University of Southern California Dissertations and Theses

Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted tri...

(USC Thesis Other)

Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted tri...

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

DEOXYNUCLEOTIDE ANALOG PROBES AND MODEL COMPOUNDS FOR STUDYING DNA
POLYMERASE STRUCTURE AND MECHANISM: SYNTHESIS AND EVALUATION OF ALKYL-,
AZIDO-, AND HALOMETHYLENE BISPHOSPHONATE-SUBSTITUTED TRIPHOSPHATES

by

Brian Thomas Chamberlain

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

August 2012

Copyright 2012 Brian Thomas Chamberlain
ii

DEDICATION

To my parents,
for their incredible love and support.

iii

ACKNOWLEDGEMENTS
First and foremost I would like to thank Professor Charles McKenna for the
opportunity to study chemistry with him and his lab. I wandered into Charles’ office with
little more than a pre-med’s knowledge of chemistry looking to learn more about drug
discovery. Over the years, Charles taught me about chemistry and research, the art of
writing scientific articles and giving compelling presentations, and the value of international
collaboration. I am truly grateful for his mentorship and what he has made possible for me.
Next, of course, thank you to Dr. Boris Kashemirov for working with me day-in and
day-out as I developed into an organic chemist. Simply put, the work presented here could
not have been accomplished without his help. I will miss our discussions about chemistry
and his perspectives on life.
Bioorganic chemistry is inherently interdisciplinary and as such relies on extensive
collaboration. I must recognize Professor Myron Goodman and Dr. Samuel Wilson for
having organized the NIH-funded program project of which this research was a part (NIH
grant U19CA105010). The work presented here contains contributions from scientists in the
Wilson lab, as well as the lab of Professor Debbie Crans and select members of the McKenna
lab. In Chapter 1, Elena Ferri re-synthesized the β,y-CHCl-dTTP compound so that we could
better examine the NMR properties of the compounds. Chapter 3 was published, in part, in
ChemBioChem (2011, 13 (4), 528) and features the work of several members of the Wilson
lab. Dr. Vinod Batra crystallized the a,β-dNTP-DNA-pol β ternary complexes and resolved
their structures, Dr. William Beard and David Shock determined the K
d
values for the dNTP

iv

probes, and Anastasia Kadina (McKenna lab) assisted with the synthesis of precursors and
performed the molecular docking calculations. The kinetic results incorporated into Chapter
4 are from the analytical studies of Dr. Ernestas Gaidamauskas, a visiting scholar, and Jorge
Osuna of the McKenna lab. The experimental methods and data from the hydrolysis
experiments featured in this chapter will be presented in Jorge Osuna’s dissertation. Also
for Chapter 4, Jorge synthesized the post-column HPLC reagent and Boris performed the ab
initio calculations used to design the supplementary model compounds.
Thank you to Myron and Professor Ian Haworth for service on my dissertation
committee, as well as Professor Travis Williams and Professor Peter Qin for membership in
my qualifying committee. I would like to give a special thank you to Travis and Professor
Surya Prakash for their support throughout my graduate career.
Allan Kershaw faithfully maintained the USC NMR facility which, especially as this
work was mainly developed using a vintage Bruker AMX-500, is worthy of recognition. The
chemistry department administrative team always kept things running smoothly so that I
could stay on track in lab: Marie de la Torre, Michele Dea, Katie McKissick, and Inah Kang,
thank you to all.
Finally, thank you to entire McKenna lab. In particular, thank you to Dr. Joy Bala, Dr.
James Hogan, Professor Larryn Peterson, and Dr. Tom Upton for making my first years in the
group so welcoming. I am indebted to Tom for starting such a fruitful project and to Larryn
for always being there for advice and support. Thanks are due to Yue Wu, Dr. Valeria
Zakharova, Ivan Krylov, and Anastasia for chemistry discussions, friendship and lending me
glassware even with full knowledge that it would never be returned. Also, thank you to Yue,

v

Shuting Sun and Dr. Feng Ni for helping me (and laughing with me) as I dabbled in Mandarin
Chinese.
Including my undergraduate study, I was enrolled at USC for almost 9 years. Looking
from where I started when I was 19, it would have been impossible to predict that I would
be here now, writing my dissertation on chemistry. From the bottom of my heart, thank
you to Christina Chen for supporting me through all the late nights in the lab and on the
computer as I finished my research and composed this dissertation. My time at USC was
filled with wonderful people that made the experience so worthwhile. To my teachers,
friends, and teammates thank you and fight on!

vi

TABLE OF CONTENTS

DEDICATION............................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................................ iii
LIST OF TABLES ......................................................................................................................... ix
LIST OF FIGURES ........................................................................................................................ x
LIST OF SCHEMES .................................................................................................................... xiv
ABSTRACT ................................................................................................................................ xv

CHAPTER 1. Synthesis of a Series of Halogenated Thymidine 5’-Triphosphate β,γ-
Phosphonate dNTP Analogs ...................................................................................................... 1
1.1 Introduction .................................................................................................................... 1
1.2 Results and discussion .................................................................................................... 2
1.3 Conclusion ...................................................................................................................... 7
1.4 Experimental ................................................................................................................... 8
1.4.1 Material and methods .............................................................................................. 8
1.4.2 Synthesis of 2’-deoxythymidine-5’-triphosphate β,γ-CXY analogs .......................... 9
1.5 Chapter references ....................................................................................................... 14

CHAPTER 2. α-Azido Bisphosphonates: Synthesis, pKa Determination, Nucleotide
Analogs, and Reactions ............................................................................................................ 17
2.1 Introduction .................................................................................................................. 17
2.2 Results and discussion ................................................................................................... 18
2.2.1 Synthesis of α-azido bisphosphonates ................................................................... 18
2.2.2 Determination of α-azido bisphosphonic acid stability constants by
potentiometric titration .................................................................................................. 23
2.2.3 Synthesis of α-azido bisphosphonate containing nucleotide analogs ................... 26
2.2.4 Subsequent reactions of α-azido bisphosphonates ............................................... 29
2.3 Conclusion .................................................................................................................... 31
2.4 Experimental ................................................................................................................. 32

vii

2.4.1 Materials and methods .......................................................................................... 32
2.4.2 Synthetic procedures .............................................................................................. 33
2.4.3 Potentiometric titration of 3 and 4 ........................................................................ 40
2.5 Chapter references ....................................................................................................... 42

CHAPTER 3. α,β-CXY dATP Probes for DNA Polymerase β: Binding Affinities and
Structural Evidence for a Non-covalent C-F Interaction ......................................................... 48
3.1 Introduction .................................................................................................................. 48
3.2 Results and discussion .................................................................................................. 50
3.2.1 Synthesis of α,β-CXY-dATP analogs ....................................................................... 50
3.2.2 α,β-CXY-dATP binding affinities ............................................................................. 51
3.2.3 X-ray crystal structures of pol β-DNA-dATP analog ternary complexes ................ 53
3.2.4 Molecular docking studies with Autodock Vina .................................................... 55
3.3 Conclusion .................................................................................................................... 56
3.4 Experimental ................................................................................................................. 56
3.4.1 Materials and methods ......................................................................................... 56
3.4.2 Synthetic procedures ............................................................................................. 58
3.4.3 Molecular docking ................................................................................................. 62
3.5 Chapter references ....................................................................................................... 64

CHAPTER 4. Examining the Stability of the P
α
-O-P
β
Bond in Triphosphate Monoesters ....... 67
4.1 Introduction .................................................................................................................. 67
4.2 Results........................................................................................................................... 68
4.2.1 Design and synthesis of model compounds 1-3 .................................................... 68
4.2.2 Hydrolysis of 1-3 .................................................................................................... 72
4.2.3 Design and synthesis of second generation model compounds 4 and 5 .............. 73
4.2.3 Hydrolysis of 4 and 5 ............................................................................................. 75
4.3 Discussion ..................................................................................................................... 77
4.3.1 Triphosphate P
β
-O bond stability .......................................................................... 77
4.3.2 Triphosphate P
α
-O bond stability .......................................................................... 78
4.4 Conclusion .................................................................................................................... 79
4.5 Experimental ................................................................................................................. 80

viii

4.5.1 Materials and methods ......................................................................................... 80
4.5.2 Synthetic procedures ............................................................................................. 82
4.6 Chapter references ....................................................................................................... 88

BIBLIOGRAPHY ......................................................................................................................... 93

APPENDIX A. Chapter 1 Supporting Data ............................................................................. 104
APPENDIX B. Chapter 2 Supporting Data ............................................................................. 136
APPENDIX C. Chapter 3 Supporting Data.............................................................................. 193
APPENDIX D. Chapter 4 Supporting Data ............................................................................. 216

ix

LIST OF TABLES
Table 1.2.1. NMR spectral data for bisphosphonic acids 8-14 (D
2
O, Na
2
CO
3
; pH ~ 10.3). ....... 4

Table 1.2.2. Analytical SAX HPLC retention times for β,γ-CXY dTTP analogs 16-22. ............... 6

Table 2.2.1. pKa values for α-azido bisphosphonic acids 3 and 4 and known
bisphosphonic acids. ............................................................................................................... 24

Table 2.4.1. NMR data (D
2
O, Na
2
CO
3
; pH ~ 10.3) of auxiliary bisphosphonic acids used in
Figure 2.2.1. ............................................................................................................................. 41

Table 4.3.1. Activation parameters for hydrolysis of β,γ-CXY neopentyl triphosphates 1-3
and related phosphate compounds ........................................................................................ 78

Table 4.3.2. Catalytic power of enzymes catalyzing triphosphate hydrolysis at P

............... 78

Table B1. Potentiometric titration data for (1-azidoethane-1,1-diyl)bis(phosphonic
acid), 3. .................................................................................................................................. 157

Table B2. Potentiometric titration data for (azidomethanediyl)bis(phosphonic acid), 4 .... 162

x

LIST OF FIGURES
Figure 1.2.1. Representative analytical SAX HPLC trace of morpholidate coupling reaction.
Shown: reaction of 14 with 15 after 48h.. ................................................................................ 5

Figure 1.2.2.
31
P NMR spectra of the P
α
20a/b resonances A) Before Chelex
TM
treatment,
at neutral pH (162 MHz); B) After Chelex
TM
treatment at pH > 10 (162 MHz); C) After
Chelex
TM
treatment at pH > 10 at higher operating frequency (202 MHz). .............................. 7

Figure 2.1.1. Structures of α -azido bisphosphonate esters and acids. ................................. 18

Figure 2.2.1. Bisphosphonic acid pKa
4
displays a linear correlations with C
α

13
C chemical
shifts depending on the degree of methylation. ..................................................................... 25

Figure 2.2.2. Separation of α,β-CMeN
3
dADP diastereomers (14a/b) by RP semi-
preparative HPLC. .................................................................................................................... 28

Figure 2.2.3. CD spectra of α,β-CMeN
3
dATPs 7a and 7b (25° C, 0.05 mM) in H
2
O, pH 8...... 28

Figure 3.1.1. X-ray crystal structure of the DNA pol  active site containing (S)- , -CHF-
dATP ......................................................................................................................................... 50

Figure 3.2.1. DNA pol  dissociation constants of dATP and , -CXY dATP analogs. ........... 52

Figure 3.2.2. A) The ternary complex crystallographic structures of pol  with an
incoming , -CF
2
-dATP (7) and , -NH-dUTP (PDB ID 2FMS) superimposed using all 326
C  (rmsd = 0.29 Å). B) The ternary complex crystallographic structures of pol  with an
incoming 7 and , -CHF-dATP (6b) superimposed using all 326 C  (rmsd = 0.15 Å). C) A
difference density map generated using the (R)-CHF isomer shows positive and negative
density in the vicinity of the position of the proposed fluorine atom .................................... 54

Figure 3.2.3. Geometries of the lowest energy conformations calculated by docking
dATP analogs with the pol β-DNA binary complex (PDB ID 3TFS). A) 5 displays a good
overlap with the coordinates of the crystallized ligand in the absence of the water bound
to Asp276 (ID water 3). B) 6b overlaps well with the crystallized ligand even in the
presence of the water near Asp276 C) 5 does not achieve good alignment in the
presence of the water 3. ......................................................................................................... 55

Figure 4.2.1. Structure of triphosphate model compounds (1-3) and supplementary P
β
-
CXYPh compounds (4-5).. ........................................................................................................ 69

xi

Figure 4.2.2. Image and schematic of semi-preparative post-column derivatizition HPLC
system ...................................................................................................................................... 71

Figure 4.2.3.
18
O-labeled water hydrolysis experiments demonstrated that the site of
nucleophilic attack for β,γ-CXY neopentyl triphosphates 1-3 is P
β
. ........................................ 72

Figures A1-A4.
1
H-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC data for
thymidine 5’-triphosphate β,γ-CH
2
, 16. ................................................................................. 104

Figures A5-A9.
1
H-NMR,
19
F-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC data
for thymidine 5’-triphosphate β,γ-CF
2
, 17. ............................................................................ 108

Figures A10-A14.
1
H-NMR,
19
F-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC
data for thymidine 5’-triphosphate β,γ-CHF, 18a/b. ............................................................ 113

Figures A15-A18.
1
H-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC data for
thymidine 5’-triphosphate β,γ-CCl
2
, 19. ................................................................................ 118

Figures A19-A24.
1
H-NMR,
31
P-NMR (multiple field strengths and preparations), LRMS
[M-H]
-
, and analytical SAX HPLC data for thymidine 5’-triphosphate β,γ-CHCl, 20a/b. ....... 122

Figures A25-A28.
1
H-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC data for
thymidine 5’-triphosphate β,γ-CBr
2
, 21. ............................................................................... 128

Figures A29-A32.
1
H-NMR,
31
P-NMR, LRMS [M-H]
-
, and analytical SAX HPLC data for
thymidine 5’-triphosphate β,γ-CHBr, 22a/b. ........................................................................ 132

Figures B1-B4.
1
H-NMR,
13
C-NMR,
31
P-NMR, and FTIR spectra of tetraisopropyl (1-
azidoethane-1,1-diyl)bis(phosphonate),1. ............................................................................ 136

Figures B5-B8.
1
H-NMR,
13
C-NMR,
31
P-NMR, and FTIR spectra of tetraisopropyl
(azidomethanediyl)bis(phosphonate), 2. .............................................................................. 140

Figures B9-B14.
1
H-NMR,
13
C-NMR,
31
P-NMR, FTIR, LC-MS PDA, and HRMS data for (1-
azidoethane-1,1-diyl)bis(phosphonic acid), 3. ...................................................................... 144

Figures B15-B20.
1
H-NMR,
13
C-NMR,
31
P-NMR, FTIR, LC-MS PDA, and HRMS data of
(azidomethanediyl)bis(phosphonic acid), 4. ......................................................................... 150

Figure B21. Potentiometric titration curve for (1-azidoethane-1,1-diyl)bis(phosphonic
acid), 3. .................................................................................................................................. 156

xii

Figure B22. Potentiometric titration curve for (azidomethanediyl)bis-
(phosphonic acid), 4 .............................................................................................................. 161

Figure B23. Sample Hyperquad2006 output after refinement calculations. Shown,
(azidomethanediyl) bis(phosphonic acid), 4. ........................................................................ 166

Figures B24-B27.
1
H-NMR,
31
P-NMR (multiple field strengths and enlarged P
β
), and
HRMS data for 2’-deoxyguanosine 5’-triphosphate β,γ-C(CH
3
)N
3
, 5a/b............................... 167

Figures B28-B30.
1
H-NMR,
31
P-NMR, and HRMS data for 2’-deoxyguanosine 5’-
triphosphate β,γ-CHN
3
, 6a/b. ................................................................................................ 171

Figures B31-B32.
1
H-NMR and
31
P-NMR spectra of 2’-deoxyadenosine 5’-triphosphate
α,β-C(CH
3
)N
3
, 7a. ................................................................................................................... 174

Figures B33-B34.
1
H-NMR and
31
P-NMR spectra of 2’-deoxyadenosine 5’-triphosphate
α,β-C(CH
3
)N
3
, 7b .................................................................................................................... 176

Figure B35 UV/VIS spectra of 2’-deoxyadenosine 5’-triphosphate α,β-C(CH
3
)N
3
7a and
7b in H
2
O, pH 8. Est. conc. ~0.05 mM (E = 15300). .............................................................. 178

Figures B36-B37.
1
H NMR and
31
P-NMR spectra of 2’-deoxyadenosine 5’-triphosphate
α,β-CHN
3
, 8a/b. ..................................................................................................................... 179

Figures B38-B44.
1
H NMR, gCOSY 2-D NMR,
13
C-NMR, gHSQC(AD) 2-D NMR,
31
P-NMR,
FTIR, and LRMS [M+Na]
+
data for tetraisopropyl (1-aminoethane-1,1-diyl)bis
(phosphonate), 16 ................................................................................................................. 181

Figures B45-B49.
1
H-NMR,
13
C-NMR, gHSQC(AD) 2-D NMR,
31
P-NMR, and LRMS [M+H]
+

and [M+Na]
+
data for diisopropyl (1-([bis(propan-2-
yloxy)phosphoryl]amino)ethenyl)phosphonate, 18. ............................................................. 188

Figure C1.
31
P NMR spectrum of α,β-C(Me)
2
dADP, 3. ......................................................... 193

Figures C2-C5.
31
P NMR and
19
F-NMR (with simulation) spectra of α,β-CHF-dADP, 4a/b. .. 194

Figure C6. Representative HPLC trace of enzymatic phorsphorylation reaction mixture. 198

Figures C7-C11.
1
H-NMR, gCOSY 2-D NMR,
31
P-NMR, anayltical SAX HPLC, and HRMS
[M-H]
-
data for α,β-C(Me)
2
-dATP, 5. ..................................................................................... 199

xiii

Figures C12-C20.
1
H-NMR, gCOSY 2-D NMR,
19
F-NMR (different field strengths and with
simulation),
31
P-NMR (different field strengths), analytical SAX HPLC, and LRMS [M-H]
-

data for α,β-CHF-dATP, 6a/b. ................................................................................................ 204

Figure C21. Gel analysis of DNA synthesis background of dual-pass HPLC purified 8a/b
(2 ′-deoxyadenosine 5 ′-diphosphate, α,β-CHN
3
), 9a and 9b (2 ′-deoxyadenosine 5’-
diphosphate, α,β-C(CH
3
)N
3
) ................................................................................................... 213

Figure C22. Inhibition of dATP incorporation by 8a/b (α,β-CHN
3
-dATP), 9a
(α,β-C(CH
3
)N
3
-dATP 1), and 9b (α,β-C(CH
3
)N
3
-dATP 2). ........................................................ 214

Figure C23. Analysis of dATP incorporation inhibition by α,β-CHN
3
-dATP 8a/b ................. 215

Figures D1-D3.
1
H-NMR,
31
P-NMR, and HRMS [M-H]
-
data for 2,2-dimethylpropyl β,γ-
methylene-triphosphate, 1.................................................................................................... 216

Figures D4-D7.
1
H-NMR,
19
F-NMR,
31
P-NMR, and HRMS [M-H]
-
data for 2,2-
dimethylpropyl β,γ-(fluoro)methylene-triphosphate, 2. ...................................................... 219

Figures D8-D11.
1
H-NMR,
19
F-NMR,
31
P-NMR, and HRMS [M-H]
-
data for 2,2-
dimethylpropyl β,γ-(difluoro)methylene-triphosphate, 3. ................................................... 223

Figures D12-D15.
1
H-NMR,
13
C-NMR,
31
P-NMR, and LRMS [M-H]
-
data for
benzylphosphonic (2,2-dimethylpropyl phosphoric) anhydride, 4. ...................................... 227

Figures D16-D19.
1
H-NMR,
19
F-NMR,
31
P-NMR, and LRMS [M-H]
-
data for
[difluoro(phenyl)methyl]phosphonic (2,2-dimethylpropyl phosphoric) anhydride, 5. ........ 231

Figure D20. Calculated bond lengths for β,γ-CXY neopentyl triphosphates 1-3. ................. 235

Figure D21. Calculated bond lengths for P
β
-CXYPh model compounds 4 and 5. ................. 236

Figure D22. Sample output from Spartan ’08 ab initio calculations of P
β
-CF
2
Ph model
compound 5. .......................................................................................................................... 237

xiv

LIST OF SCHEMES
Scheme 1.2.1. Synthesis of halogenated β,γ-dTTP analogs 16-22. .......................................... 3

Scheme 2.2.1. Synthesis of tetraisopropyl (1-azidoethane-1,1-diyl)bis(phosphonate), 1..... 19

Scheme 2.2.2. Synthesis of tetraisopropyl (azidomethanediyl)bis(phosphonate) (2)
showing crude yields as determined by
31
P NMR. .................................................................. 20

Scheme 2.2.3. Base induced decomposition of tetraisopropyl
(azidomethanediyl)bis(phosphonate) (2). ............................................................................... 21

Scheme 2.2.4. Synthesis of α,β-CXN
3
(7a, 7b, 8a/b) and β,γ-CXN
3
(5a/b, 6a/b) nucleotide
analogs ..................................................................................................................................... 26

Scheme 2.2.5. Reduction of (1-azidoethane-1,1-diyl)bis(phosphonate), 1. .......................... 30

Scheme 2.2.6. UV photolysis of (1-azidoethane-1,1-diyl)bis(phosphonate), 1. .................... 31

Scheme 3.2.1. Synthesis of α,β-CXY-dATP analogs 5 and 6a/b. ............................................ 51

Scheme 4.2.1. Synthesis of P
β
-CXYPh model compounds 4 and 5. ........................................ 75

Scheme 4.2.2. The products observed in the
31
P NMR resulting from the hydrolysis of 5.
These products demonstrate that the P-C bond of 5 is unstable under the hydrolysis
conditions. ............................................................................................................................... 75

xv

ABSTRACT
A variety of triphosphate analogs that replace a natural phosphate anhydride P-O-P
linkage with a phosphonate P-CXY-P moiety have been synthesized for the characterization
of DNA polymerase β (pol β) mechanism and structure. A suite of β,γ-CXY-dTTP compounds
(X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of
β,γ-CXY-dGTP analogs. The high purity of the β,γ-CXY-dTTP following dual-pass HPLC
purification is demonstrated by NMR, MS, and HPLC data and illustrates the merit of the
DCC-mediated morpholidate coupling approach to P
β
-CXY-P
γ
triphosphate mimics.
Novel α-azido bisphosphonates [(RO)
2
P(O)]
2
CXN
3
(R = i-Pr, X = Me; R = i-Pr, X = H; R =
H, X = Me; and R = H, X = H) were developed to examine the impact of the unique azido
steric profile on the synthesis and bioactivity of α,β-CXN
3
and β,γ-CXN
3
dNTP analogs. For
one example, α,β-CMeN
3
-dATP, RP HPLC separated dADP precursors that were used to
generate the first examples of nucleoside triphosphate analogs with isolated
stereochemistry at an asymmetrically substituted bridging carbon. The acidity constants of
the α-azido bisphosphonic acids were determined by potentiometric titration and selected
reactions including reduction and UV photolysis are introduced.
The α,β-C(Me)
2
-dATP and (R/S)-α,β-CHF-dATP analogs were synthesized and,
together with the α,β-CXN
3
dATP analogs, the binding affinities (K
d
) with pol β were
determined. (R/S)-α,β-CHF-dATP was crystallized with a DNA-pol β binary complex. X-ray
structure analysis revealed only the (S)-α,β-CFH-dATP diastereomer was present in the
enzyme active site. Analysis of this structure provides evidence for a non-covalent

xvi

stabilizing interaction between an active site water bound to Asp276 and the fluorine in (S)-
α,β-CFH-dATP. Molecular docking studies further demonstrate the importance of this
structural water by suggesting a steric clash with the methyl group of α,β-C(Me)
2
-dATP.
A series of novel β,γ-CH
2
, -CHF, and -CF
2
bisphosphonophosphate alkyl monoesters
were synthesized and used as model compounds intended to estimate the activation
parameters for the non-enzymatic hydrolysis of the triphosphate P
α
-O-P
β
moiety.
18
O-
labeled water experiments showed that the exclusive site of nucleophilic attack in these
systems is at P
β
. Thus, the catalytic efficiency for a small class of diphosphokinases could be
approximated and the upper limit for the rate of non-enzymatic hydrolysis at P
α
established.
Supplementary compounds that increase P
β
-O bond stability by substituting P
γ
with a phenyl
ring are introduced. Preliminary hydrolysis experiments of these compounds highlight the
relative stability of the P
α
-O anhydride bond in triphosphates.

1

CHAPTER 1. Synthesis of a Series of Halogenated Thymidine
5’-Triphosphate β,γ-Phosphonate dNTP Analogs

1.1 Introduction
2’-Deoxynucleoside 5’-triphosphate (dNTP) analogs in which the natural P
β
-O-P
γ

bridging oxygen is replaced by a non-hydrolysable bisphosphonate motif are of continuing
interest as probes of DNA polymerase mechanism and fidelity.
1-3
These compounds, while
mimicking native dNTPs, are resistant to dephosphorylation, offer increased specificity for
certain enzymes, may reveal novel active site binding interactions, and for polymerases,
allow modulation of leaving group aptitude.
2
The use of β,γ-CXY dGTP bisphosphonate
analogs as probes of polymerase β (pol β) mechanism and fidelity was recently reported.
3,4

By systematically changing the X,Y-substitution, the bisphosphonate leaving group aptitude
could be adjusted to assess whether P-O bond cleavage was rate-determining in the overall
nucleotidyl transfer reaction mechanism of pol β. In support of a rate-determining chemical
step, a negative correlation was revealed by plots of log(k
pol
) vs the bisphosphonate leaving
group pKa
4
.
3,4
Unexpectedly, when the incoming dGTP bisphosphonate was mispaired (dT
instead of dC in the template), the linear free energy relationship (LFER) data for analogs
incorporating a dihalogenated bisphosphonate correlated to a different trend line than the
other analogs tested and indicated a leaving group effect on fidelity.
3

In 2009, Kamerlin and co-workers published a computational study that examined
whether this “di-halo effect” observed in the incorporation of β,γ-CXY dGTP analogs by pol β

2

was a general feature of β,γ-CXY triphosphate hydrolysis that would also be observed in the
uncatalyzed solution reactions.
5
Using model compounds that replaced the nucleoside with
a methyl group, they approximated the partitioning observed in the base mismatch reaction
when the simulation was performed with an aqueous solution model, but found a single
trend line, similar to what was observed in the base match reaction, when hydrolysis was
modeled in the gas phase. These results indicated that differences in the solvation energies
of the analogs when bound in the active site of pol β were the source of the larger di-halo
effect observed in mismatch incorporation, and thus implied that base-pair mismatch
results in an enzyme binding-pocket that is more “open” than the correct pair complex and
thereby more resembles bulk solvent.
The influence of the incoming dNTP base structure remains to be investigated in
biochemical experiments, prompting expansion of the nucleotide probe “toolkit” to include
examples of pyrimidine-based analogs. Here detailed synthetic procedures and
characterization data for the β,γ-CXY (X, Y = H, F, Cl, Br) analogs of 2’-deoxythymidine 5’-
triphosphate (dTTP) (16-22) are provided. This pyrimidine dNTP toolkit could test whether
the split LFER observed in earlier biochemical studies
3
and computations
5
is indeed a
general feature of β,γ-CXY triphosphate hydrolysis.

1.2 Results and discussion
A few examples of β,γ-CXY dTTP derivatives were prepared in the late 1990's in
connection with AZT triphosphate studies.
6-8
Although full synthetic procedures were not
given, the authors reported using 1,1'-carbonyldiimidazole activation of thymidine 5'-

3

monophosphate
9
(dTMP) to prepare the CF
2
(17), CHF (18), CFMe, and CBr
2
(21) analogs
with yields of 11-47%.
8,9
Compounds 17 and 18a/b were characterized solely by
31
P NMR
8

while 21 was characterized by
1
H and
31
P NMR
9
alone, with no indication of compound
purity. Our preferred synthetic approach, as presented here, is to use DCC-mediated
morpholidate coupling
10,11
of the commercially available dTMP with the tributylammonium
salt of the requisite bisphosphonic acid 8-14
12
(Scheme 1.2.1). Though this method requires
relatively long reaction times (up to 48 h) and furnishes the desired products in modest
isolated yields, it avoids the generation of difficult to remove side-products. As was the
case for the dGTP series,
3,4
dual-pass (SAX then RP-18) HPLC provided the dTTP analogs 16–
22 in high purity, free of detectable contaminating nucleotides, making them suitable for
reliable kinetics studies of polymerases.
Scheme 1.2.1. Synthesis of halogenated β,γ-dTTP analogs 16-22.
NH
N
O
OH
O P HO
OH
O
O P N
O
O
O
morpholine,
DCC
t-BuOH:
H
2
O
P
O
i-PrO P
O
Oi-Pr
i-PrO Oi-Pr
P
O
i-PrO P
O
Oi-Pr
i-PrO Oi-Pr
NH
O
O N
O
OH
O P O
O
O
P
O
P
O
HO
O O
8, 16: X, Y = H
2, 9, 17: X, Y = F
3, 10, 18a/b: X, Y = H, F
4, 11, 19: X, Y = Cl
5, 12, 20a/b: X, Y = H, Cl
6, 13, 21: X, Y = Br
7, 14, 22a/b: X, Y = H, Br
a
a) 2, 3: NaH, Selectfluor/ THF:DMF, -42
o
C; 4: NaOCl, 0
o
C; 5: (from 4) Na
2
SO
3
/ H
2
O: EtOH, 0
o
C; 6: Br
2
,
NaOH/ H
2
O, 0
o
C; 7: (from 6): SnCl
2
/ H
2
O: EtOH, 0
o
C. b) for 1-5, HCl reflux; for 6 and 7, HBr, reflux. c)
Bu
3
N/ H
2
O: EtOH
16-22
O
O
1
2-7
15
8-14
b
c
X Y
P
O
HO P
O
OH
HO OH
X Y
X Y

The bisphosphonate tetraester precursors 2-7 were prepared by halogenation of
tetraisopropyl methylenebis(phosphonate), 1.
13,14
Electrophilic fluorination of the
carbanion of 1 was performed with Selectfluor
TM
under conditions that provided useable
amounts of both the mono- and difluorinated esters, 2 and 3, in a single reaction.
14
Esters

4

4-7 were synthesized following known procedures.
13
Briefly, the dichloro- and
dibromomethylenebisphosphonate esters 4 and 6 were obtained in quantitative yield
without the need for chromatographic purification, by oxidation of 1 with the corresponding
hypohalite reagent.
13
Esters 4 and 6 were reduced to the monohalogenated derivatives 5
and 7 in ~97% yield as determined by
31
P NMR.
13
In the synthesis of 6, maintaining the
temperature below 0 °C and adding the bromine slowly
13,15
are essential for successful
hypobromite formation.
Esters 1-7 were hydrolyzed in refluxing HCl (or HBr in the case of 6 and 7) to cleanly
provide the tetraacids 8-14. Published NMR spectral data for the halogenated
bisphosphonic acids 9-14 are either incomplete or for differing salt forms.
14,16-19

1
H,
13
C,
19
F,
and
31
P NMR data for 8-14, acquired under self-consistent solution conditions (Table 1.2.1)
are compiled here.
Table 1.2.1. NMR spectral data for bisphosphonic acids 8-14 (D
2
O, Na
2
CO
3
; pH ~ 10.3, J = Hz). Literature
values are either incomplete or for differing salt forms.
14,16-19

1
H (499.8 MHz)
13
C (125.7 MHz)
19
F (470.2
MHz)
31
P (202.3 MHz)
8 (CH
2
) 1.86 (t,
2
J
HP
19.2) 34.8 (t,
1
J
CP
117.8) - 16.6
9 (CF
2
)
-
127.4 (tt,
1
J
CF
271.3,
1
J
CP
156.9)
-116.8 (t,
2
J
FP

76.8)
7.4 (t,
2
J
PF
76.8)
10 (CHF)
4.53 (dt,
2
J
HF
45.3,
2
J
HP
11.4)
98.0 (dt,
1
J
CF
170.5,
1
J
CP
135.7)
-216.8 (dt,
2
J
FP

57.6,
2
J
FH
45.7)
11.5 (d,
2
J
PF

57.5)
11 (CCl
2
) - 88.5 (t,
1
J
CP
119.0) - 12.1
12 (CHCl)
3.60 (t,
2
J
HP
14.8) 56.5 (t,
1
J
CP
121.7) - 13.0
13 (CBr
2
) - 72.4 (t,
1
J
CP
108.2) - 11.7
14 (CHBr) 3.51 (t,
2
J
HP
13.9) 48.8 (t,
1
J
CP
117.8) - 12.7

5

2’-Deoxythymidine 5’-phosphomorpholidate 15 was prepared from the acid form of
the commercially available dTMP by DCC activation.
10,11
The reaction proceeded with better
than 90% conversion (as monitored by
31
P NMR) and required minimal purification before
advancing to the coupling step. To prepare the dTTP analogs 16-22, the tri-n-
butylammonium salts of the corresponding bisphosphonic acids were reacted with 15 in
anhydrous DMSO at room temperature.
20
The reaction was conveniently monitored by
analytical SAX HPLC with the starting material, dTMP side-product, and desired triphosphate
all being well separated in the chromatogram (Figure 1.2.1). After 48 h, a typical reaction
was approximately 60% complete with < 10% thymidine 5’-monophosphate formed. The
desired products were purified by dual-pass HPLC with an SAX column eluted with a 0-0.5 N
TEAB gradient, followed by passage through a RP-C18 column eluted with 0.1 N TEAB 4%
acetonitrile buffer. As seen in Table 1.2.2, nucleotide analogs incorporating a more acidic
bisphosphonate moiety
3
have longer retention on the SAX HPLC column. The final products
16-22 were obtained on milligram scale as triethylammonium salts in ~20% yield,
determined by UV absorbance at λ
267
(ε= 9600).
21

Figure 1.2.1. Representative analytical SAX HPLC trace of morpholidate coupling reaction. Shown:
reaction of 14 with 15 after 48h. Peak 1 = 15, peak 2 = TMP, peak 3 = 22a/b. Conditions: Pump A: H
2
O;
pump B: 0.5 M (0-50% linear) LiCl gradient over 30 min; flow rate: 4 mL/min.

