University of Southern California Dissertations and Theses

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

(USC Thesis Other)

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

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

Nucleotide Analogs and Molecular Probes for LFER, Time-Resolved Crystallography,
FRET, and EPR Studies of Human DNA Polymerase:
Probing Mechanism, Conformation and Structure

By

Jorge Osuna

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)

December 2013

Copyright 2013 Jorge Osuna
ii

DEDICATION

The work presented here is dedicated to my loving family for all their support,
financially, emotionally, and spiritually throughout my academic career. To my hard working
dad, Domingo, who for over 20 years would wake up at 3:30 a.m. every weekday without taking
a single sick day in order to provide for his family. To my loving mother, Maria, who gave up
working a secular job in order to take care and nurture her children every day. To my one and
only sibling, Veronica, my best and worst sister all in one, you have always been my best friend
and a pillar of support. To my wife, Mariana, thanks for your love, communication and all the
help you provide me. All of you played a major role in shaping the person I was to become and
to achieve this milestone in my life. Lastly to my three nephews, Alex, Andrew, and Adrian, I
hope that the work I present here as well as my achievement serves as inspiration for all you to
succeed.

iii

ACKNOWLEDGMENTS

Meditating on the journey that I have gone through in order to write and defend this
dissertation brings to mind the proverb “It takes a village to raise a child” , or in my case, it took
the support, help and motivation of a lot of people for me have achieved a Ph.D. Foremost, I
would like to thank Prof. Charles E. McKenna for allowing me to join his lab on Dec 2006 as a
participant of the CSULA and NIH sponsored Biomed PREP program. Even in those early years
prior to being admitted to USC as a graduate student, you gave me the resources, tools, and
support in order to be a successful chemist. I look back and am truly grateful that I have been a
part of the McKenna group.
Next I would like to thank Dr. Boris A. Kashemirov for his tremendous help in the lab.
I recognize that I would have struggled a lot more if it wasn’t for your willingness to listen and
provide input into any synthetic or lab related problem I encountered. Although at times there
would be a line of people waiting to talk to you with their own synthetic challenges, you were
always ready to provide your expertise to the next student no matter how insignificant the
problem might seem.
I enjoyed and learned enormously from the PPG project that I was a part of. Its success
and mine are attributed to the highly skilled, intelligent, and diverse members that compose it.
Crystallographers, theoreticians, chemists, molecular biologists, computational chemists, and
other fields united in order to study structure and mechanism of DNA polymerase β. Thank you
Prof. Myron F. Goodman for financial support, Dr. Samuel H. Wilson, Dr. William A. Beard,
Dr. Vinod K. Batra for obtaining crystal structures of analogs I synthesized, Dr. Ernestas
Gaidamauskas for your mentorship during the kinetic experiments, and to everyone who
iv

participated in the annual PPG retreats to further my understanding of the importance of studying
DNA pol β.
At USC I also interacted with a few members of the chemistry department some of who
were willing to a part of my qualifying exam and dissertation defense committee. I would like to
thank both Prof. Richard L. Brutchey and Prof. Matthew R. Pratt for giving me the opportunity
of being a teaching assistant in Chem. 203 of which both of you exceptionally taught. I would
like to thank Prof. G. K Surya Prakash, Prof. Susumu Takahashi, Prof. Matthew R. Pratt, and
Prof. Malancha Gupta for being a part of my stressful but rewarding screening and qualifying
exam committee.
Thinking back into when I first began college as an undergrad I would have never
imagined that I would have gone to graduate school and become the first member in my family
in obtaining a Ph.D. degree. The motivation in pursuing such a degree in the first place comes
from my undergrad P.I. Prof. Linda M. Tunstad and Prof. Carlos G. Gutierrez program director
of the MORE programs at CSULA. Prof. Tunstad, thank you for asking me that one day after
organic chemistry class if I would like to do research in your lab. Prof. Gutierrez, thank you for
having me become a part of the RISE, MARC, and Biomed PREP programs, you are a true
inspiration and I never forget the words that you once told me, “There are many good chemists.
Strive not to be just a good chemist. Be a good chemist and a good person.” Prof. Gutierrez,
you are a great person and an exceptional chemist.
I would also like to thank all the members in the chemistry department that keep
everything in working order, be it instrumental or administrative, which allowed me to complete
my graduate school career. Allan Kershaw for your time in NMR, MALDI, and fluorimeter
training, Lewis Ross for fixing many instruments in our lab, Bruno Herreros and Jaime Avila for
v

computer and network support, Marie de la Torre for your cheerful greeting everyday and kind
words, and Michele Dea for helping me fill out paperwork, reserving rooms for presentations,
answering administrative questions that I had, and so much more that you did on a personal note
and for the department, thank you.
Finally, I would like to thank all the people I came across within the lab that became
not only my coworkers but my friends. Many of you helped me on a day to day basis, either
emotionally, secularly, or administrative. I would like to thank past members such as Dr.
Thomas G. Upton who was my first mentor at the McKenna group and guided me through the
pK
a
project, thank you for your patience and kindness. Past post-doc, Dr. Michaela Serpi, thank
you for being always cheerful and answering mechanism questions that I would have.
I would also like to thank all current McKenna group members. In particular to
Anastasia Kadina for your help in the synthesis of FRET compounds and to Dana Mustafa,
Melissa M. Williams, Inah Kang, Elena Ferri, Kim Nguyen, Candy Hwang all of you provided
many joyous occasions of laugher and sparked many interesting conversations, thank you.
Special thanks to Inah for all your help in many various fields, although not a chemist in training,
you are very knowledgeable in many areas and pillar in the McKenna group.
In closing, I look back at the time that has passed and the interactions I’ve had with
many remarkable people, and feel great appreciation and joy for coming across many mentors,
friends, and coworkers that have made my journey not only a successful one but a delightful one.
Thanks.

vi

TABLE OF CONTENTS

DEDICATION .............................................................................................................................................. ii
ACKNOWLEDGMENTS ........................................................................................................................... iii
LIST OF TABLES ........................................................................................................................................ x
LIST OF FIGURES ..................................................................................................................................... xi
LIST OF SCHEMES ................................................................................................................................ xviii
ABSTRACT ............................................................................................................................................... xix
Chapter 1 Probing the Chemical Step of Pol β by Construct of Linear Free Energy Relationship Plot: pKa
Determination of the Respective Bisphosphonic Acid of β,γ -Substituded dNTP Analogs .......................... 1
1.1 Introduction ................................................................................................................................ 1
1.2 Results and Discussion .............................................................................................................. 2
1.3 Conclusion ................................................................................................................................. 6
1.4 Experimental .............................................................................................................................. 7
1.5 References .................................................................................................................................. 9
Chapter 2 Synthesis of γ -phosphate and 3’ -O ‘caged’ Nucleotide Analogues for Time-Resolved
Crystallographic Analysis. .......................................................................................................................... 10
2.1 Introduction .............................................................................................................................. 10
2.2 Results and Discussions ........................................................................................................... 11
2.3 Conclusions .............................................................................................................................. 21
2.4 Experimental ............................................................................................................................ 23
2.4.1 Materials and Methods .......................................................................................................... 23
2.4.2 Synthesis of adenosine 5'-tetraphosphate .............................................................................. 23
2.4.3 Synthesis of hydrazone of 2-nitroacetophenone ................................................................... 24
2.4.4 Synthesis 1-(2-nitrophenyl)diazoethane ............................................................................... 25
2.4.5 Synthesis of P
3
-1-(2-nitrophenyl)ethyl adenosine triphosphate ............................................ 25
2.4.6 Synthesis of γ -Caged dATP .................................................................................................. 25
2.4.7 Synthetic attempt of 3’,5’-di-O-trifluoroacylate .................................................................. 26
2.4.8 Synthesis of 3’,5’ -Di-O-acetylated deoxyadenosine............................................................. 26
2.4.9 Synthesis of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-chloropurine ................. 27
2.4.10 Synthesis of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine .............................. 27
vii

2.4.11 Synthesis of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’deoxyribofuranosyl]-6-chloropurine
....................................................................................................................................................... 28
2.4.12 Synthesis of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-
2’deoxyribofuranosyl]-6-chloropurine........................................................................................... 28
2.4.13 Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine ....................................................... 28
2.4.14 Synthesis of 3’,5’ -Bis-O-[(tert-butyl)dimetthylsilyl]-2’-deoxyadenosine .......................... 29
2.4.15 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine .............................................................................................................................. 29
2.4.16 Synthesis of N
6
, N
6
-Bis-(tert-butyloxycarbonyl) -2’-deoxyadenosine by deprotection of
TBMS protective groups ................................................................................................................ 30
2.4.17 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine .............................................................................................................................. 30
2.4.18 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 3’,5’-O-(acetyl)-2’-deoxyadenosine ........ 31
2.4.19Synthesis of N
6
, N
6
-Bis-(tert-butyloxycarbonyl) -2’-deoxyadenosine by deprotection of
acetyl protective groups ................................................................................................................. 31
2.4.20 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine .............................................................................................................................. 32
2.4.21 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-3’-(2-
nitrophenyl)-2’-deoxyadenosine .................................................................................................... 32
2.4.22 Synthesis of 5’ -O-[(tert-butyl)dimethylsilyl]-3’-O-(2-nitrophenyl)-2’-deoxyadenosine .... 33
2.4.23 Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine ....................................................... 33
2.4.24 Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate .................................. 34
2.4.25 Photolysis of 3’ -O-(2-nitrophenyl)-2’-dATP by UV irradiation ........................................ 35
2.4 References ................................................................................................................................ 35
Chapter 3 Synthesis of Alexa 555 and 7-DEAC Linked dNTP Analogues for Fluorescence Resonance
Energy Transfer Studies .............................................................................................................................. 38
3.1 Introduction .............................................................................................................................. 38
3.2 Results and Discussions ........................................................................................................... 40
3.3 Conclusions .............................................................................................................................. 43
3.4 Experimental ............................................................................................................................ 44
3.4.1 Material and Methods ........................................................................................................... 44
3.4.2 Synthesis of 2’-deoxyadenosine-5’-tosylate ......................................................................... 44
3.4.3 Synthesis of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate ......................................... 45
3.4.4 Synthesis of Fmoc-4-aminobutylphosphate .......................................................................... 46
3.4.5 Synthesis of aminobutyl-PPCH
2
PdA .................................................................................... 47
viii

3.4.6 Synthesis of Alexa555 aminobutyl PPCH2PdA ................................................................... 47
3.4.7 Synthesis of 7-DEAC-aminobutyl PPCH2PdA .................................................................... 48
3.5 References ................................................................................................................................ 50
Chapter 4 Non-Enzymatic Hydrolysis Studies of Nucleotide Mimics for the Evaluation of the Catalytic
Efficiency of Polymerase β. ........................................................................................................................ 52
4.1 Introduction .............................................................................................................................. 52
4.2 Results and Discussion ............................................................................................................ 53
4.3 Conclusion ............................................................................................................................... 62
4.4 Experimental ............................................................................................................................ 65
4.4.1 NpPOPCH
2
P stock solution standardization. ........................................................................ 65
4.4.2 Reaction volume optimization .............................................................................................. 65
4.4.3 Hydrolysis reaction stoichiometry. ....................................................................................... 66
4.4.4 Kinetic runs of NpPOPCH
2
P, NpPOPCHFP, and NpPOPCF
2
P hydrolysis in the temperature
range from 90ºC to 143ºC. ............................................................................................................. 67
4.4.5 NpPOPCH
2
P hydrolysis kinetics in the 0.2 M KOH. ........................................................... 67
4.4.6 NpPOPCHFP hydrolysis kinetics in the 0.2 M KOH. .......................................................... 68
4.4.7 NpPOPCF
2
P hydrolysis kinetics in the 0.2 M KOH. ............................................................ 68
4.4.8 NpPOPCH
2
P, NpPOPCHFP and NpPOPCH
2
P hydrolysis in
18
O-enriched H
2
O. ................ 68
4.4.9 Benzyl diphosphate neopentyl .............................................................................................. 69
4.4.10 Benzyl difluoro diphosphate neopentyl .............................................................................. 70
4.4.11 NpPOPNp ........................................................................................................................... 71
4.5 References ................................................................................................................................ 73
Chapter 5 Probing the Coordinating Metals in Polymerase β: Synthesis of Spin Labeled dATP Analogues
for Electron Paramagnetic Studies .............................................................................................................. 74
5.1 Introduction .............................................................................................................................. 74
5.2 Results and Discussion ............................................................................................................ 77
5.3 Conclusion ............................................................................................................................... 81
5.4 Experimental ............................................................................................................................ 81
5.4.1 Synthesis of 2,5-Dioxopyrrolidinyl 2,2,6,6-tetramethyl-1-oxypiperidine-4-carboxylate
(Succinimidyl ester activated TEMPO) ......................................................................................... 82
5.4.2 Synthesis of TEMPO-aminobutyl PPCH
2
PdA...................................................................... 82
5.4.3 Synthesis of P
γ
-(TEMPO) α,β -CH
2
-adenosine triphosphate ................................................. 83
5.5 References ................................................................................................................................ 83
Bibliography ............................................................................................................................................... 85
ix

Appendix A ................................................................................................................................................. 95
Appendix B ............................................................................................................................................... 134
Appendix C ............................................................................................................................................... 169
Appendix D ............................................................................................................................................... 194
Appendix E………………………………………………………………………………………………2 19

x

LIST OF TABLES

Table 1.1. Titration condition optimization. Using smaller surface are pH electrode with KOH as titrant
gave pKa values closer to that found in literature.
15
..................................................................................... 3

Table 1.2. Acidity constants obtained by potentiometric titration on BP, using 0.1 M KOH and 0.1 M
KCl. ............................................................................................................................................................... 4

Table 4.1. ,-Substituted Neopentyl Triphosphates Hydrolysis Reaction Activation Parameters

(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.) ................................................................. 59

Table A1. Potentiometric titration data for methylenebisphosphonic acid, 1. ........................................... 96

Table A2. Potentiometric titration data for (difluoromethylene)bis(phosphonic acid), 2. ....................... 101

Table A3. Potentiometric titration data for (monofluoromethylene)bis(phosphonic acid), 3. ................. 105

Table A4. Potentiometric titration data for (dichloromethylene)bis(phosphonic acid), 4. ...................... 110

Table A5. Potentiometric titration data for (monochloromethylene)bis(phosphonic acid), 5. ................ 114

Table A6. Potentiometric titration data for (dibromomethylene)bis(phosphonic acid), 6. ..................... 118

Table A7. Potentiometric titration data for (monobromomethylene)bis(phosphonic acid), 7. .............. 122

Table A8. Potentiometric titration data for (1-fluoro-1,1-ethanediyl)bis(phosphonic acid), 8. .............. 126

Table A9. Potentiometric titration data for [chloro(fluoro)methylene]bis(phosphonic acid), 9. ........... 130

Table D1. The hydrolysis reaction rate constants (in min
-1
) obtained at constant temperatures. The data
for individual runs are shown. The initial substrate concentration in 0.2 M KOH is shown in parentheses.
.................................................................................................................................................................. 203

xi

LIST OF FIGURES

Figure 1.1. Structures of bisphosphonic acids for which pK values were determined in this work. ........... 3

Figure 1.2. Brønsted correlations of log(kpol) vs leaving-group pKa4. Data for dGTP and analogs,
incorporated opposite (A) the correct template base C, and (B) the mispairing template base T. ................ 5

Figure 2.1. Structure of proposed "caged" molecules P3-1-(2-nitro)phenylethyl 2’ -deoxyadenosine
triphosphate (1) and 3’O -(2-nitrobenzyl) 2’ -deoxyadenosine triphosphate (2) .......................................... 12

Figure 2.2. Diagram of incorporation of 3’ -O-caged dATP and photolytic cleavage. (A) Primer requires
the incorporation of two adenosine nucleosides (B) the 3’ -O of the primer incorporates the incoming
caged nucleoside with the photolabile group nitrobenzyl (NB) by nucleophilic attack on the Pα
succesfully (C) the other nucleoside cannot be incorporated until the NB moiety is cleaved by UV
irradiation thus providing a free 3’ -OH group on the primer (D) the second nucleoside is incorporated (E)
primer extension can be repeated for several rounds or terminated. ........................................................... 12

Figure 2.3. Adenosine tetraphosphate, 3, and ‘caged’ ATP, 4 ................................................................... 13

Figure 2.4. Gel showing incorporation of incoming nucleotide, 1. ........................................................... 15

Figure 2.5. Crystal structure of incorporated 3’ -O-caged dA into primer strand. ...................................... 20

Figure 3.1. Crystal structure representation of Alexa 555 dATP bound within pol β and the fluorophore
Alexa 647—5’ amino DNA. ....................................................................................................................... 40

Figure 3.2. Phospholinked non-hydrolyzable fluorescent dATP. .............................................................. 41

Figure 3.3. Gapped DNA insertion assay with DNA pol β for dATP analogs. The purity of the analogues
was assessed from their failure to be inserted into a gapped DNA substrate. Primer (n) extension was
assayed in the presence of low or high pol β and [MDCC -dATP] = 1 mM [AF555-dATP] = 0.8 mM for 5
min. Insertion assay performed by Dr. William A. Beard, NIEHS. .......................................................... 43

Figure 4.1. Nucleotide mimics for the non-enzymatic hydrolysis of Pα. ................................................... 53

Figure 4.2. Non-enzymatic nucleophilic site of attack of labeled
18
O. ...................................................... 55

Figure 4.3. Computational modeling showing the bond lengths of Pα -O and Pβ -O for model compounds
1-5. .............................................................................................................................................................. 56

Figure 4.5. Representative analytical data showing reaction progress (1, 0.2 M KOH, 110 °C) as
monitored by
1
H NMR (left) and the best fit to first order reaction kinetics (right). A: 1, B: NpP, C:
NpOH, open symbols: [NpPPCH
2
P]/[NpPPCH
2
P]
0
, and full symbols: 1-[NpP]/[NpPOPCH
2
P]
0
. ............ 57
xii

Figure 4.6. Arrhenius plot of the for , --substituted triphosphate neopentyl ester hydrolysis in 0.2 M
KOH rate constant (s
-1
) logarithm as a function of reciprocal temperature (K
-1
). Error bars are three
standard deviations of three replicates. ....................................................................................................... 58

Figure 4.7. LRMS of
18
O hydrolysis of 1-3 indicating that site of attack was exclusively at P
β.
............... 60

Figure 5.1. Proposed nitroxide spin label dNTP analogs. .......................................................................... 77

Figure A1. Titration curve for methylenebisphosphonic acid, 1, with KOH. ............................................ 95

Figure A2. Titration curve for (difluoromethylene)bis(phosphonic acid), 2, with KOH. ....................... 100

Figure A3. Titration curve for (monofluoromethylene)bis(phosphonic acid), 3, with KOH. ................. 104

Figure A4. Titration curve for (dichloromethylene)bis(phosphonic acid), 4, with KOH ........................ 109

Figure A5. Titration curve for (monochloromethylene)bis(phosphonic acid), 5, with KOH. ................ 113

Figure A6. Titration curve for (dibromomethylene)bis(phosphonic acid), 6, with KOH. ...................... 117

Figure A7. Titration curve for (monobromomethylene)bis(phosphonic acid), 7, with KOH. ................ 121

Figure A8. Titration curve for (1-fluoro-1,1-ethanediyl)bis(phosphonic acid), 8, with KOH. ............... 125

Figure A9. Titration curve for [chloro(fluoro)methylene]bis(phosphonic acid), 9, with KOH. ............. 129

Figure A10 A) Sample Hyperquad2006 output after refinement calculations. Shown,
methylenebis(phosphonic acid), 1. B) Sample Hyperquad2006 analysis showing calculated and
experimental titration curves for methylenebisphosphonic acid, 1. .......................................................... 133

Figure B1. HPLC Chromatogram of 1) ATP and 2) reaction mixture. Retention time of product 30.6
min. SAX Column Nucleogel 0.5 M TEAB pH 8. Flow Rate 9 mL/min λ
max
= 245nm. ....................... 134

Figure B2. TEA salt of ATP
1
H NMR. Varian 400 MHz. D
2
O. ............................................................ 135

Figure B3.
31
P NMR of adenosine tetraphosphate. Varian 400 MHz D
2
O. ............................................ 136

Figure B4. Low resolution mass spectra of P
3
-1-(2-nitro)phenylethyl 2’ -deoxyadenosine triphosphate.
592 [+ Li
+
], 598 [+ 2 Li
+
], After SAX column. ....................................................................................... 137

Figure B5.
1
H NMR of hydrazone, 8 (CDCl
3
) ......................................................................................... 138

Figure B6. UV-vis spectra monitoring the conversion of hydrazone to diazoethane, solvent ethanol. .. 139
xiii

Figure B7. HPLC Trace of 1) Reaction mixture 2) ATP 3) Spiked ATP in reaction mixture. Retention
time of ATP 9.35 min, retention time of caged ATP 10.28 min. Analytical SAX column, Flow 3 ml/min
0.5 M LiCl 0-100% B in 30 min. .............................................................................................................. 139

Figure B8.
31
P NMR of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1. ............................... 140

Figure B9.
1
H NMR of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1. .............................. 141

Figure B10. LRMS of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1.................................. 142

Figure B11. Absorption Spectra of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1. ............ 143

Figure B12. LRMS revealing glycosidic cleavage................................................................................... 144

Figure B13,
1
H NMR of 3’,5’ -Di-O-acetylated deoxyadenosine, 17 ....................................................... 145

Figure B14.
1
H NMR of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-chloropurine, 18 .......... 146

Figure B15. LRMS of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-chloropurine, 18 ............... 147

Figure B16.
1
H NMR (D
2
O) of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine, 12 ................ 148

Figure B17. LRMS of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine, 12 ............................. 149

Figure B18.
1
H NMR of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’deoxyribofuranosyl]-6-chloropurine, 13
.................................................................................................................................................................. 150

Figure B19. LRMS (+ mode) of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’deoxyribofuranosyl]-6-
chloropurine, 13. ....................................................................................................................................... 151

Figure B20. 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-2’deoxyribofuranosyl]-6-
chloropurine, 14. ....................................................................................................................................... 152

Figure B21. LRMS of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-
2’deoxyribofuranosyl]-6-chloropurine, 14. ............................................................................................... 153

Figure B22.
1
H NMR (CD
3
OD) of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 15. .................................. 154

Figure B23.
1
H NMR (DMSO-d6) of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 15. .............................. 155

Figure B24.
1
H NMR of 3’,5’ -Bis-O-[(tert-butyl)dimetthylsilyl]-2’-deoxyadenosine. ........................... 156

Figure B25.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine. ........................................................................................................................................ 157
xiv

Figure B26.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine, 19 ................................................................................................................................... 158

Figure B27.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-2’-deoxyadenosine, 20. .............................. 159

Figure B28.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine, 21 ................................................................................................................................... 160

Figure B29.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-3’-(2-
nitrophenyl)-2’-deoxyadenosine, 22 ......................................................................................................... 161

Figure B30.
1
H NMR of 5’ -O-[(tert-butyl)dimethylsilyl]-3’-O-(2-nitrophenyl)-2’-deoxyadenosine, 23.
.................................................................................................................................................................. 162

Figure B31.
1
H NMR of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 24. .................................................. 163

Figure B32.
1
H NMR of Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2 ............ 164

Figure B33.
31
P NMR of Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2 ........... 165

Figure B34. LRMS of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2 .................................... 166

Figure B35. HPLC Chromatogram of dATP, 2, and a mixture of dATP and 2. ...................................... 167

Figure B36. UV spectrum of compound 2. .............................................................................................. 167

Figure B37. Photolysis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2 .............................. 168

Figure C1.
1
H NMR of 2’ -deoxyadenosine-5’-tosylate, 2 ....................................................................... 169

Figure C2. HPLC Chromatogram of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3. ................. 170

Figure C3. LRMS of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3........................................... 171

Figure C4.
1
H NMR of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3 ....................................... 172

Figure C5.
31
P NMR (203 MHz, D
2
O) 2’ -deoxyadenosine-5’-tosylate, 2 .............................................. 173

Figure C6. LRMS of Fmoc-6-aminohexano-phosphate, 5. ..................................................................... 174

Figure C7.
1
H NMR (500 MHz, D
2
O) Fmoc-6-aminohexano-phosphate, 5. .......................................... 175

Figure C8.
31
P NMR (203 MHz, D
2
O) Fmoc-6-aminohexano-phosphate, 5. ......................................... 176

xv

Figure C9. LRMS of reaction progress. ................................................................................................... 177

Figure C10 LRMS of aminobutyl-PPCH
2
PdA, 6. ................................................................................... 178

Figure C11.
1
H NMR (600 MHz, D
2
O) aminobutyl-PPCH
2
PdA, 6. ...................................................... 179

Figure C12.
31
P NMR (203 MHz, D
2
O) aminobutyl-PPCH
2
PdA, 6. ...................................................... 180

Figure C13. LRMS of Alexa Fluor 555 succinimidyl ester. .................................................................... 181

Figure C14. HPLC Chromatogram of AF555NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA ............ 182

Figure C15. Emission and Excitation Spectra of AF555NH(CH
2
)
4
ppCH
2
pdA. ...................................... 183

Figure C16. LRMS Spectra of AF555NH(CH
2
)
4
ppCH
2
pdA. ................................................................. 184

Figure C17.
1
H NMR of AF555NH(CH
2
)
4
ppCH
2
pdA............................................................................ 185

Figure C18.
1
H NMR Spectra of AF555-NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA ................... 186

Figure C19
31
P NMR Spectra AF555-NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA ........................ 186

Figure C20. HPLC Chromatogram of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA .... 187

Figure C21. Emission and Excitation spectra of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA. ............................... 188

Figure C22. LRMS of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (negative mode) ............................................. 189

Figure C23. LRMS of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (positive mode). ............................................. 190

Figure C24.
1
H NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (zoomed in region 6.05-8.35ppm). ............ 191

Figure C25.
1
H NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (full spectra). .............................................. 192

Figure C26.
31
P NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA. .................................................................. 193

Figure D1.
1
H NMR spectra of a typical run of neopentyl ,-methylene triphosphate (1 = NpPPCP). . 194

Figure D2. NpPPCH
2
P hydrolysis in 0.2 M KOH reaction data as [NpPPCH
2
P]/[NpPPCH
2
P]
0
(open
symbols), and 1-[NpP]/[NpPOPCH
2
P]
0
(full symbols). The best fit to the first order kinetic equation is
shown. ....................................................................................................................................................... 195

xvi

Figure D3. NpPPCHFP hydrolysis in 0.2 M KOH reaction data as [NpPPCHFP]/[NpPPCHFP]
0
(open
symbols), and 1-[NpP]/[NpPOPCHFP]
0
(full symbols). The best fit to the first order kinetic equation is
shown. ....................................................................................................................................................... 197

Figure D4. NpPPCF
2
P hydrolysis in 0.2 M KOH reaction data as [NpPPCF
2
P]/[NpPPCF
2
P]
0
(open
symbols), and 1-[NpP]/[NpPOPCF
2
P]
0
(full symbols). The best fit to the first order kinetic equation is
shown. ....................................................................................................................................................... 199

Figure D5. MS spectra of NpPPCXYP complete hydrolysis products in 0.2 M KOH in the
18
O-enriched
H
2
O (50% enrichment).............................................................................................................................. 201

Figure D6. Fragmentation MS/MS spectra of NpPPCH
2
P complete hydrolysis products in 0.2 M KOH in
the
18
O-enriched H
2
O (50% enrichment). ................................................................................................. 202

Figure D7. Fragmentation MS/MS spectra of NpPPCHFP complete hydrolysis products in 0.2 M KOH
in the
18
O-enriched H
2
O (50% enrichment). ............................................................................................. 203

Figure D8. Benzyl diphosphate neopentyl
1
H NMR spectra of hydrolysis. ............................................. 204

Figure D9. LRMS of Benzyl diphosphate neopentyl after 200
o
C for 1h 30 min. ................................... 205

Figure D10. LRMS of Benzyl diphosphate neopentyl hydrolysis with MeOH. ...................................... 206

Figure D11. LRMS of Benzyl diphosphate neopentyl hydrolysis with labeled water. ............................ 207

Figure D12.
31
P NMR of 4 after 1h at 200
o
C. ......................................................................................... 208

Figure D13. HPLC purification of 5. ....................................................................................................... 208

Figure D14.
1
H NMR spectra of hydrolysis of compound 4 and 5. ........................................................ 209

Figure D15. Hydrolysis of 5 with MeOH. .............................................................................................. 210

Figure D16. Hydrolysis of 5 with labeled water. ..................................................................................... 210

Figure D17.
31
P NMR of 5. ...................................................................................................................... 211

Figure D18. HPLC Chromatogram of NpPOPNp .................................................................................... 211

Figure D19.
1
H NMR and
31
P NMR of NpPOPNp ................................................................................. 212

Figure D20. LRMS of NpPOPNp. ........................................................................................................... 213

Figure D21. Hydrolysis of NpPOPNp at 200
o
C for 30 min. ................................................................... 214
xvii

Figure D22. Hydrolysis of NpPOPNp at 200
o
C for 3 hours ................................................................... 215

Figure D23. NMR and Hydrolysis study of NpPhosphate. ...................................................................... 216

Figure E1. TLC of reaction progress of succinimidyl activated spin label. ............................................ 218

Figure E2. LRMS of succinimidyl activated nitroxide spin label. ........................................................... 219

Figure E3. LRMS of TEMPO-aminobutyl PPCH
2
PdA. .......................................................................... 220

Figure E4. EPR spectra of 200 μM of TEMPO-aminobutyl PPCH
2
PdA. ............................................... 221

xviii

LIST OF SCHEMES

Scheme 2.1 Synthesis of adenosine tetraphosphate, 3. ............................................................................... 13

Scheme 2.2 Synthesis of P3-1-(2-nitro)phenylethyl 2’ -deoxyadenosine triphosphate, 1 ........................... 15

Scheme 2.3 Synthetic scheme for 3’ -O-caged dATP, 2, from 2’ -deoxyinsoine, 10. ................................. 17

Scheme 2.4 Synthetic scheme to 6-Chloropurine, 12. ................................................................................ 17

Scheme 2.5 Successful synthetic scheme for 3’ -O-(2-nitrobenzyl)-2’-deoxyadenosine, 2. ....................... 19

Scheme 3.1 Phosphorylation of 4-(Fmoc-amino)-1-butanol to yield 5. ..................................................... 42

Scheme 3.2 Synthesis of Dye-aminobutyl-dAPPCP. ................................................................................. 42

Scheme 4.1 Synthesis of neopentyl triphosphate analogs. ......................................................................... 54

Scheme 5.1 Sythesis of TEMPO-hydoxysuccinimide. ............................................................................... 79

Scheme 5.2 Synthesis of TEMPO-aminobutyl-dAPPCP. .......................................................................... 79

Scheme 5.3 Synthesis of P
1
-adenosine P
3
-(2,2,6,6-tetramethylpiperidine-N-oxyl) triphosphate. .............. 80

xix

ABSTRACT

A variety of molecular probes were synthesized and characterized with the goal of
gaining structural, conformational, and mechanistic information on polymerases. The leaving
group effects of β,γ -CXY deoxynucleotide probes was investigated in order to determine the
rate-determining step in the nucleotidyl mechanism of pol β. Due to inconsistencies in literature
the pKa
(2–4)
values for nine bisphosphonic acids used to comprise the β,γ−CXY toolkit were
determined under identical conditions. Applying these acidity constants to the analysis of kinetic
results obtained with a series of dGTP-β,γ−CXY analogues clarified the relationship between the
rate of single-gap nucleotide insertion and BP pKa
4
values. Through construct of Brønsted
LFER plot results seem to indicate that chemistry and not conformation is the rate-limiting step
in the nucleotidyl transfer mechanism.
Modification on the P
γ
of α,β -CH
2
non-hydrolyzable dATP analogs were synthesized to
probe the dynamics of conformation upon substrate binding. These include fluorescent linked P
γ

dATP analogs for fluorescence resonance energy transfer studies and nitroxide P
γ
linked dATP
analogs for electron paramagnetic studies. Compounds for FRET analysis such as the one
employing Alexa555 will be useful into gauging distances between a substrate bound in the
active site and an acceptor nucleotide on the DNA strand, while 7-DEAC linked nucleotide can
provide active site environment information. Compounds for EPR analysis will probe the Mg
2+

metals that bind dNTP and catalyze 3’ -O primer nucleophilic attack on P
α
.
A suite of model compounds were synthesized and studied in an attempt to mimic P
α

hydrolysis achieved by the enzyme. Through these model compounds it was hoped to gain
insight into the efficiency of catalysis by pol β by mimicking the reaction non -enzymatically.
xx

Results indicated that in the absence of enzyme phosphate hydrolyzes typically occur on the P
β
in
contrast to P
α
by a nucleophile. Second generation compounds were able to shift some of the site
of attack to P
β
but not by much, indicating the catalytic power of pol β.
3’-O-(2-nitrobenzyl)-2’-deoxyadenosine, “caged” compound, was synthesized in order to
be utilized in ultra-fast Laue X-ray crystallographic. The proposed work would have the 3’-O-
caged dATP bound into a crystalline ternary complex but due to the photolabile tag render
additional nucleoside incorporation inactive. A pulse of laser light irradiation photochemically
will cleave the caging group, initiating turnover at a defined zero time, thus providing time-
resolved crystallographic “snapshots” of the DNA pol β catalyzed hydrolysis of dNTPs.
In conjunction these probes will provide structural, conformational, and mechanism
insight into pol β which in turn can be used to synthesize inhibitors for therapeutic use.

