3 days. The reaction
can be monitored by
31
P NMR to ascertain completion. After the reaction has reached
completion, the solvent is removed under reduced pressure and the excess HBr is coe-
vaporated with CH2Cl2. The brownish residue is then dissolved in 2 mL of H2O and al-
lowed to sit at room temperature for 10 minutes and then the solution is washed with di-
27
ethyl ether twice. The solvent is removed and 15 is dried to a brownish white foam (yield
= 520 mg - 100% conversion).
31
P: 2.6 (t),
19
F: -122.8 (t) compared to the respective lit.
13

values of 3.7 and -121. Anal. calc. for CH4F2O6P2 ⋅H2O: C, 5.67; H, 2.62. Found: C, 5.71;
H, 2.65.
Synthesis of tetraisopropyl (fluoromethylene)bis(phosphonate), 16
A total of 4.53 g (12.8 mmol) of Selectfluor is weighed out and separated into two test
tubes. 390 mg (10.2 mmol) of NaH (60% oil immersion) is weighed out in a dried three-
necked flask. While under N2, the flask is cooled down to 0
o
C and then 7.5 mL of dried
and distilled THF and 7.5 mL of anhydrous DMF is added with a dried 20 mL air-tight
glass syringe. 0.927 mL (2.91 mmol) of TiPMBP is then added dropwise with a dried 1
mL gas-tight syringe. The reaction is carried out under N2 and allowed to react at 0
o
C.
After 20 minutes, the first portion of Selectfluor is added. After 10 minutes, the second
portion of Selectfluor is added. After 30 minutes, the reaction is brought back to room
temperature. The reaction is then monitored by
31
P NMR until it is determined that the
reaction is complete (roughly 1 hr). After completion, the reaction is quenched with a
saturated NH4Cl solution (until a transplant solution is achieved). The reaction mixture is
washed with ice-cold CH2Cl2 and the organic layer is extracted. The solvent is removed
via reduced pressure and the oily residue is dissolved in minimum amount of hexane and
wet-loaded onto a column of hexane and silica gel. The 16 is eluted with a 50% mixture
of EtOAc/hexanes. Fractions containing the desired bisphosphonate are combined and the
solvent is removed under reduced pressure and then the yellowish oil is dried under vac-
28
uum (yield = 588 mg - 56%).
31
P: 10.8 (d);
19
F: -226.1 (dt) compared to the respective
lit.
13
values of 10.7 and -221.
Synthesis of (fluoromethylene)bis(phosphonic acid), 17
336 mg (0.928 mmol) of 16 is dissolved in ~1 mL of BTMS (neat) and placed in a small
flask and sealed. The reaction is carried out at room temperature for 3 days. The reaction
can be monitored by
31
P NMR to ascertain completion. After the reaction has reached
completion, the solvent is removed under reduced pressure and the excess HBr is coe-
vaporated with CH2Cl2. The brownish residue is then dissolved in 2 mL of H2O and al-
lowed to sit at room temperature for 10 minutes and then the solution is washed with di-
ethyl ether twice. The solvent is removed and MFBP is dried to a brownish white foam
(yield = 179 mg - 100% conversion).
31
P: 9.6 (d);
19
F: -226.4 (q);
1
H (D2O): 4.8 (dt) com-
pared to the respective lit.
13
values of 10.5 and -225. Anal. calc. for CH5FO6P2 ⋅(CH3OH)
0.2: C, 7.19; H, 2.92. Found: C, 7.26; H, 2.98.
Synthesis of tetraisopropyl [chloro(fluoro)methylene]bis(phosphonate), 18
A total of 2.4 g (6.78 mmol) of Selectfluor is weighed out. 127 mg (5.29 mmol) of NaH
(95% oil immersion) is weighed out in a dried three-necked flask. While under N2, the
flask is cooled down to 0
o
C and then 15 mL of anhydrous DMF is added with a dried 20
mL air-tight glass syringe. 2 g (5.29 mmol) of 16 is then added dropwise with a dried 1
mL gas-tight syringe. The reaction is carried out under N2 and allowed to react at 0
o
C.
After 20 minutes, the Selectfluor is added. After 30 minutes, the reaction is brought back
29
to room temperature. The reaction is then monitored by
31
P NMR until it is determined
that the reaction is complete (roughly 1 hr). After completion, the reaction is quenched
with a saturated NH4Cl solution (until a transparent solution is achieved). The reaction
mixture is washed with ice-cold CH2Cl2 and the organic layer is extracted. The solvent is
removed via reduced pressure and the colorless oily residue 18 is dried under vacuum
(yield = 2.1 g - 78%).
31
P: 5 (d);
19
F: -145.8 (t) compared to the lit.
6
value of 4.9.
Synthesis of [chloro(fluoro)methylene]bis(phosphonic acid), 19
2 g (2.42 mmol) of 18 is dissolved in 15 mL of conc. HCl. The reaction is carried out at
reflux for 24 hrs. The reaction is monitored by
31
P NMR. After the reaction has reached
completion, the solvent is removed under reduced pressure and redissolved in water and
then extracted with diethyl ether twice. The solvent is removed and 19 is dried to a
brownish white foam (yield = 1.1 g - 96%).
31
P: 4.4 (d);
19
F: -145.4 (t).
Synthesis of tetraisopropyl (1-fluoro-1,1-ethanediyl)bis(phosphonate), 20
A total of 786 mg (2.15 mmol) of Selectfluor is weighed out. 61.5 mg (2.56 mmol) of
NaH (95%) is weighed out in a dried three-necked flask. While under N2, the flask is
cooled down to 0
o
C and then 5 mL of anhydrous DMF is added with a dried 20 mL air-
tight glass syringe. Then a mixture of 9 and 11 (containing ~500 mg - 1.40 mmol of 11)
in 5 mL of dried and distilled THF is then added dropwise with a dried 1 mL gas-tight
syringe. The reaction is carried out under N2 and allowed to react at 0
o
C until complete
carbanion formation is confirmed by
31
P NMR. Then, the Selectfluor is added. After 30
30
minutes, the reaction is brought back to room temperature. The reaction is then monitored
by
31
P NMR until it is determined that the reaction is complete (roughly 1 hr). After com-
pletion, the reaction is quenched with a saturated NH4Cl solution (until a transparent so-
lution is achieved). The reaction mixture is washed with ice-cold CH2Cl2 and the organic
layer is extracted. The solvent is removed via reduced pressure and the product mixture is
purified by column chromatography (100% hexane). The colorless oily residue 20 is
dried under vacuum (yield after chromatography = 283 mg - 53%).
31
P: 13.1 (d);
19
F:
-186.6 (m);
1
H (CDCl3): 1.25 (d), 1.65 (dt), 4.75 (m).
Synthesis of tetraethyl (1-fluoro-1,1-ethanediyl)bis(phosphonate), 21
A total of 1 g (2.73 mmol) of Selectfluor is weighed out. 50 mg (2.08 mmol) of NaH
(95%) is weighed out in a dried three-necked flask. While under N2, the flask is cooled
down to 0
o
C and then 3 mL of anhydrous DMF is added with a dried 20 mL air-tight
glass syringe. 565 mg (1.87 mmol) of 12 is then added dropwise with a dried 1 mL gas-
tight syringe. The reaction is carried out under N2 and allowed to react at 0
o
C. After car-
banion formation is confirmed by
31
P NMR, the Selectfluor is added. After 30 minutes,
the reaction is brought back to room temperature. The reaction is then monitored by
31
P
NMR until it is determined that the reaction is complete (roughly 1 hr). After completion,
the reaction is quenched with a saturated NH4Cl solution (until a transparent solution is
achieved). The reaction mixture is washed with ice-cold CH2Cl2 and the organic layer is
extracted. The solvent is removed via reduced pressure and product mixture is purified on
silica (50% EtOAc/hexane). The colorless oily residue 21 is dried under vacuum (yield =
31
480 mg - 80%).
31
P: 14 (d);
19
F: -186.1 (m);
1
H (CDCl3): 1.45 (t), 1.85 (dt), 4.35 (m)
compared to the respective lit.
6
values of 14.85, -109, and 1.3, 1.8, 4.18.
Synthesis of (1-fluoro-1,1-ethanediyl)bis(phosphonic acid), 22
480 mg (1.50 mmol) of 20 or 21 is dissolved in 15 mL of conc. HCl. The reaction is car-
ried out at reflux for 24 hrs. The reaction is monitored by
31
P NMR. After the reaction has
reached completion, the solvent is removed under reduced pressure and redissolved in
water and then extracted with diethyl ether twice. The solvent is removed and 22 is dried
to a brownish white foam (yield = 293 mg - quantitative).
31
P: 14 (d);
19
F: -186.2;
1
H
(D2O): 1.32 (dt). Anal. calc. for C2H7FO6P2 ⋅(CH3OH)0.15 ⋅(H2O)0.15: C, 11.98; H, 3.69.
Found: C, 11.93; H, 3.72.
32
CHAPTER 2 REFERENCES
1. Ahlmark, M. J.; Vepsalainen, J. J. Tetrahedron 2000, 56, 5213-5219.
2. Blackburn, G. M.; England, D. A.; Kolkmann, F. Journal of the Chemical Society,
Chemical Communications 1981, 930-932.
3. Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R. M. J. Fluorine
Chem. 1982, 20, 617-626.
4. Grabenstetter, R. J.; Quimby, O. T.; Flautt, T. J. J. Phys. Chem. 1967, 71, 4194-
4202.
5. Iorga, B.; Savignac, P. J. Organomet. Chem. 2001, 624, 203-207; Romanenko, V .
D.; Kukhar, V . P. Chemical Reviews 2006, 106, 3868-3935.
6. McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y .; Pham, P.; Bongartz, J. P. Phospho-
rus Sulfur and Silicon and the Related Elements 1988, 37, 1-12.
7. Popov, K.; Ronkkomaki, H.; Lajunen, L. H. J. Pure Appl. Chem. 2001, 73, 1641-
1677.
8. Vanura, P.; Jedinakova-Krizova, V .; Hakenova, L.; Munesawa, Y . J. Radioanal.
Nucl. Chem. 2000, 246, 689-692.
9. Popov, K.; Niskanen, E.; Ronkkomaki, H.; Lajunen, L. H. J. New J. Chem. 2000,
246, 689-692.
10. Kabachnik, M. I.; al., e. Doklady Akademii Nauk SSSR 1967, 177, 582.
11. Matczak-Jon, E.; Videnova-Adrabinska, V . Coord. Chem. Rev. 2005, 249, 2458-
2488.
12. Marma, M. S.; Khawli, L. A.; Harutunian, V .; Kashemirov, B. A.; McKenna, C. E.
J. Fluorine Chem. 2005, 126, 1467-1475.
13. McKenna, C. E.; Shen, P. D. J. Org. Chem. 1981, 46, 4573-4576.
14. Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V . K.; Martinek, V .; Xiang,
Y .; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.;
Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461-471.
33
15. Claessens, R. A. M. J.; Vanderlinden, J. G. M. J. Inorg. Biochem. 1984, 21, 73-82.
16. Dietsch, P.; Gunther, T.; Rohnelt, M. Zeithschrift Fur Naturforschung C-A Journal
of Biosciences 1976, 31, 661-663.
17. Grossmann, G.; Burkov, K. A.; Hagele, G.; Myund, L. A.; Hermens, S.; Verwey, C.;
Arat-ool, S. M. Inorg. Chim. Acta 2004, 357, 797-808.
18. Reed, W. A.; Rao, L. F.; Zanonato, P.; Garnov, A. Y .; Powell, B. A.; Nash, K. L. In-
org. Chem. 2007, 46, 2870-2876.
19. Aboujaoude, E. E.; Lietje, S.; Collignon, N.; Teulade, M. P.; Savignac, P. Tetrahe-
dron Lett. 1985, 26, 4435-4438.
34
CHAPTER 3
DETERMINATION OF PKAS OF A SERIES OF METHYLENEBISPHOSPHONIC
ACIDS BY POTENTIOMETRIC TITRATION
INTRODUCTION
The acidity constants for pyrophosphate and MBP have been determined by sev-
eral methods including potentiometric as well as NMR titrations and as expected they
show that pyrophosphate is more acidic than MBP.
1-8
Several α-haloderivatives of MBP
have been proposed and synthesized by various researchers.
1,4,5,9,10
In some cases, acidity
constants have been determined for these compounds; however, most pKas are deter-
mined under different conditions (counter ion, ionic strength, base, titration environment,
etc.).
1-9
In an ongoing study with DNA polymerases and deoxynucleoside triphosphos-
phate (dNTP) analogues,
11
we have employed the versatility or tunable aspect of MBP to
create a series of α-substituted bisphosphonates (BPs) P-CXY-P (X,Y = Br, Cl, F, H,
CH3), as well as the respective β,γ-dNTP analogues, with varying acidity compared to the
“natural” pyrophosphate (see FIGURE 2.1). In these particular studies, kinetics and fi-
delity of DNA polymerase β (Pol β) are being examined for the incorporation of a single
dNTP by the enzyme.
11
Pyrophosphate or BP is the leaving group in the chemistry step of
Pol β and the rate at which the BP leaves can be adjusted by the introduction of various
electron withdrawing groups at the bridging C. This property allows us to examine BPs
35
that are either higher, lower, or similar in acidity to pyrophosphate. As mentioned previ-
ously, the pKas for some of these compounds are known
1-9,12
and in some cases, there are
several different pKas for the same compound (see TABLE 3.1).
1-8,13
This disparaging
finding has compelled us to perform a study to determine the acidity constants of this se-
ries under consistent conditions.
Table 3.1 List of pKas reported in literature
BP pKa4 Ref.
MBP
9.89 8
10.00 14
10.42 5
10.75 2
10.75 15
DCBP
8.84 3
9.5 7
9.72 2
9.78 4
DFBP
7.63 1
8.00 9
MFBP
9.35 9
9.44 N. D. Leswara, Ph.D. Thesis, USC, 1983
MBBP 10.00 4
DMBP 11.76 4
EBP 11.67 4
36
MATERIALS AND METHODS
All chemicals were purchased from Sigma Aldrich except tetraisopropyl methyle-
nebisphosphonate (TiPMBP) which was generously provided by Albright and Wilson
Americas, Inc. and used in the synthesis of the series of BPs (detailed in CHAPTER 2).
The BPs were analyzed using either a Varian 400 MHz or Bruker 500 MHz NMR. Ele-
mental analysis was provided by Galbraith Laboratories. Two types of pH electrodes
were used to measure pH, Aldrich Z113441 glass combination electrode 183 mm x 3.5
mm and Beckman 150 mm x 7 mm combination electrode.
All acidity constants are obtained by potentiometric titration and environments are
kept free of CO2 and at a constant temperature of 25
o
C (using a THERMOSTAT). Pre-
liminary titrations were carried out with 0.1 M NaOH (standardized with 0.1 M KHP and
HCl) and 5 mM solutions of BP in 0.1 M NaCl. All solutions were prepared with CO2-
free H2O (see Table 2). Methods were optimized with commercially available MBP. Ti-
trant was added to the MBP solutions in 10-20 µL aliquots via automated Schott Titrator
Basic under Ar. All titrations were carried out at least three times at 25
o
C under Ar. The
titration data was analyzed by Hyperquad2006.
16
Hyperquad2006 and its preceding ver-
sions have been used to derive stability constants from potentiometric or spectrographic
data or batch measurements.
13,16,17
Factoring in the amount of acid and base used, one can
employ Hyperquad2006 to fit titration data to a calculated titration curve by adjusting the
model. pKa/Log β values (see FIGURE 3.1) were calculated by Hyperquad2006 (titra-
37
tions with Χ
2
values greater than 12.60 are discarded due to confidence levels lower than
95%).
Figure 3.1 Calculation of pKas from log βs
pKa4 = log β1
pKa3 = log β2 - log β1
pKa2 = log β3 - log β2
Table 3.2 Optimization of our methods with MBP as compared to Kabachnik et al.
5
Electrode Conditions pKa4 pKa3 pKa2
Smaller SA
0.1M NaCl
0.086M NaOH
10.39 6.94 2.78
Larger SA
0.1M NaCl
0.089M NaOH
10.06 6.89 2.73
Larger SA
0.1M KCl
0.091M KOH
10.39 7.06 2.61
Smaller SA
0.1M KCl
0.091M KOH
10.52 7.35 2.78
Kabachnik et. al.
0.1M KCl
0.1M KOH
10.42 7.33 2.75
Careful analysis of our methods (see TABLE 3.2) shows that in comparison to a
titration under conditions similar to ours (0.1 M buffer and 0.1 M titrant)
5
, a definite
background electrolyte interference is observed and is more evident in the readings ac-
quired with a pH electrode with a greater surface area (SA). This effect can be reduced by
two practices: 1) reduce the area of the glass surface interacting with the solutions and 2)
increase background counter ion size to help better distinguish from hydrogen concentra-
tion
6,7
. Many of the previously determined pKas of BPs had been determined with NaCl
and NaOH which exemplifies the importance of this study. According to our studies, the
conditions employing 0.1 M KCl and 0.1 M KOH show consistent and comparable val-
38
ues to the model
5
and are conditions designed to greatly reduce the background interfer-
ence by sodium
6,7
and closely resemble the optimal conditions for electrode accuracy and
low uncertainty for measurement of acidity constants. For the remaining BPs our general
procedure was as follows: approximately 30-50 mg of the free acid form of a BP was dis-
solved in 50 mL of 0.1 M KCl in CO2–free H2O and titrated with ~0.1 M KOH in CO2–
free H2O using a Schott Instruments Titrator Basic by 10–20 µL aliquots. KOH solutions
were standardized with 0.1 M standard solutions of KHP and HCl. All titrations were car-
ried out at least three times at 25
o
C under Ar using the electrode with the smaller surface
area. The titration data was analyzed by Hyperquad2006.
16
and the electrode was cali-
brated using the glass electrode calibration software GLEE
18
.
RESULTS AND DISCUSSION
Each BP was dried under vacuum at 50
o
C. Titrations were performed under con-
sistent and reliable conditions and repeated at least three times. All acidity constants were
calculated with Hyperquad2006 and found to be consistent with errors within 0.01-0.2 of
the average. A summary of our findings can be found in TABLE 3.3. A sample of the
Hyperquad2006 analysis of the titration data for MBP is in FIGURE 3.2.
Coinciding with the chemistry used in the synthesis of this series of BPs was the
modernized methods and consistent conditions practiced in the determination of the dis-
cussed acidity constants. We can report with great confidence and repeated reproducibil-
ity that the methods practiced herein were those that produce the most reliable results.
6-8

The determined acidity constants of the BPs, particularly pKa4, follow the expected
39
Table 3.3 Stability constants for a series of BPs. All values are the average of at least three titrations
at 25
o
C under Ar
BP pKa4 pKa3 pKa2
CF2 7.76 5.63 1.69
CFCl 8.36 5.58 1.52
CCl2 8.84 5.77 1.90
CHF 9.01 6.16 1.31
CBr2 9.30 5.93 1.98
CHCl 9.50 6.31 2.27
CHBr 9.91 6.15 2.34
CFCH3 10.21 6.21 1.84
CHCH3 11.59 7.05 2.73
C(CH3)2 12.24 7.67 2.88
Figure 3.2 Sample of Hyperquad2006 analysis
40
trends as related to electronegativity and acidity. To date, a complete study of the acidity
constants of these BPs had not been performed. Therefore, this practical study is quite
important as it provides the pKas of a complete series of BPs under the self-consistent
conditions. As all pKas were determined with 0.1 M KCl, these values can be accepted as
a reliable relative index for the BPs listed.
6
EXPERIMENTAL SECTION
Methylenebisphosphonic (MBP) acid that was purchased from Sigma Aldrich was
labeled to be 98% pure. The standardized 0.1 M HCl, pH standardized buffers, KCl and
KOH were purchased from VWR. The synthesis of the remaining BPs in the series and
detailed NMR analysis of product purity is described in CHAPTER 2. The purity of all
BPs was determined to be ≥ 5%. All relevant titration data can be found in APPENDIX
C.
41
CHAPTER 3 REFERENCES
1. Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R. M. J. Fluorine
Chem. 1982, 20, 617-626.
2. Claessens, R. A. M. J.; Vanderlinden, J. G. M. J. Inorg. Biochem. 1984, 21, 73-82.
3. Dietsch, P.; Gunther, T.; Rohnelt, M. Zeithschrift Fur Naturforschung C-A Journal
of Biosciences 1976, 31, 661-663.
4. Grabenstetter, R. J.; Quimby, O. T.; Flautt, T. J. J. Phys. Chem. 1967, 71, 4194-
4202.
5. Kabachnik, M. I.; al., e. Doklady Akademii Nauk SSSR 1967, 177, 582.
6. Popov, K.; Niskanen, E.; Ronkkomaki, H.; Lajunen, L. H. J. New J. Chem. 2000,
246, 689-692.
7. Popov, K.; Ronkkomaki, H.; Lajunen, L. H. J. Pure Appl. Chem. 2001, 73, 1641-
1677.
8. Vanura, P.; Jedinakova-Krizova, V .; Hakenova, L.; Munesawa, Y . J. Radioanal.
Nucl. Chem. 2000, 246, 689-692.
9. Blackburn, G. M.; England, D. A.; Kolkmann, F. Journal of the Chemical Society,
Chemical Communications 1981, 930-932.
10. Marma, M. S.; Khawli, L. A.; Harutunian, V .; Kashemirov, B. A.; McKenna, C. E.
J. Fluorine Chem. 2005, 126, 1467-1475; McKenna, C. E.; Khawli, L. A.; Ahmad,
W. Y .; Pham, P.; Bongartz, J. P. Phosphorus Sulfur and Silicon and the Related
Elements 1988, 37, 1-12; McKenna, C. E.; Shen, P. D. J. Org. Chem. 1981, 46,
4573-4576; Ahlmark, M. J.; Vepsalainen, J. J. Tetrahedron 2000, 56, 5213-5219.;
Ahlmark, M. J.; Vepsalainen, J. J. Tetrahedron 2000, 56, 5213-5219.
11. Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V . K.; Martinek, V .; Xiang,
Y .; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.;
Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461-471.
12. Grossmann, G.; Burkov, K. A.; Hagele, G.; Myund, L. A.; Hermens, S.; Verwey, C.;
Arat-ool, S. M. Inorg. Chim. Acta 2004, 357, 797-808.
42
13. Reed, W. A.; Rao, L. F.; Zanonato, P.; Garnov, A. Y .; Powell, B. A.; Nash, K. L. In-
org. Chem. 2007, 46, 2870-2876.
14. Sanna, D.; Bodi, I.; Bouhsina, S.; Micera, G.; Kiss, T. Journal of the Chemical
Society-Dalton Transactions 1999, 3275-3282.
15. Deluchat, V .; Serpaud, B.; Caullet, C.; Bollinger, J. C. Phosphorus Sulfur and Sili-
con and the Related Elements 1995, 104, 81-92.
16. Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739-1753.; Gans, P.; Sabatini,
A.; Vacca, A. Talanta 1996, 43, 1739-1753.
17. Norman, S.; Maeder, M. Crit. Rev. Anal. Chem. 2006, 36, 199-209; Le Bris, N.;
Bernard, H.; Tripier, R.; Handel, H. Inorg. Chim. Acta 2007, 360, 3026-3032.; Le
Bris, N.; Bernard, H.; Tripier, R.; Handel, H. Inorg. Chim. Acta 2007, 360, 3026-
3032.
18. Gans, P.; O'Sullivan, B. Talanta 2000, 51, 33-37.
43
CHAPTER 4
SYNTHESIS OF α,β-dNTP AS INHIBITORS OF DNA POL β
INTRODUCTION
DNA polymerases play a crucial role in replicating and maintaining the fidelity of
genetic information. In an ongoing multidisciplinary study of structure and function of
DNA polymerase β, a eukaryotic enzyme primarily involved in filling short DNA gaps
1
,
we required a series of α,β-methylene-substituted analogues of deoxynucleoside 5’-
triphosphates (dNTPs). When the Pα-O-Pβ bridging oxygen in a natural mononucleotide
substrate is replaced by an imido (NH)
2-4
or methylene (CXY)
5-8
group, the P-N or P-C
bond should resist cleavage in the nucleotidyl transfer reaction catalyzed by the enzyme.
As a result, these analogues will remain intact in stable ternary DNA complexes with the
polymerase and therefore should be useful to probe pre-chemistry enzyme-complex func-
tion and structure, as recently shown with in an X-ray crystallographic study of α,β-NH
dUTP with DNA pol β.
9
Information about such complexes provides a reference point for
theoretical analysis of the chemical mechanism
10
for the complete transfer of a mono-
phosphate nucleoside donor to the sugar acceptor in the active site. As probes for the
mechanism of polymerase catalysis and its relationship to polymerase fidelity, α,β-
methylene dNTP analogues permit exploration of stereoelectronic effects on active site
interactions, by making appropriate substitutions X,Y on the adjacent Pα CXY bridging
carbon. The largest obtainable electron-withdrawing effect with minimal steric perturba-
tion can be achieved using X,Y = F, resulting in analogues in which the bisphosphonate
44
group is expected to be less basic than the pyrophosphate moiety in the natural dNTPs.
To obtain representative combinations of correct and incorrect base pairing at the nascent
base pair binding site, a set of analogues comprising both purine and pyrimidine bases is
desirable.
Here, we describe the first synthesis of α,β-CF2-dCTP, using a modified chemical-
enzymatic approach that also can be applied to synthesis of α,β-CF2-dATP, affording
these compounds in sufficient purity (≥ 99%) to eliminate detectable contaminating sub-
strate activity in polymerase inhibition kinetics. DNA pol β Kd values have been deter-
mined for these analogues, and compared to Kd values for the natural substrates (dATP,
dCTP, dTTP), α,β-NH-dUTP, and the α,β-CH2-dATP and dGTP analogues. We also de-
scribe the configuration of α,β-CF2-dTTP bound with template-primer DNA into a ternary
complex with DNA polymerase β, determined by X-ray crystallography, and discuss its
structural implications.
An obvious route to α,β-methylene-dNTP analogues entails coupling a particular
methylenebisphosphonate (MBP) derivative to the target nucleoside, followed by phos-
phorylation to add the terminal γ-phosphate. Several α,β-CXY-NDPs (X,Y = H or F) were
previously synthesized via tosylation of the 5’-OH in the protected ribonucleoside with
tosyl chloride and (dimethylamino)-pyridine, followed by deprotection and displacement
of the tosyl moiety with the appropriate tris(tetrabutylammonium)
11
bisphosphonate, a
similar approach was used to prepare a α,β-CF2-dGDP (N
2
-[p-N
n
-butylphenyl]-
derivative)
12
, dATP and dTTP,
6
and related nucleotide analogues.
7
Alternatively,
45
methylene-bisphosphonic acid was coupled to 2,3-protected cytidine with dicyclohexyl-
carbodiimide (DCC), but the reaction required a week, a tenfold excess of coupling rea-
gent, and gave the product in low yield.
13
Direct 5’-coupling of two to six equivalents of
methylenebis(phosphonic dichloride) with unprotected nucleosides also has been
demonstrated.
14

Chemical phosphorylation of α,β-CH2-dADP and dTTP has been carried out via
the p-nitrobenzyl-phosphoromorpholidates.
6
Unfortunately, subsequent deprotection by
hydrogenolysis of the p-nitrobenzyl-group restricts the pyrimidine substrate, because the
cytosine ring is prone to reduction under these conditions.
15
Side products from the phos-
phorylation step typically contaminate the α,β-methylene-dNTP analogue product. Car-
bonyldiimidazole (CDI) activation of the dGDP derivative referred to above has been
utilized as an alternative route.
12
We found phosphorylation of our dNDP substrates using
CDI to be problematic due to the reactivity of the imidazolate, which tended to give un-
wanted nucleoside phosphorylation at the 3’-OH and other side products.
As an alternative to syntheses of α,β-imido dNTPs using CDI to activate the NDP
analogue for coupling with tributylammonium phosphate,
3,4
Kenyon proposed enzymatic
phosphorylation by either creatine kinase (CK)
2,4
or pyruvate kinase (PK) and phos-
phonenolpyruvate (PEP)
4
. However, inadequate CK and PK activity in attempted PEP
phosphorylation of our dNDP intermediates, led us finally to consider adaptation of a
method for phosphorylation of azole carboxamide riboxynucleoside 5’-diphosphates
(NDPs) by ~stoichiometric ATP and nucleoside diphosphate kinase (NDPK),
13,16
in which
46
PEP and PK are included to recycle the ADP produced in the phosphoryl transfer and
drive the reaction to completion. Wu et al.,
16
noting that removal of excess ATP from the
product mixture was difficult to achieve by conventional ion exchange chromatography,
accomplished this by using a boronate affinity column.
MATERIALS AND METHODS
SYNTHESIS OF α,β-dNTPS
We first converted dA or N
4
-benzoyl-dC 7 to the corresponding 5’-tosylates, 1 or
4 respectively (see SCHEME 4.1), by reaction with tosyl chloride in pyridine (75 -
80%).
6
The NH2-group of dC was protected via facile microwave-induced reaction with
benzoic anhydride and diisopropylethylamine (DIEA) in pyridine, (2 min at 300 W)
17
to
give 7 (76%). When purifying 4 from the reaction mixture, we initially used conventional
extraction with NaHCO3 aq. (pH ~ 8.7), but obtained low isolated yields (~20%). The pKa
of the N
4
-proton in 4 was estimated (ACDLabs pKaDB 8.01 software program) to be ~8.
Accordingly, we changed to a slightly acidic aqueous work-up (citric acid, pH ~ 4.5), to
avoid 4 from being extracted by the aqueous wash which increased the isolated yield to
~70%.
47
Scheme 4.1 Synthesis of α,β-methylene dNTP analogues
O
OH
O
BASE
P P
O
HO
OH
O
OH
CF
2
O
OH
O
BASE
P P
O
O
HO
O
OH
CF
2
P
O
HO
HO
O
OH
TsO
BASE
O
OH
HO
BASE
TsCl, pyridine
0
o
C
(Bu
3
NH)
3
-O
3
P-CF
2
-PO
3
H,
CH
3
CN
PK, PEP,
NDPK, ATP
cat
1, 4
2, 5
3, 6
1, 2, and 3: BASE = adenine, 4: BASE = N
4
-Bz-cytosine, 5 and 6: BASE = cytosine. [Note: Before tosyla-
tion, dC was converted to N
4
-Bz-cytosine 7 with benzoic anhydride, DIEA in pyridine, microwave irradia-
tion (2 min – 300W) prior to the enzymatic phosphorylation, the N
4
-protected 5 was dealkylated to the cor-
responding unprotected dCDP analogue 8 using methanolic ammonia. Compounds 1 – 6 are shown in fully
protonated form for convenience.]
The purified tosylates were converted to the dNDP α,β-difluoromethylene ana-
logues 2 or 5 via condensation with the tris(tetrabutylammonium) salt
6
of
difluoromethylene-bis(phosphonic acid) (DFBP). The N
4
-benzoyl-dCDP analogue 8 ob-
tained as the direct tosylation product from 4 was then deprotected in methanolic ammo-
nia to give 5 quantitatively, which was purified by preparative ion-exchange HPLC.
DFBP was obtained by bromotrimethylsilane (BTMS) dealkylation
18
of tetraisopropyl
difluoromethylenebisphosphonate (TiPDFBP)
19
, synthesized using Selectfluor to fluori-
nate the carbanion of tetraisopropylmethylenebisphosphonate.
10,20
48
γ-Phosphorylation of the analogues by different methods was evaluated by ion-
exchange HPLC using a 0-100% gradient of 0.5 M TEAB buffer eluting a 7 µm 500 Å
SAX PUREGEL column, which distinguished monophosphate, diphosphate and triphos-
Figure 4.1 Analytical HPLC (on SAX) of the synthesis of α,β-methylene dNTP analogues: bottom
(red) - before the addition of phosphorylation enzymes and rest - sample of reaction mixture over 24 hours.
t=24hrs
t=18hrs
t=15min
Before PK, PEP, &
ATP, NDPK
!,"-CF2-
dCDP
!,"-CF2-
dCTP
ATP
HPLC ANALYSIS OF THE CONVERSION OF
!,"-CF
2
-dCDP TO !,"-CF
2
-dCTP
49
phate nucleotides (example in FIGURE 4.1). This sensitive and rapid HPLC analysis
was supplemented by
31
P NMR analysis to identify reaction components. As noted above,
CDI activation followed by treatment with orthophosphate gave overly complex mix-
tures, and direct enzymatic phosphorylation by PEP catalyzed by CK or PK was unsuc-
cessful. However, we found that phosphorylation to the dNTP analogues 3 or 6 was
cleanly achieved using NDPK and a catalytic amount of ATP, regenerated with 2.5
equivalents of PEP with PK in 50 mM HEPES buffer (HPLC - FIGURE 4.1). Impor-
tantly, the latter modification removes the unnecessary use of an affinity column to purify
the product from excess ATP.
PURIFICATION OF α,β-dNTPS
In order to obtain product completely free of biochemically detectable nucleotide
contaminants, we found that separation on DEAE Sephadex or Dowex
3,6-8,12,21-28
or
single-pass preparative HPLC using a C-18
29
or ion-exchange
3,6-8,12,21-30
column was not
sufficient. We adapted our analytical HPLC method to a preparative scale using the re-
cently available Macherey-Nagel SP150/25 NUCLEOGEL SAX 1000-10 anion exchange
column. The UV-detected fraction containing the desired dNTP analogue (retention time
= 20 - 30 min) was collected and lyophilized. It was then rechromatographed (2%
CH3CN - 0.1 N TEAB buffer, retention time 15 - 20 min) using RP HPLC (Varian 250 x
21.4 mm MICROSORB 100-5 C18) to remove remaining traces of nucleotide contami-
nants, and the final products obtained as triethylammonium salts by lyophilization: struc-
ture and purity confirmed by HPLC, NMR and HRMS.
50
RESULTS AND DISCUSSION
This overall synthesis/purification scheme has the advantages of being applicable
to both purine and pyrimidine examples, including the previously unavailable α,β-CF2-
dCTP analogue. The reactions are very clean (particularly compared to CDI phosphoryla-
tion), do not require protection/deprotection chemistry in the phosphorus moieties, elimi-
nate the problem of excess ATP in the enzymatic phosphorylation, and thanks to the two-
stage HPLC purification, provide exceptionally pure analogues suitable for polymerase
inhibition studies (purity > 99.5%). It should be noted that the dual HPLC purification
protocol was also quite effective in eliminating biochemically detectable nucleotide con-
taminants in α,β-CF2-dTTP 9 prepared by the p-nitrobenzyl-phosphomorpholidate phos-
phorylation method
6
.
Figure 4.2 Gapped DNA insertion assay with DNA pol β for dNTPs (dATP, dTTP) and α,β-CXY
analogues. The purity of the analogues was assessed from their failure to be inserted into a gapped DNA
substrate. Primer (n) extension was assayed in the presence of low (L, 0.5 nM) or high (H, 50 nM) pol β
and 1 mM analogue for 5 or 10 min., respectively. The mobility of the extension product (n+1) with dATP
or dTTP is shown in the last two lanes where most of the primer (200 nM) is extended after a 21 min incu-
bation with 50 nM enzyme and 100 µM dNTP. Insertion assay performed by Dr. Wlliam A. Beard, NIEHS.