6

Table 1.2.2. Analytical SAX HPLC retention times for β,γ-CXY dTTP analogs 16-22.

Compound RT (min)
16 (CH
2
) 9.1
22 (CHBr) 9.4
20 (CHCl) 9.8
21 (CBr
2
) 9.8
29 (CCl
2
) 9.9
28 (CHF) 10.5
17 (CF
2
) 11.8

By analytical SAX HPLC analysis, the target compounds account for >99% of the
detected UV absorbance (Appendix A). The
31
P NMR spectra are free of any significant
monophosphate or other nucleotide side product signals and the
1
H NMR spectra show a
clean aromatic region, demonstrating the integrity of the nucleobase (Appendix A). As was
the case for dGTP-β,γ-CXN
3
analogs,
22
under appropriate conditions the
31
P NMR resonances
of dTTP β,γ-CXY diastereomers resulting from introduction of a pro-chiral bisphosphonate
(X ≠ Y) can be resolved. For example, Chelex
TM
treatment of 20a/b followed by addition of
Na
2
CO
3
(raising the pH to ~10), narrows the line width of the resonances, revealing two
doublets for P
α
and two doublets of doublets for P
β
(Figure 1.2.2; Appendix A, figures A20-
A22). As a result, the relative concentrations of the individual stereoisomers can be
monitored in solution. The δ and J assignments were unambiguously confirmed by
comparing spectra acquired at different operating frequencies.

7

Figure 1.2.2.
31
P NMR spectra of the P
α
20a/b resonances A) Before Chelex
TM
treatment, at neutral pH
(162 MHz); B) After Chelex
TM
treatment at pH > 10 (162 MHz); C) After Chelex
TM
treatment at pH > 10 at
higher operating frequency (202 MHz).

1.3 Conclusion
DCC-mediated morpholidate coupling has been used to synthesize 7 β,γ-CXY-dTTP
analogs that span a large range of leaving group capacities. As was the case for the
previously described β,γ-CXY-dGTP analogs, this methodology afforded compounds that
were free from detectable amounts of contaminating nucleotides or phosphorus containing
compounds after dual-pass HPLC purification. These compounds constitute a pyrimidine-
based tool-kit useful for the study of DNA polymerase β mechanism. It will be interesting to
see if the LFER plots observed with the β,γ-CXY-dGTP tool-kit are a general feature of pol β
catalysis or if the individual nucleobases are incorporated through mechanisms that vary in
sensitivity to leaving group effects.

8

1.4 Experimental
1.4.1 Material and methods
Reagents were obtained from Sigma-Aldrich, Inc. RP HPLC purification was
conducted using a Varian 21.4 mm x 250 mm MICROSORB 100-5 C-18 column eluted
isocratically with 0.1 N triethylammonium bicarbonate (TEAB) pH 7.5 buffer containing 4%
acetonitrile pumped at 10 mL/min. Strong anion exchange (SAX) HPLC chromatography was
performed using a Macherey-Nagel 21.4 mm x 250 mm SP15/25 Nucleogel column eluted
with A: H
2
O, B: 0.5 N TEAB pH 7.5 using a gradient that was increased from 0-40% over 10
min, maintained at 40% from 10-15 min, and then increased to 100% from 15-25 min using
an 8 mL/min flow rate. Analytical HPLC analysis was conducted on a Varian PureGel SAX 10
mm x 100 mm 7 μL column eluted with an A: H
2
O, B: 0.5 M (0-50% linear) LiCl gradient over
30 min at a 4 mL/min flow rate. The final products, obtained as the triethylammonium salts,
were quantitated by UV absorbance using the extinction coefficient (λ
267
, ε= 9600) of the
natural dTTP compound at pH 7.
21

NMR operating frequencies are listed with their respective assignments.
1
H NMR
spectra were referenced to residual CHCl
3
(δ 7.24) in CDCl
3
or to HDO (δ 4.79) in D
2
O.
23

13
C
NMR spectra were referenced to internal CO
3
2-
(δ 168.9).
23

31
P NMR spectra are proton
decoupled and referenced to external 85% H
3
PO
4
(δ 0.00).
19
F NMR spectra were
referenced to external hexafluorobenzene, C
6
F
6
in benzene (δ -164.9). All chemical shift
values (δ) are reported in ppm. All J values are reported in Hz. NMR sample pH values were
measured in 99.9% D
2
O and are reported without deuterium isotope correction. Low

9

resolution mass spectra were obtained with a Thermo-Finnagan Deca XP Plus mass
spectrometer using the ESI probe.
1
H-NMR spectra (16-22),
19
F-NMR spectra (17 and 18),
31
P-NMR spectra (16-22), LR
mass spectra (16-22), and analytical HPLC chromatograms (16-22) are presented in
Appendix A. The
31
P-NMR spectra of 20a/b are presented at a variety of field strengths and
sample preparations.

1.4.2 Synthesis of 2’-deoxythymidine 5’-triphosphate β,γ-CXY analogs
Tetraisopropyl (difluoromethylene)bis(phosphonate), 2, and Tetraisopropyl
(fluoromethylene)bis(phosphonate), 3. NaH (0.52 g as a 60% oil immersion, 13 mmol) was
added to 15 mL of dry THF/ dry DMF 50:50 and stirred at 0°C. 1 (2.00 g, 5.8 mmol) was
added and stirred for 10 min. Next, Selectfluor
TM
(3.08 g, 8.7 mmol) was added to the
reaction mixture which was stirred for 30 min. A second 3.08 g portion of Selectfluor
TM
was
added and the reaction mixture was brought to rt and stirred for an additional hour. NH
4
Cl
(35 mL) was added to the reaction mixture, which was then extracted with EtOAc (3 x 50 mL)
to give a mixture of compounds with 1, 2, and 3 in a ratio of 13: 39: 48 upon concentration.
Purification by silica gel chromatography afforded 662 mg (30%) of 2 and 756 mg (36%) of 3.
2: δ
H
(CDCl
3
, 500 MHz): 1.32 (m, 24H), 4.81 (m, 4H); δ
F
(CDCl
3
, 470 MHz): -124.1 (t, J
FP
87.2);
δ
P
(CDCl
3
, 202 MHz): 2.7 (t, J
PF
87.3). Lit
14
: δ
H
(CDCl
3
): 1.40 (d), 4.93 (m); δ
F
(neat): -121 (t, J
FH

85); δ
P
(neat): 2.80 (d, J
PF
85); 3: δ
H
(600 MHz, CDCl
3
): 1.33 (m, 24H), 4.82 (m, 5H); δ
F
(564
MHz, CDCl
3
): -228.5 (dd, J
FP
65, J
FH
45); δ
P
(243 MHz, CDCl
3
): 10.1 (d, J
PF
65). Lit
14
: δ
H
(CDCl
3
):
1.26 (d), 4.77 (m), 4.82 (dt); δ
F
(neat): - 221 (dt, J 63, J 44); δ
P
(neat): 10.7 (d, J 63).

10

Tetraisopropyl (dichloromethylene)bis(phosphonate), 4; Tetraisopropyl
(chloromethylene)bis(phosphonate), 5; Tetraisopropyl (dibromomethylene)-
bis(phosphonate), 6; Tetraisopropyl (bromomethylene)bis(phosphonate), 7. These
compounds were prepared by literature procedures with characterization data (
1
H,
31
P NMR)
in agreement with reported values.
13
In the synthesis of 6, maintaining a temperature
below 0°C is essential for successful sodium hypobromite formation. In the synthesis of 5,
31
P NMR analysis showed >99% purity and the compound was used without further
purification. After extraction and concentration,
31
P NMR analysis indicated that 7 was >95%
pure. Purification by column chromatography on silica gel eluted with ACE: DCM (2:3)
provided the final compound (90%).
General procedure for synthesis of bisphosphonic acids 8-14. Approximately 500
mg of corresponding ester 1-5 was dissolved in ~3 mL of conc. HCl and refluxed for 4 - 8 h.
Solvent was then removed under reduced pressure with rotovap and the remaining residue
repeatedly dissolved in water, which was removed under reduced pressure with heating.
The resulting oily white solid was dissolved in 4 mL of EtOH and H
2
O (1:1) and stirred at rt.
Tri-n-butylamine (1/4 equiv based on weight of starting ester) was added. The solvent was
removed under reduced pressure with rotovap and the remaining oil co-evaporated with
anhydrous DMF several times. In preparation of the acids 13 and 14 conc. HBr was used
instead of conc. HCl. For self-consistency, the NMR data of 9-14 are reported for solutions
with the pH adjusted to ~10.3 (sodium carbonate) (Table 1.2.1).
2’-Deoxythymidine 5’-phosphoromorpholidate, 15. Procedure adapted from the
original synthesis of nucleoside 5’-phosphoromorpholidates.
10,11
Thymidine 5’-

11

monophosphate, acid form (200 mg, 0.621 mmol), was dissolved in 7 mL t-BuOH: H
2
O 1:1.
Morpholine (162 mg, 1.86 mmol) was added to the solution which was set to reflux. DCC
(384 mg, 1.86 mmol) was dissolved in 4 mL t-BuOH and 1/8 of this solution was added drop-
wise to the reaction mixture every 15 min for 2 h. After 2.5 h, the reaction was complete
(single peak in the
31
P NMR spectrum). The solvent was then removed and the resulting oil
dissolved in water. The solution was filtered and water was removed under reduced
pressure to yield 15 as an amorphous yellow solid which was used without further
purification. δ
P
(D
2
O, pH neutral, 162 MHz): 8.1 (s).
General coupling procedure. As previously reported for β,γ-CXY dGTP
analogs,
3,4,12,20
15 (1 equiv, ~100 mg) was dissolved in 2 mL anhydrous DMSO. Tri-n-butyl
ammonium bisphosphonic acid (3 equiv) was dissolved in anhydrous DMSO and added by
syringe at rt. After 48 h, volatiles were removed on the rotovap and the reaction mixture
was dissolved in 0.5 N TEAB buffer, pH 7.5. The solution was then purified by dual-pass
preparative HPLC (SAX then RP) to yield the final compounds (~20%). Analytical SAX HPLC
analysis demonstrated that purity was ≥99% (UV detection; Appendix A). Retention times
for 16-22 are presented in Table 1.2.2.
2’-Deoxythymidine 5’-triphosphate β,γ-CH
2
, 16. δ
H
(D
2
O, pH 8, 400 MHz): 1.92 (s,
3H), 2.21 (2H, t, J
HP
20.0), 2.30-2.42 (2H), 4.13-4.21 (3H), 4.62-4.65 (1H), 6.34 (1H, t, J 7.2),
7.74 (d, 1H); δ
P
(D
2
O, pH 9.5, 202 MHz): -10.6 (d, J
 
26.9, P

), 12.1, (dd, J
γ
7.5, J
 
27.0, P
β
),
12.9 (d, J
γ 
7.9, P
γ
); MS [M-H]
-
: calcd for C
12
H
20
N
2
O
13
P
3
: 479.0. Found: 479.0 m/z.
2’-Deoxythymidine 5’-triphosphate β,γ-CF
2
, 17. δ
H
(D
2
O, pH 8, 400 MHz): 1.88 (s,
3H), 2.27-2.38 (2H), 4.13-4.22 (3H), 4.60-4.63 (1H), 6.32 (1H, t, J 7.2), 7.70 (d, 1H); δ
F
(D
2
O,

12

pH 8, 376 MHz): -119 (dd, J
FP1
90.4, J
FP2
75.3); δ
P
(D
2
O, pH 8, 162 MHz): -10.4 (d, J
 
32, P

),
-2.0 (m, P

), 4.2 (dt, J
γβ
56, J
PF
71, P
γ
); MS [M-H]
-
: calcd for C
11
H
17
F
2
N
2
O
13
P
3
:

515.0. Found:
514.9. Lit
8
(pH not reported): δ
P
: -10.2 (d, J 30.1), -3.1 m, 4.3 (dt, J 66.0).
2’-Deoxythymidine 5’-triphosphate β,γ-CHF, 18a/b. δ
H
(D
2
O, pH 8, 400 MHz): 1.88
(s, 3H), 2.25-2.37 (2H), 4.11-4.20 (3H), 4.61 (1H), 6.33 (1H, J 7.2), 7.68 (1H); δ
F
(D
2
O, pH 8,
376 MHz): -219 (bs); δ
P
(D
2
O, pH 8, 162 MHz): -10.5 (d, J
αβ
29, P

), 5.1 (m, P

), 7.9 (dd, J
βγ

11.9, J
PF
54.3, P
γ
); MS [M-H]
-
: calcd for C
11
H
17
FN
2
O
13
P
3
: 497.0. Found: 497.0. Lit
8
(pH not
reported): δ
P
: -10.3 (d, 27.3), 2.64 m, 8.9 (dt, J 60.2).
2’-Deoxythymidine 5’-triphosphate β,γ-CCl
2
, 19. δ
H
(D
2
O, pH 8, 400 MHz): 1.93 (s,
3H), 2.30-2.43 (2H), 4.17-4.25 (2H), 4.31-4.36 (1H), 4.71-4.75 (1H), 6.36 (1H, t, J 7.2), 7.77 (d,
1H); δ
P
(D
2
O, pH 8, 162 MHz): -10.4 (d, J
 
32, P

), 2.3 (dd, J
 
32, J
βγ
17, P

), 8.5 (d, J
βγ
17, P
γ
);
MS [M-H]
-
: calcd for C
11
H
16
Cl
2
N
2
O
13
P
3
: 546.9 (100%), 548.9 (64%). Found: 547.0 (100%),
549.0 (63%).
2’-Deoxythymidine 5’-triphosphate β,γ-CHCl, 20a/b. δ
H
(D
2
O, pH 8, 400 MHz): 1.92
(s, 3H), 2.33-2.37 (2H), 3.92 (2 dd, 1H), 4.17-4.28 (3H), 4.65-4.69 (1H), 6.39 (1H, t, J 7.2), 7.68
(1H, 2 d); δ
P
(D
2
O, 162 MHz, pH 8): -10.3 (d, J
 
26, P

), 8.9 (m, P

), 10.1 (bs, P
γ
); MS [M-H]
-
:
calcd for C
11
H
17
ClN
2
O
13
P
3
: 513.0 (100%), 515.0 (32%). Found: 513.0 (100%), 515.0 (33%).
After Chelex
TM
treatment: δ
P
(D
2
O, pH 10.3, 202 MHz): -10.63 (d, J
αβ
28.3, P
,
), -10.68 (d, J
αβ

28.3. P
,
), 6.9 (dd, J
αβ
28.3, J
βγ
6.2, P
β
), 8.9 (d, J
βγ
6.2, P
γ
).
2’-Deoxythymidine 5’-triphosphate β,γ-CBr
2,
21. δ
H
(D
2
O, pH 8, 400 MHz): 1.91 (s,
3H), 2.30-2.40 (2H), 4.16 (s, 1H), 4.21-4.26 (1H), 4.34-4.40 (1H), 4.72-4.75 (1H), 6.39 (1H),

13

7.68 (1H); δ
P
(D
2
O, 162 MHz, pH 8): -10.5 (d, J
 
32, P

), 2.1 (dd, J
 
32, J
βγ
13, P

), 8.0 (d, J
βγ

13, P
γ
); MS [M-H]
-
: calcd for C
11
H
16
Br
2
N
2
O
13
P
3
: 636.8 (100%), 634.8 (51%), 638.8 (49%).
Found: 636.8 (100%), 634.9 (51%), 638.8 (52%). Lit
9
(pH not reported): δ
H
(D
2
O): 7.63 (1H, q,
J
6,5-Me
1, H-6), 6.23 (1H, t, J
1’-2’
7, H-1’), 4.56 (1H, m, H-3’), 4.11 (3H, m, H-4’, 5’), 2.26 (2H, m,
H-2’), 1.81 (3H, d, 5-CH
3
); δ
P
: -11.0 (d, P
α
), -0.9 (dd, J
βα
24), 7.4 (d, J
γβ
14, P
γ
).
2’-Deoxythymidine 5’-triphosphate β,γ-CHBr, 22a/b. δ
H
(D
2
O, pH 8, 400 MHz):
1.93 (s, 3H), 2.30-2.43 (2H), 3.93 (1H, t, J
HP
15.6), 4.18-4.26 (3H), 4.65-4.68 (1H), 6.35 (1H, t, J
7.2), 7.77 (1H, 2 doublets); δ
P
(D
2
O, 162 MHz, pH 8): -10.8 (d, J
 
26, P

), -10.9 (d, J
 
26, P

),
5.9 (d, J
 
26, P

), 8.4 (bs, P
γ
); MS [M-H]
-
: calcd for C
11
H
17
BrN
2
O
13
P
3
: 556.9 (100%), 558.9
(97%). Found: 556.9 (100%), 558.9 (96%).

14

1.5 Chapter references
(1) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.;
Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-Fluoromethylene-dGTP-DNA Ternary
Complex with DNA Polymerase β, J. Am. Chem. Soc. 2007, 129, 15412-13.
(2) McKenna, C. E.; Kashemirov, B. A.; Peterson, L. W.; Goodman, M. F. Modifications to
the dNTP triphosphate moiety: From mechanistic probes for DNA pol to antiviral and anti-
cancer drug design, Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 1223-30.
(3) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA Polymerase β
Fidelity: Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis
Reveal Differences at the Chemical Transition State, Biochemistry 2008, 47, 870-79.
(4) 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. Modifying the β,γ LG Bridging -O- Alters Nucleotide Incorporation Efficiency,
Fidelity, and the Catalytic Mechanism of DNA Pol β, Biochemistry 2007, 46, 461-71.
(5) Kamerlin, S. C. L.; McKenna, C. E.; Goondman, M. F.; Warshel, A. A Computational
Study of the Hydrolysis of dGTP Analogues with Halomethylene-Modified Leaving Groups in
Solution: Implications for the Mechanism of DNA Pol, Biochemistry 2009, 48, 5963-71.
(6) Viktorova, L. S.; Arzumanov, A. A.; Shirikova, E. A.; Yas'ko, M. V.; Aleksandrova, L. A.;
Shipitsyn, A. V.; Skoblov, A. Y.; Krayevsky, A. A. Modified nucleoside 5'-triphosphates with
elevated stability against dephosphorylating enzymes, Mol. Biol. (Mosk) 1998, 32, 162-71.
(7) Victorova, L. S.; Semizarov, D. G.; Shirokova, E. A.; Alexandrova, L. A.; Arzumanov, A.
A.; Jasko, M. V.; Krayevsky, A. A. Human DNA polymerases and retroviral reverse
transcriptases: selectivity in respect to dNTPs modified at triphosphate residues,
Nucleosides Nucleotides 1999, 18, 1031-32.
(8) Martynov, B. I.; Shirokova, E. A.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A. A. Effect
of triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity
towards various DNA polymerases, FEBS Lett. 1997, 410, 423-27.

15

(9) Alexandrova, L. A.; Skoblov, A. Y.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A. A. 2-
Deoxynucleoside 5'-triphosphates modified at α-, β- and γ-phosphates as substrates for
DNA polymerases, Nucleic Acids Res. 1998, 26, 778-86.
(10) Moffatt, J. G.; Khorana, H. G. Nucleoside polyphosphates. X. The synthesis of
nucleoside 5'-phosphoromorpholidates and related compounds. Improved methods for the
preparation of nucleoside 5'-polyphosphates, J. Am. Chem. Soc. 1961, 83, 649-58.
(11) Moffatt, J. G. General synthesis of nucleoside 5'-triphosphates, Can. J. Chem. 1964,
42, 599-604.
(12) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T.
G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA
Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding
of the Fluoromethylene Analogues, J. Am. Chem. Soc. 2010, 132, 7617-25.
(13) McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y.; Pham, P.; Bongartz, J. P. Synthesis of α-
halogenated methanediphosphonates, Phosphorus, Sulfur Silicon Relat. Elem. 1988, 37, 1-12.
(14) McKenna, C. E.; Shen, P.-D. Fluorination of methanediphosphonate esters by
perchloryl fluoride. Synthesis of fluoromethanediphosphonic acid and
difluoromethanediphosphonic acid, J. Org. Chem. 1981, 46, 4573-76.
(15) McKenna, C. E.; Khawli, L. A. Synthesis of halogenated phosphonoacetate esters, J.
Org. Chem. 1986, 51, 5467-70.
(16) Blackburn, G. M.; England, D. A.; Kolkmann, F. Monofluoro- and
difluoromethylenebisphosphonic acids: isopolar analogs of pyrophosphoric acid, J. Chem.
Soc., Chem. Commun. 1981, 930-32.
(17) Hutchinson, D. W.; Thornton, D. M. A simple synthesis of
monofluoromethylenebis(phosphonic acid), J. Organomet. Chem. 1988, 340, 93-99.
(18) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. Synthesis of nucleotide 5'-
diphosphates from 5'-O-tosyl nucleosides, J. Org. Chem. 1987, 52, 1794-801.

16

(19) Burton, D.; Pietrzyk, D.; Ishihara, T.; Fonong, T.; Flynn, R. Preparation, stability and
acidity of difluoromethylene bis phosphonic acid, J. Fluorine Chem. 1982, 20, 617-26.
(20) McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M. Product class 16: phosphoric
acid and derivatives, Sci. Synth. 2009, 42, 779-921.
(21) Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical
Research, third ed.; Clarendon: Oxford, England, 1986.
(22) Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E. α-Azido
Bisphosphonates: Synthesis and Nucleotide Analogs, J. Org. Chem. 2011, 76, 5132-36.
(23) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 1997, 62, 7512-15.

17

CHAPTER 2. α-Azido Bisphosphonates: Synthesis, pKa Determination,
Nucleotide Analogs, and Reactions
*

2.1 Introduction
dNTP analogs that replace a natural P-O-P anhydride with a phosphonate moiety
present a range of tunable steric and electronic properties depending on the CXY (X,Y = H, F,
Cl, Br, Me) substitution at phosphonate carbon.
1
When incorporated into the β,γ position,
the quality of the leaving group can be altered relative to natural pyrophosphate
2,3
while
incorporation into the α,β position prevents polymerase catalysis and allows for
crystallization of fully competent enzymes. Using existing chemistry,
4,5
prochiral
bisphosphonates are coupled to nucleotides and nucleosides without stereocontrol and
furnish products as a mixture of diastereomers. The consequences of using this mixture for
biochemical assays has received little attention until recently when X-ray analysis of CXF
containing α,β
6
and β,γ
7,8
nucleotide analogs displayed stereospecific binding with pol β-
DNA complexes. In an effort to better understand the implications of phosphonate
stereochemistry on the synthesis and bioactivity of nucleoside triphosphate analogs, we
wished to construct β,γ (5–6) and α,β (7–8) dNTPs that incorporate a bridging CXN
3
group.
As a pseudohalide,
9
the azido functionality could usefully extend the tunability of the analog

*
Sections 2.2.1, 2.2.3, and related experimental procedures are reproduced with permission from
Chamberlain, B. T., Upton, T. G., Kashemirov, B. A., McKenna, C. E. Journal of Organic Chemistry. 2011, 76
(12), 5132-5136. Copyright 2011 American Chemical Society.

18

series, providing more information on molecular interactions at the active site arising from
the stereochemistry at the bridging carbon. Introduction of the azido group might also
facilitate separation of individual CXY diastereomers, which has not been accomplished with
the various X,Y = H, methyl or halide derivatives prepared thus far.
7,10
These considerations,
combined with the prospect of developing a new and potentially useful bisphosphonate
synthon, motivated an investigation of α-azido bisphosphonic esters and acids, a previously
unknown class of compounds (Figure 2.1.1).
P RO
O
RO
P
O
OR
OR
X
N
N
N
1: X = CH
3
; R = i-Pr
2: X = H; R = i-Pr
3: X = CH
3
; R = H
4: X = H; R = H

Figure 2.1.1. Structures of -azido bisphosphonate esters and acids.

2.2 Results and discussion
2.2.1 Synthesis of α-azido bisphosphonates
Electrophilic azido transfer was explored as a methodology offering a direct route to
1 and 2 from readily available starting materials. In this approach, a sulfonyl azide reacts
with a carbanion species to generate a triazene intermediate that can fragment into either
diazo or azido products.
11
Various factors influence the fragmentation pathway, but the
nature of the sulfonyl azide is particularly important. Sulfonyl azides with sterically bulky
and electron-donating substituents, such as 2,4,6-triisopropylbenzenesulfonyl azide (trisyl

19

azide; TrN
3
), favor azido transfer
11,12
whereas those containing strongly electron-
withdrawing moieties, for example trifluoromethylsulfonyl azide (triflyl azide; TfN
3
), favor
diazo transfer.
12,13

Although the methylene group of a bisphosphonate ester such as tetraisopropyl
methylenebis(phosphonate), 9, is exposed in principle to either pathway, replacement of
one α-H by a methyl group, as in tetraisopropyl ethylidenebis(phosphonate) (10) will
enforce azido transfer. In the synthesis of 1 from 10, methylation of 9 with potassium tert-
butoxide (t-BuOK) followed by methyl iodide gave a mixture of 78% 10 and 20% α,α-
dimethyl side product. Fortunately, this crude 10 preparation could be directly treated with
t-BuOK and then p-toluenesulfonyl azide (tosyl azide; TsN
3
) in DMSO at room temperature
to give the azido ester 1 in 70% yield after purification by column chromatography (Scheme
2.2.1).
Scheme 2.2.1. Synthesis of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
9
t-BuOK; MeI
CH
3
CN
10
1) t-BuOK; TsN
3
2) AcOH
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
N
3
1
DMSO

Attempts to synthesize 2 from 9 using tosyl azide led to the unwanted diazo transfer
product. Reaction of a carbanion derived from an activated methylene group with a
sulfonyl azide is a known route to tetraalkyl diazomethylenebis(phosphonates) and trialkyl
α-diazo phosphonoacetates.
14
However, use of trisyl azide with careful optimization of
conditions (THF, −78 °C, potassium counterion, 5-fold azide reagent excess, immediate

20

quenching by AcOH) generated 2 from the carbanion of 9 in greater than 95% yield by
31
P
NMR (Scheme 2.2.2). Despite the efficient conversion, purification of 2 proved to be
problematic initially due to difficulty in removing the large excess of trisyl azide and a minor
side product, tetraisopropyl diazomethylenebis(phosphonate) (11), by preparative
chromatography on silica gel. Pure 2 (50% yield) was eventually obtained by crystallization
of remaining trisyl azide, filtration, and selective oxidation of 11 with t-butyl hypochlorite
15

in wet ethyl acetate
16
prior to chromatography.

Scheme 2.2.2. Synthesis of 2 showing crude yields as determined by
31
P NMR.
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
9
1) KHMDS; TrN
3
2) AcOH
THF -78
o
C
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
N
3
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
N
2
+
2
11
> 95% < 5%

Care must be taken during workup of 2, which retains a relatively acidic α-H,
because it decomposes slowly on silica gel, more quickly in aqueous solution at pH 6, and
rapidly at pH 8. This contrasts with the reported stability of the malonate derivative
17
and
presumably reflects the resonance stabilization available in the enolate form of the latter
compound.
Exposure of 2 to bases such as BuLi, NaH, KHMDS, and t-BuOK results in the release
of N
2
with formation of diisopropyl phosphorocyanidate (12)
18
and diispropyl phosphite (13)
(Scheme 2.2.3). The base-induced elimination of nitrogen from α-azido esters,
19

malonates,
17
and sulfones
20
has been previously reported and transition metal-catalyzed
versions of this reaction have found synthetic utility.
21,22
In contrast to diethyl

21

azidomalonate, in which the carbanion was further derivatized at −15 °C without the loss of
N
2
,
17
2 loses N
2
rapidly upon exposure to a variety of bases in organic solvents at −78 °C and
does not give appreciable amounts of adducts in the presence of a large excess of N-
chlorosuccinimide.

Scheme 2.2.3. Base induced decomposition of 2.
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
N
3
2
Base
P i-PrO
O
i-PrO
CN
12
HP
O
Oi-Pr
Oi-Pr
13
+

The structures of 1–4 are supported by their suite of IR, NMR, and MS data. Esters 1
and 2 are both colorless oils with strong IR absorbances in the azido region (1: 2123, 2086
cm
-1
; 2: 2104, sh. at 2134 cm
-1
). The
1
H NMR spectrum of 1 includes a CH
3
triplet at δ 1.60
ppm (J
HP
15 Hz) and that of 2 has an α-H triplet at δ 3.53 ppm (J
HP
21 Hz). The
13
C NMR
spectrum of 1 has resonances at δ 17.6 (CH
3
) and δ 59.8 ppm (C
α
, t, J
CP
150 Hz). The C
α
of 2
resonates at δ 55.0 ppm (t, J
CP
145 Hz). The
31
P chemical shifts of 1 (δ 17.0 ppm) and 2 (δ
13.3 ppm) are consistent with a previously observed 3–4 ppm downfield effect of α-
methylation in other bisphosphonate esters.
23,24

Reaction of 1 and 2 with bromotrimethylsilane (BTMS)
25,26
in acetonitrile at room
temperature quantitatively gave the tetrakis(trimethylsilyl) esters, which display the
expected upfield
31
P shifts, Δδ +(16–17) ppm. Desilylation with aqueous ethanol produced
the acids 3 and 4 (>98% overall) as hygroscopic white solids. These acids are stable in dilute
aqueous solution pH 2–12 for at least several days at room temperature. They have IR

22

bands at 2126, 2085 (sh) cm
-1
(3) and 2113 cm
-1
(4) attributed to the azido group. The
1
H
NMR spectra include resonances at δ 1.43 ppm (C(CH
3
)N
3
, t, J
HP
13 Hz) for 3 and δ 3.26 ppm
(CHN
3
, t, J
HP
17 Hz) for 4. The
13
C NMR displays resonances at δ 23.5 (CH
3
) and δ 68.4 ppm
(C
α
, t, J
CP
129 Hz) for 3, and at δ 65.1 ppm (C
α
, t, J
CP
124 Hz) for 4. The
31
P chemical shifts are
in the expected regions, δ 18.3 ppm (3) and δ 13.5 ppm (4). The HRMS m/z values for the
[M – H]
-
anion of 3 and 4 match calculated values within error, confirming the assigned
structures.
The present study and our previous interest in diazo transfer chemistry
27,28

prompted us to reinvestigate a reported azido transfer to triethyl phosphonoacetate using
triflyl azide in the presence of triethyl amine.
29
Triflyl azide is known to be a powerful diazo
transfer reagent
11
and attempts by other authors to extend the procedure to benzocyclic β-
keto esters were unsuccessful;
12
nevertheless, the original reaction has been cited as a
viable azido transfer reaction in recent reviews.
30,31
In our hands, the procedure of
Hakimelahi and Just
29
produced triethyl diazophosphonoacetate, characterized by
1
H,
31
P
NMR and MS (ESI and APCI) and generated no detectable amounts of azido transfer product.
In light of these results and analysis of the literature,
†
we propose that the reaction of triflyl

†
The IR band at 2100 cm
-1
for the putative azido product
29
is within the range of literature values for
triethyl diazophosphonoacetate
32
including a sample prepared by a methodology that could not generate
an azido product.
33
The
1
H NMR data do not positively identify a PC(HX)C hydrogen. The α-proton is
distinguishable in analogous H-F,
34
H-Cl, and H-Br
35
triethyl phosphonoacetates and should be apparent in
authentic trialkyl azidophosphonoacetate. The C.I. MS data in the original report identifies a m/z 223
peak which was assigned to a [M – N
3
]
+
species. However, this peak also corresponds to a prominent
fragment which we observe in the MS/MS of the triethyl diazophosphonoacetate parent peak [M + 1]
+
at
m/z 251. Finally, the authors supported their structure by reducing their product to the
aminophosphonoacetate with H
2
(Pd/C) in EtOH. t-Butyl diazo(diethoxyphosphoryl)acetate is converted

23

azide with triethylphosphonoacetate is consistent with known diazo transfer chemistry and
is not an anomalous case of azido transfer.