1

Chapter 1
Probing the chemical step of pol β by construct of Linear Free Energy
Relationship plot: pKa determination of the respective bisphosphonic acid of
β,γ-substituded dNTP analogs

1.1 Introduction

DNA polymerases are vital in DNA replication and in maintaining the fidelity of genetic
information. Eukaryotic cells contain at least 15 DNA polymerases, which are grouped based on
sequence homology.
1
One polymerase group, the X family, contains the repair enzyme
polymerase β (pol β) which plays a crucial role in maintaining genome integrity. Through its
role in base excision repair (BER) pol β is able to repair lesions in the DNA strand caused by
reactive oxygen species or alkylating agents.
1, 2
However, recent studies have shown that over
30% of human tumors express DNA pol β variant proteins. DNA pol β has also been shown to
be over expressed in a variety of tumors.
3
In order to develop inhibitors for pol β and provide
novel cancer therapies it is of prime importance to fully understand the dynamics, function, and
key steps that the enzyme is involved in.
4

One interest had been to probe the rate limiting step in the nucleotidyl transfer
mechanism of BER. The mechanism of binding and incorporation in nucleotidyl transfer consist
of a large conformational change brought about by having the incoming 2’-deoxynucleoside
triphosphate (dNTP) bind to the active site and a subsequent chemical step involving 3’ -OH
deprotonation of the primer, nucleophilic attack on the α -phosphate of the incoming dNTP, and
pyrophosphate leaving group elimination.
5, 6

2

Substituting the β,γ bridging oxygen (P
γ
-O-P
β
) of the dNTP analog to a carbon (P
γ
-CXY-P
β
; X,
Y = F, Cl, Br, H) provides a pyrophosphate analog with different electronic and steric properties
that can be used to probe the transition state (TS) of the nucleotidyl transfer mechanism.
5, 6
By
determining the acid dissociation constant of the leaving group a parameter is obtained that can
be used in a Brønsted type LFER plot (log k
pol
vs leaving group pK
a
) to quantify relative leaving
group ability
7
.
Here we provide the rigorous data from pKa determination through potentiometric
titration.
1.2 Results and Discussion

Acidity constants for a few individual bisphosphonic acids (BP) had been determined by
different methods, including calculations from potentiometric titrations and
31
P NMR shifts,
involving variations in the counter ion, ionic strength, and other conditions that may account for
the differences of nearly 1 unit in the pK
4
values reported for both 1 and 5.
8–11
In this work, all
the acidity constants for a variety of BP (Figure 1.1) were obtained by potentiometric titration
under with exclusion of atmospheric CO
2
and a constant temperature of 25 °C. The titration data
was analyzed by Hyperquad2006
12,13
and the electrode calibrated with calibration software
GLEE.
13,14

3

Figure 1.1. Structures of bisphosphonic acids for which pK values were determined in this work.

A comparison of preliminary titrations carried out with 0.1 M NaOH and those carried
out later with 0.1 M KOH revealed that a background electrolyte interference is observed and
made more evident with pH electrodes with a greater surface area (SA) (Table 1.1). This effect
was found to be reduced by two routines: 1) reducing the area of the glass surface interacting
with the solution and 2) increasing background counter ion size to help distinguish from
hydrogen concentration.
10
Many of the previously determined pK
a
’s of bisphosphonic acids
(BP) had been determined with NaCl and NaOH which demonstrates the importance of this
study.
Electrode Conditions pKa
4
pKa
3
pKa
2
Smaller SA
0.1 M NaCl
0.086 M NaOH
10.39 6.94 2.78
Larger SA
0.1 M NaCl
0.089 M NaOH
10.06 6.89 2.73
Larger SA
0.1 M KCl
0.091 M KOH
10.39 7.06 2.61
Smaller SA
0.1 M KCl
0.091 M KOH
10.52 7.35 2.78
Kabachnik et.
al.
0.1 M KCl
0.1 M KOH
10.42 7.33 2.75

Table 1.1. Titration condition optimization. Using smaller surface are pH electrode with KOH
as titrant gave pKa values closer to that found in literature.
15

According to our investigations, the conditions employing 0.1 M KCl and 0.1 M KOH
showed consistent and comparable values to the model
15
and are designed to greatly reduce the
4

background interference caused by sodium.
10,13, 16

Initial construct of an LFER plot with four β ,γ dG analogs and their respective BP (1-4)
5

offered compelling evidence supporting “chemistry” as the rate limiting step for rig ht (G•C) and
wrong (G•T) incorporation for pol β, a conclusion based on the negative linearity of Brønsted
correlations of log (k
pol
) vs leaving group pK
a4.
5,17
A break in the linearity of a Brønsted plot
could reveal a change in mechanism
18
involving a transition from one rate limiting step to
another in the nucleotide insertion reaction.
5
Therefore, additional pyrophosphate analogs were
synthesized and their acidity constants determined (Table 1.2).
- CXY Previously reported pK
a4
pK
a4
pK
a3
pK
a2

2 CF
2
7.63
19
, 8.00
20
, 8.14
21
, 8.16
22
7.77 ± .04 5.63 ± .04 1.69 ± .2
9 CFCl ---- 8.36 ± .03 5.58 ± .02 1.58 ± .1
4 CCl
2
8.84
23
, 9.5
10
, 9.72
8
, 9.78
20
8.84 ± .1 5.83 ± .1 1.95 ± .1
3 CHF 9.35
20
, 9.44
21
8.96 ± .08 6.13 ± .04 1.53 ± .4
6 CBr
2
---- 9.27 ± .003 5.87 ± .006 1.83 ± .03
5 CHCl ---- 9.58 ± .06 6.32 ± .02 1.19 ± .4
7 CHBr 10.00
9
9.91 ± .009 6.16 ± .03 2.33 ± .1
8 CFCH
3
---- 10.21 ± .03 6.16 ± .03 1.96 ± .07
1 CH
2

9.89
11
, 10.00
24
, 10.42
15
,
10.75
8
, 10.75
25

10.44 ± .1 6.95 ± .03 2.75 ± .08
10 CHCH
3
11.67
9
11.92 ± .5 7.05 ± .008 2.73 ± .06
11 C(CH
3
)
2
11.76
9
12.23 ± .04 7.67 ± .004 2.88 ± .02

Table 1.2. Acidity constants obtained by potentiometric titration on BP, using 0.1 M KOH and
0.1 M KCl.

Construct of a new Brønsted plot with additional BP (4-7, 9) acidity constants revealed
that there was no break in linearity, instead the mono- and dihalo derivatives were split into two
different lines (Figure 1.2).
6

5

Figure 1.2. Brønsted correlations of log(kpol) vs leaving-group pKa4. Data for dGTP and
analogs, incorporated opposite (A) the correct template base C, and (B) the mispairing template
base T.

The separate lines occurred for both right and wrong insertions, with significantly larger
distances separating the two lines for the wrong insertions. A computational study performed by
a collaborating group examined whether the di-halogen effect observed in the incorporation of
β,γ−CXY dGTP analogues by pol β was a general feature of β,γ−CXY triphosphate hydrolysis
that would also be observed in the un-catalyzed solution reactions
17
. 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
pK
a4
, leaving group
8 9 10 11
log(k
pol
)
-2.0
-1.5
-1.0
-0.5
0.0
T-G mispairing
CH
2
CBr
2
CF
2
CCl
2
CHF
CHBr
CFCl CHCl
O
B
8 9 10 11
log(k
pol
)
0.0
0.5
1.0
1.5
2.0
C-G correct pairing
CH
2
CBr
2
CF
2
CCl
2
CHF
CHBr
CFCl
CHCl
O
A
6

hydrolysis was modeled in the gas phase. These results indicated that differences in the
solvation energies of the analogues 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” th an the correct pair complex
and thereby more resembles bulk solvent.
1.3 Conclusion

Literature values for the acidity constants of a series of BPs varied greatly from one
source to another (1 and 4) and in some cases had never been reported (5, 6, 8, and 9), due more
likely because of inconsistent experimental conditions such as with respect to counter-ion, ionic
strength, method of electrode calibration and so on. An internally consistent set of pK values for
all the existing bisphosphonic acid components is essential for reliably rationalizing
experimental kinetic results. Therefore, the pKa
(2–4)
values for the nine BPs used to comprise the
β,γ−CXY toolkit were determined under identical conditions.
We validated our approach by comparing our pKa
4
-pKa
2
values of 1 and 4 with literature
values conducted under similar conditions (I = 0.10 M KCl; t = 25°C). While optimizing
titration conditions we noted a background electrolyte effect with the sodium cation that became
more evident with larger electrode surface area. By reducing the surface area of the electrode
and using a potassium counterion we were able reduce interference while making counterion
complexation to the analyte less significant.
Lastly, applying these acidity constants to the analysis of kinetic results obtained with a
series of dGTP-β,γ−CXY analogues clarified the relationship between the rate of single-gap
7

nucleotide insertion and BP pKa
4
values.
5,6
These results indicate that chemistry and not
conformation is the rate-limiting step in the nucleotidyl transfer mechanism.
1.4 Experimental

All chemicals were obtained by Sigma Aldrich. All acidity constants were obtained by
potentiometric titration and environments were kept free of CO
2
and kept at a constant
temperature of 25 °C via thermostat. A semi-micro combined glass electrode (Aldrich Z113441
O.D. 3.5 mm) and precision pH-meter (Orion 720A+) were utilized. Titrant was added with an
automated Schott Titrator Basic under a flow of Ar or N
2.
For all runs, a 10 mL (± 0.01 mL) solutions of ~5 mM bisphosphonic acid and 0.1 M KCl
were 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. 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 using GLEE software, and 20 μL aliquots of ~0.1
M KOH (standardized with potassium hydrogenphthalate and a phenolphthalein indicator) were
delivered at the surface of the analyte solution using a Schott Titrator Basic titration device.
All titrations were carried out at least three times and the calculated pKa values were
averaged. The error is reported as the estimated standard deviation. Titration curves were fitted
to log β values using Hyperquad 2006 (Figure A10) which optimized the amount of analyte
(constrained to 1BP: 4H
+
). In general, refinement included pH values reflecting bisphosphonic
tetraacid concentrations of up to ~95%. This procedure was not practical with bisphosphonic
acids possessing a pKa
4
greater than 10 (ie. 10, 11) and in these cases upper pH data were
8

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.

9

1.5 References

(1) Yamtich, J.; Sweasy, J. B. Biochim. Biophys. Acta BBA - Proteins Proteomics 2010, 1804,
1136–1150.
(2) DNA repair and mutagenesis; 2nd ed.; ASM Press: Washington, D.C, 2006.
(3) Starcevic, D.; Dalal, S.; Sweasy, J. B. Cell Cycle Georget. Tex 2004, 3, 998–1001.
(4) Barakat, K. H.; Gajewski, M. M.; Tuszynski, J. A. Drug Discov. Today 2012, 17, 913–
920.
(5) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martínek, V.; Xiang, Y.;
Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florián, J.; Warshel, A.;
Goodman, M. F. Biochemistry (Mosc.) 2007, 46, 461–471.
(6) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florián, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. Biochemistry
(Mosc.) 2008, 47, 870–879.
(7) Williams, A. Free energy relationships in organic and bio-organic chemistry; RSC:
Cambridge, UK, 2003.
(8) Claessens, R. A. M. J.; van der Linden, J. G. M. J. Inorg. Biochem. 1984, 21, 73–82.
(9) Grabenstetter, R. J.; Quimby, O. T.; Flautt, T. J. J. Phys. Chem. 1967, 71, 4194–4202.
(10) Popov, K.; Rönkkömäki, H.; Lajunen, L. H. J. Pure Appl. Chem. 2001, 73, 1641–1677.
(11) Vaňura, P.; Jedináková -Křížová, V.; Hakenová, L.; Munesawa, Y. J. Radioanal. Nucl.
Chem. 2000, 246, 689–692.
(12) Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739–1753.
(13) Koort, E.; Gans, P.; Herodes, K.; Pihl, V.; Leito, I. Anal. Bioanal. Chem. 2006, 385, 1124–
1139.
(14) Gans, P. Talanta 2000, 51, 33–37.
(15) Kabachnik, M. I.; Lastovskii, R. P.; Medved, T. Y.; Medyntsev, V. V.; Kolpakova, I. D.;
Dyatlova, N. M. Doklady Akademii Nauk SSSR 1967, 177, 582–585.
(16) Iuliano, M.; Ciavatta, L.; De Tommaso, G. J. Colloid Interface Sci. 2007, 310, 402–410.
(17) Kamerlin, S. C. L.; McKenna, C. E.; Goodman, M. F.; Goondman, M. F.; Warshel, A.
Biochemistry (Mosc.) 2009, 48, 5963–5971.
(18) Jencks, W. P. In Nucleophilicity; Harris, J. M.; McManus, S. P., Eds.; American Chemical
Society: Washington, DC, 1987; Vol. 215, pp. 155–167.
(19) Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R. M. J. Fluor. Chem. 1982,
20, 617–626.
(20) Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc. Chem. Commun. 1981,
930.
(21) Leswara, N. D. Alpha -fluoromethanediphosphonic Acids and Derived ATP Analogs;
University of Southern California, 1982.
(22) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649–658.
(23) Dietsch, P.; Guenther, T.; Roehnelt, M. J. Biosci. 1979, 31C, 661–663.
(24) Sanna, D.; Micera, G.; Bugly � , P.; Kiss, T. J. Chem. Soc. Dalton Trans. 1996, 87.
(25) Deluchat, V.; Serpaud, B.; Caullet, C.; Bollinger, J.-C. Phosphorus Sulfur Silicon Relat.
Elem. 1995, 104, 81–92.

10

Chapter 2
Synthesis of γ-phosphate and 3’-O ‘caged’ nucleotide analogues for time-
resolved crystallographic analysis
2.1 Introduction

After binding a DNA substrate, DNA polymerases preferably bind a nucleoside
triphosphate that preserves Watson-Crick hydrogen bonding complementary to the template
base. Upon binding the correct dNTP, the ternary complex undergoes numerous conformational
changes, including subdomain motions, protein side-chain repositioning, and DNA structural
alterations. Following the rate-limiting chemical step of nucleotide insertion (Chapter 1), the
ternary product complex undergoes conformational changes that facilitates pyrophosphate (PPi)
release and either extended product release (DNA
+1
) or another round of nucleoside insertion.
1

An innovative approach to visually follow these large amplitude and subtle conformational
changes would be to explore the transition-state (TS), using time-resolved crystallography aided
by a UV-activated “caged” dNTP analog.
This approach relies on the synthesis of “caged” dNTPs that are designed for cleavage via
photolysis. The modified “caged” dNTP show in activity with the photo labile group but upon
irradiation with light, the tag is cleaved and the compound’s original activity is restored. The
main advantage is that a clean start can be made to studies of repair, as the substrate can be de-
caged with a laser flash at a controlled time point initiating activity.
2, 3
The ultimate goal being
to produce atomic resolution movies of working proteins, so that their mechanisms of action can
be visualized and understood.
11

A few critical conditions must be met for successful implementation of caged molecule to
study biological systems: 1) the byproducts as a result of photolysis should not affect the rate of
reaction, 2) the rate of un-caging must be faster than the process under study, 3) photolysis
should occur quickly in order to avoid long irradiation times and negative effects to the
biological sample.
4
The choice of caging agents released by light is still limited to a small
number of molecules (2-nitrobenzyl,
5,6,7
nitrophenyl alkyl derivatives,
8,9,10
and coumarin
derivatives
4,11,12
). Of these photo labile molecules the nitro aryl moiety is more advantages than
the coumarin derivatives since coumarin has the disadvantage of being bulkier, thus requiring
long irradiation times for cleavage and possessing lower water solubility than the nitro aryl
derivatives.
4

One of the first photo labile precursors of an important biological substrate was the 1-(2-
nitrophenyl)ethyl P
3
-ester of ATP, synthesized in the late 70’s, that on photolysis releases ATP at
220 s
-1
at pH = 7 and 21 °C.
3,13,14
Since then, this approach has been used to study several
proteins and enzyme
15,16

,17
including polymerases
9,18
.
2.2 Results and Discussions

Previously our group had made use of a photolabile group in order to induce
phosphorylation under mild conditions
19
. Our new approach for time-resolved crystallographic
snapshots required the synthesis of a dNTP analog which would render the nucleotidyl transfer
step in pol β mechanism inactive until photolysis. The goal was to couple the photolabile group
to the γ-phosphate of dATP (1) for proof of principle and then to the 3’O -hydoxy of dATP (2)
(Figure 2.1) to block incorporation of the incoming nucleotide on the second round of insertion
(Figure 2.2).
12

Figure 2.1Structure of proposed "caged" molecules P3-1-(2-nitro)phenylethyl 2’ -
deoxyadenosine triphosphate (1) and 3’O -(2-nitrobenzyl) 2’ -deoxyadenosine triphosphate (2)

Figure 2.2Diagram of incorporation of 3’ -O-caged dATP and photolytic cleavage. (A) Primer
requires the incorporation of two adenosine nucleosides (B) the 3’ -O of the primer incorporates
the incoming caged nucleoside with the photolabile group nitrobenzyl (NB) by nucleophilic
attack on the Pα succesful ly (C) the other nucleoside cannot be incorporated until the NB moiety
is cleaved by UV irradiation thus providing a free 3’ -OH group on the primer (D) the second
nucleoside is incorporated (E) primer extension can be repeated for several rounds or terminated.
In order to gain experience in performing chemistry on the γ phosphate of a nucleotide
and to work with photolabile groups two additional structures (3 and 4) were proposed (Figure
2.3).
13

Figure 2.3 Adenosine tetraphosphate, 3, and ‘caged’ ATP, 4

Tetraphosphates are known to be better inhibitors of pol β with improved Kd compared to
analogs that have a moiety attached at the γ phosphate.
20,21
Synthesis was carried out by
imidazole activation on P
γ
,6, and subsequent coupling with tris(triethylammonium)phosphate
(Scheme 2.1). Product 3 was obtained after SAX HPLC purification using 0.5 M of TEAB pH 8
as buffer. 3 elutes slower than 5 due to the additional negative charge added by δ phosphate
(Figure B1). Purity and confirmation of our desired product was observed by
1
H,
31
P NMR and
mass spectrometry (Figure B2-4).

Scheme 2.1 Synthesis of adenosine tetraphosphate, 3.
14

Compound 4 was synthesized by two steps following a literature procedure of alkylating
the weakly ionizing P
γ
by the alkylating agent 1-(2-nitrophenyl)diazoethane
3,22

2-nitroacetophenone hydrazone, 7, and 1-(2-nitrophenyl)diazoethane, 8, were synthesized
according to the methods of Walker (Scheme 2.2).
3
The observation of elution profiles for
caged compounds frequently show two components arising from the presence of a chiral carbon
at the 2-position of the ethyl residue of the caging molecule, thus the dual nature of the elution
peaks is explained by the presence of diastereoisomers of the caged molecules (Figure B7).
9

An incorporation experiment was performed with compound 1 and pol β in order to
confirm that the nitrobenzyl group by itself would not hinder placement into the active site. It
was observed that the primer strand of pol β successfully incorporated the γ -caged nucleotide
likely by having a caged pyrophosphate analog as a leaving group (Figure 2.4). Indeed, the
caged molecule was incorporated slightly slower than the native, but almost identical.
15

Scheme 2.2. Synthesis of P3-1-(2-nitro)phenylethyl 2’ -deoxyadenosine triphosphate, 1
Zero –
DNA + pol β
native dA
caged dA
Time (s)
10 60
dA 5uM dA 20uM dA 80uM cage 5uM cage 20uM cage 80uM
10 86.351 89.664 90.635 61.684 85.562 88.329
15 88.893 90.018 91.214 74.935 87.924 89.523
30 89.317 90.351 91.282 85.404 88.545 89.972
45 89.708 90.284 91.28 87.862 88.809 90.03
60 89.38 90.587 91.659 89.318 89.857 91.324
5μM 5μM 20μM 20μM 80μM 80μM
50nM DNA, 300nM pol β, 37°C
GCGTCAG
32
P CGC GTC

Figure 2.4. Gel showing incorporation of incoming nucleotide, 1.
16

Our final goal was to devise a route for the synthesis of 3’-O-(2-nitrobenzyl)-dATP, 2. A
simple route was described in literature
23
where 2’ -deoxyadenosine was dissolved in DMF and
2-nitrobenzyl bromide in the presence of NaH, followed by phosphate coupling on the 5’ -OH.
Interestingly enough it was determined 13 years later that the 3’ -O was not alkylated as
described but instead had alkylated the N
6
of the purine.
5
Thus, the ‘simple’ route reported did
not lead to synthesis of 3’ -O-(2-nitrobenzyl)-2’-deoxyadenosine but instead yielded N
6
,N
6
-bis-
(2-nitrobenzyl)-2’-deoxyadenosine.
Indeed it was particularly challenging to synthesize 3’ -O-(2-nitrobenzyl)-dNTPs because
the nucleophilic nitrogen on the base preferentially reacts with the 2-nitrobenzyl group. Thus, it
was clear that protection groups would have to be employed on N
6

of the purine as well as on the
3’ and 5’ -OH groups.
Initial attempts were made with starting 2’ -deoxyinosine, 10, as starting material
(Scheme 2.3). According to literature, one pot trifluoroacetylation of 10 would give 3’,5’ -bis-O-
trifluoroacetate, 11, and then subsequent reaction of the electronegatively substituted
deoxynucloside with thionyl chloride in refluxing methylene chloride would yield 6-chloropurine
2’-deoxyriboside, 12, after deblocking.
24
However, in our hands the deprotection was
unsuccessful, instead of desired compound, 12, LRMS revealed glycosidic cleavage had
occurred (Figure B11). Several possibilities can explain the lack of success in reproducing the
procedure: 1) procedure calls for extremely dry conditions and although steps were taken to
avoid moister, starting material was not dried for 15 h at 75 °C 0.5 mm as suggested since this
would decompose nucleoside, 2) trifluoracetic anhydride was not distilled because “purification
by distilling over KMnO
4
is extremely dangerous due to the possibility of explosion”.
25
3)
Lastly, a literature search revealed that the reaction was not easily reproduced in other groups
17

since under acidic conditions, chlorination with POCl
3
and SOCl
2
result in glycosidic bond
cleavage.
26,27,28

Scheme 2.3 Synthetic scheme for 3’ -O-caged dATP, 2, from 2’ -deoxyinsoine, 10.

A second attempt was to protect the 3’, 5’ -OH of 2’ -deoxyadenosine, 16, by acetylation,
with acetic anhydride. Acetic acid is a weaker acid compared to trifluoroacetic acid
(trifluoroacetic acid pKa = - 0.25, acetic acid pKa = 4.8) thus minimizing the chances of
glycosidic cleavage. The synthetic route included a three-step reaction: 1) protection of the
hydroxyl groups, 17 2) reductive deamination, 18 and 3) deprotection 12 (Scheme 2.4).
29,30

Scheme 2.4. Synthetic scheme to 6-Chloropurine, 12.
18

Another benefit from starting with 2’deoxyadenosine is that halo -dediazoniations of
aminopurines can be performed under milder conditions than halo-deoxygentation of oxopurine
derivatives. A problem encountered is that the diazotization/dediazoniation mechanisms can be
complex and is influenced to a significant degree by minor changes in reaction conditions.
27,31
In
fact he exact pathway for the conversion of a diazonium salt to aryl halide is not certain or is
debated, resulting in uncertainty regarding the intermediacy of cation versus radical and other
intermediates in dediazoniation reactions of electron-deficient rings such as purines.
31- 34
This
uncertainty in reaction pathway also leads to production of the undesired 6-oxopurine derivatives
during halo-dediazoniations step.
31

Due to low yields and unstable 6-chloro purine intermediates, another synthetic scheme
was investigated (Scheme 2.5). Although, literature procedure called for 3’, 5’ -O protection
with tert-butyldimethylsilyl (TBMS),
5
protection with acetyls was favored. This in part was a
result of the problems resulting in removing 2 TBMS moieties. Although the same tetra-n-
butylammonium fluoride deprotection is common to all reports using TBMS protection, an
inconsistency is observed regarding the length of time required for desilylation, ranging
anywhere from 4 hours to 24 hours.
35,36
The efficiency of desilylation by 1 M TBAF in THF is
adversely effected by the presence of water.
35
Even in unopened bottles of TBAF water content
has been reported to be in the range of 5 to 8 %,
35
leading to incomplete deprotection.
However, acetyl protection groups are readily removed with NH
3
dissolved in MeOH.
37

Another interesting note is that N
6
cannot be Boc protected if 3’ and 5’ are left in their
unprotective OH protonated form.
38
Therefore, while it would seem logical to selectively
protect the amine and then the 5’ -OH to yield intermediate 21, to our knowledge synthetic
procedures for it have not been reported.
19

Alkylation of intermediate 21 with 2-nitrobenzyl bromide, using phase transfer catalysis
under basic conditions, gave the desired 3’-O-alkylated intermediated 22. The bis-Boc groups
were removed by heating on silica gel under vacuum to give compound 23 followed by the
removal of the 5’ -O- TBS group with tetra(n-butyl)ammonium fluoride to give compound 24 in
64 % yield. Synthesis of the triphosphate was performed using the ‘one -pot’ pro cedure
described by Ludwig
39
followed by SAX and C18 HPLC purification to yield 25 as a
triethylammonium salt.

Scheme 2.5. Successful synthetic scheme for 3’ -O-(2-nitrobenzyl)-2’-deoxyadenosine, 2.
20

The removal of the 2-nitrobenzyl moiety is readily accomplished by laser irradiation at
355 nm,
6
365 nm,
5
or 308 nm
7
. Typically, a laser pulse operating at 25 mJ has been used to
induce photolysis.
14
In our case to test decaging a UV table lamp was used operating at 365 nm
was sufficient to induce photolysis as monitored by LRMS.
Preliminary experiments are currently underway in the Wilson group with ternary
complex crystals containing dATP with a blocked 3’O group, for which conventional structures
have been obtained (Figure 2.5).

Figure 2.5. Crystal structure of incorporated 3’ -O-caged dA into primer strand.

21

2.3 Conclusions

In order to study subtle conformational changes of pol β through time resolved
crystallography caged nucleotides were desired. This would have a photocleavable group that
would restore activity once irradiated with UV light. The easier to synthesize caged γ phosphate
dA (1) was to serve as a control group still susceptible for primer 3’O nucleophilic attack to the
P
α
, while the 3’O cage dA (2) would serve as a reversible terminator being incorporated into the
primer but stopping an additional round of incorporation until the photolabile tag would be
removed by photolysis.
The rationale of using caged compounds is straightforward: the molecule of interest is
rendered biologically inactive (caged) by chemical modification with a protecting group that can
be removed with light by irradiation of a suitable wavelength. This promotes the release of the
biologically active molecule, generating a time-controlled burst in concentration with tight
spatial control.
4,6

The synthesis of 3’ -O-modified dNTPs was particularly challenging due to few literature
example on the synthesis of 3’ -O caged dNTP as well as inconsistencies regarding desired
product formation (ie. synthetic route leading to N
6
,N
6
-bis-(2-nitrobenzyl)-2’-deoxyadenosine
instead of desired of 3’ -O-(2-nitrobenzyl)-2’-deoxyadenosine).
5, 23
Synthesis of useful synthetic
intermediate 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine (12) was troublesome due
to glycosidic cleavage (N9-C’1) occ urring under acidic conditions as well as halo-
dediazotization resulting in in 6-oxopurine and instability of the intermediates.
29, 30, 31, 40,41

A successful synthetic route was devised in which the readily available and stable 2’ -
deoxyadenosine was used as starting material. Acetyl protection was carried out on the 3’, 5’ -O
22

in order to allow bis-Boc protection on the N
6
.
Acetyl deprotection with NH
3
and selective 5’ -O
protection with TBMS yielded intermediate 21 containing a free 3’ -OH which reacted with 2-
nitrobenzyl bromide to give the desired 3’-O-alkylated intermediated, 22. The Boc protective
groups as well as the 5’-O- TBS group were removed by heating on silica gel and with TBAF
respectively to yield precursor 24. Synthesis of the triphosphate was performed using the ‘one -
pot’ procedure de scribed by Ludwig.
39

Although literature reports that chromatography is typically inadequate for the complete
removal of the 2‘ -deoxyadenosine from 3’ -O-modified-dNTP synthesis,
42
our reverse-phase
high-performance liquid chromatography trace revealed that our product was free from dATP
contamination (Figure B21).
Preliminary experiments to cleave the photolabile tag with a UV table lamp operating at
365 nm were sufficient to induce photolysis, as monitored by LRMS. Our collaborators at
NIEHS have successfully incorporated a 3’ -O cage nucleoside into the primer strand of pol β and
have obtained a crystal structure of it.
Future experiments will utilize ultra-fast Laue X-ray crystallographic along with a rapid
laser pulse to initiate the reaction. This technique will make it possible to observe enzyme
reaction intermediates at atomic resolution using 3’-O caged dNTP analogs. The proposed work
would have the 3’-O-(2-nitrobenzyl)-2’-deoxyadenosine bound into a crystalline ternary
complex but due to the photolabile tag rendor additional nucleoside incorporation inactive. A
pulse of laser light irradiation photochemically will cleave the caging group, initiating turnover
at a defined zero time, thus providing time-resolved crystallographic “snapshots” of the DNA pol
β catalyzed hydrolysis of dNTPs.
23

2.4 Experimental
2.4.1 Materials and Methods

High performance liquid chromatography was performed using a Dynamax solvent
delivery system Model SD-200 equipped with a Shimadzu SPD-10A UV-vis detector.
Purification was done utilizing the column Macherey-Nagel Sp 150/25 Nucleogel for SAX and a
Phenomenex Luna 5μ C18 250 x 21.20 mm column.
2.4.2 Synthesis of adenosine 5'-tetraphosphate, 3
43

Adenosine-5'-triphosphate sodium salt (300 mg, 0.544 mmol) was dissolved in 3:5:1
mixture of MeOH: H
2
O:TEA and passed through 6 mL of Dowex® 50Wx8 200-400 mesh resin
in order to convert to TEA salt of ATP (466 mg).
1
H NMR (Figure B2)
TEA salt of ATP (80 mg, 0.1 mmol) was dissolved in DMF (1.2 mL) and TEA (14 μL)
followed by addition of imidazole (34 mg, 0.5 mmol) and 2,2’ -dithiodipyridine (44 mg, 0.2
mmol). After a few minutes of stirring at room temperature triphenylphosphine (52 mg, 0.2
mmol) was added and left stirring for two days. The solution was a clear yellowish color.
After two days of stirring the mixture was centrifuged and sodium perchlorate (49 mg, 0.4
mmol) dissolved in acetone (6 mL) was added resulting in precipitation.
After cooling for 2 hours in a refrigerator, the mixture was centrifuged and the
supernatant discarded. The precipitate was washed with a new portion of acetone, cooled and
centrifuged. The process was repeated 3 times. The precipitate was dried in a vacuum
desiccator over P
2
O
5
.
24

Tris(triethylammonium)phosphate was prepared by titrating 20% solution of phosphoric
acid to TEA (7 mL) in approximate a 3:1 ratio while cooling. The clear colorless solution was
dried on vacuum desiccator over P
2
O
5
.
The imidazolide previously obtained was dissolved in DMF (1.2 mL) followed by
addition of tris(triethylammonium)phosphate (200 mg). Finally ZnCl
2
(80 mg) were added and
stirred at room temperature over 6.5 h.
Reaction mixture was poured into 15 mL of an aqueous solution containing EDTA (250
and neutralized with NaHCO
3
.
Compound was purified by SAX HPLC chromatography using a gradient of 0.5 M TEAB
pH 8 from 0-100% in 30 min with a flow rate of 9 ml/min and possessing a retention time of
product 30.6 min. Retention time of adenosine triphosphate 25.4 min.