51
INHIBITION ASSAYS
Initial DNA polymerase β inhibition studies on the preparation of 9 as obtained
via the p-nitrobenzyl phosphoromorpholidate method
6
using 50 nM pol β showed mas-
sive incorporation of a contaminating dNMP into the primer strand, by gel electrophoretic
analysis (FIGURE 4.2). After dual-HPLC purification, this artifact was no longer ob-
servable. At the same enzyme level, contaminating activity was also detectable in a
commercial sample of α,β-CH2-dATP, while little or none is detectable in the synthesized
α,β-CF2-dATP, 3.
DNA synthesis was assayed on single-nucleotide gapped DNA substrates where
the templating base was varied to be complementary to the incoming nucleotide. Specific
reaction conditions have been described previously.
9
Reactions were initiated with 0.5
nM enzyme at room temperature and stopped with EDTA mixed with formamide dye.
The substrates/products were separated on 15% denaturing polyacrylamide gels and
quantified in the dried gels by phosphorimagery.
Inhibition of natural (Oα,β) nucleotide insertion by the respective analogues was
determined at various concentrations of the analogue and sub-saturating concentrations of
the natural nucleotide. The inhibition constant, Ki, was determined by Dixon analysis.
The -CF2-analogues readily inhibit pol β single-nucleotide insertion (FIGURE 4.3).
However, their binding affinities are at least an order of magnitude lower than those of
the natural nucleotides. Interestingly, the -NH- and -CH2- analogue exhibited the tightest
52
binding (~10-fold tighter than natural dNTPs), despite polarity differences between the
α,β-NH and α,β-CH2 groups.
Figure 4.3 Analogue Binding Affinity. A comparison of the inhibition constants for the commer-
cially available NH- and CH2-α,β-analogues with the CF2-derivatives. The binding affinities of the natural
dNTPs determined by single-turnover analysis are also shown. Inhibition assay performed by Dr. Wlliam
A. Beard, NIEHS.
Crystallization and analysis of α,β-modified dNTP analogues complexed with
DNA primer and DNA pol β. The α,β-modified dNTP analogues were soaked into a bi-
nary complex crystal form of a one nucleotide gapped DNA substrate with an unpaired
deoxyadenosine and DNA polymerase β. The crystal structure of the ternary complex of
pol β with the incoming analogue 3, 6, and 9 opposite dA represents the pre-catalytic
state of the nucleotidyl transfer reaction for correct incorporation, containing all atoms
required for catalysis including the two catalytic metal ions. As expected, the substitution
of the CF2 for the bridging oxygen prevented dissociation of the pyrophosphate leaving
53
Figure 4.4 Superposition of a) the active site of the ternary complex of DNA Polymerase β with in-
coming α,β-CF2-dTTP 9 and the ternary complex with α,β-NH-dUTP (shown in yellow) bound. For the
crystal structure with 9 bound the three active site aspartic acid residues are shown in khaki, the metal ions
are green, and waters are shown as red spheres. The fluoride atoms are colored cyan. Electron density for
the 2Fo-Fc simulated annealing omit map (blue) is shown contoured at 5σ; b) the α,β-CF2-dATP 3 and α,β-
CF2-dCTP 6 (silver) [enzyme-complex residues have been removed for clarity]. X-ray crystallographic
studies performed by S. H. Wlison, et al; NIEHS.
D192
D190
D256
P10
α,β-CF
2
dCTP
v. α,β-NH dUTP
DNA pol β
α,β-CF2-dTTP
vs
α,β-NH-dUTP
a)
b)
α,β-CF2-dCTP (silver) superimposed with α,β-CF2-dATP
54
Figure 4.5 X-ray crystallographic study of α,β-CF2-dCTP 6 highlighted opposite its base pair G
(top) and its mispair A (bottom). This is the first report of a mispair structure of DNA pol β in ternary
complex with DNA and incoming dNTP substrate. The mismatch structure was obtained with Mn
2+
and
also is the first report of a structure with Mn
2+
replacing the binding metal-cation Mg
2+
- performed by S.
H. Wlison, et al; NIEHS.
55
group, trapping the complex. All correlated atoms in the active site of this structure super-
impose well with previously determined ternary complex structures of pol β where the
reaction was trapped by deletion of the nucleophilic 3’-OH on the primer terminus
31
or by
using a nitrogen in place of the α,β bridging oxygen
1,5,9
(FIGURE 4.4). The α,β-CF2-
dCTP 6 was also used with Mn
2+
to successfully obtain a structure opposite its mispair A
(see FIGURE 4.5). This is the first report of a mismatched structure with DNA pol β in
ternary complex with DNA and incoming dNTP as well as the first report with evidence
Mn
2+
replacing the binding metal cation Mg
2+
in the complex.
This crystallographic evidence suggests that 3, 6, and 9 are good non-
hydrolyzable mimics of the natural substrate dNTP and prove to be very useful in obtain-
ing crystal structures of correct and incorrect base pairs in different DNA polymerases in
the nascent base pair binding site, an essential aid in deciphering mechanistic details and
furthering our understanding of what dictates fidelity for different polymerase systems.
The very high purity of the analogues will also benefit exploration of structural influ-
ences on fidelity using inhibition kinetics where the normal nucleotide is also present,
competing as a substrate.
CONCLUSION
In summary, we have synthesized a series of α,β-CF2-dNTP analogues. Two of
these, 3 and 6, were synthesized by an advantageous chemical-enzymatic method that
was used to obtain the previously unavailable dCTP analogues. The methods used to iso-
late these analogues provides dNTP samples in the highest purity. Examination of the
56
binding and active site interactions of these “probes” has alluded to the important binding
characteristics that can be achieved by modification of the Pα-O-Pβ bridging position
while maintaining the analogous binding orientation the natural substrate.
EXPERIMENTAL SECTION
All necessary reagents including enzymes were purchased from Sigma Aldrich. HPLC
analytical and preparative separations were carried out using a Varian ProStar 210 pump
and injector system equipped with a Shimadzu SPD-10A VP UV Vis detector, on a) a
Varian C-18 (ODS) Microsorb-MV 4.6 mm x 25 cm, 5 µm analytical column; b) a Dy-
namax C-18 21.4 mm x 25 cm, 5 µm preparative column; c) a Varian PureGel SAX 10
mm x 10 cm, 7 µm analytical column; d) Macherey-Nagel Nucleogel SAX 1000-10 25
mm x 15 cm preparative column. NMR spectra were recorded (D2O solutions, pH ~8) on
Bruker AM-360 or Varian Mercury 400 spectrometers. HRMS were obtained at the UCR
mass spectrometry facility (Dr. Ron New). A complete collection of HPLC analysis,
NMR, and HRMS of synthesized compounds can be found in APPENDIX D. 9
6
was
provided by Prakash and coworkers as a crude product mixture and then purified by dual-
pass preparative HPLC. A detailed description of the purification and characterization of
9 can be found in CHAPTER 9 and APPENDIX H.
57
Synthesis of N
4
-Benzoyl-2’-deoxycytidine, 7
Method A: Microwave-assisted synthesis (small scale)
In a dried 5 mL microwave test tube with cap, 100 mg of deoxycytosine and 294 mg of
benzoic anhydride is weighed out and dissolved in 2 mL of pyridine. 180 µL of DIEA is
added and the test tube is sealed. The solution is irradiated at 300 W for 45 seconds after
which it is analyzed by TLC. The solution is irradiated at 45 second intervals and moni-
tored by TLC until the reaction is complete. The solvent is removed under reduced pres-
sure and the benzoylated product is isolated by column chromatography (10%
MeOH:CH2Cl2 and then 4:1 CH2Cl2:MeOH) as a white powder. 125 mg (82% yield)
1
H:
8.60 (d), 8.00 (t), 7.66 (m), 7.58 (t), 7.44 (m), 7.39 (t), 6,28 (t), 4.89 (d), 4.43 (d), 3.84
(m), 2.56 (m), 2.24 (d).
Method B: Standard synthetic approach (large scale)
In a dried round bottom flask, 1 g of deoxycytosine and 937 mg of benzoic anhydride is
weighed out and dissolved in pyridine. The reaction is set to reflux at 90
o
C for 2 hours
after which the reaction is analyzed by TLC. The mixture is reacted at 90
o
C until com-
pletion. When the reaction is completed, the solution is cooled to room temperature after
which the solvent is removed in vacuo and the benzoylated product is isolated via column
chromatography (4:1 CH2Cl2:MeOH) as a white powder. Alternatively, the product mix-
ture can be suspended in water and the benzoic acid is removed by extraction with ether
two times. The aqueous mixture is dried under reduced pressure and the product is recrys-
58
tallized in ethanol. 850 mg (61% yield)
1
H: 8.60 (d), 8.00 (t), 7.66 (m), 7.58 (t), 7.44 (m),
7.39 (t), 6,28 (t), 4.89 (d), 4.43 (d), 3.84 (m), 2.56 (m), 2.24 (d).
General synthesis of 2’-deoxynucleoside 5’-tosylates
1.1 equivalent of nucleoside is weighed out in a dried round bottom flask and dried with
pyridine. The dried nucleoside is then dissolved in pyridine and cooled to 0 ºC. Then, 1.1
equivalents of p-toluenesulfonyl chloride is added to the stirring nucleoside solution. The
reaction is monitored by
1
H NMR. When the reaction has completed, the mixture is
warmed to room temperature after which it can be dried under reduced pressure and puri-
fied by chromatography or it may be two-thirds concentrated in vacuo and purified by
extraction. The product is dried and is obtained as a colorless foam.
Synthesis of 2’-deoxyadenine-5’-tosylate, 1
857 mg (3.4 mmol) of 2’-deoxyadenosine is weighed out in a dried round bottom flask
and dried with pyridine. The dried nucleoside is then dissolved in 30 mL of anhydrous
pyridine and cooled to 0ºC. Then, 839 mg (3.4 mmol) of p-toluenesulfonyl chloride is
added to the stirring nucleoside solution. The reaction is monitored by
1
H NMR. When
the reaction has completed (18 hrs), the mixture is warmed to room temperature after
which it is two-thirds concentrated in vacuo and added to 20 ml of ice-cold H2O. 100 ml
of dried, chilled ethyl acetate is added and the organic layer is extracted and washed with
basic H2O (chilled). The organic layer is dried over MgSO4. The solvent is removed un-
der reduced pressure and the product
6
is dried and obtained as a colorless foam (501 mg -
59
37%).
1
H: 8.31 (d), 8.06 (s), 7.74 (d), 7.27 (d), 6.46 (t), 4.77 (d), 4.61 (d), 4.11 (d), 3.82
(m), 2.43 (m), 2.40 (m).
Synthesis of N
4
-benzoyl-2’-deoxycytidine-5’-tosylate, 4
639 mg (1.93 mmol) of N
4
-benzoyl-2’-deoxycytidine is weighed out in a dried round bot-
tom flask and dried with pyridine. The dried nucleoside is then dissolved in 1.6 mL of
anhydrous pyridine and cooled to 0 ºC. Then, 331 mg (1.74 mmol) of p-toluenesulfonyl
chloride is added to the stirring nucleoside solution. The reaction is monitored by
1
H
NMR. When the reaction has completed (18 hrs), the solvent is removed in vacuo and
added to 20 mL of ice-cold 0.25 M citric acid buffer (pH = 4.5). 200 mL of dried, chilled
CHCl3 is added and the organic layer is extracted and washed with 10 mL citric acid
buffer (chilled). The organic layer is dried over Na2SO4. The solvent is removed under
reduced pressure and the product is dried and obtained as a colorless foam (600 mg -
64%). 8.60 (d), 8.00 (t), 7.66 (m), 7.58 (t), 7.44 (m), 7.39 (t), 6,28 (t), 4.89 (d), 4.43 (d),
3.84 (m), 2.56 (m), 2.24 (d).
Synthesis of tris(tetrabutylammonium) salt of (difluoromethylene)bis(phosphonate)
The (difluoromethylene)bis(phosphonic acid)
19
is dissolved in H2O. The aqueous solution
is titrated to pH 7.6 with 40% tetrabutylammonium hydroxide. The solvent is removed
under reduced pressure and dried leaving a colorless foam.
60
General synthesis of α,β-CF2 nucleoside 5'-diphosphate analogues
1.1 equivalents of dried 5’-deoxynucleoside tosylate are added to a dried round bottom
flask and cooled to 0 ºC. The dried tosylate is then dissolved in dried and distilled aceto-
nitrile. In a separate flask, 1.1 equivalents of the dried tris(tetrabutylammonium) salt of
the methylenebisphosphonate is dissolved in dried and distilled acetonitrile. The methyl-
enebisphosphonate solution is added to the tosylate solution dropwise. The reaction is
monitored by ion-exchange HPLC. After completion, the reaction mixture is diluted with
0.5 M TEAB buffer (pH ~ 8) and purified via two-stage preparative ion-exchange HPLC
on SAX. The nucleoside diphosphate analogue is obtained as a triethylammonium salt.
Synthesis of deoxyadenine 5'-(difluoromethylene)bis(phosphonate), 2
150 mg (0.4 mmol) of dried 2’-deoxyadenine-5’-tosylate is added to a dried round bottom
flask and cooled to 0 ºC. The dried tosylate is then dissolved in 1.5 mL of dried and dis-
tilled acetonitrile. In a separate flask, 525 mg (0.6 mmol) of the dried tris(tetrabutylam-
monium) salt of DFBP is dissolved in 1.5mL dried and distilled acetonitrile. The DFBP
solution is added to the tosylate solution dropwise. The reaction is monitored by ion-
exchange HPLC. After completion (48 hrs), the reaction mixture is diluted with 0.5 M
TEAB buffer (pH ~ 8) and purified via two-stage preparative ion-exchange HPLC on
SAX followed by repurification on the C-18 column. α,β-CF2-dADP
6
2 is obtained as a
triethylammonium salt. (71.4 mg - 40% yield).
1
H: 8.31 (s), 8.06 (s), 6.32 (t), 4.63 (d),
4.11 (d), 2.43 (m), 2.40 (m);
31
P: 6.54 (m), 3.57 (m);
19
F: -116.3 (dd).
61
Synthesis of N
4
-benzoyldeoxycytidine 5'-(difluoromethylene)bis(phosphonate)
100 mg (0.2 mmol) of dried N
4
-benzoyl-2’-deoxycytidine-5’-tosylate is added to a dried
round bottom flask and cooled to 0 ºC. The dried tosylate is then dissolved in dried and
distilled acetonitrile. In a separate flask, 266 mg (0.3 mol) of the dried tris(tetrabutylam-
monium) salt of DFBP is dissolved in dried and distilled acetonitrile. The DFBP solution
is added to the tosylate solution dropwise. The reaction is monitored by ion-exchange
HPLC. After completion (18 hrs), the reaction mixture is diluted with 0.5M TEAB buffer
(pH ~ 8) and purified via two-stage preparative ion-exchange HPLC on SAX followed by
separation on C-18. α,β-CF2-BzdCDP is obtained as the triethylammonium salt. (45 mg -
43% yield).
1
H: 8.53 (d), 7.83 (d), 6.13 (t), 4.44 (d), 4.10 (m), 3.95 (d), 2.22 (m), 2.13
(m);
31
P: 6.66 (m), 3.36 (m);
19
F: -116.6 (dd).
Dealkylation of benzoyl-protecting group (to give α,β-CF2-dCDP), 5
10 mg (0.09 mmol) of α,β-CF2-BzdCDP added to a round bottom flask and dissolved in
half-saturated methanolic ammonia. The cloudy solution is stirred at room temperature
overnight. The reaction can be monitored by the repeated ion-exchange HPLC conditions
and shows a shift of the Bz-dCDP peak (11 min) to the dCDP peak (8 min). The solvent
is removed under reduced pressure and the white residue is washed with diethyl ether.
The white powder is dried under vacuum and then dissolved in 2 mL of 0.5 M TEAB
buffer (pH ~ 8) and purified via two-stage HPLC (SAX then C18). α,β-CF2-dCDP 5 is
isolated as the triethylammonium salt (7 mg).
1
H: 7.83 (d), 6.13 (t), 5.93 (d), 4.44 (d),
4.10 (m), 3.95 (d), 2.22 (m), 2.13 (m);
31
P: 6.66 (m), 3.36 (m);
19
F: -116.6 (dd).
62
General synthesis of α,β-methylene-nucleoside triphosphate analogues
1.1 equivalents of α,β-methylene-nucleoside diphosphate are added to a dried round bot-
tom flask and 5.6 equivalents of KCl and 1.8 equivalents of MgCl2 as well as 2.5 equiva-
lents of phosphoenol pyruvic acid (PEP) are added and prepared in 50 mM HEPES
buffer. In a separate eppendorf tube, appropriate units (approx. concentration of 2 mg/
mL) of pyruvate kinase (PK) are prepared and dissolved in HEPES buffer. In a separate
eppendorf, appropriate units of nucleoside diphosphate kinase (NDPK) is prepared in
HEPES buffer, as well. All of the solutions are kept at 0 ºC prior to incubation. The PK
solution is added to the dNDP solution. A catalytic amount of ATP is added to the NDPK
solution prior addition to the reaction mixture. The reaction is incubated at 37 ºC. The
reaction is monitored by ion-exchange HPLC. However, before analysis the enzymes are
removed from the product mixtures by passing them through an Amicon YM-10 micron-
filter. After approximately 24 - 48 hours, the enzymes are removed and the reaction mix-
ture is purified via preparative ion exchange HPLC on SAX.
Synthesis of α,β-difluoromethylene-adenosine triphosphate (α,β-CF2-dATP), 3
35 mg (0.08 mmol) of 2 are added to a dried round bottom flask and 24.5 mg of KCl and
21 mg of MgCl2 as well as 25 mg of PEP are added and prepared in 1mL of 50 mM HE-
PES buffer. In a separate eppendorf tube, 1 unit of PK are prepared and dissolved in 200
µL HEPES buffer. In a separate eppendorf, 1 unit of NDPK is prepared in 200 µL HEPES
buffer, as well. All of the solutions are kept at 0 ºC prior to incubation. The PK solution is
added to the dNDP solution. A catalytic amount of ATP is added to the NDPK solution
63
prior addition to the reaction mixture. The reaction is incubated at 37 ºC. The reaction is
monitored by ion-exchange HPLC. However, before analysis the enzymes are removed
from the product mixtures by passing them through an Amicon YM-10 micron-filter. Af-
ter approximately 48 hours, the enzymes are removed via filtration and the reaction mix-
ture is purified via two-stage preparative ion exchange HPLC on SAX and then on C-18.
α,β-CF2-dATP
6
3 is isolated as the triethylammonium salt (15 mg).
1
H: 8.33 (s), 8.06 (s),
6.33 (t), 4.63 (d), 4.11 (d), 2.43 (m), 2.40 (m);
31
P: 4.29 (m), -5.33 (d), -6.20 (m);
19
F:
-119.6 (dd).
Synthesis of α,β-difluoromethylene-cytidine triphosphate (α,β-CF2-dCTP), 6
33 mg (0.08 mmol) of 5 are added to a dried round bottom flask and 33 mg of KCl and
28 mg of MgCl2 as well as 47 mg of PEP are added and prepared in 10 mL of 50 mM
HEPES buffer. In a separate eppendorf tube, ~5 units of PK are prepared and dissolved
in 200 µL HEPES buffer. In a separate eppendorf, ~5 units of NDPK are prepared in 200
µL HEPES buffer, as well. All of the solutions are kept at 0 ºC prior to incubation. The
PK solution is added to the dNDP solution. A catalytic amount of ATP is added to the
NDPK solution prior addition to the reaction mixture. The reaction is incubated at 37 ºC.
The reaction is monitored by ion-exchange HPLC. However, before analysis the enzymes
are removed from the product mixtures by passing them through an Amicon YM-10
micron-filter. After approximately 24 hours, the enzymes are removed via filtration and
the reaction mixture is purified via two-stage preparative ion exchange HPLC on SAX
and then on C-18. α,β-CF2-dCTP 6 is isolated as the triethylammonium salt (5 mg).
1
H:
64
7.85 (d), 6.15 (t), 5.95 (d), 4.47 (d), 4.13 (m), 3.99 (d), 2.23 (m), 2.15 (m);
31
P: 4.6 (m),
-5.75 (d), -7.1 (m);
19
F: -119.6 (dd).
Preparation and determination of concentrations of dNTP stock solutions
The TEA salts of 3, 8 and 9 are lyophilized to dryness and then dissolved in 500 µL of 50
mM TrisCl buffer (pH = 8.0). The solutions were passed through an Amicon YM-30 filter
and the total nucleotide concentration determined by UV-Vis spectroscopy (reference:
dCTP, ε=9100 at 272 nm; dATP, ε=15300 at 259 nm
32
). The solutions are passed through
an Amicon (YM-30) filter to remove any dust or large insoluble particles. Because the
measured weight of TEA salts does not really translate to the actual amount of dNTP ana-
logue (due to the uncertainty of either being mono-, bis-, or tris-salts), determination of
the concentrations of the TrisCl solutions by UV-Vis spectroscopy is an acceptable alter-
native method. The concentrations are determined by diluting 10 µL and 20 µL aliquots of
the stock solutions to 2.0 mL. The method was initially validated with a solution of dGTP
with a known concentration. The extinction coefficient for dGTP (ελmax=13700; λmax=262
nm
32
) was used as an acceptable approximation for the extinction coefficients of our syn-
thesized dGTP analogues. The absorbance was determined for every dilution and the av-
erage concentration for the stock solution is calculated. These dNTP analogues have been
found to be relatively stable in solution at pH 8 and are usually stored at -20 ºC.
65
Crystallization of the pol β substrate complexes (performed by Wilson, et al;
NIEHS)
Human DNA polymerase β was over-expressed in E. coli and purified.
33
The binary com-
plex crystals with one-nucleotide gapped DNA and adenine as a templating base were
grown as previously described.
34
These crystals were then soaked in artificial mother liq-
uor (100 mM imidazole, pH 8.0, 20% PEG3350, 90 mM sodium acetate, 200 mM
MgCl2) with 5 mM α,β-CF2-dNTP and cryoprotectant (12% ethylene glycol) resulting in
a ternary complex.
Data collection and structure determination (performed by Wilson, et al; NIEHS)
Data were collected at 100 K on a CCD detector system mounted on a MicroMax007HF
(Rigaku Corporation) rotating anode generator. Data were integrated and reduced with
HKL2000 software.
35
Ternary substrate complex structures were determined by molecu-
lar replacement with a previously determined structure of pol β complexed with one-
nucleotide gapped DNA and an incoming dUMPNPP (PDB ID 2FMS).
9
The crystal
structures have similar lattices and are sufficiently isomorphous to determine the
molecular-replacement model using CNS and manual model building using O. The fig-
ures were prepared using Molscript/Raster3D.
36
The parameters and topology files for the
analogues were prepared using the program XPLO2D.
37
66
Table 4.1
Sample of crystallographic statistics: data presented for 9, Wlison, et al; NIEHS.
Data Collection

Space Group P2 1

a (Å) 50.5

b (Å) 79.7

c (Å) 55.3

β (˚) 107.9

dmin (Å) 2.20

R merge (%)
a
9.8 (43.8)
b

Completeness (%) 86.4 (82.2)

1/σ 9.2 (2.3)

Unique reflections 18597 (1757)

Total reflections 41942
Refinement

R.m.s. Deviations

Bond angles (˚) 1.12

Bond lengths (Å) 0.005

R work (%)
c
20.76

R free (%)
d
26.57

Ramachandran analysis (%)
e

Favored (%) 96.9

Allowed (%) 100

Average B-factors (Å
2
)

Protein 27.7

Adduct 33.3
a
R merge = 100 x S h S i | I h,i – Ih | /S h S i Ih,i where I h is the mean intensity of symmetry-
related
reflections I h,i.
b
Numbers in parentheses refer to the highest resolution shell of data (10%).
c
R work = 100 x S | |F obs| - | F calc || / S |F obs|
d
R free for a 5% subset of reflections.
e
As determined by MolProbity.
8
67
CHAPTER 4 REFERENCES
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2006, 106, 361-382.
2. Ma, Q. F.; Kenyon, G. L.; Markham, G. D. Biochemistry 1990, 29, 1412-1416.
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15. Otmar, M.; Masojfdkova, M.; V otruba, I.; Holy, A. Collect. Czech. Chem. Commun.
2001, 66, 500-506.
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cleotides & Nucleic Acids 2002, 21, 393-400.
18. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M.-C. Tetrahedron Lett.
1977, 155-158.
19. McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573-4576.; McKenna, C. E.;
Shen, P.-D. J. Org. Chem. 1981, 46, 4573-4576.
20. Marma, M. S.; Khawli, L. A.; Harutunian, V .; Kashemirov, B. A.; McKenna, C. E.
J. Fluorine Chem. 2005, 126, 1467-1475.
21. Blackburn, G. M.; Kent, D. E.; Kolkmann, F. Journal of the Chemical Society,
Chemical Communications 1981, 1188-1190.
22. Blackburn, G. M.; England, D. A.; Kolkmann, F. Journal of the Chemical Society,
Chemical Communications 1981, 930-932.
23. Victorova, L.; Sosunov, V .; Skoblov, A.; Shipytsin, A.; Krayevsky, A. FEBS Lett.
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24. Hamilton, C. J.; Roberts, S. M.; Shipitsin, A. Chemical Communications (Cam-
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25. Shipitsin, A. V .; Victorova, L. S.; Shirokova, E. A.; Dyatkina, N. B.; Goryunova, L.
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the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry
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FEBS Lett. 1997, 410, 423-427.
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70
CHAPTER 5
SYNTHESIS OF β,γ-METHYLENEBISPHOSPHONATE
NUCLEOSIDE TRIPHOSPHATE ANALOGUES AS PROBES FOR KINETICS AND
FIDELITY OF DNA POLYMERASE β
INTRODUCTION
DNA polymerases are crucial to replicating and maintaining the fidelity of en-
coded genetic information. In an ongoing study of structure and function in DNA po-
lymerase β, a eukaryotic enzyme primarily involved in repair of single-stranded DNA
gaps smaller than 6 nucleotides, we required a series of β,γ-methylene analogues
1-3
of de-
oxynucleoside triphosphates (dNTPs), with varying stereoelectronic properties in the β,γ-
methylene bridge. The method by which these β,γ-methylene dNTP analogues are ob-
tained should be applicable to the synthesis of both purine or pyrimidine-dNTPs (such as
β,γ-dGTP and β,γ-dCTP analogues, respectively). For accurate bioanalysis of kinetic and
fidelity measurements, these dNTP analogues must be of the utmost purity (>99%). The
dNTPs must be absent of any other nucleoside triphosphate or methylene bisphosphonate
side-products/impurities since these may interfere with kinetic measurements; some of
these compounds are known to be substrates
4
for pol β.
These dNTP analogues have been modified along the triphosphate chain in the
β,γ-position, to be stable against hydrolysis at this site. It has been explained that the rate-
limiting step of nucleotide incorporation by polymerases is at the “chemistry” step in its
71
mechanism. The native leaving group in this “chemistry” step is pyrophosphate. By sub-
stituting -O- with -CH2-, the pyrophosphate analogue becomes non-hydrolyzable and
modifications of the methylene-linker (i.e substitutions by one or two halogens) can
achieve
3,5,6
analogues isopolar and/or isosteric to the natural pyrophosphate. This idea has
been explored previously in perturbing NTP metal binding
3
as well as reverse
transcriptase
7-9
, DNA polymerase α
1,8,10,11
, and other G protein activity
10,12
.
There are two general methods of synthesizing β,γ-dNTP analogues which involve
activation of the nucleoside monophosphate using either a morpholidate or imidazolate
intermediate. The morpholidate method
2,3,6,8,13
, used to synthesize the HIV-1 reverse tran-
scriptase inhibitors in CHAPTER 1, employs a two-step process which involves conver-
sion of the nucleoside monophosphate to the corresponding morpholidate followed by
conjugation with the appropriate pyrophosphate analogue. The imidazolate method
1,8,14-17

is usually performed in one step as a one-pot synthesis or a two-step method similar to
the morpholidate method; the nucleoside monophosphate is converted to the imidazolate
(in situ for the one-pot reaction) followed by the addition of the appropriate pyrophos-
phate analogue. The main drawback to the morpholidate method compared to the imida-
zolate counterpart is the time required for a complete reaction; 2.5 - 3 days compared to
2.5 - 3 hours, respectively. The significant discrepancy in reaction time combined with
proposed enhancement in isolated yields
15
and considering a suspected low nucleo-
philicity of halogenated methylenebisphosphonates
3,15
, the imidazolate method is widely
used as the more popular route to β,γ-dNTP analogues. To date, there is little to no dis-
cussion of the difficult to separate side-products or impurities that contaminate reaction
72
mixtures, as well as the end product that arise from each method. It is likely that the imi-
dazolate, being the more reactive intermediate, should give rise to a larger amount of nu-
cleotide products, in a nucleobase and sugar-moiety (ribose, deoxy- and dideoxyribose)
dependent manner. Also, in order to drive the selectivity of the correct imidazolate inter-
mediate, there is the necessity for protection/ deprotection tactics.
15,16,18,19
These aspects
of the imidazolate method are what make it unattractive to us for the synthesis of com-
pletely pure β,γ-dNTP analogues that are to be used as probes to study DNA polymerase
β.
A list of known β,γ-methylene-NTP analogues and the references related to the
methods by which they have been synthesized is summarized in TABLE 5.1. Those NTP
analogues listed in the table as “--” are compounds that have not been synthesized or no
data concerning characterization is available.
In order to successfully synthesize the series of halogenated and unhalogenated
β,γ-methylene-dNTP analogues, the tributylammonium salts of the BPs must be provided.
As described in CHAPTER 2, the series of BPs was synthesized beginning from
TIPMBP or TEEBP followed by dealkyllation with BTMS or ester hydrolysis with HCl
or HBr.
27,28
The tributylammonium salts are obtained by dissolving the BPs in 50%
EtOH:H2O and reacting with 1.5 equivalents of tributylamine for 30 minutes. The solvent
is removed and the BP salts are redissolved in EtOH and the BPs are dried via coevapora-
tion with EtOH or DMF.
73
Table 5.1 Summary of known methylene-NTP analogues. The corresponding reference(s) for each
NTP is listed in the table below.
B
O
R OH
O P O
OH
O
P X P
O O
O O
O
X = CH2 CF2 CHF CCl2 CHCl CBr2 CHBr CFCH3
C(OH)
CH3
B=A
R=OH
2,3,12,15,
20-23
2,3,12,15 2,3,15 2,3,12 12 12,24 12 -- 20,22
B=U
R=OH
15,25 15 15 15 -- -- -- -- 20,22
B=C
R=OH
-- -- -- -- -- -- -- -- 22
B=G
R=OH
3,21,26 3 3,26 3 -- -- -- -- --
B=A
R=H
20,23,25 -- -- -- -- -- -- -- 20
B=T
R=H
11,23,24 11,24 11,24 -- -- 11,24 -- 11,24 --
B=C
R=H
-- -- -- -- -- -- -- -- --
B=G
R=H
23 1 -- -- -- -- -- -- --
Following a method adapted from Khorana’s procedure for morpholidate activa-
tion of nucleoside monophosphates
13
and the synthesis of β,γ-nucleotide analogues, we
synthesized morpholidates (1 and 2) from the commercially available nucleoside mono-
phosphates, dGMP and dCMP, using dicyclohexylcarboxydiimide (DCC) coupling chem-
istry. After 100% conversion was observed by
31
P NMR, the reaction is quenched and
then purified. Product purification requires no chromatography, only filtration, to remove
the dicyclohexylurea side-product, and extraction with diethylether to remove any unre-
acted morpholine and DCC. To prepare the β,γ-CF2, CHF, CCl2, CHCl, CFCl, CBr2,
CHBr, CFCH3, CHCH3, C(CH3)2, and CH2 analogues of dGTP and dCTP, the tribu-
74
tylammonium salts of methylenebisphosphonic acid derivatives (DFBP, MFBP, DCBP,
MCBP, FClBP, DBBP, MBBP, FMBP, EBP, DMBP, and MBP, respectively) are dried and
added to the appropriate solution of morpholidate, either 1 or 2, dissolved in DMSO and
the reaction is carried out at room temperature for approximately 30 - 60 hrs (see
SCHEME 5.1). This reaction was quenched by neutralization with triethylammonium
bicarbonate (TEAB) buffer (0.5 M, pH = 8.0) or a saturated NaHCO3 solution.
Scheme 5.1 Synthesis of β,γ-methylene bisphosphonate dNTP analogues; a) DCC, morpholine, 50%
tBuOH/H2O; b) [O3P-X-PO3] [Bu3NH
+
], DMSO; X = CH2, CHF, CF2, CHCl, CCl2, CBr2, CHBr, CFCH3,
CHCH3, C(CH3)2. dNMP-Morph - 1: BASE = guanidine, 2: BASE = cytidine. β,γ-dNTP analogues - 3:
BASE = guanidine, X = CH2; 4: BASE = guanidine, X = CF2; 5(R)/6(S): BASE = guanidine, X = CHF; 7:
BASE = cytidine, X = CH2; 8: BASE = cytidine, X = CF2; 9: BASE = guanidine, X = CCl2; 10(R)/11(S):
BASE = guanidine, X = CHCl 12(R)/13(S): BASE = guanidine, X = CFCl; 14: BASE = guanidine, X =
CBr2; 15(R)/16(S): BASE = guanidine, X = CHBr; 17(R)/18(S): BASE = guanidine, X = CFCH3; 19(R)/
20(S): BASE = guanidine, X = CHCH3; 21: BASE = guanidine, X = C(CH3)2.
O
OH
O P
-
O
O
O
-
O
OH
O P
-
O
O
N
O
O
OH
O P O
O
O
-
P
O
O
-
X P
O
O
-
-
O
P P
O
-
O
O
-
O
O
-
O
-
[Bu
3
NH
+
] [ ]
DMSO
anh
morpholine,DCC
tBuOH:H
2
O
X
X = CH
2
, CHF, CF
2
, CCl
2
,
CHCl, CFCl, CBr
2
, CHBr,
C(CH
3
)
2
, CHCH
3
, CFCH
3
dNMP
dNMP-Morph
BASE BASE
BASE
75
Figure 5.1 Analytical HPLC (SAX) of a) the conversion of dNMP-Morph to β,γ-dNTP; b) β,γ-dNTP
after dual-phase preparative HPLC purification.
dNMP-
Morph
dNMP
β,γ- dNTP
a) b)
Analogous to the synthesis of the reverse transcriptase inhibitors in CHAPTER 1
and α,β-dNTPs in CHAPTER 4
27
, the conversion to the desired dNTP analogue was
monitored by analytical ion-exchange HPLC using a gradient of 0 - 100% 0.5M TEAB
buffer (pH = 8) or 0 - 50% 0.5M LiCl (pH = 8) and SAX PureGEL column (see FIGURE
5.1). The unique feature of the ion-exchange HPLC analysis method that was created as
previously mentioned is that we can discern morpholidates from monophosphates, di-
phosphonate analogues, and triphosphate analogues as well as observe diphosphonates
and tetraphosphonates (the impurities or side-products from different synthetic ap-
proaches). The general method of analysis for these reactions is by
31
P NMR.
7,8,10,11,15