2.2.2 Determination of α-azido bisphosphonic acid stability constants by potentiometric
titration
The acid dissociation constant is an established parameter used in LFER studies to
quantify relative leaving group ability.
2,3
Determining the pKa values of 3 and 4 using
conditions and procedures consistent with previous studies examining bisphosphonate pKa
and β,γ-dNTP k
pol
2
was essential for including 3 and 4 in the catalog of bisphosphonic acids
comprising the dNTP tool-kit. The potentiometric titration conditions employed here are
the same as those used in the original study
2
(0.1 M KCl counterion, small electrode surface
area, mathematical analysis with Hyperquad 2006
37
) that were found to reduce background
interference, make counterion complexation to the analyte less significant,
38
while also
providing optimal electrode accuracy.
39
The choice and optimization of conditions are
discussed elsewhere.
40,41
The acid dissociation constants (pKa
2-4
) for 3 and 4 are presented
in Table 2.2.1 along with the values of select halogenated bisphosphonic acids for
comparison.
40
As the table shows, 3 and 4 are less acidic than the analogous fluorinated
bisphosphonic acids, while 4 is more acidic than monochlorobisphosphonic acid.

to a cognate PCH-(NH
2
)C product under these conditions,
36
demonstrating that the reduction would not
necessarily distinguish the azido compound from triethyl diazophosphonoacetate.

24

Table 2.2.1. pKa values for α-azido bisphosphonic acids 3 and 4 and known bisphosphonic acids.

CXY pKa
4
pKa
3
pKa
2

CHF 9.01 ± .03 6.15 ± .02 1.30 ± .4
CBr
2
9.27 ± .003 5.87 ± .006 1.83 ± .03
CHN
3
(4) 9.39 ± .008 6.26 ± .02 2.08 ± .05
CHCl 9.58 ± .06 6.32 ± .02 1.19 ± .4
CHBr 9.91 ± .009 6.16 ± .03 2.33 ± .1
C(CH
3
)F 10.20 ± .01 6.21 ± .003 1.86 ± .02
C(CH
3
)N
3
(3) 10.50 ± .05 6.40 ± .01 2.33 ± .02
CH
2
10.52 ± .007 6.92 ± .001 2.75 ± .05

Although rationalizing the pKa trends resulting from methylation or halogenation is
qualitiatively straightforward, assessing the reasonableness of the pKa values for 3 and 4 is
complicated by poor definition of the electronegativity of the azido group in aliphatic
systems. Certain older references indicate that the electron withdrawing capacity of an
aliphatic azido group is intermediate of bromine and iodine,
42,43
but careful analysis of the
literature reveals that this characterization relies on an antique synthesis
44
and conductivity

25

study
45
of azidoacetic acid - a compound that was too unstable to isolate and
characterize.
46‡

The pKa values for 3 and 4 obtained from potentiometric titration are supported by
the linear relationship observed between the C
α

13
C chemical shift (δ) and the pKa
4
of the
halogenated bisphosphonic acids (Figure 2.2.1). It is well-established that in contrast to
halogenation, methylation decreases relative acidity while also causing a downfield shift in
the carbon NMR chemical shift;
48
accordingly, methylation results in deviations from the
trend observed with the halogenated compounds. Nevertheless, the monomethylated
compounds appear to form a trend line of their own. The trend observed for the non-
methylated series supports our titration data indicating that the inductive strength of the
azido group in bisphosphonic acids is greater than that of chlorine.

Figure 2.2.1. Bisphosphonic acid pKa
4
displays a linear correlations with C
α

13
C chemical shifts presented
in Tables 1.2.1 and 2.4.1 depending on the degree of methylation.

‡
Although, Charton’s list of σ
I
states that all of the values for substituted acetic acid pKas were taken from
an IUPAC compilation,
47
azidoacetic acid is not contained within that publication. Indeed, the pKa used in
Charton’s calculations, 3.03, matches the values of the 1908 study
45
and the resulting σ
I
is therefore
unreliable.

26

2.2.3 Synthesis of α-azido bisphosphonate containing nucleotide analogs
With acids 3 and 4 in hand, we then proceeded to the synthesis of β,γ (5, 6) and α,β
(7, 8) azido methylene nucleoside triphosphate analogs. The β,γ-CXN
3
dGTP analogs 5 and 6
were prepared by coupling
2,7
the Bu
3
NH
+
salts of 3 and 4 with dGMP-morpholidate.
4,49
α,β-
CXN
3
analogs of dATP (7, 8) were synthesized by reaction of the Bu
4
N
+
salt of 3 or 4 with dA-
5′-tosylate followed by enzymatic phosphorylation of the resulting α,β-CXN
3
dADP
intermediates (14, 15) by ATP-phosphoenolpyruvate (PEP) catalyzed by pyruvate kinase (PK)
and nucleoside diphosphate kinase (NDPK) in HEPES buffer, pH 7.5
10
(Scheme 2.2.4). The
final compounds were purified by dual-pass (strong-anion exchange, SAX, followed by
reverse phase, RP) HPLC to ≥97% as determined by
31
P NMR and analytical HPLC and were
characterized by NMR (
1
H,
31
P) and HRMS.
Scheme 2.2.4. Synthesis of α,β-CXN
3
(7a, 7b, 8a/b) and β,γ-CXN
3
(5a/b, 6a/b) nucleotide analogs.
3, 4
5a/b, 6a/b
14a/b, 15a/b
PK, PEP
NDPK, ATP
cat
7a, 7b, 8a/b
O
OH
O P N
O
-
O
O
G
O
OH
TsO
A

Nucleoside triphosphate α,β-CXY or β,γ-bisphosphonate analogs with an
asymmetrically substituted bridging carbon (X ≠ Y) are obtained as mixtures of
diastereomers by existing synthetic procedures starting from prochiral bisphosphonate

27

precursors, and thus far none have been separated chromatographically.
7,8,10

Diastereomeric β,γ-fluoromethylene dNTPs can be individually observed in the synthetic
mixture by
19
F NMR.
7,8
In addition to the expected nucleoside resonances for dGuo, the
1
H
NMR of 5a/b shows a pair of doublets centered at δ 1.58–1.59 ppm (C(CH
3
)N
3
) and that of
6a/b an apparent triplet at δ 3.40 ppm (CHN
3
). However, although the proton-decoupled P
α

and P
γ

31
P NMR signals of 5a/b are doublets at δ −9.45 and 14.5 ppm respectively, two
resolvable (Δδ 0.03 ppm) diastereomeric pairs of doublets (J = 34, 19 Hz) are observed for P
β
.
The δ vs J assignments were confirmed by comparing
31
P spectral data obtained at 162 and
202 MHz.
A similar pattern is seen with 6a/6b except that resolvable diastereomeric multiplets
are observed for both P
α
(δ −9.78, −9.81 ppm) and P
β
(δ 9.04, 9.06 ppm), but not for P
γ
(10.4
ppm). This difference between 5a/b and 6a/b with respect to the sensitivity of the P
α

chemical shift to the P
βγ
CXN
3
chiral center invites further investigation.
In the preparation of the α,β-C(CH
3
)N
3
dATP analogs 7a and 7b, we found that the
corresponding dADP bisphosphonate intermediates (14a/b) could be isolated individually by
isocratic RP HPLC (Figure 2.2.2). Subsequent enzymatic phosphorylation as described above
then generated the individual diastereomers 7a and 7b. CD spectra measured for 7a and 7b
showed prominent features of opposite polarity centered near 260 nm (7a, (−); 7b (+);
Figure 2.2.3). As the UV absorption spectra of these compounds are identical (Appendix B,
figure B35), the distinct response to circularly polarized light indicates that 7a and 7b have
different stereochemical features. 7a and 7b are diastereoisomers and are not expected to
give true mirror image CD spectra as is observed with enantiomers. Curiously, the α,β-CHN
3

28

dADP intermediate (15a/b) did not separate under similar conditions and its
phosphorylation yielded the triphosphates 8a/b as a mixture of diastereomers which could
not be distinguished by
31
P NMR.

Figure 2.2.2. Separation of α,β-CMeN
3
dADP diastereomers (14a/b) by RP semi-preparative HPLC. Left:
31
P NMR spectrum of 14a/b as synthesized displays two pairs of doublets for both P
α
and P
β
. Right:
31
P
NMR (14a and 14b) spectra of separated diastereomers show single pairs of doublets for P
α
and P
β
.

Figure 2.2.3. CD spectra of α,β-CMeN
3
dATPs 7a and 7b (25° C, 0.05 mM) in H
2
O, pH 8.

29

2.2.4 Subsequent reactions of α-azido bisphosphonates
The azido group is a powerful synthetic precursor that, through retention of the N
3
unit
or expulsion of nitrogen, provides access to a diverse range of heterocycles and amines.
50,51

In general, the reactions of alkyl azides include reductions, cycloadditions, and reactions
generating reactive iminophosphorane or nitrene intermediates.
50,51
The synthetic
applications of α-azidoalkylphosphonates have been reviewed,
30
and here we present
preliminary efforts to harness α-azido bisphosphonates as synthons.
The use of azides to introduce (often regio- or stereoselectively) and protect primary
amines is a common strategy used in the synthesis of carbohydrates and other sensitive
compounds.
50,52
Although the amino analogs of 1-4 are conveniently prepared by three-
component approaches,
53-55
the synthesis of α-aminobisphosphonates via reduction of α-
azidobisphosphonates could be an attractive approach to the synthesis of α-amino
bisphosphonates with fragile esters or α-substituents. Although several of the screened
conditions were either unreactive or completely removed the azido group, sodium
borohydride with cobalt (II) catalyst in water
56
quickly provides 16 in 82% isolated yield
(Scheme 2.2.5). This reaction has been shown to be chemoselective for the azido function
in the presence of a variety of functional groups in the original report and citing
references.
56

30

Scheme 2.2.5. Reduction of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.
P
O
P
O
Oi-Pr
Oi-Pr
i-PrO
i-PrO
N
3
NaBH
4
, CoCl
2
(cat.)
H
2
O
1
P
O
P
O
Oi-Pr
Oi-Pr
i-PrO
i-PrO
NH
2
16
82%

The structure of 16 is supported by the IR, NMR, and MS data. The IR spectrum shows
the disappearance of the azido band (~2100 cm
-1
) and the formation of a new primary
amino band at 3584 cm
-1
. The NMR data display the same number of resonances in similar
regions as the starting material (1), but with distinct chemical shifts. Relative to 1, the CH
3

resonances are shifted 0.16 ppm upfield in the
1
H spectrum and 2.3 ppm downfield in the
13
C spectrum. The C
α
triplet is shifted upfield by 7 ppm and the
31
P singlet is shifted 5.6 ppm
downfield. The LRMS m/z value for the [M+Na]
+
ion was observed. The available literature
preparations of 16 (and the tetraethyl ester) do not provide spectral data for
comparison.
54,57

Photo-irradiation of organoazides provides convenient access to intra- and
intermolecular nitrene insertion reactions.
50
It was demonstrated previously that the
deoxygenation of α-nitroso, α-methyl triethylphosphonoacetate proceeds with exclusive
1,2-C,N phosphoryl migration to form an imine intermediate that tautomerizes to form the
corresponding enphosphamide.
58
Similarly, formation of the α-nitrene via photo-irradiation
of 1 in methanol induces a 1,2-C,N phosphoryl shift to form 17 in approximately 85%
conversion by
31
P NMR. 17 is stable for days in methanol at room temperature and has
been characterized from the reaction mixture by NMR. Acidic conditions (i.e. photolysis in

31

dichloromethane or purification on silica gel) resulted in complete tautomerization to the
enphosphamide, 18, which was isolated (Scheme 2.2.6).

Scheme 2.2.6. UV photolysis of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.
P i-PrO
O
i-PrO
P
O
Oi-Pr
Oi-Pr
N
3
1
MeOH
UV
P i-PrO
O
i-PrO
N P
O
Oi-Pr
Oi-Pr
P i-PrO
O
i-PrO
N P
O
Oi-Pr
Oi-Pr
17 18
H

The UV photolysis of 1 generates two doublets in the
31
P NMR spectrum (δ 18.3, d, J
48.8 Hz; δ 2.9, d, J 48.8 Hz) in better than 80% conversion assigned to 17. The
1
H NMR of
the reaction mixture containing 17 features a ~3H doublet (14.4 Hz) in the methyl region
(1.67 ppm) that is replaced by 2 1H doublets (J 18.5 Hz, J 42.3 Hz) confirmed by gHSQC(AD)
2-D NMR to be connected to the same carbon, and a broad 1H apparent triplet assigned to
the alkene and amine protons of 18 respectively. Upon isolation of 18 by thin layer silica gel
chromatography, the
31
P NMR is shifted to δ 11.5 ppm (d, J 43.5 Hz) and δ -0.6 ppm (d, J
43.5 Hz). The structure of 18 is corroborated by
13
C-NMR, gHSQC(AD)-NMR, and mass
spectroscopy.

2.3 Conclusion
In conclusion, we have described the first examples of α-azido bisphosphonate
esters and acids. The latter compounds can be used to synthesize novel nucleotide analogs
containing a CHN
3
or C(Me)N
3
at either the α,β or β,γ bridging position. The ability to obtain

32

for the first time the individual diastereomers of the α,β-C(Me)N
3
dADP (14a, 14b) and
corresponding dATP analogs (7a, 7b) provides more refined probes for stereochemical
interactions of these compounds with appropriate enzymes. The pKa

values of 3 and 4 have
been determined by potentiometric titration and therefore make β,γ-CXN
3
nucleotide
analogs suitable for inclusion with existing nucleotide analog toolkits. The use of these new
bisphosphonates as synthetic intermediates is introduced with the development of
protocols for reduction and UV photolysis of 1.

2.4 Experimental
2.4.1 Materials and methods
The nucleoside triphosphate analogs were prepared by adapting our previously
published methods for β,γ-CXY
2,7,8
and α,β-CXY
10
dNTP analogs. Analytical HPLC analysis
was conducted on a Varian PureGel SAX 10 mm × 100 mm 7 μL column eluted with an A:
H
2
O, B: 0.5 M (0–50%) LiCl linear gradient over 30 min at a 4 mL/min flow rate. Products
were detected at 259 nm for adenosine derivatives and at 253 nm for guanosine derivatives.
All dNTP derivatives were prepared as triethylammonium salts.
2,7,8,10
CD spectra were
obtained on a JASCO J-815 spectropolarimeter.
Proton spectra were referenced to residual CHCl
3
(δ 7.24) in CDCl
3
or to HDO (δ
4.79).
59

13
C spectra were referenced to internal CDCl
3
(δ 77.0) or internal carbonate (δ
168.9).
59

31
P NMR spectra were referenced to external 85 % H
3
PO
4
(capillary, δ 0.0) and are
proton decoupled unless otherwise noted. pH values are reported without a deuterium
isotope correction. NMR δ values are reported in ppm. IR samples were prepared for 1 and

33

2 as films on NaCl disks, and for 3 and 4 as solid KBr pellets. High resolution mass spectra
were obtained by the UC Riverside mass spectrometry facility.
Images of
1
H-NMR spectra (1-8, 16, 18),
13
C-NMR (1-4, 16, 18),
31
P-NMR spectra (1-8,
16, 18), gCOSY (16, 18), gHSQC(AD) (16, 18), FTIR spectra (1-4, 16), HR mass spectra (3-6), LR
mass spectra (16, 18), titration curves for 3 and 4, sample HYPERQUAD analysis, and UV
spectra of 7a and 7b are presented in Appendix B. The
31
P-NMR spectra of 5a/b are
presented at a variety of field strengths and sample preparations.

2.4.2 Synthetic procedures
Preparation of azido transfer reagents. The desired sulfonyl chloride (1 equiv) was
dissolved in acetone and mixed with a solution of 3.5 equiv of sodium azide in water, then
stirred overnight at room temperature. The sulfonyl azide was precipitated by the addition
of excess water, extracted into hexanes, dried over MgSO
4
, and concentrated under
reduced pressure. The sulfonyl azide reagents were used without further purification.
Tetraisopropyl (1-Azidoethane-1,1-diyl)bis(phosphonate), 1. Tetraisopropyl
methylenebis(phosphonate) (1.75 g, 5 mmol) was treated with 1.2 equiv of t-BuOK and 1.2
equiv of MeI in anhydrous acetonitrile overnight at rt. Volatiles were removed at reduced
pressure and the residue was dissolved in water, extracted into DCM, and dried over MgSO
4
.
Evaporation of the solvent left an oil (1.84 g) that contained 20% dimethylated product,
2% unmethylated material, and 78% monomethylated product. This mixture ( 4 mmol
monomethylated product) was dissolved in 20 mL of DMSO and stirred with 0.689 g (6
mmol) of t-BuOK. TsN
3
(1.05 g, 5 mmol) was added drop-wise. The reaction mixture was

34

stirred for 30 s, quenched with 1.5 mL of AcOH, and then allowed to stir for several hours at
rt. The reaction mixture was extracted with hexanes and the organic phase was dried over
MgSO
4
. Following concentration by evaporation, the product was purified on silica gel using
ACE/DCM (1:3), R
f
= 0.69, giving 1 as a colorless oil. Yield: 1.45 g (70% overall). IR: v (film)
2123, 2086 cm
-1
(N
3
). δ
H
(399.8 MHz; CDCl
3
): 1.33–1.37 (24 H, m, 4 × OCH(CH
3
)
2
), 1.60 (3H, t,
J
HP
15.2 Hz, PCCH
3
N
3
P), 4.83 (4 H, m, 4 × OCH(CH
3
)
2
); δ
C
(100.5 MHz; CDCl
3
): 17.6 (t, J
CP
3.9
Hz, PCCH
3
N
3
P), 23.7–23.8, 24.3–24.4 (OCH(CH
3
)
3
, 59.8 (t, J
CP
150.3 Hz, PCP), 72.6–72.8
((OCH(CH
3
)
3
; δ
P
(161.9 MHz; CDCl
3
; ext. 85% H
3
PO
4
): 17.0 (s; coupled J
PH
15.2 Hz (qm), J
PC

150 Hz (satellites)).
Tetraisopropyl (Azidomethanediyl)bis(phosphonate), 2. In a 500 mL RB flask under
N 2, 6 g (19 mmol) of TrN
3
were dissolved in 30 mL of anhydrous THF and stirred vigorously
at −78 °C. A solution of 1.52 g (4.41 mmol) of tetraisopropyl methylenebis(phosphonate)
and 4.41 mL of 1 M KHMDS in 10 mL THF was mixed at rt, cooled to −78 °C and added at
once to the TrN
3
solution by syringe. The solution was stirred for 10 s and then quenched
with 2.5 mL AcOH. The flask was moved to a −20 °C freezer and allowed to stand for 48 h,
or until the triazene had decomposed as determined by
31
P NMR. The precipitate was
filtered and volatiles removed at reduced pressure. The reaction mixture was then seeded
with solid TrN
3
and allowed to sit overnight at 0 °C. The resulting precipitate was washed
with H
2
O and filtered off. After removing water by rotovap, the residue was dissolved in 20
mL wet EtOAc. Ten drops of t-BuOCl were added and stirred at rt until evolution of N
2
ceased. EtOAc was washed with a saturated aqueous NH
4
Cl solution, dried, and
concentrated. The residue was purified on silica gel eluted with ACE/DCM (1:9), R
f
= 0.36.

35

Contaminating phosphite was removed by rotary evaporation (oil pump) with gentle
warming to afford 0.83 g of 2 as a colorless oil (yield: 50% overall). IR: v (film) 2134 (m),
2104 cm
-1
(N
3
). NMR: δ
H
(399.8 MHz; CDCl
3
): 1.31–1.34 (24 H, m, 4 × OCH(CH
3
)
2
), 3.53 (1H, t,
J
HP
20.8 Hz, PCHN
3
P), 4.79 (4 H, m, 4 × OCH(CH
3
)
2
); δ
C
(100.5 MHz; CDCl
3
): 23.7–23.8, 24.1–
24.2 (OCH(CH
3
)
3
, 55.0 (t, J
CP
144.8 Hz, PCP), 72.7, 72.9 (2t, J
CP
3.5 Hz OCH(CH
3
)
3
); δ
P
(161.9
MHz; CDCl
3
; ext. 85% H
3
PO
4
): 13.3 (s; coupled J
PH
20.5 Hz (dm), J
PC
145 Hz (satellites)).
Base-Induced Decomposition of 2. 5 mg (13 μmol) of 2 was dissolved in 1.5 mL of
anhydrous THF and cooled to −78 °C. t-BuOK (1.5 equiv) was added at once, causing rapid
evolution of gas. Identical results were obtained with BuLi and KHMDS. NMR: δ
P
(202.5
MHz; CDCl
3
; ext. 85% H
3
PO
4
): 22.3 (diisopropyl phosphorocyanidate),
18
4.8 (diispropyl
phosphite) with integration 1:1.
(1-Azidoethane-1,1-diyl)bis(phosphonic acid), 3, and (Azidomethanediyl)-
bis(phosphonic acid), 4. The appropriate bisphosphonate ester was dissolved in 2 mL
anhydrous acetonitrile and 6 equiv of BTMS were added by syringe. Stirring overnight gave
the tetrakis(trimethylsilyl) esters (100% by
31
P NMR). From 1: δ
P
(202.5 MHz; CH 3CN, ext. 85%
H
3
PO
4
) 0.9. From 2: δ
P
−3.2 (s). Hydrolysis with 1 mL of EtOH/H
2
O (1:1) and removal of
volatiles by prolonged evaporation at low pressure provided the acids as hygroscopic white
solids (>98% overall yield). The acids were further dried under vacuum in a desiccator
containing P
2
O
5
.
3: IR: v (KBr): 2085 (m), 2126 cm
-1
(N
3
). NMR: δ
H
(399.8 MHz; D
2
O; pH 10.88): 1.43 (t,
J
HP
13.2 Hz, PCCH
3
N
3
P); δ
C
(100.5 MHz; D
2
O; ext. pH 10.88): 23.5 (s, PCCH
3
P), 68.4 (t, J
CP
129
Hz, PCP); δ
P
(161.9 MHz; D
2
O; 85% ext. H
3
PO
4
; pH 10.88): 18.3 (s; coupled, J
PH
13.2 Hz (q); J
PC

36

129 Hz (satellites)). HRMS (ESI/APCI) [M – H]
-
: calcd for C
2
H
6
N
3
O
6
P
2
: 229.9737. Found:
229.9734.
4: IR: v (KBr): 2113 cm
-1
(N
3
). δ
H
(399.8 MHz; D
2
O; pH 10.88): 3.26 (t, J
HP
16.8 Hz,
PCHN
3
P); δ
C
(100.5 MHz; D 2O; pH 10.88): 65.1 (t, J
CP
124 Hz, PCP); δ
P
(161.9 MHz; D
2
O; 85%
ext. H
3
PO
4
; pH 10.88): 13.5 (s; coupled, J
PH
16.5 Hz (d); J
PC
122 Hz (satellites)); HRMS
(ESI/APCI) [M – H]
-
: calcd for CH
4
N
3
O
6
P
2
, 215.9581. Found: 215.9584.
Ethyl diazo(diethoxyphosphoryl)acetate from trifyl azide and
ethyl(diethylphosphoryl)acetate. Following the procedure of Hakimelahi and Just,
29
72 mg
of NaN
3
(1.1 mmol) was suspended in 20 mL of anhydrous DMF and 106 μL (1.0 mmol) of
CF
3
SO
2
Cl was added under N
2
. After several min, a solution of 222 mg (1.0 mmol) of ethyl
(diethylphosphoryl)acetate and 101 mg (1.0 mmol) of Et
3
N in 5 mL anhydrous DMF was
added drop wise. After 40 min, a mixture of compounds were detected by
31
P NMR with δ
P

(DMF) 20.8, 18.3, 10.4 in a ratio of 5:4:1. Gentle heating for an additional 30 min gave a
binary mixture of the compounds at δ
P
10.4 (40%) and 20.8 (60%). Ether (30 mL) was added
and the reaction mixture was washed 5× with 5 mL water. The organic layer was dried over
MgSO
4
and evaporated. A portion of the residue was purified by preparative TLC on silica
gel eluted with ACE/DCM (1:9) giving a single mobile band (UV), R
f
= 0.65, which was
identified as ethyl diazo(diethoxyphosphoryl)acetate. NMR: δ
H
(400.2 MHz; CDCl
3
): 1.32 (3H,
t, J
HH
7 Hz), 1.38 (6H, dt, J
HH
7 Hz, J
HP
0.8 Hz), 4.22 (4H, m), 4.29 (2H, q, J
HH
7 Hz); δ
P
(202.5
MHz, CDCl
3
; 85% H
3
PO
4
) 10.3 (s). MS (APCI, m/z), 251 (M + 1)
+
; an MS/MS analysis of the
m/z 251 species gave a major fragment at m/z 223.
14

37

2′-Deoxyguanosine 5′-triphosphate β,γ-C(CH
3
)N
3
, 5a/b. Compound 3 (2 equiv of
the 1.5 tributylammonium salt) was coupled to 100 mg (0.242 mmol) of dGuo-morpholidate
in anhydrous DMSO to yield a white solid, 32.5 mg (23.8%) after dual pass (SAX then RP)
HPLC purification.
2
Analytical HPLC: retention time = 9.6 min; purity = 98%. NMR: δ
H
(399.8
MHz; D
2
O; pH 10.88): 1.58 (dd, PCCH
3
N
3
P) 2.48–2.54, 2.72–2.79, 4.11–4.21, 4.12, 6.32 8.05.
δ
P
(161.9 MHz; D
2
O; 85% H
3
PO
4
; pH 10.88): −9.5 (d, J
αβ
33.7 Hz, P
α
), 13.28 (dd, J
αβ
33.7
Hz, J
βγ
19.4 Hz, P
β
), 13.31 (dd, J
αβ
33.7 Hz, J
βγ
19.4 Hz, P
β
) 14.5 (d, J
βγ
19.4 Hz, P
γ
). HRMS
(ESI/APCI) [M−H]
-
: calcd for C
12
H
18
N
8
O
12
P
3
, 559.0263. Found: 559.0266.
2′-Deoxyguanosine 5′-Triphosphate β,γ-CHN
3
, 6a/b. Compound 4 (2 equiv of the
1.5 tributylammonium salt) was coupled to 100 mg (0.242 mmol) of dGuo-morpholidate in
anhydrous DMSO to yield a white solid, 31.1 mg (23.5%) 6a/b after dual pass (SAX then RP)
HPLC purification.
2
Analytical HPLC: retention time = 10.3 min, purity = 98%. NMR: δ
H
(499.8
MHz; D
2
O; pH 10.88): 2.48–2.53, 2.71–2.76, 3.53 (ddd, PCHN
3
P), 4.10–4.20, 4.25, 6.31
(t, J 6.0 Hz) 8.03, 8.03; δ
P
(161.9 MHz; D
2
O; ext. 85% H
3
PO
4
; pH 10.88): −9.78 (d, J
αβ 27.8 Hz,
P
α
), −9.81 (d, J 27.8 Hz P
α
), 9.04 (dd, J
αβ
27.8 Hz, J
βγ
12.9 Hz, P
β
), 9.06 (dd, J
αβ
27.8 Hz, J
βγ
12.9
Hz, P
β
) 10.41 (d, J
βγ
12.9 Hz, P
γ
). HRMS (ESI/APCI) [M – H]
-
: calcd for C
11
H
16
N
8
O
12
P
3
, 545.0106.
Found: 545.0121.
2′-Deoxyadenosine 5′-Diphosphate, α,β-C(CH
3
)N
3
, 14a/b. The
tris(tetrabutylammonium) salt from 60 mg (0.260 mmol) of 3 was allowed to react with 115
mg ( 1.1 equiv) of dA-5′-Ts in anhydrous acetonitrile
10
to yield 65.1 mg of the nucleoside
diphosphonate diasteromers in a ratio of 2:3 (54% crude conversion), which could be
separated by RP HPLC. 14a: NMR: δ
H
(399.8 MHz; D
2
O; pH 10.88): 1.50 (dd, J
HP
12.4, J
HP
15.2

38

Hz, PCCH
3
N
3
P), 2.57–2.63, 2.82–2.91, 4.10–4.16, 4.22–4.27, 6.48 (t, 6.4 Hz), 8.25, 8.52;
δ
P
(161.9 MHz; D
2
O; 85% H
3
PO
4
; pH 10.88) 14.5 (d, J 15 Hz, P
β
); 21.7 (d, J 15 Hz, P
α
). 14b:
NMR: δ
H
(399.8 MHz; D
2
O; pH 10.88): 1.50 (dd, J
HP
12.8, 15.2 Hz, PCCH
3
N
3
P), 2.57–2.63,
2.82–2.88, 4.10–4.16, 4.22–4.27, 6.47 (t, 6.4 Hz), 8.22, 8.50; δ
P
(161.9 MHz; D
2
O; ext. 85%
H
3
PO
4
; pH 10.88) 14.4 (d, J 15 Hz, P
β
); 21.6 (d, J 15 Hz, P
α
).
2′-Deoxyadenosine 5′-Triphosphate α,β-C(CH
3
)N
3
, 7a/b. Enzymatic
phosphorylation
10
of 14a and 14b followed by HPLC purification of each product gave 17.3
mg (yield: 12.2% overall from starting acid) of 7a and 14.9 mg (10.5%) of 7b. 7a:Analytical
HPLC: retention time = 9.5 min; purity = 98%. NMR: δ
H
(499.8 MHz; D
2
O; pH 10.88): 1.58
(t, J
HP
15.0 Hz, PCCH
3
N
3
P), 2.58–2.63, 2.83–2.89, 4.21–4.29, 6.50 (t, 6.5 Hz), 8.25, 8.54; δ
P

(202.5 MHz; D
2
O; 85% H
3
PO
4
; pH 10.88) −4.5 (d, J
βγ
31.9 Hz, P
γ
); 7.0 (dd, P
β
); 18.51
(d, J
αβ
19.0 Hz, P
α
). HRMS (ESI/APCi) [M – H]
-
: calcd for C
12
H
18
N
8
O
11
P
3
, 543.0313. Found:
543.0333. 7b: Analytical HPLC: retention time = 9.5 min; purity = 96%. NMR: δ
H
(499.8 MHz;
D
2
O; pH 10.88): 1.59 (t, J
HP
15 Hz, PCCH
3
N
3
P), 2.58–2.63, 2.85–2.90, 4.17–4.22, 6.51 (t, 6.5
Hz), 8.26, 8.56; δ
P
(202.3 MHz; D
2
O; ext. 85% H
3
PO
4
; pH 10.88) −4.4 (d, J
βγ
30.1 Hz, P
γ
); 8.0
(dd, P
β
); 18.47 (d, J
αβ
18.2 Hz, P
α
). HRMS (ESI/APCI) [M – H]
-
: calcd for C
12
H
18
N
8
O
11
P
3
,
543.0315. Found: 543.0315.
2′-Deoxyadenosine 5′-Diphosphate, α,β-CHN
3
, 15a/b. The
tris(tetrabutylammonium) salt was prepared from 60 mg (0.28 mmol) of 4 and allowed to
react with 120 mg ( 1.1 equiv) of dAdo-5′-Ts in anhydrous acetonitrile
10
to yield a white
solid, 32 mg (26%). NMR: δ
P
(242.8; D
2
O; pH 10.88): 9.9 (P
β
); 17.2 (P
α
).