31
P NMR (Figure B3), LRMS (Figure B4)
2.4.3 Synthesis of hydrazone of 2-nitroacetophenone, 8
3,22

To a round bottom flask 95% ethanol (12.5 ml), 2-nitroacetophenone (0.8 ml, 6.1 mmol),
glacial acetic acid (0.38 mL, 6.0 mmol) and hydrazine hydrate (0.66 mL, 13.5 mmol) were
added. Reaction mixture was refluxed for 3 h at 105 °C. After solvent was removed by vacuum
yellowish oil remained. The oil was extracted with CHCl
3
and H
2
O collecting the organic layer
and drying with MgSO
4
. Evaporating solvent under vacuum yielded 8 (243 mg).
1
H NMR (Figure B5).

25

2.4.4 Synthesis 1-(2-nitrophenyl)diazoethane, 9
3,22

8 (243 mg, 1.4 mmol) and MnO
2
(580 mg, 6.7 mmol) were added to a flask and dissolved
with CHCl
3
(6 mL) while stirring vigorously for 5 min. Effort was made to protect reaction from
sunlight. Progress was followed by UV-vis spectrometer revealing new peaks at λ
max
405 nm
and 272 nm corresponding to 1-(2-nitrophenyl)diazoethane (Figure B6). MnO
2
and Mn(OH)
2

were removed by filtration with a Buchner funnel using gentle suction.
2.4.5 Synthesis of P
3
-1-(2-nitrophenyl)ethyl adenosine triphosphate, 4
3

ATP was acidified by Dowex® 50Wx8 200-400 mesh resin at a pH of 5.9. To a flask
containing an aqueous solution of acidified ATP (300 mg, 0.544 mmol), CHCl
3
(5 mL) and 9
(237 mg, 1.34 mmol) were added and stirred at room temperature. Product was purified by SAX
HPLC with a flow rate of 3 mL/min and a 0.5M LiCl 0-100% gradient monitored at λ
max
=
254nm. (Figure B7)
2.4.6 Synthesis of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1
3,22

2’-deoxyadenosine triphosphate (30 mg, 0.06 mmol) was acidified by Dowex® 50Wx8
200-400 mesh resin. 9 (16 mg, 0.09 mmol) was then added in CHCl
3
adjusting the pH to 4 with
4 drops of 1N NaOH. Product was purified by SAX HPLC with a flow rate of 9 mL/min and
0.5 M TEAB buffer 0-100% gradient pH = 7.6. Retention time of 1 was 23 min. After
evaporation of solvent another pass at HPLC was performed this time using a C18 column 0.1M
TEAB pH 7.1, flow rate 8 mL/min and 5% acetonitrile. 5.3 mg of 1 was isolated after
26

evaporation of solvent.
31
P NMR (Figure B8),
1
H NMR (Figure B9), LRMS (Figure B10) and
UV-Vis spectra (Figure B11) taken.
2.4.7 Synthetic attempt of 3’,5’-di-O-trifluoroacylate , 11
24

To a oven dried three neck round bottom flask, flushed with N
2
, 2’-deoxyinosinie, 10
(500 mg, 2 mmol) was added. Ice cold distilled CH
2
Cl
2
(20 mL) and trifluoroacetic anhydride (3
mL, 22 mmol) were added and stirred in ice bath for 30 min followed by allowing the reaction to
reach room temperature. Solvent was evaporated with a stream of N
2
until a viscous clear liquid
remained. After leaving flask overnight mixture had turned pink. By means of a dropping
funnel DMF (2 mL), thionyl chloride (2 mL) and CH
2
Cl
2
(40 mL) were added to the reaction
mixture and refluxed at 60 °C. LRMS of reaction mixture revealed glycosidic cleavage (Figure
B12).
2.4.8 Synthesis of 3’,5’-Di-O-acetylated deoxyadenosine, 17
27, 29, 44

To a round bottom flask 16 (2.56, 10 mmol) anhydrous pyridine (35 mL) and catalytic
amount of 4-(dimethylamino)pyridine were added. Distilled acetic anhydride (5.5 mL, 53 mmol)
was added and stirred under N
2
at room temperature. Monitored by TLC (8.8:1.2 CH
2
Cl
2
:MeOH,
rf = 0.48). Reaction was quenched after 3 h with ice cold water which was subsequently
removed by vacuum. Toluene was then added in three separate portions and removed by
vacuum to yield yellowish oil.
1
H NMR (Figure B13).
27

2.4.9 Synthesis of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-
chloropurine, 18
27

To a round bottom flask containing distilled TMSCl (3.5 mL, 28 mmol) approximately 3
mL of C
3
H
7
ONO (prepared by mixing NaNO
2
, H
2
SO
4
and C
3
H
7
OH in stoichiometric amounts
and extracting the organic layer) were added and refluxed. Solution turned bright red, and the
reddish gas that evolved was condensed at -78 °C and dissolved in CH
2
Cl
2
, to yield NOCl.
To a separate round bottom flask 17 (335 mg, 1 mmol) was dissolved with distilled
CH
2
Cl
2
(30 ml). NOCl was then added to the flask containing 17 turning the solution dark
yellow and producing gas. After 30-45 min stirring the solution became a light yellow cloudy
color. Reaction monitored by LRMS.
Desired product was isolated by semiprep glass TLC (9:1 CH
2
Cl
2
:MeOH, rf = 0.60).
Solvent was evaporated and
1
H NMR taken in CDCl
3
(Figure B14) LRMS (Figure B15).
2.4.10 Synthesis of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine, 12
30

2 M NH
3
/MeOH was prepared by refluxing NH
4
OH and condensing NH
3
gas produced
in a known amount of MeOH. A round bottom flask containing 18 (217 mg, 0.6 mmol) was
cooled via ice bath to which 2 M NH
3
/MeOH (8.11 mL) was added and stirred at 0 °C. Solution
was a pink/orange color turning cloudy after a few minutes. Solvent was evaporated by vacuum
and deprotected product identified by
1
H NMR (Figure B16) and LRMS using APCI probe
(Figure B17). Product isolated by semiprep TLC (10:1 CH
2
Cl
2
:MeOH, rf = 0.56).
28

2.4.11 Synthesis of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-
2’deoxyribofuranosyl]-6-chloropurine, 13
6

To a stirred solution of 12 (89.7 mg, 0.33 mmol), imidazole (50 mg, 0.73 mmol)
dissolved in anhydrous DMF (2 mL) was added, followed by addition tert-butyldimethylsilyl
chloride (54 mg, 0.36 mmol). Reaction mixture was stirred at room temperature for 24 h.
Solvent was then removed by vacuum and purified by semiprep TLC (2:1 EtOAc:Hex, rf = 0.61)
1
H NMR (Figure B18) and LRMS (positive mode) (Figure B19).
2.4.12 Synthesis of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-
2’deoxyribofuranosyl]-6-chloropurine, 14
6

To a stirred solution of 13 (58.2 mg, 0.15 mmol) in CH
2
Cl
2
(5mL), tetra-butylammonium
bromide (0.025 mg, 0.08 mmol), 2-nitrobenzyl bromide (105 mg, 0.45 mmol) and a 40 %
aqueous solution of NaOH (3 mL) were added and stirred for 1.5 h. Monitored by TLC (2:1
EtOAc:Hex, rf = 0.35). After 1.5 h. EtOAc (14 mL) was added and the reaction mixture
extracted with EtOAc (2 x 5 mL). Organic layer was washed with saturated aqueous NaHCO
3

and dried over Na
2
SO
4
. Product isolated by preparative TLC.
1
H NMR taken in CDCL
3
(Figure
B20) and LRMS (negative mode) (Figure B21).
2.4.13 Synthesis of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine, 15
5, 6, 45

To a round bottom flask containing 14 (33.6 mg, 0.06 mmol), THF (1.54 mL) was added,
followed by addition of 1 M TBAF/THF solution (71 μL) . The clear colorless solution turned
orange. Monitored by TLC (1:15 MeOH:CH
2
Cl
2
). After 45 min stirring solvent was removed
by vacuum and dissolved with dioxane (0.56 mL). To the solution 7 M MeOH/NH
3
(1.1 mL)
29

were added and stirred at 85-88 °C overnight under reflux. The mixture was then placed into a
teflon liner and sealed within a stainless steel PARR vessel with a portion of NaOH, heated on an
oil bath at 140 °C. Product isolated by preparative TLC (1:14 MeOH: CH
2
Cl
2
) to yield 15 (12
mg). Product was dried in a desiccator over P
2
O
5
and
1
H NMR obtained in CD
3
OD (Figure
B22) and DMSO-d6 (Figure B23).
2.4.14 Synthesis of 3’,5’-Bis-O-[(tert-butyl)dimetthylsilyl]-2’-deoxyadenosine
5

A solution of 16 (1.25 g, 5 mmol), imidazole (2.25 g, 33 mmol) and TBDMSCl (2.41 g,
16 mmol) in anhydrious DMF (14 mL) was stirred at room temperature overnight with N
2
. The
reaction was then quenched with methanol (20 mL) added to quench reaction and concentrated
by rotovap to yield a white solid. CH
2
Cl
2
(50 mL) was then added turning the solution cloudy
white. Mixture was extracted with an aqueous solution of saturated NH
4
Cl and CH
2
Cl
2
. The
organic layer was dried over Na
2
SO
4
and solvent evaporated to yield desired product 333 mg.
1
H NMR taken (Figure B24)
2.4.15 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine
5

3’, 5’ -Bis-O-[(tert-butyl)dimetthylsilyl]-2’-deoxyadenosine (336 mg, 0.7 mmol) was co-
evaporated with DMF and then dissolved in anhydrous DMF (10 mL). To this solution di-tert-
butyl dicarbonate (3.30g, 15.1 mmol) and DMAP (1.83 g, 14.9 mmol) were added and stirred
overnight at room temperature under N
2
. After18 h of stirring solvent was removed by vacuum
and orange residue extracted with (100 mL) and saturated NH
4
Cl solution (100 mL). Organic
layer was dried over Na
2
SO
4
and purified by silica column chromatography using 3:1 to 2:1
30

Hex:EtOAc as the mobil phase. TLC 3:1 Hex:EtOAc, rf = 0.7. After removal of solvent 2.498 g
(74 %) clear colorless foam obtained.
1
H NMR (Figure B25).
2.4.16 Synthesis of N
6
, N
6
-Bis-(tert-butyloxycarbonyl) -2’-deoxyadenosine by
deprotection of TBMS protective groups, 20
5,

35

A solution of tetra-butylammonium fluoride (3.4 g, 11 mmol) in THF (12 mL) was added
to a solution of intermediate N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine (2.5g, 3.7 mmol) in THF (12 mL) at 0 °C allowing
temperature to gradually rise to room temperature. After 24 h of stirring under N
2
solvent was
removed by vacuum. The residue was dissolved in CH
2
Cl
2
(80 mL) and washed twice with
saturated NH
4
Cl solution. Organic layer was dried over Na
2
SO
4
, concentrated in vacuo, and
purified by silica gel column chromatography (1:1 Hex:EtOAc to. 100 % EtOAc) to yield 1.324
g (80 %) of bis-Boc protected dA.
1
H NMR (Figure B27).
2.4.17 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine, 21
5

20 (1.324 g, 2.9 mmol) was dissolved in anhydrous DMF (6 mL) and stirred at 0 °C.
Imidazole (400 mg, 5.8 mmol) was added to the solution followed by addition of TBDMSCl
(0.57 g, 3.8 mmol) dissolved in DMF (2 mL). Solution was allowed to gradually reach room
temperature and left over the weekend stirring. Monitored by TLC (2:1 EtOAc:Hex)
31

Reaction mixture was extracted with EtOAc (3 x 20 mL) and organic phase washed with
saturated NH
4
Cl solution. Organic layer was dried over Na
2
SO
4
and concentrated by vacuo.
1
H NMR (Figure B28).
2.4.18 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 3’,5’-O-(acetyl)-2’-
deoxyadenosine, 19

17 (1.66 g, 5 mmol) was dissolved in anhydrous DMF (10 mL) followed by addition of
DMAP (1.81 g, 15 mmol) and contents stirred under N
2
until fully dissolved. Di-tert-
butydicarbonate (3.24 g, 15 mmol) was added turning the solution from yellow-orange to dark
orange and lastly a dark brown color. Monitored by TLC (2:1 EtOAc:Hex, rf = 0.52). Solvent
was removed by vacuum after 24 h and purified by silica gel column chromatography (2:1 Hex:
EtOAc up to 1:1 Hex: EtOAc) to yield 1.85 g (70 %) of 19.
1
H NMR (Figure B26).
2.4.19Synthesis of N
6
, N
6
-Bis-(tert-butyloxycarbonyl) -2’-deoxyadenosine by
deprotection of acetyl protective groups, 20

19 (1.852 g, 3.5 mmol) was dissolved in MeOH (10 mL) and stirred at 0 °C. 2 M
NH
3
:MeOH (13 mL) were added and left stirring overnight at 4 °C. Reaction was monitored by
TLC (2:1 EtOAc:Hex, rf = 0.36). Purified by silica gel column chromatography (1:1 EtOAc:
Hex to 100 % EtOAc) to yield 1.42 g (91 %) of 20 as a white foam.
1
H NMR (Figure B27).
32

2.4.20 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine, 21
5

20 (1.42 g, 3 mmol) was coevaporated with anhydrous DMF followed by dissolving in
anhydrous DMF (20 mL). To the solution imidazole (0.43 g, 6.3 mmol) followed by TBSCl
(0.62 g, 4.1 mmol) were added. Reaction was monitored by TLC (1:1 EtOAc: Hex, rf = 0.48).
Solvent was removed by vacuo after 2 days of stirring. Product 21 was isolated by silica gel
chromatography (1:1 EtoAc: Hex) to yield 1.409 g (79% yield)
1
H NMR (Figure B28)
2.4.21 Synthesis of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-
butyl)dimethylsilyl]-3’-(2-nitrophenyl)-2’-deoxyadenosine, 22
6

To a flask containing 21 (1.35 g, 2.39 mmol), CH
2
Cl
2
(50 mL) was added and stirred
under N
2
. Tert-butylammonium bromide (385 mg, 1.2 mmol) was added followed by addition of
40% aqueous solution of NaOH (50 mL). Lastly 2-nitrobenzylbromide (1.66 g, 7.2 mmol) was
dissolved in CH
2
Cl
2
(50 mL) and added dropwise. Reaction was monitored by TLC (1:2
EtOAc:Hex, rf = 0.44). Reaction was complete in 1 h to which EtOAc (200 mL) was added and
washed with aqueous solution of saturated NaHCO
3
:NaCl. Organic layer was dried over Na
2
SO
4

and solvent evaporated. Product 22 was isolated by silica gel column chromatography (3:1 Hex:
EtOAc to 2:1 Hex:EtOAc) to yield 1.272 g (76% yield) of a white foam.
1
H NMR (Figure B29).
33

2.4.22 Synthesis of 5’-O-[(tert-butyl)dimethylsilyl]-3’-O-(2-nitrophenyl)-2’-
deoxyadenosine, 23
5,46,47

Silica gel 62 (10 g, 60-200 mesh) was activated by placing in a vacuum oven with drierite
and heating under vacuum for 24 h at 80 °C. Oven was cooled to 40 °C and silica added to a
round bottom flask containing 22 (1.27 g, 1.8 mmol) and CH
2
Cl
2
(50 mL). Contents were
concentrated by vacuum and then placed in oven at 70-80 °C under reduced pressure for 48 h.
Silica was washed with MeOH (3 x 50 mL) and solvent collected. Product 23 was isolated by
silica gel column chromatography (1:1 EtOAc:Hex to 2:1 EtOAc: Hex) to yield 0.552 g (61 %).
1
H NMR (Figure B30).
2.4.23 Synthesis of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine, 24

23 (0.52 g, 1.04 mmol) was co-evaporated with CH
3
CN and then dissolved in dry THF
(3 mL). To the yellow solution TBAF (491 mg, 1.56 mmol) dissolved in THF (2 mL) was added
drop wise turning the solution orange. Contents were stirred at room temperature and monitored
by TLC (100 % EtOAc, rf = 0.33). After 4 h stirring solvent was removed by vacuum to yield
reddish/violet oil. Product 24 was isolated by silica gel chromatography (100 % CH
2
Cl
2
to 100
% EtOAc) to yield 0.284 g (68 % yield) of a white powder which was placed in vacuo oven over
P
2
O
5
and drierite.
1
H NMR (Figure B31).

34

2.4.24 Synthesis of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2
48

24 (269 mg, 0.706 mmol) was dissolved in trimethyl phosphate (5 mL) turning mixture a
white cloudy color. POCl
3
(88 μL, 0.92 mmol) was added drop wise while stirring at 0 °C.
Sodium pyrophosphate decahydrate (3.0 g, 6.7 mmol) was dissolved in H
2
O (75 mL) and
added to Dowex® 50Wx8 resin, stirring for 30 min. In a separate flask ethanol (20 mL) and tri-
n-butylamine (3.2 mL) was put in an ice bath. Dowex mixture was filtered allowing filtrate to go
into a ethanol:tributylamine solution. The solvent was evaporated by vacuum and co-evaporated
with ethanol and then with DMF.
Reaction mixture containing 29 had gone from cloudy white to a slightly yellow cloudy
solution. Half the contents of a solution of bis-tri-n-butylammonium pyrophosphate dissolved in
DMF (10 mL) were transferred out to the flask containing 29. A solution of tributlyamine (0.75
mL) and DMF (2.5 mL) was poured in one portion to the reaction mixture. After 10 min of
stirring 1 M triethylammonium bicarbonate buffer (25 mL, pH = 7.5) was added and reaction
flask placed in refrigerator. Product was confirmed by LRMS 625 m/z (-) mode.
Product 2 was isolated by two phase HPLC purification: 1) SAX HPLC utilizing
Machery-Nagel SP 150-25 Nucleogel SAX 1000-10 column and a gradient of 0.5 M TEAB (pH
= 7.3 , flow rate 9 mL/min) in 30 min. Retention time of product was 29.2 min. 2) C18 HPLC
utilizing Phenomenex 250 x 21.20 mm column and a gradient of 0.1M TEAB 10 % CH
3
CN (
pH = 7) to 50 % CH
3
CN in 40 min. Retention time of product was 20.6 min (Figure B35).
After removal of solvent concentration of 2 was determined to be 42 mg by UV-vis (ε = 15,200
in 0.1 M phosphate buffer, pH = 7) (Figure B36). 2 was characterized by
1
H (Figure B32),
31
P
NMR (Figure B33), and LRMS (Figure B34).
35

2.4.25 Photolysis of 3’-O-(2-nitrophenyl)-2’-dATP by UV irradiation

LRMS of 2 protected from light 625.3 m/z (- mode). Upon irradiation with a UV-vis
table lamp for 2 min at 365 nm new peaks observed 490.2 m/z (dATP). Irradiation for 20 min at
365 nm led to peak 490.2 m/z being more pronounced.
Irradiation at 254 nm for 5 min did not result in substantial amount of dATP. This leads
one to believe that 254 nm is not an appropriate wavelength for photolysis and although more
dATP is observed at 365 nm this wavelength is not optimal and lacks power. According to
literature λ = 302 nm is optimal.
5
(Figure B37)

36

2.4 References

(1) Beard, W. A.; Wilson, S. H. Chem. Rev. 2006, 106, 361–382.
(2) Meldrum, R. A.; Chittock, R. S.; Wharton, C. W. Methods Enzymol. 1998, 291, 483–495.
(3) Walker, J. W.; Reid, G. P.; McCray, J. A.; Trentham, D. R. J. Am. Chem. Soc. 1988, 110,
7170–7177.
(4) Pinheiro, A. V.; Baptista, P.; Lima, J. C. Nucleic Acids Res. 2008, 36, e90.
(5) Wu, W.; Stupi, B. P.; Litosh, V. A.; Mansouri, D.; Farley, D.; Morris, S.; Metzker, S.;
Metzker, M. L. Nucleic Acids Res. 2007, 35, 6339–6349.
(6) Wu, J.; Zhang, S.; Meng, Q.; Cao, H.; Li, Z.; Li, X.; Shi, S.; Kim, D. H.; Bi, L.; Turro, N.
J.; Ju, J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16462–16467.
(7) Chaulk, S. G.; MacMillan, A. M. Nat. Protoc. 2007, 2, 1052–1058.
(8) Mayer, G.; Kröck, L.; Mikat, V.; Engeser, M.; Heckel, A. ChemBioChem 2005, 6, 1966–
1970.
(9) A, M. R.; S, S.; R, T. D.; W, W. C. Kinetics and mechanism of DNA repair. Preparation,
purification and some properties of caged dideoxynucleoside triphosphates.
http://www.biochemj.org/bj/266/bj2660885.htm (accessed Jul 18, 2013).
(10) Corrie, J. E. T. J. Label. Compd. Radiopharm. 1996, 38, 403–410.
(11) Muller, C.; Even, P.; Viriot,, M.-L.; Carré, M.-C. Helv. Chim. Acta 2001, 84, 3735–3741.
(12) Geissler, D.; Kresse, W.; Wiesner, B.; Bendig, J.; Kettenmann, H.; Hagen, V.
Chembiochem Eur. J. Chem. Biol. 2003, 4, 162–170.
(13) Kaplan, J. H.; Forbush, B.; Hoffman, J. F. Biochemistry (Mosc.) 1978, 17, 1929–1935.
(14) McCray, J. A.; Herbette, L.; Kihara, T.; Trentham, D. R. Proc. Natl. Acad. Sci. U. S. A.
1980, 77, 7237–7241.
(15) Schlichting, I.; Almo, S. C.; Rapp, G.; Wilson, K.; Petratos, K.; Lentfer, A.; Wittinghofer,
A.; Kabsch, W.; Pai, E. F.; Petsko, G. A.; Goody, R. S. Nature 1990, 345, 309–315.
(16) Schlichting, I.; Rapp, G.; John, J.; Wittinghofer, A.; Pai, E. F.; Goody, R. S. Proc. Natl.
Acad. Sci. 1989, 86, 7687–7690.
(17) Stoddard, B. L.; Koenigs, P.; Porter, N.; Petratos, K.; Petsko, G. A.; Ringe, D. Proc. Natl.
Acad. Sci. 1991, 88, 5503–5507.
(18) Wharton, C. W.; Meldrum, R. A.; Reason, C.; Boone, J.; Lester, W. Biochem. J. 1993,
293, 825–828.
(19) Carrick, J. M.; Kashemirov, B. A.; McKenna, C. E. Tetrahedron 2000, 56, 2391–2396.
(20) Kumar, S.; Sood, A.; Wegener, J.; Finn, P. J.; Nampalli, S.; Nelson, J. R.; Sekher, A.;
Mitsis, P.; Macklin, J.; Fuller, C. W. Nucleosides Nucleotides Nucleic Acids 2005, 24,
401–408.
(21) Sood, A.; Kumar, S.; Nampalli, S.; Nelson, J. R.; Macklin, J.; Fuller, C. W. J. Am. Chem.
Soc. 2005, 127, 2394–2395.
(22) Walker, J. W.; Reid, G. P.; Trentham, D. R. Methods Enzymol. 1989, 172, 288–301.
(23) Metzker, M. L.; Raghavachari, R.; Richards, S.; Jacutin, S. E.; Civitello, A.; Burgess, K.;
Gibbs, R. A. Nucleic Acids Res. 1994, 22, 4259–4267.
(24) Robins, M. J.; Basom, G. L. Can. J. Chem. 1973, 51, 3161–3169.
(25) Armarego, W. L. F.; Perrin, D. D. Purification of laboratory chemicals; Butterworth
Heinemann, 1997.
(26) De Napoli, L.; Messere, A.; Montesarchio, D.; Piccialli, G.; Santacroce, C.; Varra, M. J.
Chem. Soc. [Perkin 1] 1994, 923.
37

(27) Francom, P.; Robins, M. J. J. Org. Chem. 2003, 68, 666–669.
(28) Liu, J.; Janeba, Z.; Robins, M. J. Org. Lett. 2004, 6, 2917–2919.
(29) Van der Wenden, E. M.; von Frijtag Drabbe Kuenzel, J. K.; Mathot, R. A. A.; Danhof, M.;
IJzerman, A. P.; Soudijn, W. J. Med. Chem. 1995, 38, 4000–4006.
(30) Ikejiri, M.; Saijo, M.; Morikawa, S.; Fukushi, S.; Mizutani, T.; Kurane, I.; Maruyama, T.
Bioorg. Med. Chem. Lett. 2007, 17, 2470–2473.
(31) Francom, P.; Janeba, Z.; Shibuya, S.; Robins, M. J. J. Org. Chem. 2002, 67, 6788–6796.
(32) Lee, J. G.; Cha, H. T. Tetrahedron Lett. 1992, 33, 3167–3168.
(33) Nair, V.; Richardson, S. G. J. Org. Chem. 1980, 45, 3969–3974.
(34) Zollinger, H. Angew. Chem. Int. Ed. Engl. 1978, 17, 141–150.
(35) Hogrefe, R. I.; McCaffrey, A. P.; Borozdina, L. U.; McCampbell, E. S.; Vaghefi, M. M.
Nucleic Acids Res. 1993, 21, 4739–4741.
(36) Westmanu, E.; Stromberg, R. Nucleic Acids Res. 1994, 22, 2430–2431.
(37) Krock, L.; Heckel, A. Angew. Chem. Int. Ed. 2005, 44, 471–473.
(38) Sikchi, S. A.; Hultin, P. G. J. Org. Chem. 2006, 71, 5888–5891.
(39) Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131–133.
(40) Srivastava, P. C.; Robins, R. K.; Jr, R. B. M. In Chemistry of Nucleosides and
Nucleotides; Townsend, L. B., Ed.; Springer US, 1988; pp. 113–281.
(41) Zoltewicz, J. A.; Clark, D. F.; Sharpless, T. W.; Grahe, G. J. Am. Chem. Soc. 1970, 92,
1741–1750.
(42) Metzker, M. L.; Raghavachari, R.; Burgess, K.; Gibbs, R. A. BioTechniques 1998, 25,
814–817.
(43) Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. RNA 2001, 7,
1486–1495.
(44) Ciuffreda, P.; Casati, S.; Manzocchi, A. Magn. Reson. Chem. 2007, 45, 781–784.
(45) Orji, C. C.; Michalczyk, R.; Silks III, L. A. "Pete J. Org. Chem. 1999, 64, 4685–4689.
(46) apelqvist, T.; Wensbo, D. Tetrahedron Lett. 1996, 37, 1471–1472.
(47) Ravindranath, N.; Ramesh, C.; Ravinder Reddy, M.; Das, B. Adv. Synth. Catal. 2003, 345,
1207–1208.
(48) Calleri, E.; Ceruti, S.; Cristalli, G.; Martini, C.; Temporini, C.; Parravicini, C.; Volpini, R.;
Daniele, S.; Caccialanza, G.; Lecca, D.; Lambertucci, C.; Trincavelli, M. L.; Marucci, G.;
Wainer, I. W.; Ranghino, G.; Fantucci, P.; Abbracchio, M. P.; Massolini, G. J. Med.
Chem. 2010, 53, 3489–3501.

38

Chapter 3
Synthesis of Alexa 555 and 7-DEAC linked dNTP analogues for Fluorescence
Resonance Energy Transfer studies
3.1 Introduction

Another approach into gaining visual insight into the mechanism of DNA pol β is by use
of fluorescent resonance energy-transfer (FRET) studies. This technique has been broadly
applied in studies of protein conformational change and protein interactions including in the
study of DNA polymerase systems.
1–3

Nucleotide substrates are typically labeled with fluorescent dyes at the purine or
pyrimidine base for genomic applications. These modified dNTPs add the labeled nucleotide
base into the primer chain of DNA polymerase which can then be read by determining the
fluorescence of the dye.
4–7
However, these types of nucleotide modifications cannot be used for
real-time sequencing because they are poorly incorporated into consecutive positions by DNA
polymerase.
8,9

A more appropriate approach is to have the fluorescent dye linked to the P
γ
, of a dNTP
analog. A phospholinked nucleotide would be catalyzed by the DNA polymerase resulting in
release of the fluorophore from the incorporated nucleotide, thus generating natural, unmodified
DNA and enabling visualization of the fluorescent tag as it is cleaved.
10–12

Another structural modification that can be done on dNTP is replacement of the bridging
oxygen of the triphosphate to a carbon. Previously, it was described how β,γ -CXY dNTP
analogs are incorporated by pol β providing a way to study leaving group effects by LFER
39

(Chapter 1). However, the same type of modification can be done on the bridging oxygen of
α,β phosphorus yielding a non -hydrolyzable and stable dNTP analog that is ideal for X-ray
structural studies.
13, 14
Ternary structures of pol β with natural dNTP and non -hydrolyzable α,β -
CXY-dNTP as incoming substrates have been compared and found to have negligible difference
in active site geometry.
15,16
Therefore, labeling a non-hydrolyzable dNTP with a fluorescent dye
could prove useful in studying conformational changes and enzyme motions by FRET analysis.
Two novel non-hydrolyzable dNTP compounds possessing a fluorescent dye at the
terminal phosphate was proposed which in turn could be used for two different types of
experiments, FRET and environmental sensitivity. 1) For FRET analysis, the stable fluorophore,
Alexa 555 will be coupled to the P
γ
of a non-hydrolyzable dATP. An acceptor partner, such as
Alexa 647 would be strategically placed within the DNA, thus allowing to probe distance
measurements (Figure 3.1). 2) For environmental sensitivity experiments, the conformational
and environmentally sensitive coumarin probe, 7-diethylamino-3-((((2-
maleimidyl)ethyl)amino)carbonyl)coumarin (7-DEAC),
17,18
will be linked to the P
γ
of a non-
hydrolyzable dATP. Coumarins are sensitive to environmental conditions, usually experiencing
a decrease in fluorescence intensity and a shift in emission to longer wavelengths when placed in
an environment with a thiol or metal. In the past it has been successfully used to characterize the
conformational changes of a phosphate binding protein from E.coli.
19

40

Figure 3.1. Crystal structure representation of Alexa 555 dATP bound within pol β and the
fluorophore Alexa 647—5’ amino DNA.
3.2 Results and Discussions

In order to measure the conformational changes of pol β upon substrate binding and
catalysis, two novel non-hydrolyzable dATP compounds in which the terminal phosphate is
attached to a fluorescent label through a four carbon linker were synthesized (Figure 3.2). When
used in conjunction with a fluorescently labeled enzyme-DNA complex, enzyme motions can be
analyzed by FRET studies. Additionally, conformational changes can be studied with the
environment sensitive 7-DEAC linked dATP, 8.
41

Figure 3.2. Phospholinked non-hydrolyzable fluorescent dATP.

The fluorophores were attached at the terminal phosphate via a four carbon linker
scaffold as opposed to directly on the P
γ
of the triphosphate since K
d
for dNTP substrates tend to
worsen if a moiety is directly coupled to the P
γ
.
10,11,20
Indeed, even at five atoms away the
experimental K
d
value for 8 was determined to be ~15 µM, a reduced affinity of around 15-fold
(K
d
for AMPcPP was 1 µM). Additionally it has also been observed that fluorophore residues
can be quenched by the proximal adenine, thus providing better fluorescent signal if the
fluorophore is distant to the base of the nucleoside.
20

Synthesis of 7 and 8 involved activating 2’-deoxyadenosine (1) with p-toluenesulfonyl
chloride in order to obtain 5’-O-tosylated 2’ -deoxyadenosine (2). Methylene-bridged
disphosphonate (3) is acquired through nucleophilic displacement of 5’ -O-tosyl nucleoside with
tris(tetra-n-butylammonium) at room temperature.
21
Fmoc-6-aminobutanol (4) was
phosphorylated (5) (Scheme 3.1) and then non-enzymatically coupled to the 3 using CDI as a
condensing agent to yield a non-hydrolyzable triphosphate nucleoside analog with a four carbon
linker coupled to the P
γ
(6).
22,

23
The final two steps of the synthesis consisted of Fmoc-
deprotection and coupling of the free amine (6) with the succinimidyl ester of the desired
42

fluorescent dye, Alexa Flour® 555 or 7-DEAC, under basic conditions to yield the desired
product, 7, 8.
23, 24
Through this chemistry any succinimidyl ester activated fluorophore could
potentially be coupled to 6 to acquire an array of fluorescent dNTPs with different fluorescent
properties. (Scheme 3.2).

Scheme 3.1. Phosphorylation of 4-(Fmoc-amino)-1-butanol to yield 5.

Scheme 3.2 Synthesis of Dye-aminobutyl-dAPPCP.