This analytical method [by NMR] is suitable for large-scale reactions because the product
concentrations are well above the sensitivity levels for
31
P nuclei, but in our case, for
small-scale reactions, longer acquisition times or larger concentrations are generally re-
quired for appropriate NMR analysis. As witnessed throughout the course of these syn-
76
theses, this presents an undesired opportunity for decreased yields and unpractical analy-
sis for this reaction. This HPLC method is relatively quick (≤15 min.) and a very sensi-
tive technique that only requires 2-3 µL of reaction mixture to successfully determine
product concentrations. The triphosphate analogues are isolated from the product mixture
by ion-exchange chromatography. This is generally accomplished on DEAE Sephadex
and Dowex.
1-5,7-13,17
However, use of these resins requires tedious swelling and column
packing procedures as well as lengthy purification trials. Here, we adapted our useful
analytical HPLC method to a preparative scale on the preparative NUCLEOGEL SAX
anion exchange column. The peak corresponding to the triphosphate analogue (retention
time = 15-30 min, depending on the nucleobase and MBP derivative) was isolated via
preparative anion-exchange HPLC and lyophilized. It has been noted by others
15
that
when isolating these triphosphate analogs by ion-exchange chromatography that any ex-
cess methylene disphosphonate derivative elutes along with the dNTP. However, our
method distinguishes monophosphates from diphosphates and triphosphates and separates
them exclusively by charge. Should there be the possibility of any other dNTP contami-
nant, the dNTP analogue is purified further on C-18, to refine the purity or polish the ana-
logue for bioanalysis. Using an HPLC method adapted from the analytical method we
have used previously for the analysis nucleoside analogues, a 0.1N TEAB buffer (pH =
7.5) containing 4% acetonitrile is used to elute the dNTP analogue (retention time 9-15
minutes) and following lyopholization, the desired dNTP is obtained as the TEA salt with
product purity ≥ 99% (see FIGURE 5.1b). This two-stage purification method is much
more convenient and efficient compared to separation on DEAE Sephadex or Dowex and
77
removes any chance of contamination from MBP derivatives or more importantly other
dNTP compounds that are not addressed by other methods, particularly on those using C-
18
15
or ion-exchange
1-5,7-13,17
only.
Table 5.2 Summary of synthesized dNTP analogues
Nucleotide
31
P-NMR (ppm)
19
F-NMR (ppm)
Pα Pβ Pγ
dG 3 β,γ-CH2dGTP* -11 (d) 11.7 (d) 13.5 (m) -----
4 β,γ-CF2dGTP -11 (d) -3 (m) 3.5 (m) -117.8 (dd)
5/6 β,γ-CHFdGTP* -11 (d) -4.7 (m) 7 (m) -218.7 (m)
9 β,γ-CCl 2dGTP* -11 (d) 1.7 (m) 7.8 (d) -----
10/11 β,γ-CHCldGTP* -11 (d) 8.3 (m) 9.5 (d) -----
12/13 β,γ-CFCldGTP* -11 (d) -0.9 (m) 5.5 (m) -136.5 (m)
14 β,γ-CBr 2dGTP* -11 (d) 2.9 (m) 8.1 (d) -----
15/16 β,γ-CHBrdGTP* -11 (m) 7.7 (m) 8.6 (d) -----
17/18 β,γ-CFCH 3dGTP* -11 (d) 10 (m) 11.5 (dd) -176.5 (m)
19/20 β,γ-CHCH 3dGTP* -11 (d) 18.5 (m) 17 (d) -----
21 β,γ-C(CH 3) 2dGTP* -11 (d) 19.5 (m) 23 (d) -----
dC 7 β,γ-CF 2dCTP* -11 (d) -3 (m) 3.5 (m) -117.6 (dd)
8 β,γ-CH 2dCTP* -11 (d) 11 (d) 12.5 (m) -----
A summary of synthesized dNTP analogues and novel analogues are highlighted by “*” .
RESULTS AND DISCUSSION
HPLC, HRMS, and NMR analysis support successful synthesis of the deoxynu-
cleotide triphosphate analogues 3 - 21. See TABLE 5.2. Compounds 3 and 5 - 21 are
novel compounds. The usefulness of this synthetic approach as well as the two-stage pu-
rification is evident in the range of compounds that they can be synthesized. The most
important factor of this method is that it is clean and straightforward; avoiding the intro-
duction of any additional, unnecessary chemical contaminants or protection/
78
deprotection
15,16,18,19
steps, regardless of nucleoside while avoiding complex reaction
mixtures that are difficult to purify. This clean and austere approach also empowers the
advantage of our analytical HPLC and two-stage HPLC purification methods as they con-
sider the biological relevance of such approaches.
Even though the overall reaction requires a bit more time and is lower yielding
than the imidazole approach, the morpholidate method is specific for phosphorylation
only at the activated α-phosphate without recently reported protection or deprotection tac-
tics and is therefore cleaner (no additional organic chemical contaminants). This ap-
proach is not discriminatory of one base (purine or pyrimidine) over another; it does not
show different reactivity with different bases. In the case of protection/deprotection pro-
cedures using trifluoroacetic anhydride
15,16
(TFAA), for 100% conversion, reactions re-
quire heating
18
(contrary to the reported rapid protection procedure
15
) or overnight reac-
tion (for successful deprotection, not recognized in the recent communication
15
). The
previous suspicion that fluorinated-MBPs display decreased nucleophilicty
3,15
was not
observed in the synthesis of 3 - 21 (all morpholidates were converted to β,γ-dNTP ana-
logues within 30 - 60 hrs). Whereas the imidazole method, particularly Jakeman’s,
15
is
attractive for purely synthetic purposes (i.e. industrial relevance), our morpholidate pro-
cedure combined with the two-stage purification provides a more practical, product-
specific and purity driven approach (significant to biochemists).
79
EXPERIMENTAL SECTION
All reagents are purchased from Sigma-Aldrich. HPLC analysis and separation was car-
ried out on a Varian ProStar 210 (pump/injector) and Shimatzu SPD-10A VP (UV Vis de-
tector) using a Varian Microsorb-MV 4.6 mm x 250 mm - 5 µm analytical column, Varian
Dynamax 100A C-18 21.4 mm x 250 mm - 5 µm preparative column, Varian PureGel
SAX 10 mm x 100 mm - 7 µm analytical column, and Macherey-Nagel Nucleogel SAX
1000-10 25 mm x 150 mm - preparative column. NMR analysis was carried out on either
Bruker AM-360 MHz FT NMR or Varian Mercury 400 NMR instruments. HRMS was
acquired via an outside source (at UCLA and UCR). A detailed collection of HPLC,
HRMS, and NMR data can be found in APPENDIX E.
Synthesis of tributylammonium salts of methylene bisphosphonic acids
The methylene bisphosphonic acids are dissolved in 50% EtOH/H2O and placed in a
conical flask. 1.5 equivalents of tributylamine are added dropwise and the solution is
placed at room temperature for 30 minutes. The solvent is removed and the remaining
Bu3N is coevaporated with ethanol. The tributylammonium salts of MBP, DFBP, MFBP,
DCBP, MCBP, FClBP, DBBP, and MBBP are dried by coevaporation with DMF under
vacuum.
General synthesis of nucleoside-monophosphate morpholidate
1.1 equivalents of dNMP are weighed out in a dried round bottom flask and dissolved in a
50% tBuOH:H2O solution. Complete dissolution of sodium salts of dNMP is achieved by
80
lowering the pH to 2. Three equivalents of morpholine are added dropwise. The solution
is left at ambient temperature for 15 minutes then set to reflux. After a steady reflux, 3
equivalents of DCC dissolved in tBuOH is added dropwise over 2 hours. After 2.5 hours,
the reaction is examined by
31
P NMR. If the reaction is incomplete, an appropriate
amount of DCC in tBuOH is added and allowed to react for another hour to drive the re-
action to completion. When the reaction is completed, the solvent is removed under re-
duced pressure. The light brown or yellowish-white residue is then dissolved in H2O and
the suspension is filtered to remove the dicyclohexylurea. The aqueous solution is then
washed three times with diethyl ether. The solvent is then removed under reduced pres-
sure and the brownish white residue is dried under vacuum.
Synthesis of 2’-deoxy-5’-guanosinemonophosphate morpholidate, 1
107.6 mg (0.310 mmol) of dGMP is weighed out in a dried round bottom flask and is dis-
solved in 10mL of a 50% tBuOH:H2O solution. The pH is adjusted to 2. 64 µL (0.930
mmol) of distilled morpholine is added dropwise using a 100 µL gas-tight syringe. The
solution is reacted at ambient temperature for 15 minutes then set to reflux. After a steady
reflux 198 mg (0.930 mmol) of DCC dissolved in 2 mL of tBuOH is added dropwise over
2 hours. After 2.5 hours, the reaction is examined by
31
P NMR. When the reaction is
completed, the solvent is removed by reduced pressure. The light brown or yellowish-
white residue is then dissolved in H2O and the suspension is filtered to remove the dicy-
clohexylurea. The solvent is then removed under reduced pressure and the brownish
81
white residue is dried under vacuum. dGMP-Morph 1 is obtained in good yield (yield =
130 mg - 95.5%).
31
P: 7 (s);
1
H: 8 (d), 6.2 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m).
Synthesis of 2’-deoxy-5’-cytidinemonophosphate morpholidate, 2
100 mg (0.326 mmol) of dCMP is weighed out in a dried round bottom flask and dis-
solved in a 50% tBuOH:H2O solution. Complete dissolution of dCMP is achieved by
lowering the pH to 2. 85 µL (0.978 mmol) of morpholine are added dropwise. The solu-
tion is reacted at ambient temperature for 15 minutes then set to reflux. After a steady re-
flux, 202 mg (0.978 mmol) of DCC is dissolved in tBuOH is added dropwise over 2
hours. After 2.5 hours, the reaction is monitored by
31
P NMR. When the reaction is com-
pleted, the solvent is removed under reduced pressure. The light brown or yellowish-
white residue is then dissolved in H2O and the suspension is filtered to remove the dicy-
clohexylurea. The solvent is then removed under reduced pressure and the brownish
white foam 2 is dried under vacuum. (112 mg, 91% yield)
31
P: 7 (s);
1
H: 7.8 (d), 6.07 (t),
5.8 (d), 4.4 (m), 4 (m), 2.1 (m), 2.05 (m), 2.0 (dd).
General procedure for the synthesis of β,γ-methylene-deoxynucleoside triphosphate
analogues
1.1 equivalents of dried dNMP-Morph is dissolved in freshly distilled anhydrous DMSO.
In a separate flask, 4.4 equivalents of methylene bisphosphonate derivative (tributylam-
monium salt) are dissolved in anhydrous DMSO. The [Bu3NH
+
]BP solution is added
slowly to the dNMP-Morph. The reaction is monitored using analytical HPLC on an SAX
82
ion exchange column and a 0 - 100% 0.5 M TEAB buffer (pH = 8) gradient or 0 - 50%
0.5 M LiCl (pH = 8) gradient. After the reaction has reached completion, the solvent is
removed under reduced pressure. The yellowish oil is then dissolved in 1.5 mL of 0.5 M
TEAB buffer. The product is isolated from the reaction mixture via two-stage preparative
HPLC; first by SAX (0 - 100% 0.5 M TEAB gradient) and then on C-18 (0.1 N TEAB
4% CH3CN). The fractions containing the β,γ-dNTP analogue are collected and combined
and then lyophilized to the TEA salt.
Synthesis of 2’-deoxy-5’-β,γ-difluoromethylene-cytidine triphosphate (dCMP-β,γ-
DFBP, 8)
90 mg (0.239 mmol) of dried 2 is transferred to a sealed flask and is dissolved in 2 mL of
distilled anhydrous DMSO. In a separate sealed flask, the trisbutylammonium salt of
DFBP (1.05 mmol) is dissolved in 2 mL of distilled anhydrous DMSO. The
[Bu3NH
+
]DFBP solution is added slowly to the dCMP-Morph solution using a dried 5
mL air-tight glass syringe. After approximately 55 hrs, the reaction is stopped and the
solvent is removed under reduced pressure. The yellowish oil is then dissolved in 1.5 mL
of 0.5 M TEAB buffer. The product is isolated from the reaction mixture via two-stage
preparative HPLC; once on SAX and then on C-18. The fractions containing dCMP-β,γ-
DFBP are collected and combined and then lyophilized. dCMP-β,γ-DFBP is obtained as
the TEA salt (yield = 27 mg - 23%).
31
P: -11 (d), -3 (m), 3.5 (m);
31
P (decoup. to F): -11
(d), -3 (dd), 3.5 (d);
1
H: 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m), 2.1 (m), 2.05 (m), 2.0
(dd);
19
F: -117.6 (dd).
83
Synthesis of 2’-deoxy-5’-β,γ-methylene-cytdine triphosphate (dCMP-β,γ-MBP, 7)
120 mg (0.318 mmol) of dried 2 is transferred to a sealed flask and is dissolved in 2mL of
distilled anhydrous DMSO. In a separate sealed flask, the trisbutylammonium salt of
MBP (1.40 mmol) is dissolved in 2mL of distilled anhydrous DMSO. The [Bu3NH
+
]MBP
solution is added slowly to the dCMP-Morph solution using a dried 5 mL air-tight glass
syringe. After approximately 55 hrs, the reaction is stopped and the solvent is removed
under reduced pressure. The yellowish oil is then dissolved in 1.5 mL of 0.5 M TEAB
buffer. The product is isolated from the reaction mixture via two-stage preparative HPLC;
once on SAX and then on C-18. The fractions containing dCMP-β,γ-MBP are collected
and combined and then lyophilized. dCMP-β,γ-MBP is obtained as the TEA salt (yield =
31 mg - 28%).
31
P: -11 (d), 11 (d), 12.5 (dd);
1
H: 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m),
2.1 (m), 2.05 (m), 2.0 (dd).
Synthesis of 2’-deoxy-5’-β,γ-difluoromethylene-guanosine triphosphate (dGMP-β,γ-
DFBP, 4)
Following the general procedure, 47 mg (0.107 mmol) of dried 1 is dissolved in 2 mL of
freshly distilled anhydrous DMSO. The tributylammonium salt of DFBP (0.472 mmol) is
dissolved in 2 mL of freshly distilled anhydrous DMSO. The product is isolated from the
reaction mixture via two-stage preparative HPLC; once on SAX and then on C-18. The
fractions containing dGMP-β,γ-DFBP are collected and combined and then lyophilized.
84
dGMP-β,γ-DFBP
1
is obtained as the TEA salt (yield = 15 mg - 26%).
31
P: -11 (d), -3 (m),
3.5 (m);
31
P (decoup. to F): -11 (d), -3 (dd), 3.5 (d);
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m),
2.75 (m), 2.4 (m);
19
F: -117.8 (dd).
Synthesis of 2’-deoxy-5’-β,γ-monofluoromethylene-guanosine triphosphate (dGMP-
β,γ-MFBP, 5/6)
Following the general procedure, 125 mg (0.298 mmol) of dried 1 is dissolved in 2 mL of
freshly distilled anhydrous DMSO. The tributylammonium salt of MFBP (1.31 mmol) is
dissolved in 2 mL of freshly distilled anhydrous DMSO. The product is isolated from the
reaction mixture via our two-stage preparative HPLC methods. The fractions containing
dGMP-β,γ-MFBP are collected and combined and then lyophilized. dGMP-β,γ-MFBP is
obtained as the TEA salt (yield = 30 mg - 19%).
31
P: -11 (d), -4.7 (m), 7 (m);
1
H: 8 (d),
6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m);
19
F: -218.7 (m).
Synthesis of 2’-deoxy-5’-β,γ-methylene-guanosine triphosphate (dGMP-β,γ-MBP, 3)
Following the general procedure, 47 mg (0.112 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of MBP (0.494 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture via two-stage preparative HPLC. The frac-
tions containing dGMP-β,γ-MBP are collected, combined, and then lyophilized. dGMP-
β,γ-MBP is obtained as the TEA (yield = 28 mg - 50%).
31
P: -11.0 (d), 11.7 (d), 13.5 (dd);
1
H: 8 (d), 6.25 (t), 4.5 (m), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m).
85
Synthesis of 2’-deoxy-5’-β,γ-dichloromethylene-guanosine triphosphate (dGMP-β,γ-
DCBP, 9)
Following the general procedure, 60 mg (0.144 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of DCBP (0.634 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture via two-stage preparative HPLC. The frac-
tions containing dGMP-β,γ-DCBP are collected, combined, and then lyophilized. dGMP-
β,γ-DCBP is obtained as the TEA salt (yield = 12 mg - 15%) - [low yield attributed to not
allowing reaction to go to completion, apparent in analytical HPLC].
31
P: -11.0 (d), 1.73
(dd), 7.81 (d);
1
H: 8.0 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m).
Synthesis of 2’-deoxy-5’-β,γ-monochloromethylene-guanosine triphosphate (dGMP-
β,γ-MCBP, 10/11)
Following the general procedure, 60 mg (0.144 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of MCBP (0.634 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture via two-stage preparative HPLC. The frac-
tions containing dGMP-β,γ-MCBP are collected, combined, and then lyophilized. dGMP-
β,γ-MCBP is obtained as the TEA salt (yield = 9 mg - 12%) - [as with dGMP-β,γ-DCBP,
low yield attributed to not allowing reaction to go to completion, apparent in analytical
HPLC].
31
P: -11.0 (d), 8.26 (dd), 9.24 (d);
1
H: 8 (d), 6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m),
2.75 (m), 2.4 (m).
86
Synthesis of 2’-deoxy-5’-β,γ-fluorochloromethylene-guanosine triphosphate (dGMP-
β,γ-FClBP, 12/13)
Following the general procedure, 70 mg (0.167 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of FClBP (0.668 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture via two-stage preparative HPLC. The frac-
tions containing dGMP-β,γ-FClBP are collected, combined, and then lyophilized. dGMP-
β,γ-FClBP is obtained as the TEA salt (yield = 26 mg - 28%).
31
P: -11 (d), 1.8 (m), 3.5
(m);
31
P (decoup. to F): -11.0 (d), -0.91 (dt), 5.5 (dd);
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m),
2.75 (m), 2.4 (m);
19
F: -136.5 (m).
Synthesis of 2’-deoxy-5’-β,γ-dibromomethylene-guanosine triphosphate (dGMP-β,γ-
DBBP, 14)
Following the general procedure, 60 mg (0.144 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of DBBP (0.573 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture and the fractions containing dGMP-β,γ-
DBBP are collected, combined, and then lyophilized. dGMP-β,γ-DBBP is obtained as the
TEA salt (yield = 38 mg - 40%).
31
P: -11.0 (d), 2.91 (dd), 8.12 (d);
1
H: 8 (d), 6.25 (t), 4.2
(s), 4.1 (m), 2.75 (m), 2.4 (m).
87
Synthesis of 2’-deoxy-5’-β,γ-monobromomethylene-guanosine triphosphate (dGMP-
β,γ-MBBP, 15/16)
Following the general procedure, 60 mg (0.144 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of MBBP (0.634 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture via two-stage preparative HPLC and the
fractions containing dGMP-β,γ-MBBP are collected, combined, and then lyophilized.
dGMP-β,γ-MBBP is obtained as the TEA salt (yield = 33 mg - 40%).
31
P: -11.0 (dd), 7.73
(m), 8.62 (dd);
1
H: 8 (d), 6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m).
Synthesis of 2’-deoxy-5’-β,γ-[2,2-propanediyl]-guanosine triphosphate (dGMP-β,γ-
DMBP), 21
Following the general procedure, 60 mg (0.144 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of IBP (0.634 mmol) in freshly distilled anhydrous DMSO. The prod-
uct is isolated from the reaction mixture and the fractions containing dGMP-β,γ-DMBP
are collected, combined, and then lyophilized. dGMP-β,γ-DMBP is obtained as the TEA
salt (yield = 15 mg - 20%).
31
P: -11.0 (d), 19.5 (m), 23 (d);
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1
(m), 2.75 (m), 2.4 (m).
Synthesis of 2’-deoxy-5’-β,γ-[1,1-ethanediyl]-guanosine triphosphate (dGMP-β,γ-
EBP), 19/20
Following the general procedure, 90 mg (0.215 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of EBP (0.859 mmol) in freshly distilled anhydrous DMSO. The prod-
88
uct is isolated from the reaction mixture and the fractions containing dGMP-β,γ-EBP are
collected, combined, and then lyophilized. dGMP-β,γ-EBP is obtained as the TEA salt
(yield = 43 mg - 39%).
31
P: -11.0 (d), 17 (d), 18.5 (dd);
1
H: 8 (d), 6.25 (t), 4.8 (s), 4.3 (s),
4.2 (s), 3.8 (s), 2.75 (m), 2.4 (m), 2.2 (m), 1.4 (m).
Synthesis of 2’-deoxy-5’-β,γ-[1-fluoro-1,1-ethanediyl]-guanosine triphosphate
(dGMP-β,γ-FMBP), 17/18
Following the general procedure, 80 mg (0.192 mmol) of dried 1 is reacted with the tribu-
tylammonium salt of FMBP (0.769 mmol) in freshly distilled anhydrous DMSO. The
product is isolated from the reaction mixture and the fractions containing dGMP-β,γ-
FMBP are collected, combined, and then lyophilized. dGMP-β,γ-FMBP is obtained as the
TEA salt (yield = 13 mg - 13%).
31
P: -11.0 (d), 10 (m), 11.5 (dd);
1
H: 8 (d), 6.25 (t), 4.8
(s), 4.2 (s), 4.1 (m), 2.75 (m), 2.5 (m), 1.7 (dt);
19
F: -176.5 (m).
89
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script in preparation,
28. 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-
15413; McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y .; Pham, P.; Bongartz, J. P.
Phosphorus and Sulfur and the Related Elements 1988, 37, 1-12; McKenna, C. E.;
Shen, P.-D. J. Org. Chem. 1981, 46, 4573-4576.
92
CHAPTER 6
β,γ-METHYLENEBISPHOSPHONATE NUCLEOSIDE TRIPHOSPHATE
ANALOGUES AS PROBES FOR KINETICS AND FIDELITY OF DNA PO-
LYMERASE β
*Excepts taken from ref. 1
INTRODUCTION
The molecular-level characterization of DNA polymerase β (pol β)-catalyzed nu-
cleotidyl transfer is an active area of structural
2-6
, kinetic
7-10
, and computational
11
re-
search as pol β is considered a model system for understanding the nature of polymerase
substrate specificity
12
, and thus a key component of maintaining genomic integrity. Inter-
est in pol β is underscored because of its role in removing damaged nucleotides and aba-
sic sites via the base excision repair pathway, a series of enzymatic steps critical in repair-
ing damage to DNA from endogenous or exogenous reactive oxygen species and alky-
lating agents
13
. Mutant forms of pol β are found in a high percentage of human carcino-
mas
14
, suggesting a link between pol β activity and carcinogenesis which is yet to be fully
understood.
Pol β has a 31 kDa polymerase domain with three subdomains having separate
roles in binding and orienting Mg
2+
cofactors, dsDNA, and the nascent base pair. X-ray
crystallography on pol β in various liganded states
2,4,6,15-17
has identified large-amplitude
movements of the subdomains when the nascent base pair conforms to Watson-Crick ge-
93
ometry, forming a closed ternary pol-DNA-dNTP complex. Mismatched nascent base
pairs leading to single base substitution errors have proven more difficult to characterize
in ternary complexes, but are believed to be incorporated from a structurally distinct
conformation.
18,19
A comprehensive review of the structural and mechanistic studies on
pol β, spanning some 35 years, has recently appeared
18
. Consensus from numerous spec-
troscopic studies of conformational dynamics,
9,10,20
is that the large-amplitude conforma-
tional change associated with initial dNTP binding is too fast to be rate-limiting in the pol
β mechanism. Thus, attention has focused on more subtle side chain rearrangements near
the active site, and the chemical steps in the reaction pathway (primer 3’ O-, nucleophilic
attack on the dNTP α-phosphorus, and pyrophosphate leaving-group elimination).
Until recently, the only direct probe of chemical steps was thio-substitution for a
non-bridging oxygen on Pα of the dNTP.
18,21
Thio-substitution should destabilize nucleo-
philic attack on Pα and therefore raise the activation barrier of chemistry relative to non-
bridging oxygen,
22
allowing for a test of whether or not chemical activation barriers
dominate the reaction pathway. As has been described in detail,
18,21
however, interpreta-
tion of thio-effect data is complicated by pol β stereoselectivity for the Sp isomer of
dATPαS and steric considerations that lead to uncertainty in assigning a proper reference
thio-effect
23
with which to compare that measured in the enzyme. In a recent article, we
introduced the use of dNTP analogues modified at the β,γ-bridging oxygen as a probe of
the chemical step of leaving group elimination (Pα-Oαβ bond breaking) in pol β for both
correct and mispair incorporation.
8
Analyzing the transient kinetics of a series of such
94
compounds according to a linear free energy relationship (LFER) offers key advantages
over dNTPαS substrates.
Unlike thio-substitution which is limited to comparing the differential activity of
oxygen versus sulfur substituents, β,γ modification has been shown to accommodate a
range of halomethylene groups having diverse steric and electronic properties.
8
The β,γ-
bridging position is observed to protrude into a solvent exposed cleft so that, within some
bounds, the leaving-group ability and steric profile can be varied to gain a high degree of
“leverage” on the Pα-Oαβ bond-breaking activation barrier without grossly perturbing ac-
tive site geometry (FIGURE 6.1A). Additionally, the β,γ-bridging position is not in the
coordination sphere of Mg
2+
cofactors, unlike one of the non-bridging oxygens of Pα
which stereospecifically interacts with both metal cations.
16,18
In the previous work with a limited number of analogues, we observed Brønsted
plots (log kpol versus leaving group pKa) with a linear region of steep slope adjacent to a
region of reduced catalytic sensitivity at stronger stabilization of leaving group elimina-
tion,
8
leading to a proposed LFER model where two or more activation barriers lie close
in height. Trends for both correct and mispair incorporation appeared similar. Here, we
expand the analysis to include a much broader range of alkyl- or halomethylene replace-
ments for Oβγ (FIGURE 6.1B), which significantly increases the number of interpolating
points and the steric/electronic properties at the β,γ-bridging position. We find additional
evidence supporting the existence of regions of steep slope and thus a rate-limiting
chemical step. Importantly, however, the different behavior of a subset of the analogues
95
comparing correct and mispair incorporation points to structural differences in the active
site of these two processes, possibly in the conformation of the triphosphate moiety in
coordination with Mg
2+
and/or the position of Arg183 relative to Oβγ.
Figure 6.1 (A) Schematic diagram* of the pol β active site based on atomic positions from crystal
diffraction,
2
showing the positions of the primer and incoming nucleotides, catalytic Mg
2+
ions, and key
aspartate residues. In this study the β,γ-bridging triphosphate oxygen is replaced with a series of halo-
methylene groups, and the pH and solvent isotope content varied. Mechanistic effects on both correct (C-G)
and mispair (T-G) incorporation in terms of substituent electron withdrawing ability (indicated by rouge
arrow) and other steric/electronic properties (indicated in red) at the β,γ-bridging position or proton-
exchangeable sites are examined. (B) Structures of dGTP and analogues, 1-12, and bisphosphonic acids,
13-21, prepared for kinetic and thermodynamic analysis. Compound numbers follow the trend in the sub-
stituent effect on pKa4 of the bisphosphonic acids compared to pyrophosphate (see FIGURE 2). X = CF2
(1, 10); CFCl (2, 11); CCl2 (3, 12); O (4, 13); CHF (5, 14); CBr2 (6, 15); CHCl (7, 16); CHBr (8, 17); CH2
(9, 18), CFCH3 (10, 19), CHCH3 (11, 20), and C(CH3)2 (12, 21). *From C. A. Sucato, Ph. D. Dissertation,
University of Southern California as well as ref. 1.
1-12
13-21
1-12
96
MATERIALS AND METHODS
High purity solution dGTP and T4 Polynucleotide kinase (Optikinase) were pur-
chased from GE Healthcare. DNA synthesis reagents and protected deoxyribonucleoside
phosphoramidites were purchased from Applied Biosystems. Radiolabeled [γ-
32
P]ATP
was purchased from MP Biomedicals. Other buffer components and chemicals for dGTP-
analogue synthesis were purchased from Sigma-Aldrich or GE. Recombinant human WT
pol β was expressed in E. coli and purified as described previously.
24
SYNTHESIS OF β,γ-METHYLENE-dGTP ANALOGUES
The dGTP analogues were synthesized and characterized as described
5,8
previ-
ously in CHAPTER 5. Briefly, analogues of dGTP were prepared from dGMP morpholi-
date and the corresponding substituted-methylenebisphosphonic acid, purified by two-
stage preparative HPLC (> 99%), and characterized by
1
H,
31
P,
19
F NMR and UV-visible
spectra, analytical HPLC (Varian PureGel R00087E1PE 7 µM 500Å SAX), and negative
ion FAB or MALDI HRMS. The monomethyl- and monohalogenated analogues are dia-
stereomeric; racemic mixtures of the R and S configuration at the β,γ-bridging carbon
were used in kinetic assays.
DNA SYNTHESIS/PURIFICATION, RADIOLABELING, AND ANNEALING
Primer (TAT TAC CGC GCT GAT GCG C), template (GCG TTG TTC CGA
CMG CGC ATC AGC GCG GTA ATA; M=C, T), and 5'-end-phosphorylated downstream
(GTC GGA ACA ACG C) oligomers were synthesized on a solid phase DNA synthesizer
97
and purified as described previously.
8
Primer DNA (1 molar equiv) was 5'-end labeled
using T4 polynucleotide kinase (0.4 U/µL) and [γ-
32
P]ATP (~0.7 molar equiv) using the
supplied buffer. The kinase was inactivated after 30 min by heating at 90 °C for 10 min,
and the reaction mixture was used for annealing without further purification. Annealing
was carried out by mixing primer, template (1.2 molar equiv), and downstream (1.5 molar
equiv) oligomers, heating to 90 ºC, and then cooling slowly to room temperature.
BUFFER PREPARATION
The assay buffers used to study dGTP or analogue incorporation kinetics con-
sisted of 50 mM Tris-Cl, 20 mM KCl, 20 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 6 v/
v % glycerol. Buffers were prepared at various acidities by adjustment with HCl or
NaOH solution and monitoring pH with a Tris-compatible electrode.
SINGLE-TURNOVER GAP-FILLING ASSAYS
Single-nucleotide gapped DNA (template C or T) was preincubated for ~2 min at
37 °C with pol β in assay buffer and then mixed with a solution of substrate (dGTP or
analog in assay buffer at 37 °C). Concentrations after mixing were: 300 nM pol β, 50 nM
DNA, 0.5 - 20 µM substrate (C-G incorporation) or 50-1500 µM substrate (T-G incorpo-
ration). The reaction mixture was quenched by ~3-fold dilution with 500 mM EDTA. Re-
action time ranges were 0.09 - 0.5 s (C-G) and 0.5 - 100 s (T-G). Reaction times under 20
s were carried out with a rapid-mixing chemical quench apparatus (KinTek model RQF-
3), otherwise mixing was done manually with a micropipette. The radiolabeled 19-mer
98
primer and 20-mer product were separated by denaturing polyacrylamide gel electropho-
resis (39×33×0.035 cm, 20 w/v % polyacrylamide, 8 M urea gels run at 65 W, 4.5 h).
Table 6.1 Kinetic and thermodynamic parameters for incorporation of β,γ-bridging dGTP analogues in
pol β-catalyzed single-turnover assays. Results described in C. A. Sucato, Ph. D. Dissertation, University of
Southern California as well as ref. 1.
kpol/Kd
M-N -X- kpol (s
-1
) Kd (µM) (s
-1
M
-1
)×10
5
fidelity
C-G
b
CF2 21.9 ± 0.9 2.9 ± 0.3 75
C-G CFCl 14.9 ± 0.3 3.4 ± 0.5 44
C-G CCl2 8.4 ± 1.0 3.7 ± 1.3 23
C-G
b
O 14.9 ± 2.3 1.7 ± 0.7 87
C-G
b
CHF 14.6 ± 2.3 2.2 ± 1.5 65
C-G CBr2 3.1 ± 0.2 11.4 ± 1.4 2.7
C-G CHCl 8.1 ± 3.0 3.0 ± 1.7 27
C-G CHBr 6.9 ± 0.8 2.1 ± 0.2 33
C-G CFCH3 3.1 ± 0.2 1 ± 0.2 31
C-G
b
CH2 1.7 ± 0.2 0.9 ± 0.2 19
C-G CHCH3 0.3 ± 0.01 1 ± 0.14 3
C-G C(CH3)2 0.27 ± 0.04 4.3 ± 3.6 0.6
T-G
b
CF2 0.76 ± 0.01 380 ± 50 0.02000 3700
T-G CFCl 0.23 ± 0.03 390 ± 80 0.00590 7600
T-G CCl2 0.05 ± 0.00 800 ± 70 0.00063 37000
T-G
b
O 1.34 ± 0.10 200 ± 7 0.06600 1300
T-G
b
CHF 0.93 ± 0.06 270 ± 130 0.03400 1900
T-G CBr2 0.02 ± 0.01 500 ± 200 0.00036 7600
T-G CHCl 0.23 ± 0.03 410 ± 50 0.00560 4800
T-G CHBr 0.14 ± 0.02 470 ± 130 0.00300 11000
T-G CFCH3 0.22 ± 0.02 108 ± 8 0.02037 1500
T-G
b
CH2 0.12 ± 0.03 470 ± 110 0.00250 7100
T-G CHCH3 0.01 ± 0 745 ± 180 0.00016 3
T-G C(CH3)2 0 ± 0 1044 ± 66 0.00004 0.6
a
Assays were performed by rapid mixing chemical quench at a pH of 8.0 (23 °C). M-N is the template-
incoming base-pair and -X- is the β,γ-bridging group. Values of kpol and Kd are reported as the mean ± stan-
dard deviation of three replicates, and error in the efficiencies (kpol/Kd) and fidelity, (kpol/Kd)CG/(kpol/Kd)TG,
are calculated according to the standard rules for error propagation.
b
Data from Ref 8 used in average.
99
Percentage of primer extended was measured by exposure of the dehydrated gel to a stor-
age phosphor screen followed by detection of phosphorescence emission on an imaging
system (Molecular Dynamics STORM 860). For β,γ-methylene-analogues work, three
replicates were performed for each substrate/template base combination and the best-fit
values for kpol and Kd are reported in TABLE 6.1 as the mean ± standard error. The ana-
logue assays were performed at a pH of 8.0.
DATA ANALYSIS
Kinetic assays were performed under single-turnover conditions (E>>S) where
the time evolution of product formation fits a single exponential: [P]t = [P]final(1-exp[-
kobst]), where P is the extended primer product and kobs is the observed catalytic rate con-
stant. The observed rate constants were plotted against the substrate concentrations, and
the data fit to the hyperbolic equation: kobs = kpol[S]/([S] + Kd), where S is dNTP substrate
(dGTP or analogue), kpol is an overall rate constant for the nucleotide insertion step(s),
and Kd the dissociation constant for substrate binding. The pH dependence of kpol was fit
to a sigmoidal function describing a single ionization: kpol = klim(1+10
pKa–pH
)-1 + ko;
where klim and ko define kpol at the limits of pH. Fidelity for dGTP or analogue incorpora-
tion was calculated as (kpol/Kd)CG/(kpol/Kd)TG, where the subscripts refer to correct (C-G)
and mispair (T-G) incorporation. The above terms can be rearranged to (kpol,CG/kpol,TG)
(Kd,TG/Kd,CG), to reflect contributions from catalytic and binding terms, respectively.
100
RESULTS AND DISCUSSION
The use of dNTP analogues modified at the β,γ-bridging oxygen as a probe of
nucleotidyl-transfer mechanism has been demonstrated with pol β
8
using compounds hav-
ing CH2, CHF, and CF2 at the Oβ,γ (1, 5, and 9 in FIGURE 6.1B). The analysis is now
expanded to include alkylsubstituted and halogenated moieties (2, 3, 6-12) at the β,γ-
bridging position having a more extensive set of steric and electronic properties. The con-
jugate acids of the respective pyrophosphate analogue leaving groups (13-21) were also
prepared for systematic evaluation of their stability constants. Detailed procedures for the
synthesis and purification of Oβγ-modified dNTP analogues and corresponding bisphos-
phonates are given elsewhere
5
(see CHAPTERS 2 & 5). Presently, our expanded set of
Oβγ-modified dGTP analogues makes necessary the characterization of bisphosphonic
acid protonation equilibria for heretofore unreported species, as well as the compilation
of such values for the entire series under a set of identical, well-defined conditions for
consistency in LFER determinations. Using the bisphosphonic acids 10-18 as analytes,
potentiometric titrations for the series of compounds with KOH and identical conditions
of temperature and ionic strength have been performed(detailed in CHAPTER 3).
26

These data are indicated in the legend of FIGURE 6.2. The trend in pKa values along
with the number of electronegative halogens on the bridging group, and a comparison
with available literature values, is discussed elsewhere.
26
101
Figure 6.2 Brønsted correlations of log(kpol) versus leaving-group pKa4. Data for dGTP and ana-
logues, 1-12, incorporated opposite (A) the correct template base C, and (B) the mispairing template base T.
For both C-G and T-G curves the data for mono-halogenated analogues, the methylene analogue, and native
dGTP conform to a linear relationship between pKa4 9-11 (βlg,CG = -0.56 and βlg,TG = -0.68). The catalytic
sensitivity to leaving group ability in this region is similar to that reported for other enzymatic P-O bond
cleavages,
23,25
and suggests leaving-group elimination is rate-limiting (or near rate-limiting) for incorpora-
tion of dGTP opposite either template. The di-halogenated analogues (connected by separate solid lines)
show deviation from the other data which is more pronounced for incorporation of the mispair (B). pKa4
values obtained by potentiometric titration at 25 ºC and 0.1 M ionic strength, ref. 26: X = CF2 (7.8); CFCl
(8.4); CCl2 (8.8); CHF (9.0); CBr2 (9.3); CHCl (9.5); CHBr (9.9); CFCH3 (10.2); CH2 (10.5); CHCH3
(11.6); C(CH3)2 (12.2). The pKa4 value for pyrophosphate (8.9) is from Ref. 27. Results described in C. A.
Sucato, Ph. D. Dissertation, University of Southern California as well as ref. 1.
102
Pol β-catalyzed nucleotidyl transfer using Oβγ-modified dGTP analogues
Our primary objective in designing a series of halo-substituted dNTP analogues is
to slow down or speed up the chemical step of leaving group elimination in order to
probe its relative prominence in the overall scheme of nucleotidyl transfer. The LFER
established with such a series of substrates is a well-known tool for deciphering reaction
mechanisms in solution and in enzymatic reactions.
28-30
In order to be effective in an
LFER analysis, the Oβγ modification must alter the activation barrier for Pα-Oαβ bond
breaking and the subsequent product state to the exclusion of other local transition or
ground states along the reaction coordinate, including any conformational states involv-
ing protein or DNA moieties. Key evidence in favor of these assumptions is that X-ray
crystal structures of the CH2- and CF2-analogues in ternary complex with pol β and DNA
are in excellent overlap with the conformation of native dNTP, and catalytic active-site
components are properly aligned;
8
Kd data on previously assayed analogues are also
comparable with native dNTP binding.
8
Despite these positive assertions, implications of
the previous work on Oβγ modification rested on relatively few analogues between the
extremes of leaving group acidity (i.e. between CF2- and CH2-analogues). In this work
we increase the number of interpolating points from two to ten, varying charge and size,
so as to arrive at new and more general mechanistic insights.
Single-turnover transient kinetic assays of pol β-catalyzed incorporation of ana-
logues 2, 3, 6-12 into 1-nt gapped DNA were performed in a standard manner by rapid
mixing chemical quench. Two different template DNA strands were studied (templating
base C or T). Three independent trials were performed with each analogue to obtain the
103
parameters kpol and Kd, the overall catalytic rate- and dNTP dissociation constants, re-
spectively. These parameters are shown in TABLE 6.1 (FIGURE 6.3 depicts the result
of a typical assay). Brønsted plots are shown in FIGURE 6.2 according to the relation
log(kpol) = βlg(pKa4) + C. The constant βlg is a measure of the sensitivity of kpol to leaving
group ability, and C (the intercept) is a constant of no physical significance.
Figure 6.3 Single-turnover assay of CHBr-analogue (racemic mixture of compound 8, FIGURE 6.1)
kinetics for insertion opposite template C, showing saturation of the observed single-exponential rate con-
stant, kobs, as a function of substrate concentration. Plots of kobs versus substrate concentration were least-
squares fit to the hyperbolic equation (see Data Analysis) to obtain the catalytic rate constant kpol, and the
substrate dissociation constant Kd. Inset: The percentage of
32
P 5'-end-labeled primer extended during the
course of the nucleotidyl transfer reactions. The curves are the best least-squares fits of the data to a single
exponential, yielding kobs at each substrate concentration. Experiments were done in triplicate for all sub-
strates 1-12 opposite template C or T, and the results are tabulated in TABLE 6.1. The plots here are repre-
sentative of the typical signal-to-noise and fluctuations observed in the data acquisition. Results described
in C. A. Sucato, Ph. D. Dissertation, University of Southern California as well as ref. 1.
k
104
Kinetics of correct-base incorporation: dGTP analogues opposite template base C
The chief finding of our earlier work
8
primarily with fluoro-methylene bridging
groups was a biphasic LFER suggesting a change in rate limiting step due to modification
at Oβγ; the position for the oxo-bridging compound near the apparent intersection of two
linear regions suggested that for the natural dNTP two or more activation barriers lie ap-
proximately equal in height on the reaction coordinate. The present work adds interpolat-
ing points to the Brønsted plot for incorporation opposite template C that allows for more
detailed evaluation (FIGURE 6.2A).
First, the mono-halogenated compounds studied now comprise CHBr, CHCl, and
CHF groups. Data points for these compounds fall between the O- and CH2-analogue
points and are colinear. The βlg calculated for the region between pKa4 9–11 is relatively
steep, -0.56, consistent with significant contribution of the elimination step to the rate-
limiting transition state. Second, the recent pKa study of bisphosphonic acids
26
indicates
that Claessens' pKa4 value for the CCl2-analogue,
31
upon which we relied in our previous
work,
8
is high by about 1 unit; we have adjusted this value to 8.8 as shown in FIGURE
6.2. At a pKa4 of 9.7 the CCl2-analogue position fell colinear with that for O-, CHF-, and
CH2-dNTPs; with the present value—that better represents the trend in halo-
substitution
26
—this point now deviates from the rightmost line in FIGURE 6.2A, as does
that for the bulky CBr2-bridging compound. Additionally, the points for the CF2- and
CFCl-analogues deviate in the same direction (reduction in kpol relative to a single linear
105
trend). Furthermore, the β,γ-alkylmethylene-analogues follow the expected trend for
alkyl-substitution as it relates to pKa4 of the methylene-bridged leaving group.
The systematic reduction in catalytic activity with dihalogen-substituted ana-
logues raises the possibility that steric or electronic properties may affect kpol, and the
log(kpol) vs. pKa4 data for these compounds, suggesting that they may more appropriately
be treated separately from the others. Although the deviation between the linear relation-
ships drawn for the dihalogen compounds (leftmost line in FIGURE 6.2A) and the others
(rightmost line) appears modest, such separate treatment will be justified when the kinet-
ics of mispair incorporation are considered below.
The Kd values for binding with the new analogues (TABLE 6.1) confirm previous
structural and binding equilibrium data suggesting that a wide range of alkyl-and halo-
methylene groups are accepted at the β,γ-bridging position with minimal perturbation of
substrate binding. For incorporation opposite template C the Kd values, with the excep-
tion of the CBr2-analogue, fall in the narrow range 1 - 4 µM, in excellent agreement with
that of native dGTP.
Kinetics of mispair incorporation: dGTP analogues opposite template base T
Prior to expanding the series of β,γ-bridging analogues to include new alkyl- and
halo-substitutions, the shape of Brønsted correlations for C-G and T-G incorporation ap-
peared similar both in βlg in the catalytically sensitive region and in the position of the
break in the biphasic plots,
8
suggesting that both processes, i.e., right compared with
106
wrong incorporations, are catalyzed via similar rate limiting chemical steps, as opposed
to a dominant conformational change barrier. Here, we find that addition of points for the
expanded set of analogues affirms that a catalytically-sensitive linear region exists at pKa4
≥ 9 (βlg = -0.68). The considerably diminished activity for dihalogen compounds opposite
template T, however, underscores important differences between correct and mispair in-
corporation mechanisms.
While the kinetics of the mono-halogenated analogues put their respective points
tightly colinear with those of O- and CH2- compounds (FIGURE 6.2B), as was the case
in FIGURE 6.2A, the new dihalogenated analogues (CFCl-, CCl2-, CBr2-) show devia-
tions to a significantly greater extent than observed in correct base incorporation, pre-
sumably due to the increased steric bulk of the halogen substituents. However, the argu-
ment for steric bulk is not supported by the data presented by the methyl-substituted ana-
logues (CFCH3-, CHCH3-, C(CH3)2-) which allude to greater electronic effect in the
dihalo-analogues. This is illustrated by comparing dihalogen lines with the rightmost
lines in each of FIGURES 6.2A and 6.2B. The trend in the dihalogen data, besides show-
ing diminished activity, is linear and parallel to the rightmost line in FIGURE 6.2B. In-
voking a “dihalogen correction factor” could reconcile all points onto a single LFER line,
by shifting the dihalogen points upward, but the physical basis for such a correction is not
well defined. If plausible, it would suggest that the LFER behavior of T-G mispair incor-
poration, after dihalo- correction, would extend with negative slope over the entire pKa4
range shown and not exhibit a break in linearity near the pKa4 of the native pyrophos-
phoric acid. The same procedure could in principle be applied to the correct incorporation
107
plots in FIGURE 6.2A, with the same result albeit with smaller presumed correction fac-
tor.
In a theoretical model, if the activation barrier to leaving group elimination is suf-
ficiently high relative to other barriers near the transition state, perturbation of its height
(i.e. by Oβγ modification) would result in a single linear plot in the LFER formalism. On
the other hand, if the leaving group elimination barrier lies close in height to its neigh-
bors, sufficient stabilization (at the far left of FIGURES 6.2A and 6.2B) would be pre-
dicted to result in a break in linearity with a catalytically insensitive (zero slope) region at
low leaving group pKa. Reconciling the experimentally observed plots with either theo-
retical model (single line versus biphasic) is difficult, even with the expanded set of al-
kyl- and halomethylene-bridged analogues, because the CF2-methylene species remains
the strongest leaving group, and in both correct and T-G mispairing incorporation its
point falls roughly level with that of the native compound, so a theoretical biphasic pro-
file cannot be ruled out. Therefore, future work in this area should address this hypothe-
sis. Regardless of theoretical model, the significantly stronger dihalogen-dependent re-
duction in catalysis for T-G mispairing suggests energetic and structural differences com-
pared to correct incorporation.
The Kd values for analogues in mispair assays (TABLE 6.1) do not show the
same trend in deviation as observed for log(kpol). The CCl2-analogue has a Kd approxi-
mately 4-fold higher than that of dGTP, but those for CBr2- and CFCl- analogues are no
higher than Kd values for mono-halogenated analogues. Therefore, the differences im-
108
plied by FIGURE 2 likely occur at the chemical transition state as opposed to conforma-
tionally more stable intermediates. The intermolecular steric/electrostatic effects may be
triggered as small geometric distortions, propagated from the base moiety of the mispair
to the catalytic site.
32
Effect of Oβγ modification on fidelity
Fidelity in this work is computed as the ratio of catalytic efficiencies (kpol/Kd) for
substrates with the same nucleobase (dGTP or analogue), opposite two different template
bases, C or T. This “template-variable” fidelity (TABLE 6.1) contrasts with polymerase
selectivity in vivo, where four different nucleobases compete for incorporation opposite
the same template base. Since the environment of the template base and that of the dNTP
are different in the active site, selectivity measured by template-variable fidelity may not
be the same as the in vivo process. In either case, however, the enzyme preferentially
catalyzes the correct-pairing reaction, so template-variable fidelity is a valid measure of
polymerase mechanism. Reckoning fidelity in this manner allows for comparison with
computational simulations and facilitates further computational analysis of Oβγ modifica-
tions, where a template-variable model is adopted to minimize inaccuracies in calculated
parameters.
8,33
Our results with the expanded series of methyl- and halo-substituted analogues
indicate that overall, Oβγ modification results in an increase in fidelity for T-G mispair
incorporation (roughly 27-fold for the CCl2-analogue). The strongest enhancement in dis-
crimination is a direct result of the large reduction in kpol for incorporation of the mispair
109
with the dihalogen-substituted compounds. This enhancement is further illustrated in
FIGURE 6.4, where the separate catalytic (kpol,CG/kpol,TG) and binding (Kd,TG/Kd,CG) con-
tributions to fidelity are graphed. The strong effect on pol β fidelity with the CCl2-
analogue is due to a favorable catalytic contribution. Because of its poor binding opposite
template C, the CBr2-analogue does not have the strongest effect on fidelity, even though
its catalytic term is larger than that of the CCl2-analogue.
Figure 6.4 Effect of Oβγ modification on fidelity. The kpol and Kd ratios (discussed in the text) for
each analogue are plotted relative to that of native dGTP, and account for contributions to fidelity (right)
from catalysis and substrate binding, respectively. Error bars are calculated by propagation of error in the
corresponding kpol and Kd terms. The dominant effect is a roughly 27-fold increase in fidelity for the CCl2-
analogue due to marked reduction in kpol for mispair incorporation. Surprisingly, an equal-or-greater effect
is not observed with the CBr2-analogue despite a favorable kpol ratio. This can be explained by relatively
poor binding of the CBr2-analogue opposite the correct template base C. Results described in C. A. Sucato,
Ph. D. Dissertation, University of Southern California as well as ref. 1.
0
5
10
15
20
25
30
35
40
45
relative units








TG pol
CG pol
k
k
,
,








CG d
TG d
K
K
,
,
CCl
2
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
CCl
2
CBr
2
fidelity
dGTP analog
CBr
2
The roughly 27-fold increase in fidelity for the CCl2-analogue is notable, consid-
ering that the leaving group for this substrate, compound 10, is one of the most investi-
gated of the first generation bisphosphonate class of drugs (clodronate), used clinically
110
for over 15 years in the treatment of hypercalcemia and bone metastases.
34,35
Simple
bisphosphonates, those that closely resemble pyrophosphate, are thought to act via intra-
cellular conversion into β,γ-bridged analogues of ATP, which are likely inhibitors of ATP-
utilizing enzymes.
34
To our knowledge there is no documentation of “simple” bisphos-
phonate conversion to the analogous deoxy-ribonucleotide substrates in humans, so the
potential for clodronate targeting polymerase activity in human cells remains to be dem-
onstrated, despite the significant effect observed here when clodronate is synthetically
linked to dGMP.
X-ray crystal diffraction studies of pol β in various liganded states,
2,4,6,15-17
begun
over a decade ago, have provided the structural insight as to how protein residues could
facilitate conformational changes and the chemical steps of nucleotidyl transfer. Recent
structures obtained with non-hydrolysable dNTP analogues
6
that allow the crucial primer
3’-OH and Mg
2+
ions to be retained largely confirmed and extended the earlier findings
employing abortive dideoxy-terminated substrates.
16
In terms of geometry near the site of
chemical steps (FIGURE 6.1A), the nucleotide-binding Mg
2+
coordinates nonbridging
oxygens on all three phosphates of the correct incoming dNTP (α,β,γ tridentate), whereas
the other, so-called “catalytic” Mg
2+
, coordinates the same non-bridging oxygen on Pα,
and directly coordinates the primer 3’ oxygen, activating it for in-line nucleophilic attack.
The implication of the Mg
2+
coordination scheme depicted in FIGURE 6.1A is that the
negative charge developing on Oα,β is not directly stabilized by coordination with either
metal cation. Moreover, inspection of high resolution crystal structures shows no amino
acid residue likely to provide such stabilization
17
. It is important to emphasize that these
111
structural findings regarding conformational changes and the positioning of active site
participants are limited to Watson-Crick nascent base pairs. The ternary pol-DNA-dNTP
complexes which provide structural information that is currently the most relevant to the
chemical transition state (in the absence of a transition state analogue structure), remain
an elusive target when the nascent base pair does not conform to Watson-Crick base pair-
ing,
18
impeding efforts to characterize the insertion mechanism of base substitution er-
rors.
The primary motivation for applying LFER methodology to leaving group elimi-
nation in the pol β system is to establish a direct probe of the energetics of the chemical
steps of nucleotidyl transfer, and to potentially distinguish between different models of
LFER behavior which relate the height of leaving group elimination barriers to other
steps in the pathway. The diminished catalytic activity observed for dihalo-methylene
analogues, with greater magnitude for T-G mispair incorporation compared to C-G incor-
poration, was unexpected. This observation complicates interpretation of the experimen-
tal LFERs of FIGURE 6.2 in terms of ideal model behavior, but offers an opportunity to
infer structural differences between correct and mispair incorporation from a perspective
unavailable from x-ray crystallography alone.
FIGURE 6.5 depicts the structure of the triphosphate portion of the CF2-bridging
analogue bound in the ternary pol β complex, viewed from an angle where Arg183 is in
the foreground and the nucleotide binding metal in the background. C-F bonds are di-
rected out toward the viewer. Two possible interactions between β,γ-bridging group halo-
112
gens and active-site atoms are illustrated in FIGURE 6.5 that may account for the di-
minished activity observed with the dihalogen groups.
Figure 6.5 An illustration of possible interactions between bulky halogen atoms at the β,γ-bridging
position and other elements of the active site. Atomic positions in the pol β active site are from a crystal
structure of an abortive ternary complex with dGTP(β,γ)CF2 10 opposite correct template base C.
8
Repul-
sion between the pro-S halogen and Oαβ (arrow 1) or a catalytically-debilitating steric/electrostatic interac-
tion between the pro-R halogen and Arg183 (arrow 2) may be more pronounced in the mispair, indicative
of active-site structural differences at the chemical transition state. The magnesium ion, shown in green,
coordinates non-bridging oxygens on the triphosphate moiety and does not interact directly with the β,γ-
bridging group. Study described in C. A. Sucato, Ph. D. Dissertation, University of Southern California as
well as ref. 1.
P
C
P
Mg
P
Oαβ
δ−
δ−
δ−
Arg183
δ−
α
β
γ
δ+
δ+
F
F
(1)
(2)
First, intramolecular repulsion between the negative charge developing on Oαβ
and the pro-S halogen may hinder leaving group elimination. All-atom empirical valence-
bond/free-energy perturbation (EVB/FEP) simulations in pol β and aqueous solution car-
ried out for incorporation of the CF2-analogue
8
suggested heightened chemical activation
113
barriers for this compound with contributions from intramolecular steric and electrostatic
repulsion involving Oαβ and one of the two fluorine atoms. Since this contribution (arrow
1, FIGURE 6.5) is larger in the transition state than in the reactant state it may cause the
diminished catalytic activity observed for the dihalogen compounds. Upon going from F
to larger but less electronegative Cl and Br atoms we expect the electrostatic repulsion to
decrease and steric repulsion to increase. This offsetting trend may explain the small de-
pendence of the dihalogen effect on the van der Waals size of the halogen atom and the
good fits to linearity for dihalogen-bridged compounds in FIGURE 6.2.
Theoretical analyses have indicated that the LFER for phosphoryl transfer reac-
tions are expected to be similar in enzymes and in solutions as long as the rate limiting
steps are similar,
36
regardless of the structural details of the enzyme active site. Although
anomalous behavior when, for example, product states containing negatively charged
leaving groups with lower pKa would be less stable than groups with higher pKa cannot
be completely excluded, the fact that pol β active-site structure shows the absence of any
metal cation or enzyme group capable of directly stabilizing the negative charge that de-
velops on Oαβ during leaving group elimination
17
greatly reduces the chance of such
anomaly by eliminating quantum chemical effects associated with strong hydrogen bond-
ing or metal-ligand complexes. Thus, we expected to observe a LFER with a slope simi-
lar to the slope observed in model diester hydrolysis reactions in aqueous solution.
28-30,37