39

2′-Deoxyadenosine 5′-Triphosphate α,β-CHN
3
, 8a/b. Enzymatic
phosphorylation
10
of 15a/b above followed by HPLC purification gave 33.2 mg (22.7%) 8a/b.
Analytical HPLC: retention time = 10.4 min; purity = 98%. NMR: δ
H
(400.2 MHz; D
2
O; pH
10.88): 2.57–2.63, 2.82–2.90, 3.86 (dt, PCHN
3
P), 4.11–4.25, 4.29, 6.50 (t, J 6.5 Hz), 8.24,
8.528, 8.532; δ
P
(202.3 MHz; D
2
O; ext. 85% H
3
PO
4
; pH 10.88) −5.1 (d, J
βγ
25.1 Hz, P
γ
); 2.8 (dd,
P
β
); 14.3 (d, J
αβ
13.8 Hz, P
α
). HRMS (ESI/APCI) [M – H]
-
: calcd for C
11
H
16
N
8
O
11
P
3
, 529.0157.
Found: 529.0147.
Tetraisopropyl (1-aminoethane-1,1-diyl)bis(phosphonate), 16. 75 mg of 1 (0.188
mmol) and 4.5 mg CoCl
2
.
6H
2
O (0.0188 mmol) was dissolved in 5 mL H
2
O and formed a
homogeneous solution. This was added drop wise at room temperature to vigorously
stirring solution of 14 mg (3.76 mmol) NaBH
4
in 2 mL H
2
O. The reaction was stirred for 10
minutes, extracted with EtOAc (3x10 mL), dried over sodium sulfate and purified by column
chromatography on silica eluted with 10% MeOH, 40% ACE, and 50% DCM to yield 58 mg
(0.155 mmol) of 16 in 82% yield. IR v (thin film): 3584, 2978, 2933, 1452, 1385, 1245, 1178,
1142, 1107, 987, 886 cm
-1
. 
H
(399.8 MHz; CDCl
3
): 1.32 (24 H, m, 4 × OCH(CH
3
)
2
), 1.44 (3H, t,
J
HP
16.3 Hz, PCCH
3
NH
2
P), 4.76 (4 H, m, 4 × OCH(CH
3
)
2
); 
C
(100.5 MHz; CDCl
3
): 19.9 (t, J
CP
3.8
Hz, PCCH3NH
2
P), 23.9 (dt, OCH(CH
3
)
3
), 24.3 (t, J
CH
1.6 Hz, OCH(CH
3
)
3
), 24.4 (bs, OCH(CH
3
)
3
),
52.8 (t, J
CP
148 Hz, PCP), 71.5 and 71.8 (t, J
CH
3.9 Hz, (OCH(CH
3
)
3
; 
P
(161.9 MHz; CDCl
3
; 85%
H
3
PO
4
): 22.6. LRMS [M + Na]
+
: calcd for C
14
H
33
NNaO
6
P
2
,396.2. Found: 396.0.
Diisopropyl (1-([bis(propan-2-yloxy)phosphoryl]amino)ethenyl)phosphonate, 18.
5 mg (12.3 μmol) of 1 was dissolved with 2 mL of MeOH in a pyrex glass tube and irradiated
with broad spectrum UV light for 2 h. Methanol was removed under reduced pressure and

40

the reaction mixture was dissolved in EtOAc and purified by TLC (EtOAc: EtOH, 3:1) to yield
~3.5 mg 18 (~75% yield). 
H
(399.8 MHz; CDCl
3
): 1.28-1.35 (~24 H, m, 4 × OCH(CH
3
)
2
), 4.59-
4.70 (4 H, m, 4 × OCH(CH
3
)
2
), 4.91 (1 H, bt, J 10.5 Hz, NH), 5.20 (1 H, d, J
HP
18.5), 5.30 (1 H, d,
J
HP
42.3). 
C
(100.6 MHz; CDCl
3
): 23.54, 23.59, 23.66, 23.71, 23.78, 23.83, 23.97, 24.01
(CH(CH
3
)
2
), 71.7 (d, J
CP
5.2 Hz, CH(CH
3
), 71.9 (d, J
CP
5.6 Hz, CH(CH
3
)
2
, 106.0 (d, 13.8 Hz, CCH
2
);

P
(202.5 MHz; CDCl
3
; 85% H
3
PO
4
): -0.6 (d, J
PP
43.5), 11.5 (d, J
PP
43.5). LRMS calcd for [M H]
+

C
14
H
32
NO
6
P
2
, 372.2; [M + Na]
+

C
14
H
31
NNaO
6
P
2
, 394.2. Found: 372.1, 394.1.

2.4.3 Potentiometric titration of 3 and 4
For each titration, a 10 mL (±0.01 mL) solutions of ~5 mM bisphosphonic acid and
0.1 M KCl was prepared with DI water purged of CO
2
(boiled and then cooled under nitrogen)
was placed into a dry 50 mL round bottom flask equipped with a small stir bar and kept
under the flow of nitrogen. The flask was placed in a water bath attached to a thermostat
and maintained at 25 °C. The pH was monitored (Aldrich Z113441 electrode (O.D. 3.5 mm),
calibrated with standard buffers and 20 μL aliquots of ~0.1 M KOH (standardized with
potassium hydrogenphthalate and phenolphthalein indicator) were delivered at the surface
of the analyte solution using a Schott Titrator Basic titration device.
Titrations were carried out three times per compound and the calculated pKa values
were averaged with the error reported as the estimated standard deviation. Titration
curves were fitted to log β values using Hyperquad 2006.
37
The difficulty of drying
bisphosphonic acids is well documented,
60
and accordingly, it was difficult to obtain

41

elemental analysis results that agreed with the calculated values. The purity of our samples
was assayed by
1
H,
13
C,
31
P NMR, and LC-MS (Appendix B). The effect of non-acidic
contamination in our samples was minimized by allowing the amount of analyte
(constrained to 1BP: 4H
+
) to be optimized by Hyperquad.
For 4, refinement included pH values reflecting bisphosphonic tetraacid
concentrations of up to ~95%. This procedure was not possible with 3 which possesses a
pKa
4
greater than 10, and in this case upper pH data was included as necessary to obtain
good agreement with experimental and calculated titration curves. The pKa values were
derived from the log β values using the equations pKa
4
= log β
1
; pKa
3
= log β
2
- log β
1;
pKa
2

= log β
3
- log β
2.
The
13
C NMR values for compounds not listed in Table 1.2.1 acquired on compounds
prepared previously
40
using identical conditions and instrumentation as described in
Chapter 1 and listed in Table 2.4.1.
Table 2.4.1. NMR data (D
2
O, Na
2
CO
3
, pH ~10.3, J = Hz) of auxiliary bisphosphonic acids used in Figure 2.2.1.
CXY
1
H (499.8 MHz)
13
C (125.7 MHz)
19
F (470.2
MHz)
31
P (202.3
MHz)
CFCl -
115.3 (dt,
1
J
CF

261.0,
1
J
CP
135.7)
-137.2 (t,
2
J
FP

68.4)
9.8 (d,
2
J
PF

68.5)
C(CH
3
)H
1.24 (dt,
3
J
HH
7.4,
3
J
HP
16.2, CH
3
),
1.81 (tq,
2
J
HP

21.5,
3
J
HH
7.4,
PCHMeP)
37.1 (t,
1
J
CP
116.9,
C
α
), 13.9 (t,
2
J
CP

5.4, CH
3
)
- 21.4
C(CH
3
)
2
1.23 (t,
3
J
HP
15.0)
38.6 (t,
1
J
CP
116.0,
C
α
), 23.7 (t,
2
J
CP

3.9, CH
3
)
- 26.6
C(CH
3
)F
1.56 (dt,
2
J
HP
13.0,
3
J
HF
27.5)
104.3 (dt,
1
J
CF

166.6,
1
J
CP
138.1,
C
α
), 25.6 (d,
2
J
CF

20.5, CH
3
)
-177.3 (tq,
2
J
FP

64.9,
3
J
FH
27.5)
15.5 (d,
2
J
PF

65.2)

42

2.5 Chapter references
(1) McKenna, C. E.; Kashemirov, B. A.; Peterson, L. W.; Goodman, M. F. Modifications to
the dNTP triphosphate moiety: From mechanistic probes for DNA polymerases to antiviral
and anti-cancer drug design, Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 1223-
30.

(2) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA Polymerase β
Fidelity: Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis
Reveal Differences at the Chemical Transition State, Biochemistry 2008, 47, 870-79.

(3) 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. Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide
Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β,
Biochemistry 2007, 46, 461-71.

(4) Moffatt, J. G. General synthesis of nucleoside 5'-triphosphates, Can. J. Chem. 1964,
42, 599-604.

(5) Mohamady, S.; Jakeman, D. L. An Improved Method for the Synthesis of Nucleoside
Triphosphate Analogs, J. Org. Chem. 2005, 70, 10588-91.

(6) Chamberlain, B. T.; Batra, V. K.; Beard, W. A.; Kadina, A. P.; Shock, D. D.; Kashemirov,
B. A.; McKenna, C. E.; Goodman, M. F.; Wilson, S. H. Stereospecific Formation of a Ternary
Complex of (S)-α,β-Fluoromethylene-dATP with DNA Pol β, ChemBioChem 2012, 13, 528-30.

(7) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T.
G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA
Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding
of the Fluoromethylene Analogues, J. Am. Chem. Soc. 2010, 132, 7617-25.

(8) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.;
Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-Fluoromethylene-dGTP-DNA Ternary
Complex with DNA Polymerase β, J. Am. Chem. Soc. 2007, 129, 15412-13.

(9) Ellis, G. P.; Luscombe, D. K.; Editors Progress in Medicinal Chemistry, Vol. 31; Elsevier,
1994.

43

(10) Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.;
Kultyshev, R.; Batra, V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. α,β-
Difluoromethylene Deoxynucleoside 5'-Triphosphates: A Convenient Synthesis of Useful
Probes for DNA Polymerase β Structure and Function, Org. Lett. 2009, 11, 1883-86.

(11) Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. The asymmetric synthesis of α-
amino acids. Electrophilic azidation of chiral imide enolates, a practical approach to the
synthesis of (R)- and (S)-α-azido carboxylic acids, J. Am. Chem. Soc. 1990, 112, 4011-30.

(12) Benati, L.; Nanni, D.; Spagnolo, P. Reactions of Benzocyclic β-Keto Esters with
Sulfonyl Azides. 2. Further Insight into the Influence of Azide Structure and Solvent on the
Reaction Course, J. Org. Chem. 1999, 64, 5132-38.

(13) Wurz, R. P.; Lin, W.; Charette, A. B. Trifluoromethanesulfonyl azide: an efficient
reagent for the preparation of α-cyano-α-diazo carbonyls and an α-sulfonyl-α-diazo
carbonyl, Tetrahedron Lett. 2003, 44, 8845-48.

(14) Jaszay, Z. M.; Pham, T. S.; Gonczi, K.; Petnehazy, I.; Toke, L. Efficient solid/liquid
phase-transfer catalytic diazo transfer synthesis, Synth. Commun. 2010, 40, 1574-79.

(15) Mintz, M. J.; Walling, C. Tert-butyl hypochlorite, Org. Syn. 1969, 49, 9-12.

(16) McKenna, C. E.; Kashemirov, B. A. Recent progress in carbonylphosphonate
chemistry, Top. Curr. Chem. 2002, 220, 201-38.

(17) Delacotte, J. M.; Galons, H. Diethyl azidomalonate: revised synthesis and reactivity
of the anion with electrophiles, J. Chem. Res., Synop. 1991, 64-5.

(18) Sun, D.; Shi, E.; Xiao, J.; Pei, C. An efficient synthesis of dialkyl phosphorocyanidates,
Phosphorus, Sulfur Silicon Relat. Elem. 2005, 180, 2155-61.

(19) Manis, P. A.; Rathke, M. W. Reaction of α-azido esters with lithium ethoxide:
synthesis of dehydroamino esters and α-keto esters, J. Org. Chem. 1980, 45, 4952-4.

(20) Jarvis, B. B.; Nicholas, P. E. Reactions of α-azido sulfones with bases, J. Org. Chem.
1980, 45, 2265-8.

(21) Chiba, S.; Zhang, L.; Ang, G. Y.; Hui, B. W.-Q. Generation of Iminyl Copper Species
from α-Azido Carbonyl Compounds and Their Catalytic C-C Bond Cleavage under an Oxygen
Atmosphere, Org. Lett. 2010, 12, 2052-55.

44

(22) Ciez, D. A direct preparation of N-unsubstituted pyrrole-2,5-dicarboxylates from 2-
azidocarboxylic esters, Org. Lett. 2009, 11, 4282-85.

(23) McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y.; Pham, P.; Bongartz, J. P. Synthesis of α-
halogenated methanediphosphonates, Phosphorus, Sulfur Silicon Relat. Elem. 1988, 37, 1-12.

(24) Hutchinson, D. W.; Semple, G. Synthesis of alkylated methylene bisphosphonates via
organothallium intermediates, J. Organomet. Chem. 1985, 291, 145-51.

(25) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C. The facile dealkylation of
phosphonic acid dialkyl esters by bromotrimethylsilane, Tetrahedron Lett. 1977, 155-8.

(26) McKenna, C. E.; Schmidhauser, J. Functional selectivity in phosphonate ester
dealkylation with bromotrimethylsilane, J. Chem. Soc., Chem. Commun. 1979, 739.

(27) McKenna, C. E.; Levy, J. N. α-Keto phosphonoacetates, J. Chem. Soc., Chem. Commun.
1989, 246-7.

(28) Khare, A. B.; McKenna, C. E. An improved synthesis of tetraalkyl
diazomethylenediphosphonates and alkyl diazo(dialkoxyphosphoryl)acetates, Synthesis
1991, 405-6.

(29) Hakimelahi, G. H.; Just, G. Two simple methods for the synthesis of trialkyl α-
aminophosphonoacetates. Trifluoromethanesulfonyl azide as an azide-transfer agent, Synth.
Commun. 1980, 10, 429-35.

(30) Gajda, A.; Gajda, T. Synthesis and reactivity of azidoalkylphosphonates, -
phosphinates and -phosphine oxides, Curr. Org. Chem. 2007, 11, 1652-68.

(31) Sobhani, S.; Tashrifi, Z. Synthesis of α-functionalized phosphonates from α-
hydroxyphosphonates, Tetrahedron 2010, 66, 1429-39.

(32) Regitz, M.; Anschuetz, W.; Liedhegener, A. Reactions of CH-active compounds with
azides. XXIII. Synthesis of α-diazophosphonic acid esters, Chem. Ber. 1968, 101, 3734-43.

(33) Khokhlov, P. S.; Kashemirov, B. A.; Mikityuk, A. D.; Strepikheev, Y. A.; Chimishkyan, A.
L. Diazotization of α-aminophosphonylacetates, Zh. Obshch. Khim. 1984, 54, 2785-7.

45

(34) Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E.
Synthesis of α-fluorinated phosphonoacetate derivatives using electrophilic fluorine
reagents: Perchloryl fluoride versus 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor), J. Fluorine Chem. 2005,
126, 1467-75.

(35) McKenna, C. E.; Khawli, L. A. Synthesis of halogenated phosphonoacetate esters, J.
Org. Chem. 1986, 51, 5467-70.

(36) Shiraki, C.; Saito, H.; Takahashi, K.; Urakawa, C.; Hirata, T. Preparation of
amino(diethoxyphosphoryl)acetic esters. Catalytic hydrogenation of diazo compounds to
amines, Synthesis 1988, 399-401.

(37) Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination
of equilibrium constants with the HYPERQUAD suite of programs, Talanta 1996, 43, 1739-53.

(38) Popov, K.; Ronkkomaki, H.; Lajunen, L. Critical evaluation of stability constants of
phosphonic acids (IUPAC technical report), Pure Appl. Chem. 2001, 73, 1641-77.

(39) Koort, E.; Gans, P.; Herodes, K.; Pihl, V.; Leito, I. Acidity constants in different media
(I=0 and I=0.1 M KCl) from the uncertainty perspective., Anal. Bioanal. Chem. 2006, 385,
1124-39.

(40) Upton, T. G. Design and synthesis of a series of methylenebisphosphonates: A
nucleotide analogue toolkit to probe nucleic acid polymerase structure and function, Ph.D.,
dissertation, University of Southern California, 2008.

(41) Osuna, J. Ph.D., dissertation, University of Southern California , In preparation.

(42) Charton, M. Definition of "inductive" substituent constants, J. Org. Chem. 1964, 29,
1222-7.

(43) Boyer, J. H.; Canter, F. C. Alkyl and aryl azides, Chem. Rev. (Washington, DC, U. S.)
1954, 54, 1-57.

(44) Forster, M. O.; Fierz, H. E. The Triazo Group. Part I. Triazoacetic Acid and
Triazoacetone (Acetonylazoimide), J. Chem. Soc., Trans. 1908, 93, 72-85.

(45) Philip, J. C. The Dissociation Constants of Triazoacetic and α-Triazopropionic Acids, J.
Chem. Soc., Trans. 1908, 93, 925-7.

(46) Boyer, J. H.; Hamer, J. The acid-catalyzed reaction of alkyl azides upon carbonyl
compounds, J. Am. Chem. Soc. 1955, 77, 951-4.

46

(47) Kortum, G.; Vogel, W.; Andrussow, K. Dissociation constants of organic acids in
aqueous solution, Pure Appl. Chem. 1961, 1, 190-536.

(48) Wiberg, K. B.; Pratt, W. E.; Bailey, W. F. Nature of substituent effects in nuclear
magnetic resonance spectroscopy. 1. Factor analysis of carbon-13 chemical shifts in aliphatic
halides, J. Org. Chem. 1980, 45, 4936-47.

(49) Moffatt, J. G.; Khorana, H. G. Nucleoside polyphosphates. X. The synthesis and some
reactions of nucleoside 5'-phosphoromorpholidates and related compounds. Improved
methods for the preparation of nucleoside 5'-polyphosphates, J. Am. Chem. Soc. 1961, 83,
649-58.

(50) Braese, S.; Gil, C.; Knepper, K.; Zimmermann, V. Organic azides. An exploding
diversity of a unique class of compounds, Angew. Chem., Int. Ed. 2005, 44, 5188-240.

(51) Scriven, E. F. V.; Turnbull, K. Azides: their preparation and synthetic uses, Chem. Rev.
1988, 88, 297-368.

(52) Amantini, D.; Fringuelli, F.; Pizzo, F.; Vaccaro, L. Selected methods for the reduction
of the azido group, Org. Prep. Proced. Int. 2002, 34, 109,11-47.

(53) Kaabak, L. V.; Kuz'mina, N. E.; Khudenko, A. V.; Tomilov, A. P. Improved synthesis of
1-aminoethylidenediphosphonic acid, Russ. J. Gen. Chem. 2006, 76, 1673-74.

(54) Orlovskii, V. V.; Vovsi, B. A. Reaction of dialkyl phosphites with nitriles, Zh. Obshch.
Khim. 1976, 46, 297-300.

(55) Kantoci, D.; Denike, J. K.; Wechter, W. J. Synthesis of aminobisphosphonate, Synth.
Commun. 1996, 26, 2037-43.

(56) Fringuelli, F.; Pizzo, F.; Vaccaro, L. Cobalt(II) chloride-catalyzed chemoselective
sodium borohydride reduction of azides in water, Synthesis 2000, 646-50.

(57) Midrier, C.; Lantsoght, M.; Volle, J.-N.; Pirat, J.-L.; Virieux, D.; Stevens, C. V.
Hydrophosphonylation of alkenes or nitriles by double radical transfer mediated by
titanocene/propylene oxide, Tetrahedron Lett. 2011, 52, 6693-96.

(58) Kashemirov, B. A.; Skoblikova, L. I.; Savenkov, N. F.; Khokhlov, P. S. 1,2-C →N
migration of a phosphoryl group during deoxygenation of 1-(methoxycarbonyl)-1-nitroso-1-
(diethoxyphosphoryl)ethane, Zh. Obshch. Khim. 1990, 60, 1184-5.

47

(59) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 1997, 62, 7512-15.

(60) Burton, D.; Pietrzyk, D.; Ishihara, T.; Fonong, T.; Flynn, R. Preparation, stability and
acidity of difluoromethylene bis phosphonic acid, J. Fluorine Chem. 1982, 20, 617-26.

48

CHAPTER 3. α,β-CXY dATP Probes for DNA Polymerase β: Binding
Affinities and Structural Evidence for a Non-covalent C-F Interaction
*

3.1 Introduction
Fluorinated compounds account for an ever-growing proportion of medicines and
bioactive compounds and, as such, the non-covalent bonding interactions of compounds
containing C-F functionalization are of broad interest.
1
As the evidence for the unique
biochemical behavior of fluorinated ligands continues to mount,
1,2
controversy persists as to
whether these experimental findings are due C-F···H-X hydrogen bonding, or if more general
phenomena, such as electrostatic interactions of the C-F dipole
3
and/or favorable changes in
entropy accompanying fluorine desolvation (i.e. polar hydrophobic interactions)
4
are at play.
The division on this issue is dramatic: on one hand, some structural biologists often identify
and classify fluorine hydrogen bonding solely on the basis of short intermolecular C-F···H-X
bond distances;
5
while on the other, certain theoreticians claim that short C-F···H-X bond
distances can be energetically insignificant
6
and that fluorine "hardly ever" accepts
hydrogen bonds.
7

An interesting note recently added to the fluorine hydrogen bonding discussion is
the notion that different types of fluorine have different potentials for hydrogen bonding.
8

*
Excerpts reproduced with permission from Chamberlain, B. T., Batra, V. K., Beard, W. A., Kadina, A. P.,
Shock, D. D., Kashemirov, B. A., McKenna, C. E., Goodman, M. F., Wilson, S. H. ChemBioChem. 2012, 13 (4),
528-530. Copyright 2012 John Wiley and Sons Inc. Information on author contributions can be found in
the Acknowledgements section. The original publication is cited in this chapter to refer to biochemical or
structural details contained in the original publication’s supporting information.

49

Simply put, the organofluorine electron density determines a compound’s ability to
participate in C-F bonding interactions. Fluorinated ligands with high electron density, such
as our previously studied β,γ-CXF dGTP analogs
9
have a greater capacity to participate in
dipolar interactions than electron-poor C-F groups (like those found in difluorotoluene
10
).
Previously several α,β-methylene-dNTP analogs were studied as probes for the
capture of pre-insertion substrate complexes with pol β and it was found that α,β-CF
2
-
analogs have an apparent equilibrium dissociation constant (K
d
) that is two orders of
magnitude greater than the corresponding α,β-CH
2
-analogs and the β,γ-dGTP-CF
2
-
analog.
11,12
To further explore the molecular basis of pol β nucleotide binding, the
dimethylated (α,β-C(Me)
2
-, 5) and the monofluorinated (R/S)-(α,β-CHF-, 6a/b) dATP analogs
were synthesized and, together with the available α,β-CXN
3
- (8a/b: X = H, 9a: X = Me, 9b: X=
Me) dATP analogs,
13
their K
d
values were determined. The X-ray crystallographic structures
of ternary substrate complexes of pol β and DNA primer template formed from α,β-CF
2
-
dATP
11
(7) and 6a/b were solved. Interestingly, only one diastereomer of 6 is found in the
active site which provides evidence for a non-covalent C-F···H-X interaction with a structural
water bound to pol β active site Asp 276 (Figure 3.1.1).

50

Figure 3.1.1. X-ray crystal structure of the DNA pol  active site containing (S)- , -CHF-dATP (yellow
carbons). The solvent-excluded pol  active-site surface is colored according to atom (carbon, gray;
nitrogen, light blue; oxygen, pink; magnesium, dark blue spheres). The surface of Asp276 has been made
transparent. The fluorine atom (light green) is 2.8 Å from the oxygen of a conserved water molecule
(small red sphere) that is within hydrogen bonding distance (green solid lines) of the oxygen of an
adjacent water (2.6 Å), the carboxyamide nitrogen (2.8 Å) and a carboxylate anion oxygen of Asp276 (2.9
Å), as well as the phosphoryl oxygen of P

(2.8 Å), which accepts a hydrogen bond from the 3’-OH (2.8 Å).
The 3´-primer terminal nucleoside is shown (gray carbons), but its backbone is omitted for clarity.

3.2 Results and discussion
3.2.1 Synthesis of α,β-CXY-dATP analogs
5 and 6a/b were prepared by reaction of 2’-deoxyadenosine 5’-tosylate with the
tris(tetrabutylammonium) salt of the corresponding bisphosphonic acid followed by
enzymatic phosphorylation of the resulting dA 5’-bisphosphonate
11
(Scheme 3.2.1). In a
previously reported synthesis of 6a/b, in which (R/S)- , -CHF-dADP (4a/b) was
phosphorylated with p-nitrobenzyl phosphormorpholidate,
14
an approximately 1:1 mixture
of , -CHF-dATP diastereomers was observed in the
19
F NMR spectrum. Similarly,

51

phosphorylation of 4a/b using nucleoside diphosphate kinase and a catalytic amount of ATP
generates both diastereomers, as observed in the
19
F and
31
P (P
α
) NMR spectra (Appendix C;
figures C4, C5, C14-C18). Although precise quantification is not possible due to signal
overlap, according to
19
F NMR analysis, the isomers are in roughly similar abundance; this
indicates that the enzymatic phosphorylation did not significantly enrich a particular isomer.
Scheme 3.2.1. Synthesis of α,β-CXY-dATP analogs 5 and 6a/b..
P
O
P
O
OH
O
O
O
X
Y
3(Bu
4
N
+
)
O
OH
O
A
P
O
O
P
O
X
Y
HO
O
O
OH
O P
O
O
P
O
X
Y
O P
O
O
HO
O
N
N
N
N
NH
2
P
O
P
O
Oi-Pr
Oi-Pr
i-PrO
i-PrO
dA-5'-OTs
PK, PEP
NDPK, ATP
cat.
1: X = Y = Me
2: X = H, Y = F
3: X = Y = Me
4a/b: X = H, Y = F
5: X = Y = Me; 6a/b: X = H, Y = F

3.2.2 α,β-CXY-dATP binding affinities
As expected, 5, 6a/b, 8a/b, 9a, and 9b are not pol β substrates and inhibit gap-filling
DNA synthesis by pol β in a concentration dependent manner
15
(Appendix C, figure C22).
The apparent equilibrium binding constant for 6a/b is approximately 10 μM, similar to that
of the natural substrate and significantly lower than that of the α,β-CF
2
-dATP analog (Figure
3.2.1).
11
The , -CH
2
-dATP analog binds with approximately tenfold higher affinity
11,16
than
6a/b; this is consistent with a correlation between the basicity of the phosphonate moiety
and the strength of inhibitor binding.
11
However, , -C(Me)
2
-dATP (5) which has two
electron-donating substituents on the , -methylene, has a K
d
of approximately 1100 µM
and does not conform to this trend.

52

Although the K
d
values for 9a and 9b were only estimated,
†
they display
unmistakably distinct K
d
values (~470 μM and ~8000 μM respectively). When considered
together with the poor binding affinity of 5, these values emphasize the adverse effect on
binding with pol β caused by bulky substitution in α,β-CXY dATP analogs. Remarkably, the
pronounced steric profile of the azido group in 8a/b does not prohibit binding with the pol β
active site (K
d
= 5.9 μM). This value conforms to a potential trend between phosphonate
basicity and the strength of dNTP binding while adding the qualification that a single non-
bulky substituent in the α,β position of dNTP analogs might be allowable while still
maintaining potent activity.

Figure 3.2.1. DNA pol  dissociation constants of dATP and , -CXY dATP analogs. The data for dATP, , -
CH
2
-dATP, and , -CF
2
-dATP were previously published.
11,17
The K
i
(i.e., K
d
) values were determined as
described previously.
18
dAMP-CHN
3
-PP (*) is a provisional value.
ƚ

†
Whereas the K
d
values for 5 and 6a/b were determined using an optimized concentration of dATP and
were measured using at least 10 different concentrations of the inhibitor, the K
d
values for 9a and 9b
were determined from a single inhibitor concentration in the presence of 1 μM dATP (Appendix C, figure
C22). These conditions are suitable for estimating activity, but should be compared with the more
accurately determined values with caution. The observed K
d
for 8a/b determined from 5 inhibitor
concentrations in the presence of 1 μM dATP is a provisional value (Appendix C, figure C23).

53

3.2.3 X-ray crystal structures of pol β-DNA-dATP analog ternary complexes
The crystallographic structures, resolved at 2.00 Å,
15
of ternary complexes of pol 
with incoming , -CF
2
-dATP (7; PDB ID 3TFR) or ,-CHF-dATP (6; PDB ID 3TFS) opposite a
templating thymidine were next determined. All corresponding atoms in the active site of
these structures superimpose well with previously determined ternary complex structures
of pol  in which the reaction was stopped by deletion of the nucleophilic 3’-OH on the
primer terminus or by using a nitrogen in place of the , -bridging oxygen (Figure 3.2.2A).
18

Thus, , -CHF-dATP and 7 are well accommodated in the polymerase active site as has been
shown previously for , -CF
2
-dTTP.
11

Incubation of crystals of a binary DNA complex of pol β with the ,β-CHF-dATP
diastereomer mixture (6a/b) resulted in a complex in which only the (S)-CHF-stereoisomer
(6b) could be found by X-ray analysis (Figures 3.2.1 and 3.2.2B). Modeling (R)- ,β-CFH-dATP
(6a) into the electron density failed to account for the observed density (Figure 3.2.2C). The
close overlap of the CF
2
and the (S)-CHF conformations indicates that exclusion of 6a analog
is not the result of an unattainable binding conformation or destabilizing steric interaction
(Figure 3.2.2B). As was reasoned previously,
19,20
stabilization of as little as ~1 kcal/mol could
be sufficient to generate the selectivity observed in the crystal structures.

54

Figure 3.2.2. A) The ternary complex crystallographic structures of pol  with an incoming , -CF
2
-dATP
(7; gray carbons) and , -NH-dUTP (light blue carbons; PDB ID 2FMS)
18
were superimposed using all 326
C  (rmsd = 0.29 Å). The incoming nucleoside triphosphates are shown as well as the primer terminal
nucleoside (O3´ is labeled). The position of the active site Mg
2+
(spheres) are colored according to the
respective carbons. A and B refer to the catalytic and nucleotide binding metal, respectively. B) The
ternary complex crystallographic structures of pol  with an incoming 7 (gray carbons) and , -CHF-dATP
(6b; yellow carbons) were superimposed using all 326 C  (rmsd = 0.15 Å). C) A difference density map
generated using the (R)-CHF isomer shows positive (green, contoured at 3.2 ) and negative density (red,
contoured at 4.0 ) in the vicinity of the position of the proposed fluorine atom; this indicates that the
orientation for the (R)-isomer is not correct.

In an earlier study, a series of stereospecifically formed pol β-DNA-dGTP ternary
complexes were reported that provided evidence for a stabilizing polar interaction between
the active site Arg183 and the chiral phosphonate C-F in CHF, CMeF, and CClF dGTP-β,γ
analogs.
19,20
Unlike the β,γ-CXF-dGTP-DNA-pol β ternary complexes, in this structure no
direct stabilizing interactions with active site residues can be identified. The structure does,
however, reveal that the S isomer fluorine is within 2.8 Å (B-factor: 15.4 Å
2
)

of an oxygen in
a structural water molecule, present in both structures (for CF
2
distance = 2.9 Å, B-factor:
15.1 Å
2
), that is within hydrogen bonding distance of several key atoms (a carboxylate anion
oxygen and carboxyamide nitrogen of Asp276, phosphoryl oxygen of P
β
; Figure 3.1.1).
Observing no similar interactions with the pro-R-fluorine in the CF
2
-structure, one might
consider a weak interaction of the S fluorine in 6b with structural water to be the source of

55

its stabilization in the complex relative to 6a; however, other factors might be invoked
6
and
further work will be necessary to clarify the origin of the observed stereospecificity.

3.2.4 Molecular docking studies with Autodock Vina
Molecular docking of 5 and 6 with pol β-DNA binary complex using Autodock Vina
21

suggests that the unexpectedly low binding affinity of 5 can be attributed to an unfavorable
steric interaction, specifically a clash of the methyl groups of 5 with a structural water
bound to Asp276 (PDB ID 3TFS, water 3). When docked in the absence of this structural
water, the lowest energy conformations for both 5 and 6 overlap well with the coordinates
of the 6 found in the crystal structure, whose conformation is congruent with those of DNA
pol β-bound ,β-CF
2
-dTTP
11
and ,β-NH-dUTP.
18
However, when docking with a
macromolecule file prepared to include the water bound to Asp276, 5 is unable to achieve
the experimentally observed conformation, whereas the geometry of 6 is unperturbed
(Figure 3.2.3).

Figure 3.2.3. Geometries of the lowest energy conformations calculated by docking dATP analogs with
the pol β-DNA binary complex (PDB ID 3TFS). The coordinates of the crystallized ligand are shown in grey,
the docked molecule is multi-colored, Asp276 is colored cyan, magnesium ions are green, and the oxygen
atoms of water are shown in red. A) 5 displays a good overlap with the coordinates of the crystallized
ligand in the absence of the water bound to Asp276 (ID water 3). B) 6b overlaps well with the crystallized
ligand even in the presence of the water near Asp276 (highlighted with the black arrow). C) 5 does not
achieve good alignment in the presence of the water 3.

56

3.3 Conclusion
In summary, our results examine the factors affecting the nucleotide binding
properties of DNA pol β. K
d
determinations for a series of ,β-CXY substituted dATP analogs
display a correlation with the basicity of the bisphosphonate moiety that seems valid in the
absence of two bulky X,Y substituents; ,β-C(Me)
2
-dATP and both ,β-CMeN
3
-dATP analogs
display larger K
d
values than their pKa values would predict, whereas the K
d
values of ,β-
CHF-dATP and ,β-CHN
3
-dATP are consistent with their relative acidity. The larger than
expected K
d
of ,β-C(Me)
2
-dATP is attributed to an unfavorable steric interaction of the
methyl substituents with the structural water bound to Asp276 by molecular docking. The
X-ray crystal structure of ,β-CHF-dATP in complex with pol β and DNA contains only one
diastereomer, despite the presence of both diastereomers in the crystallization mixture.
Comparison of this structure with that of ,β-CF
2
-dATP raises the possibility of a non-
covalent weak interaction between the ligand and the active-site structural water proximal
to Asp276. Our structural observations may be worth taking into account in PDB
studies
7,22,23
searching for fluorine/protein interactions.