43

Both target compounds were purified by reverse phase HPLC using dual UV detectors to
monitor simultaneously the purine and fluorophores in the nucleotide. The labeled dATP
analogs were characterized by
1
H NMR,
31
P NMR, and LRMS. Excitation and emission spectra
were obtained using a Fluorolog Tau-3 Lifetime System Fluorimeter (FL-3-222-Tau) and a
Beckman Coulter DU®. Purity and non-incorporation of 7 and 8 were assessed by a gapped
DNA insertion assay with DNA pol β ( Figure 3.3).
a, -CH
2
-dATP
L H
-MDCC- -AF555-
H L –
(Primer) n

Figure 3.3. Gapped DNA insertion assay with DNA pol β for dATP analogs. The purity of the
analogues was assessed from their failure to be inserted into a gapped DNA substrate. Primer (n)
extension was assayed in the presence of low or high pol β and [MDCC -dATP] = 1 mM
[AF555-dATP] = 0.8 mM for 5 min. Insertion assay performed by Dr. William A. Beard,
NIEHS.
Preliminary FRET studies are in progress by the Wilson group.
3.3 Conclusions

Two novel non-hydrolyzable α ,β-substituted nucleosides with a fluorescent tag at the
terminal phosphate were synthesized in order to visualize conformational changes, measure
distance, and probe environment of the active site. To demonstrate feasibility, two dyes were
selected, Alexa Fluor 555 and 7-DEAC, and coupled to the P
γ
via a four carbon linker. By
means of a Horiba NanoLog Spectrofluorometer System the excitation and emission of 7 was
44

determined to be λ
(max)
= 550 nm and λ
(max)
= 564 nm respectively while for 8 λ
(max)
= 430 nm
and λ
(max)
= 475 nm.
After dual pass HPLC purification, SAX and C18, 855 μg of 7 and 450 μg of 8 were
obtained. Both compounds were given to Wilson’s group at NIEHS for further studies with pol
β. The rationale being using Alexa555-dATP as the incoming nucleotide and placing a modified
base pair within the DNA structure surrounding the gap to measure conformational changes of
the molecule as a function of metal ions, gap size, etc.
3.4 Experimental
3.4.1 Material and Methods

HPLC purification was done using Dynamax Solvent Delivery System Model SD-200
equipped with Dynamax Absorbance Detector Model UV-D II and/or Shimadzu UV-Vis
Detector SPD-10A. The SAX column utilized was (SP 150/25 Nucleogel SAX 1000-10,
Macherey-Nagel). C18 column utilized was Beckman Ultrasphere 5 μ 10 mm × 25 cm. Horiba
NanoLog Spectrofluorometer System was used to measure excitation and emission wavelengths.
Molecular Probes Alexa Fluor® 555 carboxylic acid succinimidyl ester was acquired
from Invitrogen™. All other chemicals obtained through Sigma Aldrich or VWR.
3.4.2 Synthesis of 2’-deoxyadenosine-5’-tosylate, 2
21,14

2’-deoxyadenosine monohydrate (1) (547.5 mg, 2.0 mmol) was co-evaporated with
anhydrous pyridine (3 mL × 3). The dried nucleoside was then dissolved in anhydrous pyridine
45

(15 mL) and brought to 0 °C by ice bath. To the stirring solution p-toluenesulfonyl chloride (499
mg, 2.62 mmol) was added drop wise turning the solution bright yellow and brought slowly to
room temperature.
After 24 h of stirring the mixture was concentrated by vacuo followed by addition of ice
cold H
2
O (15 mL). Mixture was extracted with cold ethyl acetate (25 mL× 3) and the organic
layer dried with MgSO
4
. After removal of solvent by reduced pressure 2 was obtained as a white
foam (587 mg, 67 % yield).
1
H: 8.12 (2H, 2, 8), 7.44 (4H, C
2
H
4
), 6.26 (1H, 1’), 4.37 (2H, 5’), 4.25 (1H, 4’), 2.88 (1H, 2’),
2.56 (1H, 2’) , 2.26 (3H, CH
3
) (Figure C1).
3.4.3 Synthesis of α, β-methylene 2’-deoxyadenosine-5’-diphosphate, 3
14

Tris(tetrabutylammonium) methylene bisphosphonate was prepared by dissolving
methylene bishophonic acid (110 mg, 0.63 mmol) in H
2
O (2 mL) and adjusting pH to 8.5 with
tetrabutyl ammonium hydroxide.
21,25
Solvent was then removed by vacuo and product co-
evaporated with acetonitrile to yield a pink/yellow oily substance.
2 (250 mg, 0.62 mmol) was co-evaporated with acetonitrile (2 mL × 3) before dissolving
in acetonitrile (5 mL). A solution of tris(tetrabutylammonium) methylene bisphosphonate
dissolved in acetonitrile was added drop wise while stirring under N
2
. Mixture became a
blue/violet color. Left stirring overnight to which the solution became a bright pink/red color.
Solvent was removed by vacuo and dissolved with H
2
O turning the solution yellow.
Product was purified via dual pass preperative HPLC (SAX followed by RP-C18 with
λ
max
= 260 nm). 1) SAX: Gradient 0-100% 0.5 M TEAB pH = 7.5. 2) C18: Isocratic TEAB 0.1
46

M 10% acetonitrile pH = 7.3 (Figure C2). Isolated product 164 mg by weight, by UV/Vis 72
mg (ε = 15,300, λ
max
= 260 nm), LRMS 408 m/z (- mode) (Figure C3)
1
H NMR (500 MHz, D
2
O): 8.48 (1H, 8), 8.21 (1H, 2), 6.48 (1H, 1’), 4.76 (1H, 4 ’), 4.27 (1H, 3’),
4.08 (2H, 5’), 2.84 (1H, 2’ ), 2.58 (1H, 2’) , 2.14 (2H, PCH
2
P) (Figure C4).
31
P NMR (203 MHz, D
2
O) P
α
18.54 ppm, P
β
14.54 ppm (Figure C5).
3.4.4 Synthesis of Fmoc-4-aminobutylphosphate, 5
23

Fmoc-4-aminobutanol (0.5 g, 1.5 mmol) was co-evaporated with anhydrous acetonitrile
(10 mL × 2) then suspended in anhydrous triethylphosphate (5 mL). Phosphorus oxychloride
(275 μL, 2.9 mmol) was added to the stirring suspension followed by placing reaction vessel in
refrigerator (4 °C) for 2 hours. Monitored by TLC 9:1 CHCl
3
: MeOH, R
f
= 0.94.
To the reaction mixture a solution of NaHCO
3
(8 mL, 0.8 M) was until pH = 6.8. The
reaction mixture was extracted with CHCl
3
and H
2
O. The aqueous layer was collected and
extracted with ethyl ether and then exchanged on Dowex 50W x 8 to a tributylammonium form.
Product was purified by preparative C18 HPLC with TEAB 0.1 M as buffer and a
gradient of 5% to 20% CH
3
CN in 25 min. Flow rate 8 mL/min, λ
max
= 260 nm. Product eluted
from column after an isocratic wash of 60:40 CH
3
CN:H
2
O, retention time 10.7 min. LRMS 390
m/z (- mode) (Figure C6). Isolated product 57 mg by UV/Vis. (ε = 20,800, λ
max
= 263 nm)
1
H NMR (500 MHz, D
2
O): 7.64 (2H, 2, 10), 8.5 (2H, 5, 13), 7.29 (4H, 1, 6, 11, 12), 4.32 (2H,
14), 4.04 (1H, 9), 3.78 (2H, 19), 2.99 (2H, 22), 1.49 (2H, 21), 1.41 (2H, 20) (Figure C7).
31
P NMR: 3.56 (Figure C8).
47

3.4.5 Synthesis of aminobutyl-PPCH
2
PdA, 6

To a round bottom flask containing 5 (57 mg, 0.147 mmol) anhydrous DMF (2 mL) was
added and stirred under N
2
. To the solution CDI (95 mg, 0.59 mmol) was added and stirred at
room temperature. After 5 hours MeOH (25 μL) was added to decompose unreacted CDI.
Lastly, a solution of 3 as a TBA salt in DMF (1 mL) was added followed by addition of ZnCl
2

(29 mg, 0.21 mmol). Left stirring overnight and analyzed by LRMS (Figure C9).
Product was purified by preparative SAX HPLC with a gradient of 20% to 70% 0.5 M
TEAB in 30 min and an eluent of 30% CH
3
CN. Flow rate 8mL/min, λ
max
= 256 nm, retention
time 16.9 min.
Fmoc deprotection was carried out by stirring recovered product in 10% TEA for 16 h,
extracting with ether and removing solvent by vacuo.
LRMS 559 m/z (- mode) (Figure C10). Isolated product 2 mg by UV/Vis. (ε = 15,300, λ
max
=
260 nm)
1
H NMR (600 MHz, D
2
O): 8.58 (1H, 16), 8.31 (1H, 10), 6.53 (1H, 21), 4.27 (1H, 23), 4.15 (2H,
25), 4.00 (3H, 19, 30), 3.06 (2H, 33), 2.90(1H, 20), 2.60 (1H, 20), 2.38 (2H, PCH
2
P), 1.82 (2H,
31), 1.75 (2H, 32) (Figure C11).
31
P NMR: 17.09, 7.11, -10.80 (Figure C12).
3.4.6 Synthesis of Alexa555 aminobutyl PPCH2PdA, 7

In a conical vial the sodium salt of 6 (7 mg, 12.5 μmol ) was dissolved in H
2
O (150 μL).
The pH was adjusted to 8 by addition of NaCO
3
and with a gentle stream of CO
2
. Alexa Fluor®
48

555 (1 mg) was dissolved in anhydrous DMF (25 μL) and transferred to the vial containing 6.
Reaction was monitored by TLC using methanol as eluent.
Product was purified by semi-preparative C18 HPLC purification using a Beckman
Ultrasphere 5 micron particle size, 10 mm × 25 cm column. The mobile phase consisted 0.1 M
TEAB pH = 7.2 with a gradient of 20% to 50% MeOH in 60 min, flow rate 3 mL/min.
26
Dual
UV-Vis detectors were used 1) λ
max
= 260 nm 2) λ
max
= 546 nm. Product possessing both
wavelengths was collected, retention time 16.6 min (Figure C14). Solvent was then removed
and concentration determined by UV-vis (ε = 150,000, λ
max
= 550 nm) to be 855 μg. Converted
to Na
+
salt by addition of Na
2
CO
3
and lyophilized to yield pink crystals.
Excitation and emission determined on a Horiba NanoLog Spectrofluorometer System to
be λ
max
= 550 nm and λ
max
= 564 nm respectively (Figure C15).
LRMS 1375 m/z (+ mode) (Figure C16),
1
H NMR (Figure C17, C18), and
31
P NMR (Figure
C19) acquired.
3.4.7 Synthesis of 7-DEAC-aminobutyl PPCH2PdA, 8

In a conical vial the sodium salt of 6 (2 mg, 3 μmol ) was dissolved in H
2
O (150 μL). 7-
DEAC (2.5 mg, 5.6 mmol) was dissolved in anhydrous DMF (25 μL) and transferred to the vial
containing 6. Reaction was monitored by TLC using methanol as mobile phase.
Product was purified by semi-preparative C18 HPLC purification using a Beckman
Ultrasphere 5 micron particle size, 10 mm × 25 cm column. The mobile phase consisted 0.1 M
TEAB pH = 7.2 with a gradient of 20% to 70% MeOH in 30 min, flow rate 3 mL/min. Dual UV-
Vis detectors were used 1) λ
max
= 260 nm 2) λ
max
= 420 nm. Product possessing both
49

wavelengths was collected, retention time 33.0 min (Figure C20). Solvent was then removed
and concentration determined by UV-vis (ε = 48,000, λ
max
= 430 nm) to be 660 μ g. LRMS in
negative (Figure C22) and positive mode acquired (Figure C23). Converted to Na
+
salt by
addition of Na
2
CO
3
and lyophilized to yield yellow crystals.
Excitation and emission determined on a Horiba NanoLog Spectrofluorometer System to
be λ
max
= 550 nm and λ
max
= 564 nm respectively (Figure C21).
Further characterized by
1
H NMR (Figure C24 and C25) and
31
P NMR (Figure C26).

50

3.5 References
(1) Sexton, D. J.; Carver, T. E.; Berdis, A. J.; Benkovic, S. J. J. Biol. Chem. 1996, 271,
28045–28051.
(2) Alley, S. C.; Abel-Santos, E.; Benkovic, S. J. Biochemistry (Mosc.) 2000, 39, 3076–3090.
(3) Allen, D. J.; Benkovic, S. J. Biochemistry (Mosc.) 1989, 28, 9586–9593.
(4) Braslavsky, I. Proc. Natl. Acad. Sci. 2003, 100, 3960–3964.
(5) Harris, T. D.; Buzby, P. R.; Babcock, H.; Beer, E.; Bowers, J.; Braslavsky, I.; Causey, M.;
Colonell, J.; DiMeo, J.; Efcavitch, J. W.; Giladi, E.; Gill, J.; Healy, J.; Jarosz, M.; Lapen,
D.; Moulton, K.; Quake, S. R.; Steinmann, K.; Thayer, E.; Tyurina, A.; Ward, R.; Weiss,
H.; Xie, Z. Science 2008, 320, 106–109.
(6) Levene, M. J. Science 2003, 299, 682–686.
(7) Ju, J.; Kim, D. H.; Bi, L.; Meng, Q.; Bai, X.; Li, Z.; Li, X.; Marma, M. S.; Shi, S.; Wu, J.;
Edwards, J. R.; Romu, A.; Turro, N. J. Proc. Natl. Acad. Sci. 2006, 103, 19635–19640.
(8) Eid, J.; Fehr, A.; Gray, J.; Luong, K.; Lyle, J.; Otto, G.; Peluso, P.; Rank, D.; Baybayan,
P.; Bettman, B.; Bibillo, A.; Bjornson, K.; Chaudhuri, B.; Christians, F.; Cicero, R.; Clark,
S.; Dalal, R.; deWinter, A.; Dixon, J.; Foquet, M.; Gaertner, A.; Hardenbol, P.; Heiner, C.;
Hester, K.; Holden, D.; Kearns, G.; Kong, X.; Kuse, R.; Lacroix, Y.; Lin, S.; Lundquist,
P.; Ma, C.; Marks, P.; Maxham, M.; Murphy, D.; Park, I.; Pham, T.; Phillips, M.; Roy, J.;
Sebra, R.; Shen, G.; Sorenson, J.; Tomaney, A.; Travers, K.; Trulson, M.; Vieceli, J.;
Wegener, J.; Wu, D.; Yang, A.; Zaccarin, D.; Zhao, P.; Zhong, F.; Korlach, J.; Turner, S.
Science 2009, 323, 133–138.
(9) Korlach, J.; Bjornson, K. P.; Chaudhuri, B. P.; Cicero, R. L.; Flusberg, B. A.; Gray, J. J.;
Holden, D.; Saxena, R.; Wegener, J.; Turner, S. W. Methods Enzymol. 2010, 472, 431–
455.
(10) Sood, A.; Kumar, S.; Nampalli, S.; Nelson, J. R.; Macklin, J.; Fuller, C. W. J. Am. Chem.
Soc. 2005, 127, 2394–2395.
(11) Kumar, S.; Sood, A.; Wegener, J.; Finn, P. J.; Nampalli, S.; Nelson, J. R.; Sekher, A.;
Mitsis, P.; Macklin, J.; Fuller, C. W. Nucleosides Nucleotides Nucleic Acids 2005, 24,
401–408.
(12) Mulder, B. A.; Anaya, S.; Yu, P.; Lee, K. W.; Nguyen, A.; Murphy, J.; Willson, R.;
Briggs, J. M.; Gao, X.; Hardin, S. H. Nucleic Acids Res. 2005, 33, 4865–4873.
(13) 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.
Org. Lett. 2009, 11, 1883–1886.
(14) Liang, F.; Jain, N.; Hutchens, T.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; Chiarelli, M.
P.; Cho, B. P. J. Med. Chem. 2008, 51, 6460–6470.
(15) Batra, V. K.; Beard, W. A.; Shock, D. D.; Pedersen, L. C.; Wilson, S. H. Mol. Cell 2008,
30, 315–324.
(16) Batra, V. K.; Beard, W. A.; Shock, D. D.; Krahn, J. M.; Pedersen, L. C.; Wilson, S. H.
Struct. Lond. Engl. 1993 2006, 14, 757–766.
(17) He, G.; Zhang, X.; He, C.; Zhao, X.; Duan, C. Tetrahedron 2010, 66, 9762–9768.
(18) Katritzky, A.; Abdelmajeid, A.; Tala, S.; Amine, M.; Steel, P. Synthesis 2010, 2011, 83–
90.
51

(19) Jin, J. The Molecular Basis of Nucleotide Recognition for T7 DNA Polymerase; ProQuest,
2008.
(20) Kozlov, M.; Bergendahl, V.; Burgess, R.; Goldfarb, A.; Mustaev, A. Anal. Biochem. 2005,
342, 206–213.
(21) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org. Chem. 1987, 52, 1794–
1801.
(22) Mohamady, S.; Jakeman, D. L. J. Org. Chem. 2005, 70, 10588–10591.
(23) Korlach, J.; Bibillo, A.; Wegener, J.; Peluso, P.; Pham, T. T.; Park, I.; Clark, S.; Otto, G.
A.; Turner, S. W. Nucleosides Nucleotides Nucleic Acids 2008, 27, 1072–1082.
(24) Blackburn, B. M.; Langston, S. P. Tetrahedron Lett. 1991, 32, 6425–6428.
(25) Calleri, E.; Ceruti, S.; Cristalli, G.; Martini, C.; Temporini, C.; Parravicini, C.; Volpini, R.;
Daniele, S.; Caccialanza, G.; Lecca, D.; Lambertucci, C.; Trincavelli, M. L.; Marucci, G.;
Wainer, I. W.; Ranghino, G.; Fantucci, P.; Abbracchio, M. P.; Massolini, G. J. Med.
Chem. 2010, 53, 3489–3501.
(26) Khandazhinskaya, A. L.; Kukhanova, M. K.; Jasko, M. V. Russ. J. Bioorganic Chem.
2005, 31, 352–356.

52

Chapter 4
Non-enzymatic hydrolysis studies of nucleotide mimics for the evaluation of
the catalytic efficiency of polymerase β.
4.1 Introduction

Reactions involving dNTP are central to biology and are involved in energy storage and
transfer, signaling processes, and DNA/ RNA replication and repair.
1
Recently, a series of X,Y-
substituted ,-methylene bisphosphonate analogues of dGTP were used to examine the leaving
group effect on pol  catalysis and fidelity.
4,5
In addition, metabolism of bisphosphonate drugs
have been studied, to explore their stability and potential cytotoxicity.
6
As characterizing the
rate and temperature dependence of a biologically relevant reaction in the absence of a catalyst is
of fundamental theoretical interest,
2
we attempted to use modified triphosphate compounds,
similar to those studied with pol β, to determine the stability of the P
α
-O-P
β
bond in solution.
Indeed, evaluating the catalytic efficiency achieved by an enzyme requires the reaction
quantification in solution in the absence of enzyme.
3

While non-enzymatic hydrolysis studies of ATP and other phosphoanhyrides have
characterized the solution stability and activation parameters of P
γ
solvolysis,
3,4
extending these
studies to the P
α
-O-P
β
moiety is inherently obscured by the initial cleavage at P
γ
and nucleoside
decomposition.
5,6
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
.

2, 6
Here, we present hydrolysis kinetics for a series triphosphate model
compounds that similarly block P
γ
hydrolysis but also mimic the native bridging oxygen
7
while
53

eliminating competing side reactions and potential catalysis from the sugar or nucleobase
(Figure 4.1).

Figure 4.1. Nucleotide mimics for the non-enzymatic hydrolysis of Pα.

Model compounds incorporating a fluorinated bisphosphonate moiety (2, 3, and 5) probe
whether having a better leaving group results in detectable hydrolysis through nucleophilic attack
at P
α
.
3–5
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
.
When considered in the context of the corresponding enzymatic reactions, this data sets the
upper-limit for the rate of triphosphate hydrolysis by nucleophilic attack at P
α
, and allows for
evaluation of catalytic reactions directed to P
β
.
4.2 Results and Discussion

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,
2,6
C-O ester bond cleavage,
7
and any possible catalysis from nitrogen
54

moieties.
8
The neopentyl ester is a common surrogate used in phosphate ester hydrolysis studies
that meets these criteria and does not significantly impact nucleophilic attack at the phosphorus
center.
9,10
Additionally, the bulky neopentyl group (C(CH
3
)
3
) disfavors alternative attack at the
ester carbon and still resemble the neighboring ribose while as an added bonus provides a robust
and uncomplicated
1
H NMR spectrum useful for monitoring reaction progress (Figure D1).
Together these models span a wide range of electronic properties with pK
a4
values spanning
nearly 3 pK
a
units (with CF
2
7.8, CHF 9.0, CH
2
10.4)
5,11
and might allow for potential leaving
group effects to emerge. The non-hydrolyzable CHF bisphosphonate (pK
a
of 9.0) mimics the
acidity of the natural pyrophosphate (pK
a
of 8.9).
Synthesis of 1-3 was accomplished by coupling the requisite bisphosphonate with the
neopentyl monophosphate-N-methylimidazolide generated in situ from neopentyl trifluoracyl
phophate
12
. Neopentyl monophsphate 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
13
and acidification on Dowex ™ cation exchange resin ( Scheme 4.1).
Following the coupling reaction, phosphate-containing compounds were precipitated from
ethanol with sodium iodide and then purified by semi-preperative HPLC equipped with a post-
column derivitazation of the eluent with a phosphate selective fluorescent chemosensor.
14

Scheme 4.1. Synthesis of neopentyl triphosphate analogs.

55

The findings from the hydrolysis studies of 1-3 in isotopically labeled water (H
2
18
O)
showed that nucleophilic attack occurred exclusively via breakage of the P
β
-O anhydride bond
(Figure 4.2), and that 1(X,Y=H) was significantly less stable than 3 (X,Y=F).

Figure 4.2. Non-enzymatic nucleophilic site of attack of labeled
18
O.
Next, we tested how much higher the free energy of activation for P
α
might be and what
modification 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
, decreased the charge density on the P
γ

oxygens, and reduced the electron density on the P
γ
oxygens. 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 the compounds possessing a longer phosphate
anhydride bond than the complementary phosphonate bridging oxygen bond (Figure 4.3).
Additionally, a potential advantage of removing the P
γ
phosphonate is that it allows for the
incorporation of phosphonate units with lower pK
a
values than neopentyl phosphate. Ultimately,
we incorporated a phenyl ring into the second generation model compounds 4 and 5, as this
moiety was consistent with all of the above considerations and provided a weak chromophore
that is useful for detection during purification. Benzyl phosponic acid has a pK
a2
value of 7.5
56

(lower than difluorobisphosphonic acid; pK
a4
= 7.8)
11
and fluorinated benzyl phosphonic
acidsnwill certainly have a pK
a2
value lower than that of the monoalkyl phosphates.

Figure 4.3. Computational modeling showing the bond lengths of P
α
-O and P
β
-O for model
compounds 1-5.
Compounds 4 and 5 were prepared by coupling the requisite benzyl phosphonic acid with
neopentyl phosphate using the same coupling chemistry employed in the synthesis of 1-3 and
purified by RP C-18 HPLC.
For each analog 1-3 the hydrolysis data points were obtained for 0.5, 1, 1.5, 2, 2.5, and 3
half-lives at 4 different temperatures in triplicate. Hydrolysis reactions were also carried out in
18
O-enriched H
2
O for determination of the site of nucleophilic attack. LRMS revealed that
57

hydrolysis proceeded with attack on the P
β
and with the CXY moiety with the lowest pKa4
hydrolyzing the slowest a complete reversal of what is observed with pol β which cleaves the P
α
.
Kinetic runs were conducted by dissolving a stock solution (1.2-5.0 mM for CH
2
, 0.8-4.2
mM for CHF, and 0.84-4.0 mM for CF
2
) in 0.2 M KOH in PTFE lined stainless steel reactors.
After a reaction time of 0.5, 1, 1.5, 2, 2.5, or 3 half-lives at a particular temperature the reaction
mixture was allowed to cool to RT and 100 μL of the reaction mixture was combined with 600
μL of NpOH standard in D
2
O. The
1
H NMR spectrum was acquired on a Varian 400 MR using a
PRESAT pulse sequence, a saturation delay of 12s, a 90° pulse width, and 64 scans.
Quantification of starting material and reaction product was obtained by integration of the
neopentyl group methyl resonance (Figure 4.5).

Figure 4.5. Representative analytical data showing reaction progress (1, 0.2 M KOH, 110 °C) as
monitored by
1
H NMR (left) and the best fit to first order reaction kinetics (right). A: 1, B: NpP,
C: NpOH, open symbols: [NpPPCH
2
P]/[NpPPCH
2
P]
0
, and full symbols: 1-
[NpP]/[NpPOPCH
2
P]
0
.

58

We determined hydrolysis reaction rate, nucleophile attack site, and investigated the
potential change in mechanism by modifying the leaving group at pH where the triphosphate
monoester is completely deprotonated. The resulting linear and parallel Arrenhius plots (Figure
4.6) were used to determine the reaction activation parameters and were extrapolated to estimate
the rate constant at 25° C (Table 4.1).

Figure 4.6. Arrhenius plot of the for ,--substituted triphosphate neopentyl ester hydrolysis in
0.2 M KOH rate constant (s
-1
) logarithm as a function of reciprocal temperature (K
-1
). Error bars
are three standard deviations of three replicates.

59

CH
2
(1) CHF (2) CF
2
(3) ATP(4-) (P
γ
)
3
MeP(-1)
5
Np
2
P
-
(H
2
O)
5

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

Table 4.1 ,-Substituted Neopentyl Triphosphates Hydrolysis Reaction Activation Parameters

(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.)

Comparison of the rate constants obtained in this study with that of natural ATP
4-
, in
which 1.5% of the reaction was shown to proceed through P
β
attack,
3
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. Comparison
with other classes of phosphates shows that the rate of hydrolysis of ,-CXY-triphosphate
tetraanions is significantly faster than that of monomethyl phosphate dianion and the dineopentyl
phosphate monoanion; and is faster than, but comparable to, the rate of monoester monoanion
hydrolysis.
5
The negative entropies of activation for 1-3 are less than those generally reported
for phosphate monoester monoanions but more than the values obtained for phosphate di- or tri-
esters. The enthalpies are similar to those observed for both mono- and diester monoanions.
18
O-labeled hydrolysis studies were conducted in 50:50 H
2
18
O. The resulting products
were exchanged on DOWEX to the corresponding acids, dissolved in methanol and analyzed by
LRMS in negative mode. For compounds 1-3, there was no detectable
18
O-substituted neopentyl
phosphate indicating that the reaction proceeds with attack on the P
β
completely (Figure 4.7).
60

O
18
Hydrolysis Mass Spectra

Figure 4.7 LRMS of
18
O hydrolysis of 1-3 indicating that site of attack was exclusively at P
β.

In an attempt to have nucleophilic attack on the P
α
a second generation model compounds
4 and 5 were synthesized. These compounds not only increase the length of the P
α
-O bond but in
the case of phenyl CF
2
provide a much more stable leaving group.
The hydrolysis of 1-6 was conducted in 0.2 M KOH solution. The reaction mixtures were
heated in PTFE liners sealed in stainless steel vessels
10
for various time intervals the in a
vigorously circulating oil bath with precision temperature control. Reaction mixtures were
analyzed by proton NMR after 7-fold dilution with D
2
O, to which neopentanol was added as an
integration standard (CH
3
(s), 0.734 ppm, CH
2
(s), 3.13 ppm) for experiments pertaining to 1-3.
The only hydrolysis reaction products detected by
1
H NMR were neopentyl monophosphate
(CH
3
(s), 0.758 ppm, CH
2
(d), 3.26 ppm) and the corresponding substituted bisphosphonate
61

(CH
2
(t), 1.74 ppm, CHF(dt,
2
J
PH
= 12 Hz,
2
J
FH
= 45 Hz) 4.42 ppm). The reaction progress was
followed up to 87% conversion. Both the disappearance of the methyl signal (0.788 ppm) in
neopentyl triphosphate, and appearance of the methyl signal (0.758 ppm) in neopentyl
monophosphate were quantified. Changes in substrate and product both proceeded with first-
order kinetics.
Negative mode ionization MS of the hydrolysis reaction products in 0.2 M KOH in
18
O-
enriched water (50% enrichment) shows that all three triphosphates bridging groups react via the
nucleophilic attack at P

. This observation is consistent with the decreased electron density
associated with the P

compared to P
a
as reflected in the
31
P NMR chemical shifts
11
and in
agreement with previous report for ,-CMeOH and ,-CH
2
substituted nucleoside
triphosphates at pH 8.
6
Hydrolysis experiments conducted in solvent mixtures ruled out the
formation of metaphosphonate-like intermediate. There was no evidence for a change in the
attack site of the triphosphate despite the wide range in pKa values for the leaving groups used in
this study. The possibility that bifurcated LFER for pol  observed
5
originated from a change in
mechanism of the reaction in solution has been ruled out.
The substitution at the triphosphate neopentyl ester takes place at the P

site and no
change is observed as the leaving group pKa-value change. Polymerases catalyze reactions with
attack on the P
a
of the incoming dNTP or NTPs. The attack on the model system is different
from the attack at P

by the 3’ -OH of the primer ribose ring in pol , resulting in dNTP
insertion.
14
We estimate based on the fact that no
18
O substituted neopentyl phosphate ester was
observed in our experiments that the free energy of the reaction at P
a
is at least 3 kcal higher.
Our hydrolysis reaction parameters thus define a lower limit for the attack at P
a
in solution.
62

Combined these facts suggest that the preorganized complex is important to the reaction in pol 
and may be key to the fidelity of these enzymes.
Though not as prevalent as enzymatic reactions occurring at P
γ
or P
α
, several
diphosphokinase
13
and Nudix hydrolase enzymes are known to catalyze reactions at P
β
of a
triphosphate. Of these at least 2 bacterial Nudix hydrolases, MutT
14,15
and Orf17,
16,17
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. Nudix
hydrolases provide moderate rate enhancements
2
over of the uncatalyzed reactions.
4.3 Conclusion

It was discovered that the mechanism for solution hydrolysis of triphosphate analogs 1-3
proceeds through a different mechanism than reactions catalyzed by pol β . That is, in solution,
the preferred site of nucleophilic attack is the P
β
rather than the P
α
as would be anticipated
through extension of DNA base incorporation reaction. Additionally, the effect of substituents
on the bisphosphonic acid is the reverse of what is observed when the attack is directed to the P
α
.
In this case, the substituents with greater electronegativity are hydrolyzed more slowly.
Furthermore, it was demonstrated that adjusting the Pα-O

bond length and altering leaving group
pK
a
to essentially yield a better leaving group can transfer the site of nucleophilic attack to P
α.

However, in doing so high temperature is required causing P-C bond cleavage.
By comparing the temperature and amount of hydrolysis between the 1-3 analogues and
those of 4-5 an interesting trend was observed. As the bond length of P
α
-OP
β
decreased by 0.03
Å an increase in temperature was required to achieve the same amount of hydrolysis (roughly
63

50%) at the same incubation time (30 min). This of course is attributed to having a shorter
(stronger) P
α
-OP
β
bond and thus requiring more energy to break.
An exception to this trend was found for 5 which although possessing the shortest
(strongest) P
α
-OP
β
bond underwent hydrolysis at a much faster rate. A possible explanation to
this deviation in trend is that 5 has a better leaving group BnzCF
2
, with a 1 pK
a
difference
compared to NpPO
4
. It was reasonable to conclude that with a better leaving group and a
difference in rate, 5 was undergoing a different mechanism compared to the other analogues.
Hydrolysis studies using labeled
18
O water showed that 5 had underwent some percentage
of P
α
attack which hadn’t been observed with 1-3 analogues. However, the site of attack was not
exclusive; in fact more P
β
nucleophilic attack was observed compared to P
α
. An explanation to
this is that at those elevated temperatures C-P
α
bond breakage is occurring as the anion is
stabilized by the benzyl group. To probe into the ease of having P
α
attack another analogue was
synthesized 6. The reasoning behind this compound was that now both phosphate groups would
be identical and thus considered P
α
.
There were few mentions of the synthesis of 6 in literature.
1,2
We devised two methods
for the synthesis of 6 and purified each by preparative HPLC using C18. Previously the 1-3
analogues had been purified by an elaborate HPLC method using a post derivatization pump and
a phosphate binding chromophore. In order to facilitate the purification step a conductivity
detector was implemented. Lastly the desired compound was converted to Na
+
salt form through
Dowex.
Hydrolysis studies on 6 showed that nucleophilic attack on P
α
is achieved but high
temperature is required. At 200
o
C (30 min) only about 6-9% of hydrolysis occurs. Interestingly
64

this is also close to the percentage of P
α
attack that is observed with labeled
18
O water on 5 by
MS.
Longer incubation time at 200
o
C (180 min) resulted in decomposition of hydrolysis
product, NpPO4 to NpOH. In order to probe what occurred first, P-OC hydrolysis vs POP
hydrolysis, NpPO
4
was incubated for 180 min at 200
o
C. The observation is that NpPO
4
quickly
decomposes to NpOH and PO
4
. An explanation is that NpPO
4
as a monoester dianion quickly
undergoes metaphosphate formation to yield NpOH and PO
4
. On the other hand 6 first goes
POP hydrolysis and in turn the hydrolysis product undergoes decomposition.
1
H and
31
P NMR
of the 180 min incubation of 6 clearly shows more NpOH and PO
4
formation compared to the
initial hydrolysis product in support of the previously mentioned mechanism.
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 monesters. For compounds 1-3 hydrolysis
occurred exclusively through nucleophilic attack at P
β
. The activation parameters have been
extrapolated to 25 °C and thus establish a reference for the estimation of the catalytic efficiency
of enzymes that catalyze reactions at P
β
while establishing the upper-limit for the rate of
hydrolysis through attack at P
α
. These results show that increasing the basicity of the
phosphonate moiety increases the rate of reaction and opens the question of whether this trend is
merely a reflection of increasing P
β
-O bond strength, or if the basicity of P
γ
plays a role in the
mechanism.