Since this LFER is obtained (FIGURE 6.2) it is likely that P-O bond breaking contrib-
utes significantly to the rate-limiting step.
114
A second possibility, not mutually exclusive of intramolecular repulsion with Oαβ,
is that an interaction between the pro-R halogen and the nearby guanidino group of
Arg183 is catalytically debilitating (arrow 2, FIGURE 6.5). Arg183 forms an H-bond
with the β phosphate in correct incorporation of natural dNTPs and plays an important
role in transition state stabilization;
38
the presence of additional negative charge density
nearby, in the form of the pro-R halogen, may interrupt this transition state stabilization.
Recently, it has been shown that a stereospecific pol β ternary complex forms with the R
isomer of the CHF-analogue (5), despite both R and S isomers being present in the crys-
tallization mixture,
5
suggesting the R isomer is stabilized by a preferential H-F interaction
with Arg183. What effect such H-F interactions would have on catalysis is unclear from
crystal structures since hydrogen atom positions are uncertain. The precise nature of in-
teractions between Cl or Br atoms and the Arg183 guanidino group is also unknown, ab-
sent from ternary crystal structures with these analogues.
The same interactions described above and in FIGURE 6.5 may also occur to
some extent with the monohalogenated analogues. The finding that catalysis is severely
diminished for dihalogenated species, whereas monohalogenated compounds are collin-
ear with non-halogenated O- and CH2-bridged compounds, may suggest that only when a
combination of halo-effects from both the pro-R and pro-S positions are present is the
LFER perturbed.
The report of stereospecific pol β ternary complex formation with the R isomer of
the CHF-analogue,
5
under crystallization conditions, also raises the question whether an
115
analogous preference for the R isomer occurs under conditions of our kinetic assays,
since the monohalogenated analogues used here are racemic mixtures of the R and S con-
figurations at the β,γ-bridging position. Interestingly, while the CHF-bridging group
shows a stereospecific preference under crystallization conditions, the CHCl- and CHBr-
groups do not.
5
Careful inspection of the time courses and saturation curves for primer
extension (FIGURE 6.3) can also provide limited clues as to the behavior of the two dia-
stereomers. That Kd data for monohalogenated analogues opposite template C fall in the
same narrow range as dihalogenated analogs and the native dGTP, and time courses giv-
ing kobs are good fits to single exponentials rather than double exponentials, argues
against very large differences in binding or catalysis behavior for the R versus S configu-
ration under the reaction conditions used for this work. This does not exclude the possi-
bility that more subtle differences between isomers exist. The availability of pure dia-
stereomers (requiring a clever strategy for purification of diastereomeric dNTP analogues
or chiral bisphosphonate derivatives) would help resolve this question.
CONCLUSION
The probes of nucleotidyl transfer chemistry described here have been used in the
pol β system to separately evaluate the chemical steps of correct and mispair incorpora-
tion. The magnitude of diminished activity using dihalogenated leaving groups, depend-
ent on the identity of the template base, is a key finding and may help to identify impor-
tant structural differences in transition states for correct and mispair incorporation, and to
suggest future protein active-site mutagenesis experiments and computer simulations that
116
can be used to verify and refine hypotheses on the nature of structural differences. The
fidelity of pol β is intermediate between the highly accurate replicative polymerases and
the most error-prone;
39
further progress in understanding nucleotidyl transfer mechanism
may therefore come in applying the methodology described here to other polymerases,
and to other combinations of nascent base pairs including mispairs less stable than the
relatively facile T-G mispair.
117
CHAPTER 6 REFERENCES
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120
CHAPTER 7
β,γ-METHYLENEBISPHOSPHONATE NTP ANALOGUES - DNA IN TERNARY
COMPLEX WITH DNA POLYMERASE β: EVIDENCE OF STEREOSELECTIVE
BINDING DUE TO AN INTERESTING C-F···H-N INTERACTION WITH ARG183
*Excerpts taken from McKenna, et al; J. Am. Chem. Soc. 2007, 129, 15412-15413.
INTRODUCTION
DNA polymerases are crucial to maintaining the fidelity of genetic information
encoded into DNA, and failure to repair aberrant bases in damaged DNA strands is noto-
riously implicated in oncogenesis.
1-10
During base excision repair (BER)
11,12
, DNA po-
lymerase β (pol β) typically inserts a single deoxynucleoside triphosphate (dNTP) replac-
ing an excised damaged or mismatched nucleoside residue with release of pyrophosphate.
In the ongoing effort to elucidate mechanistic details of these processes, pol β, the small-
est eukaryotic cellular DNA polymerase, has been the subject of extensive studies exam-
ining its key role in BER
12
and cancer
1-10,13
.
Designed modifications in the structures of natural dNTPs or NTPs can provide
information on molecular interactions with nucleic acid polymerases.
12-23
Often the ana-
logues have been modified in their purine or pyrimidine bases or (deoxy)ribose moieties.
However, changes in the triphosphate group
14-18,20-23
are of particular interest because
they involve the locus of chemical transformation catalyzed in the polymerase active site.
Replacement of the Pα-O-Pβ bridging oxygen by a carbon atom will prevent hydrolysis,
121
whereas a Pβ-CXY-Pγ modification alters the leaving group properties depending on the
nature of substituents X and Y . The introduction of these substituents may also enable
entirely new bonding (or repulsive) active site interactions, not present with the natural
nucleoside triphosphate, and thus could inform mechanistic insight as well as inhibitor
design seeking to exploit pol β as a drug target.
Figure 7.1 β,γ-methylene analogues of dGTP. X = CH2 (1), CF2 (2), CHF (3/4), CCl2 (5), CBr2 (6),
CHCl (7/8); CHBr (9/10), CHCH3 (11/12), C(CH3)2 (13), CFCH3 (14/15), CFCl (16/17).
NH
N
N
N
O
O
OH
O P O
O
O
P P
O
O
O
O
O
X
Y
NH
2
Recently, a series of unfluorinated (X,Y = H; 1) and fluorinated (X,Y = F; 2, and
X=F and Y=H; 3 (R) and 4 (S)) β,γ-methylenebisphosphonate analogues of dNTP (see
FIGURE 7.1) were used to probe leaving group effects on pol β catalysis and fidelity.
24

These analogues are substrates of the polymerase, but release an X,Y-
methylenebisphosphonate in place of the natural pyrophosphate leaving group. Several
β,γ-CXY-NTP analogues were previously investigated as inhibitors of viral RNA or
RNA-directed DNA polymerases
15,18,25
, but the structures of the putative complexes
formed during turnover were not determined. In all cases, CHX analogues were used as
the diastereomer mixtures obtained synthetically, and the potential for a stereospecific
interaction with the enzyme active site has received little attention. Here, we report X-ray
122
crystallographic studies and computer-based docking simulations of terminal ddNMP
primer-template-pol β complexes with dGMP-β,γ-fluoromethylenebisphosphonates 3 and
4, leading to the first demonstration of stereospecific dNTP-analogue-enzyme complex
formation determined by the β,γ-CHX configuration of the analogue. Further investiga-
tion with the more complete series of dGTP analogues (5-17), supports evidence for
stereospecific dNTP-analogue-enzyme complex formation due to specific bridging inter-
actions between the β,γ-CXF-dGTP-C-F···H-N-Arg183.
MATERIALS AND METHODS
β,γ-dGTP analogs (see FIGURE 7.1) were prepared by conjugation of dG 5’-
phosphoramidate with the appropriate methylenebisphosphonic acid. For a detailed pro-
cedure with experimental data see CHAPTER 5. For protein crystallography, human pol
β was over-expressed in E. coli and purified as described previously.
26
The double-
stranded DNA substrate consisted of a 16-mer template (5’-
CCGACCGCGCATCAGC–3’), a complementary 9-mer primer (5’-GCTGATGCG-3’),
and a 5-mer downstream oligonucleotide (5’-pGTCGG-3’), thus creating a two-
nucleotide gap with annealed primer. The analogues were added to a solution of the pre-
formed protein-DNA complex.
Molecular docking calculations
27
have proved valuable in studying protein-ligand
interactions and prediction of experimental outcomes, especially in enzymatic inhibition
and catalysis studies. Prior to obtaining this structure, we carried out an exploratory
ligand docking study using the 2FMS structure of pol β.
28
Ligands and the macromole-
123
cule were prepared and docked using standard docking procedures.
29
and used to formu-
late docking calculations using Autodock 3.0.
30,31
The original ligand (α,β-NH dUTP)
28

was converted
32
to the corresponding β,γ-difluoromethylene-dTTP and energy-minimized
using Hartree-Fock 3-21G* theory. Docking runs predicted a preferred triphosphate chain
orientation similar to that of natural dNTPs, but notably this placed one of the two dia-
stereotopic F atoms close to Arg183 in the active site environment. Redocking using the
similarly modeled R-β,γ-fluoromethylene-dGTP analogue and our recently published
2ISO structure
24
revealed a clustering of solutions placing the F atom within H-bridging
proximity of the Arg183, in contrast to the S diastereoisomer where such an interaction
was predicted to be less favored (visually represented using VMD 1.8.3
33
).
124
Figure 7.2 Comparison of DNA pol β active site complex structures for dGTP and analogues ob-
tained from crystallographic data and predicted by computational docking studies. A) 3, B) dGTP (modeled
with SPARTAN ‘02), C) 1, D) 2. (left column: from crystallographic data; right column: from Autodock
3.0.5 simulations) [crystal H2Os have been turned on in some viewings for analysis of hydrogen bonding]
125
Figure 7.3 Comparison of DNA pol β active site binding and docking calculations of the dGTP ana-
logues 3 (left) and 4 (right). The distribution of the green (fluorine atoms) aids in determining the overall fit
for the ligands. All X-ray crystal structures were solved by S. H. Wilson, et al; NIEHS.
Figure 7.4 (on the following page) X-ray crystal structures solved for DNA pol β:DNA complex
soaked with β,γ-dGTPs. A) 2:DNA:pol β complex, B) 3:DNA:pol β complex, C) 1:DNA:pol β complex. All
X-ray crystal structures were solved by S. H. Wilson, et al; NIEHS.
126
Figure 7.4 X-ray crystal structures solved for DNA pol β:DNA complex soaked with β,γ-dGTPs. A) 2:DNA:pol β complex, B) 3:DNA:pol β com-
plex, C) 1:DNA:pol β complex. All X-ray crystal structures were solved by S. H. Wilson, et al; NIEHS.
A B C
127
Figure 7.5 X-ray crystallographic structure A) of α,β-imido-dUTP (in teal) superimposed with the
2:DNA:pol β complex; B) of the 1:DNA:pol β (yellow), 2:DNA:pol β (red), and 3:DNA:pol β (blue) com-
plexes superimposed. All X-ray crystal structures were solved by S. H. Wilson, et al; NIEHS.
A B
128
Figure 7.6 X-ray crystallographic structures of a) 3:DNA:pol β complex; b) 5:DNA:pol β complex;
c) 9/10:DNA:pol β complex; d) 7/8:DNA:pol β complex; e) 13:DNA:pol β complex; f) 11/12:DNA:pol β
complex. All X-ray crystal structures were solved by S. H. Wilson, et al; NIEHS.
MB
DC
DM
MC
MM
MF
3.1 Å
a) b)
c) d)
e) f)
129
Figure 7.7 X-ray crystallographic structures of a) 15:DNA:pol β complex; b) overlaid with
3:DNA:pol β complex (yellow); c) 17:DNA:pol β complex; d) overlaid with 3:DNA:pol β complex (yel-
low) and 15:DNA:pol β complex (purple). All X-ray crystal structures were solved by S. H. Wilson, et al;
NIEHS.
3.4 Å
c) d)
FCl overlaid w/ MF (yellow) and
FM (purple)
FCl
FM
3.1 Å
FM overlaid w/ MF (yellow)
a) b)
RESULTS AND DISCUSSION
The crystal structures of the resulting complexes were resolved at 2.1 Å. (FIG-
URES 7.4-7.7). It is significant that an overlay of the nucleoside backbones and phospho-
bisphonsphonate moieties of 1, 2 and 3 within the DNA pol β complexes (FIGURE 7.5)
reveal them to be highly congruent, confirming that introduction of the F atom(s) does
not perturb the overall fit of the substrate to the active site. Despite both stereoisomers
130
being present in the crystallizing mixture (in the case of the mono-substituted or mixed
halo-methylene-bridged analogues, electron density was only observed for the (R)-CHF 3
(see FIGURES 7.4B and 7.6a), (S)-CFCH3 15 and (S)-CFCl 17 (see FIGURE 7.7)
stereoisomers in the enzyme active site position normally occupied by dGTP. In contrast,
the corresponding CHCl-, CHBr-, and CHCH3-analogue stereoisomers (7/8, 9/10, and 11/
12, respectively; see FIGURE 7.6) populate the DNA pol β active site about equally. Ex-
clusion of the (S)-CHF analogue as well as the other stereoisomers of other analogues is
not the result of an unattainable conformation since the crystal structure of the CF2-
analogue
24
indicates that the geometry of the fluorine atoms superimposes well with the
(R)-stereoisomer 3. However, the fluorine atoms in the 3, 15, and 17 complexes are lo-
cated ~3.1-3.3 Å from an Arg183 guanidine N, suggesting that Arg183 could contribute
to stabilizing the preferred stereoisomer through a potential hydrogen bridge. H··F bridg-
ing has been previously discussed and invoked in other biochemical contexts, such as the
apparent fluorophilicity
44
of arginine.
34,35,44
CONCLUSION
In conclusion, the stereoisomers 3, 15, and 17 are preferentially bound into a pol β
complex, plausibly due,
38,44
at least in part, to a CXF··H “bridge” to Arg183. Docking
simulations indicate that formation of this bond is favored in the R-diastereomer 3, rela-
tive to the S-diastereomer 4 which was not detected in the crystal complex. Introduction
of a single fluorine atom at the bridging carbon atom of a dNTP methylenebisphosphon-
ate analogue does not merely adjust the analogue pKa to more closely mimic the parent
131
nucleotide
36
, but also can result in stereospecific binding to an enzyme, determined by the
CXF chirality. This finding supports the increasing evidence of the fluorophilicity of ar-
ginine within enzyme binding sites.
44
EXPERIMENTAL SECTION
Crystallization of the pol β substrate complexes (performed by Wilson, et al;
NIEHS)
Human DNA polymerase β was over-expressed in E. coli and purified.
26
The DNA sub-
strate consisted of a 16-mer template, a complementary 9-mer primer strand, and a 5-mer
downstream oligonucleotide. The annealed 9-mer primer creates a two-nucleotide gap.
The sequence of the downstream oligonucleotide was 5’-GTCGG–3’ and the 5’-terminus
was phosphorylated. The oligonucleotides (1:1:1) were dissolved in 20 mM MgCl2 in
100 mM Tris/HCl, pH 7.5, and annealed using a PCR thermocycler by heating for 10 min
at 90 °C and cooling to 4 °C (1 °C min-1) resulting in 1 mM gapped duplex DNA. This
solution was mixed with an equal volume of pol β (15 mg/ml in 20 mM Bis-Tris, pH 7.0)
at 4 ˚C, the mixture warmed to 35 °C and then gradually cooled to 4 ˚C. A four-fold ex-
cess of 2’,3’-dideoxycytosine 5’-triphosphate (ddCTP) was added to obtain a 1-
nucleotide gap complex with a dideoxy primer terminus. Pol β-DNA complexes were
crystallized by sitting drop vapor diffusion by mixing protein 1 to 1 with mother liquor
consisting of 16% PEG-3350, 350 mM sodium acetate, and 50 mM imidazole, pH 7.5 at
18 °C. The trays were streak seeded after 1 day. Crystals (2 to 4 days after seeding) were
soaked in a solution consisting of 200 mM MgCl2, 90 mM sodium acetate, 4.5 mM of the
132
analogue, 20% PEG-3350, and 12% ethylene glycol, and then flash frozen in liquid nitro-
gen. The template sequence was 5’-CCGACCGCGCATCAGC–3’ and the primer se-
quence was 5’-GCTGATGCG-3’. Oligonucleotides were dissolved in 20 mM MgCl2,
100 mM Tris/HCl, pH 7.5.
Data collection and structure determination (performed by Wilson, et al; NIEHS)
Data were collected at 100K on a Saturn92 CCD detector system mounted on a
MicroMax-007HF (Rigaku Corporation) rotating anode generator. Data were integrated
and reduced with HKL2000 software.
39
Ternary substrate complex structures were deter-
mined by molecular replacement with a previously determined structure of pol β com-
plexed with one-nucleotide gapped DNA and a complementary incoming ddCTP (PDB
accession 2FMP).
40
The crystal structures have similar lattices and are sufficiently iso-
morphous to determine the molecular-replacement model using CNS
41
and manual model
building using O. The figures were prepared in Molscript/Raster3D.
42
The parameters and
topology files for the analogues were prepared using the program XPOL2D.
43
For results,
see FIGURES 7.4-7.7 and TABLE 7.1.
Docking experiments
In silico binding experiments were performed using the interpreted crystallographic data
and AUTODOCK 3.0.5.
30
There have been several reports that have correlated docking
energies and clusters with similar experimental results.
27
In order to minimize bias in the
docking experiments, the dNTP analogue in the crystal structure was extracted from the
structure and labeled as the Ligand.pdb and the protein:DNA complex (including all in-
133
corporated metals) was extracted to a separate Macromolecule.pdb. SPARTAN ’02
32
is
used to modify and minimize the energy of the Ligand.pdb and the DNA strands of the
Macromolecule.pdb. The autogrid and autodock scripts were modified as instructed
29
to
accomodate phosphorus and fluorine atoms. All procedures were carried out as specified
in the manual.
29
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. A grid map for the space to be docked within the
protein: DNA complex was set at a maximum of 81 X 81 X 81 points. 100 docking runs
were generated by lamarckian genetic algorithims (LGA). Those results that differed by
less than 2.0 Å in root-mean-square deviation in space were clustered and defined as hav-
ing the most favorable free energy of binding. The results can be found in APPENDIX F
TABLES F.1-10 and in visual representations, in FIGURE 7.2 and 7.3.
134
Table 7. 1 Example of crystallographic Statistics for 3. Provided by S. H. Wilson, et al; NIEHS.
Complex dGMPPCFHP
____________________________________________________________________________
Data Collection
Space Group P21
a (Å) 50.6
b (Å) 80.2
c (Å) 55.4
b (°) 107.8
dmin (Å) 2.10
Rmerge (%)
a
11.9 (44.5)
Completeness (%) 99.9 (99.9)
Unique Reflections 24711 (2459)
Total Reflections 90165
I/s 7.0 (3.0)
___________________________________________________________________________________
Refinement
R.m.s. Deviations
Bond lengths (Å) 0.005
Bond angles (°) 1.10
Rwork (%)
c
20.61
Rfree (%)
d
27.04
Average B Factor (Å
2
)
Protein 28.7
DNA 40.1
Ramachandran analysis (%)
e
Favored 98.2
Allowed 100
a
Rmerge=100 x ShSi|Ih,i-Ih| ShSi Ih,j, where Ih is the mean intensity of symmetry related reflections Ih,j.
b
Numbers in the parentheses refer to the highest resolution shell of data (10%).
c
Rwork = 100 x S ||Fobs|-|Fcalc||/S|Fobs|
d
Rfree for a 10% subset of reflections.
e
As determined by Molprobity (Lovell et al., 2003)
__________________________________________________________________________________
Lovell, S. C., Davis, I. W., Arendall, W. B., III, de Bakker, P. I. W., Word, J. M., Prisant, M. G., Richardson,
J. S., and Richardson, D. C. (2003). Structure validation by Cα geometry: φ,ψ and Cβ deviation. Proteins:
Structure, Function, and Genetics 50, 437-450.
135
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139
CHAPTER 8
SYNTHESIS AND CHARACTERIZATION OF CF3-BPS
INTRODUCTION
As discussed in CHAPTER 6, in the theoretical model of DNA polymerase β
mediated nucleotidyl-transfer
1,2
, if the activation barrier to leaving group elimination is
sufficiently high relative to other barriers near the transition state, perturbation of its
height (i.e. by Oβγ modification) would result in a single linear plot in the LFER formal-
ism. On the other hand, if the leaving group elimination barrier lies close in height to its
neighbors, sufficient stabilization would be predicted to result in a break in linearity with
a catalytically insensitive (zero slope) region at low leaving group pKa. Reconciling the
experimentally observed plots with either theoretical model (single line versus biphasic)
is difficult, even with the expanded set of alkyl- and halomethylene-bridged analogues,
because the CF2-methylene species remains the strongest leaving group, and in both cor-
rect and T-G mispairing incorporation its point falls roughly level with that of the native
compound, so a theoretical biphasic profile cannot be ruled out.
To examine this notion further, requires the use of methylenebisphosphonate de-
rivatives that are more electronegative and more acidic than the natural pyrophosphate,
preferably with a pKa4 between 7-9; therefore, providing better leaving groups. Examina-
tion of the literature shows the existence of a small collection of methylenebisphosphon-
ates that theoretically fit the criteria for this study: CF2
1-11
, CFCl
2,3,5,8
, CFBr
8
, CC=O
12,13
.
140
CCl2
1-3,5,8,12,14
, and CHF
1-3,5-11
(four of which have already been used for this study). At-
tempts at synthesizing the CFBr-derivative of TiPMBP from TiPMBBP (CHBr) by elec-
trophilic fluorination with Selectfluor
1-3,5,6,9,10,15
afforded TiPCFBr in low yield with sev-
eral side products. This result is due to the suspected instability of the
monobromomethylene-carbanion. The carbonylbisphosphonate derivative (CC=O) can be
synthesized from the dichlorobisphosphonate and depending on the pH of its environ-
ment, carbonylbisphosphonate exists in equilibrium with dihydroxybisphosphonate
(C(OH)2).
12,13

Since, the synthesis and isolation of CFBr proves difficult and the existing inven-
tory of worthy bisphosphonates has already been exhausted, new target bisphosphonates
(BP) need to be considered, particularly incorporating electronegative groups such as
CF3. Using ACD/Labs Online (I-Lab) pKa prediction
16
, a list of possible target BPs was
created (FIGURE 8.1). Of this list, only one BP has been proposed and synthesized pre-
viously, the trifluoromethylhydroxybisphosphonate C(OH)CF3 or trifluoroetidronate.
17

Figure 8.1 Proposed target BPs. pKa4s predicted using ACD Labs Online (I-Lab 8.0)
R = H, F , Cl, CF3, CH3, OH
pKa4 = 7 - 9
P P
O O
HO OH
OH OH
R CF
3
141
Convenient procedures for two new BPs to be added to our series is presented, herein; the
previously known trifluoroetidronate
17
and the unknown (1,1,1-trifluoro-2,2-
propanediyl)bis(phosphonate).
MATERIALS AND METHODS
ELECTROPHILIC TRIFLUOROMETHYLATION
We set out to conveniently synthesize these new CF3-BPs using approaches simi-
lar to the previously synthesized alkyl- and halo-derivatives in this work (see CHAPTER
2). Electrophilic trifluoromethylation
18-20
could be valuable in the synthesis of BPs of this
nature; however, there is no existing literature on the trifluoromethylation of bisphos-
phonates as well as phosphono-acetates. Although there is some possibility this approach
may work due to the success of Pirat et al.
21
on electrophilic trifluoromethylation of a
phosphono-keto derivative. Following the standard procedure for electrophilic trifluoro-
methylation with Umemoto’s reagent (see SCHEME 8.1), the carbanion is generated
from the methylenebisphosphonate ester in the presence of base (NaH) under N2 at 0
o
C.
Scheme 8.1 Synthesis of CF3-BPs by electrophilic trifluoromethylation using Umemoto’s reagent
P P
O O
RO
RO OR
OR
X
P P
O O
RO
RO OR
OR
X CF
3
0
o
C, THF
NaH, Umemoto reagent
X = H, F , Cl, CH3, CF3
Umemoto
reagent
=
S
CF
3
OTf
142
After carbanion formation is confirmed via
31
P NMR, the temperature is lowered to -78
o
C. Two equivalents of Umemoto’s reagent is added. The reaction is allowed to warm to
room temperature over 5 hours. Reaction mixtures are monitored by
31
P NMR.
SYNTHESIS OF CF3-ETIDRONATE
Breuer’s reported synthesis of CF3-etidronate involves the reaction of trifluoroa-
cetyl chloride with tris(trimethylsilyl)phosphite.
17
Trifluoroacetyl chloride is not a con-
venient chemical to work with as it is a gas and the reaction requires low temperature to
ensure adequate TFA/tris(trimethylsilyl)phosphite adduct as the acid chloride is relatively
unstable under the reaction conditions. This perceived inconvenience is avoided by using
“modified Kabachnik” chemistry where hydroxybisphosphonic acids are synthesized
from the reaction of a carboxylic acid with H3PO3 and PCl3.
22,24
This method is known to
yield several contaminating phosphonate and phosphites. When attempted with tri-
fluoroacetic acid (see SCHEME 8.2, several contaminating (~70%) phosphates, phos-
phonates, and phosphites are visible in the product mixture.
Scheme 8.2 Synthesis of CF3-etidronate from trifluoroacetic acid
F
3
C OH
O
i. H
3
PO
3
, PCl
3
P P HO OH
O O
OH HO
HO CF
3
ii. H
+
Considering the super-solubility of the calcium salt of trifluoroetidronate and the relative
insolubility of calcium phosphates, phosphonates and phosphites, the product mixture can
be adjusted to pH = 10 with Ca(OH)2 and then separated from the contaminating side
products, leaving only the calcium salt of trifluoroetidronate which can be exchanged to
143
the free acid on DOWEX. The low yield of this method is suspected to be due to the in-
sufficient trifluoroacetyl chloride produced in situ. Revisiting Breuer’s method for tri-
fluoroetidronate, an improvement to this reaction can be made as an adaptation with Le-
couvey’s method to synthesize etidronic acid from reaction of acetic anhydride with
tris(trimethylsilyl)phosphite.
23
Reacting tris(trimethylsilyl)phosphite with trifluoroacetic
anhydride (see SCHEME 8.3) yields predominantly trifluoroetidronate in a relatively
short period of time.
Scheme 8.3 Synthesis of CF3-etidronate from trifluoroacetic anhydride
P P Me
3
SiO OSiMe
3
O O
OSiMe
3
Me
3
SiO
Me
3
SiO
CF
3
P(OSiMe
3
)
3
MeOH
P P HO OH
O O
OH HO
HO CF
3
F
3
C O
O O
CF
3
RESULTS AND DISCUSSION
Umemoto chemistry
Attempts at electrophilic trifluoromethylation were carried out on various sub-
strates according to SCHEME 8.1. Due to the suspected low nucleophilicity of attempted
carbanions, there was little to no product formed using Umemoto’s reagent (see TABLE
8.1) and substituted-methylenebisphosphonate. Reactions with the
tetraisopropylmonochloromethylene-bisphosphonate (TiPMCBP) showed no desired
product by
31
P and
19
F NMR. A series of experiments were carried out on monomethyl-
substituted BPs to examine the scope of this reaction (TABLE 8.1). The low yield ob-
served with trifluoromethylation is presumably due to a combination of weak nucleophile
and steric hinderance due to the isopropyl ester groups in the case of tetraisopropylethy-
lidenebisphosphonate (1% conversion). This observation is supported by a slight in-
144
crease in the yield for the less sterically-hindered tetraethylester (7% conversion). An in-
crease in the nucleophilicity of the carbanion in the ethylidenephosphono acetate is ob-
served by an increase in yield of trifluoromethylated product (~30% yield). This trend is
expected as it compares to the substrates with similar reactivity to the phosphonoacetate,
methylmalonate and α-keto-phosphonate, reported by Umemoto
18,20
and Pirat
21
, respec-
tively.
Table 8.1 Summary of attempts at electrophilic trfluoromethylation using Umemoto’s reagent
(note: yields determined by
31
P and
19
F NMR)
P P
O O
O OiPr
OiPr OiPr
CH
3
iPr
P P
O O
EtO OEt
OEt OEt
CH
3
P C
O O
EtO OEt
OEt
CH
3
BP used Base Solvent Result
NaH THF 1-3% yield
NaH THF 7% yield
NaH THF ~30% yield
CF3-Etidronate
Trifluoroetidronate was successfully synthesized by the “modified Kabachnik”
method.
22,24
Trifluoroacetic acid was reacted with H3PO3 in toluene, followed by treat-
ment with PCl3 at -78
o
C. After reflux in HCl, the solvent is removed under reduced pres-
sure. After workup, several contaminating (~70%) phosphates, phosphonates, and phos-
phites are visible in the product mixture (by
31
P NMR). Considering the super-solubility
of the calcium salt of trifluoroetidronate
17
and the relative insolubility of calcium phos-
145
phates, phosphonates and phosphites, the product mixture was adjusted to pH = 10 with
Ca(OH)2 and then filtered to remove the white precipitate. The product mixture is con-
centrated under vacuum and in a minimum amount of H2O is centrifuged to remove the
remaining precipitate leaving only the calcium salt of trifluoroetidronate which is ex-
changed to the free acid on DOWEX (< 25% yield).
An alternative and more convenient method was also employed to obtain tri-
fluoroetidronate in ideal purity and acceptable yields. Trifluoroacetic anhydride is reacted
with tris(trimethylsilyl)phosphite (neat) and allowed to react at room temperature for 1
hr. After work up in methanol, trifluoroetidronate is isolated in > 80% purity. This proce-
dure is an adaption of Lecouvey
23
and Beuer’s
17
methods which is an improvement on the
existing method using convenient and readily available materials. NMR and LC-MS-MS
analysis of trifluoroetidronate synthesized from both methods confirms successful syn-
thesis of trifluoroetidronate compared to the previously reported data
17
.
CONCLUSION
Reconciling the experimentally observed plots of our study of pol β-catalyzed
nucleotidyl-transfer with either theoretical model (single line versus biphasic) is difficult,
even with the expanded set of alkyl- and halomethylene-bridged analogues, because the
CF2-methylene species remains the strongest leaving group, and in both correct and T-G
mispairing incorporation its point falls roughly level with that of the native compound, so
a theoretical biphasic profile cannot be ruled out. To examine this further, requires the use
of methylenebisphosphonate derivatives that are more electronegative and more acidic
than the natural pyrophosphate, preferably with a pKa4 between 7-9; therefore, providing
146
better leaving groups. Attempts at the synthesis and isolation of a CFBr-derivative proved
difficult and the existing inventory of worthy bisphosphonates had already been ex-
hausted; therefore, new target bisphosphonates (BP) needed to be considered, particularly
incorporating electronegative groups such as CF3. Using ACD/Labs Online (I-Lab) pKa
prediction
16
, a list of possible target BPs was created (FIGURE 8.1). Of this list, only
one BP has been proposed and synthesized previously, the trifluoromethylhydroxy-
bisphosphonate C(OH)CF3 or trifluoroetidronate.
17

Convenient procedures for two new BPs to be added to our series is presented,
herein; the previously known trifluoroetidrone
17
and trifluoromethylethylidene-
bisphosphonate. This is the first report examining the scope of electrophilic trifluoro-
methylation on bisphosphonates as well as phosphonoacetates and is the first report of
trifluormethylethylidene (both tetraisopropyl and tetraethyl esters). Although electrophilic
trifluoromethylation is low yielding, sufficient quantities can be collected following
scale-up of the procedure. The esters (tetraisopropyl and tetraethyl) can be removed by
hydrolysis in conc. HCl
8
or dealkylation in BTMS
7,8
. This is also the first report of two
new efficient and convenient approaches to trifluoroetidronate based on practical proce-
dures for the synthesis of α-hydroxybisphosphonates
17,23
. Trifluoroetidronate can be syn-
thesized from TFA but unfortunately with a great deal of contaminating phosphates and
phosphonates which can be removed as the calcium salt. Trifluoroetidronate can also be
synthesized in greater purity (> 80%) using TFA anhydride. These two methods present
relatively inexpensive alternative routes to a known BP that is in our target list. Using
sufficient quantities of these new BPs, dGTP-analogues can be synthesized. Trifluoroeti-
147
dronate can theoretically converted into other useful BPs in our target list. The α-hydroxy
group can be removed by reaction with Raney Nickel.
12
The deshydroxy-derivatives can
be used to synthesize other new BPs, such as ditrifluoromethyl-, monotrifluoro-, fluoro-
trifluoromethyl-, and chlorotrifluoromethyl-substituted methylenebisphosphonates. Once
obtained the new BPs are used for pKa4 determination by potentiometric titration as well
in the synthesis of corresponding β,γ-dGTP analogues (described in CHAPTER 9).
EXPERIMENTAL SECTION
All reagents are purchased from Sigma-Aldrich except TFAA which was so graciously
offered by Nicos Petasis. NMR analysis was carried out on either Varian Mercury 400
NMR instruments or Bruker AMX-500 MHz FT NMR. LC-MS-MS is carried out on
ThermoScientific FINNIGAN LCQ DECA XP MAX. Any relevant NMR or MS data is
present in APPENDIX G.
General procedure for electrophilic trifluoromethylation
1.5 equivalents of NaH (95 %) is weighed out in a dry-three-necked flask and dissolved
in THF at 0
o
C. 1 equivalent of bisphosphonate in THF is added dropwise. Once carban-
ion formation is confirmed by
31
P NMR, the reaction mixture is cooled to -78
o
C then 1.5
equivalents of Umemoto reagent is added. The reaction mixture is allowed to react at -78
o
C for 30 minutes and then allowed to warm to room temperature and monitored by
31
P
NMR. When the reactionhas reach equilibrium (no new product formation), it is
quenched with a NH4Clsat solution until the reaction mixture is transparent. 25 mL of
CH2Cl2 (2 X 25 mL). The organic layers are collected and dried with MgSO4 and then the
solvent is removed under reduced pressure.
148
Synthesis of tetraisopropyl (1,1,1-trifluoro-2,2-propanediyl)bis(phosphonate) or
(CF3TiPEBP)
50 mg (2.08 mmol) NaH (95 %) is weighed out in a dry-three-necked flask and dissolved
in 1.5 mL of THF at 0
o
C. A mixture of TiPEBP with TiPDMBP (containing ~220 mg -
0.61 mmol.) in 1.5 mL THF is added dropwise. Once carbanion formation is confirmed
by
31
P NMR, the reaction mixture is cooled to -78
o
C then 370 mg (0.92 mmol) of Ume-
moto reagent is added. The reaction mixture is allowed to react at -78
o
C for 30 minutes
and then allowed to warm to room temperature and monitored by
31
P NMR. When the
reaction has reach equilibrium (no new product formation), it is quenched with a NH4Clsat
solution until the reaction mixture is transparent. 25 mL of CH2Cl2 (2 X 25 mL). The or-
ganic layers are collected and dried with MgSO4 and then the solvent is removed under
reduced pressure. (NMR yield 1%).
31
P: 12 (q),
19
F: -66 (t).
Synthesis of tetraethyl (1,1,1-trifluoro-2,2-propanediyl)bis(phosphonate) or
(CF3TEEBP)
26 mg (1.08 mmol) NaH (95 %) is weighed out in a dry-three-necked flask and dissolved
in 1.5 mL of THF at 0
o
C. A mixture of TEEBP with TEFMBP (containing ~220 mg -
0.72 mmol.) in 1.5 mL THF is added dropwise. Once carbanion formation is confirmed
by
31
P NMR, the reaction mixture is cooled to -78
o
C then 370 mg (0.92 mmol) of Ume-
moto reagent is added. The reaction mixture is allowed to react at -78
o
C for 30 minutes
and then allowed to warm to room temperature and monitored by
31
P NMR. When the
reaction has reach equilibrium (no new product formation), it is quenched with a NH4Clsat
solution until the reaction mixture is transparent. 25 mL of CH2Cl2 (2 X 25 mL). The or-
149
ganic layers are collected and dried with MgSO4 and then the solvent is removed under
reduced pressure. (NMR yield 7%).
31
P: 12 (q),
19
F: -65 (t).
Synthesis of ethyl 2-(diethoxyphosphoryl)propanoate or (triethyl)methylphos-
phonoacetate (TEMPAA)
188 mg (60% emersion in oil) is weighed out in a dried 3-necked flask and cooled to 0
o
C
and then dissolved in 10 mL of dry and distilled THF. 0.88 mL (4.5 mmol) of (trieth-
yl)phosphonoacetate (TEPAA) is added dropwise. 433 µL (5.3 mmol) is added dropwise.
After there is little or no visible starting TEPAA, 10mL of NaHO3 solution, 10 mL of
H2O, and 50 mL of CH3Cl is added and the organic layers are collected (3 x 50 mL). The
solvent is removed under reduced pressure. Left with a 900 mg sample containing 54%
monomethylated - TEMPAA (480 mg, 45% yield,
31
P: 21.1); 43% - dimethylated - ethyl
2-(diethoxyphosphoryl)-2-methylpropanoate or TEDMPAA (382 mg, 34% yield,
31
P:
24.5); 1% - starting material - TEPAA; 2% - other impurities.
Synthesis of ethyl 2-(diethoxyphosphoryl)-3,3,3-trifluoro-2-methylpropanoate or
(triethyl)trifluoromethylmethylphosphonoacetate (CF3TEMPAA)
8 mg (1.78 µmol) NaH (95 %) is weighed out in a dry-three-necked flask and dissolved in
1.5 mL of THF at 0
o
C. A 50% mixture of TEMPAA with TEDMPAA (containing ~37.5
mg - 0.15 mmol.) in 0.5 mL DMF is added dropwise. Once carbanion formation is con-
firmed by
31
P NMR, the reaction mixture is cooled to -78
o
C then 95 mg (0.24 mmol) of
Umemoto reagent is added. The reaction mixture is allowed to react at -78
o
C for 30 min-
utes and then allowed to warm to room temperature and monitored by
31
P NMR. When
the reaction has reached equilibrium (no new product formation), it is quenched with a
150
NH4Clsat solution until the reaction mixture is transparent. 25 mL of CH2Cl2 (2 X 25 mL).
The organic layers are collected and dried with MgSO4 and then the solvent is removed
under reduced pressure. (NMR yield = 30%).
31
P: 12.7 (q),
19
F: -70.4 (d).
Synthesis of (2,2,2-trifluoro-1-hydroxy-1,1-ethanediyl)bis(phosphonic acid) or tri-
fluoroetidronate (CF3-etidronate)
17
Method A. “Modified Kabachnik” Chemistry
22,24
6.48 g of H3PO3 is dissolved in 120 mL of dried and distilled toluene and is cooled to -78
o
C. 1.95 mL of TFA is added to the cooled solution. 8.04 mL of distilled PCl3 is added.
The reaction mixture is allowed to warm to room temperature over 4 hours. The reaction
mixture is then set to reflux for 4-5 hours at which a bright orange precipitate begins to
form. Once cooled to room temperature, the toluene is decanted and ~100 mL of 6M HCl
is added to the orange residue. The mixture is set to reflux overnight. The solvent is re-
moved under vacuum then the orange residue is washed with acetone and methanol and
then filtered. The organic fractions are combined and the product mixture is dried under
reduced pressure yielding a colorless oil [8.8 g containing 20% CF3-etidronate;
31
P (pH <
5): 10.5-12 (b);
19
F (pH < 5): -73.5 (bt)]. The contaminating phosphonate, phosphate, and
phosphites can be removed by increasing the pH to 10 with Ca(OH)2(sat) and then filtering
off the white precipitate. The aqueous fraction is concentrated under reduced pressure and
centrifuged. The supernatant is collected and dried leaving the calcium salt of CF3-
etidronate [colorless oil - 90% pure,
31
P (pH = 10.7): 8.5 (q);
19
F (pH = 10.7): -74.9 (t)].
LC-MS (in D2O, [M-2H+D]
-
= 261; in Ca(OH)2, [M-H+2Ca]
-
= 358)
151
Method B. Trifluoromethylacetic Anhydride Method
1.2 mL (3.59 mmol) of tris(trimethylsilyl)phosphite (neat) is added to a dried 10 mL
round bottom flask under N2. 0.25 mL (1.79 mmol) of TFAA is added to the tris(trimeth-
ylsilyl)phosphite dropwise under N2. The reaction is allowed to react at room temperature
for 1 hour. After which, the volatiles are removed under vacuum at 50
o
C. Then ~ 2 mL of
MeOH is added and reacted at room temperature for 1 hr. The solvent is removed under
reduced pressure and dried at 50
o
C yielding a waxy oil [CF3-etidronate - 181 mg purity
~75%, 37% yield,
31
P: 9.88 (q);
19
F: -70.2 (bt)]. LC-MS = [M-H]
-
= 259
152
CHAPTER 8 REFERENCES
1. Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V . K.; Martinek, V .; Xiang,
Y .; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.;
Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461-471.
2. Sucato, C. A.; Upton, T. G.; Osuna, J.; Oertell, K.; Kashemirov, B. A.; Beard, W. A.;
Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F. Biochem-
istry 2008, 47, 870-879.
3. Upton, T. G.; Osuna, J.; Kashemirov, B. A.; McKenna, C. E. in preparation 2008,
4. Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R. M. J. Fluorine
Chem. 1982, 20, 617-626.
5. Upton, T. G.; Kashemirov, B. A.; McKenna, C. E. in preparation 2008,
6. Mohamady, S.; Jakeman, D. L. J. Org. Chem. 2005, 70, 10588-10591.
7. McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573-4576.
8. McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y .; Pham, P.; Bongartz, J. P. Phospho-
rus and Sulfur and the Related Elements 1988, 37, 1-12.
9. 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-
15413.
10. McKenna, C. E., T. G. Upton, B. A. Kashemirov, M. F. Goodman, G. K. S. Prakash,
R. Kultyshev, V . Batra, L. C. Pedersen, and S. Wilson. manuscript in preparation
2008,
11. Blackburn, G. M.; England, D. A.; Kolkmann, F. Journal of the Chemical Society,
Chemical Communications 1981, 930-932.
12. Grabenstetter, R. J.; Quimby, O. T.; Flautt, T. J. J. Phys. Chem. 1967, 71, 4194-
4202.
13. Kashemirov, B. A.; Roze, C. N.; McKenna, C. E. Phosphorus, Sulfur and Silicon
and the Related Elements 2002, 177, 2275; McKenna, C. E.; Kashemirov, B. A.;
Roze, C. N. Bioorg. Chem. 2002, 30, 383-395.
153
14. Dietsch, P.; Gunther, T.; Rohnelt, M. Zeithschrift Fur Naturforschung C-A Journal
of Biosciences 1976, 31, 661-663.
15. Marma, M. S.; Khawli, L. A.; Harutunian, V .; Kashemirov, B. A.; McKenna, C. E.
J. Fluorine Chem. 2005, 126, 1467-1475.
16. Labs, A. C. D. http://ilab.acdlabs.com/ 2004,
17. Schoth, R. M.; Lork, E.; Seifert, F. U.; Roeschenthaler, G. V .; Cohen, H.; Golomb,
G.; Breuer, E. Naturwissenschaften 1996, 83, 571-574.
18. Umemoto, T.; Adachi, K. J. Org. Chem. 1994, 59, 5692-5699.
19. Umemoto, T.; Ishihara, S. J. Am. Chem. Soc. 1993, 115, 2156-2164.
20. Umemoto, T. Chem. Rev. 1996, 96, 1757-1777.
21. Pirat, J.-L.; Marat, X.; Clarion, L.; Van, d. L., Arie; V ors, J.-P.; Cristau, H.-J. J. Or-
ganomet. Chem. 2005, 690, 2626-2637.
22. Kieczykowski, G. R.; Jobson, R. B.; Melillo, D. G.; Reinhold, D. F.; Grenda, V . J.;
Shinkai, I. J. Org. Chem. 1995, 60, 8310-8312.
23. Guenin, E.; Degache, E.; Liquier, J.; Lecouvey, M. Eur. J. Org. Chem. 2004, 2983-
2987.
24. Krueger, F.; Bauer, L.; Michel, W. Ger. Offen. 1973, 8. CODEN: GWXXBX DE
2130794 19730111 CAN 78:84528 AN 1973:84528 CAPLUS,
154
CHAPTER 9
SYNTHESIS, ANALYSIS, AND PURIFICATION OF OTHER NTPS
INTRODUCTION
Congruent with the creation of this dNTP toolkit was the development of syn-
thetic approaches and analysis and purification methods that are applicable to other nu-
cleotide analogues. The synthetic endeavors practiced herein have been conveniently
adapted for the synthesis of some known nucleotide analogues
1-7
as well as new nucleo-
side triphosphate analogues
8,9
. The analytical methods used in the production of these
analogues have proven utility in the development of new analogues in this group and by
others. The purification methods used to obtain these nucleotide analogues with excep-
tional purity have been employed for the refinement of analogues synthesized by varying
strategies which have been identified difficulties in purification. Presented here is a sum-
mary of this work. The nucleotide analogues described herein as in the preceding reports
exemplify the contribution of this work to the ongoing study of polymerases.
SYNTHESIS OF KNOWN AND NOVEL NUCLEOSIDE TRIPHOSPHATE ANA-
LOGUES
As previously mentioned in CHAPTERS 4 and 5, the synthetic strategies were
developed with utility in mind. This is evident by the variety of α,β- and β,γ-analogues
that have been synthesized by these methods. Currently, the combined chemical-
enzymatic procedure is being used for the synthesis of α,β-CF2-dUTP that will be exam-
155
ined as an inhibitor of RNA polymerases by our colleagues Benkovic and coworkers at
Penn State University. The morpholidate method has been employed for this synthesis of
a few known β,γ-NTP analogues (including β,γ-CH2-dTTP
4,10
, β,γ-CF2-ATP
2,3,5,6,11
, β,γ-
C(OH)CH3-GTP
7
) as well as new analogues (including β,γ-C(OH)CH3-dGTP, β,γ-
Risedronate-dGTP, and β,γ-C(OH)CF3-dGTP). These analogues have been synthesized to
examine their affect on polymerase function in various projects. The corresponding nu-
cleotide monophosphate was converted to the morpholidate
12-14
followed by condensation
with the tributylammonium salt of the corresponding bisphosphonate (SCHEME 9.1).
The β,γ-NTP analogues are then isolated by dual-pass preparative HPLC (SAX then
C18).(see APPENDIX H for detailed experimental data).
Scheme 9.1 Synthesis of β,γ-NTP analogues
O
OH
O P
-
O
O
O
-
O
OH
O P
-
O
O
N
O
O
OH
O P O
O
O
-
P
O
O
-
X P
O
O
-
-
O
P P
O
-
O
O
-
O
O
-
O
-
[Bu
3
NH
+
] [ ]
DMSO
anh
morpholine,DCC
tBuOH:H
2
O
X
X = CH
2
, CHF, CF
2
, CCl
2
, CHCl,
CFCl, CBr
2
, CHBr, C(CH
3
)
2
,
CHCH
3
, CFCH
3
, C(OH)CH
3
,
C(OH)-2'pyridinyl, C(OH)CH
3
dNMP
dNMP-Morph
BASE BASE
BASE
R R
R R
R R
R = H, OH
156
Figure 9.1 HPLC analysis of the β,γ-CF3-Etidronate-dGTP reaction mixture
dGMP
β,γ-CF3Etid-
dGTP
ANALYSIS AND PURIFICATION OF NEW NTP ANALOGUES
Several of the analytical and purification methods used in the synthesis and char-
acterization of the nucleoside triphosphate analogues described in the preceding chapters
have been applied to analysis and purification of new nucleotide analogues synthesized
by our colleagues. The analytical HPLC method developed with SAX proved its useful-
ness when examining complex reaction mixtures as well as analysis of several isolated
nucleotide analogues to determine product purity, characterization, and stability. As well
as being employed in the aforementioned procedures (see FIGURE 9.1 APPENDIX H
for experimental data), this method was used in the analysis of α,β-CF2-dTTP synthesized
by our colleagues Prakash and Kultyshev (mentioned in CHAPTER 4). Prior to our col-
157
laboration NMR and DNA polymerase β-mediated nucleotidyl-transfer assay analysis
was performed on this analogue, in which small organic (non-NTP and NTP) were ob-
served. Further analysis on analytical HPLC (C18 and SAX) proved that additional puri-
fication was necessary for this sample to be suitable for biochemical analysis (see FIG-
URE 9.2).
Figure 9.2 Analytical HPLC analysis of α,β-CF2-dTTP; a) analysis on SAX shows α,β-CF2-dTDP
contaminating the sample as well as other small impurities; b) analysis (C18) of α,β-CF2-dTTP after prepa-
rative purification on SAX shows small NTP impurity (left) and α,β-CF2-dTTP sample spiked with dTTP
(right); d) analysis (C18) of α,β-CF2-dTTP after dual-pass HPLC (SAX then C18).
e)
b)
α,β-CF2-dTTP
?
d)
α,β-CF2-dTPP α,β-CF2-
dTPP
dTPP