3.4 Experimental
3.4.1 Materials and methods
All chemicals and reagents were purchased from Sigma-Aldrich Inc and used as
received. RP HPLC purification was conducted using a Varian 21.4 mm x 250 mm
MICROSORB 100-5 C-18 column eluted with isocratic 0.1 N triethylammonium bicarbonate

57

(TEAB) pH 7.5 buffer containing 4% acetonitrile pumped at 10 mL/min. Strong Anion
Exchange (SAX) HPLC chromatography was performed using a Macherey-Nagel 21.4 mm x
250 mm SP15/25 Nucleogel column eluted with A: H
2
O, B: 0.5 N TEAB pH7.5 using a
gradient that increased from 0-40% over 10 minutes, was level at 40% from 10-15 min, and
then increased to 100% from 15-25 minutes using an 8 mL/min flow rate. Analytical HPLC
analysis was conducted on a Varian PureGel SAX 10 mm x 100 mm 7 μL column eluted with
A: H
2
O, B: 0.5 M (0-50% linear) LiCl gradient over 30 min at a 4 mL/min flow rate. Products
were detected at 259 nm. Mass of final products was determined by UV absorbance using
the extinction coefficient of dATP, ε = 15300.
24

1
H NMR spectra were referenced to residual CHCl
3
(δ 7.24) in CDCl
3
or to HDO (δ
4.79) in D
2
O.
25

31
P NMR spectra were referenced to external H
3
PO
4
(δ 0.00) and
19
F NMR
spectra were referenced to external hexafluorobenzene, C
6
F
6
in benzene (δ -164.9). pH
values measured in 99.9% D
2
O and reported as measured without deuterium isotope
correction. NMR δ values are reported in ppm while coupling constants (J) are reported in
Hz. 1D NMR spectra processing and 1D NMR simulations were performed with NUTS Pro for
Windows (Acorn, Inc.) while gCOSY spectra were processed using MestReNova v. 6.2.1
(Mestrelab Research S.L.) and used to support proton assignments. High resolution mass
spectra were obtained by the UC Riverside mass spectrometry facility.
Information regarding DNA synthesis assays, K
d
determinations, and crystallographic
procedures and statistics is available in the supporting information of the original
publication.
15
Images of
1
H-NMR spectra (5, 6 a/b);
19
F-NMR, with simulations (4a/b, 6a/b);
31
P-NMR spectra (3-6a/b); gCOSY (5, 6a/b); HPLC of enzymatic phosphorylation reaction

58

mixture, 5, 6a/b; HR mass spectra (5); LR mass spectra (6a/b); and gel analysis of 8a/b, 9a,
and 9b incorporation into single gap DNA and inhibition of dATP incorporation are
presented in Appendix C.

3.4.2 Synthetic procedures
Propane-2,2-diylbis(phosphonic acid) tris(tetrabutylammonium) salt, 1. To a
solution of 0.50 g (1.45 mmol) tetraisopropyl methylenebis(phosphonate) in 10 mL
anhydrous DMSO, 0.49 g (3 equiv, 4.37 mmol) potassium tert-butoxide powder was added
at rt. After several minutes of stirring 0.20 mL MeI (2.2 equiv, 0.453 g, 3.19 mmol) was
added and stirred at rt for 2 h. Reaction mixture was extracted with hexanes (3 x 25 mL)
and dried over Na
2
SO
4
. Solvent was removed on rotovap to yield 0.52 g (95%) of
tetraisopropyl propane-2,2-diylbis(phosphonate) which was used without further
purification. δ
H
(600 MHz, CDCl
3
): 1.33 (m, 24H), 1.37 (t, J
HP
16.2 Hz, 6H), 4.77 (m, 4H);

δ
P

(293 MHz, CDCl
3
): 26.4 (s). Lit.
26
: δ
H
1.12-1.68 (unresolved from P-C(CH
3
)-P (t) and
(CH
3
)
2
CHO-, 30 H), 4.81 (m, 4H).
0.52 g (1.38 mmol) tetraisopropyl propane-2,2-diylbis(phosphonate) was then
dissolved in 4 mL HCl (conc.) and refluxed for several hours to yield the bisphosphonic acid
in quantitative yield. After removing solvent on rotovap, water was added and removed
three times. δ
H
(600 MHz, D
2
O, pH < 1): 1.40 (t, J
HP
16.4 Hz); δ
P
(293 MHz, D
2
O, pH1.0): 27.8
(s). Lit.
26
: δ
H
(CH
3
CN std.):

~1

.32. The acid was then dissolved in 2 mL of H
2
O and titrated to
pH 11.7 (pKa
4
12.2) with tetrabutylammonium hydroxide (40% solution in H
2
O), solvent was
removed and the salt was dried by co-evaporation with anhydrous acetonitrile.

59

(Fluoromethanediyl)bis(phosphonic acid) tris(tetrabutylammonium) salt, 2. To a
solution of 2.50 g (7.26 mmol) tetraisopropyl methylenebis(phosphonate) in 20 mL
anhydrous DMF, 8.7 mmol NaH suspended in DMF was added at 0° . The mixture was
stirred at 0° C for 5 min, then at rt for 1 h, and then cooled to 0° C for 10 min. 3.08 g (8.70
mmol) of solid Selectfluor
TM
was rapidly added to the mixture which was then allowed to
warm to rt. After 2 h the reaction mixture cooled to 0 °C and quenched by addition of a
saturated solution of ammonium chloride (25 mL) and extracted with CH
2
Cl
2
(3 x 50 mL)
.

The organic layer was dried over MgSO
4
, concentrated and purified by silica gel
chromatography to yield 0.85 g (32%) of tetraisopropyl
(fluoromethanediyl)bis(phosphonate). δ
H
(500 MHz, CDCl
3
): 1.33 (m, 24H), 4.82 (m, 5H);

δ
F
(564 MHz, CDCl
3
): -228.5 (dd, J
FP
65 Hz, J
FH
45 Hz); δ
P
(243 MHz, CDCl
3
): 10.1 (d, J
PF
65 Hz).
Lit
27
: δ
H
: 1.26 (d), 4.77 (m), 4.82 (dt); δ
F
(neat): 221 (dt, 63 Hz, 44 Hz); δ
P
(neat): 10.7 (d, 63
Hz).
0.24 g (0.66 mmol) of tetraisopropyl (fluoromethanediyl)bis(phosphonate) was
dissolved in 2 mL HCl (conc.) and refluxed for 5 h to yield the bisphosphonic acid in
quantitative yield. After removing solvent on rotovap, water was added and removed
several times. δ
H
(400 MHz, D
2
O, pH < 1): 4.65 (dt, J
HF
45.0 Hz, J
HP
12.5 Hz); δ
P
(162 MHz,
D
2
O, pH1.0): 10.3 (d, J
PF
64.8 Hz). Lit.
27
: δ
P
(D
2
O) 10.5 (d, J
PF
64 Hz). The acid was then
dissolved in 2 mL H
2
O and titrated to pH 8.5 (pKa
4
9.0) tetrabutylammonium ammonium
hydroxide (40% solution in H
2
O), solvent was removed and the residue was dried by co-
evaporation with acetonitrile.

60

2’-Deoxyadenosine 5’-diphosphate α,β-C(Me)
2
-dADP, 3, and 2’-deoxyadenosine 5’-
diphosphate α,β-CHF-dADP, 4a/b. 0.50 g 2’-deoxyadenosine monohydrate (mw = 269.3;
1.9 mmol) was first dissolved in water and then diluted with pyridine and the solvent
removed on rotovap. The resulting film was dried by evaporation of anhydrous pyridine
three times. The film was then dissolved 15 mL anhydrous pyridine and stirred on ice bath.
400 mg (1.1 equiv) TsCl was dissolved in 2 mL anhydrous pyridine and added at once by
syringe under N
2
causing the solution to turn pale yellow. After 1 h of stirring the mixture
became completely dissolved. After several hours on ice bath the solution was brought to rt
and stirred overnight.
1
H NMR showed 88% conversion (5’-CH
2
OH ≈ 3.8 ppm; 5’-CH
2
OTs ≈
4.3 ppm). Pyridine was removed on rotovap and the concentrate was dissolved in 20 mL
cold H
2
O, extracted into ethyl acetate, and washed with NaHCO
3
solution. The organic layer
was dried over Na
2
SO
4
and concentrated to yield 328 mg of crude compound as a pale
yellow foam which was used without further purification.
150 mg of the crude 2’-deoxyadenosine 5’-tosylate was dissolved in 3 mL anhydrous
acetonitrile and stirred under nitrogen. To this, a solution of tris-tetrabutylammonium salt
of the requisite bisphosphonic acid (estimated 1.25 equiv as related to the 5’-tosylate) in 1
mL anhydrous acetonitrile was added by syringe at room temperature. After 24 h, water
was added and the reaction mixture was concentrated on rotovap and purified by dual pass
HPLC to yield the α,β-dADP intermediates as the triethylammonium salts. α,β-C(Me)
2
-dADP
(3): 91 mg (apprx 15% from the starting dA); δ
P
(162 MHz, D
2
O, pH 10.8): 21.5 (d, J
αβ
4 Hz,
P
β
), 31.9 (d, J
αβ
4Hz, P
α
). α,β-CHF-dADP (4a/b): 57 mg (apprx 10% from the starting dA); δ
F
(376 MHz, D
2
O, pH 10.8): -218.5 (ddd, J
FPα
63 Hz, J
FPβ
56 Hz, J
FH
45 Hz), -218.8 (ddd, J
FPα
63

61

Hz, J
FPβ
56 Hz, J
FH
45 Hz); δ
P
(162 MHz, D
2
O, pH 10.8): 7.8 (dd, J
PF
56 Hz, J
αβ
12 Hz, P
β
), 14.6
(dd, J
PF
63 Hz, J
αβ
12 Hz, P
α
).
2’-Deoxyadenosine 5’-triphosphate α,β-C(Me)
2
, 5. 27.9 mg (0.064 mmol) as
determined by UV absorbance of α,β-C(Me)
2
-dADP is dissolved in 2 mL 50 mM HEPES buffer
pH 7.5 with 37 mg PEP trisodium salt hydrate (2.5 equiv), 27 mg KCl (5.6 equiv), and 23 mg
MgCl
2
hexahydrate (1.8 equiv). To this solution, 3 units pyruvate kinase, 3 units of
nucleoside diphosphate kinase, and 3 mol % ATP (0.96 mg) dissolved in 200 μL HEPES buffer
was added and shaken at 37° C for 48 h.
28
Analytical HPLC showed ~80% conversion to the
triphosphate analog. Solutions were filtered through Pall Nanosep 10K Omega filters and
purified by dual-pass HPLC (SAX then C-18 RP) yielding 15.5 mg of isolated final product
(47%). Triethyl amine was removed by adding Na
2
CO
3
and removing volatiles on rotovap.
pH was readjusted to 8 with dilute HCl. Analytical HPLC: retention time: 10.9 min; >99%
purity. δ
H
(400 MHz, D
2
O, pH 7.2): 1.30 (t, J
HP
15.5 Hz, 6H), 2.62 (m, 1H), 2.86 (m, 1H), 4.13
(m, 1H), 4.20 (m, 1H), 6.49 (t, 6.4 Hz, 1H), 8.25 (s, 1H), 8.52 (s, 1H) 8.53 (s, 1H); δ
P
(162 MHz,
D
2
O, pH 10.8): -4.5 (d, J
γβ
31 Hz, P
γ
), 18.0 (dd, J
βα
5Hz, J
βγ
31 Hz, P
β
), 28.3 (d, J
αβ
5Hz, P
α
);
HRMS (ESI/ APCI) [M-H]
-
: calcd for C
13
H
21
N
5
O
4
P
3
, 516.0456; found, 516.0432 m/z.
2’-Deoxyadenosine 5’-triphosphate α,β-CHF, 6a/b. 32.4 mg (0.076 mmol) as
determined by UV absorbance of α,β-CHF-dADP was dissolved in 2mL 50 mM HEPES buffer
pH 7.5 with 44.3 mg PEP trisodium salt hydrate (2.5 equiv), 32 mg KCl (5.6 equiv), and 28 mg
MgCl
2
hexahydrate (1.8 equiv). To this solution, 3 units pyruvate kinase, 3 units of
nucleoside diphosphate kinase, and 3 mol % ATP (1.3 mg) dissolved in 200 μL HEPES buffer
was added and shaken at 37°C for 48 h. Analytical HPLC showed ~48% conversion to the

62

triphosphate analog. Solutions were filtered through Pall Nanosep 10K Omega filters and
purified by dual-pass HPLC (SAX then C-18 RP) yielding 4.7 mg of isolated final product (12%).
Triethyl amine was removed by adding Na
2
CO
3
and removing volatiles on rotovap. pH was
readjusted to 8 with dilute HCl. Analytical HPLC: retention time: 13.8 minutes; >99% purity.
δ
H
(600 MHz, D
2
O, pH 8.2): 2.60 (m, 1H), 2.83 (m, 1H), 4.19 (m, 1H) 4.24 (m, 1H), 4.29 (m,
1H), 5.15 (dt, J
HF
45.3 Hz, J
HP
12.6 Hz, 1H), 6.51 (t, J 6.6 Hz, 1H), 8.24 (s, 1H), 8.51 (s, 1H); δ
F
(564 MHz, D
2
O, pH 10.8 [pH 7.2]): -220.85 (dt, J
FP
60 Hz, J
FH
45 Hz), [-219.6], -221.15 (dt, J
FP

60 Hz, J
FH
45 Hz), [-219.9]; δ
P
(162 MHz, D
2
O, pH 10.8 [pH 7.2]): -4.7 (d, J
γβ
24 Hz, P
γ
), [-6.0],
0.4 (ddd, J
PF
60 Hz, J
βγ
24 Hz, J
βα
14 Hz, P
β
), [0.4], 11.97 (dd, J
PF
61 Hz, J
αβ
14.5 Hz, P
α2
), 12.03
(dd, J
PF
61 Hz, J
αβ
14.5 Hz, P
α1
), [11.7]; Lit.
14
(pH not reported): δ
F
: -219.2, -219.4(J
FP
60.9, J
FP
60.1); δ
P
-5.1 (J
αβ
15.8 Hz), -0.6, 11.3 (J
γβ
25.4Hz); LRMS [M-H]
-
: calcd C
11
H
16
FN
5
O
11
P
3
, 506.1;
found, 506.0 m/z.

3.4.3 Molecular docking
Docking studies were performed using Autodock Vina 1.0.2 software.
21
The
exhaustiveness parameter value was set to 64 and all other parameters were left as default
values. The cell dimensions were 30 Å
3
centered in the active site with coordinates x=2.181,
y=4.089, z=16.352. Receptor was kept rigid while all rotatable bonds of the ligand were
allowed to rotate freely. Every calculation was repeated at least 3 times.
The crystal structure of ternary complex of polymerase β-DNA-α,β-
fluorodeoxyadenosine triphosphate (PDB ID 3TFS) was used for docking studies. Water was
removed from the files except for waters 4, 171, and 235 which were left in the active site

63

for all calculations. The influence of water number 3 was tested by including it in some
calculations and omitting it in others as described. Receptor file was prepared with
Autodock Tools 1.5.4 and Accelrys Discovery Studio 2.5.
Ligands were prepared by calculating the optimum geometry and atomic charges
with density functional calculations using the B3LYP 6-31G* basis set (Spartan’08 software).
Total charge of the ligand set to -3. Using Autodock Tools 1.5.4, AD4 type assignment was
applied to all ligand atoms before converting the file to pdbqt format. The images were
prepared using Chimera 1.5.3.
29

64

3.5 Chapter references
(1) Mueller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals: Looking Beyond
Intuition, Science (Washington, DC, U. S.) 2007, 317, 1881-86.

(2) Berkowitz, D. B.; Karukurichi, K. R.; de, l. S.-B. R.; Nelson, D. L.; McCune, C. D. Use of
fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical
advantages, J. Fluorine Chem. 2008, 129, 731-42.

(3) O'Hagan, D. Understanding organofluorine chemistry. An introduction to the C-F
bond, Chem. Soc. Rev. 2008, 37, 308-19.

(4) Biffinger, J. C.; Kim, H. W.; DiMagno, S. G. The polar hydrophobicity of fluorinated
compounds, ChemBioChem 2004, 5, 622-27.

(5) Xia, S.; Konigsberg, W. H.; Wang, J. Hydrogen-Bonding Capability of a Templating
Difluorotoluene Nucleotide Residue in an RB69 DNA Polymerase Ternary Complex,J. Am.
Chem. Soc. 2011, 133, 10003-05.

(6) Dunitz, J. D.; Schweizer, W. B. Molecular pair analysis: C-H···F interactions in the
crystal structure of fluorobenzene? And related matters, Chem.--Eur. J. 2006, 12, 6804-15.

(7) Dunitz, J. D.; Taylor, R. Organic fluorine hardly ever accepts hydrogen bonds, Chem.--
Eur. J. 1997, 3, 89-98.
(8) Dalvit, C.; Vulpetti, A. Intermolecular and Intramolecular Hydrogen Bonds Involving
Fluorine Atoms: Implications for Recognition, Selectivity, and Chemical Properties,
ChemMedChem 2012, 7, 262-72.

(9) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T.
G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA
Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding
of the Fluoromethylene Analogues, J. Am. Chem. Soc. 2010, 132, 7617-25.

(10) Kool, E. T.; Sintim, H. O. The difluorotoluene debate-a decade later, Chem. Commun.
(Cambridge, U. K.) 2006, 3665-75.

(11) Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.;
Kultyshev, R.; Batra, V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. α,β-
Diflurormethylene deoxynucleoside 5'-triphosphates: A convenient synthesis of useful
probes for DNA polymerase β structure and function, Org. Lett. 2009, 11, 1883-86.

65

(12) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA polymerase β
fidelity: Halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal
differences at the chemical transition state, Biochemistry 2008, 47, 870-79.

(13) Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E. α-Azido
Bisphosphonates: Synthesis and Nucleotide Analogs, J. Org. Chem. 2011, 76, 5132-36.

(14) Blackburn, G. M.; Langston, S. P. Novel P1,P2-substituted phosphonate analogs of 2'-
deoxyadenosine and thymidine 5'-triphosphates, Tetrahedron Lett. 1991, 32, 6425-8.

(15) Chamberlain, B. T.; Batra, V. K.; Beard, W. A.; Kadina, A. P.; Shock, D. D.; Kashemirov,
B. A.; McKenna, C. E.; Goodman, M. F.; Wilson, S. H. Stereospecific Formation of a Ternary
Complex of (S)-α,β-Fluoromethylene-dATP with DNA Pol β, ChemBioChem 2012, 13, 528-30.

(16) Liang, F.; Jain, N.; Hutchens, T.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; Chiarelli, M.
P.; Cho, B. P. α,β-Methylene-2'-deoxynucleoside 5'-triphosphates as noncleavable
substrates for DNA polymerases: Isolation, characterization, and stability studies of novel 2'-
deoxycyclonucleosides, 3,5'-cyclo-dG, and 2,5'-cyclo-dT, J. Med. Chem. 2008, 51, 6460-70.

(17) Radhakrishnan, R.; Arora, K.; Wang, Y.; Beard, W. A.; Wilson, S. H.; Schlick, T.
Regulation of DNA repair fidelity by molecular checkpoints: "Gates" in DNA polymerase β's
substrate selection, Biochemistry 2006, 45, 15142-56.

(18) Batra, V. K.; Beard, W. A.; Shock, D. D.; Krahn, J. M.; Pedersen, L. C.; Wilson, S. H.
Magnesium induced assembly of a complete DNA polymerase catalytic complex, Structure
(Camb) 2006, 14, 757-66.

(19) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T.
G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-methylene- and ethylidene-dGTP-DNA
ternary complexes with DNA polymerase β: Structural evidence for stereospecific binding of
the fluoromethylene analogues, J. Am. Chem. Soc. 2010, 132, 7617-25.

(20) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.;
Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-fluoromethylene-dGTP-DNA ternary
complex with DNA polymerase β, J. Am. Chem. Soc. 2007, 129, 15412-13.

(21) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem.
2010, 31, 455-61.

66

(22) Dalvit, C.; Vulpetti, A. Fluorine-Protein Interactions and 19F NMR Isotropic Chemical
Shifts: An Empirical Correlation with Implications for Drug Design, ChemMedChem 2011, 6,
104-14.

(23) Carosati, E.; Sciabola, S.; Cruciani, G. Hydrogen Bonding Interactions of Covalently
Bonded Fluorine Atoms: From Crystallographic Data to a New Angular Function in the GRID
Force Field, J. Med. Chem. 2004, 47, 5114-25.

(24) Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical
Research, third ed.; Clarendon: Oxford, England, 1986.

(25) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 1997, 62, 7512-15.

(26) Menge, M.; Muenzenberg, K. J.; Reimann, E. 2,2-Propane diphosphonic acid, Arch.
Pharm. (Weinheim, Ger.) 1981, 314, 218-22.

(27) McKenna, C. E.; Shen, P.-D. Fluorination of methanediphosphonate esters by
perchloryl fluoride. Synthesis of fluoromethanediphosphonic acid and
difluoromethanediphosphonic acid, J. Org. Chem. 1981, 46, 4573-76.

(28) Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.;
Kultyshev, R.; Batra, V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. α,β-
Difluoromethylene Deoxynucleoside 5'-Triphosphates: A Convenient Synthesis of Useful
Probes for DNA Polymerase β Structure and Function, Org. Lett. 2009, 11, 1883-86.

(29) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng,
E. C.; Ferrin, T. E. UCSF Chimera-A visualization system for exploratory research and analysis,
J. Comput. Chem. 2004, 25, 1605-12.

67

CHAPTER 4. Examining the Stability of the P
α
-O-P
β
Bond in Triphosphate
Monoesters
*

4.1 Introduction
Reactions involving triphosphates are central to biology and are involved in energy
storage and transfer, signaling processes, and DNA/ RNA replication and repair.
1
Our
interest in DNA polymerase β (pol β) mechanism and fidelity led us to investigate the rate of
incorporation of a series of 2’-deoxynucleoside triphosphate analogs that contained a
modified pyrophosphate leaving group and could thus probe the importance of P
α
-O bond
breakage relative to other steps in the nucleotidyl transfer reaction.
2
As characterizing the
rate and temperature dependence of a biologically relevant reaction in the absence of a
catalyst is of fundamental theoretical interest,
3
we attempted to use modified triphosphate
compounds, similar to those studied with pol β, to determine the stability of the
triphosphate P
α
-O bond in solution.
The stability of phosphate esters and anhydrides has been persistently investigated
since at least the 1940’s
4-6
with an intense interest due as much to the topic’s fundamental
biochemical importance as its fascinating mechanistic implications.
7
In his seminal paper,
1

Frank Westheimer describes how the remarkable hydrolytic stability of this class of
compounds, together with the ease in which enzymes are capable of performing the

*
The synthesis of 1-3 was presented at the 18
th
International Conference of Phosphorus Chemistry,
Wroclaw, Poland, July 11-15 (2010); abstract published, Chamberlain, B.T., Osuna, J., Kashemirov, B. A.,
McKenna, C. E. Phosphorus, Sulfur, Silicon Relat. Elem. 2011, 186, 966-967. Collaborators and their
contributions to this work are listed in the Acknowledgements section.

68

analogous reactions, is central to why nature “chose” phosphates as the vital linkage for
life’s genetic material. Determining the uncatalyzed rates of biological reactions gives
insight into how an enzyme stabilizes a reaction’s transition state, gives context to the rate
enhancements developed by natural selection, can guide the choice of drug targets and
structures of transition state analogs,
3,8
and provides an experimental foundation for
computational simulations.
9

While non-enzymatic hydrolysis studies of ATP and other phosphoanhyrides have
characterized the solution stability and activation parameters of P
γ
solvolysis,
10,11
extending
these studies to the P
α
-O-P
β
moiety is inherently obscured by the initial cleavage at P
γ
and
nucleoside decomposition brought about by the elevated temperatures required to
accelerate the reaction into an observable time-frame.
12,13
Here we present the design,
synthesis, site of nucleophilic attack and activation parameters for model compounds that
allow unobscured observation of P
α
-O-P
β
(CXYP
γ
) anhydride bond hydrolysis. Finally,
supplementary model compounds exploring how further modifications might affect the site
of nucleophilic attack (P
α
versus P
β
) are synthesized and their hydrolysis kinetics introduced.

4.2 Results
4.2.1 Design and synthesis of model compounds 1-3
Model compounds 1-3 were designed to mimic natural nucleoside triphosphates by
retaining the immediate methylene 5’-sugar linkage while also eliminating the potential for
nucleoside decomposition,
8,13
C-O ester bond cleavage,
14
and any possible catalysis from
nitrogen moieties
15
(Figure 4.2.1). The neopentyl ester is a common surrogate used in

69

phosphate ester hydrolysis studies that meets these criteria and does not significantly
impact nucleophilic attack at the phosphorus center.
12,16
Additionally, the neopentyl
C(CH
3
)
3
moiety provides a robust and uncomplicated
1
H-NMR spectrum useful for
monitoring reaction progress.
Previously, it was demonstrated with β,γ-CH
2
and β,γ-CH(OH) substituted 5’-
triphosphates that P
β
-P
γ
phosphonate substitution blocks P
γ
hydrolysis and shifts the site of
nucleophilic attack to P
β.
13
Model compounds 2-3 expand upon this design by incorporating
fluorinated bisphosphonates into the P
β
-P
γ
position and thus better approximate the native
P
α
-O-P
β
bond.
17-19
Together with 1, these models span a wide-range of electronic properties
(pKa
4
values spanning nearly 3 pKa units)
20,21
and might allow for potential leaving group
affects to emerge if they exist.
O P
O
HO
O P
O
HO
X
Y
1: X = H, Y = H, Z = PO
3
H
2
2: X = H, Y = F, Z = PO
3
H
2
3: X = F, Y = F, Z = PO
3
H
2
4: X = H, Y = H, Z = Ph
5: X = F, Y = F, Z = Ph
Z

Figure 4.2.1. Structure of triphosphate model compounds (1-3) and supplementary P
β
-CXYPh compounds
(4-5).

Synthesis of 1-3 was accomplished by coupling the requisite bisphosphonate with
the neopentyl monophosphate-N-methylimidazolide generated in situ from neopentyl
trifluoroacyl phosphate.
22
Neopentyl monophosphate was prepared from phosphorus
oxychloride using a limiting amount of neopentanol in the presence of triethylamine
followed by basic work-up with sodium hydroxide in water
23
and acidification on Dowex
TM

cation exchange resin. Following the coupling reaction, phosphate containing compounds

70

were precipitated from ethanol with sodium iodide and then purified by semi-preparative
HPLC. Exchange to the sodium salt and final precipitation from ethanol furnished the target
compounds in greater than 99% purity according to
1
H and
31
P NMR.
Although replacement of the nucleoside portion of our model compounds was
essential for precluding undesirable side reactions, a practical consequence of the
neopentyl ester modification was elimination of the UV/Vis chromophore provided by the
nucleobase. An alternative means of detection was therefore necessary to facilitate HPLC
purification and obtain the compounds in high-purity. A post-column HPLC derivatization
method was elaborated that introduced a phosphate-specific dinuclear anthracene
bis(zinc(II)-dipicolylamine) complex
24
into a portion of the mobile phase after
chromatographic separation and thus allowed fluorescent detection of the otherwise
unnoticeable phosphate compounds.
The use of post-column derivatization is a common technique used in sample
analysis as well as biological screening and profiling assays,
25
but its application to
preparative separations is more uncommon. The key component of the semi-preparative
post-column HPLC system (Figure 4.2.2) is the high pressure mixing tee positioned after the
column that diverts an ‘analytical’ flow (~ 1mL/ min) to the reactor while directing the bulk
of the eluate (~ 11 mL/ min) into a reagent free path for collection. Achieving this
partitioning required the use of specialty HPLC PEEK tubing with an extremely small inner-
diameter (0.0025”, 0.0635 mm ID) in the analytical segment of the system and several
meters of additional HPLC plumbing (standard ID: 0.020”, 0.508 mm) in the preparative
pathway. Synchronization was validated with a UV active bisphosphonate that reacted with

71

the chemosenor in the analytical pathway while also registering absorbance with a UV
detector at the end of the preparative pathway. The merit of this approach is demonstrated
by the purity of 1-3, which possess
1
H,
19
F, and
31
P NMR spectra free of foreign resonances
and were obtained on the 100 mg scale without an undue number of injections.

Figure 4.2.2. Image and schematic of semi-preparative post-column derivatization HPLC system.
Components not common to standard HPLC are underlined (top) or shown in red (bottom). Elution
system: pump A: 0.1 N TEAB, 2.5% CH
3
CN, pH 7.4, Flow = 12.0 mL/ min; Pump B: 50:50 H
2
O/ CH
3
CN Flow =
0.0 – 1.0 mL/ min; Flow rate through path A = ~ 1.0 mL/ min; Flow rate through path B = ~11.0 mL/ min.

As per design, 1-3 present uncomplicated
1
H-NMR spectra with the neopentyl (CH
3
)
3

resonating as a 9H singlet at 0.84 ppm and the methylene CH
2
registering as a 5.5 Hz
doublet that migrates slightly from 3.52 to 3.57 ppm upon increased fluorine substitution.

72

The signals from the bisphosphonate protons in 1 and 2 do not interfere with the neopentyl
resonances and possess chemical shift values of 2.07 (dd, J 19.0 Hz, 21.4 Hz) and ~4.7 (m)
respectively. The (CH
3
)
3
proton resonance in 1-3 is distinct from the neopentyl
monophosphate hydrolysis product (0.81 ppm) and the neopentanol integration standard
(0.79 ppm).

4.2.2 Hydrolysis of 1-3
It was determined previously that the rate of hydrolysis of β,γ-CH
2
and β,γ-CH(OH)
substituted 5’-nucleoside triphosphates increases as the pH is raised from 5 to 7 and then
becomes pH independent upon passing 7.
13
Hydrolysis of 1-3 tetraanions was conducted in
0.2 M KOH solution in PTFE lined stainless steel vessels
26
heated to 90 - 145° C by immersion
in a vigorously circulating oil bath with precision temperature control using a system that
was calibrated by reproducing the Arrhenius plot for the phenyl phosphate dianion defined
by Wolfenden and co-workers.
26

The appearance of neopentyl monophosphate and the disappearance of the model
compounds all follow first-order kinetics as monitored by
1
H NMR. No reaction products
other than neopentyl monophosphate and the examined bisphosphonate were detected by
1
H NMR,
31
P NMR and MS. Compound 1 is hydrolyzed fastest (first order rate constant k=
0.0134 min
-1
), compound 2 shows intermediate stability (k= 0.00580 min
-1
), and 3 is the
most stable (0.00245 min
-1
) as compared at 110 °C. The resulting linear and parallel
Arrhenius plots were used to determine the reaction activation parameters and were
extrapolated to estimate the rate constant at 25 °C (Table 4.3.1).

73

Performing hydrolysis in 0.2 M KOH 50%
18
O-water solution, and analyzing the
reaction by negative mode ionization MS, demonstrated that the site of reaction for 1-3 was
exclusively at P
β
(Figure 4.2.3). No signs of hydrolysis via P
α
attack, either as isotopically
labeled neopentyl monophosphate or inorganic phosphate of any kind, could be detected.
Performing the hydrolysis of 3 in a 0.2 KCO
3
H 50%
18
O-water solution gave identical results.
This site of attack is consistent with the observed relative rates of hydrolysis in which 1 is
hydrolyzed fastest and 3 is hydrolyzed slowest.

P
O
O
O
O P
O
P
O
O O
O
Y
X
H
2
18
O
P
O
O
O
O
18
O
P
O
P
O
O O
O
Y
X
+

Figure 4.2.3.
18
O-labeled water hydrolysis experiments demonstrated that the site of nucleophilic attack
for β,γ-CXY neopentyl triphosphates 1-3 is P
β
.