65

4.4 Experimental
4.4.1 NpPOPCH
2
P stock solution standardization.

The 53 mg of the initial compound (presumably, trisodium monohydrogen salt of
NpPOPCH
2
P, MW = 392 g/moL) were dissolved in 25.0 mL of deionized water to yield ~27
mM solution. The 100 µL aliquot was combined with 200 µL of 41.3 mM sodium acetate
standard, 400µL of D
2
O, and 70µL of 2.08 M KOH solution for quantitative
1
H NMR.The
sodium acetate standard solution was cross-checked with NpOH standard solution to ensure
integration accuracy of proton resonances. The 100 µL of 27.3 mM NpOH were combined with
200 µL of 41.3 mM sodium acetate standard, 400µL of D
2
O, and 70µL of 2.08 M KOH solution
for quantitative
1
H NMR. The integration of methyl signals of neopentyl group shows, that the
amount of NpPOPCH
2
P (as trisodium monohydrogen salt) in the solid starting material is equal
to 87.6%. This yields MW=447 g/moL for NpPOPCH
2
P. To account for the 55 g/moL
difference, we can estimate 3 molecules of water in the starting material (3·18 = 54 g/moL). The
similar procedure was applied for other two neopentyl triphosphates.
As a result, the optimal parameters for quantitative
1
H NMR were selected as follows:
standard PRESAT pulse sequence from the Varian library, saturation delay of 12 s, 90º pulse
width, ss=1, nt=64.
4.4.2 Reaction volume optimization.
The 1.0 mg of neopentyl phosphate (as monosodium, monohydrogen salt) was dissolved
in 1.5 mL of water. The 150 µL of 2.08 M KOH were added to the solution to yield the 0.19 M
concentration. The final solution was divided into three parts: (1) 100 µL were taken for further
66

analysis without any treatment, (2) 500 µL were placed into one reactor, and (3) 1000 µL were
placed into a second reactor. Both reactors were kept in the oil bath at 125 ºC for 69 minutes. 100
µL from each part were combined with 600 µL of 0.41 mM NpOH stock in D
2
O for quantitative
1
H NMR spectroscopy. The methylene and methyl resonances were integrated using NpOH as an
internal standard. The integration in 0.5 mL volume sample shows the ~11% increase for both
signals, whereas integration in the 1.0 mL sample is identical to the initial stock solution. As a
result, the minimal reaction volume required for quantitative work is 1.0 mL, and all the kinetic
runs will be run with at least 1.0 mL sample. The amount of water as saturated vapor in
equilibrium with the liquid phase as a function of temperature was also calculated for 1 mL of
water in the 23 mL closed volume. The data shown suggest, that the solution volume decrease
due to water evaporation is below 5 % at all temperatures used in this study.
4.4.3 Hydrolysis reaction stoichiometry.

Upon hydrolysis all three triphosphates yield the neopentyl phosphate and the
methylenediphosphonate, fluoromethylenediphosphonate, and difluoromethylenediphosphonate
(this derivative is still to be done), respectively. No other decomposition products, for instantce,
neopentanol were detected by
1
H NMR. The reaction stoichiometry was confirmed by
quantitative
1
H NMR, yielding the 1±0.04 moL of neopentyl phosphate and 1±0.04 moL of
corresponding methylenediphosphonate forming from 1 moL of triphosphate. Neopentanol (0.70
mM; CH
3
(s) 0.73 ppm, CH
2
(s) 3.13 ppm) was added as the internal integration standard.

67

4.4.4 Kinetic runs of NpPOPCH
2
P, NpPOPCHFP, and NpPOPCF
2
P
hydrolysis in the temperature range from 90ºC to 143ºC.

The 1.0 mL aliquots of the 1.2-5.0 mM (CH
2
), 0.8-4.2 mM (CHF), and 0.84-4.0 mM
(CF
2
) solution in 0.2 KOH were transferred into six PTFE liners, sealed inside stainless steel
reactors, and placed into the oil bath at controlled temperature. The reactors were placed in two
loads by three reactors in each load. The time difference between two loads usually was 5
seconds, which were added to the second batch to make the reaction time equal to all samples.
As it was shown previously, 15 min are required to reach the reaction mixture at the target
temperature, therefore, 15 min were subtracted from the total time reactor spent in the oil bath to
obtain pure reaction time. A reactor was removed from the oil bath, cooled down with a stream
of cold air (for ~1min), and placed into the water bath at ambient temperature under a stream of
running water to cool down to room temperature. The 100 µL of reaction mixture was combined
with 600 µL of NpOH standard in D
2
O (the standard concentration was 0.2, 0.41, and 0.82 mM
depending on the initial triphosphate concentration), and the
1
H NMR spectrum was acquired
under optimal conditions. Spectrum of the initial solution was also acquired under identical
conditions for control purposes. The amounts of starting material and reaction product were
obtained by integration of neopentyl group methyl resonances due to the best signal to noise ratio
in a spectrum.
1
H spectra of a typical run of NpPOPCH
2
P hydrolysis at 110ºC are shown in the
Figure D1.
4.4.5 NpPOPCH
2
P hydrolysis kinetics in the 0.2 M KOH.

Concentration of reactant and reaction product as a function of time in the temperature
ranged from 90 ºC to 125 ºC. Correlation R
2
values are shown next to each linear fit in Figure
68

D2. The reaction rate constants obtained in each run are summarized in the Table D1. Both
initial triphosphate (k
a
) hydrolysis and neopentyl phosphate appearance (k
p
) are shown in the
Table D1.
4.4.6 NpPOPCHFP hydrolysis kinetics in the 0.2 M KOH.

Concentration of reactant and reaction product as a function of time in the temperature
ranged from 95 ºC to 130 ºC. Correlation R
2
values are shown next to each linear fit in Figure
D3. The reaction rate constants obtained in each run are summarized in the Table D1. Both
initial triphosphate (k
a
) hydrolysis and neopentyl phosphate appearance (k
p
) are shown in the
Table D1.
4.4.7 NpPOPCF
2
P hydrolysis kinetics in the 0.2 M KOH.

Concentration of reactant and reaction product as a function of time in the temperature
ranged from 95ºC to 130ºC. Correlation R
2
values are shown next to each linear fit in Figure D4.
The reaction rate constants obtained in each run are summarized in the Table D1. Both initial
triphosphate (k
a
) hydrolysis and neopentyl phosphate appearance (k
p
) are shown in the Table D1.
4.4.8 NpPOPCH
2
P, NpPOPCHFP and NpPOPCH
2
P hydrolysis in
18
O-
enriched H
2
O.

Neopentyl triphosphates were hydrolyzed completely in the 0.2 M KOH solution in 50%:
50% H
2
18
O: H
2
O. The resulted reaction mixture was checked by
1
H NMR to ensure complete
hydrolysis. In case of NpPOPCH
2
P, the starting material was observable indicating that the
69

reaction was not complete. In case of NpPOPCHFP, no starting material could be detected by
1
H
NMR. The final mixture was exchange on strongly acidic Dowex cation exchange resin, until the
solution pH 2. The resin was filtered off, and the solution was used further diluted with methanol
and subjected to the MS. The MS spectra in negative mode of NpPOPCH
2
P and NpPOPCHFP
hydrolysis products are shown in Figures D5, D6, and D7. The molecular species can be readily
identified as
18
O-subsituted methylenediphosphonate and methylenefluorodiphosphonate. No
18
O-subsituted neopentyl phosphate could be detected in reaction mixture, indicating that the
reaction proceeds via the H
2
18
O attack at P

with subsequent P

-O bond scission. In case of
NpPOPCHFP the hydrolysis reaction was also conducted in 0.2 M NaHCO
3
(pH 9) at 143ºC for
3 hours. In the MS spectrum of reaction mixture only P

- attack products can be detected, as in
case of 0.2 M KOH reaction medium.
4.4.9 Benzyl diphosphate neopentyl, 4
2.83 mM solution of 4 made by dissolving 0.9 mg of sample with 1 mL of 0.2 M KOH.
Contents transferred to PTFE liner and placed in a stainless steel vessel. Vessel transferred to oil
bath circulator at 120
o
C. After 28 m 10 s vessel removed from oil bath 300 μL mixed with 600
μL D
2
O. No Decomposition observed by
1
H NMR.
700 μL of the same sample reheated for an additional 30 min at 143
o
C with no
decomposition observed. 500 μL of the same sample heated for an additional 30 min at 160
o
C
with no decomposition apparent.
A new batch of sample was heated for 180
o
C for 30 min and then at 200
o
C for an
additional 30 min. A
1
H NMR was taken for each temperature reading revealing that between
180-200
o
C benzyl diphosphate neopentyl begins to undergo hydrolysis (Figure D8).
70

1.1 mg of 4 dissolved in 1 mL of 0.2 M KOH and placed in an oil bath at 200
o
C for 1 h
30 m. After cation Dowex exchange MS reveals 167.2 and 171.2 (Figure D9).
Hydrolysis by MeOH was performed by dissolving 0.6 mg of 4 in 500 μL of 0.2 M KOH
and 500 μL of MeOH. After 30 min at 200
o
C and cation Dowex exchange LRMS was taken not
revealing any traces of P
α
nucleophilic attack, instead 185 m/z methylated benzyl phosphonate
an expected product of P
β
was observed (Figure D10).
Hydrolisis with H
2
18
O was attempted by dissolving 1.1 mg of 4 in 225 μL of H
2
18
O and
225 μL of 0.2 M KOH added. After 30 min at 200
o
C and cation Dowex exchange LRMS was
taken revealing peaks 167, 169, 171, and 173 m/z (Figure D11). Note that neopentyl phosphate
may be decomposing as it appears by
1
H NMR and
31
P NMR (Figure D12).
4.4.10 Benzyl difluoro diphosphate neopentyl, 5

5 was purified by HPLC using a gradient and the following conditions: Buffer A 0.1 M
TEAB pH = 7.7, Buffer B 60:40 CH
3
CN pH = 6.7. Method: 0-100% B in 20 min. C18
Phenomenex Column. Collected peaks 16-19.5 min (Figure D13).
1 mg of 5 was dissolved in H
2
O was heated at 200
o
C for 30 min and it’s
1
H NMR
compared to compound 4 under similar conditions (Figure D14).
Hydrolysis by MeOH was performed by dissolving 5 in 500 μL of 0.2 M KOH and 500
μL of MeOH. After 30 min at 200
o
C and cation Dowex exchange LRMS was taken (Figure
D15).
71

Hydrolysis with H
2
18
O was attempted by dissolving 5 in 225 μL of H
2
18
O and 225 μL of
0.2 M KOH added. After 30 min at 200
o
C and cation Dowex exchange, LRMS was taken
(Figure D16). Note 5 hydrolyzes faster than 4 thus leading to greater P-C cleavage which can be
seen by
31
P NMR (Figure D17)
4.4.11 NpPOPNp

1 g of phosphorus oxychloride was dissolved in 5 mL of dry toluene and chilled with
stirring on ice bath. To this solution 0.6 g of neopentanol was dissolved in 4 mL of toluene and
added dropwise. N
2
was bubbled in and gas was passed through a solution of NaOH.
Added 500 μL TEA then centrifuged to r emove precipitate. The supernatant was removed
and then concentrated by vacuo. 1 M NaOH added to oily reaction mixture until pH=12.
Removed solvent by vacuo once more and added MeOH. The organic layer was extracted from
the white ppt. 50 mg of NpPO
4
was then transferred into a round bottom flask and suspended in
a mixture of acetonitrile (1 mL) and triethylamine (480 μL, 3.44 mmol) cooled in ice bath.
White fumes were produced. The mixture was warmed and stirred to room temp for 15 min
resulting in a yellowish/orange solution. Volatiles were removed by vacuum.
To the yellowish syrup a solution of cold n-methlimidazole (72 μL, 0.9 mmol) in
acetonitrile (300 μL) and triethylamine (315 μL, 2.26mmol) was added dropwise while also in a
cold water bath.
Product was purified by HPLC using 0.02M TEAB, 25% CH
3
CN, pH = 6.5, and a
Beckman Ultrasphere C18 column using a conductivity detector with Range 10,000, Zero
supp/coarse: 2, Temp comp: 0 and Fine: 1(50). Flow rate was 8 mL /min and the product had a
72

retention time of 10.5 min (Figure D18).
1
H NMR and
31
P NMR (Figure D19), LRMS (Figure
D20) .
Hydrolysis of NpPOPNp attempted by dissolving 10.3 mg 1 mL 0.2 M KOH. Placed in a
vessel and heated to 200
o
C for 30min. 250 μL sample taken and mixed with 350 μL D
2
O for
NMR (Figure D21).
Another 2 mg of sample was dissolved in 1 mL 0.2 M KOH and heated at 200
o
C for 3
hours. 300 μL sample taken and mixed with 300 μL D
2
O for NMR (Figure D22).
It was concluded that P-O bond breaks and with prolonged time C-P bond is breaking as
well. At 200
o
C 30 min NpPhosphate is observed, however at 3 hours we see neopentanol peaks
(Figure D23).

73

4.5 References

(1) Westheimer, F. H. Science 1987, 235, 1173–1178.
(2) Ora, M.; L nnberg, T.; Florea-Wang, D.; innen, S.; Karpeisky, A.; L nnberg, H. J. Org.
Chem. 2008, 73, 4123–4130.
(3) Berkowitz, D. B.; Bose, M. J. Fluor. Chem. 2001, 112, 13–33.
(4) McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573–4576.
(5) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc. Chem. Commun. 1981, 1188.
(6) Wolfenden, R. Chem. Rev. 2006, 106, 3379–3396.
(7) Wolfenden, R.; Ridgway, C.; Young, G. J. Am. Chem. Soc. 1998, 120, 833–834.
(8) Kirby, A. J.; Lima, M. F.; da Silva, D.; Roussev, C. D.; Nome, F. J. Am. Chem. Soc. 2006,
128, 16944–16952.
(9) Williams, N. H.; Wyman, P. Chem. Commun. 2001, 1268–1269.
(10) Schroeder, G. K. Proc. Natl. Acad. Sci. 2006, 103, 4052–4055.
(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.
Org. Lett. 2009, 11, 1883–1886.
(12) Mohamady, S.; Jakeman, D. L. J. Org. Chem. 2005, 70, 10588–10591.
(13) Quin, L. D.; Wu, X.-P.; Sadanani, N. D.; Lukes, I.; Ionkin, A. S.; Day, R. O. J. Org.
Chem. 1994, 59, 120–129.
(14) Ojida, A.; Mito-Oka, Y.; Inoue, M.-A.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 6256–
6258.

74

Chapter 5
Probing the coordinating metals in polymerase β:
Synthesis of spin labeled dATP analogues for Electron Paramagnetic
Resonance studies
5.1 Introduction

Electron spin resonance (ESR) spectroscopy can provide information similar to that
obtained from fluorescence. Both ESR and FRET use a pair of molecular probes to measure
distances in macromolecules, however there are a number of features and advantages in ESR. In
ESR, distances are measured using a pair of chemically identical nitroxide probes, which
simplifies the labeling procedure as compared to attaching two chemically distinct fluorophores.
Nitroxides are smaller than most fluorophores, causing less structural perturbation as compared
with larger probes. Interpretation of distances measured using ESR is also facilitated by the fact
that the unpaired electron is localized to the nitroxyl group in a nitroxide, whereas relationships
between the dipole moment and the chemical structure of the label vary with the choice of
fluorophore. Fluorescence measurements also suffer from several limitations, such as
overlapping of both absorption and emission spectra of interacting molecules, weak quenching, a
long dead-time during stopped-flow experiments, and photobleaching of molecular probes.
1

ESR can be applied both for steady-state studies and for measurement of fast kinetics by means
of the stopped-flow technique.
2,3
On the other hand, single molecule FRET measurements in
biological systems are routine and established, while single-molecule detection of electron spins
is still being developed.
4

75

Nucleic acids are also usually labeled with radioactive
32
P to give sensitive hybridization
probes. However, the disadvantages of radio-labeling typically entail the intricate nature of
autoradiographic detection methods, the instability of isotopes such as
32
P, and problems with
radioactive contamination.
5
Nitroxides, such as those containing a moiety of either R=1-oxyl-
2,2,5,5-tetramethyl-3-pyrroline or R’ = 1 -oxyl-2,2,6,6-tetramethylpiperidine, are commonly used
as spin labels in studies of proteins and nucleic acids. These probes are stable radicals and
contain one unpaired electron that localizes primarily within the nitroxyl group. This unpaired
electron has a spin quantum number S = 1/2. The physics describing the behavior of this
unpaired electron in the presence of a magnetic field is analogous to that proton NMR describing
the behavior of a proton (I = ½) in the presence of a magnetic field.
4

The general strategy of site-directed spin labeling is to introduce the nitroxide probe
possessing a stable unpaired electron at a selected site in the macromolecule and interpret the
EPR spectrum of the labeled molecule in terms of structure and dynamics.
6–9
Three fundamental
types of information are obtained from the EPR spectrum of a nitroxide in a native
macromolecule: 1) the dynamics of the nitroxide; 2) the accessibility of the nitroxide to collision
with a paramagnetic reagent in solution, essentially the solvent accessibility; and 3) the distance
of the nitroxide from a second nitroxide or a bound metal ion.
10

EPR and nitroxides have been used for studying structure and dynamics of proteins and
nucleic acids
4,9
and as a sensitive tool for the study of intermediates in enzyme-catalyzed
reactions.
11,12
Although, much more limited in the study o polymerases it has nonetheless been
used to study DNA-dependent and RNA polymerase from E. coli by having the spin label
attached to N
8
of adenosine.
12

76

Our goal was to synthesize and characterize novel spin-labeled dATP analogs that: (i) act
as non-hydrolyzable pol β substrate analogs; and (ii) when assembled into pol β ternary
complexes (either right or wrong substrate), place the unpaired electron (localized at the NO of
the nitroxide moiety) at different predictable locations with respect to the substrate α -phosphorus
and consequently the metal ion binding site. We will compare nitroxide continuos-wave EPR
spectra obtained between pol β bound with Mg
2+
(EPR silent) and that with Mn
2+
(EPR active) to
determine line-broadening induced by nitroxide Mn
2+
dipolar coupling.
13
This will yield
distances between the metal ions and various reference nitroxide attachment points at the
substrate, thus revealing the metal binding site conformation. While these measurements likely
have a lower “structural” resolution due to size of t he nitroxide, they can be carried out in
aqueous solution a physiological temperature to measure spin-spin distance up to 20 Å,
14
thus
complementing crystallography and ENDOR measurements. They also open up the possibility
of future studies of dynamics, such as the transition from the open to closed complex. This could
transform our understanding of metal ion binding and its role in the pol β mechanism.
Concerns over the ability of pol β to accept substrates of spin -labeled nucleoside
triphosphates were dissipated when it was revealed for the first time the proficiency to be
accepted by DNA polymerase of eukaryotic and prokaryotic origin in literature.
15,16
Some
methods apply the nitroxide label on the triphosphate through a modification on a phosphate to a
phosphorothioate group.
17,18
In other work succinimidyl activated TEMPO was used to label
thymidine through a amino propyl linker,
5
and on the thymdinie via Sonogashira cross-coupling
reaction.
15
We propose to link the nitroxide spin label directly to the P
γ
of triphosphate
2’deoxyadenosine ( Figure 5.1).
1,19,

20
Probe 1 will be synthesized according to a procedure
elaborated for preparation of fluorescently-labeled dNTP (Chapter 3).
77

Figure 5.1 Proposed nitroxide spin label dNTP analogs.
Attachment through a rigid linker is necessary to minimize the effect of the motion of the
spin label on the spectrum and at the same time to maximize the backbone dynamics of the
biomacromolecule. However, the use of rigid linkers generates the risk of perturbating the native
structure because the label cannot adapt to the steric requirements of its environment.
15

Therefore, the two structures proposed are contrasted by having the spin label coupled to flexible
linker (1) and a non-flexible spin labeled dNTP analog (2).
5.2 Results and Discussion

Polymerase and substrate conformational changes are essential for high-fidelity DNA
synthesis. Pol β in complex with DNA has an open conformation that upon binding to the
nucleotide substrate closes around the nascent base pair with Mg
2+
.
23
Binding of the catalytic
divalent ion to the ternary DNA polymerase complex is thought to represent the final step in the
assembly of the catalytic complex and is consequently a critical determinant of replicative
fidelity. Although the role of the metals in catalysis has been described, their role in the
conformational activation that couples base pairing with catalysis is not well understood. It is of
fundamental importance to identify factors that affect metal binding and the structural,
thermodynamic and kinetic consequences. A recent NMR study on metal-induced DNA
78

polymerase conformational activation indicated that metal-binding can promote translocation of
the primer terminus base pair into the active site, and motions of polymerase subdomains that
close the active site.
22
In this study, DNA binding and catalytic activation were monitored in
solutions with NMR signals of
13
C labeled methionine residues. However, the metals were not
monitored directly. Through the synthesis of 1 and 2 the study of metal coordination movements
in the active site in relation to the phosphate atoms in the nucleotide triphosphate group are to be
analyzed. To date EPR has not been used to study pol β, and is an ideal complement to our
NMR, fluorescence, and X-ray crystallography approaches.
Electron paramagnetic resonance is a powerful tool for studying mobility changes of the
reporter groups.
21
The changes in the EPR spectra of the spin labeled analogs are functions of
the motion that the probe experiences and the polarity of the environment surrounding the probe.
These techniques are applicable in disordered systems, more sensitive than NMR measurements,
and provide more detailed information than techniques based on optical excitation.
15
High-
resolution structural studies using a variety of divalent metal cations will reveal amino acid
residues likely to be important in TS stabilization. Computational studies, using the structural
data, will investigate divalent metal cation contributions to catalysis and fidelity.
The proposed studies will focus primarily on detecting structural variation in the metal
ion binding site when right or wrong dNTP substrates are bound in the enzyme active site. This
will complement information from the crystallography, kinetics, and computational studies to
significantly advance our understanding of the role of the constitutive metal ions in the chemical
mechanism of pol β, particularly on incorporating the right vs. wrong substrates.
79

The carboxylated spin label 2,2,6,6-tetramethyl-1-oxy-piperidine-4-carboxylate (4) was
converted to the more active condensing agent 5 after reaction with N-hydroxysuccinimide
(3).
24,25
Although, literature described a temperature of 50 °C for 18 h for the synthesis of 3 we
found success in carrying out the reaction in an ice bath (Scheme 5.1). 5 was used in the
subsequent reaction without any purification (Scheme 5.2).

Scheme 5.1. Sythesis of TEMPO-hydoxysuccinimide.

Scheme 5.2. Synthesis of TEMPO-aminobutyl-dAPPCP.

80

Electron spin resonance spectroscopy was used to check whether the nitroxide radical
from 4-phosphooxy-TEMPO had been incorporated into the final product. A characteristic
signal broadening due to a partially hampered rotation of the nitroxide radical covalently bound
to the cap analogue. This is in contrast to the control spectrum of the unbound free radical,
TEMPOL, shown for comparison.
The spin-labeled cap analogue, P
1
-adenosine P
3
-(2,2,6,6-tetramethylpiperidine-N-oxyl)
triphosphate was synthesized by literature (2).
1
By coupling reaction of the intermediate
adenosine 5’ -diphosphate imidazolate (8) under aqueous condition in the presence of
manganese(II) salt with commercially available TEMPO derivative (4-hydroxy-2,2,6,6-
tetramethyl-1-piperidinyloxy phosphate ester), 2 was obtained (Scheme 5.3).

Scheme 5.3. Synthesis of P
1
-adenosine P
3
-(2,2,6,6-tetramethylpiperidine-N-oxyl) triphosphate.
81

5.3 Conclusion

In order to fully understand and exploit the results obtained with the new dNTP probes,
we had been lacking one critical tool in our program, namely a spectroscopic method directly
investigating the coordination environment of the Mg
2+
divalent cation that contributes to
activation of the leaving group for departure. X-ray crystallographic studies within our program
by the Wilson laboratory have defined the position of the metal ion, and also the second Mg
2+

which interacts with O3’ of the primer terminus within 3.4 Å of the P
α
of an incoming dNTP, in
the pre-catalytic complex. In this structure, the nucleotide-binding Mg
2+
coordinates non-
bridging oxygen’s from each phosphate (α,β,γ -tridentate) in a distorted octahedral geometry.
We also propose to synthesize novel spin-labeled dNTP probes, which will allow EPR
measurements of metal-spin label distances in solution at room temperature. We will probe pol β
metal binding site by measuring Mn
2+
induced EPR spectral broadening of a nitroxide spin label
at physiological temperature. We plan to synthesize three different nitroxide analogs of non-
hydrolizable dNTPs, locating the nitroxide label at different places in the “triphosphate” portion
of the nucleotide, which will incorporate an α,β -CXY bridging methylene group to prevent
turnover. The label is attached in probe 1 at the P
γ
through a flexible linker, and probe 2 at a
much closer distance to triangulate metal spin-spin coupling from two different distances with no
problems in radical stability. This design allows the nitroxide label to be placed within a non-
hydrolyzable dNTP analog at various known lengths and rigidity.

82

5.4 Experimental
Bruker ELEXSYS E580 X-band pulse spectrometer with two microwave excitation sources
(observation and pump, respectively). The spectrometer is fitted with a 2-mm split-ring
resonator and a liquid helium cryostat (CF935, Oxford Instruments).
5.4.1 Synthesis of 2,5-Dioxopyrrolidinyl 2,2,6,6-tetramethyl-1-oxypiperidine-4-
carboxylate (Succinimidyl ester activated TEMPO), 5
25

2, 2, 6, 6-tetramethylpiperidine-N-oxyl (4) (50 mg, 250 μmol) was dissolved with DMF
(1.00 mL) and cooled to 0 °C. While stirring N-hydroxysuccinimide (3) (28.8 mg, 250 μmol)
was added followed by dropwise addition of N,N’-dicyclohexylcarbodiimide (52.0 mg, 250
μmol). Reaction mixture was stirred in an ice bath for 4 h and allowed to reach room
temperature over the course of 18 h. Reaction mixture was a bright orange color. Monitored by
TLC (1:19 MeOH:CHCl
3
) (Figure E1). LRMS APCI probe. 298.2 m/z (Figure E2).
5.4.2 Synthesis of TEMPO-aminobutyl PPCH
2
PdA, 1

6, (2 mg, 3 μmole) was dissolved in H
2
O (300 μL) and stirred in a conical vial. Na
2
CO
3
was added adjusting the pH from 4.3 to 8.5. A solution of 5 (3 mg, 10 μmol) dissolved in DMF
(50 μL) was transferred to vial containing 6 and stirred under N
2
.
Reaction progress was monitored by placing 5 μL of reaction mixture into eppendorf vial
containing DOWEX® 50Wx8 (NH
4
+
) along with H
2
O (100 μL) and MeOH (100 μL),
centrifuged, filtered, and run through LRMS (ESI) m/z 741.2 (- mode) (Figure E3).
83

Product was purified by preparative C18 HPLC purification using a Phenomenex Luna 5
micron particle size 250 x 21.20 mm column. The mobile phase consisted 0.1 M TEAB pH =
8.0 with a gradient of 10% CH
3
CN to 70% CH
3
CN in 40 min, flow rate 7 mL/min. UV-Vis
detectors set at λ
max
= 260 nm, retention time 21.2 min. Solvent was then removed and
concentration determined by UV-vis (ε = 15,300, λ
max
= 258 nm) to be 2 mg of 5. EPR signal
acquired (Figure E4).
5.4.3 Synthesis of P
γ
-(TEMPO) α,β-CH
2
-adenosine triphosphate, 2
1

In a round bottom flask 7 (10 mg, 25 μmol) was dissolved with DMF (500 μL) and
stirred. CDI (16.3 mg, 100 μmol) dissolved DMF (200 μL) was subsequently added an d stirred.
After 12 h, MeOH (4 μL) was added to quench unreacted CDI. Solvent was removed by vacuo
and filtered through cotton wool.
0.2 M of N-ethylmorphonline/HCl buffer (10 mL, pH = 6.7) was prepared by titrating N-
ethylmorpholine (250 μL) with HCl and then diluting to 10 mL with H
2
O. 400 μL of buffer
placed in round bottom flask containing 8. Lastly, 4-phosphonoxy-2,2,6,6-tetramethyl-1-
piperidinyloxy hydrate (5 mg) added followed by manganese sulfate hydrate (13 mg) stirred
under N2 at room temperature.
1

HPLC purification: 0.1M TEAB 10% CH
3
CN pH = 7.2 Buffer B 0.1M TEAB 50%
CH
3
CN pH = 7.5 Gradient 0-100 in 30 min. lambda 260 nm. Phenomenex C18 column. Flow
Rate 8 mL/min. Retention time 10.6 min. Followed by HPLC utilizing a SAX Column: Flow rate
8 ml/min. Buffer A 100 % H
2
O. Buffer B 15% CH
3
CN 0.5 M TEAB pH = 8. Gradient 0-100%
B in 40 min retention time 21.2 min 112 μg acquired.
84

5.5 References
(1) Stepinski, J.; Wojcik, J.; Sienkiewicz, A.; Niedzwiecka, A. J. Phys. Condens. Matter
2007, 19, 285202.
(2) Sienkiewicz, A.; da Costa Ferreira, A. M.; Danner, B.; Scholes, C. P. J. Magn. Reson. San
Diego Calif 1997 1999, 136, 137–142.
(3) Qu, K.; Vaughn, J. L.; Sienkiewicz, A.; Scholes, C. P.; Fetrow, J. S. Biochemistry (Mosc.)
1997, 36, 2884–2897.
(4) Sowa, G. Z.; Qin, P. Z. In Progress in Nucleic Acid Research and Molecular Biology;
Elsevier, 2008; Vol. 82, pp. 147–197.
(5) Pauly, G. T.; Bobst, E. V.; Bruckman, D.; Bobst, A. M. Helv. Chim. Acta 1989, 72, 110–
116.
(6) Hubbell, W. L.; Altenbach, C. Curr. Opin. Struct. Biol. 1994, 4, 566–573.
(7) Hubbell, W. L.; Mchaourab, H. S.; Altenbach, C.; Lietzow, M. A. Struct. Lond. Engl.
1993 1996, 4, 779–783.
(8) Hubbell, W. L.; Gross, A.; Langen, R.; Lietzow, M. A. Curr. Opin. Struct. Biol. 1998, 8,
649–656.
(9) Hubbell, W. L.; Cafiso, D. S.; Altenbach, C. Nat. Struct. Biol. 2000, 7, 735–739.
(10) Qin, P. Z.; Butcher, S. E.; Feigon, J.; Hubbell, W. L. Biochemistry (Mosc.) 2001, 40,
6929–6936.
(11) Johnston, L. S.; Neuhaus, F. C. Biochemistry (Mosc.) 1977, 16, 1251–1257.
(12) Tyagi, S. C. J. Biol. Chem. 1991, 266, 17936–17940.
(13) Taylor, J. S.; Leigh, J. S.; Cohn, M. Proc. Natl. Acad. Sci. U. S. A. 1969, 64, 219–226.
(14) Altenbach, C.; Oh, K. J.; Trabanino, R. J.; Hideg, K.; Hubbell, W. L. Biochemistry
(Mosc.) 2001, 40, 15471–15482.
(15) Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Angew. Chem. Int. Ed. 2008, 47, 6782–
6785.
(16) Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Nucleic Acids Symp. Ser. 2008, 52, 373–
374.
(17) Grant, G. P. G.; Qin, P. Z. Nucleic Acids Res. 2007, 35, e77.
(18) Koteiche, H. A.; Narasimhan, C.; Runquist, J. A.; Miziorko, H. M. Biochemistry (Mosc.)
1995, 34, 15068–15074.
(19) Kedik, S. A.; Zhdanov, R. I. Bull. Acad. Sci. USSR Div. Chem. Sci. 1978, 27, 1477–1477.
(20) Shelke, S. A.; Sigurdsson, S. T. Eur. J. Org. Chem. 2012, 2012, 2291–2301.
(21) Hoppe, J.; Wagner, K. G. Eur. J. Biochem. 1974, 48, 519–525.
(22) Kirby, T. W.; DeRose, E. F.; Cavanaugh, N. A.; Beard, W. A.; Shock, D. D.; Mueller, G.
A.; Wilson, S. H.; London, R. E. Nucleic Acids Res. 2011, 40, 2974–2983.
(23) Beard, W. A.; Wilson, S. H. Chem. Rev. 2006, 106, 361–382.
(24) Heitzmann, H.; Richards, F. M. Proc. Natl. Acad. Sci. U. S. A. 1974, 71, 3537–3541.
(25) Toppin, C. R.; Pauly, G. T.; Devanesan, P.; Kryak, D.; Bobst, A. M. Helv. Chim. Acta
1986, 69, 345–349.