Chrom. 1 0.0 mins. 34.7 mins.
1
α,β-CF2-dTTP
α,β-CF2-dTDP
a)
158
Scheme 9.2 Proposed synthesis of α-CF2H-dTMP to α-CF2H-dTTP using ADK, PEP, PK, ATPcat.
O P O
O
CF
2
H
P O P O
O O
OH OH
α−CF
2
H-dTTP
O
OH
N
NH
O
O
O P O
O
CF
2
H
P O
O
OH
α−CF
2
H-dTDP
O
OH
N
NH
O
O
O P O
O
CF
2
H
α−CF
2
H-dTMP
O
OH
N
NH
O
O
ADK, PK, PEP, ATP
cat
HEPES
Our collaborators (Prakash and Beier,
8,9
) asked us to examine the possibility to
enzymatically-phosphorylate a nucleoside monophosphate, α-CF2H-dTMP (see
SCHEME 9.2), to its triphosphate derivative. The analytical HPLC (SAX) method was
used to study this hypothesis. The method used to phosphorylate α,β-CF2-dNDPs to α,β-
CF2-dNTPs is specific for monophosphorylation and cannot be employed in this matter.
We chose Whitesides’ method for enzymatic phosphorylation of NMPs to the correspond-
ing nucleoside triphosphates to accomplish the required round of phosphorylation.
15

Adenosine Kinase (AK) is used in tandem with pyruvate kinase (PK) and phosphoe-
nolpyruvate (PEP) and ATP as phosphate sources to phosphorylate the α-CF2H-dTMP to
α-CF2H-dTTP. Under these reaction conditions, the reagent concentrations we are using
are low and analysis of such mixtures would prove difficult by NMR. We are presented
with an opportunity to apply the analytical HPLC method on SAX for examining reaction
mixtures. HPLC analysis (on SAX) of the α-CF2H-dTMP sample shows a relatively clean
sample with small impurities (FIGURE 9.3A). HPLC (SAX) analysis of the enzymatic-
phosphorylation reaction mixture (FIGURE 9.3B) shows very little phosphorylated
159
Figure 9.3 HPLC analysis (on SAX) of a) α-CF2H-dTMP; b) enzymatic-phosphorylation reaction
mixture (1.5 hrs after adding ADK, PK, PEP, and ATPcat to α-CF2H-dTMP).
α-CF2-dTMP
? ?
a)
α-CF2-dTMP
? ATP
α-CF2-dTTP
(8%)
?
b)
160
Figure 9.4 HPLC analysis (on SAX): a) 19.5 hrs after addition of enzymes; b) 24 hrs after adding
more ADK, PK, PEP, and ATPcat.
α-CF2-dTMP
? ? ATP
α-CF2-dTTP (<1%)
a)
α-CF2-dTMP
? ?
ATP
α-CF2-dTTP
(<10%)
b)
161
product after 1.5 hrs (~8% α-CF2H-dTTP). After approximately 20 hrs, the reaction mix-
ture is analyzed by HPLC (SAX), < 1% α-CF2H-dTTP (FIGURE 9.4A). Additional por-
tions of ADK, PK, PEP, and ATP were added and after 24 hrs, the reaction mixture was
again analyzed by HPLC (FIGURE 9.4B). The reaction mixture shows approximately
10% α-CF2H-dTTP. The enzymatic phosphorylation has proven to be an efficient method
to engender NTPs from NMPs; however, the low amount of α-CF2H-dTTP alludes to
product stability issues. Additional ADK and ATP was added and after 5 hours the prod-
uct mixture was sampled and the enzyme is removed by YM-30 microcon filter. The
sample is analyzed over an hour at room temperature over 30 minute intervals (FIGURE
9.5). At initial analysis, 18% α-CF2H-dTTP is observed (FIGURE 9.5A). Within 30 min-
utes at room temperature (FIGURE 9.5B), α-CF2H-dTTP had decomposed to α-CF2H-
dTDP and α-CF2H-dTMP with 10% α-CF2H-dTTP remaining. After an hour at room
temperature (FIGURE 9.5C), < 1% α-CF2H-dTTP remains. This confirms α-CF2H-dTTP
can be formed by enzymatic phosphorylation; however, due to the relative stability of α-
CF2H-dTTP under the conditions of the reaction, product recovery and isolation is ren-
dered useless.
Figure 9.6 α,β,β,γ-bis-(CF2)-dNTPs
BASE
O
OH
O P
F
2
C
OH
O
P
F
2
C P
O
O
OH
O
OH
BASE = T: α,β,β,γ-bis(CF
2
)-dTTP
BASE = A: α,β,β,γ-bis(CF
2
)-dATP
162
Figure 9.5 HPLC analysis (on SAX) of the CF2H-dTTP reaction mixture 5 hours after the addition
of more ADK and ATP - A; 30 minutes after the removal of enzymes - B; 1 hr after removal of enzymes -
C.
1hr on
bench w/o
enz
30min on
bench w/o
enz
@ 5hrs
(18%)
(10%
)
α-CF2-dTTP
(<1%)
α-CF2-dTDP
α-CF2-dTMP
A
B
C
ATP
? ?
We were also asked to help with the analysis and purification of other analogues.
Prakash and Zibinsky have synthesized a new triphosphate analogue, bis-(CF2)-
methylene-triphosphonate,
8
which they used to synthesis α,β,β,γ-bis-(CF2)-dNTPs
(α,β,β,γ-bis-(CF2)-dTTP and α,β,β,γ-bis-(CF2)-dATP; see FIGURE 9.6). They were expe-
riencing difficulty in purifying the dNTP analogues. We analyzed their reaction mixtures
by our analytical HPLC method (on SAX - FIGURE 9.7) and determined acceptable
product yields and used our dual-pass preparative method to successfully isolate the
α,β,β,γ-bis-(CF2)-dNTPs with exceptional purity (FIGURE 9.8).
163
Figure 9.7 HPLC analysis (on SAX) of: A - α,β,β,γ-bis-(CF2)-dTTP reaction mixture; B - α,β,β,γ-bis-
(CF2)-dATP reaction mixtures analyzed using 0-100% 0.5M TEAB; pH = 8 (top) and 0-50% 0.5M LiCl;
pH = 8 (bottom).
Chrom. 1 0.0 mins. 20.5 mins.
1
A B
α,β,β,γ-
bis(CF2)-dTTP
α,β,β,γ-bis(CF2)-
dATP
Figure 9.8 HPLC analysis (on SAX) of: A - α,β,β,γ-bis-(CF2)-dATP after preparative purification on
SAX; B - α,β,β,γ-bis-(CF2)-dTTP (1) and α,β,β,γ-bis-(CF2)-dATP (2) after dual-pass preparative HPLC
(SAX then C18).
A
B
164
RESULTS AND DISCUSSION
Most of the isolated analogues were analyzed and characterized by analytical
HPLC, NMR, and LC-MS. The synthetic, analytical, and purification methods used to
generate the dNTP toolkit have been applied to several known as well as novel nucleotide
analogues. β,γ-CH2-dTTP
4,10
, β,γ-CF2-ATP
2,3,5,6,11
, β,γ-C(OH)CH3-GTP
7
, β,γ-C(OH)CH3-
dGTP, β,γ-Risedronate-dGTP, and β,γ-C(OH)CF3-dGTP were all successfully synthesized
using the morpholidate
12
method. β,γ-CH2-dTTP and β,γ-CF2-ATP are being used in a
separate toolkit study.
β,γ-C(OH)CH3-GTP
7
was synthesized as a model system to synthesize β,γ-dNTP
with known bisphosphonate drugs to test as anti-cancer agents or drug leads affecting po-
lymerase function. Our interest in such analogues was based largely on our finding with a
dNTP analogue containing another known bisphosphonate, clodronate, β,γ-CCl2-dGTP.
13,14
β,γ-CCl2-dGTP showed a 27-fold increase in fidelity, showing possible anti-
mutagenic potential. Perhaps, β,γ-dGTP analogues with other known bisphosphonate
drugs will show similar activity. Alkyl- and halo-methylenbisphosphonate drugs (such as
clodronate), compete for NTP binding sites in critical enzymatic pathways.
16
Bisphos-
phonates are known to specifically target bone and display anti-tumor activity by more
than one pathway in tumor metastasis.
17
Therefore, dNTP analogues of these and other
BPs as potent inhibitors of DNA pol β and other human tumor-associated mutants needs
to be explored. Anti-tumor activity was observed in breast cancer cell assays with eti-
dronate (a known BP drug) and a nucleotide drug, Ara.
18
BPs and β,γ-NTP analogues
165
containing known BPs, non-nitrogen (NNBPs) and nitrogen (NBPs) containing, inhibit
T7 RNA polymerase and HIV 1 RT.
7
When synthesis of the known etidronate-GTP was
confirmed, we set out synthesize novel β,γ-dGTP analogues with known bisphosphonates
(NNBPs and NBPs) such as etidronate, pamidronate, risedronate, and zolendronate. β,γ-
C(OH)CH3-dGTP and β,γ-Risedronate-dGTP were successfully synthesized by this
method. There is a product stability issue that arises from the incorporation of α-
hydroxybisphosphonates. These analogues are not stable at a pH > 8. During lypholiza-
tion of the TEA salt of these analogues the pH fluctuates to an extremely basic pH at
which these β,γ-analogues decompose. This was taken into account during the purifica-
tion of the dGTP analogue derived from our newly available CF3-etidronate (see CHAP-
TER 8, β,γ-C(OH)CF3-dGTP. After stability analysis in varying buffer solutions, β,γ-
C(OH)CF3-dGTP was found to be very stable at pH 4 but at other pH ranges was found to
readily decompose to dGMP. Therefore, using the same dual-pass preparative purification
method by HPLC (SAX then C18) and changing the buffer to a triethylammonium
(TEAA) acetate buffer (pH = 8 for SAX and pH = 4.5 for C18), β,γ-C(OH)CF3-dGTP
was purified and isolated as the TEA salt.
The analytical and preparative HPLC methods used in the synthesis of these ana-
logues proved useful in the analysis and purification of other novel dNTP analogues. In
collaboration with Prakash and coworkers, we were able to successfully examine and pu-
rify several dNTP analogues. Analysis of α,β-CF2-dTTP revealed several contaminating
organic side-products. After dual-pass preparative HPLC, α,β-CF2-dTTP was obtained
free of any contaminating side-products. The enzymatic phosphorylation
15
of α-CF2H-
166
dTMP
8,9
to α-CF2H-dTTP was also examined. α-CF2H-dTTP synthesis was confirmed by
analytical HPLC; however, it was determined that the product was very unstable under
the reaction conditions (> 90% decomposition within 1 hour). Non-hydrolyzable dNTP
analogues
8
were synthesized by Prakash and Zibinsky using their newly available α,β,β,γ-
bis-(CF2)-triphosphonate. α,β,β,γ-bis-(CF2)-dTTP and α,β,β,γ-bis-(CF2)-dATP reaction
mixtures were analyzed and then purified using dual-pass preparative HPLC (SAX then
C18) and isolated as TEA salts. These non-hydrolyzable dNTP analogues are envisioned
to be effective inhibitors of polymerases and are current being explored as such.
EXPERIMENTAL SECTION
All reagents are purchased from Sigma-Aldrich. HPLC analysis and separation was car-
ried out on a Varian ProStar 210 (pump/injector) and Shimatzu SPD-10A VP (UV Vis de-
tector) using a Varian Microsorb-MV 4.6 mm x 250 mm - 5 µm analytical column, Varian
Dynamax 100A C-18 21.4 mm x 250 mm - 5 µm preparative column, Varian PureGel
SAX 10 mm x 100 mm - 7 µm analytical column, and Macherey-Nagel Nucleogel SAX
1000-10 25 mm x 150 mm - preparative column. NMR analysis was carried out on either
Bruker AMX-500 MHz FT NMR or Varian Mercury 400 NMR instruments. LC-MS was
determined using ThermoScientific LCQ-X and Excaliber Workstation software. HRMS
was acquired via an outside source (at UCLA and UCR). A detailed collection of HPLC,
HRMS, and NMR data can be found in APPENDIX H.
167
General synthesis of nucleoside-monophosphate morpholidate
1.1 equivalents of dNMP are weighed out in a dried round bottom flask and dissolved in a
50% tBuOH:H2O solution. Complete dissolution of sodium salts of dNMP is achieved by
lowering the pH to 2. Three equivalents of morpholine are added dropwise. The solution
is left at ambient temperature for 15 minutes then set to reflux. After a steady reflux, 3
equivalents of DCC dissolved in tBuOH is added dropwise over 2 hours. After 2.5 hours,
the reaction is examined by
31
P NMR. If the reaction is incomplete, an appropriate
amount of DCC in tBuOH is added and allowed to react for another hour to drive the re-
action to completion. When the reaction is completed, the solvent is removed under re-
duced pressure. The light brown or yellowish-white residue is then dissolved in H2O and
the suspension is filtered to remove the dicyclohexylurea. The aqueous solution is then
washed three times with diethyl ether. The solvent is then removed under reduced pres-
sure and the brownish white residue is dried under vacuum.
Synthesis of 2’-deoxy-5’-guanosinemonophosphate morpholidate
107.6 mg (0.310 mmol) of dGMP is weighed out in a dried round bottom flask and is dis-
solved in 10mL of a 50% tBuOH:H2O solution. The pH is adjusted to 2. 64 µL (0.930
mmol) of distilled morpholine is added dropwise using a 100 µL gas-tight syringe. The
solution is reacted at ambient temperature for 15 minutes then set to reflux. After a steady
reflux 198 mg (0.930 mmol) of DCC dissolved in 2 mL of tBuOH is added dropwise over
2 hours. After 2.5 hours, the reaction is examined by
31
P NMR. When the reaction is
completed, the solvent is removed by reduced pressure. The light brown or yellowish-
168
white residue is then dissolved in H2O and the suspension is filtered to remove the dicy-
clohexylurea. The solvent is then removed under reduced pressure and the brownish
white residue is dried under vacuum. dGMP-Morph 1 is obtained in good yield (yield =
130 mg - 95.5%).
31
P: 7 (s).
Synthesis of 2’-deoxy-5’-thymidinemonophosphate morpholidate
50 mg (0.16 mmol) of dTMP is weighed out in a dried round bottom flask and is dis-
solved in 10mL of a 50% tBuOH:H2O solution. The pH is adjusted to 2. 41 µL (0.48
mmol) of distilled morpholine is added dropwise using a 100 µL gas-tight syringe. The
solution is reacted at ambient temperature for 15 minutes then set to reflux. After a steady
reflux 96 mg (0.48 mmol) of DCC dissolved in 2 mL of tBuOH is added dropwise over 2
hours. After 2.5 hours, the reaction is examined by
31
P NMR. When the reaction is com-
pleted, the solvent is removed by reduced pressure. The light brown or yellowish-white
residue is then dissolved in H2O and the suspension is filtered to remove the dicyclohexy-
lurea. The solvent is then removed under reduced pressure and the brownish white resi-
due is dried under vacuum. dTMP-Morph is obtained in good yield (yield = 61.5 mg -
98%).
31
P: 7 (s).
Synthesis of 5’-adenosinemonophosphate morpholidate
90mg (0.26 mmol) of AMP is weighed out in a dried round bottom flask and is dissolved
in 10mL of a 50% tBuOH:H2O solution. The pH is adjusted to 2. 68 µL (0.78 mmol) of
distilled morpholine is added dropwise using a 100 µL gas-tight syringe. The solution is
169
reacted at ambient temperature for 15 minutes then set to reflux. After a steady reflux 240
mg (0.78 mmol) of DCC dissolved in 2 mL of tBuOH is added dropwise over 2 hours.
After 2.5 hours, the reaction is examined by
31
P NMR. When the reaction is completed,
the solvent is removed by reduced pressure. The light brown or yellowish-white residue
is then dissolved in H2O and the suspension is filtered to remove the dicyclohexylurea.
The solvent is then removed under reduced pressure and the brownish white residue is
dried under vacuum. AMP-Morph
5
is obtained in good yield (yield = 105 mg - 98%).
31
P:
7 (s).
Synthesis of 5’-guanosinemonophosphate morpholidate
90 mg (0.25 mmol) of GMP is weighed out in a dried round bottom flask and is dissolved
in 10mL of a 50% tBuOH:H2O solution. The pH is adjusted to 2. 65 µL (0.75 mmol) of
distilled morpholine is added dropwise using a 100 µL gas-tight syringe. The solution is
reacted at ambient temperature for 15 minutes then set to reflux. After a steady reflux 206
mg (1.01 mmol) of DCC dissolved in 2 mL of tBuOH is added dropwise over 2 hours.
After 2.5 hours, the reaction is examined by
31
P NMR. When the reaction is completed,
the solvent is removed by reduced pressure. The light brown or yellowish-white residue
is then dissolved in H2O and the suspension is filtered to remove the dicyclohexylurea.
The solvent is then removed under reduced pressure and the brownish white residue is
dried under vacuum. GMP-Morph is obtained in good yield (yield = 102 mg - 95%).
31
P:
7 (s).
170
General procedure for the synthesis of β,γ-nucleoside triphosphate analogues
1.1 equivalents of dried NMP-Morph is dissolved in freshly distilled anhydrous DMSO.
In a separate flask, 4.4 equivalents of bisphosphonate (tributylammonium salt) are dis-
solved in anhydrous DMSO. The [Bu3NH
+
]BP solution is added slowly to the dNMP-
Morph. The reaction is monitored using analytical HPLC on an SAX ion exchange col-
umn and a 0 - 100% 0.5 M TEAB buffer (pH = 8) gradient or 0 - 50% 0.5 M LiCl (pH =
8) gradient. After the reaction has reached completion, the solvent is removed under re-
duced pressure. The yellowish oil is then dissolved in 1.5 mL of 0.5 M TEAB buffer. The
product is isolated from the reaction mixture via two-stage preparative HPLC; first by
SAX (0 - 100% 0.5 M TEAB gradient) and then on C-18 (0.1 N TEAB 4% CH3CN). The
fractions containing the β,γ-dNTP analogue are collected and combined and then lyophi-
lized to the TEA salt.
Synthesis of 2’-deoxy-5’-β,γ-methylene-thymidine triphosphate (β,γ-CH2-dTTP)
Following the general procedure, 90 mg (0.23 mmol) of dTMP-Morph was reacted with
the trisbutylammonium salt of MBP (0.92 mmol) in 4 mL of distilled anhydrous DMSO.
The product is isolated from the reaction mixture via two-stage preparative HPLC; once
on SAX and then on C-18. β,γ-CH2-dTTP
1
is obtained as the TEA salt (yield = 25 mg -
23%).
31
P: -11 (d), 11.2 (d), 13.2 (m);
1
H (D2O, pH = 10): 7.7 (d), 6.4 (t), 4.6 (d), 4.3 (m),
4.2 (s), 2.2 (t), 1.8 (s).
171
Synthesis of 5’-β,γ-difluoromethylene-adenosine triphosphate (β,γ-CF2-ATP)
Following the general procedure, 90 mg (0.22 mmol) of AMP-Morph was reacted with
the trisbutylammonium salt of DFBP (0.86 mmol) in 4 mL of distilled anhydrous DMSO.
The product is isolated from the reaction mixture via two-stage preparative HPLC; once
on SAX and then on C-18. β,γ-CF2-ATP
5
is obtained as the TEA salt (yield = 45 mg -
36%).
31
P: -11 (d), -3 (m), 3.5 (m);
1
H (D2O, pH = 8): 8.8 (b), 8.2 (s), 6.3 (d), 4.65 (t), 4.5
(s), 4.4 (s), 4.2 (m);
19
F: -117.1 (dd).
Synthesis of 5’-β,γ-etidronate-guanosine triphosphate (β,γ-etidronate-GTP)
Following the general procedure, 100 mg (0.23 mmol) of GMP-Morph was reacted with
the trisbutylammonium salt of etidronate (9.2 mmol) in 4 mL of distilled anhydrous
DMSO. The product is isolated from the reaction mixture via two-stage preparative
HPLC; once on SAX and then on C-18. β,γ-etidronate-GTP
7
is obtained as the TEA salt
(yield = 61 mg - 48%).
31
P: -11 (d), 12.5 (m), 16 (m);
1
H (D2O, pH = 10.5): 8.25 (s), 6.07
(d), 4.55 (m), 4.35 (m), 4.25 (m), 1.6 (t).
Synthesis of 2’-deoxy-5’-β,γ-etidronate-guanosine triphosphate (β,γ-etidronate-
dGTP)
Following the general procedure, 45 mg (0.11 mmol) of dGMP-Morph was reacted with
the trisbutylammonium salt of risedronate (0.43 mmol) in 4 mL of distilled anhydrous
DMSO. The product is isolated from the reaction mixture via two-stage preparative
HPLC; once on SAX and then on C-18. β,γ-etidronate-dGTP is obtained as the TEA salt
172
(yield = 18 mg - 32%).
31
P: -11 (d), 12.5 (m), 16 (m);
1
H (D2O, pH = 10.5): 8.0 (s), 6.25
(t), 4.25 (m), 4.1 (m), 2.8 (m), 2.4 (m).
Synthesis of 2’-deoxy-5’-β,γ-risedronate-guanosine triphosphate (β,γ-risedronate-
dGTP)
Following the general procedure, 45 mg (0.10 mmol) of dGMP-Morph was reacted with
the trisbutylammonium salt of risedronate (0.42 mmol) in 4 mL of distilled anhydrous
DMSO. The product is isolated from the reaction mixture via two-stage preparative
HPLC; once on SAX and then on C-18. β,γ-risedronate-dGTP is obtained as the TEA salt;
however it decomposes during lyopholization.
31
P (D2O, pH = 10): -11 (dd), 12.8 (m), 14
(m).
Synthesis of 2’-deoxy-5’-β,γ-CF3-etidronate-guanosine triphosphate (β,γ-CF3-
etidronate-dGTP)
Following the general procedure, 90 mg (0.21 mmol) of dGMP-Morph was reacted with
the trisbutylammonium salt of CF3-etidronate (0.86 mmol) in 4 mL of distilled anhydrous
DMSO. The product is isolated from the reaction mixture via two-stage preparative
HPLC; once on SAX (0-100% 0.5 TEAA; pH = 8) and then on C-18 (0.1 N TEAA - 4%
CH3CN; pH = 4.5). β,γ-CF3-etidronate-dGTP is obtained as the TEA salt (yield = 28 mg -
22%).
31
P: -11 (d), 0.5 (m), 8.1 (m);
1
H (D2O, pH = 4.5): 8.0 (s), 6.25 (t), 4.25 (m), 4.1
(m), 2.8 (m), 2.4 (m);
19
F: -69.6 (bt).
173
Purification 2’-deoxy-5’-α,β-difluoromethylene-thymidine triphosphate (α,β-CF2-
dTTP)
α,β-CF2-dTTP
1
was isolated as the TEA salt after dual-pass preparative HPLC.
31
P: -11
(d), -3 (m), 3.5 (m);
1
H (D2O, pH = 8): 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m), 2.1 (m),
2.05 (m), 2.0 (dd);
19
F: -119.2 (dt).
Purification 2’-deoxy-5’-α,β,β,γ-bis(CF2)-thymidine triphosphate (α,β,β,γ-bis(CF2)-
dTTP)
α,β,β,γ-bis-(CF2)-dTTP was isolated as the TEA salt after dual-pass preparative HPLC.
31
P: 3.25 (m), 3.5 (m), 13.5 (m);
1
H (D2O, pH = 8): 7.8 (d), 6.45 (t), 4.7 (m), 4.3 (m), 4 .2
(m), 2.3 (m), 1.9 (s);
19
F: -117.8 (t), -118.6 (dt).
Purification 2’-deoxy-5’-α,β,β,γ-bis(CF2)-adenosine triphosphate (α,β,β,γ-bis(CF2)-
dATP)
α,β,β,γ-bis-(CF2)-dATP was isolated as the TEA salt after dual-pass preparative HPLC.
31
P: 3.25 (m), 3.5 (m), 13.6 (m);
1
H (D2O, pH = 8): 8.61 (s), 8.28 (s), 6.52 (t), 5.8 (d),
4.75 (m), 4.3 (m), 4 (m), 3.39 (m), 2.85 (m), 2.65 (m);
19
F: -117.8 (t), -118.7 (dt)
174
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190
APPENDIX A
Carbovir monophosphate (2)
NMR A.1 -
31
P: 3.75 (s)
NMR A.2 -
1
H (CD3OD): 7.61 (s), 6.10 (m), 5.80 (m), 5.43 (m), 3.51 (t), 2.91 (m), 2.64
(dt), 1.60 (dt)
191
Table A.1 Analytical HPLC of Carbovir derivatives
Compound Retention Time Column (Buffer)
CBV 12 min C-18
CBV-MP
3 min
4 min
C-18
SAX
CBV-Morph
13 min
3 min
C-18
SAX
CBV-TP
2.5 min
12 min
C-18
SAX
Carbovir triphosphosphate (4) - LC-MS (m/z) = 486 [M-H
-
]
NMR A.3 - Tenofovir diisoproxil fumarate (5)
192
Tenofovir (6)
NMR A.4 -
1
H (D2O): 8.40 (s), 8.38 (s), 6.81 (s), 4.50 (m), 4.30 (m), 3.75 (m), 1.2 (d)
Table A.2 Analytical HPLC of Tenofovir derivatives
Compound Retention Time Column (Buffer)
TNF 3 min C-18
TNF-MP
3 min
4 min
C-18
SAX
TNF-Morph
5 min
3.5 min
C-18
SAX
TNF-DP
2.5 min
11.5 min
C-18
SAX
193
APPENDIX B
tetraisopropyl (dichloromethylene)bis(phosphonate), 1
31
P: 7.9 (s)
(dichloromethylene)bis(phosphonic acid), 2
NMR B.2 -
31
P: 7.5 (s)
tetraisopropyl (chloromethylene)bis(phosphonate), 3
194
NMR B.3 -
31
P: 12.6 (s)
chloromethylenebis(phosphonic acid), 4
NMR B.4 -
31
P: 11.8 (s)
NMR B.5 -
1
H (D2O): 3.8 (dd)
195
tetraisopropyl (dibromomethylene)bis(phosphonate), 5
NMR B.6 -
31
P: 7.4 (s)

(dibromomethylene)bis(phosphonic acid), 6
NMR B.7 -
31
P: 7.3 (s)
196
tetraisopropyl (bromomethylene)bis(phosphonate), 7
NMR B.8 -
31
P: 12.1 (s)

(bromomethylene)bis(phosphonic acid), 8
NMR B.9 -
31
P: 11.4 (s)
197
NMR B.10 -
1
H (D2O): 3.5 (dd)
tetraisopropyl 2,2-propanediylbis(phosphonate), 9
NMR B.11 -
31
P: 26 (s)
198
2,2-propanediylbis(phosphonic acid), 10
NMR B.12 -
1
H (D2O): 1.8 (td)
NMR B.13 -
31
P: 26.9 (s)
199

tetraisopropyl 1,1-ethanediylbis(phosphonate), 11
NMR B.14 -
31
P: 22.8 (t)
tetraethyl 1,1-ethanediylbis(phosphonate), 12
200
NMR B.15 -
31
P: 24 (s)
1,1-ethanediylbis(phosphonic acid), 13
NMR B.16 -
31
P: 22.5 (s)

NMR B.17 -
1
H (D2O): 1.1 (td), 2.1 (dq)
201

tetraisopropyl (difluoromethylene)bis(phosphonate), 14
NMR B.18 -
31
P: 3.0 (t)
NMR B.19 -
19
F: -123.3 (t)
202
(difluoromethylene)bis(phosphonic acid), 15
NMR B.20 -
31
P: 2.8 (t)
NMR B.21 -
19
F: -122.8 (t)
203
tetraisopropyl (fluoromethylene)bis(phosphonate), 16
NMR B.22 -
31
P: 10.8 (d)
NMR B.23 -
19
F: -226.1 (dt)
204
(fluoromethylene)bis(phosphonic acid), 17
NMR B.24 -
31
P: 10.5 (d)
NMR B.25 -
19
F: -226.4 (dt)
205
NMR B.26 -
1
H (D2O): 4.8 (dt) (other peaks @ 0.8, 0.9, 3.2 are impurities from ethanol)
tetraisopropyl [chloro(fluoro)methylene]bis(phosphonate), 18
206
NMR B.27 -
31
P: 5 (d)
[chloro(fluoro)methylene]bis(phosphonic acid), 19
NMR B.28 -
31
P: 4.4 (d)

NMR B.29 -
19
F: -145.4 (t)
207

tetraisopropyl (1-fluoro-1,1-ethanediyl)bis(phosphonate), 20
NMR B.30 -
31
P: 13.1 (d)
NMR B.31 -
19
F: -186.6 (m)
208
NMR B.32 -
1
H (CDCl3): 1.25 (d), 1.65 (dt), 4.75 (m) [small impurity peaks are from
ethyl acetate (1,2, 1.9, 4.0) and hexane (0.8, 1.4) and CDCl3 (7.26)]
tetraethyl (1-fluoro-1,1-ethanediyl)bis(phosphonate), 21
209
NMR B.33 -
31
P: 14 (d)
NMR B.34 -
19
F: -186.6 (m)
210
NMR B.35 -
1
H (CDCl3): 1.45 (t), 1.85 (dt), 4.35 (m) [H2O peak (1.7) in CDCl3 (7.26)]
fluoroethylidene bisphosphonic acid (22)
NMR B.36 -
31
P: 12 (d)
NMR B.37 -
19
F: -186.2
211