4.2.3 Design and synthesis of supplementary model compounds 4 and 5
The findings from the hydrolysis studies of 1-3 showed that nucleophilic attack
occurred exclusively via breakage of the P
β
-O

anhydride bond and that 1 (X, Y = H) was
significantly less stable than 3 (X, Y= F). Next, we tested how much higher the free energy of
activation for P
α
might be and what modifications, if any, might shift the site of nucleophilic
attack from the phosphonate to the phosphate phosphorus. Computational modeling of 1-3
indicated that increasing fluorine substitution shortened the P
β
-O bond, increased the
charge on P
β
, and decreased the negative charge density on the P
γ
oxygens (Appendix D,

74

figure D20). Further calculations showed that replacement of the P
γ
phosphonate with an
electron-withdrawing substituent continued the trend of shortening P
β
-O bond length and
can result in compounds possessing a longer phosphate anhydride bond than the
complementary phosphonate bridging oxygen bond (Appendix D, figure D21). Additionally,
a potential advantage of removing the P
γ
phosphonate is that it allows for the incorporation
of phosphonate units with lower pKa values than neopentyl phosphate.
†
Ultimately, we
incorporated a phenyl ring into the second generation model compounds 4 and 5 (Figure
4.2.1) as this moiety was consistent with all of the above considerations and provided a
weak chromophore useful for detection during purification. Benzyl phosphonic acid, 6, has
a pKa
2
value of 7.5
28
(lower than difluorobisphosphonic acid; pKa
4
= 7.8)
20,21
and fluorinated
benzyl phosphonic acids, such as 9, will certainly have a pKa
2
value lower than that of
monoalkyl phosphates.
Compounds 4 and 5 were prepared by coupling the requisite benzyl phosphonic acid
(7, 9) with neopentyl phosphate using the same coupling chemistry employed in the
synthesis of 1-3 (Scheme 4.2.1). Benzyl phosphonate diethyl ester, 6, was synthesized on
the multi-gram scale from an Arbuzov reaction of triethyl phosphite and benzyl chloride.
Electrophilic fluorination afforded 9 in modest yield using KHMDS and NFSI in THF.
29
In our
hands, work-up of 9 led to significant ester hydrolysis which further diminished product
recovery. Interestingly, electrophilic fluorination was unsuccessful with the Selectfluor
TM

†
n-Butyl phosphate pKa
2
= 6.8; n-propyl phosphate pKa
2
= 6.7 (I= 0.35 mM Na
+
, t= 25° C).
27

75

reagent which did not provide any detectable 9 in the reaction mixture as determined by
31
P
NMR analysis. Ethyl esters of 6 were cleaved to the corresponding acid 7 in refluxing HCl. 9
was unstable to this condition and was dealkylated via reaction with bromotrimethylsilane
followed by aqueous work-up. Coupling with trifluoroacetic anhydride mediated N-
methylimidazole activated neopentylphosphate proceeded with similar conversion as
observed in the synthesis of 1-3. 4 was purified by RP C-18 HPLC and was obtained in
similar yield and purity as 1-3. As the stability of 5 was uncertain, it was synthesized on the
milligram scale and purified via a single RP C-18 HPLC injection.

Scheme 4.2.1. Synthesis of P
β
-CXYPh model compounds 4 and 5.
P
X
X
O
OH
OH
a
Conc. HCl
reflux
PhCF
2
PO(OEt)
2
P
O
OEt
OEt
6 8
O P
O
O
HO
P
O
HO
X
X
4: X = H; 5: X = F
c
b
9: X = F
7: X = H
a) KHMDS, NFSI/ THF; b) 1. BTMS/ CH
3
CN, 2. H
2
O: EtOH; c) 1. TFAA, Et
3
N, 2. N-MeIm, Et
3
N, 3. NpP / CH
3
CN

4.2.3 Hydrolysis of 4 and 5
Compound 4 was dissolved in 0.2 M KOH and incubated in PTFE lined sealed
stainless steel vessels. 30 minutes of incubation at 200 °C yielded approximately ~50%
decomposition has determined by
1
H NMR. This compound is significantly more stable than
the first generation series (1-3) in which the most stable example, 3, underwent similar
decompostion at 143 °C (30 minutes incubation).

76

Hydrolysis of 4 in 50% MeOH:H
2
O solution followed by Dowex
TM
exhange to the
ammonium salt and MS analysis indicated at least a portion of nucleophilic attack occurs via
nucleophilic attack at P
β
. In this experiment the masses for 4, neopentyl phosphate, 7, and
the monomethyl ester of 7 were all observed in the MS. No traces of the monomethylated
version of neopentyl phosphate could be detected in the instrument. Also, no inorganic
phosphate, a possible decomposition product of the methyl, neopentyl phosphate diester
could be observed in the
31
P NMR. Further experiments with
18
O-water are necessary to
conclusively determine if there is also hydrolysis through P
α
in this system.
Hydrolysis of 5 was conducted for 30 minutes at 160 °C and 30 minutes at 200 °C. A
few percent decomposition was observed at 160 °C and ~70% decomposition was observed
at 200 °C. Interestingly, 10 was not observed in the products by
31
P-NMR; instead the
spectra contained resonances corresponding to 5, and equal amounts of neopentyl
monophosphate and P
i
(Scheme 4.2.2). Investigating the stability of 10 at 200 °C for 30
minutes generated complete decomposition of the phosphonic acid and demonstrated the
instability of the P-C bond in difluorinated benzyl phosphonates. The P-C bond breakage
observed in the hydrolysis of 5 makes analysis of the reaction mechanism difficult (P-C bond
breakage, P
β
or P
α
attack) especially if neopentyl phosphate is portion of 5 is further
decomposed to inorganic phosphate in the event of nucleophilic attack at phosphorus.

77

Scheme 4.2.2. The products observed in the
31
P NMR resulting from the hydrolysis of 5. These products
demonstrate that the P-C bond of 5 is unstable under the hydrolysis conditions.

O P
O
O
O
P
O
O
F
F
O P
O
O
O
O P
O
O
O
+
5
0.2 M KOH
200
o
C

4.3 Discussion
4.3.1 Triphosphate P
β
-O bond stability
Comparing triphosphate P
β
-O bond cleavage with the stability of other types of
phosphate bonds shows that the rate of hydrolysis of , -CXY-triphosphate tetra-anions is
significantly faster than that of monomethyl phosphate dianion and the dineopentyl
phosphate monoanion; and is faster than, but comparable to, the rate of phosphate
monomethyl monoanion hydrolysis (Table 4.3.1).
12
The negative entropy of activation for 1-
3 (ranging from approximately -1.4 to -2.0 kcal/mol at 25° C) is less than generally reported
for phosphate methyl monoester monoanion (-0.6 kcal/mol at 25° C)
12
but more than the
values obtained for phosphate neopentyl diester (-8.6 kcal/mol at 25° C).
12
The enthalpy of
activation for 1-3 (ranging from 27.2 to 27.7 kcal/mol) is similar to that observed for both
the phosphate methyl monoester monoanion and phosphate neopentyl diester monoanion
(30.0 and 29.5 kcal/mol, respectively).
12
Comparison of the rate constants of 1-3 with that of natural ATP
4-
, in which 1.5% of
the reaction was shown to proceed through P
β
attack,
10
suggest that difluorophosphonate is
a reasonable mimic for the native non-bridging oxygen as the rate of hydrolysis for this
compound is slightly slower than the rate of ATP P
γ
hydrolysis.

78

Table 4.3.1. Activation parameters
a

for the hydrolysis of β,γ-CXY neopentyl triphosphates 1-3 and related
phosphate compounds

CH
2
(1) CHF (2) CF
2
(3) ATP(4-)
(P
γ
)
10

MeP
(-1)
12

Np
2
P
-
(H
2
O)
12

H

kcal/mol
27.2 (±0.3) 27.1 (±0.3) 27.7 (±0.3) 27.9 30.0 29.5
T S

25C

kcal/mol
-1.4 (±0.4) -2.0 (±0.5) -2.0 (±0.5) -1.0 -0.6 -8.6
G

25C

kcal/mol
28.6 (±0.3) 29.1 (±0.4) 29.7 (±0.4) 28.9 30.6 38.1
k
25
, s
-1
(6.6±3.8)·10
-9
(3.0±2.0)·10
-9
(1.0±0.7)·10
-9
1.5·10
-9
2.4·10
-10
7·10
-16
a
Parameters were obtained from rate constants in 0.2 M KOH. Errors given are three standard
deviations estimated by linear least square regression of Arrhenius plots.

Though not as prevalent as enzymatic reactions occurring at P
γ
or P
α
, several
diphosphokinase
30
and Nudix hydrolase enzymes are known to catalyze reactions at P
β
of a
triphosphate. Of these at least 2 bacterial Nudix hydrolases, MutT
31,32
and Orf17
33,34
,
catalyze hydrolysis via nucleophilic attack at P
β
. Using the parameters obtained for 3, we
estimate the rate enhancement, log k
cat
/k
non
, of these enzymes to be 8.4 and 7.7
respectively (Table 4.3.2).
Table 4.3.2. Catalytic power of enzymes catalyzing hydrolysis triphosphate at P


Enzyme t, C k
cat

s
-1

k
i
non
s
-1
-log (k
cat
/k
non
)
MutT
a
23 4.0 1.5·10
-8
8.4
Orf17
b
37 5.2 1.0·10
-7
7.7
a
MutT enzyme
32
(dGTP + H
2
O  dGMP + PP
i
),
b
orf17 enzyme
34
(dATP + H
2
O  dAMP + PP
i
) ,
i
k
non

is calculated for CF
2
derivative hydrolysis at a given temperature.

4.3.2 Triphosphate P
α
-O bond stability
Our kinetic data place an upper-limit on the rate of triphosphate hydrolysis through
P
α
attack similar to the reaction catalyzed by polymerase enzymes. Experiments with
18
O-

79

labeled water revealed no detectable hydrolysis through attack at P
α
(1-3). Given the
stability of 4, it is conservative to estimate the free energy of activation for P

-O bond
breakage to be at least 3 kcal/mol greater than P
β
-O hydrolysis for triphosphate model
compound (3). Thus, using hydrolysis as a reference state, the -log (k
cat
/k
non
) catalytic
efficiency of a polymerase such as DNA polymerase β
35
must be at least 12±1. Although this
initial estimate places the rate enhancement of P
α
-O bond cleavage catalyzed by pol β a
several orders of magnitude shy of the rate enhancement conferred by
phosophodiesterases (10
17
-fold rate enhancement
12
), it nevertheless highlights the
incredible stability of the P
α
-O bond in triphosphates. It is fascinating that polymerases
operate with such incredible fidelity, but also do so by cleaving the most stable phosphate
anhydride bond in the system. Future studies might provide more precise estimations of
the native stability of the P
α
-O anhydride bond.

4.4 Conclusion
In conclusion, we have synthesized a suite of model compounds and used them to
study the stability of the P
α
-O-P
β
bond of triphosphate monoesters. Our results provide rate
constants and activation parameters for hydrolysis of the P
α
-O-P
β
(CXYP
γ
) anhydride bond
and define the site of nucleophilic attack to occur exclusively at P
β
. The activation
parameters were extrapolated to 25°C and thus establish a reference for the estimation of
the catalytic efficiency of enzymes that catalyze reactions at P
β.
When considered in the
context of nucleophilic attack at P
α
in which a pyrophosphate leaving group is displaced, this
data sets the upper-limit for the rate of triphosphate hydrolysis by nucleophilic attack at P
α

80

in solution. Supplementary model compounds were synthesized in order to explore how
much higher the free energy of activation for hydrolysis through nucleophilic attack at P
α

might be. Even with increased P
β
-O bond stability nucleophilic attack proceeding through P
β

attack is still observed.

Taken together, these results illustrate the stability of the P
α
-O bond
in triphosphate anhydrides and underscore the incredible catalysis performed by
polymerase enzymes.

4.5 Experimental
4.5.1 Materials and methods
All chemicals and reagents were purchased from Sigma-Aldrich Inc and used as
received. Difluoro- and fluoromethylene bisphosphonic acid were synthesized as described
in Chapter 1. The 9,10-Bis[(2,2’-dipicolylamino)methyl]anthracene zinc complex was
synthesized according to literature procedure.
24

1
H NMR spectra were referenced to residual CHCl
3
(δ 7.24) in CDCl
3
or to HDO (δ
4.79) in D
2
O.
36

13
C NMR spectra were referenced to external methanol (δ 49.0).
36

31
P NMR
spectra were proton decoupled and referenced to external H
3
PO
4
(δ 0.00) and
19
F NMR
spectra were referenced to external hexafluorobenzene, C
6
F
6
in benzene (δ -164.9). pH
values measured in 99.9% D
2
O and reported as measured without deuterium isotope
correction. NMR operating frequencies are listed with their respective assignments.
Chemical shifts (δ) are reported in ppm. Low resolution mass spectra were obtained with a
Thermo-Finnagan Deca XP Plus mass spectrometer using the ESI probe. High resolution
mass spectra were obtained by the UC Riverside mass spectrometry facility.

81

HPLC purification was conducted on a system that split the mobile phase into an
analytical path and a preparative path using a Upchurch Scientific high pressure mixing tee
immediately after the RP-C18 column (Varian Dynamax 250 x 21.4 mm (L x ID) microsorb
(100-5)). The analytical path was directed to a Pickering Laboratory Vector PCX post-column
reactor pump and a Shimadzu RF-10AxL fluorescent detector (ex 380 nm; em 434 nm) using
0.0025” ID PEEK tubing and was rigged with a 100 psi back pressure regulator. The
preparative path was plumbed with 0.020” ID PEEK tubing which terminated in a UV/Vis
absorbance detector used to validate the synchronization of the two paths. Optimization of
path length was conducted using risedronic acid. The preparative path required ~2 meters
of additional PEEK tubing in order to elute simultaneously with the analytical pathway.
The HPLC system was eluted at 12 mL/ min with at a 0.1 N triethylammonium
bicarbonate pH 7.4 buffer. Acetonitrile content was increased for 2.5% to 10% over the first
10 min and then was held constant for the rest of the run. The flow rate from the analytical
portion of the system was ~1 mL/ min while the flow rate from the preparative path was ~
11 mL/ min. The post column reactor pumped the anthracene zinc complex (50 μM) at a
rate of 0.4 mL/min. Up to 100 mg of reaction mixture could be injected with good
resolution.
HPLC for 4 and 5 was conducted using a Phenomenex LUNA C18 (250 x 21.20 mm)
column, eluted with a 0.1 N TEAB pH 7 mobile phase incorporating a linear 0-60% CH
3
CN
gradient over 20 min. Products were visualized at 257 nm. Initially all fractions were
collected and analyzed by MS (or
19
F NMR in the case of 5) to identify the retention time of
target compound.

82

Bond lengths for 1-5 were derived from geometry optimization calculations on the
tetraanions in the gas phase using Spartan ’08 using the density functional 6-31G(D) RB3LYP
basis set.
Hydrolysis studies including rate constant determination (1-3),
18
O-enriched H
2
O
hydrolysis experiments (1-3), and kinetic evaluation of 4 and 5 are the work of Ernestas
Gaidamauskas and Jorge Osuna and will be presented in Jorge Osuna’s Ph.D. dissertation (in
preparation). NMR and mass spectra for 1-5 and bond length calculations (1-5) are
presented in Appendix D.

4.5.2 Synthetic procedures
2,2-Dimethylpropyl dihydrogen phosphate. 7.4 g phosphorus oxychloride (55
mmol, 1.1 equiv) was dissolved in 30 mL of dry toluene and chilled with stirring on ice-bath.
To this solution, 4.4 g neopentanol (50 mmol, 1 equiv) and 6.8 mL triethylamine (49 mmol,
0.98 equiv) in 30 mL toluene was added drop-wise. Volatiles were then removed under
reduced pressure and the remaining oil was dissolved in 100 mL H
2
O: Acetone (1:1). This
mixture was titrated with 1M NaOH to pH > 12 and then water and acetone were removed
by evaporation at reduced pressure. The resulting salt was acidified on Dowex
TM
cation
exchange resin as needed for coupling reactions. δ
H
(500 MHz, D
2
O, pH ~ 2): 3.58 (d, J 4.8
Hz, 2H), 0.95 (s); δ
P
(202 MHz, D
2
O, pH ~2): 1.28 (s).
General coupling procedure for 2,2-Dimethylpropyl β,γ-methylene-triphosphates,
1-3; Target bisphosphonic acid (~ 400 mg, for methylene bisphosphonic acid: 2.27 mmol)
was dissolved in 2 mL H
2
O : EtOH (1:1) and titrated to neutral pH with tetrabutyl ammonium

83

hydroxide (40 % solution). Volatiles were removed and the remaining salt was repeatedly
dissolved in anhydrous acetonitrile which was removed under vacuum with heating.
Neopentyl dihydrogen phosphate (0.380 g, 1.80 mmol) was dried in a similar manner and
taken up in 3 mL dry acetonitrile. The solution was stirred at room temperature under
nitrogen while triethylamine (0.364 g, 3.60 mmol) and trifluoroacetic anhydride (1.89 g, 9.00
mmol) were added by syringe. After ten minutes, volatiles were removed on rotovap and
the resulting syrup was dissolved in 3 mL dry acetonitrile. Triethylamine (0.364 g, 3.60
mmol) and 1-methylimidazole (0.739 g, 9.00 mmol) were added by syringe. After 15 min,
this mixture was added by syringe to a 5 mL dry acetonitrile solution of the tetrabutyl
ammonium salt of methylene bisphosphonic acid. After completion as determined by
31
P
NMR (typical reaction time < 1 hr), the solution was diluted with water, concentrated under
reduced pressure, and dissolved in ethanol. The phosphate containing compounds were
then precipitated via addition of a solution of sodium iodide in ethanol. The resulting white
precipitate was washed with ethanol, dissolved in water and purified by HPLC (see 4.4.1).
After collection of HPLC peaks, solvent was removed and the products were exchanged to
the sodium salts on Dowex
TM
cationic exchange resin. Finally, the salt was dissolved in a
minimum amount of water and precipitated by addition of ethanol to remove trace
amounts of neopentyl phosphate monoester. The final compounds were filtered through
centrifugal filters and lyophilized. Typical yields were around 100 mg (~15%).
2,2-Dimethylpropyl β,γ-methylene-triphosphate, 1: δ
H
(400 MHz, D
2
O, pH > 10.3):
0.84 (s, 9H, (CH
3
)
3
), 2.07 (dd,
2
J
HP
19.0 Hz,
2
J
HP’
21.4 Hz, 2H, PCH
2
P), 3.52 (d,
3
J
HP
5.5 Hz, 2H,
CH
2
C(CH
3
)
3
); δ
P
(203 MHz, D
2
O, pH > 10.3): -9.2 (d,
2
J
αβ
26.8 Hz, P
α
), 12.5 (d,
2
J
γβ
6.7 HZ, P
γ
),

84

13.8 (dd,
2
J
βγ
6.6 Hz,
2
J
βα
26.6 Hz, P
β
); HRMS: Calc for C
6
H
16
O
9
P
3
[M-H]
-
: 325.0013. Found:
325.0012.
2,2-Dimethylpropyl β,γ-(fluoro)methylene-triphosphate, 2: δ
H
(400 MHz, D
2
O, pH >
10.3): 0.84 (s, 9H,(CH
3
)
3
), 3.55 (d,
3
J
HP
5.6 Hz, 2H CH
2
C(CH
3
)
3
), ~4.7 (m); δ
F
(376 MHz, D
2
O,
pH > 10.3): -218.3 (ddd,
2
J
FH
45.7 Hz,
2
J
FPγ
56.0 Hz,
2
J
FPβ
65.2 Hz); δ
P
(203 MHz, D
2
O, pH >
10.3): -9.6 (d,
2
J
αβ
29.0 Hz, P
α
), 4.7 (ddd,
2
J
βα
29.0 Hz,
2
J
βγ
15.4 Hz,
2
J
βF
65.4 Hz, P
β
), 7.7 (dd,
2
J
γβ

15.4 Hz,
2
J
γF
56.1 Hz, P
γ
); HRMS: Calc for C
6
H
15
O
9
FP
3
[M-H]
-
: 342.9918. Found: 342.9908.
2,2-Dimethylpropyl β,γ-(difluoro)methylene-triphosphate, 3: δ
H
(400 MHz, D
2
O,
pH > 10.3): 0.84 (s, 9H,(CH
3
)
3
), 3.57 (d,
3
J
HP
5.6 Hz, 2H OCH
2
C(CH
3
)
3
); δ
F
(376.1 MHz, D
2
O,
pH > 10.3): -120.0 (dd,
2
J
F
72.5 Hz,
2
J
FPγ
89.4 Hz); δ
P
(161.9 MHz, D
2
O, pH > 10.3): -9.2 (d,
2
J
αβ

32.0 Hz, P
α
), -2.4 (ddt,
2
J
βα
32.0 Hz,
2
J
βγ
57.1 Hz,
2
J
βF
89.4 Hz, P
β
), 4.7 (dt,
2
J
γβ
57.1 Hz,
2
J
γF
72.5
Hz, P
γ
); HRMS: Calc for C
6
H
15
O
9
FP
3
[M-H]
-
: 360.9824. Found: 360.9815.
Diethyl benzylphosphonate, 6: Benzyl chloride (6 g, 4.17 mL, 35 mmol, 1 equiv) and
triethylphosphite (6.4 g, 6.62 mL, 0.39 mmol, 1.1 equiv) were distilled, mixed and brought to
reflux (temperature of sand bath set to 185 °C) for 4 h.
31
P NMR of the reaction mixture
indicated 81 % formation of 6 and no remaining phosphite. The reaction mixture was
cooled and distilled under vacuum (82-87 ° C/0.02 torr; lit
37
: 91-97° C/0.05 torr). 5.7 g (25
mmol), 71 %. δ
H
(600 MHz, CDCl
3
): 1.22 (t, 7.0 Hz, 6H), 3.13 (d, 21.6 Hz, 2H), 3.99 (m, 4H),
7.23 (m, 1H), 7.29 (m, 4H); δ
P
(243 MHz, CDCl
3
): 27.1 (s). Lit: δ
H
(CDCl
3
): 7.30 (s. 5H, C
6
H
5
),
3.99 (m, 4H, CH
2
CH
3
), 3.13 (d, J
HP
22Hz, 2H, CH
2
P), 1.22 (t, J
HH
7.4 Hz, 6H, CH
3
).
Benzylphosphonic acid, 7: 1.0 g (4.4 mmol) of 6 was dissolved in 4 mL conc HCl
solution and set to reflux. 1 mL fresh HCl solution was added every 3 h. Reaction took 10 h

85

to reach completion as determined by
31
P NMR. Solution was concentrated under reduced
to yield a white solid in quantitative yield. δ
H
(500 MHz, D
2
O, pH ~2): 3.20 (d, J
HP
21.2 Hz,
2H), 7.33-7.39 (m, 5H). δ
P
(202 MHz, D
2
O, pH ~2): 25.8 (s). To prepare the
tetrabutylammonium salt of 7, the acid was dissolved in 1 mL H
2
O:EtOH (1:1) and titrated to
neutral pH with tetrabutylammonium hydroxide (40% solution). The volatiles were
removed at reduced pressure and the resulting syrup was dried by adding and removing
anhydrous acetonitrile several times.
[(Diethylphosphono)difluoromethyl]benzene, 8: 100 mg 6 (0.44 mmol, 1 equiv)
was dissolved in 2 mL dry THF and cooled on a dry ice-acetone bath and stirred for 15 min
under N
2
. 0.96 mL of a 1 M KHMDS in THF solution (2.2 equiv) was added drop-wise by
syringe and formation of carbanion was confirmed by
31
P NMR. 347 mg (1.1 mmol, 2.5
equiv) NFSI was dissolved in 2 mL THF and added to the reaction mixture by syringe. After
30 min, the reaction mixture showed ~35% conversion to the monofluorinated product and
~27% of target compound, 8. This ratio did not improve after further stirring at rt. 4 mL
NH
4
Cl solution was added to the reaction mixture which was extracted with EtOAc, dried
over Na
2
SO
4
and purified by silica gel chromatography eluted with 47% EtOAc, 47% hexanes,
and 6% MeOH to yield 23 mg 8, 20 %. δ
H
(500 MHz, CDCl
3
): 1.28 (t, J
HH
7.0 Hz, 6H), 4.08-4.23
(m, 4H), 7.41-7.42, 7.56- 7.61 (m, 5H); δ
F
(470 MHz, CDCl
3
): -110.6 (d, J
FP
116.4); δ
P
(202
MHz, CDCl
3
): 6.9 (t, J
PF
117 Hz). Lit
29
: δ
H
7.64 (m, 2H, aryl), 7.48 (m, 2H, aryl, 4.18 (m, 4H,
2(CH
2
)), 1.43 (t, J 7.1 Hz, 6H, 2(CH
3
)); δ
F
-32.6 (d, 116.0 Hz); δ
P
4.36 (t, J
PF
116.0 Hz).
[Difluoro(phenyl)methyl]phosphonic acid, 9: 20 mg of 8 was dissolved in 200 μL
dry CH
3
CN and 6 equiv BTMS was added at rt and stirred for 36 hr. H
2
O: EtOH (1:1) was

86

added and volatiles were removed on rotovap. Tetra-n-butyl ammonium salt was obtained
as described above. δ
H
(500 MHz, D
2
O, pH neutral): 7.51-7.53 (m, 3H), 7.63-7.65 (m, 2H); δ
F

(470 MHz, D
2
O, pH neutral): -106.8 (d, 92.6 Hz); δ
P
(202 MHz, D
2
O, pH neutral): 6.0 (t, J
PF

93.9 Hz). Lit (ammonium salt)
29
: δ
H
7.62 (m, 2H, aryl), 7.49 (m, 3H, aryl); δ
F
-27.5 (d, J
FP
93.8
Hz); δ
P
5.83 (t, J
PF
91.6 Hz).
Benzylphosphonic (2,2-dimethylpropyl phosphoric) anhydride, 4: 65 mg (0.387
mmol, 1 equiv) of neopentyl dihydrogen phosphate was dissolved in 1 mL dry acetonitrile
and ~0.5 mL triethylamine and stirred on ice. 0.27 mL trifluoroacetic anhydride (1.9 mmol,
5 equiv) was added by syringe under nitrogen. The solution became a pale yellow and, after
a few minutes of stirring, was concentrated under reduced pressure. The remaining liquid
was dissolved in 1 mL CH
3
CN and 0.5 mL triethylamine to which 0.092 mL N-
methylimidazole was added and stirred on ice for10 min. Finally, 100 mg of 7 (0.581 mmol,
1.5 equiv), as the tetrabutylammonium salt, dissolved in 1 mL CH
3
CN was added at once and
stirred at rt for 1 hr. Solvent was removed and compound was dissolved in water, filtered,
and purified by HPLC. Retention time = 16 minutes. Target HPLC fractions were collected,
concentrated and exchanged on Dowex
TM
cation exchange resin to yield 34 mg of 4 as the
disodium salt (24%). 4: δ
H
(600 MHz, D
2
O, pH nuetral): 0.92 (s, 9H, CH
3
), 3.20 (d, 21.3 Hz,
PCH
2
Bz), 3.54 (d, 5.3 Hz, POCH
2
Np), 7.31 (m, 1H), 7.39 (m 4H); δ
C
(151 MHz, D
2
O, pH nuetral):
25.2 (s, CH
3
), 30.9 (d, 7.6 Hz, CH
2
C(CH
3
)
3
), 35.4 (d, 134.4 Hz, PCBz), 75.6 (d, 5.8 Hz, POCH
2
),
125.97, 126.00, 128.23, 128.25, 129.5, 129.6, 134.46, 134.52; δ
P
(243 MHz, D
2
O, pH nuetral):
-9.5 (d, J
PP
26.8 Hz, PONp), 14.4 (d, J
PP
26.8 Hz, BzP); LRMS: Calc for C
12
H
19
O
6
P
2
[M-H]
-
:
321.1. Found: 321.2.

87

[Difluoro(phenyl)methyl]phosphonic (2,2-dimethylpropyl phosphoric) anhydride, 5:
To a solution of the tetrabutylammonium salt of 9 (2.5 mg, 6.1 μmol, 1 equiv) in 100 μL of
dry acetronitrile stirred at rt, 2.5 μL triethylamine (18.3 μmol, 3 equiv) followed by 3.4 μL of
trifluoroacetic anhydride (24.4 μmol, 4 equiv) were added and stirred for 10 min. Volatiles
were removed on rotovap and the resulting syrup was dissolved in 100 μL acetonitrile with
2.5 μL triethylamine (18.3 μmol, 3 equiv). 1.4 μL of N-methylimidazole (18.3 μmol, 3 equiv)
were added to the solution, and stirred at rt for 15 min. This solution was added by syringe
to a solution of 2 mg of 2,2-dimethylpropyl dihydrogen phosphate (11.8 μmol, 2 equiv, acid
form) in 100 μL acetonrile stirred with 4A molecular sieves. After 1 hr volatiles were
removed under reduced pressure and the reaction mixture was purified on RP-C18 HPLC in
a single injection. The fractions containing 5 were identified by
19
F NMR, concentrated and
exchanged to the disodium salt on Dowex
TM
cation exchange. A second exchange on
Dowex
TM
did not improve purity of the
1
H NMR. δ
H
(500 MHz, D
2
O, pH nuetral): 3.40 (d, J
HP
5.2 Hz, POCH
2
C(CH
3
)
3
), 7.56-7.67 (m, 5H); δ
F
(564 MHz, D
2
O, pH nuetral): -110.0 (d, J
FP
110.9
Hz); δ
P
(243 MHz, D
2
O, pH nuetral): -10.3 (d, J
PP
32.1 Hz, POPOCH
2
C(CH
3
)
3
), -5.3 (dt, J
PF
100.2,
J
PP
32.1, BzCF
2
POP); LRMS: Calc for C
12
H
17
F
2
O
6
P
2
[M-H]
-
: 357.0. Found: 357.1.

88

4.6 Chapter references
(1) Westheimer, F. H. Why nature chose phosphates, Science (Washington, D. C., 1883-)
1987, 235, 1173-8.
(2) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA Polymerase β
Fidelity: Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis
Reveal Differences at the Chemical Transition State, Biochemistry 2008, 47, 870-79.
(3) Wolfenden, R. Benchmark reaction rates, the stability of biological molecules in
water, and the evolution of catalytic power in enzymes, Annu. Rev. Biochem. 2011, 80, 645-
67.
(4) Bailly, M. C. Transposition and hydrolysis of the glycerol monoorthophosphates. III.
Hydrolysis and transposition of the β- and α-glycerophosphates, Bull. Soc. Chim. Fr. 1942, 9,
405-20.
(5) Desjobert, A. Hydrolysis of monoethyl orthophosphate, Bull. Soc. Chim. Fr. 1947,
809-12.
(6) Campbell, D. O.; Kilpatrick, M. L. A kinetic study of the hydrolysis of pyrophosphates,
J. Am. Chem. Soc. 1954, 76, 893-901.
(7) Lassila, J. K.; Zalatan, J. G.; Herschlag, D. Biological phosphoryl-transfer reactions:
Understanding mechanism and catalysis, Annu. Rev. Biochem. 2011, 80, 669-702.
(8) Wolfenden, R. Degrees of difficulty of water-consuming reactions in the absence of
enzymes, Chem. Rev. (Washington, DC, U. S.) 2006, 106, 3379-96.
(9) Braun-Sand, S.; Olsson, M. H. M.; Warshel, A. Computer modeling of enzyme
catalysis and its relationship to concepts in physical organic chemistry, Adv. Phys. Org. Chem.
2005, 40, 201-45.

89

(10) Stockbridge, R. B.; Wolfenden, R. The Intrinsic Reactivity of ATP and the Catalytic
Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine: a
thermodynamic analysis, J. Biol. Chem. 2009, 284, 22747-57.
(11) Admiraal, S. J.; Herschlag, D. Mapping the transition state for ATP hydrolysis:
implications for enzymic catalysis, Chem. Biol. 1995, 2, 729-39.
(12) Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden, R. The time
required for water attack at the phosphorus atom of simple phosphodiesters and of DNA,
Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4052-55.
(13) Ora, M.; Lonnberg, T.; Florea-Wang, D.; Zinnen, S.; Karpeisky, A.; Lonnberg, H.
Bisphosphonate Derivatives of Nucleoside Antimetabolites: Hydrolytic Stability and
Hydroxyapatite Adsorption of 5'-β,γ-Methylene and 5'-β,γ-(1-Hydroxyethylidene)
Triphosphates of 5-Fluorouridine and ara-Cytidine, J. Org. Chem. 2008, 73, 4123-30.
(14) Wolfenden, R.; Ridgway, C.; Young, G. Spontaneous Hydrolysis of Ionized Phosphate
Monoesters and Diesters and the Proficiencies of Phosphohydrolases as Catalysts, J. Am.
Chem. Soc. 1998, 120, 833-34.
(15) Kirby, A. J.; Lima, M. F.; da, S. D.; Roussev, C. D.; Nome, F. Efficient Intramolecular
General Acid Catalysis of Nucleophilic Attack on a Phosphodiester, J. Am. Chem. Soc. 2006,
128, 16944-52.
(16) Williams, N. H.; Wyman, P. Base catalyzed phosphate diester hydrolysis, Chem.
Commun. (Cambridge, U. K.) 2001, 1268-69.
(17) Berkowitz, D. B.; Bose, M. (α-Monofluoroalkyl)phosphonates: a class of isoacidic and
"tunable" mimics of biological phosphates, J. Fluorine Chem. 2001, 112, 13-33.
(18) McKenna, C. E.; Shen, P.-D. Fluorination of methanediphosphonate esters by
perchloryl fluoride. Synthesis of fluoromethanediphosphonic acid and
difluoromethanediphosphonic acid, J. Org. Chem. 1981, 46, 4573-76.