85

Bibliography
Allen, D. J.; Benkovic, S. J. Biochemistry (Mosc.) 1989, 28, 9586–9593.
Alley, S. C.; Abel-Santos, E.; Benkovic, S. J. Biochemistry (Mosc.) 2000, 39, 3076–3090.
Altenbach, C.; Oh, K. J.; Trabanino, R. J.; Hideg, K.; Hubbell, W. L. Biochemistry (Mosc.) 2001,
40, 15471–15482.
A, M. R.; S, S.; R, T. D.; W, W. C. Kinetics and mechanism of DNA repair. Preparation,
purification and some properties of caged dideoxynucleoside triphosphates.
http://www.biochemj.org/bj/266/bj2660885.htm (accessed Jul 18, 2013).
Armarego, W. L. F.; Perrin, D. D. Purification of laboratory chemicals; Butterworth Heinemann,
1997.
apelqvist, T.; Wensbo, D. Tetrahedron Lett. 1996, 37, 1471–1472
Beard, W. A.; Wilson, S. H. Chem. Rev. 2006, 106, 361–382.
Barakat, K. H.; Gajewski, M. M.; Tuszynski, J. A. Drug Discov. Today 2012, 17, 913–920.
Batra, V. K.; Beard, W. A.; Shock, D. D.; Krahn, J. M.; Pedersen, L. C.; Wilson, S. H. Struct.
Lond. Engl. 1993 2006, 14, 757–766
Batra, V. K.; Beard, W. A.; Shock, D. D.; Pedersen, L. C.; Wilson, S. H. Mol. Cell 2008, 30,
315–324.
Berkowitz, D. B.; Bose, M. J. Fluor. Chem. 2001, 112, 13–33
Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc. Chem. Commun. 1981, 930.
Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc. Chem. Commun. 1981, 1188.
Blackburn, B. M.; Langston, S. P. Tetrahedron Lett. 1991, 32, 6425–6428.
86

Braslavsky, I. Proc. Natl. Acad. Sci. 2003, 100, 3960–3964.
Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R. M. J. Fluor. Chem. 1982, 20,
617–626.
Calleri, E.; Ceruti, S.; Cristalli, G.; Martini, C.; Temporini, C.; Parravicini, C.; Volpini, R.;
Daniele, S.; Caccialanza, G.; Lecca, D.; Lambertucci, C.; Trincavelli, M. L.; Marucci, G.;
Wainer, I. W.; Ranghino, G.; Fantucci, P.; Abbracchio, M. P.; Massolini, G. J. Med. Chem.
2010, 53, 3489–3501.
Carrick, J. M.; Kashemirov, B. A.; McKenna, C. E. Tetrahedron 2000, 56, 2391–2396.
Chaulk, S. G.; MacMillan, A. M. Nat. Protoc. 2007, 2, 1052–1058
Ciuffreda, P.; Casati, S.; Manzocchi, A. Magn. Reson. Chem. 2007, 45, 781–784.
Claessens, R. A. M. J.; van der Linden, J. G. M. J. Inorg. Biochem. 1984, 21, 73–82.
Corrie, J. E. T. J. Label. Compd. Radiopharm. 1996, 38, 403–410.
Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org. Chem. 1987, 52, 1794–1801.
Deluchat, V.; Serpaud, B.; Caullet, C.; Bollinger, J.-C. Phosphorus Sulfur Silicon Relat. Elem.
1995, 104, 81–92.
De Napoli, L.; Messere, A.; Montesarchio, D.; Piccialli, G.; Santacroce, C.; Varra, M. J. Chem.
Soc. [Perkin 1] 1994, 923.
Dietsch, P.; Guenther, T.; Roehnelt, M. J. Biosci. 1979, 31C, 661–663.
Eid, J.; Fehr, A.; Gray, J.; Luong, K.; Lyle, J.; Otto, G.; Peluso, P.; Rank, D.; Baybayan, P.;
Bettman, B.; Bibillo, A.; Bjornson, K.; Chaudhuri, B.; Christians, F.; Cicero, R.; Clark, S.; Dalal,
R.; deWinter, A.; Dixon, J.; Foquet, M.; Gaertner, A.; Hardenbol, P.; Heiner, C.; Hester, K.;
Holden, D.; Kearns, G.; Kong, X.; Kuse, R.; Lacroix, Y.; Lin, S.; Lundquist, P.; Ma, C.; Marks,
P.; Maxham, M.; Murphy, D.; Park, I.; Pham, T.; Phillips, M.; Roy, J.; Sebra, R.; Shen, G.;
Sorenson, J.; Tomaney, A.; Travers, K.; Trulson, M.; Vieceli, J.; Wegener, J.; Wu, D.; Yang, A.;
Zaccarin, D.; Zhao, P.; Zhong, F.; Korlach, J.; Turner, S. Science 2009, 323, 133–138.
87

Francom, P.; Janeba, Z.; Shibuya, S.; Robins, M. J. J. Org. Chem. 2002, 67, 6788–6796.
Francom, P.; Robins, M. J. J. Org. Chem. 2003, 68, 666–669.
Friedberg, E. C.; DNA repair and mutagenesis; 2nd ed.; ASM Press: Washington, D.C, 2006.
Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739–1753.
Gans, P. Talanta 2000, 51, 33–37.
Geissler, D.; Kresse, W.; Wiesner, B.; Bendig, J.; Kettenmann, H.; Hagen, V. Chembiochem Eur.
J. Chem. Biol. 2003, 4, 162–170
Grabenstetter, R. J.; Quimby, O. T.; Flautt, T. J. J. Phys. Chem. 1967, 71, 4194–4202
Grant, G. P. G.; Qin, P. Z. Nucleic Acids Res. 2007, 35, e77
Harris, T. D.; Buzby, P. R.; Babcock, H.; Beer, E.; Bowers, J.; Braslavsky, I.; Causey, M.;
Colonell, J.; DiMeo, J.; Efcavitch, J. W.; Giladi, E.; Gill, J.; Healy, J.; Jarosz, M.; Lapen, D.;
Moulton, K.; Quake, S. R.; Steinmann, K.; Thayer, E.; Tyurina, A.; Ward, R.; Weiss, H.; Xie, Z.
Science 2008, 320, 106–109.
He, G.; Zhang, X.; He, C.; Zhao, X.; Duan, C. Tetrahedron 2010, 66, 9762–9768.
Heitzmann, H.; Richards, F. M. Proc. Natl. Acad. Sci. U. S. A. 1974, 71, 3537–3541.
Hogrefe, R. I.; McCaffrey, A. P.; Borozdina, L. U.; McCampbell, E. S.; Vaghefi, M. M. Nucleic
Acids Res. 1993, 21, 4739–4741.
Hoppe, J.; Wagner, K. G. Eur. J. Biochem. 1974, 48, 519–525
Hubbell, W. L.; Altenbach, C. Curr. Opin. Struct. Biol. 1994, 4, 566–573.
Hubbell, W. L.; Mchaourab, H. S.; Altenbach, C.; Lietzow, M. A. Struct. Lond. Engl. 1993 1996,
4, 779–783.
88

Hubbell, W. L.; Gross, A.; Langen, R.; Lietzow, M. A. Curr. Opin. Struct. Biol. 1998, 8, 649–
656.
Hubbell, W. L.; Cafiso, D. S.; Altenbach, C. Nat. Struct. Biol. 2000, 7, 735–739.
Ikejiri, M.; Saijo, M.; Morikawa, S.; Fukushi, S.; Mizutani, T.; Kurane, I.; Maruyama, T. Bioorg.
Med. Chem. Lett. 2007, 17, 2470–2473.
Jencks, W. P. In Nucleophilicity; Harris, J. M.; McManus, S. P., Eds.; American Chemical
Society: Washington, DC, 1987; Vol. 215, pp. 155–167.
Jin, J. The Molecular Basis of Nucleotide Recognition for T7 DNA Polymerase; ProQuest, 2008.
Johnston, L. S.; Neuhaus, F. C. Biochemistry (Mosc.) 1977, 16, 1251–1257.
Ju, J.; Kim, D. H.; Bi, L.; Meng, Q.; Bai, X.; Li, Z.; Li, X.; Marma, M. S.; Shi, S.; Wu, J.;
Edwards, J. R.; Romu, A.; Turro, N. J. Proc. Natl. Acad. Sci. 2006, 103, 19635–19640.
Iuliano, M.; Ciavatta, L.; De Tommaso, G. J. Colloid Interface Sci. 2007, 310, 402–410.
Kabachnik, M. I.; Lastovskii, R. P.; Medved, T. Y.; Medyntsev, V. V.; Kolpakova, I. D.;
Dyatlova, N. M. Doklady Akademii Nauk SSSR 1967, 177, 582–585.
Kamerlin, S. C. L.; McKenna, C. E.; Goodman, M. F.; Goondman, M. F.; Warshel, A.
Biochemistry (Mosc.) 2009, 48, 5963–5971.
Kaplan, J. H.; Forbush, B.; Hoffman, J. F. Biochemistry (Mosc.) 1978, 17, 1929–1935.
Katritzky, A.; Abdelmajeid, A.; Tala, S.; Amine, M.; Steel, P. Synthesis 2010, 2011, 83–90.
Kedik, S. A.; Zhdanov, R. I. Bull. Acad. Sci. USSR Div. Chem. Sci. 1978, 27, 1477–1477
Khandazhinskaya, A. L.; Kukhanova, M. K.; Jasko, M. V. Russ. J. Bioorganic Chem. 2005, 31,
352–356.
89

Kirby, T. W.; DeRose, E. F.; Cavanaugh, N. A.; Beard, W. A.; Shock, D. D.; Mueller, G. A.;
Wilson, S. H.; London, R. E. Nucleic Acids Res. 2011, 40, 2974–2983.
Kirby, A. J.; Lima, M. F.; da Silva, D.; Roussev, C. D.; Nome, F. J. Am. Chem. Soc. 2006, 128,
16944–16952.
Koort, E.; Gans, P.; Herodes, K.; Pihl, V.; Leito, I. Anal. Bioanal. Chem. 2006, 385, 1124–1139.
Korlach, J.; Bibillo, A.; Wegener, J.; Peluso, P.; Pham, T. T.; Park, I.; Clark, S.; Otto, G. A.;
Turner, S. W. Nucleosides Nucleotides Nucleic Acids 2008, 27, 1072–1082.
Korlach, J.; Bjornson, K. P.; Chaudhuri, B. P.; Cicero, R. L.; Flusberg, B. A.; Gray, J. J.; Holden,
D.; Saxena, R.; Wegener, J.; Turner, S. W. Methods Enzymol. 2010, 472, 431–455.
Koteiche, H. A.; Narasimhan, C.; Runquist, J. A.; Miziorko, H. M. Biochemistry (Mosc.) 1995,
34, 15068–15074.
Kozlov, M.; Bergendahl, V.; Burgess, R.; Goldfarb, A.; Mustaev, A. Anal. Biochem. 2005, 342,
206–213.
Krock, L.; Heckel, A. Angew. Chem. Int. Ed. 2005, 44, 471–473.
Kumar, S.; Sood, A.; Wegener, J.; Finn, P. J.; Nampalli, S.; Nelson, J. R.; Sekher, A.; Mitsis, P.;
Macklin, J.; Fuller, C. W. Nucleosides Nucleotides Nucleic Acids 2005, 24, 401–408.
Lee, J. G.; Cha, H. T. Tetrahedron Lett. 1992, 33, 3167–3168.
Leswara, N. D. Alpha -fluoromethanediphosphonic Acids and Derived ATP Analogs; University
of Southern California, 1982.
Levene, M. J. Science 2003, 299, 682–686.
Liang, F.; Jain, N.; Hutchens, T.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; Chiarelli, M. P.;
Cho, B. P. J. Med. Chem. 2008, 51, 6460–6470.
Liu, J.; Janeba, Z.; Robins, M. J. Org. Lett. 2004, 6, 2917–2919.
90

Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131–133.
Mayer, G.; Kröck, L.; Mikat, V.; Engeser, M.; Heckel, A. ChemBioChem 2005, 6, 1966–1970
McCray, J. A.; Herbette, L.; Kihara, T.; Trentham, D. R. Proc. Natl. Acad. Sci. U. S. A. 1980, 77,
7237–7241
McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573–4576
Meldrum, R. A.; Chittock, R. S.; Wharton, C. W. Methods Enzymol. 1998, 291, 483–495.
Metzker, M. L.; Raghavachari, R.; Richards, S.; Jacutin, S. E.; Civitello, A.; Burgess, K.; Gibbs,
R. A. Nucleic Acids Res. 1994, 22, 4259–4267.
Metzker, M. L.; Raghavachari, R.; Burgess, K.; Gibbs, R. A. BioTechniques 1998, 25, 814–817.
Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649–658
Mohamady, S.; Jakeman, D. L. J. Org. Chem. 2005, 70, 10588–10591.
Mulder, B. A.; Anaya, S.; Yu, P.; Lee, K. W.; Nguyen, A.; Murphy, J.; Willson, R.; Briggs, J.
M.; Gao, X.; Hardin, S. H. Nucleic Acids Res. 2005, 33, 4865–4873.
Muller, C.; Even, P.; Viriot,, M.-L.; Carré, M.-C. Helv. Chim. Acta 2001, 84, 3735–3741
Nair, V.; Richardson, S. G. J. Org. Chem. 1980, 45, 3969–3974.
Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Angew. Chem. Int. Ed. 2008, 47, 6782–6785.
Obeid, S.; Yulikov, M.; Jeschke, G.; Marx, A. Nucleic Acids Symp. Ser. 2008, 52, 373–374.
Ojida, A.; Mito-Oka, Y.; Inoue, M.-A.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 6256–6258.
Ora, M.; L nnberg, T.; Florea-Wang, D.; innen, S.; Karpeisky, A.; L nnberg, H. J. Org. Chem.
2008, 73, 4123–4130.
91

Orji, C. C.; Michalczyk, R.; Silks III, L. A. "Pete J. Org. Chem. 1999, 64, 4685–4689
Pauly, G. T.; Bobst, E. V.; Bruckman, D.; Bobst, A. M. Helv. Chim. Acta 1989, 72, 110–116.
Pinheiro, A. V.; Baptista, P.; Lima, J. C. Nucleic Acids Res. 2008, 36, e90.
Popov, K.; Rönkkömäki, H.; Lajunen, L. H. J. Pure Appl. Chem. 2001, 73, 1641–1677.
Qin, P. Z.; Butcher, S. E.; Feigon, J.; Hubbell, W. L. Biochemistry (Mosc.) 2001, 40, 6929–6936.
Qu, K.; Vaughn, J. L.; Sienkiewicz, A.; Scholes, C. P.; Fetrow, J. S. Biochemistry (Mosc.) 1997,
36, 2884–2897.
Quin, L. D.; Wu, X.-P.; Sadanani, N. D.; Lukes, I.; Ionkin, A. S.; Day, R. O. J. Org. Chem. 1994,
59, 120–129.
Ravindranath, N.; Ramesh, C.; Ravinder Reddy, M.; Das, B. Adv. Synth. Catal. 2003, 345, 1207–
1208.
Robins, M. J.; Basom, G. L. Can. J. Chem. 1973, 51, 3161–3169.
Sanna, D.; Micera, G.; Bugly � , P.; Kiss, T. J. Chem. Soc. Dalton Trans. 1996, 87.
Schlichting, I.; Rapp, G.; John, J.; Wittinghofer, A.; Pai, E. F.; Goody, R. S. Proc. Natl. Acad.
Sci. 1989, 86, 7687–7690
Schlichting, I.; Almo, S. C.; Rapp, G.; Wilson, K.; Petratos, K.; Lentfer, A.; Wittinghofer, A.;
Kabsch, W.; Pai, E. F.; Petsko, G. A.; Goody, R. S. Nature 1990, 345, 309–315.
Schroeder, G. K. Proc. Natl. Acad. Sci. 2006, 103, 4052–4055.
Sexton, D. J.; Carver, T. E.; Berdis, A. J.; Benkovic, S. J. J. Biol. Chem. 1996, 271, 28045–
28051.
Shelke, S. A.; Sigurdsson, S. T. Eur. J. Org. Chem. 2012, 2012, 2291–2301
92

Sienkiewicz, A.; da Costa Ferreira, A. M.; Danner, B.; Scholes, C. P. J. Magn. Reson. San Diego
Calif 1997 1999, 136, 137–142.
Srivastava, P. C.; Robins, R. K.; Jr, R. B. M. In Chemistry of Nucleosides and Nucleotides;
Townsend, L. B., Ed.; Springer US, 1988; pp. 113–281.
Sikchi, S. A.; Hultin, P. G. J. Org. Chem. 2006, 71, 5888–5891.
Sood, A.; Kumar, S.; Nampalli, S.; Nelson, J. R.; Macklin, J.; Fuller, C. W. J. Am. Chem. Soc.
2005, 127, 2394–2395.
Sowa, G. Z.; Qin, P. Z. In Progress in Nucleic Acid Research and Molecular Biology; Elsevier,
2008; Vol. 82, pp. 147–197.
Starcevic, D.; Dalal, S.; Sweasy, J. B. Cell Cycle Georget. Tex 2004, 3, 998–1001.
Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. RNA 2001, 7, 1486–
1495.
Stepinski, J.; Wojcik, J.; Sienkiewicz, A.; Niedzwiecka, A. J. Phys. Condens. Matter 2007, 19,
285202.
Stoddard, B. L.; Koenigs, P.; Porter, N.; Petratos, K.; Petsko, G. A.; Ringe, D. Proc. Natl. Acad.
Sci. 1991, 88, 5503–5507
Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martínek, V.; Xiang, Y.; Beard,
W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florián, J.; Warshel, A.; Goodman, M.
F. Biochemistry (Mosc.) 2007, 46, 461–471.
Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S.
H.; Florián, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. Biochemistry (Mosc.) 2008, 47,
870–879.
Taylor, J. S.; Leigh, J. S.; Cohn, M. Proc. Natl. Acad. Sci. U. S. A. 1969, 64, 219–226
Toppin, C. R.; Pauly, G. T.; Devanesan, P.; Kryak, D.; Bobst, A. M. Helv. Chim. Acta 1986, 69,
345–349.
93

Tyagi, S. C. J. Biol. Chem. 1991, 266, 17936–17940
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. Org.
Lett. 2009, 11, 1883–1886.
Van der Wenden, E. M.; von Frijtag Drabbe Kuenzel, J. K.; Mathot, R. A. A.; Danhof, M.;
IJzerman, A. P.; Soudijn, W. J. Med. Chem. 1995, 38, 4000–4006.
Vaňura, P.; Jedináková -Křížová, V.; Hakenová, L.; Munesawa, Y. J. Radioanal. Nucl. Chem.
2000, 246, 689–692.
Walker, J. W.; Reid, G. P.; Trentham, D. R. Methods Enzymol. 1989, 172, 288–301.
Walker, J. W.; Reid, G. P.; McCray, J. A.; Trentham, D. R. J. Am. Chem. Soc. 1988, 110, 7170–
7177.
Westheimer, F. H. Science 1987, 235, 1173–1178.
Westmanu, E.; Stromberg, R. Nucleic Acids Res. 1994, 22, 2430–2431.
Wharton, C. W.; Meldrum, R. A.; Reason, C.; Boone, J.; Lester, W. Biochem. J. 1993, 293, 825–
828.
Williams, A. Free energy relationships in organic and bio-organic chemistry; RSC: Cambridge,
UK, 2003.
Williams, N. H.; Wyman, P. Chem. Commun. 2001, 1268–1269.
Wolfenden, R. Chem. Rev. 2006, 106, 3379–3396.
Wolfenden, R.; Ridgway, C.; Young, G. J. Am. Chem. Soc. 1998, 120, 833–834.
Wu, W.; Stupi, B. P.; Litosh, V. A.; Mansouri, D.; Farley, D.; Morris, S.; Metzker, S.; Metzker,
M. L. Nucleic Acids Res. 2007, 35, 6339–6349.
94

Wu, J.; Zhang, S.; Meng, Q.; Cao, H.; Li, Z.; Li, X.; Shi, S.; Kim, D. H.; Bi, L.; Turro, N. J.; Ju,
J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16462–16467.
Yamtich, J.; Sweasy, J. B. Biochim. Biophys. Acta BBA - Proteins Proteomics 2010, 1804, 1136–
1150.
Zollinger, H. Angew. Chem. Int. Ed. Engl. 1978, 17, 141–150.
Zoltewicz, J. A.; Clark, D. F.; Sharpless, T. W.; Grahe, G. J. Am. Chem. Soc. 1970, 92, 1741–
1750.

95

Appendix A

Figure A1. Titration curve for methylenebisphosphonic acid, 1, with KOH.
Titration of CH
2
BP
0.000
2.835
5.670
8.505
11.340
0 0.7 1.4 2.1 2.8
Volume of KOH (mL)
Trial 1 and 2: 0.0860 M
Trial 3 : 0.0870 M
Trial 4 and 5: 0.0893 M
pH
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Stock solution: 45.31 mg of 1 in 50 mL 0.1 M KCl solution; trials 1-4 performed on 10 mL (±0.01)
samples, trial 5 perfomed on 6.3 mL (±0.01) sample.

96

Table A1. Potentiometric titration data for methylenebisphosphonic acid, 1.
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
Volume KOH (mL) 1 10.36 10.35 10.25 10.50 10.52
Trial 1 and 2: 0.0860 M 2 17.32 17.33 17.32 17.38 17.44
Trial 3 : 0.0870 M 3 20.13 20.20 20.28 20.04 20.16
Trial 4 and 5: 0.0893 M pH
0 2.26 2.25 2.31 2.33 2.24
0.02 2.27 2.26 2.31 2.33 2.26
0.04 2.28 2.27 2.32 2.34 2.28
0.06 2.29 2.28 2.33 2.34 2.3
0.08 2.3 2.29 2.34 2.34 2.32
0.1 2.31 2.3 2.35 2.35 2.34
0.12 2.32 2.31 2.37 2.36 2.36
0.14 2.33 2.32 2.38 2.37 2.38
0.16 2.35 2.33 2.39 2.38 2.41
0.18 2.36 2.35 2.41 2.39 2.43
0.2 2.37 2.36 2.42 2.41 2.45
0.22 2.38 2.37 2.43 2.42 2.47
0.24 2.4 2.38 2.45 2.43 2.5
0.26 2.41 2.4 2.46 2.44 2.53
0.28 2.42 2.41 2.48 2.45 2.56
0.3 2.44 2.42 2.49 2.47 2.59
0.32 2.45 2.44 2.51 2.48 2.62
0.34 2.47 2.45 2.52 2.5 2.65
0.36 2.48 2.47 2.54 2.51 2.69
0.38 2.5 2.48 2.56 2.53 2.72
0.4 2.52 2.5 2.57 2.54 2.76
0.42 2.53 2.52 2.59 2.56 2.8
0.44 2.55 2.53 2.61 2.58 2.84
0.46 2.57 2.55 2.63 2.6 2.89
0.48 2.59 2.57 2.64 2.62 2.94
0.5 2.6 2.58 2.66 2.63 2.99
0.52 2.62 2.6 2.68 2.65 3.04
0.54 2.64 2.62 2.7 2.68 3.11
0.56 2.66 2.64 2.73 2.7 3.18
0.58 2.68 2.66 2.75 2.72 3.26
0.6 2.71 2.68 2.77 2.74 3.35
0.62 2.73 2.7 2.79 2.77 3.47
97

Volume KOH (mL)
Trial 1 and 2: 0.0860 M
Trial 3 : 0.0870 M
Trial 4 and 5: 0.0893 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
1 10.36 10.35 10.25 10.50 10.52
2 17.32 17.33 17.32 17.38 17.44
3 20.13 20.20 20.28 20.04 20.16
pH
0.64 2.75 2.72 2.82 2.79 3.61
0.66 2.78 2.74 2.84 2.82 3.81
0.68 2.8 2.76 2.87 2.85 4.15
0.7 2.83 2.79 2.9 2.87 5
0.72 2.85 2.81 2.93 2.9 5.69
0.74 2.88 2.83 2.96 2.93 6
0.76 2.91 2.86 2.99 2.96 6.2
0.78 2.94 2.88 3.02 3 6.36
0.8 2.97 2.91 3.05 3.03 6.48
0.82 3 2.94 3.09 3.07 6.6
0.84 3.04 2.97 3.13 3.11 6.71
0.86 3.08 3 3.17 3.16 6.8
0.88 3.12 3.03 3.21 3.2 6.9
0.9 3.16 3.07 3.26 3.26 7
0.92 3.21 3.1 3.31 3.31 7.11
0.94 3.25 3.14 3.36 3.38 7.22
0.96 3.31 3.18 3.42 3.45 7.35
0.98 3.37 3.22 3.49 3.53 7.49
1 3.43 3.27 3.57 3.63 7.66
1.02 3.51 3.32 3.66 3.76 7.89
1.04 3.59 3.38 3.77 3.93 8.22
1.06 3.7 3.44 3.9 4.18 8.74
1.08 3.83 3.51 4.09 4.67 9.21
1.1 4.01 3.59 4.38 5.36 9.5
1.12 4.29 3.69 4.93 5.7 9.71
1.14 4.85 3.8 5.52 5.9 9.86
1.16 5.46 3.95 5.85 6.05 10
1.18 5.78 4.17 6.06 6.17 10.12
1.2 5.99 4.56 6.21 6.28 10.22
1.22 6.13 5.22 6.33 6.37 10.32
1.24 6.25 5.64 6.44 6.45 10.41
1.26 6.35 5.88 6.53 6.52 10.49
1.28 6.44 6.05 6.62 6.59 10.58
98

Volume KOH (mL)
Trial 1 and 2: 0.0860 M
Trial 3 : 0.0870 M
Trial 4 and 5: 0.0893 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
1 10.36 10.35 10.25 10.50 10.52
2 17.32 17.33 17.32 17.38 17.44
3 20.13 20.20 20.28 20.04 20.16
pH
1.3 6.52 6.18 6.69 6.67 10.66
1.32 6.6 6.29 6.77 6.73 10.73
1.34 6.67 6.38 6.84 6.79 10.81
1.36 6.73 6.46 6.9 6.86 10.89
1.38 6.8 6.54 6.96 6.92 10.96
1.4 6.86 6.61 7.02 6.98 11.02
1.42 6.92 6.67 7.08 7.04 11.09
1.44 6.98 6.73 7.14 7.11 11.15
1.46 7.03 6.8 7.2 7.18 11.21
1.48 7.09 6.85 7.26 7.26 11.27
1.5 7.16 6.91 7.32 7.35 11.32
1.52 7.22 6.97 7.39 7.44
1.54 7.29 7.02 7.46 7.54
1.56 7.37 7.08 7.53 7.66
1.58 7.44 7.14 7.61 7.8
1.6 7.53 7.2 7.7 7.97
1.62 7.63 7.26 7.81 8.22
1.64 7.74 7.33 7.93 8.54
1.66 7.87 7.39 8.08 8.91
1.68 8.04 7.47 8.28 9.19
1.7 8.25 7.55 8.52 9.39
1.72 8.55 7.64 8.81 9.54
1.74 8.88 7.74 9.05 9.67
1.76 9.15 7.86 9.24 9.78
1.78 9.34 8.01 9.39 9.88
1.8 9.48 8.21 9.51 9.96
1.82 9.59 8.47 9.61 10.04
1.84 9.69 8.78 9.7 10.11
1.86 9.77 9.04 9.78 10.17
1.88 9.85 9.24 9.85 10.23
1.9 9.93 9.41 9.92 10.3
1.92 9.99 9.53 9.98 10.36
1.94 10.05 9.63 10.04 10.41
99

Volume KOH (mL)
Trial 1 and 2: 0.0860 M
Trial 3 : 0.0870 M
Trial 4 and 5: 0.0893 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
1 10.36 10.35 10.25 10.50 10.52
2 17.32 17.33 17.32 17.38 17.44
3 20.13 20.20 20.28 20.04 20.16
pH
1.96 10.11 9.72 10.09 10.47
1.98 10.16 9.8 10.15 10.52
2 10.21 9.87 10.2 10.57
2.02 10.26 9.94 10.25 10.62
2.04 10.31 10 10.29 10.67
2.06 10.36 10.06 10.34 10.72
2.08 10.41 10.11 10.39 10.77

100

Figure A2. Titration curve for (difluoromethylene)bis(phosphonic acid), 2, with KOH.
Titration of CF
2
BP
0.00
2.65
5.30
7.95
10.60
0 0.45 0.9 1.35 1.8
Volume of 0.0901 M KOH (mL)
pH
Trial 1 Trial 2 Trial 3

Stock solution: 44.60 mg of 2 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.

101

Table A2. Potentiometric titration data for (difluoromethylene)bis(phosphonic acid), 2.
Trial 1 Trial 2 Trial 3

Volume KOH (mL)
Trial 1-3: 0.0901 M
log betas
1 7.80 7.80 7.72
2 13.47 13.47 13.31
3 15.28 15.28 14.72
pH
0 2.21 2.2 2.15
0.02 2.21 2.21 2.16
0.04 2.23 2.22 2.17
0.06 2.24 2.23 2.18
0.08 2.24 2.24 2.19
0.1 2.26 2.25 2.21
0.12 2.27 2.26 2.22
0.14 2.28 2.27 2.23
0.16 2.29 2.28 2.24
0.18 2.3 2.3 2.26
0.2 2.31 2.31 2.27
0.22 2.33 2.32 2.28
0.24 2.34 2.34 2.3
0.26 2.36 2.35 2.31
0.28 2.37 2.37 2.33
0.3 2.39 2.38 2.34
0.32 2.4 2.39 2.36
0.34 2.42 2.41 2.37
0.36 2.43 2.43 2.39
0.38 2.45 2.44 2.41
0.4 2.47 2.46 2.43
0.42 2.49 2.48 2.45
0.44 2.51 2.5 2.47
0.46 2.53 2.52 2.49
0.48 2.56 2.55 2.51
0.5 2.58 2.57 2.54
0.52 2.6 2.6 2.56
0.54 2.63 2.63 2.59
0.56 2.66 2.65 2.62
0.58 2.69 2.69 2.65
0.6 2.72 2.72 2.68
0.62 2.76 2.76 2.72
102

Volume KOH (mL)
Trial 1-3: 0.0901 M
Trial 1 Trial 2 Trial 3
log betas
1 7.80 7.80 7.72
2 13.47 13.47 13.31
3 15.28 15.28 14.72
pH
0.64 2.8 2.8 2.76
0.66 2.84 2.84 2.8
0.68 2.89 2.89 2.84
0.7 2.94 2.95 2.9
0.72 3.01 3.01 2.96
0.74 3.08 3.08 3.02
0.76 3.16 3.17 3.1
0.78 3.26 3.28 3.2
0.8 3.38 3.41 3.31
0.82 3.56 3.6 3.47
0.84 3.81 3.88 3.69
0.86 4.14 4.22 4
0.88 4.46 4.51 4.34
0.9 4.69 4.73 4.59
0.92 4.85 4.89 4.78
0.94 4.99 5.02 4.93
0.96 5.11 5.14 5.05
0.98 5.21 5.24 5.15
1 5.31 5.33 5.25
1.02 5.39 5.41 5.34
1.04 5.47 5.5 5.43
1.06 5.55 5.57 5.51
1.08 5.63 5.65 5.59
1.1 5.71 5.74 5.67
1.12 5.79 5.81 5.75
1.14 5.87 5.9 5.83
1.16 5.96 5.98 5.91
1.18 6.05 6.08 6
1.2 6.14 6.17 6.09
1.22 6.25 6.28 6.19
1.24 6.36 6.39 6.3
1.26 6.48 6.51 6.41
1.28 6.61 6.64 6.54
103

Volume KOH (mL)
Trial 1-3: 0.0901 M
Trial 1 Trial 2 Trial 3
log betas
1 7.80 7.80 7.72
2 13.47 13.47 13.31
3 15.28 15.28 14.72
pH
1.3 6.74 6.77 6.67
1.32 6.87 6.9 6.8
1.34 6.99 7.01 6.93
1.36 7.1 7.13 7.04
1.38 7.2 7.23 7.15
1.4 7.3 7.33 7.25
1.42 7.39 7.42 7.34
1.44 7.48 7.5 7.43
1.46 7.56 7.59 7.51
1.48 7.65 7.67 7.6
1.5 7.73 7.75 7.68
1.52 7.81 7.84 7.76
1.54 7.9 7.93 7.85
1.56 7.99 8.02 7.93
1.58 8.08 8.11 8.03
1.6 8.19 8.21 8.12
1.62 8.3 8.33 8.23
1.64 8.43 8.47 8.36
1.66 8.59 8.63 8.5
1.68 8.79 8.86 8.68
1.7 9.08 9.17 8.91
1.72 9.52 9.62 9.27
1.74 9.99 9.75
1.76 10.28
1.78 10.47
1.8 10.6

104

Figure A3. Titration curve for (monofluoromethylene)bis(phosphonic acid), 3, with KOH.