NMR B.38 -
1
H (D2O): 1.32 (dt)
212
APPENDIX C
Table C.1 Initial analysis of MBP for calibration of electrode (Aldrich - NMR electrode): 5 mM
MDP in 50 mL of 0.1M NaCl titrated with 0.099M NaOH
NaOH emf (mv) NaOH emf (mv) NaOH emf (mv)
0 276 0.86 227.3 1.84 -107.1
0.02 275.2 0.88 225.3 1.86 -119.7
0.04 274.4 0.9 223.2 1.88 -129.9
0.06 273.6 0.94 218.7 1.9 -138.3
0.08 272.8 0.96 216.3 1.84 -107.1
0.1 272 0.98 213.6 1.86 -119.7
0.12 271.2 1 210.8 1.88 -129.9
0.14 270.4 1.02 207.7 1.9 -138.3
0.16 269.5 1.04 204.3 1.92 -145.2
0.18 268.7 1.06 200.6 1.94 -151
0.2 267.9 1.08 196.3 1.96 -156.1
0.22 267 1.1 191.5 1.98 -160.7
0.24 266.1 1.12 185.7 2 -164.8
0.26 265.2 1.14 178.6 2.02 -168.6
0.28 264.2 1.16 169.1 2.04 -172.2
0.3 263.3 1.18 154.9 2.06 -175.6
0.32 262.4 1.2 128.9 2.08 -178.9
0.34 261.4 1.22 91.6 2.1 -182
0.36 260.5 1.24 69.8 2.12 -185.1
0.38 259.5 1.26 56.9 2.14 -188
0.4 258.5 1.28 47.7 2.16 -190.8
0.42 257.5 1.3 40.3 2.18 -193.8
0.44 256.4 1.32 34.1 2.2 -196.6
0.46 255.4 1.34 28.7 2.22 -199.4
0.48 254.3 1.36 24 2.24 -202.1
213
NaOH emf (mv) NaOH emf (mv) NaOH emf (mv)
0.5 253.3 1.38 19.6 2.26 -204.8
0.52 252.1 1.4 15.6 2.28 -207.6
0.54 250.9 1.42 11.8 2.3 -210.2
0.56 249.8 1.44 8.2 2.32 -212.9
0.58 248.5 1.46 4.6 2.34 -215.6
0.6 247.3 1.48 1.2 2.36 -218.3
0.62 246 1.5 -2.3 2.38 -221
0.64 244.7 1.52 -5.7 2.4 -223.7
0.66 243.4 1.54 -9 2.42 -226.4
0.68 242 1.56 -12.4 2.44 -229
0.7 240.6 1.58 -15.8 2.46 -231.5
0.72 239.1 1.6 -19.5 2.48 -234
0.74 237.6 1.62 -23.1 2.5 -236.4
0.76 236.1 1.64 -27 2.52 -238.8
0.78 234.5 1.66 -31.1 2.54 -241.1
0.8 232.8 1.68 -35.5 2.56 -243.2
0.82 231 1.7 -40.1 2.58 -245.3
0.84 229.2 1.72 -45.4
214
Figure C.1 Titration curve for MBP to calibrate electrode; standard potential is measured
-245.300
-114.975
15.350
145.675
276.000
0 0.65 1.30 1.95 2.60
Calibration Curve for Aldrich pH Electrode
Standard Potential (mv)
Amount of NaOH added (mL)
215
Figure C.2 Titration curve for titration of MBP with NaOH
Trial 1 Trial 2 Trial 3 Trial 4
0
2.874
5.747
8.621
11.494
0 0.525 1.050 1.575 2.100
Titration of MBP
pH
Amount of NaOH added (mL)
Trial 1: 5 mM MBP in 10 mL of 0.1 M NaCl with 0.1 M NaOH
Trial 2: 12.35 mg MBP in 10.85 ml 0.1 M NaCl with 0.1 M NaOH
Trial 3: 10.63 ml of stock MBP (46.6 mg in 50 ml NaCl) with 0.1 M NaOH
Trial 4: 10.27 ml of Stock MBP with 0.1 M NaOH
NaOH pH NaOH pH NaOH pH NaOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0 2.286 0 2.199 0 2.2 0 2.17
0.02 2.3 0.02 2.202 0.02 2.21 0.02 2.18
0.04 2.317 0.04 2.21 0.04 2.22 0.04 2.19
0.06 2.335 0.06 2.22 0.06 2.23 0.06 2.2
216
NaOH pH NaOH pH NaOH pH NaOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0.08 2.354 0.08 2.23 0.08 2.24 0.08 2.22
0.1 2.373 0.1 2.24 0.1 2.25 0.1 2.23
0.12 2.393 0.12 2.251 0.12 2.26 0.12 2.25
0.14 2.414 0.14 2.262 0.14 2.27 0.14 2.26
0.16 2.435 0.16 2.273 0.16 2.28 0.16 2.27
0.18 2.457 0.18 2.284 0.18 2.3 0.18 2.29
0.2 2.48 0.2 2.296 0.2 2.31 0.2 2.3
0.22 2.503 0.22 2.308 0.22 2.33 0.22 2.32
0.24 2.528 0.24 2.32 0.24 2.34 0.24 2.33
0.26 2.555 0.26 2.333 0.26 2.35 0.26 2.35
0.28 2.582 0.28 2.346 0.28 2.37 0.28 2.37
0.3 2.611 0.3 2.359 0.3 2.38 0.3 2.38
0.32 2.64 0.32 2.372 0.32 2.4 0.32 2.4
0.34 2.672 0.34 2.386 0.34 2.42 0.34 2.42
0.36 2.703 0.36 2.4 0.36 2.43 0.36 2.44
0.38 2.737 0.38 2.414 0.38 2.45 0.38 2.46
0.4 2.772 0.4 2.428 0.4 2.47 0.4 2.48
0.42 2.808 0.42 2.443 0.42 2.49 0.42 2.5
0.44 2.847 0.44 2.458 0.44 2.51 0.44 2.52
0.46 2.887 0.46 2.473 0.46 2.53 0.46 2.54
0.48 2.93 0.48 2.489 0.48 2.54 0.48 2.56
0.5 2.975 0.5 2.505 0.5 2.56 0.5 2.58
0.52 3.024 0.52 2.521 0.52 2.58 0.52 2.61
0.54 3.078 0.54 2.538 0.54 2.61 0.54 2.63
0.56 3.137 0.56 2.555 0.56 2.62 0.56 2.66
0.58 3.204 0.58 2.572 0.58 2.65 0.58 2.68
0.6 3.279 0.6 2.591 0.6 2.67 0.6 2.71
0.62 3.363 0.62 2.609 0.62 2.69 0.62 2.73
0.64 3.462 0.64 2.627 0.64 2.72 0.64 2.76
0.66 3.585 0.66 2.647 0.66 2.75 0.66 2.79
0.68 3.746 0.68 2.667 0.68 2.78 0.68 2.82
0.7 3.987 0.7 2.687 0.7 2.8 0.7 2.86
0.72 4.453 0.72 2.708 0.72 2.83 0.72 2.89
0.74 5.341 0.74 2.729 0.74 2.86 0.74 2.93
0.76 5.801 0.76 2.751 0.76 2.89 0.76 2.96
0.78 6.056 0.78 2.774 0.78 2.92 0.78 3
0.8 6.232 0.8 2.797 0.8 2.96 0.8 3.04
0.82 6.375 0.82 2.82 0.82 3 0.82 3.09
0.84 6.499 0.84 2.846 0.84 3.03 0.84 3.14
0.86 6.61 0.86 2.871 0.86 3.07 0.86 3.19
0.88 6.713 0.88 2.898 0.88 3.12 0.88 3.25
0.9 6.811 0.9 2.926 0.9 3.17 0.9 3.32
0.92 6.907 0.92 2.955 0.92 3.22 0.92 3.39
0.94 7.003 0.94 2.984 0.94 3.28 0.94 3.47
0.96 7.099 0.96 3.016 0.96 3.34 0.96 3.57
0.98 7.2 0.98 3.049 0.98 3.41 0.98 3.69
1 7.309 1 3.083 1 3.48 1 3.86
217
NaOH pH NaOH pH NaOH pH NaOH pH
Trial 1 Trial 2 Trial 3 Trial 4
1.02 7.429 1.02 3.119 1.02 3.58 1.02 4.09
1.04 7.568 1.04 3.158 1.04 3.69 1.04 4.52
1.06 7.738 1.06 3.199 1.06 3.84 1.06 5.25
1.08 7.96 1.08 3.244 1.08 4.05 1.08 5.67
1.1 8.265 1.1 3.291 1.1 4.39 1.1 5.91
1.12 8.644 1.12 3.342 1.12 5.03 1.12 6.08
1.14 8.968 1.14 3.4 1.14 5.53 1.14 6.21
1.16 9.199 1.16 3.464 1.16 5.8 1.16 6.33
1.18 9.375 1.18 3.534 1.18 5.98 1.18 6.43
1.2 9.516 1.2 3.618 1.2 6.12 1.2 6.51
1.22 9.633 1.22 3.716 1.22 6.23 1.22 6.6
1.24 9.736 1.24 3.835 1.24 6.33 1.24 6.67
1.26 9.828 1.26 3.994 1.26 6.41 1.26 6.74
1.28 9.913 1.28 4.225 1.28 6.49 1.28 6.81
1.3 9.993 1.3 4.634 1.3 6.57 1.3 6.88
1.32 10.069 1.32 5.238 1.32 6.64 1.32 6.95
1.34 10.142 1.34 5.613 1.34 6.7 1.34 7
1.36 10.212 1.36 5.838 1.36 6.77 1.36 7.07
1.38 10.28 1.38 5.998 1.38 6.83 1.38 7.14
1.4 10.346 1.4 6.122 1.4 6.89 1.4 7.21
1.42 10.41 1.42 6.224 1.42 6.95 1.42 7.28
1.44 10.474 1.44 6.314 1.44 7.01 1.44 7.36
1.46 10.55 1.46 6.395 1.46 7.07 1.46 7.45
1.48 10.6 1.48 6.468 1.48 7.14 1.48 7.54
1.5 10.66 1.5 6.536 1.5 7.2 1.5 7.65
1.52 10.715 1.52 6.599 1.52 7.27 1.52 7.77
1.54 10.764 1.54 6.659 1.54 7.35 1.54 7.92
1.56 10.814 1.56 6.717 1.56 7.43 1.56 8.12
1.58 10.864 1.58 6.773 1.58 7.52 1.58 8.38
1.6 10.91 1.6 6.828 1.6 7.62 1.6 8.67
1.62 10.954 1.62 6.881 1.62 7.74 1.62 8.92
1.64 10.994 1.64 6.935 1.64 7.88 1.64 9.12
1.66 11.032 1.66 6.988 1.66 8.06 1.66 9.27
1.68 11.068 1.68 7.041 1.68 8.29 1.68 9.39
1.7 11.101 1.7 7.093 1.7 8.56 1.7 9.5
1.72 11.132 1.72 7.147 1.72 8.81 1.72 9.59
1.74 11.162 1.74 7.204 1.74 9.02 1.74 9.68
1.76 11.19 1.76 7.262 1.76 9.18 1.76 9.76
1.78 11.217 1.78 7.323 1.78 9.31 1.78 9.83
1.8 11.241 1.8 7.387 1.8 9.42 1.8 9.89
1.82 11.264 1.82 7.455 1.82 9.51 1.82 9.95
1.84 11.286 1.84 7.527 1.84 9.59 1.84 10.01
1.86 11.306 1.86 7.606 1.86 9.67 1.86 10.07
1.88 11.325 1.88 7.696 1.88 9.73 1.88 10.13
1.9 11.345 1.9 7.803 1.9 9.8 1.9 10.18
1.92 11.362 1.92 7.929 1.92 9.87 1.92 10.23
1.94 11.379 1.94 8.081 1.94 9.93 1.94 10.28
218
NaOH pH NaOH pH NaOH pH NaOH pH
1.96 11.395 1.96 8.273 1.96 9.98 1.96 10.33
1.98 11.411 1.98 8.509 1.98 10.03 1.98 10.38
2 11.426 2 8.746 2 10.08 2 10.44
2.02 11.44 2.02 8.945 2.02 10.14 2.02 10.49
2.04 11.455 2.04 9.106 2.04 10.19 2.04 10.53
2.06 11.469 2.06 9.238 2.06 10.24 2.06 10.58
2.08 11.482 2.08 9.346 2.08 10.29 2.08 10.63
2.1 11.494 2.1 9.44 2.1 10.34 2.1 10.68
Trial 1 Trial 2 Trial 3 Trial 4
219
Figure C.3 Titration curve for titration of MBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4
0
2.835
5.670
8.505
11.340
0 0.7 1.4 2.1 2.8
Titration of MBP (KOH)
pH
Amount of NaOH added (mL)
Trial 1: 10 ml MBP (45.31 mg MBP in 50 ml 0.1 M KCl) with 0.086 M KOH
Trial 2: 10.69 ml of stock MBP with 0.086 M KOH
Trial 3: 10 ml of stock MBP with 0.087 M KOH
Trial 4: 10 ml of stock MBP with 0.0893 M KOH
220
NaOH pH NaOH pH NaOH pH NaOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0 2.26 0 2.25 0 2.31 0 2.33
0.02 2.27 0.02 2.26 0.02 2.31 0.02 2.33
0.04 2.28 0.04 2.27 0.04 2.32 0.04 2.34
0.06 2.29 0.06 2.28 0.06 2.33 0.06 2.34
0.08 2.3 0.08 2.29 0.08 2.34 0.08 2.34
0.1 2.31 0.1 2.3 0.1 2.35 0.1 2.35
0.12 2.32 0.12 2.31 0.12 2.37 0.12 2.36
0.14 2.33 0.14 2.32 0.14 2.38 0.14 2.37
0.16 2.35 0.16 2.33 0.16 2.39 0.16 2.38
0.18 2.36 0.18 2.35 0.18 2.41 0.18 2.39
0.2 2.37 0.2 2.36 0.2 2.42 0.2 2.41
0.22 2.38 0.22 2.37 0.22 2.43 0.22 2.42
0.24 2.4 0.24 2.38 0.24 2.45 0.24 2.43
0.26 2.41 0.26 2.4 0.26 2.46 0.26 2.44
0.28 2.42 0.28 2.41 0.28 2.48 0.28 2.45
0.3 2.44 0.3 2.42 0.3 2.49 0.3 2.47
0.32 2.45 0.32 2.44 0.32 2.51 0.32 2.48
0.34 2.47 0.34 2.45 0.34 2.52 0.34 2.5
0.36 2.48 0.36 2.47 0.36 2.54 0.36 2.51
0.38 2.5 0.38 2.48 0.38 2.56 0.38 2.53
0.4 2.52 0.4 2.5 0.4 2.57 0.4 2.54
0.42 2.53 0.42 2.52 0.42 2.59 0.42 2.56
0.44 2.55 0.44 2.53 0.44 2.61 0.44 2.58
0.46 2.57 0.46 2.55 0.46 2.63 0.46 2.6
0.48 2.59 0.48 2.57 0.48 2.64 0.48 2.62
0.5 2.6 0.5 2.58 0.5 2.66 0.5 2.63
0.52 2.62 0.52 2.6 0.52 2.68 0.52 2.65
0.54 2.64 0.54 2.62 0.54 2.7 0.54 2.68
0.56 2.66 0.56 2.64 0.56 2.73 0.56 2.7
0.58 2.68 0.58 2.66 0.58 2.75 0.58 2.72
0.6 2.71 0.6 2.68 0.6 2.77 0.6 2.74
0.62 2.73 0.62 2.7 0.62 2.79 0.62 2.77
0.64 2.75 0.64 2.72 0.64 2.82 0.64 2.79
0.66 2.78 0.66 2.74 0.66 2.84 0.66 2.82
0.68 2.8 0.68 2.76 0.68 2.87 0.68 2.85
0.7 2.83 0.7 2.79 0.7 2.9 0.7 2.87
0.72 2.85 0.72 2.81 0.72 2.93 0.72 2.9
0.74 2.88 0.74 2.83 0.74 2.96 0.74 2.93
0.76 2.91 0.76 2.86 0.76 2.99 0.76 2.96
0.78 2.94 0.78 2.88 0.78 3.02 0.78 3
0.8 2.97 0.8 2.91 0.8 3.05 0.8 3.03
0.82 3 0.82 2.94 0.82 3.09 0.82 3.07
0.84 3.04 0.84 2.97 0.84 3.13 0.84 3.11
0.86 3.08 0.86 3 0.86 3.17 0.86 3.16
0.88 3.12 0.88 3.03 0.88 3.21 0.88 3.2
0.9 3.16 0.9 3.07 0.9 3.26 0.9 3.26
221
NaOH pH NaOH pH NaOH pH NaOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0.92 3.21 0.92 3.1 0.92 3.31 0.92 3.31
0.94 3.25 0.94 3.14 0.94 3.36 0.94 3.38
0.96 3.31 0.96 3.18 0.96 3.42 0.96 3.45
0.98 3.37 0.98 3.22 0.98 3.49 0.98 3.53
1 3.43 1 3.27 1 3.57 1 3.63
1.02 3.51 1.02 3.32 1.02 3.66 1.02 3.76
1.04 3.59 1.04 3.38 1.04 3.77 1.04 3.93
1.06 3.7 1.06 3.44 1.06 3.9 1.06 4.18
1.08 3.83 1.08 3.51 1.08 4.09 1.08 4.67
1.1 4.01 1.1 3.59 1.1 4.38 1.1 5.36
1.12 4.29 1.12 3.69 1.12 4.93 1.12 5.7
1.14 4.85 1.14 3.8 1.14 5.52 1.14 5.9
1.16 5.46 1.16 3.95 1.16 5.85 1.16 6.05
1.18 5.78 1.18 4.17 1.18 6.06 1.18 6.17
1.2 5.99 1.2 4.56 1.2 6.21 1.2 6.28
1.22 6.13 1.22 5.22 1.22 6.33 1.22 6.37
1.24 6.25 1.24 5.64 1.24 6.44 1.24 6.45
1.26 6.35 1.26 5.88 1.26 6.53 1.26 6.52
1.28 6.44 1.28 6.05 1.28 6.62 1.28 6.59
1.3 6.52 1.3 6.18 1.3 6.69 1.3 6.67
1.32 6.6 1.32 6.29 1.32 6.77 1.32 6.73
1.34 6.67 1.34 6.38 1.34 6.84 1.34 6.79
1.36 6.73 1.36 6.46 1.36 6.9 1.36 6.86
1.38 6.8 1.38 6.54 1.38 6.96 1.38 6.92
1.4 6.86 1.4 6.61 1.4 7.02 1.4 6.98
1.42 6.92 1.42 6.67 1.42 7.08 1.42 7.04
1.44 6.98 1.44 6.73 1.44 7.14 1.44 7.11
1.46 7.03 1.46 6.8 1.46 7.2 1.46 7.18
1.48 7.09 1.48 6.85 1.48 7.26 1.48 7.26
1.5 7.16 1.5 6.91 1.5 7.32 1.5 7.35
1.52 7.22 1.52 6.97 1.52 7.39 1.52 7.44
1.54 7.29 1.54 7.02 1.54 7.46 1.54 7.54
1.56 7.37 1.56 7.08 1.56 7.53 1.56 7.66
1.58 7.44 1.58 7.14 1.58 7.61 1.58 7.8
1.6 7.53 1.6 7.2 1.6 7.7 1.6 7.97
1.62 7.63 1.62 7.26 1.62 7.81 1.62 8.22
1.64 7.74 1.64 7.33 1.64 7.93 1.64 8.54
1.66 7.87 1.66 7.39 1.66 8.08 1.66 8.91
1.68 8.04 1.68 7.47 1.68 8.28 1.68 9.19
1.7 8.25 1.7 7.55 1.7 8.52 1.7 9.39
1.72 8.55 1.72 7.64 1.72 8.81 1.72 9.54
1.74 8.88 1.74 7.74 1.74 9.05 1.74 9.67
1.76 9.15 1.76 7.86 1.76 9.24 1.76 9.78
1.78 9.34 1.78 8.01 1.78 9.39 1.78 9.88
1.8 9.48 1.8 8.21 1.8 9.51 1.8 9.96
1.82 9.59 1.82 8.47 1.82 9.61 1.82 10.04
1.84 9.69 1.84 8.78 1.84 9.7 1.84 10.11
222
NaOH pH NaOH pH NaOH pH NaOH pH
1.86 9.77 1.86 9.04 1.86 9.78 1.86 10.17
1.88 9.85 1.88 9.24 1.88 9.85 1.88 10.23
1.9 9.93 1.9 9.41 1.9 9.92 1.9 10.3
1.92 9.99 1.92 9.53 1.92 9.98 1.92 10.36
1.94 10.05 1.94 9.63 1.94 10.04 1.94 10.41
1.96 10.11 1.96 9.72 1.96 10.09 1.96 10.47
1.98 10.16 1.98 9.8 1.98 10.15 1.98 10.52
2 10.21 2 9.87 2 10.2 2 10.57
2.02 10.26 2.02 9.94 2.02 10.25 2.02 10.62
2.04 10.31 2.04 10 2.04 10.29 2.04 10.67
2.06 10.36 2.06 10.06 2.06 10.34 2.06 10.72
2.08 10.41 2.08 10.11 2.08 10.39 2.08 10.77
223
Figure C.4 Titration curve for titration of MFBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4
Trial 5 Trial 6 Trial 7
0
2.728
5.455
8.183
10.910
0 0.525 1.050 1.575 2.100
Titration of MFBP
pH
Amount of KOH added (mL)
Trial 1: 10.0 ml MFBP (47.4 mg MFBP in 50 ml KCl) with 0.0893 M KOH
Trial 2: 10.0 ml of stock MFBP with 0.0893 M KOH
Trial 3: 10 ml of stock MFBP with 0.0893 M KOH
Trial 4: 10 ml of stock MFBP with 0.0893 M KOH
Trial 5: 10 ml MFBP (44.98 mg MFBP in 50 ml 0.1 M KCl) with 0.0927 M KOH
Trial 6: 10 ml of stock MFBP with 0.0927 M KOH
Trial 7: 10 ml of stock MFBP with 0.0927 M KOH
224
KOH pH
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
0 2.15 2.08 2.02 2 2.2 2.13 2.11
0.02 2.16 2.09 2.03 2.02 2.21 2.15 2.13
0.04 2.17 2.1 2.04 2.03 2.22 2.16 2.15
0.06 2.17 2.11 2.06 2.04 2.23 2.17 2.16
0.08 2.18 2.12 2.07 2.05 2.24 2.18 2.17
0.1 2.19 2.13 2.08 2.07 2.25 2.2 2.18
0.12 2.2 2.14 2.09 2.08 2.26 2.21 2.19
0.14 2.21 2.15 2.1 2.09 2.27 2.22 2.21
0.16 2.22 2.16 2.11 2.1 2.28 2.23 2.22
0.18 2.23 2.17 2.12 2.11 2.29 2.24 2.23
0.2 2.24 2.18 2.14 2.13 2.3 2.26 2.24
0.22 2.25 2.2 2.15 2.14 2.32 2.27 2.26
0.24 2.27 2.21 2.16 2.15 2.33 2.28 2.27
0.26 2.28 2.22 2.18 2.17 2.34 2.3 2.28
0.28 2.29 2.24 2.19 2.18 2.35 2.31 2.3
0.3 2.31 2.25 2.21 2.2 2.37 2.33 2.32
0.32 2.32 2.26 2.22 2.21 2.38 2.34 2.33
0.34 2.34 2.28 2.24 2.23 2.4 2.36 2.34
0.36 2.35 2.29 2.25 2.24 2.41 2.38 2.36
0.38 2.36 2.31 2.27 2.26 2.43 2.39 2.38
0.4 2.38 2.32 2.28 2.28 2.45 2.41 2.4
0.42 2.4 2.34 2.3 2.29 2.47 2.43 2.42
0.44 2.42 2.36 2.32 2.31 2.48 2.45 2.44
0.46 2.43 2.38 2.34 2.32 2.5 2.47 2.46
0.48 2.45 2.39 2.36 2.34 2.52 2.49 2.48
0.5 2.47 2.41 2.38 2.36 2.54 2.51 2.5
0.52 2.49 2.43 2.4 2.39 2.56 2.54 2.52
0.54 2.51 2.45 2.42 2.41 2.59 2.56 2.55
0.56 2.54 2.48 2.44 2.43 2.61 2.59 2.57
0.58 2.56 2.5 2.47 2.45 2.64 2.62 2.6
0.6 2.59 2.52 2.49 2.48 2.67 2.65 2.63
0.62 2.62 2.55 2.52 2.51 2.7 2.68 2.66
0.64 2.64 2.58 2.55 2.53 2.74 2.71 2.69
0.66 2.67 2.6 2.58 2.56 2.77 2.75 2.73
0.68 2.71 2.63 2.61 2.59 2.81 2.79 2.77
0.7 2.74 2.67 2.64 2.62 2.85 2.84 2.81
0.72 2.78 2.7 2.67 2.66 2.9 2.88 2.86
0.74 2.82 2.74 2.71 2.7 2.95 2.94 2.91
0.76 2.87 2.78 2.76 2.74 3.01 3 2.97
0.78 2.92 2.83 2.8 2.79 3.08 3.07 3.03
0.8 2.97 2.88 2.85 2.84 3.15 3.15 3.11
0.82 3.04 2.94 2.91 2.89 3.25 3.25 3.2
0.84 3.11 3 2.97 2.96 3.37 3.39 3.31
0.86 3.21 3.08 3.05 3.03 3.53 3.57 3.47
0.88 3.32 3.17 3.14 3.11 3.78 3.86 3.69
0.9 3.47 3.28 3.25 3.22 4.21 4.34 4.07
0.92 3.68 3.42 3.39 3.35 4.68 4.76 4.57
225
KOH pH
0.94 4.01 3.62 3.58 3.53 4.99 5.04 4.92
0.96 4.46 3.93 3.88 3.82 5.2 5.23 5.15
0.98 4.82 4.39 4.34 4.25 5.36 5.38 5.31
1 5.06 4.79 4.76 4.69 5.49 5.5 5.45
1.02 5.23 5.06 5.03 4.97 5.59 5.61 5.56
1.04 5.37 5.25 5.22 5.18 5.69 5.7 5.66
1.06 5.49 5.39 5.37 5.33 5.78 5.79 5.75
1.08 5.6 5.52 5.5 5.46 5.87 5.88 5.84
1.1 5.69 5.62 5.61 5.57 5.95 5.96 5.92
1.12 5.78 5.72 5.7 5.67 6.02 6.03 6
1.14 5.86 5.81 5.79 5.76 6.1 6.11 6.07
1.16 5.93 5.89 5.87 5.84 6.17 6.19 6.15
1.18 6.01 5.97 5.95 5.92 6.25 6.27 6.23
1.2 6.08 6.04 6.03 6 6.33 6.34 6.31
1.22 6.16 6.12 6.1 6.07 6.4 6.43 6.38
1.24 6.23 6.19 6.17 6.14 6.49 6.51 6.46
1.26 6.3 6.26 6.25 6.22 6.58 6.6 6.55
1.28 6.37 6.33 6.32 6.29 6.67 6.7 6.65
1.3 6.45 6.41 6.39 6.36 6.78 6.82 6.75
1.32 6.53 6.48 6.47 6.44 6.9 6.95 6.87
1.34 6.62 6.56 6.55 6.52 7.03 7.09 7
1.36 6.71 6.65 6.64 6.6 7.2 7.27 7.16
1.38 6.81 6.74 6.73 6.69 7.39 7.47 7.35
1.4 6.93 6.84 6.83 6.79 7.6 7.68 7.55
1.42 7.05 6.95 6.94 6.9 7.79 7.88 7.76
1.44 7.21 7.07 7.06 7.01 7.98 8.05 7.95
1.46 7.39 7.21 7.21 7.16 8.14 8.2 8.11
1.48 7.59 7.39 7.38 7.33 8.28 8.33 8.25
1.5 7.79 7.59 7.58 7.52 8.4 8.44 8.38
1.52 7.97 7.79 7.78 7.72 8.51 8.54 8.49
1.54 8.12 7.98 7.98 7.92 8.6 8.64 8.59
1.56 8.26 8.15 8.14 8.09 8.7 8.73 8.68
1.58 8.39 8.3 8.3 8.26 8.78 8.81 8.77
1.6 8.5 8.44 8.43 8.39 8.87 8.89 8.86
1.62 8.6 8.56 8.55 8.51 8.95 8.98 8.94
1.64 8.7 8.66 8.66 8.62 9.03 9.05 9.02
1.66 8.78 8.76 8.75 8.71 9.1 9.14 9.1
1.68 8.87 8.85 8.84 8.8 9.19 9.22 9.18
1.7 8.95 8.93 8.92 8.89 9.27 9.31 9.26
1.72 9.03 9.01 9.01 8.97 9.36 9.4 9.35
1.74 9.11 9.09 9.09 9.05 9.45 9.5 9.44
1.76 9.18 9.17 9.17 9.13 9.55 9.6 9.54
1.78 9.26 9.25 9.24 9.21 9.67 9.73 9.65
1.8 9.34 9.33 9.32 9.29 9.8 9.88 9.78
1.82 9.43 9.4 9.4 9.37 9.94 10.03 9.92
1.84 9.52 9.49 9.49 9.45 10.1 10.19 10.08
1.86 9.61 9.57 9.57 9.53 10.26 10.33 10.23
1.88 9.71 9.66 9.66 9.62 10.4 10.46 10.38
226
KOH pH
1.9 9.82 9.76 9.76 9.72 10.53 10.58 10.52
1.92 9.94 9.87 9.87 9.82
1.94 10.07 9.99 9.99 9.94
1.96 10.22 10.12 10.13 10.07
1.98 10.37 10.27 10.28 10.22
2 10.52 10.43 10.44 10.37
2.02 10.68 10.59 10.6 10.54
2.04 10.8 10.73 10.75 10.68
2.06 10.91
227
Figure C.5 Titration curve for titration of DFBP with KOH
Trial 1 Trial 2 Trial 3
0
2.65
5.30
7.95
10.60
0 0.45 0.90 1.35 1.80
Titration of DFBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (44.6 mg DFBP in 50 ml 0.1 M KCl) with 0.0901 M KOH
Trial 2: 10.0 ml of stock DFBP with 0.0901 M KOH
Trial 3: 10 ml of stock DFBP with 0.0901 M KOH
228
KOH pH
Trial 1 Trial 2 Trial 3
0 2.21 2.2 2.15
0.02 2.21 2.21 2.16
0.04 2.23 2.22 2.17
0.06 2.24 2.23 2.18
0.08 2.24 2.24 2.19
0.1 2.26 2.25 2.21
0.12 2.27 2.26 2.22
0.14 2.28 2.27 2.23
0.16 2.29 2.28 2.24
0.18 2.3 2.3 2.26
0.2 2.31 2.31 2.27
0.22 2.33 2.32 2.28
0.24 2.34 2.34 2.3
0.26 2.36 2.35 2.31
0.28 2.37 2.37 2.33
0.3 2.39 2.38 2.34
0.32 2.4 2.39 2.36
0.34 2.42 2.41 2.37
0.36 2.43 2.43 2.39
0.38 2.45 2.44 2.41
0.4 2.47 2.46 2.43
0.42 2.49 2.48 2.45
0.44 2.51 2.5 2.47
0.46 2.53 2.52 2.49
0.48 2.56 2.55 2.51
0.5 2.58 2.57 2.54
0.52 2.6 2.6 2.56
0.54 2.63 2.63 2.59
0.56 2.66 2.65 2.62
0.58 2.69 2.69 2.65
0.6 2.72 2.72 2.68
0.62 2.76 2.76 2.72
0.64 2.8 2.8 2.76
0.66 2.84 2.84 2.8
0.68 2.89 2.89 2.84
0.7 2.94 2.95 2.9
0.72 3.01 3.01 2.96
0.74 3.08 3.08 3.02
0.76 3.16 3.17 3.1
0.78 3.26 3.28 3.2
0.8 3.38 3.41 3.31
0.82 3.56 3.6 3.47
0.84 3.81 3.88 3.69
0.86 4.14 4.22 4
0.88 4.46 4.51 4.34
0.9 4.69 4.73 4.59
0.92 4.85 4.89 4.78
229
KOH pH
0.94 4.99 5.02 4.93
0.96 5.11 5.14 5.05
0.98 5.21 5.24 5.15
1 5.31 5.33 5.25
1.02 5.39 5.41 5.34
1.04 5.47 5.5 5.43
1.06 5.55 5.57 5.51
1.08 5.63 5.65 5.59
1.1 5.71 5.74 5.67
1.12 5.79 5.81 5.75
1.14 5.87 5.9 5.83
1.16 5.96 5.98 5.91
1.18 6.05 6.08 6
1.2 6.14 6.17 6.09
1.22 6.25 6.28 6.19
1.24 6.36 6.39 6.3
1.26 6.48 6.51 6.41
1.28 6.61 6.64 6.54
1.3 6.74 6.77 6.67
1.32 6.87 6.9 6.8
1.34 6.99 7.01 6.93
1.36 7.1 7.13 7.04
1.38 7.2 7.23 7.15
1.4 7.3 7.33 7.25
1.42 7.39 7.42 7.34
1.44 7.48 7.5 7.43
1.46 7.56 7.59 7.51
1.48 7.65 7.67 7.6
1.5 7.73 7.75 7.68
1.52 7.81 7.84 7.76
1.54 7.9 7.93 7.85
1.56 7.99 8.02 7.93
1.58 8.08 8.11 8.03
1.6 8.19 8.21 8.12
1.62 8.3 8.33 8.23
1.64 8.43 8.47 8.36
1.66 8.59 8.63 8.5
1.68 8.79 8.86 8.68
1.7 9.08 9.17 8.91
1.72 9.52 9.62 9.27
1.74 9.99 9.75
1.76 10.28
1.78 10.47
1.8 10.6
230
Figure C.6 Titration curve for titration of DCBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4
0
2.543
5.085
7.628
10.170
0 0.395 0.790 1.185 1.580
Titration of DCBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (45.2 mg DCBP in 50 ml 0.1 M KCl) with 0.0919 M KOH
Trial 2: 10.0 ml of stock DCBP with 0.0889 M KOH
Trial 3: 10 ml of stock DCBP with 0.0889 M KOH
Trial 4: 10 ml of stock DCBP with 0.0889 M KOH
231
KOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0 2.26 2.25 2.27 2.26
0.02 2.27 2.26 2.28 2.27
0.04 2.28 2.27 2.29 2.28
0.06 2.29 2.28 2.3 2.29
0.08 2.3 2.3 2.31 2.31
0.1 2.31 2.31 2.32 2.32
0.12 2.32 2.32 2.34 2.33
0.14 2.34 2.34 2.35 2.35
0.16 2.36 2.35 2.37 2.36
0.18 2.37 2.37 2.38 2.38
0.2 2.39 2.38 2.4 2.4
0.22 2.4 2.4 2.41 2.41
0.24 2.42 2.42 2.43 2.43
0.26 2.44 2.43 2.45 2.45
0.28 2.46 2.45 2.47 2.47
0.3 2.48 2.47 2.48 2.49
0.32 2.5 2.49 2.5 2.51
0.34 2.52 2.51 2.53 2.53
0.36 2.54 2.53 2.55 2.55
0.38 2.57 2.56 2.57 2.58
0.4 2.59 2.58 2.59 2.61
0.42 2.62 2.61 2.62 2.64
0.44 2.64 2.63 2.64 2.66
0.46 2.67 2.66 2.67 2.7
0.48 2.71 2.69 2.7 2.73
0.5 2.74 2.72 2.73 2.77
0.52 2.78 2.75 2.76 2.81
0.54 2.82 2.79 2.8 2.86
0.56 2.86 2.83 2.84 2.91
0.58 2.91 2.88 2.88 2.97
0.6 2.96 2.92 2.93 3.04
0.62 3.03 2.98 2.98 3.12
0.64 3.1 3.04 3.04 3.21
0.66 3.18 3.11 3.11 3.34
0.68 3.29 3.2 3.19 3.51
0.7 3.42 3.3 3.3 3.75
0.72 3.6 3.43 3.42 4.15
0.74 3.89 3.62 3.59 4.55
0.76 4.28 3.9 3.85 4.82
0.78 4.64 4.29 4.23 5.02
0.8 4.87 4.64 4.6 5.17
0.82 5.05 4.88 4.85 5.3
0.84 5.19 5.05 5.04 5.41
0.86 5.31 5.2 5.19 5.52
0.88 5.42 5.32 5.31 5.62
0.9 5.52 5.43 5.42 5.71
0.92 5.61 5.52 5.52 5.8
232
KOH pH
0.94 5.7 5.62 5.61 5.9
0.96 5.79 5.7 5.7 6
0.98 5.88 5.79 5.79 6.1
1 5.97 5.88 5.88 6.21
1.02 6.07 5.97 5.97 6.34
1.04 6.17 6.07 6.06 6.5
1.06 6.28 6.17 6.16 6.69
1.08 6.42 6.27 6.27 6.95
1.1 6.57 6.4 6.39 7.3
1.12 6.76 6.54 6.53 7.7
1.14 7.02 6.72 6.71 7.98
1.16 7.38 6.97 6.95 8.18
1.18 7.75 7.3 7.29 8.33
1.2 8.01 7.68 7.67 8.46
1.22 8.19 7.96 7.95 8.58
1.24 8.34 8.16 8.16 8.68
1.26 8.47 8.32 8.31 8.77
1.28 8.57 8.44 8.44 8.86
1.3 8.68 8.56 8.56 8.96
1.32 8.77 8.66 8.66 9.05
1.34 8.86 8.76 8.76 9.15
1.36 8.95 8.85 8.85 9.25
1.38 9.04 8.94 8.94 9.36
1.4 9.13 9.02 9.03 9.48
1.42 9.22 9.11 9.11 9.62
1.44 9.31 9.2 9.21 9.78
1.46 9.42 9.29 9.3 9.96
1.48 9.53 9.4 9.41 10.17
1.5 9.66 9.51 9.52
1.52 9.81 9.63 9.65
1.54 9.97 9.77 9.8
1.56 10.15 9.92 9.97
1.58 10.09 10.15
233
Figure C.7 Titration curve for titration of DBBP with KOH
Trial 1 Trial 2 Trial 3
0
2.638
5.275
7.913
10.550
0 0.375 0.750 1.125 1.500
Titration of DBBP
pH
Amount oh KOH added (mL)
Trial 1: 10 ml (45.2 mg DBBP in 50 ml 0.1 M KCl) with 0.0919 M KOH
Trial 2: 10.0 ml of stock DBBP with 0.0889 M KOH
Trial 3: 10 ml of stock DBBP with 0.0889 M KOH
234
KOH pH
Trial 1 Trial 2 Trial 3
0 2.27 2.27 2.28
0.02 2.28 2.27 2.28
0.04 2.29 2.28 2.3
0.06 2.31 2.3 2.31
0.08 2.32 2.31 2.32
0.1 2.33 2.32 2.33
0.12 2.34 2.34 2.35
0.14 2.36 2.35 2.36
0.16 2.38 2.37 2.38
0.18 2.39 2.39 2.39
0.2 2.41 2.4 2.41
0.22 2.43 2.42 2.43
0.24 2.45 2.44 2.45
0.26 2.47 2.46 2.46
0.28 2.49 2.48 2.48
0.3 2.51 2.5 2.5
0.32 2.53 2.52 2.53
0.34 2.55 2.55 2.55
0.36 2.58 2.57 2.57
0.38 2.6 2.6 2.6
0.4 2.63 2.62 2.63
0.42 2.66 2.65 2.65
0.44 2.69 2.68 2.69
0.46 2.72 2.72 2.72
0.48 2.76 2.75 2.75
0.5 2.8 2.79 2.79
0.52 2.84 2.83 2.83
0.54 2.89 2.88 2.88
0.56 2.94 2.93 2.93
0.58 3 2.99 2.98
0.6 3.07 3.06 3.05
0.62 3.15 3.14 3.13
0.64 3.25 3.24 3.22
0.66 3.38 3.37 3.33
0.68 3.55 3.54 3.49
0.7 3.82 3.8 3.71
0.72 4.26 4.23 4.08
0.74 4.7 4.68 4.55
0.76 4.98 4.97 4.89
0.78 5.18 5.18 5.12
0.8 5.33 5.33 5.29
0.82 5.46 5.46 5.42
0.84 5.58 5.58 5.54
0.86 5.68 5.68 5.65
0.88 5.79 5.79 5.75
0.9 5.88 5.88 5.85
0.92 5.98 5.98 5.95
235
KOH pH
0.94 6.08 6.08 6.04
0.96 6.18 6.18 6.14
0.98 6.29 6.29 6.25
1 6.41 6.41 6.36
1.02 6.55 6.55 6.49
1.04 6.73 6.73 6.65
1.06 6.97 6.97 6.85
1.08 7.35 7.34 7.15
1.1 7.9 7.9 7.65
1.12 8.28 8.28 8.14
1.14 8.52 8.52 8.43
1.16 8.7 8.7 8.64
1.18 8.84 8.84 8.79
1.2 8.97 8.97 8.92
1.22 9.07 9.07 9.03
1.24 9.17 9.17 9.13
1.26 9.26 9.26 9.23
1.28 9.35 9.35 9.32
1.3 9.44 9.44 9.41
1.32 9.53 9.54 9.5
1.34 9.63 9.63 9.6
1.36 9.72 9.72 9.69
1.38 9.82 9.82 9.79
1.4 9.93 9.93 9.89
1.42 10.04 10.04 10
1.44 10.17 10.16 10.12
1.46 10.29 10.29 10.25
1.48 10.43 10.42 10.38
1.5 10.55 10.54 10.51
236
Figure C.8 Titration curve for titration of FClBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4
0
2.56
5.12
7.68
10.24
0 0.4 0.8 1.2 1.6
Titration of FClBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (42.8 mg FClBP in 50 ml 0.1 M KCl) with 0.0881 M KOH
Trial 2: 10.0 ml of stock FClBP with 0.0881 M KOH
Trial 3: 10 ml of stock FClBP with 0.0881 M KOH
Trial 4: 10 ml of stock FClBP with 0.0881 M KOH
237
KOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0 2.23 2.21 2.19 2.23
0.02 2.23 2.22 2.2 2.24
0.04 2.24 2.23 2.22 2.25
0.06 2.25 2.24 2.23 2.26
0.08 2.26 2.25 2.24 2.27
0.1 2.28 2.27 2.26 2.29
0.12 2.29 2.28 2.27 2.3
0.14 2.3 2.3 2.29 2.31
0.16 2.32 2.31 2.3 2.33
0.18 2.33 2.33 2.31 2.34
0.2 2.35 2.34 2.33 2.36
0.22 2.36 2.36 2.35 2.37
0.24 2.38 2.37 2.36 2.39
0.26 2.4 2.39 2.38 2.41
0.28 2.42 2.41 2.4 2.43
0.3 2.44 2.43 2.42 2.45
0.32 2.46 2.45 2.44 2.47
0.34 2.48 2.47 2.46 2.49
0.36 2.5 2.5 2.48 2.51
0.38 2.52 2.52 2.51 2.53
0.4 2.55 2.54 2.53 2.56
0.42 2.57 2.57 2.56 2.58
0.44 2.6 2.6 2.58 2.61
0.46 2.63 2.63 2.61 2.64
0.48 2.66 2.65 2.64 2.67
0.5 2.7 2.69 2.68 2.7
0.52 2.73 2.73 2.71 2.74
0.54 2.77 2.77 2.75 2.78
0.56 2.81 2.81 2.79 2.82
0.58 2.86 2.86 2.84 2.87
0.6 2.92 2.91 2.89 2.92
0.62 2.98 2.97 2.94 2.98
0.64 3.05 3.04 3.01 3.04
0.66 3.13 3.12 3.09 3.12
0.68 3.23 3.22 3.17 3.21
0.7 3.35 3.34 3.28 3.33
0.72 3.52 3.51 3.42 3.48
0.74 3.75 3.73 3.62 3.69
0.76 4.07 4.05 3.9 3.99
0.78 4.4 4.38 4.24 4.32
0.8 4.64 4.62 4.53 4.59
0.82 4.82 4.81 4.73 4.78
0.84 4.97 4.96 4.9 4.94
0.86 5.09 5.08 5.04 5.07
0.88 5.2 5.19 5.16 5.19
0.9 5.31 5.3 5.26 5.29
0.92 5.4 5.39 5.36 5.39
238
KOH pH
0.94 5.49 5.49 5.45 5.48
0.96 5.58 5.57 5.54 5.57
0.98 5.67 5.66 5.64 5.66
1 5.76 5.75 5.72 5.75
1.02 5.85 5.85 5.82 5.84
1.04 5.95 5.95 5.91 5.94
1.06 6.06 6.05 6.02 6.05
1.08 6.18 6.18 6.13 6.16
1.1 6.31 6.31 6.26 6.29
1.12 6.47 6.48 6.41 6.44
1.14 6.67 6.68 6.59 6.63
1.16 6.93 6.94 6.83 6.87
1.18 7.21 7.21 7.1 7.15
1.2 7.45 7.46 7.36 7.41
1.22 7.65 7.65 7.58 7.62
1.24 7.8 7.8 7.75 7.78
1.26 7.94 7.94 7.89 7.92
1.28 8.05 8.05 8.01 8.04
1.3 8.15 8.15 8.12 8.14
1.32 8.26 8.25 8.22 8.25
1.34 8.35 8.34 8.31 8.34
1.36 8.44 8.44 8.41 8.43
1.38 8.53 8.53 8.5 8.52
1.4 8.63 8.62 8.59 8.61
1.42 8.72 8.71 8.69 8.71
1.44 8.82 8.81 8.78 8.8
1.46 8.93 8.92 8.89 8.91
1.48 9.06 9.05 9.01 9.03
1.5 9.2 9.19 9.14 9.16
1.52 9.37 9.35 9.3 9.32
1.54 9.58 9.54 9.49 9.5
1.56 9.82 9.76 9.72 9.71
1.58 10.11 10.03 10.01 9.96
1.6 10.24
239
Figure C.9 Titration curve for titration of MBBP with KOH
Trial 1 Trial 2 Trial 3
0
2.828
5.655
8.483
11.310
0 0.4 0.8 1.2 1.6
Titration of MBBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (41.26 mg MBBP in 50 ml 0.1 M KCl) with 0.0891 M KOH
Trial 2: 10.0 ml of stock MBBP with 0.0893 M KOH
Trial 3: 10 ml of stock MBBP with 0.0893 M KOH
240
KOH pH
Trial 1 Trial 2 Trial 3
0 2.42 2.34 2.36
0.02 2.43 2.36 2.37
0.04 2.44 2.38 2.38
0.06 2.46 2.39 2.4
0.08 2.47 2.41 2.41
0.1 2.48 2.42 2.43
0.12 2.5 2.44 2.44
0.14 2.51 2.45 2.46
0.16 2.52 2.47 2.48
0.18 2.54 2.49 2.49
0.2 2.56 2.51 2.51
0.22 2.58 2.53 2.53
0.24 2.59 2.55 2.55
0.26 2.61 2.57 2.57
0.28 2.63 2.59 2.59
0.3 2.65 2.61 2.61
0.32 2.68 2.64 2.64
0.34 2.71 2.66 2.66
0.36 2.73 2.69 2.69
0.38 2.76 2.72 2.71
0.4 2.79 2.75 2.74
0.42 2.83 2.78 2.77
0.44 2.86 2.81 2.8
0.46 2.9 2.85 2.83
0.48 2.95 2.89 2.87
0.5 3 2.94 2.92
0.52 3.05 2.99 2.96
0.54 3.12 3.04 3.02
0.56 3.19 3.11 3.08
0.58 3.28 3.19 3.14
0.6 3.39 3.28 3.22
0.62 3.54 3.4 3.33
0.64 3.77 3.55 3.47
0.66 4.18 3.8 3.66
0.68 4.8 4.27 4
0.7 5.19 4.88 4.58
0.72 5.42 5.23 5.13
0.74 5.59 5.44 5.37
0.76 5.73 5.61 5.55
0.78 5.85 5.74 5.69
0.8 5.96 5.85 5.81
0.82 6.06 5.96 5.92
0.84 6.16 6.06 6.02
0.86 6.26 6.17 6.12
0.88 6.36 6.27 6.22
0.9 6.48 6.37 6.32
0.92 6.59 6.48 6.43
241
KOH pH
0.94 6.73 6.61 6.55
0.96 6.89 6.75 6.67
0.98 7.11 6.92 6.82
1 7.52 7.17 7.01
1.02 8.18 7.62 7.34
1.04 8.68 8.32 7.94
1.06 8.97 8.77 8.57
1.08 9.17 9.04 8.91
1.1 9.33 9.23 9.13
1.12 9.47 9.38 9.3
1.14 9.59 9.51 9.44
1.16 9.7 9.63 9.56
1.18 9.8 9.74 9.67
1.2 9.9 9.84 9.78
1.22 10 9.94 9.88
1.24 10.09 10.05 9.98
1.26 10.19 10.14 10.08
1.28 10.28 10.24 10.17
1.3 10.38 10.33 10.27
1.32 10.48 10.44 10.37
1.34 10.57 10.55 10.47
1.36 10.67 10.64 10.57
1.38 10.77 10.74 10.67
1.4 10.85 10.84 10.77
1.42 10.94 10.92 10.85
1.44 11.01 11 10.94
1.46 11.08 11.08 11.01
1.48 11.14 11.14 11.09
1.5 11.2 11.2 11.15
1.52 11.25 11.26 11.2
1.54 11.3 11.31 11.26
1.56 11.31
242
Figure C.10 Titration curve for titration of MCBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4
0
2.755
5.510
8.265
11.020
0 0.475 0.950 1.425 1.900
Titration of MCBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (40.8 mg MCBP in 50 ml 0.1 M KCl) with 0.0889 M KOH
Trial 2: 10.0 ml of stock MCBP with 0.0875 M KOH
Trial 3: 10 ml of stock MCBP with 0.0875 M KOH
Trial 4: 10 ml of stock MCBP with 0.0875 M KOH
243
KOH pH
Trial 1 Trial 2 Trial 3 Trial 4
0 2.15 2.2 2.09 2.14
0.02 2.16 2.2 2.1 2.15
0.04 2.17 2.21 2.11 2.16
0.06 2.18 2.22 2.12 2.17
0.08 2.19 2.23 2.13 2.18
0.1 2.21 2.24 2.15 2.19
0.12 2.22 2.26 2.16 2.2
0.14 2.23 2.27 2.17 2.21
0.16 2.25 2.28 2.19 2.23
0.18 2.26 2.3 2.2 2.24
0.2 2.27 2.31 2.22 2.25
0.22 2.29 2.33 2.23 2.27
0.24 2.31 2.34 2.25 2.29
0.26 2.33 2.36 2.27 2.3
0.28 2.34 2.38 2.28 2.32
0.3 2.36 2.39 2.3 2.34
0.32 2.38 2.41 2.32 2.36
0.34 2.4 2.43 2.34 2.37
0.36 2.42 2.45 2.36 2.39
0.38 2.44 2.47 2.38 2.42
0.4 2.46 2.49 2.4 2.44
0.42 2.49 2.51 2.43 2.46
0.44 2.51 2.53 2.45 2.49
0.46 2.54 2.56 2.48 2.51
0.48 2.57 2.59 2.5 2.54
0.5 2.59 2.61 2.53 2.57
0.52 2.62 2.64 2.56 2.6
0.54 2.66 2.67 2.59 2.63
0.56 2.69 2.7 2.62 2.66
0.58 2.73 2.74 2.66 2.7
0.6 2.77 2.77 2.7 2.74
0.62 2.81 2.81 2.74 2.78
0.64 2.86 2.86 2.78 2.83
0.66 2.91 2.9 2.84 2.88
0.68 2.97 2.96 2.89 2.94
0.7 3.04 3.01 2.96 3.01
0.72 3.12 3.08 3.03 3.08
0.74 3.21 3.16 3.11 3.18
0.76 3.32 3.25 3.22 3.3
0.78 3.48 3.36 3.35 3.45
0.8 3.69 3.51 3.52 3.66
0.82 4.05 3.73 3.79 4.04
0.84 4.66 4.1 4.29 4.65
0.86 5.11 4.7 4.86 5.08
0.88 5.38 5.12 5.2 5.33
0.9 5.57 5.37 5.43 5.51
0.92 5.71 5.55 5.59 5.66
244
KOH pH
0.94 5.84 5.69 5.72 5.78
0.96 5.95 5.81 5.84 5.89
0.98 6.05 5.92 5.94 5.99
1 6.14 6.02 6.04 6.09
1.02 6.23 6.11 6.13 6.18
1.04 6.32 6.2 6.22 6.26
1.06 6.41 6.28 6.31 6.35
1.08 6.5 6.37 6.39 6.44
1.1 6.59 6.45 6.48 6.53
1.12 6.68 6.54 6.57 6.63
1.14 6.78 6.63 6.67 6.73
1.16 6.9 6.73 6.78 6.84
1.18 7.02 6.84 6.9 6.97
1.2 7.16 6.96 7.03 7.11
1.22 7.35 7.09 7.18 7.29
1.24 7.6 7.25 7.39 7.55
1.26 7.97 7.48 7.68 7.91
1.28 8.36 7.8 8.09 8.31
1.3 8.63 8.2 8.45 8.6
1.32 8.81 8.51 8.69 8.8
1.34 8.96 8.72 8.86 8.95
1.36 9.08 8.87 9 9.07
1.38 9.18 9 9.12 9.18
1.4 9.27 9.11 9.22 9.27
1.42 9.36 9.2 9.31 9.36
1.44 9.44 9.29 9.39 9.44
1.46 9.52 9.37 9.47 9.53
1.48 9.6 9.45 9.55 9.6
1.5 9.67 9.53 9.63 9.68
1.52 9.75 9.6 9.7 9.76
1.54 9.83 9.68 9.78 9.83
1.56 9.91 9.75 9.86 9.91
1.58 9.99 9.83 9.94 9.99
1.6 10.07 9.91 10.02 10.08
1.62 10.17 9.99 10.11 10.16
1.64 10.26 10.08 10.2 10.25
1.66 10.36 10.16 10.29 10.35
1.68 10.45 10.25 10.39 10.44
1.7 10.54 10.35 10.48 10.53
1.72 10.63 10.44 10.57 10.62
1.74 10.71 10.53 10.66 10.71
1.76 10.78 10.62 10.74 10.78
1.78 10.85 10.7 10.82 10.85
1.8 10.91 10.77 10.88 10.91
1.82 10.96 10.83 10.95 10.97
1.84 11.01 10.89 11 11.02
245
Figure C.11 Titration curve for titration of EBP with KOH
Trial 1 Trial 2 Trial 3
0
2.849
5.699
8.548
11.397
0 0.625 1.250 1.875 2.500
Titration of EBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (48.84 mg EBP in 50 ml 0.1 M KCl) with 0.0847 M KOH
Trial 2: 10.0 ml of stock EBP with 0.0847 M KOH
Trial 3: 10 ml of stock EBP with 0.0847 M KOH
246
KOH pH
Trial 1 Trial 2 Trial 3
0 2.321 2.313 2.295
0.02 2.33 2.32 2.306
0.04 2.34 2.33 2.314
0.06 2.349 2.342 2.325
0.08 2.36 2.354 2.337
0.1 2.371 2.366 2.348
0.12 2.381 2.375 2.359
0.14 2.394 2.388 2.37
0.16 2.405 2.4 2.384
0.18 2.415 2.413 2.394
0.2 2.427 2.427 2.408
0.22 2.442 2.439 2.42
0.24 2.454 2.451 2.433
0.26 2.468 2.465 2.447
0.28 2.479 2.479 2.459
0.3 2.494 2.492 2.475
0.32 2.508 2.507 2.488
0.34 2.523 2.52 2.503
0.36 2.539 2.535 2.518
0.38 2.553 2.551 2.534
0.4 2.568 2.567 2.549
0.42 2.586 2.585 2.565
0.44 2.601 2.6 2.581
0.46 2.619 2.617 2.596
0.48 2.637 2.636 2.614
0.5 2.654 2.654 2.632
0.52 2.672 2.671 2.648
0.54 2.692 2.69 2.669
0.56 2.709 2.709 2.687
0.58 2.73 2.73 2.707
0.6 2.75 2.75 2.726
0.62 2.773 2.772 2.747
0.64 2.793 2.793 2.767
0.66 2.816 2.816 2.79
0.68 2.84 2.838 2.815
0.7 2.864 2.863 2.838
0.72 2.888 2.887 2.861
0.74 2.914 2.914 2.886
0.76 2.94 2.94 2.913
0.78 2.968 2.969 2.939
0.8 2.997 2.996 2.967
0.82 3.027 3.027 2.995
0.84 3.058 3.058 3.027
0.86 3.091 3.092 3.058
0.88 3.127 3.128 3.092
0.9 3.164 3.165 3.129
0.92 3.204 3.205 3.167
247
KOH pH
0.94 3.246 3.247 3.207
0.96 3.292 3.292 3.249
0.98 3.34 3.341 3.297
1 3.394 3.394 3.347
1.02 3.454 3.453 3.403
1.04 3.52 3.519 3.462
1.06 3.598 3.595 3.532
1.08 3.686 3.683 3.612
1.1 3.795 3.789 3.708
1.12 3.932 3.924 3.821
1.14 4.128 4.119 3.972
1.16 4.451 4.425 4.192
1.18 5.081 5.051 4.581
1.2 5.621 5.597 5.272
1.22 5.894 5.879 5.7
1.24 6.073 6.059 5.934
1.26 6.21 6.201 6.099
1.28 6.32 6.31 6.224
1.3 6.414 6.405 6.331
1.32 6.5 6.491 6.424
1.34 6.577 6.567 6.505
1.36 6.65 6.639 6.58
1.38 6.716 6.704 6.647
1.4 6.777 6.768 6.712
1.42 6.837 6.827 6.772
1.44 6.895 6.885 6.83
1.46 6.949 6.943 6.889
1.48 7.011 7 6.946
1.5 7.07 7.058 7.001
1.52 7.13 7.121 7.06
1.54 7.19 7.179 7.12
1.56 7.252 7.239 7.181
1.58 7.317 7.305 7.244
1.6 7.385 7.371 7.312
1.62 7.459 7.443 7.38
1.64 7.537 7.522 7.451
1.66 7.622 7.603 7.53
1.68 7.719 7.701 7.612
1.7 7.828 7.803 7.708
1.72 7.961 7.931 7.817
1.74 8.136 8.088 7.951
1.76 8.391 8.323 8.126
1.78 8.844 8.721 8.388
1.8 9.546 9.401 8.84
1.82 9.957 9.875 9.539
1.84 10.18 10.123 9.938
1.86 10.335 10.288 10.16
1.88 10.448 10.413 10.312
248
KOH pH
1.9 10.544 10.509 10.429
1.92 10.623 10.595 10.527
1.94 10.693 10.664 10.609
1.96 10.756 10.727 10.674
1.98 10.811 10.785 10.738
2 10.86 10.838 10.793
2.02 10.904 10.884 10.842
2.04 10.948 10.924 10.887
2.06 10.986 10.964 10.93
2.08 11.021 11.001 10.969
2.1 11.055 11.035 11.003
2.12 11.087 11.068 11.039
2.14 11.116 11.099 11.07
2.16 11.144 11.124 11.101
249
Figure C.12 Titration curve for titration of DMBP with KOH
Trial 1 Trial 2 Trial 3
0
2.918
5.836
8.753
11.671
0 0.6 1.2 1.8 2.4
Titration of DMBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (48.34 mg IBP in 50 ml 0.1 M KCl) with 0.0901 M KOH
Trial 2: 10.0 ml of stock IBP with 0.0901 M KOH
Trial 3: 10 ml of stock IBP with 0.0901 M KOH