90

(19) Blackburn, G. M.; England, D. A.; Kolkmann, F. Monofluoro- and
difluoromethylenebisphosphonic acids: isopolar analogs of pyrophosphoric acid, J. Chem.
Soc., Chem. Commun. 1981, 930-32.
(20) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA Polymerase β
Fidelity: Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis
Reveal Differences at the Chemical Transition State, Biochemistry 2008, 47, 870-79.
(21) Upton, T. G. Design and synthesis of a series of methylenebisphosphonates: A
nucleotide analogue toolkit to probe nucleic acid polymerase structure and function, Ph.D.
dissertation, University of Southern California, 2008.
(22) Mohamady, S.; Jakeman, D. L. An Improved Method for the Synthesis of Nucleoside
Triphosphate Analogs, J. Org. Chem. 2005, 70, 10588-91.
(23) Quin, L. D.; Wu, X.-P.; Sadanani, N. D.; Lukes, I.; Ionkin, A. S.; Day, R. O. Synthesis,
fragmentation, and photorearrangement of neopentyl and adamantyl phosphonates in the
2,3-oxaphosphabicyclo[2.2.2]octene system, J. Org. Chem. 1994, 59, 120-9.
(24) Ojida, A.; Mitooka, Y.; Inoue, M.; Hamachi, I. First artificial receptors and
chemosensors toward phosphorylated peptide in aqueous solution, J. Am. Chem. Soc. 2002,
124, 6256-58.
(25) Shi, S.-Y.; Zhang, Y.-P.; Jiang, X.-Y.; Chen, X.-Q.; Huang, K.-L.; Zhou, H.-H. Coupling
HPLC to on-line, post-column (bio)chemical assays for high-resolution screening of bioactive
compounds from complex mixtures, TrAC, Trends Anal. Chem. 2009, 28, 865-77.
(26) Lad, C.; Williams, N. H.; Wolfenden, R. The rate of hydrolysis of phosphomonoester
dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases,
Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5607-10.

91

(27) Kumler, W. D.; Eiler, J. J. The acid strength of mono and diesters of phosphoric acid.
The n-alkyl esters from Me to Bu, the esters of biological importance and the natural
guanidinephosphoric acids, J. Am. Chem. Soc. 1943, 65, 2355-61.
(28) Boehmer, V.; Vogt, W.; Chafaa, S.; Meullemeestre, J.; Schwing, M. J.; Vierling, F. (o-
Hydroxyphenyl)methylphosphonic acids: synthesis and potentiometric determinations of
their pKa values, Helv. Chim. Acta 1993, 76, 139-49.
(29) Taylor, S. D.; Kotoris, C. C.; Dinaut, A. N.; Chen, M.-J. Synthesis of
aryl(difluoromethylene phosphonates) via electrophilic fluorination of α-carbanions of
benzylic phosphonates with N-fluorobenzenesulfonimide, Tetrahedron 1998, 54, 1691-714.
(30) Mildvan, A. S.; Weber, D. J.; Abeygunawardana, C. Solution structure and
mechanism of the MutT pyrophosphohydrolase, Adv. Enzymol. Relat. Areas Mol. Biol. 1999,
73, xi-xii, 183-207.
(31) Mildvan, A. S.; Xia, Z.; Azurmendi, H. F.; Saraswat, V.; Legler, P. M.; Massiah, M. A.;
Gabelli, S. B.; Bianchet, M. A.; Kang, L. W.; Amzel, L. M. Structures and mechanisms of Nudix
hydrolases, Arch. Biochem. Biophys. 2005, 433, 129-43.
(32) Frick, D. N.; Weber, D. J.; Gillespie, J. R.; Bessman, M. J.; Mildvan, A. S. Dual divalent
cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the
metal and substrate complexes, J. Biol. Chem. 1994, 269, 1794-803.
(33) Gabelli, S. B.; Bianchet, M. A.; Xu, W.; Dunn, C. A.; Niu, Z.-D.; Amzel, L. M.; Bessman,
M. J. Structure and Function of the E. coli Dihydroneopterin Triphosphate Pyrophosphatase:
A Nudix Enzyme Involved in Folate Biosynthesis, Structure (Cambridge, MA, U. S.) 2007, 15,
1014-22.
(34) O'Handley, S. F.; Frick, D. N.; Bullions, L. C.; Mildvan, A. S.; Bessman, M. J. Escherichia
coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT
family of proteins. Cloning, purification, and characterization of the enzyme, J. Biol. Chem.
1996, 271, 24649-54.
(35) Beard, W. A.; Wilson, S. H. Structure and mechanism of DNA polymerase β, Chem.
Rev. (Washington, DC, U. S.) 2006, 106, 361-82.

92

(36) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 1997, 62, 7512-15.
(37) Gronowitz, S.; Stenhammar, K.; Svensson, L. Lithiation of some 3[2-(2'-
bromophenyl)ethyl]thiophenes and intramolecular transmetalation, Heterocycles 1981, 15,
947-59.

93

BIBLIOGRAPHY
Admiraal, S. J.; Herschlag, D. Mapping the transition state for ATP hydrolysis: implications
for enzymatic catalysis, Chem. Biol. 1995, 2, 729-39.

Alexandrova, L. A.; Skoblov, A. Y.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A. A. 2-
Deoxynucleoside 5'-triphosphates modified at α-, β- and γ-phosphates as substrates for
DNA polymerases, Nucleic Acids Res. 1998, 26, 778-86.

Amantini, D.; Fringuelli, F.; Pizzo, F.; Vaccaro, L. Selected methods for the reduction of the
azido group, Org. Prep. Proced. Int. 2002, 34, 109,11-47.

Bailly, M. C. Transposition and hydrolysis of the glycerol monoorthophosphates. III.
Hydrolysis and transposition of the β- and α-glycerophosphates, Bull. Soc. Chim. Fr. 1942, 9,
405-20.

Batra, V. K.; Beard, W. A.; Shock, D. D.; Krahn, J. M.; Pedersen, L. C.; Wilson, S. H.
Magnesium induced assembly of a complete DNA polymerase catalytic complex, Structure
(Camb) 2006, 14, 757-66.

Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.;
Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA
Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding
of the Fluoromethylene Analogues, J. Am. Chem. Soc. 2010, 132, 7617-25.

Beard, W. A.; Wilson, S. H. Structure and mechanism of DNA polymerase β, Chem. Rev.
(Washington, DC, U. S.) 2006, 106, 361-82.

Benati, L.; Nanni, D.; Spagnolo, P. Reactions of Benzocyclic β-Keto Esters with Sulfonyl
Azides. 2. Further Insight into the Influence of Azide Structure and Solvent on the Reaction
Course, J. Org. Chem. 1999, 64, 5132-38.

Berkowitz, D. B.; Bose, M. (α-Monofluoroalkyl)phosphonates: a class of isoacidic and
"tunable" mimics of biological phosphates, J. Fluorine Chem. 2001, 112, 13-33.

Berkowitz, D. B.; Karukurichi, K. R.; de, l. S.-B. R.; Nelson, D. L.; McCune, C. D. Use of
fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical
advantages, J. Fluorine Chem. 2008, 129, 731-42.

Biffinger, J. C.; Kim, H. W.; DiMagno, S. G. The polar hydrophobicity of fluorinated
compounds, ChemBioChem 2004, 5, 622-27.

94

Blackburn, G. M.; England, D. A.; Kolkmann, F. Monofluoro- and
difluoromethylenebisphosphonic acids: isopolar analogs of pyrophosphoric acid, J. Chem.
Soc., Chem. Commun. 1981, 930-32.

Blackburn, G. M.; Langston, S. P. Novel P1,P2-substituted phosphonate analogs of 2'-
deoxyadenosine and thymidine 5'-triphosphates, Tetrahedron Lett. 1991, 32, 6425-8.

Boehmer, V.; Vogt, W.; Chafaa, S.; Meullemeestre, J.; Schwing, M. J.; Vierling, F. (o-
Hydroxyphenyl)methylphosphonic acids: synthesis and potentiometric determinations of
their pKa values, Helv. Chim. Acta 1993, 76, 139-49.

Boyer, J. H.; Canter, F. C. Alkyl and aryl azides, Chem. Rev. (Washington, DC, U. S.) 1954, 54,
1-57.

Boyer, J. H.; Hamer, J. The acid-catalyzed reaction of alkyl azides upon carbonyl compounds,
J. Am. Chem. Soc. 1955, 77, 951-4.

Braese, S.; Gil, C.; Knepper, K.; Zimmermann, V. Organic azides. An exploding diversity of a
unique class of compounds, Angew. Chem., Int. Ed. 2005, 44, 5188-240.

Braun-Sand, S.; Olsson, M. H. M.; Warshel, A. Computer modeling of enzyme catalysis and
its relationship to concepts in physical organic chemistry, Adv. Phys. Org. Chem. 2005, 40,
201-45.

Burton, D.; Pietrzyk, D.; Ishihara, T.; Fonong, T.; Flynn, R. Preparation, stability and acidity of
difluoromethylene bis phosphonic acid, J. Fluorine Chem. 1982, 20, 617-26.

Campbell, D. O.; Kilpatrick, M. L. A kinetic study of the hydrolysis of pyrophosphates, J. Am.
Chem. Soc. 1954, 76, 893-901.

Carosati, E.; Sciabola, S.; Cruciani, G. Hydrogen Bonding Interactions of Covalently Bonded
Fluorine Atoms: From Crystallographic Data to a New Angular Function in the GRID Force
Field, J. Med. Chem. 2004, 47, 5114-25.

Chamberlain, B. T.; Batra, V. K.; Beard, W. A.; Kadina, A. P.; Shock, D. D.; Kashemirov, B. A.;
McKenna, C. E.; Goodman, M. F.; Wilson, S. H. Stereospecific Formation of a Ternary
Complex of (S)-α,β-Fluoromethylene-dATP with DNA Pol β, ChemBioChem 2012, 13, 528-30.

Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E. α-Azido Bisphosphonates:
Synthesis and Nucleotide Analogs, J. Org. Chem. 2011, 76, 5132-36.

95

Charton, M. Definition of "inductive" substituent constants, J. Org. Chem. 1964, 29, 1222-7.

Chiba, S.; Zhang, L.; Ang, G. Y.; Hui, B. W.-Q. Generation of Iminyl Copper Species from α-
Azido Carbonyl Compounds and Their Catalytic C-C Bond Cleavage under an Oxygen
Atmosphere, Org. Lett. 2010, 12, 2052-55.

Chopra, D. Is Organic Fluorine Really "Not" Polarizable?, Cryst. Growth Des. 2012, 12, 541-46.

Ciez, D. A direct preparation of N-unsubstituted pyrrole-2,5-dicarboxylates from 2-
azidocarboxylic esters, Org. Lett. 2009, 11, 4282-85.

Dalvit, C.; Vulpetti, A. Fluorine-Protein Interactions and 19F NMR Isotropic Chemical Shifts:
An Empirical Correlation with Implications for Drug Design, ChemMedChem 2011, 6, 104-14.

Dalvit, C.; Vulpetti, A. Intermolecular and Intramolecular Hydrogen Bonds Involving Fluorine
Atoms: Implications for Recognition, Selectivity, and Chemical Properties, ChemMedChem
2012, 7, 262-72.

Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. Synthesis of nucleotide 5'-
diphosphates from 5'-O-tosyl nucleosides, J. Org. Chem. 1987, 52, 1794-801.

Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical Research,
third ed.; Clarendon: Oxford, England, 1986.

Delacotte, J. M.; Galons, H. Diethyl azidomalonate: revised synthesis and reactivity of the
anion with electrophiles, J. Chem. Res., Synop. 1991, 64-5.

Desjobert, A. Hydrolysis of monoethyl orthophosphate, Bull. Soc. Chim. Fr. 1947, 809-12.

Dunitz, J. D.; Schweizer, W. B. Molecular pair analysis: C-H···F interactions in the crystal
structure of fluorobenzene? And related matters, Chem.--Eur. J. 2006, 12, 6804-15.

Dunitz, J. D.; Taylor, R. Organic fluorine hardly ever accepts hydrogen bonds, Chem.--Eur. J.
1997, 3, 89-98.

Ellis, G. P.; Luscombe, D. K.; Editors Progress in Medicinal Chemistry, Vol. 31; Elsevier, 1994.

Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. The asymmetric synthesis of α-amino
acids. Electrophilic azidation of chiral imide enolates, a practical approach to the synthesis
of (R)- and (S)-α-azido carboxylic acids, J. Am. Chem. Soc. 1990, 112, 4011-30.

96

Forster, M. O.; Fierz, H. E. The Triazo Group. Part I. Triazoacetic Acid and Triazoacetone
(Acetonylazoimide), J. Chem. Soc., Trans. 1908, 93, 72-85.

Frick, D. N.; Weber, D. J.; Gillespie, J. R.; Bessman, M. J.; Mildvan, A. S. Dual divalent cation
requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and
substrate complexes, J. Biol. Chem. 1994, 269, 1794-803.

Fringuelli, F.; Pizzo, F.; Vaccaro, L. Cobalt(II) chloride-catalyzed chemoselective sodium
borohydride reduction of azides in water, Synthesis 2000, 646-50.

Gabelli, S. B.; Bianchet, M. A.; Xu, W.; Dunn, C. A.; Niu, Z.-D.; Amzel, L. M.; Bessman, M. J.
Structure and Function of the E. coli Dihydroneopterin Triphosphate Pyrophosphatase: A
Nudix Enzyme Involved in Folate Biosynthesis, Structure (Cambridge, MA, U. S.) 2007, 15,
1014-22.

Gajda, A.; Gajda, T. Synthesis and reactivity of azidoalkylphosphonates, -phosphinates and -
phosphine oxides, Curr. Org. Chem. 2007, 11, 1652-68.

Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of
equilibrium constants with the HYPERQUAD suite of programs, Talanta 1996, 43, 1739-53.

Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 1997, 62, 7512-15.

Gronowitz, S.; Stenhammar, K.; Svensson, L. Lithiation of some 3[2-(2'-
bromophenyl)ethyl]thiophenes and intramolecular transmetalation, Heterocycles 1981, 15,
947-59.

Hakimelahi, G. H.; Just, G. Two simple methods for the synthesis of trialkyl α-
aminophosphonoacetates. Trifluoromethanesulfonyl azide as an azide-transfer agent, Synth.
Commun. 1980, 10, 429-35.

Hutchinson, D. W.; Semple, G. Synthesis of alkylated methylene bisphosphonates via
organothallium intermediates, J. Organomet. Chem. 1985, 291, 145-51.

Hutchinson, D. W.; Thornton, D. M. A simple synthesis of
monofluoromethylenebis(phosphonic acid), J. Organomet. Chem. 1988, 340, 93-99.

Jarvis, B. B.; Nicholas, P. E. Reactions of α-azido sulfones with bases, J. Org. Chem. 1980, 45,
2265-8.

97

Jaszay, Z. M.; Pham, T. S.; Gonczi, K.; Petnehazy, I.; Toke, L. Efficient solid/liquid phase-
transfer catalytic diazo transfer synthesis, Synth. Commun. 2010, 40, 1574-79.

Kaabak, L. V.; Kuz'mina, N. E.; Khudenko, A. V.; Tomilov, A. P. Improved synthesis of 1-
aminoethylidenediphosphonic acid, Russ. J. Gen. Chem. 2006, 76, 1673-74.

Kamerlin, S. C. L.; McKenna, C. E.; Goondman, M. F.; Warshel, A. A Computational Study of
the Hydrolysis of dGTP Analogues with Halomethylene-Modified Leaving Groups in Solution:
Implications for the Mechanism of DNA Polymerases, Biochemistry 2009, 48, 5963-71.

Kantoci, D.; Denike, J. K.; Wechter, W. J. Synthesis of aminobisphosphonate, Synth. Commun.
1996, 26, 2037-43.

Kashemirov, B. A.; Skoblikova, L. I.; Savenkov, N. F.; Khokhlov, P. S. 1,2-C →N migration of a
phosphoryl group during deoxygenation of 1-(methoxycarbonyl)-1-nitroso-1-
(diethoxyphosphoryl)ethane, Zh. Obshch. Khim. 1990, 60, 1184-5.

Khare, A. B.; McKenna, C. E. An improved synthesis of tetraalkyl
diazomethylenediphosphonates and alkyl diazo(dialkoxyphosphoryl)acetates, Synthesis
1991, 405-6.

Khokhlov, P. S.; Kashemirov, B. A.; Mikityuk, A. D.; Strepikheev, Y. A.; Chimishkyan, A. L.
Diazotization of α-aminophosphonylacetates, Zh. Obshch. Khim. 1984, 54, 2785-7.

Kirby, A. J.; Lima, M. F.; da, S. D.; Roussev, C. D.; Nome, F. Efficient Intramolecular General
Acid Catalysis of Nucleophilic Attack on a Phosphodiester, J. Am. Chem. Soc. 2006, 128,
16944-52.

Kool, E. T.; Sintim, H. O. The difluorotoluene debate-a decade later, Chem. Commun.
(Cambridge, U. K.) 2006, 3665-75.

Koort, E.; Gans, P.; Herodes, K.; Pihl, V.; Leito, I. Acidity constants in different media (I=0 and
I=0.1 M KCl) from the uncertainty perspective., Anal. Bioanal. Chem. 2006, 385, 1124-39.

Kortum, G.; Vogel, W.; Andrussow, K. Dissociation constants of organic acids in aqueous
solution, Pure Appl. Chem. 1961, 1, 190-536.

Kumler, W. D.; Eiler, J. J. The acid strength of mono and diesters of phosphoric acid. The n-
alkyl esters from Me to Bu, the esters of biological importance and the natural
guanidinephosphoric acids, J. Am. Chem. Soc. 1943, 65, 2355-61.

98

Lad, C.; Williams, N. H.; Wolfenden, R. The rate of hydrolysis of phosphomonoester dianions
and the exceptional catalytic proficiencies of protein and inositol phosphatases, Proc. Natl.
Acad. Sci. U. S. A. 2003, 100, 5607-10.

Lassila, J. K.; Zalatan, J. G.; Herschlag, D. Biological phosphoryl-transfer reactions:
Understanding mechanism and catalysis, Annu. Rev. Biochem. 2011, 80, 669-702.

Liang, F.; Jain, N.; Hutchens, T.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; Chiarelli, M. P.; Cho,
B. P. α,β-Methylene-2'-deoxynucleoside 5'-triphosphates as noncleavable substrates for
DNA polymerases: Isolation, characterization, and stability studies of novel 2'-
deoxycyclonucleosides, 3,5'-cyclo-dG, and 2,5'-cyclo-dT, J. Med. Chem. 2008, 51, 6460-70.

Manis, P. A.; Rathke, M. W. Reaction of α-azido esters with lithium ethoxide: synthesis of
dehydroamino esters and α-keto esters, J. Org. Chem. 1980, 45, 4952-4.

Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E. Synthesis of
α-fluorinated phosphonoacetate derivatives using electrophilic fluorine reagents: Perchloryl
fluoride versus 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane
bis(tetrafluoroborate) (Selectfluor), J. Fluorine Chem. 2005, 126, 1467-75.

Martynov, B. I.; Shirokova, E. A.; Jasko, M. V.; Victorova, L. S.; Krayevsky, A. A. Effect of
triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity
towards various DNA polymerases, FEBS Lett. 1997, 410, 423-27.

McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C. The facile dealkylation of
phosphonic acid dialkyl esters by bromotrimethylsilane, Tetrahedron Lett. 1977, 155-8.

McKenna, C. E.; Kashemirov, B. A. Recent progress in carbonylphosphonate chemistry, Top.
Curr. Chem. 2002, 220, 201-38.

McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M. Product class 16: phosphoric acid and
derivatives, Sci. Synth. 2009, 42, 779-921.

McKenna, C. E.; Kashemirov, B. A.; Peterson, L. W.; Goodman, M. F. Modifications to the
dNTP triphosphate moiety: From mechanistic probes for DNA polymerases to antiviral and
anti-cancer drug design, Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 1223-30.

McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.;
Beard, W. A.; Wilson, S. H. (R)-β,γ-fluoromethylene-dGTP-DNA ternary complex with DNA
polymerase β, J. Am. Chem. Soc. 2007, 129, 15412-13.

99

McKenna, C. E.; Khawli, L. A. Synthesis of halogenated phosphonoacetate esters, J. Org.
Chem. 1986, 51, 5467-70.

McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y.; Pham, P.; Bongartz, J. P. Synthesis of α-
halogenated methanediphosphonates, Phosphorus, Sulfur Silicon Relat. Elem. 1988, 37, 1-12.

McKenna, C. E.; Levy, J. N. α-Keto phosphonoacetates, J. Chem. Soc., Chem. Commun. 1989,
246-7.

McKenna, C. E.; Schmidhauser, J. Functional selectivity in phosphonate ester dealkylation
with bromotrimethylsilane, J. Chem. Soc., Chem. Commun. 1979, 739.

McKenna, C. E.; Shen, P.-D. Fluorination of methanediphosphonate esters by perchloryl
fluoride. Synthesis of fluoromethanediphosphonic acid and difluoromethanediphosphonic
acid, J. Org. Chem. 1981, 46, 4573-76.

Menge, M.; Muenzenberg, K. J.; Reimann, E. 2,2-Propane diphosphonic acid, Arch. Pharm.
(Weinheim, Ger.) 1981, 314, 218-22.

Midrier, C.; Lantsoght, M.; Volle, J.-N.; Pirat, J.-L.; Virieux, D.; Stevens, C. V.
Hydrophosphonylation of alkenes or nitriles by double radical transfer mediated by
titanocene/propylene oxide, Tetrahedron Lett. 2011, 52, 6693-96.

Mildvan, A. S.; Weber, D. J.; Abeygunawardana, C. Solution structure and mechanism of the
MutT pyrophosphohydrolase, Adv. Enzymol. Relat. Areas Mol. Biol. 1999, 73, xi-xii, 183-207.

Mildvan, A. S.; Xia, Z.; Azurmendi, H. F.; Saraswat, V.; Legler, P. M.; Massiah, M. A.; Gabelli, S.
B.; Bianchet, M. A.; Kang, L. W.; Amzel, L. M. Structures and mechanisms of Nudix
hydrolases, Arch. Biochem. Biophys. 2005, 433, 129-43.

Mintz, M. J.; Walling, C. Tert-butyl hypochlorite, Org. Syn. 1969, 49, 9-12.

Moffatt, J. G. General synthesis of nucleoside 5'-triphosphates, Can. J. Chem. 1964, 42, 599-
604.

Moffatt, J. G.; Khorana, H. G. Nucleoside polyphosphates. X. The synthesis and some
reactions of nucleoside 5'-phosphoromorpholidates and related compounds. Improved
methods for the preparation of nucleoside 5'-polyphosphates, J. Am. Chem. Soc. 1961, 83,
649-58.

Mohamady, S.; Jakeman, D. L. An Improved Method for the Synthesis of Nucleoside
Triphosphate Analogs, J. Org. Chem. 2005, 70, 10588-91.

100

Mueller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals: Looking Beyond Intuition,
Science (Washington, DC, U. S.) 2007, 317, 1881-86.

O'Hagan, D. Understanding organofluorine chemistry. An introduction to the C-F bond,
Chem. Soc. Rev. 2008, 37, 308-19.

O'Handley, S. F.; Frick, D. N.; Bullions, L. C.; Mildvan, A. S.; Bessman, M. J. Escherichia coli
orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT
family of proteins. Cloning, purification, and characterization of the enzyme, J. Biol. Chem.
1996, 271, 24649-54.

Ojida, A.; Mitooka, Y.; Inoue, M.; Hamachi, I. First artificial receptors and chemosensors
toward phosphorylated peptide in aqueous solution, J. Am. Chem. Soc. 2002, 124, 6256-58.

Osuna, J. Ph.D., dissertation, University of Southern California , In preparation.

Ora, M.; Lonnberg, T.; Florea-Wang, D.; Zinnen, S.; Karpeisky, A.; Lonnberg, H.
Bisphosphonate Derivatives of Nucleoside Antimetabolites: Hydrolytic Stability and
Hydroxyapatite Adsorption of 5'-β,γ-Methylene and 5'-β,γ-(1-Hydroxyethylidene)
Triphosphates of 5-Fluorouridine and ara-Cytidine, J. Org. Chem. 2008, 73, 4123-30.

Orlovskii, V. V.; Vovsi, B. A. Reaction of dialkyl phosphites with nitriles, Zh. Obshch. Khim.
1976, 46, 297-300.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.;
Ferrin, T. E. UCSF Chimera-A visualization system for exploratory research and analysis, J.
Comput. Chem. 2004, 25, 1605-12.

Philip, J. C. The Dissociation Constants of Triazoacetic and α-Triazopropionic Acids, J. Chem.
Soc., Trans. 1908, 93, 925-7.

Popov, K.; Ronkkomaki, H.; Lajunen, L. Critical evaluation of stability constants of
phosphonic acids (IUPAC technical report), Pure Appl. Chem. 2001, 73, 1641-77.

Quin, L. D.; Wu, X.-P.; Sadanani, N. D.; Lukes, I.; Ionkin, A. S.; Day, R. O. Synthesis,
fragmentation, and photorearrangement of neopentyl and adamantyl phosphonates in the
2,3-oxaphosphabicyclo[2.2.2]octene system, J. Org. Chem. 1994, 59, 120-9.

Radhakrishnan, R.; Arora, K.; Wang, Y.; Beard, W. A.; Wilson, S. H.; Schlick, T. Regulation of
DNA repair fidelity by molecular checkpoints: "Gates" in DNA polymerase β's substrate
selection, Biochemistry 2006, 45, 15142-56.

101

Regitz, M.; Anschuetz, W.; Liedhegener, A. Reactions of CH-active compounds with azides.
XXIII. Synthesis of α-diazophosphonic acid esters, Chem. Ber. 1968, 101, 3734-43.

Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden, R. The time required for
water attack at the phosphorus atom of simple phosphodiesters and of DNA, Proc. Natl.
Acad. Sci. U. S. A. 2006, 103, 4052-55.

Scriven, E. F. V.; Turnbull, K. Azides: their preparation and synthetic uses, Chem. Rev. 1988,
88, 297-368.

Shi, S.-Y.; Zhang, Y.-P.; Jiang, X.-Y.; Chen, X.-Q.; Huang, K.-L.; Zhou, H.-H. Coupling HPLC to
on-line, post-column (bio)chemical assays for high-resolution screening of bioactive
compounds from complex mixtures, TrAC, Trends Anal. Chem. 2009, 28, 865-77.

Shiraki, C.; Saito, H.; Takahashi, K.; Urakawa, C.; Hirata, T. Preparation of
amino(diethoxyphosphoryl)acetic esters. Catalytic hydrogenation of diazo compounds to
amines, Synthesis 1988, 399-401.

Sobhani, S.; Tashrifi, Z. Synthesis of α-functionalized phosphonates from α-
hydroxyphosphonates, Tetrahedron 2010, 66, 1429-39.

Stockbridge, R. B.; Wolfenden, R. The Intrinsic Reactivity of ATP and the Catalytic
Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine: a
thermodynamic analysis, J. Biol. Chem. 2009, 284, 22747-57.

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.
Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency,
Fidelity, and the Catalytic Mechanism of DNA Polymerase β, Biochemistry 2007, 46, 461-71.

Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S.
H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. DNA Polymerase β Fidelity:
Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis Reveal
Differences at the Chemical Transition State, Biochemistry 2008, 47, 870-79.

Sun, D.; Shi, E.; Xiao, J.; Pei, C. An efficient synthesis of dialkyl phosphorocyanidates,
Phosphorus, Sulfur Silicon Relat. Elem. 2005, 180, 2155-61.

Taylor, S. D.; Kotoris, C. C.; Dinaut, A. N.; Chen, M.-J. Synthesis of aryl(difluoromethylene
phosphonates) via electrophilic fluorination of α-carbanions of benzylic phosphonates with
N-fluorobenzenesulfonimide, Tetrahedron 1998, 54, 1691-714.

102

Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a
new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 2010, 31,
455-61.

Upton, T. G. Design and synthesis of a series of methylenebisphosphonates: A nucleotide
analogue toolkit to probe nucleic acid polymerase structure and function, Ph.D. dissertation,
University of Southern California, 2008.

Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.; Kultyshev,
R.; Batra, V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. α,β-
Difluoromethylene Deoxynucleoside 5'-Triphosphates: A Convenient Synthesis of Useful
Probes for DNA Polymerase β Structure and Function, Org. Lett. 2009, 11, 1883-86.

Victorova, L. S.; Semizarov, D. G.; Shirokova, E. A.; Alexandrova, L. A.; Arzumanov, A. A.;
Jasko, M. V.; Krayevsky, A. A. Human DNA polymerases and retroviral reverse transcriptases:
selectivity in respect to dNTPs modified at triphosphate residues, Nucleosides Nucleotides
1999, 18, 1031-32.

Viktorova, L. S.; Arzumanov, A. A.; Shirikova, E. A.; Yas'ko, M. V.; Aleksandrova, L. A.;
Shipitsyn, A. V.; Skoblov, A. Y.; Krayevsky, A. A. Modified nucleoside 5'-triphosphates with
elevated stability against dephosphorylating enzymes, Mol. Biol. (Mosk) 1998, 32, 162-71.

Westheimer, F. H. Why nature chose phosphates, Science (Washington, D. C., 1883-) 1987,
235, 1173-8.

Wiberg, K. B.; Pratt, W. E.; Bailey, W. F. Nature of substituent effects in nuclear magnetic
resonance spectroscopy. 1. Factor analysis of carbon-13 chemical shifts in aliphatic halides, J.
Org. Chem. 1980, 45, 4936-47.

Williams, N. H.; Wyman, P. Base catalyzed phosphate diester hydrolysis, Chem. Commun.
(Cambridge, U. K.) 2001, 1268-69.

Wolfenden, R. Benchmark reaction rates, the stability of biological molecules in water, and
the evolution of catalytic power in enzymes, Annu. Rev. Biochem. 2011, 80, 645-67.

Wolfenden, R. Degrees of difficulty of water-consuming reactions in the absence of enzymes,
Chem. Rev. (Washington, DC, U. S.) 2006, 106, 3379-96.

Wolfenden, R.; Ridgway, C.; Young, G. Spontaneous Hydrolysis of Ionized Phosphate
Monoesters and Diesters and the Proficiencies of Phosphohydrolases as Catalysts, J. Am.
Chem. Soc. 1998, 120, 833-34.

103

Wurz, R. P.; Lin, W.; Charette, A. B. Trifluoromethanesulfonyl azide: an efficient reagent for
the preparation of α-cyano-α-diazo carbonyls and an α-sulfonyl-α-diazo carbonyl,
Tetrahedron Lett. 2003, 44, 8845-48.

Xia, S.; Konigsberg, W. H.; Wang, J. Hydrogen-Bonding Capability of a Templating
Difluorotoluene Nucleotide Residue in an RB69 DNA Polymerase Ternary Complex, J. Am.
Chem. Soc. 2011, 133, 10003-05.

104

APPENDIX A. Chapter 1 Supporting Data

Figure A1.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CH
2
, 16.
Note: products obtained as the triethylammonium salts.

105

Figure A2.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CH
2
, 16.

106

Figure A3. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CH
2
, 16. Calc for C
12
H
20
N
2
O
13
P
3
: 479.0; found:
479.0 m/z.

107

Figure A4. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CH
2
, 16. RT = 9.1 min.

Chrom. 1 0.0 mins. 15.7 mins.
1

108

Figure A5.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CF
2
, 17.

109

Figure A6.
19
F-NMR spectrum (376 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CF
2
, 17.

110

Figure A7.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CF
2
, 17.

111

Figure A8. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CF
2
, 17. Calc for C
11
H
17
F
2
N
2
O
13
P
3
:

515.0; found:
514.9 m/z.

112

Figure A9. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CF
2
, 17. RT = 11.8 min.

Chrom. 1 0.0 mins. 15.0 mins.
2

113

Figure A10.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHF, 18a/b.

114

Figure A11.
19
F-NMR spectrum (376 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHF, 18a/b.

115

Figure A12.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHF, 18a/b.

116

Figure A13. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CHF, 18a/b. Calc for C
11
H
17
FN
2
O
13
P
3
: 497.0;
found: 497.0 m/z.

117

Figure A14. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CHF, 18a/b. RT = 10.5 min.

118

Figure A15.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CCl
2
, 19.

119

Figure A16.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CCl
2
, 19.

120

Figure A17. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CCl
2
, 19. Calc for C
11
H
16
Cl
2
N
2
O
13
P
3
: 546.9
(100%), 548.9 (64%) ; found: 547.0 (100%), 549.0 (63%) m/z.

121

Figure A18. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CCl
2
, 19. RT = 9.9 min.

122

Figure A19.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHCl, 20a/b.

123

Figure A20.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHCl, 20a/b.

124

Figure A21.
31
P-NMR spectrum (162 MHz, D
2
O, pH >10) of thymidine 5’-triphosphate β,γ-CHCl after
treatment with Chelex
TM
, 20a/b.

125

Figure A22.
31
P-NMR spectrum (202 MHz, D
2
O, pH >10) of thymidine 5’-triphosphate β,γ-CHCl after
treatment with Chelex
TM
, 20a/b. (Comparison with Figure A21 confirms J value assignments).

126

Figure A23. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CHCl, 20a/b. Calc for C
11
H
17
ClN
2
O
13
P
3
: 513.0
(100%), 515.0 (32%); found: 513.0 (100%), 515.0 (33%) m/z.

127

Figure A24. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CHCl, 20a/b. RT = 9.8 min.

Chrom. 1 0.0 mins. 14.9 mins.
1

128

Figure A25.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CBr
2
, 21.

129

Figure A26.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CBr
2
, 21.

130

Figure A27. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CBr
2
, 21. Calc for C
11
H
16
Br
2
N
2
O
13
P
3
:

636.8
(100%), 634.8 (51%), 638.8 (49%); found: 636.8 (100%), 634.9 (51%), 638.8 (52%) m/z.

131

Figure A28. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CBr
2
, 21. RT = 9.8 min.

132

Figure A29.
1
H-NMR spectrum (400 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHBr, 22a/b.

133

Figure A30.
31
P-NMR spectrum (162 MHz, D
2
O, pH neutral) of thymidine 5’-triphosphate β,γ-CHBr, 22a/b.

134

Figure A31. LRMS [M-H]
-
of thymidine 5’-triphosphate β,γ-CHBr, 22a/b. Calc for C
11
H
17
BrN
2
O
13
P
3
:

556.9
(100%), 558.9 (97%); found: 556.9 (100%), 558.9 (96%) m/z.

135

Figure A32. Analytical SAX HPLC trace for thymidine 5’-triphosphate β,γ-CHBr, 22a/b. RT = 9.4 min.
Chrom. 1 0.0 mins. 12.5 mins.
1

136

APPENDIX B. Chapter 2 Supporting Data

Figure B1.
1
H-NMR spectrum (400 MHz, CDCl
3
) of tetraisopropyl (1-azidoethane-1,1-diyl)-
bis(phosphonate), 1.

137

Figure B2.
13
C-NMR spectrum (101 MHz, CDCl
3
) of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.

138

Figure B3.
31
P-NMR spectra (162 MHz , CDCl
3
) of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.

139

Figure B4. FTIR spectrum (film, NaCl plate) of (1-azidoethane-1,1-diyl)bis(phosphonate), 1.

140

Figure B5.
1
H-NMR spectrum (400 MHz , CDCl
3
) of tetraisopropyl (azidomethanediyl)bis(phosphonate), 2.

141

Figure B6.
13
C-NMR spectrum (101 MHz , CDCl
3
) of tetraisopropyl (azidomethanediyl)bis(phosphonate), 2.

142

Figure B7.
31
P-NMR spectra (162 MHz , CDCl
3
) of tetraisopropyl (azidomethanediyl)bis(phosphonate), 2.

143

Figure B8. FTIR spectrum (film, NaCl plate) of tetraisopropyl (azidomethanediyl)bis(phosphonate), 2.

144

Figure B9.
1
H-NMR spectrum (400 MHz, D
2
O, pH 10.88) of (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

145

Figure B10.
13
C-NMR spectrum (101 MHz , D
2
O, pH 10.88) of (1-azidoethane-1,1-diyl)bis(phosphonic acid),
3.

146

Figure B11.
31
P-NMR spectra (162 MHz, D
2
O, pH 10.88) of (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

147

Figure B12. FTIR spectrum (KBr pellet) of (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

148

Figure B13. LC-MS PDA chromatogram of (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

149

Figure B14. HRMS (ESI/ACPI) of (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

150

Figure B15.
1
H-NMR spectrum (400 MHz , D
2
O, pH 10.88) of (azidomethanediyl)bis(phosphonic acid), 4.

151

Figure B16.
13
C -NMR spectrum (101 MHz , D
2
O, pH 10.88) of (azidomethanediyl)bis(phosphonic acid), 4.

152

Figure B17.
31
P-NMR spectra (162 MHz , D
2
O, pH 10.88) of (azidomethanediyl)bis(phosphonic acid), 4.

153

Figure B18. FTIR spectrum (KBr pellet) of (azidomethanediyl)bis(phosphonic acid), 4.

154

Figure B19. LC-MS PDA chromatogram of (azidomethanediyl)bis(phosphonic acid), 4.

155

Figure B20. HRMS (ESI/ACPI) of (azidomethanediyl)bis(phosphonic acid), 4.

156

Figure B21. Titration curve for (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3, with KOH. Stock solution:
49.60 mg of 3 in 50 mL 0.1 M KCl solution; Trials performed on 10 mL (±0.01) samples.

2
3
4
5
6
7
8
9
10
11
0 0.5 1 1.5 2
pH
Volume 0.0882 M KOH (mL)
Titration of C(CH
3
)N
3
BP

Trial 1
Trial 2
Trial 3

157

Table B1. Potentiometric titration data for (1-azidoethane-1,1-diyl)bis(phosphonic acid), 3.

Trial 1 Trial 2 Trial 3
log β
Volume 0.0882
M KOH (mL)
1 10.52 10.54 10.45
2 16.91 16.93 16.86
3 19.26 19.27 19.17
pH
0.02 2.27 2.26 2.27
0.04 2.29 2.27 2.28
0.06 2.3 2.28 2.29
0.08 2.31 2.29 2.3
0.1 2.32 2.3 2.32
0.12 2.33 2.32 2.33
0.14 2.35 2.33 2.34
0.16 2.36 2.34 2.35
0.18 2.37 2.36 2.37
0.2 2.39 2.37 2.38
0.22 2.4 2.39 2.4
0.24 2.42 2.4 2.41
0.26 2.43 2.42 2.43
0.28 2.45 2.43 2.45
0.3 2.46 2.45 2.46
0.32 2.48 2.46 2.48
0.34 2.5 2.48 2.5
0.36 2.52 2.5 2.52
0.38 2.54 2.52 2.54
0.4 2.56 2.54 2.56
0.42 2.58 2.56 2.58
0.44 2.6 2.58 2.6
0.46 2.62 2.61 2.63
0.48 2.65 2.63 2.65
0.5 2.67 2.66 2.68
0.52 2.7 2.68 2.71
0.54 2.73 2.71 2.74
0.56 2.76 2.74 2.77
0.58 2.79 2.77 2.8
0.6 2.82 2.81 2.84
0.62 2.86 2.85 2.88

158

Table B1, Continued.

mL pH
0.64 2.9 2.89 2.92
0.66 2.95 2.94 2.97
0.68 3 2.98 3.02
0.7 3.05 3.04 3.08
0.72 3.11 3.1 3.14
0.74 3.18 3.17 3.22
0.76 3.27 3.26 3.31
0.78 3.37 3.36 3.42
0.8 3.5 3.5 3.58
0.82 3.68 3.69 3.82
0.84 3.97 3.99 4.23
0.86 4.46 4.49 4.75
0.88 4.91 4.94 5.1
0.9 5.2 5.23 5.34
0.92 5.42 5.42 5.52
0.94 5.59 5.59 5.64
0.96 5.73 5.72 5.8
0.98 5.85 5.85 5.91
1 5.96 5.96 6.02
1.02 6.06 6.05 6.11
1.04 6.15 6.14 6.2
1.06 6.24 6.22 6.28
1.08 6.33 6.3 6.36
1.1 6.41 6.39 6.44
1.12 6.49 6.47 6.53
1.14 6.57 6.55 6.61
1.16 6.65 6.63 6.69
1.18 6.74 6.72 6.78
1.2 6.83 6.8 6.88
1.22 6.93 6.91 6.98
1.24 7.04 7.01 7.1
1.26 7.17 7.13 7.25
1.28 7.31 7.28 7.42
1.3 7.5 7.47 7.67
1.32 7.78 7.74 8.04
1.34 8.15 8.54
1.36 8.69 8.65 8.94

159

Table B1, Continued.

mL pH
1.38 9.05 9.01 9.22
1.4 9.31 9.27 9.43
1.42 9.49 9.47 9.61
1.44 9.66 9.64 9.75
1.46 9.79 9.78 9.88
1.48 9.92 9.9 9.99
1.5 10.02 10.08
1.52 10.11 10.1 10.17
1.54 10.2 10.18 10.24
1.56 10.27 10.26 10.32
1.58 10.34 10.33 10.39
1.6 10.41 10.4 10.45
1.62 10.48 10.47 10.51
1.64 10.54 10.53 10.57
1.66 10.59 10.58 10.62
1.68 10.65 10.64 10.68
1.7 10.7 10.69 10.73
1.72 10.75 10.74 10.78
1.74 10.8
1.76 10.84 10.83 10.87
1.78 10.89
1.8 10.93 10.92 10.95
1.82 10.97
1.84 11.01 11 11.03
1.86 11.04
1.88 11.08 11.07 11.09
1.9 11.11
1.92 11.14 11.14 11.16
1.94 11.17
1.96 11.2 11.19 11.22
1.98 11.23
2 11.26 11.25 11.27
2 9.46 9.44 9.43
2.02 9.53 9.51 9.5
2.04 9.59 9.57 9.56
2.06 9.65 9.63 9.62

160

Table B1, Continued.

mL pH
2.08 9.71 9.7 9.69
2.1 9.78 9.76 9.75
2.12 9.85 9.83 9.82
2.14 9.93 9.91 9.9
2.16 10 9.98 9.97
2.18 10.08 10.05 10.05
2.2 10.16 10.13 10.13
2.22 10.26 10.22 10.21
2.24 10.33 10.31 10.3
2.26 10.43 10.4 10.39
2.28 10.52 10.49 10.48
2.3 10.6 10.58 10.57
2.32 10.68 10.66 10.66
2.34 10.76 10.74 10.73
2.36 10.83 10.81 10.8
2.38 10.89 10.87 10.87
2.42 11 10.99 10.98
2.46 11.1 11.12 11.08
2.5 11.17 11.19 11.15
2.54 11.24 11.26 11.22
2.58 11.3 11.31 11.28
2.62 11.35 11.36 11.33
2.66 11.4 11.41 11.38
2.7 11.44 11.43
2.72 11.46 11.45 11.44

161

Figure B22. Titration curve for (azidomethanediyl)bis(phosphonic acid), 4, with KOH. Stock solution:
57.90 mg of 4 in 50 mL 0.1 M KCl solution; Trials performed on 10 mL (±0.01) samples.

2
3
4
5
6
7
8
9
10
11
0 0.5 1 1.5 2 2.5
pH
Volume 0.0882 M KOH (mL)
Titration of CHN
3
BP
Trial 1
Trial 2
Trial 3

162

Table B2. Potentiometric titration data for (azidomethanediyl)bis(phosphonic acid), 4.

Trial 1 Trial 2 Trial 3
Log β
Volume
0.0882 M
KOH (mL)
1
9.38 9.40 9.40
2
15.64 15.66 15.68
3
17.66 17.78 17.76
pH
0 2.15 2.14 2.14
0.02 2.16 2.14
0.04 2.16
0.06 2.17 2.16 2.16
0.08 2.18
0.1 2.19 2.18 2.18
0.12 2.2
0.14 2.21 2.2 2.2
0.16 2.21
0.18 2.22 2.22 2.22
0.22 2.24 2.24 2.24
0.26 2.27 2.26 2.26
0.3 2.29 2.29 2.28
0.34 2.32 2.31 2.31
0.38 2.34 2.34 2.33
0.42 2.37 2.36 2.36
0.46 2.4 2.39 2.39
0.5 2.43 2.42 2.42
0.54 2.46 2.45 2.45
0.58 2.49 2.49 2.48
0.62 2.53 2.53 2.52
0.66 2.57 2.56 2.56
0.7 2.61 2.6 2.6
0.74 2.66 2.65 2.65
0.78 2.71 2.7 2.7
0.8 2.74 2.73 2.73

163

Table B2, Continued.

mL pH
0.82 2.77 2.76 2.76
0.84 2.8 2.79 2.79
0.86 2.84 2.83 2.82
0.88 2.88 2.86 2.86
0.9 2.92 2.9 2.9
0.92 2.96 2.94 2.94
0.94 3.01 2.99 2.99
0.96 3.06 3.04 3.04
0.98 3.13 3.1 3.1
1 3.2 3.18 3.16
1.02 3.28 3.25 3.25
1.04 3.38 3.36 3.34
1.06 3.52 3.49 3.47
1.08 3.71 3.64 3.64
1.1 4.03 3.92 3.9
1.12 4.5 4.36 4.35
1.14 4.89 4.78 4.8
1.16 5.16 5.09 5.08
1.18 5.33 5.27 5.26
1.2 5.47 5.43 5.41
1.22 5.58 5.54 5.53
1.24 5.67 5.64 5.63
1.26 5.77 5.73 5.74
1.28 5.84 5.82 5.81
1.3 5.91 5.9 5.89
1.32 5.99 5.97 5.96
1.34 6.05 6.03 6.03
1.36 6.11 6.1 6.09
1.38 6.17 6.16 6.15
1.4 6.23 6.22 6.21
1.42 6.3 6.28 6.27
1.44 6.36 6.34 6.33
1.46 6.42 6.41 6.4
1.48 6.48 6.47 6.46
1.5 6.55 6.54 6.52

164

Table B2, Continued.

mL pH
1.52 6.61 6.6 6.58
1.54 6.68 6.66 6.65
1.56 6.76 6.74 6.73
1.58 6.84 6.82 6.81
1.6 6.92 6.91 6.9
1.62 7.02 7.01 6.99
1.64 7.13 7.12 7.1
1.66 7.26 7.23 7.21
1.68 7.42 7.4 7.37
1.7 7.61 7.59 7.55
1.72 7.86 7.81 7.77
1.74 8.11 8.06 8.02
1.76 8.32 8.26 8.24
1.78 8.48 8.45 8.44
1.8 8.63 8.61 8.59
1.82 8.76 8.73 8.71
1.84 8.86 8.84 8.83
1.86 8.96 8.94 8.92
1.88 9.04 9.02 9.01
1.9 9.12 9.1 9.09
1.92 9.19 9.18 9.16
1.94 9.26 9.25 9.23
1.96 9.33 9.31 9.3
1.98 9.4 9.38 9.37
2 9.46 9.44 9.43
2.02 9.53 9.51 9.5
2.04 9.59 9.57 9.56
2.06 9.65 9.63 9.62
2.08 9.71 9.7 9.69
2.1 9.78 9.76 9.75
2.12 9.85 9.83 9.82
2.14 9.93 9.91 9.9
2.16 10 9.98 9.97
2.18 10.08 10.05 10.05
2.2 10.16 10.13 10.13

165

Table B2, Continued.

mL pH
2.22 10.26 10.22 10.21
2.24 10.33 10.31 10.3
2.26 10.43 10.4 10.39
2.28 10.52 10.49 10.48
2.3 10.6 10.58 10.57
2.32 10.68 10.66 10.66
2.34 10.76 10.74 10.73
2.36 10.83 10.81 10.8
2.38 10.89 10.87 10.87
2.42 11 10.99 10.98
2.46 11.1 11.12 11.08
2.5 11.17 11.19 11.15
2.54 11.24 11.26 11.22
2.58 11.3 11.31 11.28
2.62 11.35 11.36 11.33
2.66 11.4 11.41 11.38
2.7 11.44 11.43
2.72 11.46 11.45 11.44

166

Figure B23. Sample Hyperquad2006 output after refinement calculations. Shown, (azidomethanediyl)
bis(phosphonic acid), 4.

167

Figure B24.
1
H-NMR spectrum (400 MHz, D
2
O, pH 10.88) of 2’-deoxyguanosine 5’-triphosphate β,γ-
C(CH
3
)N
3
, 5a/b.

168

Figure B25.
31
P-NMR spectrum (162 MHz, D
2
O, pH 10.88) of 2’-deoxyguanosine 5’-triphosphate β,γ-
C(CH
3
)N
3
,

5a/b.

169

Figure B26.
31
P-NMR of 2’-deoxyguanosine 5’-triphosphate β,γ-C(CH
3
)N
3
,

5a/b. P
β
at 203 MHz (left) and
162 MHz (right), pH 10.88 confirming δ vs. J assignments. The chemical shifts in the spectrum were
normalized to Figure B.25.

170

Figure B27. HRMS (ESI/ACPI) of 2’-deoxyguanosine 5’-triphosphate β,γ-C(CH
3
)N
3
,

5a/b.

171

Figure B28.
1
H-NMR spectrum (400 MHz, D
2
O, pH 10.88) of 2’-deoxyguanosine 5’-triphosphate β,γ-CHN
3
,
6a/b.

172

Figure B29.
31
P-NMR spectrum (162 MHz , D
2
O, pH 10.88) of 2’-deoxyguanosine 5’-triphosphate β,γ-CHN
3
,
6a/b.

173

Figure B30. HRMS (ESI/ACPI) of 2’-deoxyguanosine 5’-triphosphate β,γ-CHN
3
, 6a/b.

174

Figure B31.
1
H-NMR spectrum (500 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-
C(CH
3
)N
3
, 7a.

175

Figure B32.
31
P-NMR spectrum (203 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-
C(CH
3
)N
3
,

7a.

176

Figure B33.
1
H-NMR spectrum (500 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-
C(CH
3
)N
3
, 7b

177

Figure B34.
31
P-NMR spectrum (202 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-
C(CH
3
)N
3
, 7b.

178

Figure B35 UV/VIS spectra of 2’-deoxyadenosine 5’-triphosphate α,β-C(CH
3
)N
3
7a and 7b in H
2
O, pH 8. Est.
conc. ~0.05 mM (E = 15300).

179

Figure B36.
1
H-NMR spectrum (400 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-CHN
3
,
8a/b.

180

Figure B37.
31
P-NMR spectrum (202 MHz, D
2
O, pH 10.88) of 2’-deoxyadenosine 5’-triphosphate α,β-CHN
3
,

8a/b.

181

Figure B38.
1
H-NMR spectrum (400 MHz, CDCl
3
) of tetraisopropyl (1-aminoethane-1,1-
diyl)bis(phosphonate), 16.

182

Figure B39. gCOSY of tetraisopropyl (1-aminoethane-1,1-diyl)bis(phosphonate), 16.

183

Figure B40.
13
C-NMR spectrum (101 MHz, CDCl
3
) of tetraisopropyl (1-aminoethane-1,1-
diyl)bis(phosphonate), 16.

184

Figure B41. gHSQC(AD) of tetraisopropyl (1-aminoethane-1,1-diyl)bis(phosphonate), 16.

185

Figure B42.
31
P-NMR spectrum (161 MHz, CDCl
3
) of tetraisopropyl (1-aminoethane-1,1-
diyl)bis(phosphonate), 16.

186

Figure B43. FTIR spectrum (film, NaCl plate) of tetraisopropyl (1-aminoethane-1,1-diyl)bis(phosphonate),
16.

187

Figure B44. LRMS [M+Na]
+
of tetraisopropyl (1-aminoethane-1,1-diyl)bis(phosphonate), 16. calcd for
C
14
H
33
NNaO
6
P
2
, 396.2; found: 396.0 m/z.

188

Figure B45.
1
H-NMR spectrum (400 MHz, CDCl
3
,) of diisopropyl (1-([bis(propan-2-
yloxy)phosphoryl]amino)ethenyl)phosphonate, 18.

189

Figure B46.
13
C-NMR spectrum (101 MHz, CDCl
3
) of diisopropyl (1-([bis(propan-2-
yloxy)phosphoryl]amino)ethenyl)phosphonate, 18.

190

Figure B47. gHSQC of diisopropyl (1-([bis(propan-2-yloxy)phosphoryl]amino)ethenyl)phosphonate, 18.
(obtained on reaction mixture).

191

Figure B48.
31
P-NMR spectrum (203 MHz, CDCl
3
) of diisopropyl (1-([bis(propan-2-
yloxy)phosphoryl]amino)ethenyl)phosphonate, 18.

192

Figure B49. LRMS [M+H]
+
and [M+Na]
+

of diisopropyl (1-([bis(propan-2-
yloxy)phosphoryl]amino)ethenyl)phosphonate, 18. Calcd for [M + H]
+
C
14
H
32
NO
6
P
2
, 372.2; [M + Na]
+

C
14
H
31
NNaO
6
P
2
, 394.2; found: 372.1, 394.1 m/z.

193

APPENDIX C. Chapter 3 Supporting Data

Figure C1.
31
P-NMR spectrum (162 MHz, D
2
O, pH 10.8) of α,β-C(Me)
2
dADP, 3.

194

Figure C2.
31
P-NMR spectrum (162 MHz, D
2
O, pH 10.8) of α,β-CHF-dADP, 4a/b.
(contains residual MFBP acid)

195

Figure C3.
19
F-NMR spectrum (376 MHz, D
2
O, pH 10.8) of α,β-CHF-dADP, 4a/b.
(contains residual MFBP acid)

196

Figure C4.
19
F-NMR spectrum (376 MHz, D
2
O, pH 10.8) of α,β-CHF-dADP, 4a/b (zoomed).

197

Figure C5.
19
F-NMR spectrum (376 MHz) of α,β-CHF-dADP , 4a/b, with simulations.

198

Figure C6. Representative HPLC trace of enzymatic phorsphorylation reaction mixture. Shown α,β-
C(Me)
2
-dATP, 5, after 48h; aprx. 80% conversion. Retention time of dADP = 6.7 min, dATP = 10.8 min,
ATP = 16.5 minutes.

199

Figure C7.
1
H-NMR spectrum (600 MHz, D
2
O, pH 8.2) of α,β-C(Me)
2
-dATP, 5.
(contains Tris-Cl buffer).

200

FigureC8. gCOSY of α,β-C(Me)
2
-dATP, 5.

201

FigureC9.
31
P-NMR spectrum (162 MHz, D
2
O, pH 10.8) of α,β-C(Me)
2
-dATP, 5.

202

Figure C10. HRMS (ESI/APCI) [M-H]
-

of α,β-C(Me)
2
-dATP, 5.

203

Figure C11. Analytical SAX HPLC trace of α,β-C(Me)
2
-dATP, 5. RT = 10.9 min.

204

Fiigure C12.
1
H-NMR spectrum (600 MHz, D
2
O, pH 8.2) of α,β-CHF-dATP, 6a/b.

205

Figure C13. gCOSY of α,β-CHF-dATP, 6a/b.

206

Figure C14.
19
F-NMR spectrum (564 MHz, D
2
O, pH 10.8) of α,β-CHF-dATP, 6a/b.

207

Figure C15.
19
F-NMR spectrum (376 MHz, D
2
O, pH 10.8) of α,β-CHF-dATP, 6a/b.

208

Figure C16.
31
P-NMR spectrum (162 MHz, D
2
O, pH 10.8) of α,β-CHF-dATP, 6a/b.

209

Figure C17.
31
P-NMR spectrum (243 MHz, D
2
O, pH 10.8) of α,β-CHF-dATP, 6a/b, zoomed at P
α
.

210

Figure C18.
19
F-NMR spectrum (564 MHz) of α,β-CHF-dATP, 6a/b, with simulation.

211

Figure C19. LRMS [M-H]
-
of α,β-CHF-dATP, 6a/b. Calcd for C
11
H
16
FN
5
O
11
P
3
: 506.1; found: 506.0 m/z.

212

Figure C20. Analytical SAX HPLC trace of α,β-CHF-dATP, 6a/b. RT = 13.8 min.

Chrom. 1 0.0 mins. 19.6 mins.
1

213

Figure C21. Gel analysis of DNA synthesis background of dual-pass HPLC purified 8a/b (2′-
deoxyadenosine 5′-diphosphate, α,β-CHN
3
), 9a and 9b (2′-deoxyadenosine 5′-diphosphate, α,β-C(CH
3
)N
3
).
DNA synthesis was assayed on a single-nucleotide gapped DNA substrate where the templating base in
the gap was thymidine. The reaction mixture contained 50 mM Tris-HCl, pH 7.4, 20 mM MgCl
2
, 200 nM
single-nucleotide gapped DNA. The substrates/products were separated on 16% denaturing
polyacrylamide gels. The reactions were run for 10 min with 1 mM of the indicated analog in the presence
of 0.5 (L) or 50 nM (H) pol .

214

Figure C22. Inhibition of dATP incorporation by 8a/b (α,β-CHN
3
-dATP), 9a (α,β-C(CH
3
)N
3
-dATP 1), and 9b
(α,β-C(CH
3
)N
3
-dATP 2).

DNA synthesis was assayed on a single-nucleotide gapped DNA substrate where the
templating base in the gap was thymidine. The reaction mixture contained 50 mM Tris-HCl,
pH 7.4, 20 mM MgCl
2
, 200 nM single-nucleotide gapped DNA, 1 μM dATP (Km = 2.9 μM) and
various α,β-CXY-dATP concentrations. Reactions were initiated with 1.5 nM enzyme at rt
and stopped with EDTA. The substrates/products were separated on 16% denaturing
polyacrylamide gels and quantified in the gel.

215

Figure C23. Analysis of dATP incorporation inhibition by α,β-CHN
3
-dATP 8a/b.

216

APPENDIX D. Chapter 4 Supporting Data

Figure D1.
1
H-NMR (400 MHz, D
2
O, pH > 10.3) of 2,2-dimethylpropyl β,γ-methylene-triphosphate, 1.

217

Figure D2.
31
P-NMR (203 MHz, D
2
O, pH >10.3) of 2,2-dimethylpropyl β,γ-methylene-triphosphate, 1.

218

Figure D3. HRMS (ESI/ACPI) of 2,2-dimethylpropyl β,γ-methylene-triphosphate, 1.

219

Figure D4.
1
H-NMR (400 MHz, D
2
O, pH > 10.3) of 2,2-dimethylpropyl β,γ-(fluoro)methylene-triphosphate,
2.

220

Figure D5.
19
F-NMR (376 MHz, D
2
O, pH > 10.3) of 2,2-dimethylpropyl β,γ-(fluoro)methylene-triphosphate,
2.

221

Figure D6.
31
P-NMR (203 MHz, D
2
O, pH >10.3) of 2,2-dimethylpropyl β,γ-(fluoro)methylene-triphosphate,
2.

222

Figure D7. HRMS (ESI/ACPI) of 2,2-dimethylpropyl β,γ-(fluoro)methylene-triphosphate, 2.

223

Figure D8.
1
H-NMR (400 MHz, D
2
O, pH > 10.3) of 2,2-dimethylpropyl β,γ-(difluoro)methylene-
triphosphate, 3.

224

Figure D9.
19
F-NMR (376 MHz, D
2
O, pH > 10.3) of 2,2-dimethylpropyl β,γ-(difluoro)methylene-
triphosphate, 3.

225

Figure D10.
31
P-NMR (203 MHz, D
2
O, pH >10.3) of 2,2-dimethylpropyl β,γ-(difluoro)methylene-
triphosphate, 3.

226

Figure D11. HRMS (ESI/ACPI) of 2,2-dimethylpropyl β,γ-(difluoro)methylene-triphosphate, 3.

227

Figure D12.
1
H-NMR (600 MHz, D
2
O, pH nuetral) of benzylphosphonic (2,2-dimethylpropyl phosphoric)
anhydride, 4.

228

Figure D13.
13
C-NMR (151 MHz, D
2
O, pH nuetral, extrenal methanol reference) of benzylphosphonic (2,2-
dimethylpropyl phosphoric) anhydride, 4.

229

Figure D14.
31
P-NMR (243 MHz, D
2
O, pH nuetral) of benzylphosphonic (2,2-dimethylpropyl phosphoric)
anhydride, 4.

230

Figure D15. LRMS [M-H]
-
of benzylphosphonic (2,2-dimethylpropyl phosphoric) anhydride, 4. Calc for
C
12
H
19
O
6
P
2
[M-H]
-
: 321.1; found: 321.2 m/z.

231

Figure D16.
1
H-NMR (500 MHz, D
2
O, pH nuetral) of [difluoro(phenyl)methyl]phosphonic (2,2-
dimethylpropyl phosphoric) anhydride, 5.

232

Figure D17.
19
F-NMR (564 MHz, D
2
O, pH > 10.3) of [difluoro(phenyl)methyl]phosphonic (2,2-
dimethylpropyl phosphoric) anhydride, 5.

233

Figure D18.
31
P-NMR (243 MHz, D
2
O, pH nuetral) of [difluoro(phenyl)methyl]phosphonic (2,2-
dimethylpropyl phosphoric) anhydride, 5.

234

Figure D19. LRMS [M-H]- of [difluoro(phenyl)methyl]phosphonic (2,2-dimethylpropyl phosphoric)
anhydride, 5. Calc for C12H17F2O6P2 [M-H]-: 357.0; found: 357.1 m/z.

235

Figure D20. Calculated bond lengths for βγ-CXY neopentyl triphosphates 1-3.

236

Figure D21. Calculated bond lengths for neopentyl P
β
-CXYPh model compounds 4 and 5.

237

SPARTAN '08 MECHANICS PROGRAM: PC/x86 132
Frequency Calculation
Adjusted 5 (out of 78) low frequency modes
Reason for exit: Successful completion
Mechanics CPU Time : .11
Mechanics Wall Time: .54
SPARTAN '08 Quantum Mechanics Program: (PC/x86) Release 132v4
Job type: Geometry optimization.
Method: RB3LYP
Basis set: 6-31G(D)
Number of shells: 90
Number of basis functions: 294
Multiplicity: 1
SCF model:
A restricted hybrid HF-DFT SCF calculation will be
performed using Pulay DIIS + Geometric Direct Minimization
Optimization:
Step Energy Max Grad. Max Dist.
1 -1643.552843 0.026213 0.098940
2 -1643.560711 0.009465 0.189898
3 -1643.562463 0.006778 0.222206
4 -1643.563049 0.004939 0.148629
5 -1643.563385 0.004068 0.208163
6 -1643.563654 0.005124 0.180573
7 -1643.563922 0.005270 0.120661
8 -1643.564084 0.004018 0.063129
9 -1643.564175 0.001088 0.047851
10 -1643.564228 0.001250 0.027823
11 -1643.564251 0.000618 0.055380
12 -1643.564265 0.000412 0.017275
13 -1643.564271 0.000202 0.007514
14 -1643.564273 0.000159 0.003369
15 -1643.564273 0.000103 0.005615
Reason for exit: Successful completion
Quantum Calculation CPU Time : 58:10.23
Quantum Calculation Wall Time: 59:04.59
SPARTAN '08 Properties Program: (PC/x86) Release 132
Reason for exit: Successful completion
Properties CPU Time : 1.38
Properties Wall Time: 1.45

Figure D22. Sample output from Spartan ’08 ab initio calculations of 5.

Abstract (if available)

Abstract A variety of triphosphate analogs that replace a natural phosphate anhydride P-O-P linkage with a phosphonate P-CXY-P moiety have been synthesized for the characterization of DNA polymerase β (pol β) mechanism and structure. A suite of β,γ-CXY-dTTP compounds (X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of β,γ-CXY-dGTP analogs. The high purity of the β,γ-CXY-dTTP following dual-pass HPLC purification is demonstrated by NMR, MS, and HPLC data and illustrates the merit of the DCC-mediated morpholidate coupling approach to Pᵦ-CXY-Pᵧ triphosphate mimics. ❧ Novel α-azido bisphosphonates [(RO)₂P(O)]₂CXN₃ (R = i-Pr, X = Me

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Novel stereochemical probes for DNA polymerases: nucleoside triphosphate beta,gamma-CXY analogues

PDF

Nucleophilic fluorination of bisphosphonates and its application in PET imaging

PDF

Nucleotide analogs and molecular probes for LFER, time-resolved crystallography, FRET, and EPR studies of human DNA polymerase: probing mechanism, conformation and structure

PDF

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

PDF

Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

PDF

Fluorinated probes of enzyme mechanisms

PDF

Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

PDF

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

PDF

Design and synthesis of bisphosphonate analogs as inhibitors of farnesyl pyrophosphate synthase

PDF

Expansion of deoxynucleotide analog probes for studying DNA polymerase mechanism: synthesis of novel beta, gamma-CXY deoxycytidine series

PDF

Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications

PDF

I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

PDF

Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

PDF

The contribution of chemical steps to DNA polymerase beta catalysis and fidelity

PDF

Utilization of bisphosphonate drugs in fluorescent imaging and targeted drug delivery

PDF

Synthesis of foscarnet-peptide conjugates and detection of pyrophosphate analogues, bisphosphonates and phosphonocarboxylates

PDF

Synthesis of fluorescent conjugates of risedronate and related analogues for bone imaging

PDF

Investigating the E. Coli DNA polymerase V mutasome

PDF

Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs

PDF

Peptidomimetic prodrugs of cidofovir: design, synthesis, transport, mechanism of activation, and antiviral activity

Asset Metadata

Creator Chamberlain, Brian Thomas (author)

Core Title Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted tri...

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 05/31/2012

Defense Date 04/09/2012

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

Tag bisphosphonates,nucleotides,OAI-PMH Harvest,organoazides,polymerase

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 brian.t.chamberlain@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-44705

Unique identifier UC11288865

Identifier usctheses-c3-44705 (legacy record id)

Legacy Identifier etd-Chamberlai-871.pdf

Dmrecord 44705

Document Type Dissertation

Rights Chamberlain, Brian Thomas

Type texts

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

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

Repository Name University of Southern California Digital Library

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