Titration of CHFBP
0.000
2.728
5.455
8.183
10.910
0 0.525 1.05 1.575 2.1
Volume of KOH (mL)
Trial 1-4: 0.0893 M
Trial 5-7: 0.0927 M
pH
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7

Stock solution for trials 1-4 : 47.40 mg of 3 in 50 mL 0.1 M KCl solution; Stock solution for trials 5-7:
44.98 mg in 50 mL 0.1 M KCl; trials performed on 10 mL (±0.01) samples.

105

Table A3. Potentiometric titration data for (monofluoromethylene)bis(phosphonic acid), 3.
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
log betas
Volume KOH
(mL) 1 8.98 9.04 9.03 9.00 8.95 8.84 8.85
Trial 1-4: 0.0893 M 2 15.14 15.20 15.18 15.11 15.13 14.93 14.94
Trial 5-7: 0.0927 M 3 16.95 16.46 16.41 16.03 17.24 16.65 16.58
pH
0 2.15 2.08 2.02 2 2.2 2.13 2.11
0.02 2.16 2.09 2.03 2.02 2.21 2.15 2.13
0.04 2.17 2.1 2.04 2.03 2.22 2.16 2.15
0.06 2.17 2.11 2.06 2.04 2.23 2.17 2.16
0.08 2.18 2.12 2.07 2.05 2.24 2.18 2.17
0.1 2.19 2.13 2.08 2.07 2.25 2.2 2.18
0.12 2.2 2.14 2.09 2.08 2.26 2.21 2.19
0.14 2.21 2.15 2.1 2.09 2.27 2.22 2.21
0.16 2.22 2.16 2.11 2.1 2.28 2.23 2.22
0.18 2.23 2.17 2.12 2.11 2.29 2.24 2.23
0.2 2.24 2.18 2.14 2.13 2.3 2.26 2.24
0.22 2.25 2.2 2.15 2.14 2.32 2.27 2.26
0.24 2.27 2.21 2.16 2.15 2.33 2.28 2.27
0.26 2.28 2.22 2.18 2.17 2.34 2.3 2.28
0.28 2.29 2.24 2.19 2.18 2.35 2.31 2.3
0.3 2.31 2.25 2.21 2.2 2.37 2.33 2.32
0.32 2.32 2.26 2.22 2.21 2.38 2.34 2.33
0.34 2.34 2.28 2.24 2.23 2.4 2.36 2.34
0.36 2.35 2.29 2.25 2.24 2.41 2.38 2.36
0.38 2.36 2.31 2.27 2.26 2.43 2.39 2.38
0.4 2.38 2.32 2.28 2.28 2.45 2.41 2.4
0.42 2.4 2.34 2.3 2.29 2.47 2.43 2.42
0.44 2.42 2.36 2.32 2.31 2.48 2.45 2.44
0.46 2.43 2.38 2.34 2.32 2.5 2.47 2.46
0.48 2.45 2.39 2.36 2.34 2.52 2.49 2.48
0.5 2.47 2.41 2.38 2.36 2.54 2.51 2.5
0.52 2.49 2.43 2.4 2.39 2.56 2.54 2.52
0.54 2.51 2.45 2.42 2.41 2.59 2.56 2.55
0.56 2.54 2.48 2.44 2.43 2.61 2.59 2.57
0.58 2.56 2.5 2.47 2.45 2.64 2.62 2.6
0.6 2.59 2.52 2.49 2.48 2.67 2.65 2.63
0.62 2.62 2.55 2.52 2.51 2.7 2.68 2.66
106

Volume KOH
(mL)
Trial 1-4: 0.0893 M
Trial 5-7: 0.0927 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
log betas
1 8.98 9.04 9.03 9.00 8.95 8.84 8.85
2 15.14 15.20 15.18 15.11 15.13 14.93 14.94
3 16.95 16.46 16.41 16.03 17.24 16.65 16.58
pH
0.64 2.64 2.58 2.55 2.53 2.74 2.71 2.69
0.66 2.67 2.6 2.58 2.56 2.77 2.75 2.73
0.68 2.71 2.63 2.61 2.59 2.81 2.79 2.77
0.7 2.74 2.67 2.64 2.62 2.85 2.84 2.81
0.72 2.78 2.7 2.67 2.66 2.9 2.88 2.86
0.74 2.82 2.74 2.71 2.7 2.95 2.94 2.91
0.76 2.87 2.78 2.76 2.74 3.01 3 2.97
0.78 2.92 2.83 2.8 2.79 3.08 3.07 3.03
0.8 2.97 2.88 2.85 2.84 3.15 3.15 3.11
0.82 3.04 2.94 2.91 2.89 3.25 3.25 3.2
0.84 3.11 3 2.97 2.96 3.37 3.39 3.31
0.86 3.21 3.08 3.05 3.03 3.53 3.57 3.47
0.88 3.32 3.17 3.14 3.11 3.78 3.86 3.69
0.9 3.47 3.28 3.25 3.22 4.21 4.34 4.07
0.92 3.68 3.42 3.39 3.35 4.68 4.76 4.57
0.94 4.01 3.62 3.58 3.53 4.99 5.04 4.92
0.96 4.46 3.93 3.88 3.82 5.2 5.23 5.15
0.98 4.82 4.39 4.34 4.25 5.36 5.38 5.31
1 5.06 4.79 4.76 4.69 5.49 5.5 5.45
1.02 5.23 5.06 5.03 4.97 5.59 5.61 5.56
1.04 5.37 5.25 5.22 5.18 5.69 5.7 5.66
1.06 5.49 5.39 5.37 5.33 5.78 5.79 5.75
1.08 5.6 5.52 5.5 5.46 5.87 5.88 5.84
1.1 5.69 5.62 5.61 5.57 5.95 5.96 5.92
1.12 5.78 5.72 5.7 5.67 6.02 6.03 6
1.14 5.86 5.81 5.79 5.76 6.1 6.11 6.07
1.16 5.93 5.89 5.87 5.84 6.17 6.19 6.15
1.18 6.01 5.97 5.95 5.92 6.25 6.27 6.23
1.2 6.08 6.04 6.03 6 6.33 6.34 6.31
1.22 6.16 6.12 6.1 6.07 6.4 6.43 6.38
1.24 6.23 6.19 6.17 6.14 6.49 6.51 6.46
1.26 6.3 6.26 6.25 6.22 6.58 6.6 6.55
1.28 6.37 6.33 6.32 6.29 6.67 6.7 6.65
107

Volume KOH
(mL)
Trial 1-4: 0.0893 M
Trial 5-7: 0.0927 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
log betas
1 8.98 9.04 9.03 9.00 8.95 8.84 8.85
2 15.14 15.20 15.18 15.11 15.13 14.93 14.94
3 16.95 16.46 16.41 16.03 17.24 16.65 16.58
pH
1.3 6.45 6.41 6.39 6.36 6.78 6.82 6.75
1.32 6.53 6.48 6.47 6.44 6.9 6.95 6.87
1.34 6.62 6.56 6.55 6.52 7.03 7.09 7
1.36 6.71 6.65 6.64 6.6 7.2 7.27 7.16
1.38 6.81 6.74 6.73 6.69 7.39 7.47 7.35
1.4 6.93 6.84 6.83 6.79 7.6 7.68 7.55
1.42 7.05 6.95 6.94 6.9 7.79 7.88 7.76
1.44 7.21 7.07 7.06 7.01 7.98 8.05 7.95
1.46 7.39 7.21 7.21 7.16 8.14 8.2 8.11
1.48 7.59 7.39 7.38 7.33 8.28 8.33 8.25
1.5 7.79 7.59 7.58 7.52 8.4 8.44 8.38
1.52 7.97 7.79 7.78 7.72 8.51 8.54 8.49
1.54 8.12 7.98 7.98 7.92 8.6 8.64 8.59
1.56 8.26 8.15 8.14 8.09 8.7 8.73 8.68
1.58 8.39 8.3 8.3 8.26 8.78 8.81 8.77
1.6 8.5 8.44 8.43 8.39 8.87 8.89 8.86
1.62 8.6 8.56 8.55 8.51 8.95 8.98 8.94
1.64 8.7 8.66 8.66 8.62 9.03 9.05 9.02
1.66 8.78 8.76 8.75 8.71 9.1 9.14 9.1
1.68 8.87 8.85 8.84 8.8 9.19 9.22 9.18
1.7 8.95 8.93 8.92 8.89 9.27 9.31 9.26
1.72 9.03 9.01 9.01 8.97 9.36 9.4 9.35
1.74 9.11 9.09 9.09 9.05 9.45 9.5 9.44
1.76 9.18 9.17 9.17 9.13 9.55 9.6 9.54
1.78 9.26 9.25 9.24 9.21 9.67 9.73 9.65
1.8 9.34 9.33 9.32 9.29 9.8 9.88 9.78
1.82 9.43 9.4 9.4 9.37 9.94 10.03 9.92
1.84 9.52 9.49 9.49 9.45 10.1 10.19 10.08
1.86 9.61 9.57 9.57 9.53 10.26 10.33 10.23
1.88 9.71 9.66 9.66 9.62 10.4 10.46 10.38
1.9 9.82 9.76 9.76 9.72 10.53 10.58 10.52
1.92 9.94 9.87 9.87 9.82
1.94 10.07 9.99 9.99 9.94

108

Volume KOH
(mL)
Trial 1-4: 0.0893 M
Trial 5-7: 0.0927 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
log betas
1 8.98 9.04 9.03 9.00 8.95 8.84 8.85
2 15.14 15.20 15.18 15.11 15.13 14.93 14.94
3 16.95 16.46 16.41 16.03 17.24 16.65 16.58
pH
1.96 10.22 10.12 10.13 10.07

1.98 10.37 10.27 10.28 10.22

2 10.52 10.43 10.44 10.37

2.02 10.68 10.59 10.6 10.54

2.04 10.8 10.73 10.75 10.68

2.06 10.91

109

Figure A4. Titration curve for (dichloromethylene)bis(phosphonic acid), 4, with KOH
Titration of CCl
2
BP
0.000
2.543
5.085
7.628
10.170
0 0.395 0.79 1.185 1.58 1.975
Volume of KOH (mL)
Trial 1: 0.0919 M
Trial 2-4: 0.0889 M
pH
Trial 1 Trial 2 Trial 3 Trial 4

Stock solution: 45.20 mg of 4 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.

110

Table A4. Potentiometric titration data for (dichloromethylene)bis(phosphonic acid), 4.
Trial 1 Trial 2 Trial 3 Trial 4
log betas
Volume KOH (mL) 1 8.86 8.86 8.93 8.71
Trial 1: 0.0919 M 2 14.65 14.63 14.75 14.66
Trial 2-4: 0.0889 M 3 16.60 16.59 16.83 16.45
pH
0 2.26 2.25 2.27 2.26
0.02 2.27 2.26 2.28 2.27
0.04 2.28 2.27 2.29 2.28
0.06 2.29 2.28 2.3 2.29
0.08 2.3 2.3 2.31 2.31
0.1 2.31 2.31 2.32 2.32
0.12 2.32 2.32 2.34 2.33
0.14 2.34 2.34 2.35 2.35
0.16 2.36 2.35 2.37 2.36
0.18 2.37 2.37 2.38 2.38
0.2 2.39 2.38 2.4 2.4
0.22 2.4 2.4 2.41 2.41
0.24 2.42 2.42 2.43 2.43
0.26 2.44 2.43 2.45 2.45
0.28 2.46 2.45 2.47 2.47
0.3 2.48 2.47 2.48 2.49
0.32 2.5 2.49 2.5 2.51
0.34 2.52 2.51 2.53 2.53
0.36 2.54 2.53 2.55 2.55
0.38 2.57 2.56 2.57 2.58
0.4 2.59 2.58 2.59 2.61
0.42 2.62 2.61 2.62 2.64
0.44 2.64 2.63 2.64 2.66
0.46 2.67 2.66 2.67 2.7
0.48 2.71 2.69 2.7 2.73
0.5 2.74 2.72 2.73 2.77
0.52 2.78 2.75 2.76 2.81
0.54 2.82 2.79 2.8 2.86
0.56 2.86 2.83 2.84 2.91
0.58 2.91 2.88 2.88 2.97
0.6 2.96 2.92 2.93 3.04
111

Volume KOH (mL)
Trial 1: 0.0919 M
Trial 2-4: 0.0889 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 8.86 8.86 8.93 8.71
2 14.65 14.63 14.75 14.66
3 16.60 16.59 16.83 16.45
pH
0.62 3.03 2.98 2.98 3.12
0.64 3.1 3.04 3.04 3.21
0.66 3.18 3.11 3.11 3.34
0.68 3.29 3.2 3.19 3.51
0.7 3.42 3.3 3.3 3.75
0.72 3.6 3.43 3.42 4.15
0.74 3.89 3.62 3.59 4.55
0.76 4.28 3.9 3.85 4.82
0.78 4.64 4.29 4.23 5.02
0.8 4.87 4.64 4.6 5.17
0.82 5.05 4.88 4.85 5.3
0.84 5.19 5.05 5.04 5.41
0.86 5.31 5.2 5.19 5.52
0.88 5.42 5.32 5.31 5.62
0.9 5.52 5.43 5.42 5.71
0.92 5.61 5.52 5.52 5.8
0.94 5.7 5.62 5.61 5.9
0.96 5.79 5.7 5.7 6
0.98 5.88 5.79 5.79 6.1
1 5.97 5.88 5.88 6.21
1.02 6.07 5.97 5.97 6.34
1.04 6.17 6.07 6.06 6.5
1.06 6.28 6.17 6.16 6.69
1.08 6.42 6.27 6.27 6.95
1.1 6.57 6.4 6.39 7.3
1.12 6.76 6.54 6.53 7.7
1.14 7.02 6.72 6.71 7.98
1.16 7.38 6.97 6.95 8.18
1.18 7.75 7.3 7.29 8.33
1.2 8.01 7.68 7.67 8.46
1.22 8.19 7.96 7.95 8.58
1.24 8.34 8.16 8.16 8.68
1.26 8.47 8.32 8.31 8.77
112

Volume KOH (mL)
Trial 1: 0.0919 M
Trial 2-4: 0.0889 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 8.86 8.86 8.93 8.71
2 14.65 14.63 14.75 14.66
3 16.60 16.59 16.83 16.45
pH
1.28 8.57 8.44 8.44 8.86
1.3 8.68 8.56 8.56 8.96
1.32 8.77 8.66 8.66 9.05
1.34 8.86 8.76 8.76 9.15
1.36 8.95 8.85 8.85 9.25
1.38 9.04 8.94 8.94 9.36
1.4 9.13 9.02 9.03 9.48
1.42 9.22 9.11 9.11 9.62
1.44 9.31 9.2 9.21 9.78
1.46 9.42 9.29 9.3 9.96
1.48 9.53 9.4 9.41 10.17
1.5 9.66 9.51 9.52
1.52 9.81 9.63 9.65
1.54 9.97 9.77 9.8
1.56 10.15 9.92 9.97
1.58 10.09 10.15

113

Figure A5. Titration curve for (monochloromethylene)bis(phosphonic acid), 5, with KOH.
Titration of CHClBP
0.000
2.755
5.510
8.265
11.020
0 0.475 0.95 1.425 1.9 2.375
Volume of KOH (mL)
Trial 1: 0.0889 M
Trial 2-4: 0.0875 M
pH
Trial 1 Trial 2 Trial 3 Trial 4

Stock solution: 40.80 mg of 5 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.
114

Table A5. Potentiometric titration data for (monochloromethylene)bis(phosphonic acid), 5.
Trial 1 Trial 2 Trial 3 Trial 4
log betas
Volume KOH (mL) 1 9.54 9.52 9.61 9.65
Trial 1: 0.0889 M 2 15.86 15.83 15.91 16.00
Trial 2-4: 0.0875 M 3 17.17 17.59 16.66 16.96
pH
0 2.15 2.2 2.09 2.14
0.02 2.16 2.2 2.1 2.15
0.04 2.17 2.21 2.11 2.16
0.06 2.18 2.22 2.12 2.17
0.08 2.19 2.23 2.13 2.18
0.1 2.21 2.24 2.15 2.19
0.12 2.22 2.26 2.16 2.2
0.14 2.23 2.27 2.17 2.21
0.16 2.25 2.28 2.19 2.23
0.18 2.26 2.3 2.2 2.24
0.2 2.27 2.31 2.22 2.25
0.22 2.29 2.33 2.23 2.27
0.24 2.31 2.34 2.25 2.29
0.26 2.33 2.36 2.27 2.3
0.28 2.34 2.38 2.28 2.32
0.3 2.36 2.39 2.3 2.34
0.32 2.38 2.41 2.32 2.36
0.34 2.4 2.43 2.34 2.37
0.36 2.42 2.45 2.36 2.39
0.38 2.44 2.47 2.38 2.42
0.4 2.46 2.49 2.4 2.44
0.42 2.49 2.51 2.43 2.46
0.44 2.51 2.53 2.45 2.49
0.46 2.54 2.56 2.48 2.51
0.48 2.57 2.59 2.5 2.54
0.5 2.59 2.61 2.53 2.57
0.52 2.62 2.64 2.56 2.6
0.54 2.66 2.67 2.59 2.63
0.56 2.69 2.7 2.62 2.66
0.58 2.73 2.74 2.66 2.7
0.6 2.77 2.77 2.7 2.74
0.62 2.81 2.81 2.74 2.78
115

Volume KOH (mL)
Trial 1: 0.0889 M
Trial 2-4: 0.0875 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 9.54 9.52 9.61 9.65
2 15.86 15.83 15.91 16.00
3 17.17 17.59 16.66 16.96
pH
0.64 2.86 2.86 2.78 2.83
0.66 2.91 2.9 2.84 2.88
0.68 2.97 2.96 2.89 2.94
0.7 3.04 3.01 2.96 3.01
0.72 3.12 3.08 3.03 3.08
0.74 3.21 3.16 3.11 3.18
0.76 3.32 3.25 3.22 3.3
0.78 3.48 3.36 3.35 3.45
0.8 3.69 3.51 3.52 3.66
0.82 4.05 3.73 3.79 4.04
0.84 4.66 4.1 4.29 4.65
0.86 5.11 4.7 4.86 5.08
0.88 5.38 5.12 5.2 5.33
0.9 5.57 5.37 5.43 5.51
0.92 5.71 5.55 5.59 5.66
0.94 5.84 5.69 5.72 5.78
0.96 5.95 5.81 5.84 5.89
0.98 6.05 5.92 5.94 5.99
1 6.14 6.02 6.04 6.09
1.02 6.23 6.11 6.13 6.18
1.04 6.32 6.2 6.22 6.26
1.06 6.41 6.28 6.31 6.35
1.08 6.5 6.37 6.39 6.44
1.1 6.59 6.45 6.48 6.53
1.12 6.68 6.54 6.57 6.63
1.14 6.78 6.63 6.67 6.73
1.16 6.9 6.73 6.78 6.84
1.18 7.02 6.84 6.9 6.97
1.2 7.16 6.96 7.03 7.11
1.22 7.35 7.09 7.18 7.29
1.24 7.6 7.25 7.39 7.55
1.26 7.97 7.48 7.68 7.91
1.28 8.36 7.8 8.09 8.31
116

Volume KOH (mL)
Trial 1: 0.0889 M
Trial 2-4: 0.0875 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 9.54 9.52 9.61 9.65
2 15.86 15.83 15.91 16.00
3 17.17 17.59 16.66 16.96
pH
1.3 8.63 8.2 8.45 8.6
1.32 8.81 8.51 8.69 8.8
1.34 8.96 8.72 8.86 8.95
1.36 9.08 8.87 9 9.07
1.38 9.18 9 9.12 9.18
1.4 9.27 9.11 9.22 9.27
1.42 9.36 9.2 9.31 9.36
1.44 9.44 9.29 9.39 9.44
1.46 9.52 9.37 9.47 9.53
1.48 9.6 9.45 9.55 9.6
1.5 9.67 9.53 9.63 9.68
1.52 9.75 9.6 9.7 9.76
1.54 9.83 9.68 9.78 9.83
1.56 9.91 9.75 9.86 9.91
1.58 9.99 9.83 9.94 9.99
1.6 10.07 9.91 10.02 10.08
1.62 10.17 9.99 10.11 10.16
1.64 10.26 10.08 10.2 10.25
1.66 10.36 10.16 10.29 10.35
1.68 10.45 10.25 10.39 10.44
1.7 10.54 10.35 10.48 10.53
1.72 10.63 10.44 10.57 10.62
1.74 10.71 10.53 10.66 10.71
1.76 10.78 10.62 10.74 10.78
1.78 10.85 10.7 10.82 10.85
1.8 10.91 10.77 10.88 10.91
1.82 10.96 10.83 10.95 10.97
1.84 11.01 10.89 11 11.02
117

Figure A6. Titration curve for (dibromomethylene)bis(phosphonic acid), 6, with KOH.

Stock solution: 55.7 mg of 6 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.

0.000
2.638
5.275
7.913
10.550
0 0.375 0.75 1.125 1.5 1.875
pH
Volume of KOH (mL)
Trial 1-3: 0.0894
Titration of CBr
2
BP
118

Table A6. Potentiometric titration data for (dibromomethylene)bis(phosphonic acid), 6.
Trial 1 Trial 2 Trial 3
log betas
Volume KOH (mL) 1 9.26 9.27 9.27
Trial 1-3: 0.0894 M 2 15.14 15.14 15.14

3 17.00 16.94 16.99
pH
0 2.27 2.27 2.28
0.02 2.28 2.27 2.28
0.04 2.29 2.28 2.3
0.06 2.31 2.3 2.31
0.08 2.32 2.31 2.32
0.1 2.33 2.32 2.33
0.12 2.34 2.34 2.35
0.14 2.36 2.35 2.36
0.16 2.38 2.37 2.38
0.18 2.39 2.39 2.39
0.2 2.41 2.4 2.41
0.22 2.43 2.42 2.43
0.24 2.45 2.44 2.45
0.26 2.47 2.46 2.46
0.28 2.49 2.48 2.48
0.3 2.51 2.5 2.5
0.32 2.53 2.52 2.53
0.34 2.55 2.55 2.55
0.36 2.58 2.57 2.57
0.38 2.6 2.6 2.6
0.4 2.63 2.62 2.63
0.42 2.66 2.65 2.65
0.44 2.69 2.68 2.69
0.46 2.72 2.72 2.72
0.48 2.76 2.75 2.75
0.5 2.8 2.79 2.79
0.52 2.84 2.83 2.83
0.54 2.89 2.88 2.88
0.56 2.94 2.93 2.93
0.58 3 2.99 2.98
0.6 3.07 3.06 3.05
0.62 3.15 3.14 3.13
119

Volume KOH (mL)
Trial 1-3: 0.0894 M
Trial 1 Trial 2 Trial 3
log betas
1 9.26 9.27 9.27
2 15.14 15.14 15.14
3 17.00 16.94 16.99
pH
0.64 3.25 3.24 3.22
0.66 3.38 3.37 3.33
0.68 3.55 3.54 3.49
0.7 3.82 3.8 3.71
0.72 4.26 4.23 4.08
0.74 4.7 4.68 4.55
0.76 4.98 4.97 4.89
0.78 5.18 5.18 5.12
0.8 5.33 5.33 5.29
0.82 5.46 5.46 5.42
0.84 5.58 5.58 5.54
0.86 5.68 5.68 5.65
0.88 5.79 5.79 5.75
0.9 5.88 5.88 5.85
0.92 5.98 5.98 5.95
0.94 6.08 6.08 6.04
0.96 6.18 6.18 6.14
0.98 6.29 6.29 6.25
1 6.41 6.41 6.36
1.02 6.55 6.55 6.49
1.04 6.73 6.73 6.65
1.06 6.97 6.97 6.85
1.08 7.35 7.34 7.15
1.1 7.9 7.9 7.65
1.12 8.28 8.28 8.14
1.14 8.52 8.52 8.43
1.16 8.7 8.7 8.64
1.18 8.84 8.84 8.79
1.2 8.97 8.97 8.92
1.22 9.07 9.07 9.03
1.24 9.17 9.17 9.13
1.26 9.26 9.26 9.23
1.28 9.35 9.35 9.32
120

Volume KOH (mL)
Trial 1-3: 0.0894 M
Trial 1 Trial 2 Trial 3
log betas
1 9.26 9.27 9.27
2 15.14 15.14 15.14
3 17.00 16.94 16.99
pH
1.3 9.44 9.44 9.41
1.32 9.53 9.54 9.5
1.34 9.63 9.63 9.6
1.36 9.72 9.72 9.69
1.38 9.82 9.82 9.79
1.4 9.93 9.93 9.89
1.42 10.04 10.04 10
1.44 10.17 10.16 10.12
1.46 10.29 10.29 10.25
1.48 10.43 10.42 10.38
1.5 10.55 10.54 10.51

121

Figure A7. Titration curve for (monobromomethylene)bis(phosphonic acid), 7, with KOH.

Stock solution: 41.26 mg of 7 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.

0.000
2.828
5.655
8.483
11.310
0 0.4 0.8 1.2 1.6 2
pH
Volume 0.0893 M KOH (mL)
Titration of CHBrBP
122

Table A7. Potentiometric titration data for (monobromomethylene)bis(phosphonic acid), 7.
Trial 1 Trial 2 Trial 3
log betas
Volume KOH (mL) 1 9.92 9.90 9.91
Trial 1-3: 0.0893 M 2 16.11 16.03 16.05
3 18.57 18.27 18.35
pH
0 2.42 2.34 2.36
0.02 2.43 2.36 2.37
0.04 2.44 2.38 2.38
0.06 2.46 2.39 2.4
0.08 2.47 2.41 2.41
0.1 2.48 2.42 2.43
0.12 2.5 2.44 2.44
0.14 2.51 2.45 2.46
0.16 2.52 2.47 2.48
0.18 2.54 2.49 2.49
0.2 2.56 2.51 2.51
0.22 2.58 2.53 2.53
0.24 2.59 2.55 2.55
0.26 2.61 2.57 2.57
0.28 2.63 2.59 2.59
0.3 2.65 2.61 2.61
0.32 2.68 2.64 2.64
0.34 2.71 2.66 2.66
0.36 2.73 2.69 2.69
0.38 2.76 2.72 2.71
0.4 2.79 2.75 2.74
0.42 2.83 2.78 2.77
0.44 2.86 2.81 2.8
0.46 2.9 2.85 2.83
0.48 2.95 2.89 2.87
0.5 3 2.94 2.92
0.52 3.05 2.99 2.96
0.54 3.12 3.04 3.02
0.56 3.19 3.11 3.08
0.58 3.28 3.19 3.14
0.6 3.39 3.28 3.22
0.62 3.54 3.4 3.33
123

Volume KOH (mL)
Trial 1-3: 0.0893 M
Trial 1 Trial 2 Trial 3
log betas
1 9.92 9.90 9.91
2 16.11 16.03 16.05
3 18.57 18.27 18.35
pH
0.64 3.77 3.55 3.47
0.66 4.18 3.8 3.66
0.68 4.8 4.27 4
0.7 5.19 4.88 4.58
0.72 5.42 5.23 5.13
0.74 5.59 5.44 5.37
0.76 5.73 5.61 5.55
0.78 5.85 5.74 5.69
0.8 5.96 5.85 5.81
0.82 6.06 5.96 5.92
0.84 6.16 6.06 6.02
0.86 6.26 6.17 6.12
0.88 6.36 6.27 6.22
0.9 6.48 6.37 6.32
0.92 6.59 6.48 6.43
0.94 6.73 6.61 6.55
0.96 6.89 6.75 6.67
0.98 7.11 6.92 6.82
1 7.52 7.17 7.01
1.02 8.18 7.62 7.34
1.04 8.68 8.32 7.94
1.06 8.97 8.77 8.57
1.08 9.17 9.04 8.91
1.1 9.33 9.23 9.13
1.12 9.47 9.38 9.3
1.14 9.59 9.51 9.44
1.16 9.7 9.63 9.56
1.18 9.8 9.74 9.67
1.2 9.9 9.84 9.78
1.22 10 9.94 9.88
1.24 10.09 10.05 9.98
1.26 10.19 10.14 10.08
1.28 10.28 10.24 10.17
124

Volume KOH (mL)
Trial 1-3: 0.0893 M
Trial 1 Trial 2 Trial 3
log betas
1 9.92 9.90 9.91
2 16.11 16.03 16.05
3 18.57 18.27 18.35
pH
1.3 10.38 10.33 10.27
1.32 10.48 10.44 10.37
1.34 10.57 10.55 10.47
1.36 10.67 10.64 10.57
1.38 10.77 10.74 10.67
1.4 10.85 10.84 10.77
1.42 10.94 10.92 10.85
1.44 11.01 11 10.94
1.46 11.08 11.08 11.01
1.48 11.14 11.14 11.09
1.5 11.2 11.2 11.15
1.52 11.25 11.26 11.2
1.54 11.3 11.31 11.26
1.56 11.31

125

Figure A8. Titration curve for (1-fluoro-1,1-ethanediyl)bis(phosphonic acid), 8, with KOH.
Titration of CMeFBP
0.00
2.83
5.66
8.49
11.32
0 0.475 0.95 1.425 1.9
Volume of 0.0909 M KOH (mL)
pH
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Stock solution for trials 1-3: 40.44 mg of 8 in 50 mL 0.1 M KCl solution; Stock solution for trials 4 and 5:
40.18 mg in 50 mL 0.1 M KCl; trials performed on 10 mL (±0.01) samples.

126

Table A8. Potentiometric titration data for (1-fluoro-1,1-ethanediyl)bis(phosphonic acid), 8.
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
Volume KOH (mL) 1 10.21 10.20 10.22 10.24 10.16
0.0909 M 2 16.42 16.40 16.44 16.34 16.22
3 18.24 18.24 18.30 18.50 18.35
pH
0 2.211 2.208 2.209 2.308 2.3
0.02 2.218 2.217 2.221 2.316 2.311
0.04 2.228 2.229 2.233 2.327 2.322
0.06 2.241 2.242 2.244 2.339 2.333
0.08 2.253 2.255 2.256 2.351 2.344
0.1 2.266 2.267 2.27 2.363 2.356
0.12 2.28 2.282 2.284 2.374 2.367
0.14 2.294 2.295 2.298 2.387 2.38
0.16 2.308 2.31 2.312 2.399 2.393
0.18 2.322 2.324 2.328 2.411 2.407
0.2 2.338 2.339 2.344 2.428 2.422
0.22 2.353 2.356 2.359 2.443 2.438
0.24 2.371 2.372 2.377 2.46 2.454
0.26 2.388 2.388 2.393 2.477 2.472
0.28 2.406 2.407 2.412 2.495 2.49
0.3 2.423 2.425 2.431 2.513 2.508
0.32 2.443 2.445 2.448 2.533 2.526
0.34 2.462 2.464 2.469 2.555 2.548
0.36 2.483 2.486 2.49 2.577 2.571
0.38 2.504 2.507 2.512 2.599 2.594
0.4 2.526 2.528 2.536 2.624 2.616
0.42 2.552 2.553 2.56 2.65 2.642
0.44 2.577 2.577 2.586 2.677 2.669
0.46 2.602 2.604 2.611 2.707 2.697
0.48 2.63 2.633 2.641 2.737 2.729
0.5 2.66 2.663 2.67 2.771 2.761
0.52 2.691 2.695 2.702 2.807 2.796
0.54 2.724 2.729 2.736 2.847 2.835
0.56 2.761 2.764 2.774 2.89 2.876
0.58 2.801 2.805 2.814 2.937 2.923
0.6 2.845 2.848 2.859 2.99 2.975
0.62 2.892 2.896 2.908 3.05 3.033
127

Volume KOH (mL)
0.0909 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
1 10.21 10.20 10.22 10.24 10.16
2 16.42 16.40 16.44 16.34 16.22
3 18.24 18.24 18.30 18.50 18.35
pH
0.64 2.945 2.948 2.962 3.12 3.102
0.66 3.006 3.009 3.026 3.204 3.184
0.68 3.075 3.077 3.096 3.306 3.28
0.7 3.155 3.158 3.18 3.435 3.405
0.72 3.252 3.255 3.281 3.617 3.578
0.74 3.378 3.38 3.415 3.916 3.856
0.76 3.547 3.546 3.599 4.473 4.387
0.78 3.807 3.811 3.899 4.976 4.925
0.8 4.281 4.288 4.434 5.259 5.228
0.82 4.846 4.845 4.942 5.453 5.425
0.84 5.181 5.18 5.241 5.601 5.578
0.86 5.398 5.398 5.441 5.723 5.705
0.88 5.562 5.56 5.595 5.832 5.814
0.9 5.693 5.693 5.725 5.934 5.915
0.92 5.807 5.803 5.833 6.028 6.011
0.94 5.909 5.908 5.935 6.119 6.1
0.96 6.005 6.003 6.027 6.209 6.19
0.98 6.096 6.092 6.117 6.299 6.278
1 6.183 6.18 6.204 6.39 6.37
1.02 6.271 6.267 6.293 6.488 6.468
1.04 6.359 6.354 6.381 6.58 6.575
1.06 6.45 6.445 6.474 6.698 6.689
1.08 6.547 6.542 6.57 6.835 6.828
1.1 6.652 6.643 6.673 7.001 6.996
1.12 6.767 6.756 6.788 7.257 7.245
1.14 6.901 6.885 6.928 7.694 7.687
1.16 7.067 7.047 7.099 8.54 8.532
1.18 7.292 7.263 7.335 9.029 9.016
1.2 7.667 7.61 7.74 9.299 9.278
1.22 8.405 8.304 8.503 9.478 9.463
1.24 8.961 8.915 9.001 9.618 9.6
1.26 9.248 9.221 9.279 9.738 9.719
1.28 9.44 9.418 9.461 9.841 9.823
128

Volume KOH (mL)
0.0909 M
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
log betas
1 10.21 10.20 10.22 10.24 10.16
2 16.42 16.40 16.44 16.34 16.22
3 18.24 18.24 18.30 18.50 18.35
pH
1.3 9.586 9.569 9.607 9.934 9.917
1.32 9.71 9.694 9.727 10.021 10.004
1.34 9.815 9.803 9.836 10.104 10.086
1.36 9.909 9.898 9.927 10.181 10.163
1.38 9.995 9.986 10.013 10.255 10.237
1.4 10.076 10.065 10.092 10.327 10.306
1.42 10.149 10.142 10.168 10.394 10.375
1.44 10.222 10.213 10.238 10.46 10.441
1.46 10.294 10.286 10.312 10.527 10.509
1.48 10.362 10.355 10.379 10.59 10.572
1.5 10.429 10.423 10.447 10.655 10.635
1.52 10.495 10.49 10.514 10.714 10.696
1.54 10.561 10.555 10.579 10.775 10.758
1.56 10.624 10.618 10.643 10.831 10.812
1.58 10.686 10.68 10.704 10.887 10.868
1.6 10.745 10.742 10.763 10.939 10.922
1.62 10.805 10.801 10.821 10.989 10.973
1.64 10.861 10.858 10.88 11.035 11.019
1.66 10.913 10.913 10.933 11.079 11.063
1.68 10.963 10.964 10.985 11.12 11.106
1.7 11.012 11.013 11.035 11.157 11.143
1.72 11.056 11.059 11.08 11.194 11.18
1.74 11.098 11.103 11.122 11.227 11.216
1.76 11.14 11.145 11.162 11.261 11.249
1.78 11.179 11.184 11.201 11.291 11.279
1.8 11.213 11.222 11.236 11.32 11.309

129

Figure A9. Titration curve for [chloro(fluoro)methylene]bis(phosphonic acid), 9, with KOH.

Titration of CFClBP
0.00
2.56
5.12
7.68
10.24
0 0.4 0.8 1.2 1.6 2
Volume of 0.0881M KOH (mL)
pH
Trial 1 Trial 2 Trial 3 Trial 4

Stock solution: 42.80 mg of 9 in 50 mL 0.1 M KCl solution; trials performed on 10 mL (±0.01) samples.

130

Table A9. Potentiometric titration data for [chloro(fluoro)methylene]bis(phosphonic acid), 9.

Trial 1 Trial 2 Trial 3 Trial 4
log betas
Volume KOH (mL) 1 8.34 8.34 8.39 8.38
0.0881 M 2 13.91 13.91 14.00 13.97
3 15.52 15.43 15.75 15.42
pH
0 2.23 2.21 2.19 2.23
0.02 2.23 2.22 2.2 2.24
0.04 2.24 2.23 2.22 2.25
0.06 2.25 2.24 2.23 2.26
0.08 2.26 2.25 2.24 2.27
0.1 2.28 2.27 2.26 2.29
0.12 2.29 2.28 2.27 2.3
0.14 2.3 2.3 2.29 2.31
0.16 2.32 2.31 2.3 2.33
0.18 2.33 2.33 2.31 2.34
0.2 2.35 2.34 2.33 2.36
0.22 2.36 2.36 2.35 2.37
0.24 2.38 2.37 2.36 2.39
0.26 2.4 2.39 2.38 2.41
0.28 2.42 2.41 2.4 2.43
0.3 2.44 2.43 2.42 2.45
0.32 2.46 2.45 2.44 2.47
0.34 2.48 2.47 2.46 2.49
0.36 2.5 2.5 2.48 2.51
0.38 2.52 2.52 2.51 2.53
0.4 2.55 2.54 2.53 2.56
0.42 2.57 2.57 2.56 2.58
0.44 2.6 2.6 2.58 2.61
0.46 2.63 2.63 2.61 2.64
0.48 2.66 2.65 2.64 2.67
0.5 2.7 2.69 2.68 2.7
0.52 2.73 2.73 2.71 2.74
0.54 2.77 2.77 2.75 2.78
0.56 2.81 2.81 2.79 2.82
0.58 2.86 2.86 2.84 2.87
131

Volume KOH (mL)
0.0881 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 8.34 8.34 8.39 8.38
2 13.91 13.91 14.00 13.97
3 15.52 15.43 15.75 15.42
pH
0.6 2.92 2.91 2.89 2.92
0.62 2.98 2.97 2.94 2.98
0.64 3.05 3.04 3.01 3.04
0.66 3.13 3.12 3.09 3.12
0.68 3.23 3.22 3.17 3.21
0.7 3.35 3.34 3.28 3.33
0.72 3.52 3.51 3.42 3.48
0.74 3.75 3.73 3.62 3.69
0.76 4.07 4.05 3.9 3.99
0.78 4.4 4.38 4.24 4.32
0.8 4.64 4.62 4.53 4.59
0.82 4.82 4.81 4.73 4.78
0.84 4.97 4.96 4.9 4.94
0.86 5.09 5.08 5.04 5.07
0.88 5.2 5.19 5.16 5.19
0.9 5.31 5.3 5.26 5.29
0.92 5.4 5.39 5.36 5.39
0.94 5.49 5.49 5.45 5.48
0.96 5.58 5.57 5.54 5.57
0.98 5.67 5.66 5.64 5.66
1 5.76 5.75 5.72 5.75
1.02 5.85 5.85 5.82 5.84
1.04 5.95 5.95 5.91 5.94
1.06 6.06 6.05 6.02 6.05
1.08 6.18 6.18 6.13 6.16
1.1 6.31 6.31 6.26 6.29
1.12 6.47 6.48 6.41 6.44
1.14 6.67 6.68 6.59 6.63
1.16 6.93 6.94 6.83 6.87
1.18 7.21 7.21 7.1 7.15
1.2 7.45 7.46 7.36 7.41
1.22 7.65 7.65 7.58 7.62
1.24 7.8 7.8 7.75 7.78
132

Volume KOH (mL)
0.0881 M
Trial 1 Trial 2 Trial 3 Trial 4
log betas
1 8.34 8.34 8.39 8.38
2 13.91 13.91 14.00 13.97
3 15.52 15.43 15.75 15.42
pH
1.26 7.94 7.94 7.89 7.92
1.28 8.05 8.05 8.01 8.04
1.3 8.15 8.15 8.12 8.14
1.32 8.26 8.25 8.22 8.25
1.34 8.35 8.34 8.31 8.34
1.36 8.44 8.44 8.41 8.43
1.38 8.53 8.53 8.5 8.52
1.4 8.63 8.62 8.59 8.61
1.42 8.72 8.71 8.69 8.71
1.44 8.82 8.81 8.78 8.8
1.46 8.93 8.92 8.89 8.91
1.48 9.06 9.05 9.01 9.03
1.5 9.2 9.19 9.14 9.16
1.52 9.37 9.35 9.3 9.32
1.54 9.58 9.54 9.49 9.5
1.56 9.82 9.76 9.72 9.71
1.58 10.11 10.03 10.01 9.96
1.6 10.24

133

Figure A10. A) Sample Hyperquad2006 output after refinement calculations. Shown,
methylenebis(phosphonic acid), 1. B) Sample Hyperquad2006 analysis showing calculated and
experimental titration curves for methylenebisphosphonic acid, 1.

Figure A10B.

Note: calculated titration curve shows as dashed red line, experimental titration values shown as blue
dots. Experimental values excluded from refinement are shown as red dots.

134

Appendix B
Figure B1. HPLC Chromatogram of 1) ATP and 2) reaction mixture. Retention time of product
30.6 min. SAX Column Nucleogel 0.5 M TEAB pH 8. Flow Rate 9 mL/min λ
max
= 245nm.

135

Figure B2. TEA salt of ATP
1
H NMR. Varian 400 MHz. D
2
O.

136

Figure B3.
31
P NMR of adenosine tetraphosphate. Varian 400 MHz D
2
O.

137

Figure B4. Low resolution mass spectra of P
3
-1-(2-nitro)phenylethyl 2’ -deoxyadenosine
triphosphate. 592 [+ Li
+
], 598 [+ 2 Li
+
], After SAX column.

2 x Li
+

138

Figure B5.
1
H NMR of hydrazone, 8 (CDCl
3
)

139

Figure B6. UV-vis spectra monitoring the conversion of hydrazone to diazoethane, solvent
ethanol.

Figure B7. HPLC Trace of 1) Reaction mixture 2) ATP 3) Spiked ATP in reaction mixture.
Retention time of ATP 9.35 min, retention time of caged ATP 10.28 min. Analytical SAX
column, Flow 3 ml/min 0.5 M LiCl 0-100% B in 30 min.

-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
200 300 400 500 600
Intensity
Wavelength, nm
Hydrazone vs Diazoethane
Hydrazone
Diazoethane
Chrom. 1 0.0 mins. 21.6 mins.
3
2
1
ATP/ Reaction
Mixture

ATP

Reaction
Mixture
140

Figure B8.
31
P NMR of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1.

141

Figure B9.
1
H NMR of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1.

142

Figure B10. LRMS of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1

143

Figure B11. Absorption Spectra of P
3
-1-(2-nitrophenyl)ethyl deoxyadenosine triphosphate, 1.

-0.5
0
0.5
1
1.5
2
200 250 300 350 400 450 500
Intensity
Wavelength, nm
γ-phosphate caged dATP
144

Figure B12. LRMS revealing glycosidic cleavage.

145

Figure B13.
1
H NMR of 3’,5’ -Di-O-acetylated deoxyadenosine, 17

146

Figure B14.
1
H NMR of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-chloropurine, 18

147

Figure B15. LRMS of 9-(3’,5’-Di-O-acetyl-β-D-erythro-pentofuranosyl)-6-chloropurine, 18

148

Figure B16.
1
H NMR (D
2
O) of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine, 12

149

Figure B17. LRMS of 6-chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine, 12

150

Figure B18.
1
H NMR of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’deoxyribofuranosyl]-6-
chloropurine, 13

151

Figure B19. LRMS (+ mode) of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-2’deoxyribofuranosyl]-6-
chloropurine, 13.

152

Figure B20. 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-2’deoxyribofuranosyl]-
6-chloropurine, 14.

153

Figure B21. LRMS of 9-[β-D-5’-O-(tert-butyldimethylsilyl)-3’-O-(2-nitrophenyl)-
2’deoxyribofuranosyl]-6-chloropurine, 14.

154

Figure B22.
1
H NMR (CD
3
OD) of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 15.

155

Figure B23.
1
H NMR (DMSO-d6) of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 15.

156

Figure B24.
1
H NMR of 3’,5’ -Bis-O-[(tert-butyl)dimetthylsilyl]-2’-deoxyadenosine.

157

Figure B25.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine.

158

Figure B26.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-3’,5’-bis-O-[(tert-
butyl)dimethylsilyl]-2’-deoxyadenosine, 19

159

Figure B27.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]-2’-deoxyadenosine, 20.

160

Figure B28.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-2’-
deoxyadenosine, 21

161

Figure B29.
1
H NMR of N
6
,N
6
-Bis[(tert-butoxy)carbonyl]- 5’-O-[(tert-butyl)dimethylsilyl]-3’-
(2-nitrophenyl)-2’-deoxyadenosine, 22

162

Figure B30
1
H NMR of 5’ -O-[(tert-butyl)dimethylsilyl]-3’-O-(2-nitrophenyl)-2’-
deoxyadenosine, 23.

163

Figure B31.
1
H NMR of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine, 24.

164

Figure B32.
1
H NMR of Synthesis of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2

165

Figure B33.
31
P NMR of Synthesis of 3’ -O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2

166

Figure B34. LRMS of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2

167

Figure B35. HPLC Chromatogram of dATP, 2, and a mixture of dATP and 2.
HPLC
chromatogram
using UV detector
λ=256nm and C18
Column.
2’-deoxyadenosine
triphosphate
3’-O-(2-nitrobenzyl)- 2’-d
eoxyadenosine- 5’-tripho
sphate
1:1 mixture of 1 and 2
Chrom. 1 0.0 mins. 20.2 mins.
3
2
1
1
2
3
3.59
10.08

Figure B36. UV spectrum of compound 2.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
200 250 300 350 400 450 500
Intensity
Wavelength, nm
3'-O-caged dATP
168

Figure B37. Photolysis of 3’-O-(2-nitrophenyl)-2’-deoxyadenosine triphosphate, 2

[M-135]
-

[M+135]
-

169

APENDIX C

Figure C1.
1
H NMR of 2’ -deoxyadenosine-5’-tosylate, 2

1
H: 8.12 (2H, 2,8), 7.44 (4H, C
2
H
4
), 6.26 (1H, 1’), 4.37 (2H, 5’), 4.25 (1H, 4’), 2.8 8 (1H, 2’),
2.56 (1H, 2’) , 2.26 (3H, CH
3
).
Solvent peaks: pyridine 8.5 (2,6), 7.87 (4), 7.07 (3,5). Ethyl acetate 4.12 (CH
3
CO), 2.05
(CH
2
CH
3
), 1.22 (CH2CH
3
).
170

Figure C2. HPLC Chromatogram of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3.

Column: Varian Dynamax Microsorb C18 100-5 250 × 21.4 mm.
Eluent: Isocratic TEAB 0.1 M 10% acetonitrile pH = 7.3.
Flow Rate: 8 mL/min, λ
max
= 280 nm, retention time 9.0 min.

Chrom. 1 0.0 mins. 11.9 mins.
1
171

Figure C3. LRMS of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3.

172

Figure C4.
1
H NMR of α, β -methylene 2’ -deoxyadenosine-5’-diphosphate, 3

1
H NMR (500 MHz, D
2
O): 8.48 (1H, 8), 8.21 (1H, 2), 6.48 (1H, 1’), 4.76 (1H, 4 ’), 4.27 (1H, 3’),
4.08 (2H, 5’), 2.84 (1H, 2’ ), 2.58 (1H, 2’) , 2.14 (2H, PCH
2
P).
Other peaks: Triethylamine: 3.18 (N(CH
2
CH
3
)
3
), 1.25 (N(CH
2
CH
3
)
3
)

173

Figure C5.
31
P NMR (203 MHz, D
2
O) 2’ -deoxyadenosine-5’-tosylate, 2

P
α
18.54 ppm, P
β
14.54 ppm

174

Figure C6. LRMS of Fmoc-6-aminohexano-phosphate, 5.

175

Figure C7.
1
H NMR (500 MHz, D
2
O) Fmoc-6-aminohexano-phosphate, 5.

1
H: 7.64 (2H, 2, 10), 8.5 (2H, 5, 13), 7.29 (4H, 1, 6, 11, 12), 4.32 (2H, 14), 4.04 (1H, 9), 3.78
(2H, 19), 2.99 (2H, 22), 1.49 (2H, 21), 1.41 (2H, 20).
Other peaks: Triethylamine: 3.14 (N(CH
2
CH
3
)
3
), 1.24 (N(CH
2
CH
3
)
3
)

176

Figure C8.
31
P NMR (203 MHz, D
2
O) Fmoc-6-aminohexano-phosphate, 5.

177

Figure C9. LRMS of reaction progress.

178

Figure C10 LRMS of aminobutyl-PPCH
2
PdA, 6.

179

Figure C11.
1
H NMR (600 MHz, D
2
O) aminobutyl-PPCH
2
PdA, 6.

1
H: 8.58 (1H, 16), 8.31 (1H, 10), 6.53 (1H, 21), 4.27 (1H, 23), 4.15 (2H, 25), 4.00 (3H, 19, 30),
3.06 (2H, 33), 2.90(1H, 20), 2.60 (1H, 20), 2.38 (2H, PCH
2
P), 1.82 (2H, 31), 1.75 (2H, 32).
Other peaks: acetone: 2.24

180

Figure C12.
31
P NMR (203 MHz, D
2
O) aminobutyl-PPCH
2
PdA, 6.

181

Figure C13. LRMS of Alexa Fluor 555 succinimidyl ester.

182

Figure C14. HPLC Chromatogram of AF555NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA

183

Figure C15. Emission and Excitation Spectra of AF555NH(CH
2
)
4
ppCH
2
pdA.

AF555-NH(CH
2
)
4
ppCH
2
pdA Excitation and Emission Spectra
564 550
0
0.2
0.4
0.6
0.8
1
1.2
400 450 500 550 600 650 700
wavelength (nm)
Intensity
Emission
Excitation
184

Figure C16. LRMS Spectra of AF555NH(CH
2
)
4
ppCH
2
pdA.

[M+H]
+

[M+Na]
+
[M+2Na]
+

185

Figure C17.
1
H NMR of AF555NH(CH
2
)
4
ppCH
2
pdA.

186

Figure C18.
1
H NMR Spectra of AF555-NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA

Figure C19.
31
P NMR Spectra AF555-NH(CH
2
)
4
ppCH
2
pdA and NH
2
(CH
2
)
4
ppCH
2
pdA

187

Figure C20. HPLC Chromatogram of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA and
NH
2
(CH
2
)
4
ppCH
2
pdA

Chrom. 1 0.0 mins. 25.0 mins.
2
1
Beckman Ultrasphere 10 mm x 25 cm C18
Column
0.1 M TEAB Buffer pH 7.2
0-70% Methanol gradient

NH
2
(CH
2
)
4
ppCH
2
pdA
Retention time: 5.3 min
MS: 559 ( - mode)
7-DEAC-3-
NH(CH
2
)
4
ppCH
2
pdA
Retention time: 17.3 min
MS: 802 ( - mode)
2)  = 260 nm
1)  = 420 nm

188

Figure C21. Emission and Excitation spectra of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA.

7-DEAC-3-NH(CH2)4ppCH2pdA Excitation and Emission Spectra
0
0.2
0.4
0.6
0.8
1
1.2
300 350 400 450 500 550 600
wavelength, nm
intensity
Emission
Excitation
189

Figure C22. LRMS of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (negative mode)

O N O
H
N
O
O
P
O
OH
O
P
O
OH
P
O
OH
N
N
N
N
NH
2
O
OH
O
C
29
H
40
N
7
O
14
P
3
Exact Mass: 803.1846
Mol. Wt.: 803.5877

[M-H]
-

190

Figure C23. LRMS of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (positive mode).

O N O
H
N
O
O
P
O
OH
O
P
O
OH
P
O
OH
N
N
N
N
NH
2
O
OH
O
C
29
H
40
N
7
O
14
P
3
Exact Mass: 803.1846
Mol. Wt.: 803.5877

[M+H]
+

[M+Na]
+

191

Figure C24.
1
H NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (zoomed in region 6.05-8.35ppm).

8
6
5
4 1’
2A
O N O
H
N
O
O
P
O
OH
O
P
O
OH
P
O
OH
N
N
N
N
NH
2
O
OH
O
8
6
5 4
1'
8A
8A
2A
192

Figure C25.
1
H NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA (full spectra).

193

Figure C26.
31
P NMR of 7-DEAC-3-NH(CH
2
)
4
ppCH
2
pdA.

O N O
H
N
O
O
P
O
OH
O
P
O
OH
P
O
OH
N
N
N
N
NH
2
O
OH
O
C
29
H
40
N
7
O
14
P
3
Exact Mass: 803.1846
Mol. Wt.: 803.5877

194

Appendix D

Figure D1.
1
H NMR spectra of a typical run of neopentyl ,-methylene triphosphate (1 =
NpPPCP).

a)

b)

c)

Reaction mixtures obtained after incubation in 0.2 M KOH at 110ºC. Samples were taken out
after 31 (1), 56 (2), 81 (3), 106 (4), 131 (5), and 156 (6) minutes. Spectrum of initial reaction
solution (0) is also shown. The samples were prepared from the 100 µL of reaction mixture and
600 µL of 0.82 mM neopentanol stock in D
2
O. Solvent is a 78%D
2
O: 22%H
2
O mixture.
Resonances of substrate (NpPPCP) and reaction products neopentyl monophosphate (NpP) and
methylenebisphosphonic acid (PCP) are shown. a) Full spectrum; b) methylene region; c) and
methyl region are shown. The reaction progress was always monitored by integration of the
methyl resonances (c).

195

Figure D2. NpPPCH
2
P hydrolysis in 0.2 M KOH reaction data as [NpPPCH
2
P]/[NpPPCH
2
P]
0

(open symbols), and 1-[NpP]/[NpPOPCH
2
P]
0
(full symbols). The best fit to the first order kinetic
equation is shown.

t=90ºC, Run1

t=90ºC, Run2

t=90ºC, Run3

t=100ºC, Run1

t=100ºC, Run2

t=100ºC, Run3

t=110ºC, Run1

t=110ºC, Run2

t=110ºC, Run3

t=125ºC, Run1 t=125ºC, Run2 t=125ºC, Run3
196

197

Figure D3. NpPPCHFP hydrolysis in 0.2 M KOH reaction data as [NpPPCHFP]/[NpPPCHFP]
0

(open symbols), and 1-[NpP]/[NpPOPCHFP]
0
(full symbols). The best fit to the first order
kinetic equation is shown.

t=95ºC, Run1

t=95ºC, Run2

t=95ºC, Run3

t=110ºC, Run1

t=110ºC, Run2

t=110ºC, Run3

t=120ºC, Run1

t=120ºC, Run2

t=120ºC, Run3

t=130ºC, Run1 t=130ºC, Run2 t=130ºC, Run3
198

199

Figure D4. NpPPCF
2
P hydrolysis in 0.2 M KOH reaction data as [NpPPCF
2
P]/[NpPPCF
2
P]
0

(open symbols), and 1-[NpP]/[NpPOPCF
2
P]
0
(full symbols). The best fit to the first order kinetic
equation is shown.
t=110ºC, Run1

t=110ºC, Run2

t=110ºC, Run3

t=120ºC, Run1

t=120ºC, Run2

t=120ºC, Run3

t=130ºC, Run1

t=130ºC, Run2

t=130ºC, Run3

t=143ºC, Run1 t=143ºC, Run2 t=143ºC, Run3
200

201

Figure D5. MS spectra of NpPPCXYP complete hydrolysis products in 0.2 M KOH in the
18
O-
enriched H
2
O (50% enrichment).

202

Figure D6. Fragmentation MS/MS spectra of NpPPCH
2
P complete hydrolysis products in 0.2 M
KOH in the
18
O-enriched H
2
O (50% enrichment).

203

Figure D7. Fragmentation MS/MS spectra of NpPPCHFP complete hydrolysis products in 0.2
M KOH in the
18
O-enriched H
2
O (50% enrichment).

Table D1. The hydrolysis reaction rate constants (in min
-1
) obtained at constant temperatures.
The data for individual runs are shown. The initial substrate concentration in 0.2 M KOH is
shown in parentheses.
Substrate
t, ºC

Run1

Run2

Run3

k±3SD, min
-1

NpPPCH 2P
90

0.00175 (2.4)

0.00167 (2.5)

0.00184 (2.5)

0.00176±0.00025
100 0.00478 (2.5) 0.00487 (5.0) 0.00514 (1.2) 0.00493±0.00056
110 0.0130 (2.6) 0.0135 (2.2) 0.0136 (2.5) 0.0134±0.0010
125 0.0525 (2.5) 0.0520 (2.5) 0.0527 (2.7) 0.0524±0.0011
NpPPCHFP
95

0.00130 (1.7)

0.00130 (1.9)

0.00126 (1.9)

0.00129±0.00006
110 0.00595 (0.8) 0.00582 (1.7) 0.00564 (4.2) 0.00580±0.00046
120 0.0146 (1.7) 0.0148 (1.7) 0.0144 (1.8) 0.0146±0.0006
130 0.0365 (1.7) 0.0350 (2.0) 0.0332 (2.0) 0.0349±0.0049
NpPPCF 2P
110

0.00246 (1.8)

0.00244 (1.8)

0.00243 (0.9)

0.00245±0.00005
120 0.00638 (1.0) 0.00615 (2.0) 0.00627 (3.7) 0.00626±0.00035
130 0.0156 (1.9) 0.0163 (0.8) 0.0154 (1.0) 0.0158±0.0014
143 0.0463 (1.9) 0.0470 (1.5) 0.0474 (1.3) 0.0469±0.0017

204

Figure D8. Benzyl diphosphate neopentyl
1
H NMR spectra of hydrolysis.

205

Figure D9. LRMS of Benzyl diphosphate neopentyl after 200
o
C for 1h 30 min.

200
o
C 1h 30
min. Non-labeled
Water
206

Figure D10. LRMS of Benzyl diphosphate neopentyl hydrolysis with MeOH.

207

Figure D11. LRMS of Benzyl diphosphate neopentyl hydrolysis with labeled water.

208

Figure D12.
31
P NMR of 4 after 1h at 200
o
C.

Figure D13. HPLC purification of 5.

209

Figure D14.
1
H NMR spectra of hydrolysis of compound 4 and 5.

210

Figure D15. Hydrolysis of 5 with MeOH.

Figure D16. Hydrolysis of 5 with labeled water.

211

Figure D17.
31
P NMR of 5.

Figure D18. HPLC Chromatogram of NpPOPNp

212

Figure D19.
1
H NMR and
31
P NMR of NpPOPNp

213

Figure D20. LRMS of NpPOPNp.

214

Figure D21. Hydrolysis of NpPOPNp at 200
o
C for 30 min.

NpPOPNp
p
NpPO
4

NpPOPNp
p
NpPO
4

NpPOPNp
p
NpPO
4

215

Figure D22. Hydrolysis of NpPOPNp at 200
o
C for 3 hours

NpPOPNp
p
NpPO
4

PO
4

NpPOPNp
p
NpPO
4

NpO
H
NpPO
4

NpO
H
NpPOPNp
p
216

Figure D23. NMR and Hydrolysis study of NpPhosphate.

Oil Bath 200
o
C for 180 min

217

218

Appendix E

Figure E1. TLC of reaction progress of succinimidyl activated spin label.

219

Figure E2. LRMS of succinimidyl activated nitroxide spin label.

220

Figure E3. LRMS of TEMPO-aminobutyl PPCH
2
PdA.

221

Figure E4. EPR spectra of 200 μM of TEMPO-aminobutyl PPCH
2
PdA.

Abstract (if available)

Abstract A variety of molecular probes were synthesized and characterized with the goal of gaining structural, conformational, and mechanistic information on polymerases. The leaving group effects of β,γ-CXY deoxynucleotide probes was investigated in order to determine the rate-determining step in the nucleotidyl mechanism of pol β. Due to inconsistencies in literature the pKa₍₂₋₄₎ values for nine bisphosphonic acids used to comprise the β,γ−CXY toolkit were determined under identical conditions. Applying these acidity constants to the analysis of kinetic results obtained with a series of dGTP-β,γ−CXY analogues clarified the relationship between the rate of single-gap nucleotide insertion and BP pKa₄ values. Through construct of Brønsted LFER plot results seem to indicate that chemistry and not conformation is the rate-limiting step in the nucleotidyl transfer mechanism. ❧ Modification on the Pᵧ of α,β-CH₂ non-hydrolyzable dATP analogs were synthesized to probe the dynamics of conformation upon substrate binding. These include fluorescent linked Pᵧ dATP analogs for fluorescence resonance energy transfer studies and nitroxide Pᵧ linked dATP analogs for electron paramagnetic studies. Compounds for FRET analysis such as the one employing Alexa555 will be useful into gauging distances between a substrate bound in the active site and an acceptor nucleotide on the DNA strand, while 7-DEAC linked nucleotide can provide active site environment information. Compounds for EPR analysis will probe the Mg²⁺ metals that bind dNTP and catalyze 3’-O primer nucleophilic attack on Pα. ❧ A suite of model compounds were synthesized and studied in an attempt to mimic Pα hydrolysis achieved by the enzyme. Through these model compounds it was hoped to gain insight into the efficiency of catalysis by pol β by mimicking the reaction non-enzymatically. Results indicated that in the absence of enzyme phosphate hydrolyzes typically occur on the Pᵦ in contrast to Pα by a nucleophile. Second generation compounds were able to shift some of the site of attack to Pᵦ but not by much, indicating the catalytic power of pol β. ❧ 3’-O-(2-nitrobenzyl)-2’-deoxyadenosine, "caged" compound, was synthesized in order to be utilized in ultra-fast Laue X-ray crystallographic. The proposed work would have the 3’-O-caged dATP bound into a crystalline ternary complex but due to the photolabile tag render additional nucleoside incorporation inactive. A pulse of laser light irradiation photochemically will cleave the caging group, initiating turnover at a defined zero time, thus providing time-resolved crystallographic "snapshots" of the DNA pol β catalyzed hydrolysis of dNTPs. ❧ In conjunction these probes will provide structural, conformational, and mechanism insight into pol β which in turn can be used to synthesize inhibitors for therapeutic use.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

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

PDF

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

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

PDF

Nucleophilic fluorination of bisphosphonates and its application in PET imaging

PDF

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

PDF

Fluorinated probes of enzyme mechanisms

PDF

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

PDF

Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

PDF

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

Asset Metadata

Creator Osuna, Jorge (author)

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

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 10/07/2013

Defense Date 08/07/2013

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

Tag DNA polymerase,EPR,FRET,LFER,molecular probes,nucleotide,OAI-PMH Harvest,time-resolved crystallography

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Gupta, Malancha (committee member), Pratt, Matthew R. (committee member)

Creator Email jorgeosunaphd@gmail.com,josuna@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-335879

Unique identifier UC11296579

Identifier etd-OsunaJorge-2084.pdf (filename),usctheses-c3-335879 (legacy record id)

Legacy Identifier etd-OsunaJorge-2084.pdf

Dmrecord 335879

Document Type Dissertation

Format application/pdf (imt)

Rights Osuna, Jorge

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