250
KOH pH
0 2.384 2.361 2.345
0.02 2.39 2.373 2.355
0.04 2.397 2.385 2.366
0.06 2.41 2.399 2.38
0.08 2.423 2.411 2.391
0.1 2.435 2.426 2.405
0.12 2.447 2.439 2.418
0.14 2.461 2.453 2.432
0.16 2.475 2.469 2.447
0.18 2.491 2.484 2.462
0.2 2.505 2.5 2.477
0.22 2.519 2.515 2.491
0.24 2.536 2.533 2.508
0.26 2.553 2.549 2.526
0.28 2.571 2.566 2.542
0.3 2.588 2.583 2.559
0.32 2.605 2.603 2.578
0.34 2.625 2.621 2.598
0.36 2.644 2.642 2.616
0.38 2.664 2.662 2.637
0.4 2.685 2.682 2.659
0.42 2.706 2.702 2.68
0.44 2.727 2.725 2.702
0.46 2.75 2.747 2.724
0.48 2.775 2.772 2.749
0.5 2.798 2.794 2.772
0.52 2.822 2.82 2.796
0.54 2.849 2.847 2.822
0.56 2.876 2.873 2.848
0.58 2.903 2.9 2.878
0.6 2.933 2.929 2.907
0.62 2.964 2.96 2.935
0.64 2.996 2.99 2.967
0.66 3.029 3.022 3
0.68 3.065 3.057 3.032
0.7 3.1 3.091 3.068
0.72 3.139 3.129 3.106
0.74 3.18 3.169 3.144
0.76 3.224 3.21 3.184
0.78 3.27 3.253 3.229
0.8 3.318 3.299 3.276
0.82 3.373 3.35 3.323
0.84 3.431 3.406 3.38
0.86 3.497 3.467 3.439
0.88 3.573 3.535 3.507
0.9 3.658 3.612 3.583
0.92 3.76 3.703 3.67
0.94 3.889 3.81 3.775
251
KOH pH
0.96 4.06 3.948 3.905
0.98 4.327 4.138 4.08
1 4.92 4.458 4.365
1.02 5.971 5.306 5.039
1.04 6.384 6.132 6.017
1.06 6.623 6.466 6.403
1.08 6.787 6.672 6.62
1.1 6.919 6.823 6.78
1.12 7.03 6.944 6.902
1.14 7.127 7.048 7.01
1.16 7.218 7.144 7.111
1.18 7.304 7.23 7.199
1.2 7.383 7.313 7.283
1.22 7.458 7.387 7.361
1.24 7.53 7.462 7.435
1.26 7.602 7.533 7.507
1.28 7.672 7.602 7.577
1.3 7.743 7.672 7.644
1.32 7.817 7.743 7.716
1.34 7.894 7.813 7.782
1.36 7.973 7.888 7.86
1.38 8.057 7.965 7.936
1.4 8.15 8.045 8.017
1.42 8.25 8.132 8.103
1.44 8.366 8.228 8.198
1.46 8.503 8.337 8.303
1.48 8.68 8.467 8.428
1.5 8.922 8.631 8.584
1.52 9.304 8.851 8.787
1.54 9.818 9.178 9.095
1.56 10.196 9.668 9.559
1.58 10.42 10.096 10.016
1.6 10.585 10.359 10.309
1.62 10.701 10.532 10.497
1.64 10.792 10.661 10.627
1.66 10.872 10.755 10.736
1.68 10.938 10.823 10.818
1.7 10.996 10.9 10.89
1.72 11.047 10.962 10.949
1.74 11.092 11.019 11.006
1.76 11.134 11.069 11.057
1.78 11.173 11.112 11.096
1.8 11.21 11.153 11.143
1.82 11.244 11.188 11.176
1.84 11.276 11.221 11.212
1.86 11.303 11.253 11.244
1.88 11.327 11.281 11.271
1.9 11.353 11.31 11.3
252
KOH pH
1.92 11.376 11.333 11.325
1.94 11.397 11.358 11.349
1.96 11.419 11.382 11.373
1.98 11.439 11.402 11.396
2 11.459 11.423 11.417
2.02 11.477 11.442 11.435
2.04 11.494 11.459 11.457
2.06 11.511 11.495 11.473
2.08 11.53 11.511 11.492
2.1 11.545 11.524 11.51
2.12 11.56 11.54 11.527
2.14 11.573 11.552 11.54
2.16 11.586 11.568 11.556
2.18 11.598 11.581 11.568
2.2 11.612 11.594 11.583
2.22 11.625 11.606 11.598
2.24 11.636 11.617 11.611
2.26 11.65 11.63 11.621
2.28 11.66 11.636
2.3 11.671 11.65
253
Figure C.13 Titration curve for titration of FMBP with KOH
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
0
2.83
5.66
8.49
11.32
0 0.475 0.950 1.425 1.900
Titration of FMBP
pH
Amount of KOH added (mL)
Trial 1: 10 ml (40.44 mg FMBP in 50 ml 0.1 M KCl) with 0.0909 M KOH
Trial 2: 10.0 ml of stock FMBP with 0.0909 M KOH
Trial 3: 10 ml of stock FMBP with 0.0909 M KOH
Trial 4: 10 ml (40.18 mg FMBP in 50 ml 0.1 M KCl) with 0.0916 M KOH
Trial 5: 10 ml of stock FMBP with 0.0916 M KOH
254
KOH pH
0 2.211 2.208 2.209 2.308 2.3
0.02 2.218 2.217 2.221 2.316 2.311
0.04 2.228 2.229 2.233 2.327 2.322
0.06 2.241 2.242 2.244 2.339 2.333
0.08 2.253 2.255 2.256 2.351 2.344
0.1 2.266 2.267 2.27 2.363 2.356
0.12 2.28 2.282 2.284 2.374 2.367
0.14 2.294 2.295 2.298 2.387 2.38
0.16 2.308 2.31 2.312 2.399 2.393
0.18 2.322 2.324 2.328 2.411 2.407
0.2 2.338 2.339 2.344 2.428 2.422
0.22 2.353 2.356 2.359 2.443 2.438
0.24 2.371 2.372 2.377 2.46 2.454
0.26 2.388 2.388 2.393 2.477 2.472
0.28 2.406 2.407 2.412 2.495 2.49
0.3 2.423 2.425 2.431 2.513 2.508
0.32 2.443 2.445 2.448 2.533 2.526
0.34 2.462 2.464 2.469 2.555 2.548
0.36 2.483 2.486 2.49 2.577 2.571
0.38 2.504 2.507 2.512 2.599 2.594
0.4 2.526 2.528 2.536 2.624 2.616
0.42 2.552 2.553 2.56 2.65 2.642
0.44 2.577 2.577 2.586 2.677 2.669
0.46 2.602 2.604 2.611 2.707 2.697
0.48 2.63 2.633 2.641 2.737 2.729
0.5 2.66 2.663 2.67 2.771 2.761
0.52 2.691 2.695 2.702 2.807 2.796
0.54 2.724 2.729 2.736 2.847 2.835
0.56 2.761 2.764 2.774 2.89 2.876
0.58 2.801 2.805 2.814 2.937 2.923
0.6 2.845 2.848 2.859 2.99 2.975
0.62 2.892 2.896 2.908 3.05 3.033
0.64 2.945 2.948 2.962 3.12 3.102
0.66 3.006 3.009 3.026 3.204 3.184
0.68 3.075 3.077 3.096 3.306 3.28
0.7 3.155 3.158 3.18 3.435 3.405
0.72 3.252 3.255 3.281 3.617 3.578
0.74 3.378 3.38 3.415 3.916 3.856
0.76 3.547 3.546 3.599 4.473 4.387
0.78 3.807 3.811 3.899 4.976 4.925
0.8 4.281 4.288 4.434 5.259 5.228
0.82 4.846 4.845 4.942 5.453 5.425
0.84 5.181 5.18 5.241 5.601 5.578
0.86 5.398 5.398 5.441 5.723 5.705
0.88 5.562 5.56 5.595 5.832 5.814
0.9 5.693 5.693 5.725 5.934 5.915
0.92 5.807 5.803 5.833 6.028 6.011
0.94 5.909 5.908 5.935 6.119 6.1
255
KOH pH
0.96 6.005 6.003 6.027 6.209 6.19
0.98 6.096 6.092 6.117 6.299 6.278
1 6.183 6.18 6.204 6.39 6.37
1.02 6.271 6.267 6.293 6.488 6.468
1.04 6.359 6.354 6.381 6.58 6.575
1.06 6.45 6.445 6.474 6.698 6.689
1.08 6.547 6.542 6.57 6.835 6.828
1.1 6.652 6.643 6.673 7.001 6.996
1.12 6.767 6.756 6.788 7.257 7.245
1.14 6.901 6.885 6.928 7.694 7.687
1.16 7.067 7.047 7.099 8.54 8.532
1.18 7.292 7.263 7.335 9.029 9.016
1.2 7.667 7.61 7.74 9.299 9.278
1.22 8.405 8.304 8.503 9.478 9.463
1.24 8.961 8.915 9.001 9.618 9.6
1.26 9.248 9.221 9.279 9.738 9.719
1.28 9.44 9.418 9.461 9.841 9.823
1.3 9.586 9.569 9.607 9.934 9.917
1.32 9.71 9.694 9.727 10.021 10.004
1.34 9.815 9.803 9.836 10.104 10.086
1.36 9.909 9.898 9.927 10.181 10.163
1.38 9.995 9.986 10.013 10.255 10.237
1.4 10.076 10.065 10.092 10.327 10.306
1.42 10.149 10.142 10.168 10.394 10.375
1.44 10.222 10.213 10.238 10.46 10.441
1.46 10.294 10.286 10.312 10.527 10.509
1.48 10.362 10.355 10.379 10.59 10.572
1.5 10.429 10.423 10.447 10.655 10.635
1.52 10.495 10.49 10.514 10.714 10.696
1.54 10.561 10.555 10.579 10.775 10.758
1.56 10.624 10.618 10.643 10.831 10.812
1.58 10.686 10.68 10.704 10.887 10.868
1.6 10.745 10.742 10.763 10.939 10.922
1.62 10.805 10.801 10.821 10.989 10.973
1.64 10.861 10.858 10.88 11.035 11.019
1.66 10.913 10.913 10.933 11.079 11.063
1.68 10.963 10.964 10.985 11.12 11.106
1.7 11.012 11.013 11.035 11.157 11.143
1.72 11.056 11.059 11.08 11.194 11.18
1.74 11.098 11.103 11.122 11.227 11.216
1.76 11.14 11.145 11.162 11.261 11.249
1.78 11.179 11.184 11.201 11.291 11.279
1.8 11.213 11.222 11.236 11.32 11.309
256
APPENDIX D
Table D.1 HPLC summary of α,β-difluoromethylene bisphosphonate dNTP analogs
Compound Retention time (column) Buffer
TsdA, 1 2 min (SAX) 0-50% 0.5M LiCl gradient
α,β-CF2-dADP, 2 11 min (SAX) 0-50% 0.5M LiCl gradient
α,β-CF2-dATP, 3 15 min (SAX) 0-50% 0.5M LiCl gradient
dC 1.9 min (SAX) 0-50% 0.5M LiCl gradient
TsBzdC, 4 1.7 min (SAX) 0-50% 0.5M LiCl gradient
α,β-CF2-BzdCDP, 5 13 min (SAX) 0-50% 0.5M LiCl gradient
α,β-CF 2-dCDP, 8 10 min (SAX) 0-50% 0.5M LiCl gradient
α,β-CF 2-dCTP, 6 12 min (SAX) 0-50% 0.5M LiCl gradient
Figure D.1 HPLC analysis (on SAX) of conversion of 1 to 2.
a,b-DF-dA TsdA
t = 18hrs
t = 4hrs
t = 30min
HPLC analysis of conversion of TsdA to α,β-CF2-dADP
257
Figure D.2 HPLC analysis (on SAX) of conversion of 2 to 3.

Chrom. 1 0.0 mins. 28.1 mins.
6
5
4
3
2
1
!,"-DF-dATP
!,"-DF-dA
dA
t = 1hr
t = 2hrs
t = 24hrs
t = 30hrs and
addition of NDPK
t = 49hrs
t = 55hrs
HPLC analysis of conversion of α,β-CF2-dADP to α,β-CF2-dATP at t = 24hrs: NDPK
and ATPcat were added
258
Figure D.3 HPLC analysis (SAX) of 3 after dual-pass preparative HPLC on SAX then C18.

Chrom. 1 0.0 mins. 17.6 mins.
1
HPLC analysis of ultrapurified α,β-CF2-dATP (0-50% 0.5M LiCl)
Figure D.4 HPLC analysis (SAX) of conversion of 4 to 5.
t = 18hrs
!,"-CF2-
BzdCDP
TsBzdC
HPLC analysis of conversion of TsBzdC (top) to α,β-CF2-BzdCDP (bottom)
259
Figure D.5 HPLC analysis (SAX) of conversion of 5 to 8.
t=10min
t=18hrs
!,"-CF
2
-
dCDP
!,"-CF
2
-
BzdC
HPLC analysis of conversion of α,β-CF2-BzdCDP (bottom) to α,β-CF2-dCDP (top)
260
Figure D.6 HPLC analysis (SAX) of conversion of 8 to 6.
t=24hrs
t=18hrs
t=15min
Before PK, PEP,
& ATP, NDPK
!,"-CF2-
dCDP
!,"-CF2-
dCTP
ATP
HPLC analysis of conversion of α,β-DF-dCDP (bottom) to α,β-DF-dCTP (top)
Figure D.7 HPLC analysis (SAX) of 6 after dual-phase preparative HPLC on SAX then C18..
HPLC analysis of ultrapurified α,β-CF2-dCTP (0-50% 0.5 M LiCl)
261
N
4
-Benzoyl-2’-deoxycytidine, 7
NMR D.1 - (CD3OD)
1
H: 8.60 (d), 8.00 (t), 7.66 (m), 7.58 (t), 7.44 (m), 7.39 (t), 6.28 (t),
4.89 (d), 4.43 (d), 3.84 (m), 2.56 (m), 2.24 (d)
2’-deoxyadenine-5’-tosylate, 1
NMR D.2 - (CD3OD)
1
H: 8.31 (d), 8.06 (s), 7.74 (d), 7.27 (d), 6.01 (t), 4.77 (d), 4.61 (d),
4.11 (d), 3.82 (m), 2.43 (m), 2.40 (m)
N
4
-benzoyl-2’-deoxycytidine-5’-tosylate, 4
262
NMR D.3 - (CDCl3)
1
H: 8.60 (d), 8.00 (t), 7.66 (m), 7.58 (t), 7.44 (m), 7.39 (t), 6,28 (t),
4.89 (d), 4.43 (d), 3.84 (m), 2.56 (m), 2.24 (d)
2’-deoxyadenine 5'-(difluoromethylene)bis(phosphonate), 2
NMR D.4 - (D2O; pH = 8)
1
H: 8.31 (s), 8.06 (s), 6.32 (t), 4.63 (d), 4.11 (d), 2.43 (m),
2.40 (m)
NMR D.5 - (D2O; pH = 8)
31
P: 6.54 (m), 3.57 (m)
263
NMR D.6 - (D2O; pH = 8)
19
F: -116.3 (dd)
Dealkylation of benzoyl-protecting group (to give α,β-CF2-dCDP), 5
264
NMR D.7 - (D2O; pH = 8)
1
H: 7.83 (d), 6.13 (t), 5.93 (d), 4.44 (d), 4.10 (m), 3.95 (d),
2.22 (m), 2.13 (m)
NMR D.8 - (D2O; pH = 8)
31
P: 6.66 (m), 3.36 (m)
NMR D.9 - (D2O; pH = 8)
19
F: -116.6 (dd)
α,β-difluoromethylene-adenosine triphosphate (α,β-CF2-dATP), 3
265
NMR D.10 - (D2O; pH = 8)
1
H: 8.33 (s), 8.06 (s), 6.33 (t), 4.63 (d), 4.11 (d), 2.43 (m),
2.40 (m)
NMR D.11 - (D2O; pH = 8)
31
P: 4.29 (m), -5.33 (d), -6.20 (m)
NMR D.12 - (D2O; pH = 8)
19
F: -119.6 (dd)
266
α,β-difluoromethylene-cytidine triphosphate (α,β-CF2-dCTP), 6
NMR D.13 - (D2O; pH = 8)
1
H: 7.85 (d), 6.15 (t), 5.95 (d), 4.47 (d), 4.13 (m), 3.99 (d),
2.23 (m), 2.15 (m)
NMR D.14 - (D2O; pH = 8)
31
P: 4.6 (m), -5.75 (d), -7.1 (m)
267
NMR D.15 -
19
F: -120.6 (dt)
268
Figure D.8
19
F NMR analysis of the 3 after preparatory purification on SAX but before (top) and after (bot-
tom) purification C-18. This shows the presence of DFBP and other impurities that can be removed by purification on
C-18 proceeding SAX.
269
MS 4.1 - HRMS α,β-(CF2)-dATP, 3
270
MS 4.2 - HRMS α,β-(CF2)-dCTP, 6
271
APPENDIX E
Table E.1 HPLC summary of β,γ-methylene bisphosphonate dNTP analogs
Compound Retention time (column) Buffer
dGMP
3.5 min (C-18) 0.1N TEAB (2% CH3CN)
8 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-Morph
5.5 min (C-18) 0.1N TEAB (2% CH3CN)
5 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-MBP 16 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-DFBP 17 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-MFBP 13 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-DCBP 16 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-MCBP 17 min (SAX) 0-100% 0.5M TEAB gradient
dGMP-FClBP 12 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-DBBP 12 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-MBBP 13 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-IBP 11 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-EBP 12 min (SAX) 0-100% 0.5M LiCl gradient
dGMP-FMBP 13 min (SAX) 0-100% 0.5M LiCl gradient
dCMP 4.5 min (SAX) 0-50% 0.5M LiCl gradient
dCMP-Morph 2.6 min (SAX) 0-50% 0.5M LiCl gradient
dCMP-MBP 11 min (SAX) 0-50% 0.5M LiCl gradient
dCMP-DFBP 10 min (SAX) 0-50% 0.5M LiCl gradient
272
Figure E.1 HPLC analysis (SAX) of the conversion of 1 to 4.
HPLC analysis of the conversion dGMP-Morph to dGMP-DFBP
Figure E.2 HPLC analysis (SAX) of 4 after dual-pass prep. HPLC (on SAX then C18).
HPLC analysis (0-50% 0.5M LiCl)
273
Figure E.3 HPLC analysis (SAX) of the conversion of 1 to 3.
HPLC analysis of the conversion dGMP-Morph to dGMP-MBP
Figure E.4 HPLC analysis (SAX) of 3 after dual-pass prep. HPLC (on SAX then C18).
HPLC analysis of ultrapurified dGMP-MBP (0-100% 0.5M TEAB prep column)
274
Figure E.5 HPLC analysis (SAX) of the conversion of 1 to 5/6.
HPLC analysis of conversion of dGMP-Morph to dGMP-MFBP
Figure E.6 HPLC analysis (SAX) of 5/6 after dual-pass prep. HPLC (on SAX then C18).
HPLC analysis of ultrapurified dGMP-MFBP (0-100% 0.5M TEAB)
275
Figure E.7 HPLC analysis (SAX) of the conversion of 1 to 9 (top) and 10/11 (bottom).
dGMP!
DCBP
dGMP!
MCBP
dGMP
dGMP!
Morph
HPLC analysis of the conversion dGMP-Morph to dGMP-DCBP (top) and dGMP-MCBP (bottom)
Figure E.8 HPLC analysis (SAX) of 10/11 (top) and 9 (bottom) after dual-pass prep. HPLC.
dGMP!
DCBP
dGMP!
MCBP
HPLC analysis of twice purified dGMP-DCBP (bottom) and dGMP-MCBP (top)
276
Figure E.9 HPLC analysis (SAX) of the conversion of 1 to 12/13.
Figure E.10 HPLC analysis (SAX) of 12/13 after dual-pass prep. HPLC.
HPLC analysis of twice purified dGMP-FClBP (0-50% 0.5M LiCl)
277
Figure E.11 HPLC analysis (SAX) of the conversion of 1 to 14.
Figure E.12 HPLC analysis (SAX) of 14 after dual-pass prep. HPLC.
HPLC analysis of twice purified dGMP-DBBP (0-50% 0.5M LiCl)
278
Figure E.13 HPLC analysis (SAX) of the conversion of 1 to 15/16.
Figure E.14 HPLC analysis (SAX) of 15/16 after dual-pass prep. HPLC.
HPLC analysis of twice purified dGMP-MBBP (0-50% 0.5M LiCl)
279
Figure E.15 HPLC analysis (SAX) of the conversion of 2 to 8.
HPLC analysis of conversion of dCMP-Morph to dCMP-DFBP
Figure E.16 HPLC analysis (SAX) of 8 after dual-pass prep. HPLC.
HPLC analysis of ultrapurified dCMP-DFBP (0-50% 0.5M LiCl)
280
Figure E.17 HPLC analysis (SAX) of 7 after dual-pass prep. HPLC.
HPLC analysis of ultrapurified dCMP-MBP (0-50% 0.5M LiCl)
Figure E.18 HPLC analysis (SAX) of the conversion of 1 to 21.
HPLC analysis of conversion of dGMP-Morph to dGMP-DMBP
281
Figure E.19 HPLC analysis (SAX) of the conversion of 1 to 17/18.
HPLC analysis of conversion of dGMP-Morph to dGMP-FMBP
282
MS E.1 - LC-MS dGMP-DFBP, 4
283
MS E.2 - HRMS dGMP-MBP, 3
284
MS E.3 - HRMS dGMP-MFBP, 5/6
285
MS E.4 - HRMS dGMP-DCBP, 9
286
MS E.5 - HRMS dGMP-MCBP, 10/11
287
MS E.6 - HRMS dGMP-FClBP, 12/13
288
MS E.7 - HRMS dGMP-DBBP, 14
289
MS E.8 - HRMS dGMP-MBBP, 15/16
290
MS E.9 - HRMS dGMP-DMBP, 21
291
MS E.10 - HRMS dGMP-EBP, 19/20
292
MS E.11 - HRMS dGMP-FMBP, 17/18
293
MS E.12 - HRMS dCMP-DFBP, 8
294
MS E.13 - HRMS dCMP-MBP, 7
295
2’-deoxy-5’-guanosinemonophosphate morpholidate, 1
NMR E.1 - (D2O)
31
P: 7.1 (s)
2’-deoxy-5’-cytidinemonophosphate morpholidate, 2
NMR E.2 - (D2O)
31
P: 7 (s)
296
2’-deoxy-5’-β,γ-difluoromethylene-cytidine triphosphate (dCMP-β,γ-DFBP), 8
NMR E.3 - (D2O; pH = 8)
1
H: 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m), 2.1 (m), 2.05 (m),
2.0 (dd)
NMR E.4 - (D2O; pH = 8)
19
F: -117.6 (dd)
297
NMR E.5 - (D2O; pH = 8)

top -
31
P: -11 (d), -3 (m), 3.5 (m); bottom -
31
P (decoup. to F):
-11 (d), -3 (dd), 3.5 (d)
2’-deoxy-5’-β,γ-methylene-cytdine triphosphate (dCMP-β,γ-MBP), 7
NMR E.6 - (D2O; pH = 8)
1
H: 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m), 2.1 (m), 2.05 (m),
2.0 (dd)
298
NMR E.7 - (D2O; pH = 8)
31
P: -11 (d), 11 (d), 12.5 (dd)
2’-deoxy-5’-β,γ-difluoromethylene-guanosine triphosphate (dGMP-β,γ-DFBP), 4
NMR E.8 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
299
NMR E.9 - (D2O; pH = 8)
19
F: -117.8 (dd)
NMR E.10 - (D2O; pH = 8) top -
31
P: -11 (d), -3 (m), 3.5 (m);bottom -
31
P (decoup. to
F): -11 (d), -3 (dd), 3.5 (d)
300
2’-deoxy-5’-β,γ-monofluoromethylene-guanosine triphosphate (dGMP-β,γ-MFBP),
5/6
NMR E.11 - (D2O; pH = 10)
1
H: 8 (d), 6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m), 2.75 (m), 2.4
(m)
NMR E.12 - (D2O; pH = 10)
31
P: -11 (d), -4.7 (m), 7 (m)
301
NMR E.13 - (D2O; pH = 10)
19
F: -218.7 (m)
NMR E.14 -
19
F-NMR of 5 and 6 at pH = 10.
Blue: actual NMR spectra; red: predicted NMR for the two separate diastereomers.
302
2’-deoxy-5’-β,γ-methylene-guanosine triphosphate (dGMP-β,γ-MBP), 3
NMR E.15 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.5 (m), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
NMR E.16 - (D2O; pH = 8)
31
P: -11.0 (d), 11.1 (d), 13 (dd)
303
2’-deoxy-5’-β,γ-dichloromethylene-guanosine triphosphate (dGMP-β,γ-DCBP), 9
NMR E.17 - (D2O; pH = 8)
1
H: 8.0 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
NMR E.18 - (D2O; pH = 8)
31
P: -11.0 (d), 1.73 (dd), 7.81 (d)
304
2’-deoxy-5’-β,γ-monochloromethylene-guanosine triphosphate (dGMP-β,γ-MCBP),
10/11
NMR E.19 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
NMR E.20 - (D2O; pH = 8)
31
P: -11.0 (d), 8.26 (dd), 9.24 (d)
305
2’-deoxy-5’-β,γ-fluorochloromethylene-guanosine triphosphate (dGMP-β,γ-FClBP),
12/13
NMR E.21 - (D2O; pH = 10)
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
NMR E.22 - (D2O; pH = 10)
31
P: -11 (d), 1.8 (m), 3.5 (m)
306
NMR E.23 - (D2O; top - pH = 8, bottom = 10)
19
F: -136.5 (m)
2’-deoxy-5’-β,γ-dibromomethylene-guanosine triphosphate (dGMP-β,γ-DBBP), 14
NMR E.24 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
307
NMR E.25 - (D2O; pH = 8)
31
P: -11.0 (d), 2.91 (dd), 8.12 (d)
2’-deoxy-5’-β,γ-monobromomethylene-guanosine triphosphate (dGMP-β,γ-MBBP),
15/16
NMR E.26 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.5 (dt), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
308
NMR E.27 - (D2O; pH = 8)
31
P: -11.0 (dd), 7.73 (m), 8.62 (d)
2’-deoxy-5’-β,γ-[2,2-propanediyl]-guanosine triphosphate (dGMP-β,γ-DMBP), 21
NMR E.28 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.2 (s), 4.1 (m), 2.75 (m), 2.4 (m)
309
NMR E.29 - (D2O; pH = 8)
31
P: -11.0 (d), 19.5 (m), 23 (d)
2’-deoxy-5’-β,γ-[1,1-ethaanediyl]-guanosine triphosphate (dGMP-β,γ-EBP), 19/20
NMR E.30 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.8 (s), 4.3 (s), 4.2 (s), 3.8 (s), 2.75 (m),
2.4 (m), 2.2 (m), 1.4 (m)
NMR E.31 - (D2O; pH = 8)
31
P: -11.0 (d), 17 (d), 18.5 (dd)
310
2’-deoxy-5’-β,γ-[1-fluoro-1,1-ethanediyl]-guanosine triphosphate (dGMP-β,γ-
FMBP), 17/18
NMR E.32 - (D2O; pH = 8)
1
H: 8 (d), 6.25 (t), 4.8 (s), 4.2 (s), 4.1 (m), 2.75 (m), 2.5 (m),
1.7 (dt)
NMR E.33 - (D2O; pH = 8)
31
P: -11.0 (d), 6.5 (m), 11.5 (dd)
311
NMR E.34 - (D2O; pH = 8)
19
F: -176.5 (m)
312
APPENDIX F
Docking results for 2
Table F.1
Number of distinct conformational clusters found = 24, out of 100 runs, using an rmsd-tolerance of 1.0 Å.
Number of multi-member conformational clusters found = 9, out of 100 runs.
Cluster Rank Lowest Docked Energy Run Mean Docked Energy # in Cluster
1 -27.03 73 -25.48 53
2 -25.62 78 -24.94 10
3 -25.5 96 -23.99 4
4 -25.35 32 -24.56 5
5 -24.98 56 -24.58 2
6 -24.5 2 -23.54 3
7 -24.39 1 -24.13 4
8 -23.98 38 -23.98 1
9 -23.86 98 -23.86 1
10 -23.75 99 -23.75 1
11 -23.54 39 -23.54 1
12 -23.38 36 -23.32 2
13 -23.34 77 -22.89 2
14 -22.24 37 -22.24 1
15 -22.16 100 -22.16 1
16 -22.09 82 -22.09 1
17 -22.04 30 -22.04 1
18 -21.78 17 -21.78 1
19 -21.73 47 -21.73 1
20 -21.49 54 -21.49 1
21 -21.12 65 -21.12 1
22 -20.42 45 -20.42 1
23 -19.3 86 -19.3 1
24 -19.11 13 -19.11 1
Table F.2
RMSD Table for 2
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 1 73 -27.03 0 0.87
1 2 83 -27.02 0.23 0.86
1 3 90 -26.98 0.53 0.82
1 4 7 -26.86 0.27 0.91
1 5 25 -26.79 0.51 0.89
1 6 34 -26.63 0.88 1.06
1 7 43 -26.54 0.56 0.87
313
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 8 94 -26.5 0.75 0.94
1 9 4 -26.5 0.65 0.85
1 10 22 -26.4 0.6 0.87
1 11 80 -26.35 0.79 0.93
1 12 60 -26.28 0.38 0.89
1 13 16 -26.24 0.87 0.97
1 14 55 -26.21 0.78 0.92
1 15 3 -26.13 0.94 1.07
1 16 50 -26.08 0.91 1.07
1 17 46 -26.03 0.83 0.95
1 18 29 -26.03 0.69 0.9
1 19 31 -25.92 0.9 0.93
1 20 8 -25.9 0.89 1.07
1 21 14 -25.82 0.86 0.84
1 22 41 -25.74 0.77 1.08
1 23 88 -25.65 0.61 1.04
1 24 68 -25.63 0.49 0.98
1 25 58 -25.55 0.61 1.11
1 26 70 -25.55 0.6 0.87
1 27 74 -25.53 0.91 0.86
1 28 48 -25.47 0.85 0.82
1 29 91 -25.44 0.89 0.91
1 30 42 -25.36 0.66 0.89
1 31 19 -25.32 0.86 1.06
1 32 28 -25.31 0.83 0.87
1 33 20 -25.31 0.86 0.94
1 34 71 -25.23 0.96 0.85
1 35 81 -25.21 0.9 0.87
1 36 97 -25.2 0.84 0.93
1 37 51 -25.2 0.75 0.89
1 38 67 -25.16 0.88 0.97
1 39 69 -25.14 0.89 0.92
1 40 75 -24.95 0.91 1.07
1 41 27 -24.89 0.95 1.07
1 42 87 -24.67 0.74 0.95
1 43 63 -24.66 0.95 0.9
1 44 10 -24.52 0.8 0.93
1 45 62 -24.34 0.99 1.07
1 46 72 -24.29 0.85 0.84
1 47 92 -24.24 0.99 1.08
1 48 89 -24.03 0.86 1.04
1 49 24 -23.85 0.94 0.98
1 50 53 -23.8 0.85 1.11
1 51 49 -23.71 0.73 0.87
1 52 57 -23.66 0.73 0.86
314
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 53 21 -23.47 0.74 0.82
2 1 78 -25.62 0 0.91
2 2 59 -25.49 0.52 0.89
2 3 26 -25.37 0.92 1.06
2 4 23 -25.09 0.87 0.87
2 5 85 -24.92 0.67 0.94
2 6 6 -24.72 0.78 0.85
2 7 52 -24.72 0.87 0.87
2 8 15 -24.64 0.9 0.93
2 9 35 -24.6 0.87 0.89
2 10 9 -24.26 0.94 0.97
3 1 96 -25.5 0 0.92
3 2 40 -24.19 0.98 1.07
3 3 11 -23.74 0.94 1.07
3 4 33 -22.51 0.56 0.95
4 1 32 -25.35 0 0.9
4 2 5 -25.24 0.77 0.93
4 3 18 -24.56 1 1.07
4 4 79 -24.33 0.94 0.84
4 5 66 -23.31 0.97 1.08
5 1 56 -24.98 0 1.04
5 2 93 -24.18 0.69 0.98
6 1 2 -24.5 0 1.11
6 2 76 -24.07 0 0.87
6 3 95 -22.04 0.23 0.86
7 1 1 -24.39 0.53 0.82
7 2 84 -24.38 0.27 0.91
7 3 44 -24.26 0.51 0.89
7 4 64 -23.5 0.88 1.06
8 1 38 -23.98 0.56 0.87
9 1 98 -23.86 0.75 0.94
10 1 99 -23.75 0.65 0.85
11 1 39 -23.54 0.6 0.87
12 1 36 -23.38 0.79 0.93
12 2 61 -23.25 0.38 0.89
13 1 77 -23.34 0.87 0.97
13 2 12 -22.44 0.78 0.92
14 1 37 -22.24 0.94 1.07
15 1 100 -22.16 0.91 1.07
16 1 82 -22.09 0.83 0.95
17 1 30 -22.04 0.69 0.9
18 1 17 -21.78 0.9 0.93
19 1 47 -21.73 0.89 1.07
20 1 54 -21.49 0.86 0.84
21 1 65 -21.12 0.77 1.08
315
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
22 1 45 -20.42 0.61 1.04
23 1 86 -19.3 0.49 0.98
24 1 13 -19.11 0.61 1.11
53
10
4
5
2
3
4
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
0 10 20 30 40 50 60
Number in Cluster
1
3
5
7
9
11
13
15
17
19
21
23
Rank
Histogram of Docking Results for 2
Docking results for 3
Table F.3
Number of distinct conformational clusters found = 54, out of 100 runs, using an rmsd-tolerance of 1.0 Å.
Number of multi-member conformational clusters found = 14, out of 100 runs.
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
1 -25.86 19 -24.75 16
2 -24.32 44 -23.96 3
3 -23.97 24 -23.41 6
4 -23.61 68 -22.83 8
5 -23.36 27 -23.36 1
6 -23.02 61 -22.71 4
7 -22.97 39 -22.15 2
8 -22.78 34 -22.57 5
9 -22.66 95 -21.93 3
10 -22.56 92 -22.56 1
11 -22.56 59 -22.43 3
12 -22.36 83 -22.16 2
316
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
13 -22.29 47 -22.29 1
14 -22.22 77 -22.22 1
15 -22.19 84 -22.19 1
16 -22.07 2 -21.06 2
17 -22.02 86 -22.02 1
18 -21.81 53 -21.81 1
19 -21.31 57 -21.31 1
20 -21.29 15 -21.29 1
21 -21.08 31 -21.08 1
22 -21.05 55 -21.05 1
23 -20.96 11 -20.67 2
24 -20.83 49 -20.83 1
25 -20.49 6 -20.49 1
26 -20.48 25 -20.48 1
27 -20.47 87 -20.47 1
28 -20.38 3 -20.13 2
29 -20.34 64 -20.34 1
30 -20.27 42 -20.27 1
31 -20.1 67 -20.1 1
32 -20.06 43 -20.06 1
33 -20.05 7 -20.05 1
34 -20.04 82 -20.04 1
35 -20.03 46 -20.03 1
36 -20.01 37 -20.01 1
37 -19.9 33 -19.9 1
38 -19.77 29 -19.77 1
39 -19.66 69 -18.54 2
40 -19.61 17 -19.61 1
41 -19.44 41 -19.44 1
42 -19.42 90 -19.42 1
43 -18.97 94 -18.97 1
44 -18.86 58 -18.86 1
45 -18.79 79 -18.79 1
46 -18.73 13 -18.73 1
47 -18.59 78 -18.59 1
48 -17.71 52 -17.71 1
49 -17.57 48 -17.57 1
50 -17.45 65 -17.45 1
51 -17.35 60 -17.35 1
52 -17.15 9 -17.15 1
53 -16.95 45 -16.95 1
54 -16.73 38 -16.73 1
317
Table F.4 RMSD Table for 3
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 1 19 -25.86 0 0.55
1 2 5 -25.47 0.4 0.51
1 3 35 -25.28 0.73 0.91
1 4 10 -25.24 0.31 0.45
1 5 26 -25.21 0.55 0.58
1 6 62 -25.09 0.54 0.86
1 7 8 -25.06 0.44 0.65
1 8 93 -24.99 0.69 0.94
1 9 16 -24.97 0.4 0.49
1 10 50 -24.9 0.71 0.65
1 11 81 -24.27 0.92 1.01
1 12 22 -24.04 0.7 0.65
1 13 91 -23.99 0.69 0.85
1 14 70 -23.94 0.96 0.99
1 15 98 -23.92 0.93 1.06
1 16 75 -23.75 0.72 0.83
2 1 44 -24.32 0 1.16
2 2 1 -23.94 0.85 1.28
2 3 85 -23.61 0.92 1.24
3 1 24 -23.97 0 0.97
3 2 12 -23.66 0.32 0.96
3 3 99 -23.37 0.36 0.98
3 4 72 -23.26 0.78 1.1
3 5 36 -23.2 0.67 1.17
3 6 88 -23.03 0.69 1.23
4 1 68 -23.61 0 1.26
4 2 80 -23.37 0.45 1.23
4 3 18 -23.18 0.68 1.36
4 4 21 -23.09 0.79 1.54
4 5 76 -22.98 0.61 1.4
4 6 14 -22.61 0.74 1.25
4 7 30 -22.16 0.94 1.43
4 8 56 -21.62 0.83 1.23
5 1 27 -23.36 0 1.74
6 1 61 -23.02 0 1.15
6 2 40 -22.87 0.52 1.07
6 3 96 -22.83 0.85 0.95
6 4 71 -22.12 0.85 1.41
7 1 39 -22.97 0 1.26
7 2 63 -21.32 0.94 1.45
8 1 34 -22.78 0 1.58
8 2 54 -22.65 0.91 1.38
318
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
8 3 28 -22.5 0.96 1.43
8 4 32 -22.5 0.95 1.39
8 5 51 -22.41 0.91 1.46
9 1 95 -22.66 0 1.15
9 2 23 -21.63 0.67 1.28
9 3 73 -21.52 0.73 1.22
10 1 92 -22.56 0 1.91
11 1 59 -22.56 0 1.3
11 2 20 -22.49 0.78 1.36
11 3 74 -22.26 0.98 1.51
12 1 83 -22.36 0 1.79
12 2 89 -21.96 0.61 1.9
13 1 47 -22.29 0 1.92
14 1 77 -22.22 0 1.31
15 1 84 -22.19 0 1.9
16 1 2 -22.07 0 1.35
16 2 100 -20.06 0.88 1.47
17 1 86 -22.02 0 1.16
18 1 53 -21.81 0 1.63
19 1 57 -21.31 0 1.42
20 1 15 -21.29 0 1.53
21 1 31 -21.08 0 1.56
22 1 55 -21.05 0 1.8
23 1 11 -20.96 0 1.88
23 2 97 -20.39 0.73 2
24 1 49 -20.83 0 2.71
25 1 6 -20.49 0 1.47
26 1 25 -20.48 0 1.66
27 1 87 -20.47 0 1.68
28 1 3 -20.38 0 1.34
28 2 66 -19.89 0.7 1.26
29 1 64 -20.34 0 1.75
30 1 42 -20.27 0 1.6
31 1 67 -20.1 0 1.44
32 1 43 -20.06 0 1.64
33 1 7 -20.05 0 1.99
34 1 82 -20.04 0 2
35 1 46 -20.03 0 1.63
36 1 37 -20.01 0 1.57
37 1 33 -19.9 0 1.88
38 1 29 -19.77 0 1.74
39 1 69 -19.66 0 2.34
39 2 4 -17.41 0.96 2.63
40 1 17 -19.61 0 1.31
41 1 41 -19.44 0 1.86
319
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
42 1 90 -19.42 0 1.63
43 1 94 -18.97 0 2.94
44 1 58 -18.86 0 1.52
45 1 79 -18.79 0 2.53
46 1 13 -18.73 0 1.95
47 1 78 -18.59 0 2.22
48 1 52 -17.71 0 3.14
49 1 48 -17.57 0 3.08
50 1 65 -17.45 0 4.15
51 1 60 -17.35 0 2.45
52 1 9 -17.15 0 3.74
53 1 45 -16.95 0 3.96
54 1 38 -16.73 0 3.04
16
3
6
8
1
4
2
5
3
1
3
2
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0 2 4 6 8 10 12 14 16 18 20
Number in Cluster
1
4
7
10
13
16
19
22
25
28
31
34
37
40
43
46
49
52
Rank
Histogram of Docking Results for 3
320
Docking results for 4
Table F.5
Number of distinct conformational clusters found = 61, out of 100 runs, using an rmsd-tolerance of 1.0Å.
Number of multi-member conformational clusters found = 9, out of 100 runs.
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
1 -25.49 14 -24.27 20
2 -23.76 70 -22.98 3
3 -23.7 8 -22.81 11
4 -23.47 24 -23.47 1
5 -23.06 65 -23.06 1
6 -22.7 57 -21.9 4
7 -22.65 26 -22.06 2
8 -22.45 62 -22.45 1
9 -22.29 76 -21.7 2
10 -22.19 42 -22.19 1
11 -22.08 29 -21.66 2
12 -21.99 9 -21.99 1
13 -21.83 47 -21.83 1
14 -21.79 85 -21.79 1
15 -21.74 23 -21.74 1
16 -21.46 93 -21.21 2
17 -21.41 75 -21.41 1
18 -21.4 60 -21.4 1
19 -21.33 90 -21.33 1
20 -21.18 40 -21.18 1
21 -21.16 53 -21.16 1
22 -20.99 10 -20.38 2
23 -20.97 3 -20.97 1
24 -20.92 19 -20.92 1
25 -20.92 81 -20.92 1
26 -20.67 34 -20.67 1
27 -20.66 59 -20.66 1
28 -20.65 20 -20.65 1
29 -20.48 63 -20.48 1
30 -20.26 95 -20.26 1
31 -20.24 74 -20.24 1
32 -20.23 5 -20.23 1
33 -20.22 100 -20.22 1
34 -20.15 11 -20.15 1
35 -19.98 72 -19.98 1
36 -19.96 1 -19.96 1
37 -19.9 78 -19.9 1
38 -19.88 52 -19.88 1
39 -19.67 55 -19.67 1
321
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
40 -19.65 18 -19.65 1
41 -19.62 44 -19.62 1
42 -19.24 48 -19.24 1
43 -19.23 13 -19.23 1
44 -19.21 25 -19.21 1
45 -19.18 64 -19.18 1
46 -19.09 45 -19.09 1
47 -19.03 98 -19.03 1
48 -18.86 61 -18.86 1
49 -18.78 46 -18.78 1
50 -18.7 21 -18.7 1
51 -18.63 77 -18.63 1
52 -18.62 80 -18.62 1
53 -18.22 32 -18.22 1
54 -18.14 4 -18.14 1
55 -17.98 15 -17.98 1
56 -17.75 87 -17.75 1
57 -17.57 6 -17.57 1
58 -17.37 39 -17.37 1
59 -17.27 16 -17.27 1
60 -16.23 28 -16.23 1
61 -15.48 12 -15.48 1
Table F.6 RMSD Table for 4
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 1 14 -25.49 0 0.5
1 2 92 -25.44 0.23 0.52
1 3 36 -25.37 0.32 0.51
1 4 30 -25.34 0.23 0.52
1 5 38 -25.32 0.39 0.6
1 6 50 -25.2 0.37 0.47
1 7 99 -25.17 0.77 0.87
1 8 84 -25.05 0.57 0.77
1 9 73 -24.78 0.49 0.54
1 10 79 -24.71 0.98 1.09
1 11 83 -24.33 0.95 1.01
1 12 17 -24.06 0.93 0.96
1 13 35 -23.77 0.84 0.92
1 14 54 -23.67 0.9 1.07
1 15 67 -23.61 0.8 0.83
1 16 49 -23.41 0.75 0.84
1 17 51 -23.4 0.86 0.99
1 18 68 -22.96 0.95 0.99
322
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 19 86 -22.65 0.97 0.99
1 20 66 -21.59 0.93 1
2 1 70 -23.76 0 1.19
2 2 31 -23.45 0.25 1.18
2 3 69 -21.75 0.85 1.37
3 1 8 -23.7 0 1.04
3 2 41 -23.48 0.71 1.07
3 3 82 -23.45 0.97 1.24
3 4 43 -23.18 0.5 1.12
3 5 56 -23.14 0.79 1.21
3 6 88 -22.69 0.95 1.36
3 7 2 -22.64 0.9 1
3 8 22 -22.4 0.95 1.16
3 9 58 -22.35 0.95 1.36
3 10 96 -22.08 0.98 1.19
3 11 89 -21.78 0.79 1.29
4 1 24 -23.47 0 1.26
5 1 65 -23.06 0 1.7
6 1 57 -22.7 0 1.23
6 2 94 -22.49 0.47 1.33
6 3 97 -21.85 0.82 1.56
6 4 91 -20.57 0.83 1.38
7 1 26 -22.65 0 1.19
7 2 27 -21.47 0.97 1.4
8 1 62 -22.45 0 1.39
9 1 76 -22.29 0 1.68
9 2 7 -21.11 0.98 1.55
10 1 42 -22.19 0 1.06
11 1 29 -22.08 0 1.49
11 2 71 -21.25 0.82 1.41
12 1 9 -21.99 0 1.63
13 1 47 -21.83 0 1.69
14 1 85 -21.79 0 1.47
15 1 23 -21.74 0 1
16 1 93 -21.46 0 1.57
16 2 37 -20.95 0.57 1.62
17 1 75 -21.41 0 1.78
18 1 60 -21.4 0 1.33
19 1 90 -21.33 0 1.44
20 1 40 -21.18 0 1.65
21 1 53 -21.16 0 1.53
22 1 10 -20.99 0 1.53
22 2 33 -19.78 0.98 1.49
23 1 3 -20.97 0 1.41
24 1 19 -20.92 0 1.96
323
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
25 1 81 -20.92 0 2.65
26 1 34 -20.67 0 2.23
27 1 59 -20.66 0 2.14
28 1 20 -20.65 0 1.98
29 1 63 -20.48 0 2.09
30 1 95 -20.26 0 1.58
31 1 74 -20.24 0 1.82
32 1 5 -20.23 0 1.76
33 1 100 -20.22 0 2.67
34 1 11 -20.15 0 2.63
35 1 72 -19.98 0 1.73
36 1 1 -19.96 0 1.72
37 1 78 -19.9 0 1.42
38 1 52 -19.88 0 1.74
39 1 55 -19.67 0 1.84
40 1 18 -19.65 0 1.55
41 1 44 -19.62 0 2.31
42 1 48 -19.24 0 2.05
43 1 13 -19.23 0 2.01
44 1 25 -19.21 0 1.88
45 1 64 -19.18 0 2.67
46 1 45 -19.09 0 1.49
47 1 98 -19.03 0 4.05
48 1 61 -18.86 0 1.88
49 1 46 -18.78 0 1.76
50 1 21 -18.7 0 1.64
51 1 77 -18.63 0 2.39
52 1 80 -18.62 0 1.94
53 1 32 -18.22 0 3
54 1 4 -18.14 0 3.65
55 1 15 -17.98 0 3.12
56 1 87 -17.75 0 3.73
57 1 6 -17.57 0 2.03
58 1 39 -17.37 0 3.39
59 1 16 -17.27 0 2.76
60 1 28 -16.23 0 3.22
61 1 12 -15.48 0 4.65
324
20
3
11
1
1
4
2
1
2
1
2
1
1
1
1
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0 5 10 15 20 25
Number in cluster
1
4
7
10
13
16
19
22
25
28
31
34
37
40
43
46
49
52
55
58
61
Rank
Histogram of Docking Results for 4
Docking results for 1
Table F.7
Number of distinct conformational clusters found = 27, out of 100 runs, using an rmsd-tolerance of 1.0 Å.
Number of multi-member conformational clusters found = 7, out of 100 runs.
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
1 -27.09 23 -25.12 55
2 -25.8 48 -24.77 13
3 -25.56 44 -25.56 1
4 -25.35 36 -25.32 2
5 -24.78 88 -23.58 3
6 -24.54 95 -24.52 2
7 -24.34 45 -24.34 1
8 -23.77 32 -23.61 3
9 -23.62 49 -23.14 2
10 -23.44 25 -23.44 1
11 -22.9 29 -22.9 1
12 -22.78 46 -22.78 1
13 -22.41 69 -22.41 1
14 -22.4 57 -22.4 1
15 -22.12 72 -22.12 1
16 -21.65 54 -21.65 1
325
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
17 -21.48 87 -21.48 1
18 -21.33 83 -21.33 1
19 -21.31 81 -21.31 1
20 -21.28 93 -21.28 1
21 -21.25 59 -21.25 1
22 -21.11 28 -21.11 1
23 -20.8 31 -20.8 1
24 -20.79 76 -20.79 1
25 -20.73 7 -20.73 1
26 -19.84 41 -19.84 1
27 -19.33 47 -19.33 1
Table F.8 RMSD values for 1
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 1 23 -27.09 0 0.86
1 2 14 -26.88 0.54 0.75
1 3 27 -26.64 0.66 0.77
1 4 39 -26.58 0.27 0.91
1 5 58 -26.52 0.45 0.8
1 6 22 -26.52 0.55 0.82
1 7 85 -26.49 0.38 0.9
1 8 20 -26.2 0.55 0.76
1 9 37 -26.2 0.9 1.09
1 10 71 -26.12 0.8 0.96
1 11 70 -26.04 0.69 0.88
1 12 34 -25.95 0.69 0.91
1 13 21 -25.91 0.73 1.04
1 14 35 -25.86 0.72 0.88
1 15 63 -25.83 0.88 0.98
1 16 40 -25.77 0.72 0.88
1 17 97 -25.74 0.92 0.94
1 18 64 -25.7 0.86 1.05
1 19 8 -25.64 0.8 1
1 20 73 -25.62 0.75 1.08
1 21 10 -25.6 0.99 1
1 22 15 -25.4 0.87 1.04
1 23 67 -25.38 0.85 1.08
1 24 13 -25.31 0.78 1.08
1 25 17 -25.31 0.98 1.1
1 26 2 -25.26 0.81 1.08
1 27 5 -25.21 0.79 1.08
1 28 6 -25.08 0.79 1
1 29 38 -25.06 0.76 1.15
326
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 30 60 -25.06 0.91 1.07
1 31 4 -24.99 0.95 1.04
1 32 55 -24.97 0.78 1.04
1 33 24 -24.86 0.72 0.85
1 34 86 -24.84 0.79 1
1 35 56 -24.82 0.93 0.87
1 36 94 -24.78 0.89 0.92
1 37 100 -24.7 0.94 1.05
1 38 1 -24.65 0.88 1.2
1 39 66 -24.59 0.78 1.14
1 40 68 -24.55 0.54 0.95
1 41 9 -24.49 0.91 1
1 42 84 -24.46 0.66 1.16
1 43 50 -24.22 0.99 0.93
1 44 26 -24.19 0.81 1.08
1 45 90 -24.17 0.99 1.07
1 46 61 -24.07 0.7 0.94
1 47 78 -23.82 0.84 1.17
1 48 12 -23.73 0.59 0.96
1 49 30 -23.68 0.91 1.01
1 50 96 -23.65 0.93 1.04
1 51 89 -23.63 1 1.1
1 52 3 -23.56 0.85 1.07
1 53 62 -23.5 0.9 1.13
1 54 77 -23.42 0.97 1.1
1 55 79 -23.27 0.85 1.07
2 1 48 -25.8 0 1.19
2 2 51 -25.8 0.6 1.17
2 3 75 -25.27 0.64 1.14
2 4 92 -24.97 0.82 1.06
2 5 16 -24.94 0.46 1.19
2 6 33 -24.93 0.74 0.97
2 7 82 -24.82 0.33 1.23
2 8 53 -24.54 0.99 1.15
2 9 80 -24.5 0.93 1.13
2 10 11 -24.36 0.83 1.1
2 11 98 -24.21 0.78 1.12
2 12 43 -24.19 0.75 1.18
2 13 52 -23.7 0.82 1.07
3 1 44 -25.56 0 1.08
4 1 36 -25.35 0 1.03
4 2 99 -25.29 0.79 1.01
5 1 88 -24.78 0 1.12
5 2 91 -23.38 0.73 1.22
5 3 65 -22.58 0.88 1.21
327
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
6 1 95 -24.54 0 1.15
6 2 18 -24.5 0.48 1.15
7 1 45 -24.34 0 1.13
8 1 32 -23.77 0 2.03
8 2 19 -23.66 0.95 1.99
8 3 74 -23.41 0.46 2.14
9 1 49 -23.62 0 2.09
9 2 42 -22.66 0.72 2.18
10 1 25 -23.44 0 1.36
11 1 29 -22.9 0 1.14
12 1 46 -22.78 0 1.06
13 1 69 -22.41 0 2.33
14 1 57 -22.4 0 1.4
15 1 72 -22.12 0 2.13
16 1 54 -21.65 0 2.04
17 1 87 -21.48 0 2.36
18 1 83 -21.33 0 2.06
19 1 81 -21.31 0 2.32
20 1 93 -21.28 0 2.52
21 1 59 -21.25 0 2.2
22 1 28 -21.11 0 2.37
23 1 31 -20.8 0 2.52
24 1 76 -20.79 0 3.18
25 1 7 -20.73 0 2.46
26 1 41 -19.84 0 2.59
27 1 47 -19.33 0 2.53
55
13
1
2
3
2
1
3
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0 10 20 30 40 50 60
Number of Clusters
1
3
5
7
9
11
13
15
17
19
21
23
25
27
Rank
Histogram of Docking Results for 1
328
Docking results of dGTP
Table F.9
Number of distinct conformational clusters found = 27, out of 100 runs, using an rmsd-tolerance of 1.0 Å.
Number of multi-member conformational clusters found = 7, out of 100 runs.
Cluster Rank Lowest Docked Energy Run Mean Docked Energy Num in Clus
1 -25.68 19 -24.6 42
2 -25.41 21 -24.64 20
3 -25.11 47 -24.15 3
4 -24.38 72 -23.96 12
5 -24.25 2 -23.92 6
6 -23.69 27 -23.69 1
7 -23.63 97 -23.63 1
8 -23.48 54 -23.48 1
9 -23.42 93 -22.25 2
10 -23.4 99 -23.26 2
11 -22.86 92 -22.86 1
12 -22.31 39 -22.31 1
13 -22.17 100 -21.59 2
14 -21.12 59 -21.12 1
15 -20.76 88 -20.76 1
16 -20.56 58 -20.56 1
17 -20.47 37 -20.47 1
18 -20.45 53 -20.45 1
19 -19.97 29 -19.97 1
Table F.10 RMSD values for dGTP.
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 1 19 -25.68 0 1.03
1 2 41 -25.53 0.83 0.87
1 3 46 -25.52 0.39 1.06
1 4 15 -25.45 0.51 1.17
1 5 20 -25.41 0.82 0.91
1 6 85 -25.38 0.83 0.83
1 7 25 -25.16 0.73 1.11
1 8 10 -25.07 0.87 0.99
1 9 6 -25.02 0.86 0.89
1 10 57 -25.02 0.84 0.82
1 11 38 -25 0.85 0.82
1 12 8 -24.96 0.65 1.1
1 13 24 -24.95 0.77 1.11
1 14 18 -24.91 0.63 1.04
1 15 31 -24.91 0.95 1.42
329
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
1 16 34 -24.91 0.83 0.71
1 17 95 -24.84 0.74 0.94
1 18 69 -24.78 0.83 0.99
1 19 3 -24.73 0.92 1.34
1 20 45 -24.67 0.55 0.98
1 21 9 -24.66 0.84 1.03
1 22 7 -24.66 0.9 1.03
1 23 83 -24.65 0.99 0.96
1 24 82 -24.61 0.89 1
1 25 48 -24.59 0.61 1.05
1 26 70 -24.47 0.86 1.53
1 27 12 -24.44 0.78 0.9
1 28 80 -24.43 0.7 1.16
1 29 52 -24.41 0.94 1.02
1 30 55 -24.32 0.92 0.94
1 31 71 -24.25 0.55 1.12
1 32 42 -24.18 0.97 0.88
1 33 28 -24.13 0.93 1.04
1 34 61 -24.07 0.76 1.07
1 35 96 -24.06 0.86 0.66
1 36 79 -24.02 0.59 1.19
1 37 90 -24 0.98 1.11
1 38 87 -23.87 0.76 1.16
1 39 78 -23.85 0.89 1.2
1 40 89 -23.55 0.85 1.05
1 41 84 -23.55 0.95 0.99
1 42 14 -22.64 0.98 1.04
2 1 21 -25.41 0 1.05
2 2 17 -25.3 0.34 1
2 3 62 -25.23 0.83 0.86
2 4 86 -25.14 0.99 0.76
2 5 60 -25.02 0.37 1.08
2 6 68 -25 0.76 1.07
2 7 23 -25 0.29 1.03
2 8 50 -24.96 0.91 0.73
2 9 81 -24.76 0.46 1.05
2 10 98 -24.73 0.94 0.96
2 11 77 -24.64 0.33 1.02
2 12 63 -24.52 0.82 1.01
2 13 67 -24.45 0.9 1.02
2 14 65 -24.38 0.76 1.19
2 15 26 -24.36 0.68 1.07
2 16 33 -24.35 0.72 1.11
2 17 16 -24.09 0.9 1.07
2 18 66 -24.05 0.92 1.04
330
Rank Sub-Rank Run Docked Energy Cluster RMSD Reference RMSD
2 19 51 -24.05 0.83 1.2
2 20 36 -23.41 0.9 1.24
3 1 47 -25.11 0 0.87
3 2 11 -24.1 0.83 0.94
3 3 35 -23.24 0.94 1.04
4 1 72 -24.38 0 1.35
4 2 44 -24.31 0.56 1.42
4 3 1 -24.21 0.55 1.16
4 4 73 -24.15 0.89 1.25
4 5 75 -24.05 0.37 1.36
4 6 43 -23.95 0.92 0.91
4 7 32 -23.94 0.23 1.38
4 8 4 -23.94 0.67 1.45
4 9 64 -23.73 0.77 1.44
4 10 74 -23.73 0.6 1.3
4 11 13 -23.71 0.83 1.04
4 12 40 -23.43 0.81 1.2
5 1 2 -24.25 0 1.09
5 2 22 -24.14 0.97 1.5
5 3 56 -23.88 0.97 1.22
5 4 91 -23.85 0.95 1.04
5 5 76 -23.72 0.88 1.32
5 6 5 -23.69 0.93 1.17
6 1 27 -23.69 0 1.1
7 1 97 -23.63 0 1.15
8 1 54 -23.48 0 1.35
9 1 93 -23.42 0 1.52
9 2 49 -21.08 0.73 1.55
10 1 99 -23.4 0 1.1
10 2 30 -23.12 0.94 1.11
11 1 92 -22.86 0 1.07
12 1 39 -22.31 0 2.11
13 1 100 -22.17 0 1.96
13 2 94 -21.01 0.96 2.07
14 1 59 -21.12 0 2.31
15 1 88 -20.76 0 2.53
16 1 58 -20.56 0 2.27
17 1 37 -20.47 0 3.15
18 1 53 -20.45 0 3.09
19 1 29 -19.97 0 2.49
331
Histogram of Docking Results for dGTP
42
20
3
12
6
1
1
1
2
2
1
1
2
1
1
1
1
1
1
0 5 10 15 20 25 30 35 40 45
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Rank
Number in Cluster
332
APPENDIX G
tetraisopropyl (1,1,1-trifluoro-2,2-propanediyl)bis(phosphonate) (CF3TiPEBP
NMR G.1 -
31
P: 14.8 (q)
31
P NMR analysis of Umemoto reaction mixture
P P
CH
3
O
O
OiPr
O
OiPr O iPr
iPr
P P
H
3
C
O
O
OiPr
O
OiPr O
CF
3
iPr
iPr
NMR G.2 -
19
F: -66.4 (t)
P P
H
3
C
O
O
OiPr
O
OiPr O
CF
3
iPr
iPr
19
F NMR analysis of Umemoto reaction mixture
CF3H
333
ethyl 2-(diethoxyphosphoryl)propanoate (TEMPAA)
NMR G.5 - (1:1 mixture of TEMPAA : TEDMPAA in CDCl3)
31
P: 21.1 (s) and 24.5 (s)
ethyl 2-(diethoxyphosphoryl)-3,3,3-trifluoro-2-methylpropanoate (CF3TEMPAA)
NMR G.6 -
31
P: 12.7 (q)
334
NMR G.7 -
19
F: -70.4 (d)
(2,2,2-trifluoro-1-hydroxy-1,1-ethanediyl)bis(phosphonic acid) (CF3-etidronate)
Method A. “Modified Kabachnik” Chemistry
LC-MS (in D2O, [M-2H+D]
-
= 261; in Ca(OH)2, [M-H+2Ca]
-
= 358)
NMR G.8 -
31
P (pH <5): 10.5-12 (b)
335
NMR G.9 -
19
F (pH <5): -73.5 (bt)
NMR G.10 - after extraction with Ca(OH)2:
31
P (pH = 10.7): 8.5 (q)
336
NMR G.11 - after extraction with Ca(OH)2:
19
F (pH = 10.7): -74.9 (bt)
Method B. Trifluoromethylacetic Anhydride Method: LC-MS = [M-H]
-
= 259
NMR G.12 -
31
P: 9.88 (q)
337
NMR G.13 -
19
F: -70.2 (bt)
338
APPENDIX H
Synthesis of 2’-deoxy-5’-thymidinemonophosphate morpholidate
NMR H.1 -
31
P: 7.3 (s)
2’-deoxy-5’-β,γ-methylene-thymidine triphosphate (β,γ-CH2-dTTP)
NMR H.2 - (D2O; pH = 10)
1
H: 7.7 (d), 6.4 (t), 4.6 (d), 4.3 (m), 4.2 (s), 2.2 (t), 1.8 (s)
339
NMR H.3 - (D2O; pH = 10)
31
P: -11 (d), 11.2 (d), 13.2 (m)
5’-β,γ-difluoromethylene-adenosine triphosphate (β,γ-CF2-ATP)
NMR H.4 - (D2O; pH = 10)
1
H: 8.8 (b), 8.2 (s), 6.2 (d), 4.65 (t), 4.5 (s), 4.4 (s), 4.2 (m)
340
NMR H.5 - (D2O; pH = 10)
31
P: -11 (d), -3 (m), 3.5 (m)
NMR H.6 - (D2O; pH = 10)
19
F: -117.1 (dd)
341
5’-β,γ-etidronate-guanosine triphosphate (β,γ-etidronate-GTP)
NMR H.7 - (D2O; pH = 10)
1
H: 8.2 (s), 5.97 (d), 4.65 (t), 4.35 (s), 4.25 (m), 1.63 (t)
a
b
c
a b
c
1
H NMR analysis of GMP-Etidronate after purification on SAX
NH
N
N
O
NH
2
N
O
OH
O P O
O
OH
P P HO
OH OH
O O
OH
CH
3
OH
NMR H.8 - (D2O; pH = 10)
31
P: -11 (d), 13 (m), 17 (m)
!
GMP
"
#
Etidronate
31
P NMR analysis of GMP-Etidronate after purification on SAX
342
2’-deoxy-5’-β,γ-risedronate-guanosine triphosphate (β,γ-risedronate-dGTP)
NMR H.9 - (D2O; pH = 10)
31
P: -11 (m), 12.8 (m), 14 (m)
2’-deoxy-5’-β,γ-CF3-etidronate-guanosine triphosphate (β,γ-CF3-etidronate-dGTP)
NMR H.10 -
31
P (D2O; pH = 4.5) -11 (d), 0.5 (m), 8.1 (m)
343
NMR H.11 - (D2O; pH = 4.5)
19
F: -69.6 (bt)

2’-deoxy-5’-α,β-difluoromethylene-thymidine triphosphate (α,β-CF2-dTTP)
NMR H.12 -
1
H: (D2O, pH = 8): 7.8 (d), 6.07 (t), 5.8 (d), 4.4 (m), 4 (m), 2.1 (m), 2.05
(m), 2.0 (dd)
344
NMR H.13 - (D2O; pH = 8) 19F: -119.2 (dt)
Purification 2’-deoxy-5’-α,β,β,γ-bis(CF2)-thymidine triphosphate (α,β,β,γ-bis(CF2)-
dTTP)
α,β,β,γ-bis-(CF2)-dTTP was isolated as the TEA salt after dual-pass preparative HPLC
NMR H.14 -
31
P: 3.25 (m), 3.5 (m), 13.5 (m)
345
NMR H.15 -
1
H: (D2O, pH = 8): 7.8 (d), 6.45 (t), 4.7 (m), 4.3 (m), 4 .2 (m), 2.3 (m), 1.9
(s)
NMR H.16 -
19
F: -117.8 (t), -118.6 (dt)
Purification 2’-deoxy-5’-α,β,β,γ-bis(CF2)-adenosine triphosphate (α,β,β,γ-bis(CF2)-
dATP)
346
α,β,β,γ-bis-(CF2)-dATP was isolated as the TEA salt after dual-pass preparative HPLC.
NMR H.17 -
31
P: 3.25 (m), 3.5 (m), 13.6 (m)
NMR H.18 -
1
H: (D2O, pH = 8): 8.61 (s), 8.28 (s), 6.52 (t), 5.8 (d), 4.75 (m), 4.3 (m), 4
(m), 3.39 (m), 2.85 (m), 2.65 (m)
NMR H.19 -
19
F: -117.8 (t), -118.7 (dt)
347
Figure H.1 Prep. HPLC (SAX) trace of AMP-DFBP reaction mixture

Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
0
100
200
300
400
mAU
0
100
200
300
400
UV Detector Ch1-266nm
ab-b y-bis-CF2-dTTP ru n 1
ab-b y-bis-CF2-dTTP ru n 1.dat

Figure H.2 Analytical HPLC (SAX) of AMP-DFBP after dual-pass purification

348
Figure H.3 Prep. HPLC (SAX) trace of bis-CF2-dTTP reaction mixture

Minutes
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
mAU
0
100
200
300
400
mAU
0
100
200
300
400
UV Detector Ch1-266nm
ab-b y-bis-CF2-dTTP ru n 1
ab-b y-bis-CF2-dTTP ru n 1.dat

Figure H.4 Prep. HPLC (C18) trace of bis-CF2-dTTP mixture
Minutes
0 5 10 15 20 25 30 35 40 45 50
mAU
0
50
100
150
200
250
300
mAU
0
50
100
150
200
250
300 UV Detecto r Ch 1-266n m
ab -b y-b is-CF 2-d TTP C18 ru n 1
ab -b y-b is-CF 2-d TTP C18 ru n 1.d at

Fig-
ure H.5 Analytical HPLC (SAX) trace of β,γ-Etidronate-GMP (top) and β,γ-Etidronate-dGMP (bottom)
GMP-Etidronate
HPLC analysis (SAX) of GMP-Etidronate reaction mixture (top) compared to
dGMP-Etidronate reaction mixture (bottom)
dGMP-Etidronate
GMP
dGMP
dGMP-
Morph
GMP-
Morph
349
Figure H.6 HPLC analysis (SAX) of β,γ-Risedronate-dGMP reaction mixture
HPLC analysis (SAX) of (1) dGMP-Risedronate reaction
mixture compared to (2) Risedronate and (3) dGMP-
Risedronate reaction spiked with Risedronate
dGMP-Risedronate dGMP
dGMP-
Morph
Risedronate
Figure H.7 Analytical HPLC (SAX) trace of α,β,β,γ-bis-CF2-dTTP (top) and α,β,β,γ-bis-CF2-dATP (bot-
tom)
350
MS.1 - α,β,β,γ-bis-CF2-dATP
CF2-dATP
Measured Mass 557.9961
Element Low Limit High Limit
C 10 30
F 2 4
H 10 30
N 3 6
O 7 13
P 1 3
Formula Calculated Mass mDaError ppmError RDB
C16 H12 N4 O10 F4 P2 557.9965 -0.4 -0.7 12
C15 H14 N5 O9 F3 P3 557.9957 0.4 0.8 11.5
C12 H15 N5 O10 F4 P3 557.9968 -0.7 -1.2 7.5
C19 H11 N4 O9 F3 P2 557.9953 0.8 1.4 16
C18 H13 N5 O8 F2 P3 557.9945 1.6 2.9 15.5
C22 H10 N4 O8 F2 P2 557.9942 1.9 3.4 20
C13 H14 N3 O13 F4 P2 557.9938 2.3 4.1 7.5
-TOF MS: 0.332 to 0.350 min from 11210703t.wiff Agilent, subtracted (0.102 to 0.208 min) Max. 1.1e6 counts.
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200
m/z, amu
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
1.10e6
1.14e6
Intensity, counts
557.9961
380.9679
McKenna(USC) CF2-dATP ESI/APCI
Page 1
351
MS.2 - α,β,β,γ-bis-CF2-dTTP
CF2-dTTP
Measured Mass 548.9854
Element Low Limit High Limit
C 10 30
F 2 4
H 10 30
N 1 4
O 7 13
P 1 3
Formula Calculated Mass mDaError ppmError RDB
C12 H16 N2 O12 F4 P3 548.9852 0.2 0.3 5.5
C21 H12 N3 O7 F2 P3 548.9856 -0.2 -0.4 18
C16 H13 N O12 F4 P2 548.9849 0.5 0.9 10
C22 H10 N2 O8 F3 P2 548.9865 -1.1 -1.9 18.5
C15 H15 N2 O11 F3 P3 548.9841 1.3 2.4 9.5
C18 H13 N3 O8 F3 P3 548.9868 -1.4 -2.5 14
C19 H12 N O11 F3 P2 548.9838 1.6 3.0 14
C14 H11 N4 O11 F4 P2 548.9836 1.8 3.3 10.5
C19 H11 N2 O9 F4 P2 548.9876 -2.2 -4.0 14.5
C18 H14 N2 O10 F2 P3 548.9829 2.5 4.5 13.5
C15 H14 N3 O9 F4 P3 548.9879 -2.5 -4.6 10
-TOF MS: 0.444 to 0.462 min from 11210702t.wiff Agilent, subtracted (0.107 to 0.196 min) Max. 1.8e6 counts.
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200
m/z, amu
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e6
Intensity, counts
548.9854
380.9673
McKenna(USC) CF2-dTTP ESI/APCI
Page 1
352
Figure H.8 Analytical HPLC (SAX) of dGMP-CF3Etidronate reaction mixture after 24 hrs

dGMP
dGMP-Morph
dGMP-
CF3Etid
Figure H.9 Analytical HPLC (SAX) of dGMP-CF3Etidronate after dual-pass prep. HPLC; LC-MS: [M-
H]
-
= 588
dGMP
dGMP-β,γ-
CF3Etid
353
MS.3 - β,γ-CF3Etidronate-dGMP
354

Abstract (if available)

Abstract A stereoelectronically varied series of alpha-substituted methylenebisphosphonic acids (X,Y = H, F, Cl, Br, CH3) was synthesized and used to prepare corresponding dNTP beta,y-CXY and also certain alpha,beta-analogues. Improved analytical and preparative HPLC methods are described for the NTP analogues. The pKa4 values of the bisphosphonic acids were determined under self-consistent conditions, enabling a "toolkit" of pyrophosphate analogues for kinetic and structural studies of DNA polymerase mechanisms and fidelity. The dNTP analogues are substrates for DNA pol beta and the kpol values of the beta,y-CXY analogues were compared to the pKa4 of the relevant bisphosphonate (BP) leaving group. For BPs with a pKa4 less than or equal to that of pyrophosphoric acid, log kpol values were similar or decreased moderately with increasing pKa4. For BPs with a pKa4 greater than that of pyrophosphate a significant trend of decreasing log kpol was apparent. This observation, and the absence of an analogous effect on ground state analog binding (Kd values, except for the CBr2 analogue), points to an influence of the leaving group aptitude on the energy of the transition state. Reduced catalysis rates were observed with the dihalo-substituted substrates particularly for the T-G mispair incorporation. X-ray crystallographic studies with several of Palpha-Z-Pbeta (Z = NH, CH2, CF2) and Pbeta-CXY-Py dNTP analogues in ternary complex with DNA pol beta and DNA primer-template strand have been carried out. Evidence consistent with a docking study was obtained for a C-F⋅⋅⋅H-N-Arg interaction in the beta,y-CHF-dGTP analogue -- active site complex, in which only the (R)-diastereomer of the analogue is observed. The synthesis of new bisphosphonates incorporating a CF3-group was also investigated. The dNTP analogue "toolkit" should prove useful to probe structure and function in other DNA polymerases and to refine theoretical studies of the enzymes mechanisms.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Upton, Thomas George (author)

Core Title Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 02/04/2009

Defense Date 06/23/2008

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

Tag autodock,bisphosphonates,DNA polymerase beta,docking,fluorination,halogenation,HIV reverse transcriptase,inhibitor design,nucleotide analogues,OAI-PMH Harvest,polymerase kinetics,stereoselectivity,titrations,trifluoromethylation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Goodman, Myron F. (committee member), Haworth, Ian S. (committee member)

Creator Email tom.upton@cenomedbio.com,tupton@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1540

Unique identifier UC183843

Identifier etd-Upton-2311 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-109922 (legacy record id),usctheses-m1540 (legacy record id)

Legacy Identifier etd-Upton-2311.pdf

Dmrecord 109922

Document Type Dissertation

Rights Upton, Thomas George

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu