University of Southern California Dissertations and Theses

Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

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Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

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Content DEOXYRIBONUCLEOSIDE TRIPHOSPHATE ANALOGUES FOR INHIBITION OF
THERAPEUTICALLY IMPORTANT ENZYMES

by

Anastasia Kadina

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

December 2015

Copyright 2015 Anastasia Kadina
ii

Acknowledgements
First of all, I would like to thank Professor Charles E. McKenna for the opportunity to
be a part of his research group. During these long five years I have learned a lot, and not
only in the laboratory. I have learned chemistry, biology, some math, how to give
presentations, how to write papers, how to collaborate, and how to stay patient when all
these things do not go as I expect them to. All of these skills will need much further
improvement, but I definitely got something to begin with.
None of this work, of course, could be done if not for Dr. Boris Kashemirov. Having a
gift of problem solving, for these five years Boris was the person that I came to with issues
and problems of any size, starting from the small clarifications on the chemical procedures
and ending with global issues of chemistry and life which often do not have the right
answer. In many cases, I came to his office just to chat, bringing some chemical problem
as an excuse. I will miss that on a new place, wherever it will be.
I would like to thank our biology collaborators who worked with the compounds that
I have synthesized and provided us with biological data. Without this data, the products
prepared with so much time and emotional involvement, would stay in the lab notebooks
and never see the pages of papers in JACS and Org. Lett. that I am particularly proud of.
Thank you, Professor Myron Goodman, Dr. Sam Wilson, Dr. Vinod Batra and Keri, and also
Professor James T. Stivers and his crew at John Hopkins School of Medicine.
Former members of the McKenna group have been good mentors to the younger
students. Thank you Dr. Brian Chamberlain, it was a pleasure to work with you. Even after
iii

you graduated, I enjoyed going out with you for coffee and chat. Dr. Ivan Krylov, I
appreciate your life perspectives that you have shared, it was always useful to see a
different point of view. Thanks Dr. Yue Wu, for your patience, kindness, and friendliness.
Many hugs go to my labmates. Every single one of you have something that I could
learn from you. Our office have been an enjoyable place of good humor, friendly chat and
relaxing. Elena and Kim, I cannot appreciate enough your help in reading and re-reading
this manuscript. Carolina, since you joined the group it became a sunnier place. Soheil,
thank you for your incredible help with my computer-related struggles. Maryam, your
cook is amazing, and chatting with you about our home countries was always amusing.
Michelle, Chapter 1 of this document mostly describes your work. You were an awesome
mentee, with so much interest and enthusiasm it was such a pleasure to work with you.
Inah, you have helped with administrative stuff enormously, I don’t know how you do not
get lost in all those piles of paper.
I appreciate and immensely acknowledge the experience that I have obtained during
my undergraduate research with Professor Alexander F. Khlebnikov. Experimental basics
and critical analysis of the results – much of what I know and can comes from the time I
was doing the research at St. Petersburg State University.
Outside of the lab, I would like to thank my parents for everything they have done.
My interest to science (and you need a lot of that to do a Ph.D.) roots from the childhood,
when our family was doing simple physics experiments by generating electricity with
newspapers or flipping over the cup of water. All these five years they have been
iv

extremely supportive and understanding, talking to me when I needed to talk and not
talking when I did not want to.
I would like to thank my awesome roommate, Anastasia, who have been helping me
through so much. Hours of discussions and arguments, evenings of tea, and nights of black
humor have made these years much more enjoyable that they could possibly be without
her.
Vadim, you have become as great a friend as I could only hope for. We discussed so
much of what exist or does not exist under this sun, and we still have so much more. Our
opinions always seem opposite, but I think they complement each other nicely. We
changed each other a lot and still keep changing. I even started laughing at the same
things that make you laugh.
Of course, it is impossible to mention everyone who has played their part in my life
and whose presence in it I value as a treasure. Kirill, just thank you for everything you
have done to me and your kindness. Boris, I am not sure how I would survive in an entirely
new country without your knowledge and advice. There are many more, my friends,
colleagues, mentors and teachers – thank you all – now and forever.

v

Table of Contents
Acknowledgements ......................................................................................................... ii
List of Tables .................................................................................................................. vii
List of Figures ............................................................................................................... viii
List of Schemes ............................................................................................................. xvi
Abstract ....................................................................................................................... xvii
Chapter 1 - Synthesis of Individual Diastereomers of (β,γ)-CHCl-dTTP ..............................1
1.1 DNA polymerase β and base excision repair (BER) ..................................................1
1.2 Structure and mechanism of DNA polymerase β ....................................................3
1.3 Probing the active site of pol β ...............................................................................5
1.4 (β,γ)-CXY-dGTPs individual diastereomers as stereochemical probe
of DNA pol β.................................................................................................................7
1.5 Synthesis of individual diastereomers of (β,γ)-CHCl-dTTP .....................................10
Results and Discussion ...............................................................................................13
Experimental Section .................................................................................................18
Chapter 1 references..................................................................................................29
Chapter 2 - Synthesis of (α,β)/(β,γ)-CH 2/NH “Met-Im” Analogues of dTTP .....................32
2.1 Inhibitors of DNA pol β .........................................................................................32
2.2 Design of novel DNA pol β substrate mimic inhibitors ..........................................33
2.3 Synthesis of “Met-Im” Nucleotides .......................................................................36
Results and Discussion ...............................................................................................43
Experimental Section .................................................................................................46
Chapter 2 references..................................................................................................61
Chapter 3 - Design and Synthesis of Modified “Met-Im” Analogues
of dTTP – Potential Inhibitors of DNA pol β ...................................................................64
3.1 Design of the H-bonding “Met-Im” analogue inhibitors of DNA pol β ...................64
vi

3.2 Synthesis of “Met-Im” analogues 1a-c ..................................................................66
Results and Discussion ...............................................................................................71
Experimental Section .................................................................................................76
Chapter 3 references..................................................................................................91
Chapter 4 - Synthesis and Inhibition Mechanism of “pppCH 2dU”
– Inhibitor Probe of SAMHD1 .........................................................................................92
4.1 SAMHD1 structure and function ...........................................................................92
4.2 SAMHD1 activation mechanism............................................................................94
4.3 Design of the inhibitor probe of SAMHD1 .............................................................96
4.4 General method of synthesis of 5’-methylene (d)NTPs .........................................96
4.5 Synthesis of pppCH 2dU – inhibitor probe of SAMHD1 ...........................................98
Results and Discussion ............................................................................................. 100
Experimental Section ............................................................................................... 105
Chapter 4 references................................................................................................ 112
Bibliography ................................................................................................................ 115
Appendix A: Chapter 1 Supporting Data ....................................................................... 124
Appendix B: Chapter 2 Supporting Data ....................................................................... 145
Appendix C: Chapter 3 Supporting Data ....................................................................... 166
Appendix D: Chapter 4 Supporting Data ...................................................................... 192

vii

List of Tables
Table 1.1. k pol and K d of a (β,γ)-CXY-dTTPs (X, Y = H or Hal) library
for correct (T-A) and incorrect (T-G) pairing ...................................................................14
Table 2.1. Selected inhibitors of DNA pol β ....................................................................33
Table 2.2. Main differences in
1
H NMR spectra of α- and β-anomers
of dTMP and their α- and β-1a analogues ......................................................................42
Table 3.1. Screening of conditions of alkylation of 2 with benzyl
2-iodoethyl ether ...........................................................................................................69
Table B.1 Crystallographic Statistics ............................................................................. 164

viii

List of Figures
Figure 1.1. Base excision repair pathways
3a
.....................................................................2
Figure 1.2. Organization of DNA pol β ..............................................................................4
Figure 1.3. Scheme of the pre-transition state structure of DNA pol β .............................5
Figure 1.4. (A) (β,γ)-CXY-dNTP analogues (X,Y = H, Me, Hal, N 3);
(B) LFERs correlating log(k pol) and pK a4 of the leaving group for a
library of (β,γ)-CXY-dGTP analogues comparing right (G-C) and
wrong (G-T) pairing modes
25
............................................................................................7
Figure 1.5. (R)-(β,γ)-CHCl-dTTP (1a) and (S)-(β,γ)-CHCl-dTTP (1b) ...................................10
Figure 1.6. Brønsted plots correlating log(k pol) and the leaving group
pK a4 for (β,γ)-CHX-dTTP..................................................................................................16
Figure 1.7. Active site of DNA pol β incorporating (A) (R)-isomer and
(B) (S)-isomer of (β,γ)-CHCl-dGTP ...................................................................................18
Figure 2.1. Structures of selected inhibitors of DNA pol β ..............................................32
Figure 2.2. Singly bridge-modified dNTP analogues as (A) inhibitors and
(B) substrates of pol β
19, 25, 26b, 26c, 29, 48-49
........................................................................34
Figure 2.3. (A) (α,β)-CH 2-(β,γ)-NH-dTTP (1a) and (B) (α,β)-NH-(β,γ)-CH 2-dTTP
(1b) “Met-Im” nucleotides .............................................................................................36
Figure 2.4.
1
H –
31
P gHMBC NMR (CDCl 3, 500 MHz) of (A) 4a and (B) 4b .........................39
Figure 2.5. Inhibition of pol β by “Met-Im” dTTP analogues ...........................................44
Figure 2.6. 1a (left) and 1b (right) in ternary complex with pol β and DNA .....................45
Figure 3.1. (α,β)-NH-(β,γ)-CH(CH 2COO-)-dTTP (1a),
(α,β)-NH-(β,γ)-CH(CH 2CH 2OH)-dTTP (1b), (α,β)-NH-(β,γ)-CHF-dTTP (1c) .........................66
Figure 3.2. Stereochemistry of compounds 3a-c ............................................................67
Figure 3.3. Portion of
31
P NMR of 1b ..............................................................................72
Figure 3.4. Representative LC-MS trace of decomposition of 1b
at pH 8 and pH 5 at r.t.. .................................................................................................74
ix

Figure 4.1. GTP-dependent SAMHD1-catalyzed hydrolysis of
dNTP to PPP i and nucleoside..........................................................................................92
Figure 4.2. Crystal structure of SAMHD1 monomer unit catalytic
core (residues 109-626) from the GTP-bound SAMHD1 dimer
showing the major lobe, minor lobe and C-terminal region ...........................................93
Figure 4.3. Model for sequential activation of SAMHD1 by GTP activators
and dNTP coactivators and substrates by stepwise formation of a
long-lived activated tetramer ........................................................................................95
Figure 4.4. pppCH 2dU (1) – inhibitor probe of SAMHD1 .................................................96
Figure 4.5. Part of the 500 MHz
1
H NMR spectrum of α- and β- anomers
of 1 in D 2O (after SAX purification)............................................................................... 100
Figure 4.6. Double hit inhibition mechanism of SAMHD1 by 1 ..................................... 104
Figure A.1
1
H NMR of 2 (CDCl 3, 400 MHz) .................................................................... 125
Figure A.2
31
P NMR of 2 (CDCl 3, 162 MHz).................................................................... 125
Figure A.3
1
H NMR of 3 (CDCl 3, 400 MHz) .................................................................... 126
Figure A.4
31
P NMR of 3 (CDCl 3, 162 MHz).................................................................... 126
Figure A.5
1
H NMR of 5 (CDCl 3, 400 MHz) .................................................................... 127
Figure A.6
31
P NMR of 5 (CDCl 3, 162 MHz).................................................................... 127
Figure A.7
1
H NMR of TEA salt of 6 (D 2O, 400 MHz)...................................................... 128
Figure A.8
31
Р NMR of TEA salt of 6 (D 2O, 162 MHz) ..................................................... 128
Figure A.9
1
H NMR of 7 (CDCl 3, 500 MHz) .................................................................... 129
Figure A.10
31
P NMR of 7 (CDCl 3, 202 MHz).................................................................. 129
Figure A.11
1
H NMR of 9 (D 2O, 500 MHz) ..................................................................... 130
Figure A.12
31
P NMR of 9 (D 2O, 202 MHz) .................................................................... 130
Figure A.13 Preparative HPLC separation of diastereomers 10a and 10b ..................... 131
Figure A.14
1
H NMR of 10a (D 2O, 400 MHz) ................................................................. 132
x

Figure A.15
31
P NMR of 10a (D 2O, 162 MHz) ................................................................ 132
Figure A.16 Simulation of
31
P NMR of 10a (D 2O, 162 MHz) (blue)
superimposed with
31
P NMR (brown) .......................................................................... 133
Figure A.17
1
H NMR of 10b (D 2O, 400 MHz) ................................................................. 134
Figure A.18
31
P NMR of 10b (D 2O, 162 MHz) ................................................................ 134
Figure A.19
1
H NMR of 11a (CD 3OD, 500 MHz) ............................................................. 135
Figure A.20
31
P NMR of 11a (CD 3OD, 202 MHz) ............................................................ 135
Figure A.21
1
H NMR of 11b (CD 3OD, 500 MHz) ............................................................ 136
Figure A.22
31
P NMR of 11a (CD 3OD, 202 MHz) ............................................................ 136
Figure A.23 Preparative SAX HPLC purification of 12a .................................................. 137
Figure A.24
1
H NMR of 12a (D 2O, 500 MHz) ................................................................. 138
Figure A.25
31
P NMR of 12a (D 2O, 202 MHz) ................................................................ 138
Figure A.26 Preparative SAX HPLC purification of 12b .................................................. 139
Figure A.27
1
H NMR of 12b (D 2O, 500 MHz) ................................................................. 140
Figure A.28
31
P NMR of 12b (D 2O, 202 MHz) ................................................................ 140
Figure A.29 Preparative SAX HPLC purification of 1a .................................................... 141
Figure A.30
1
H NMR of 1a (D 2O, 500 MHz) ................................................................... 142
Figure A.31
31
P NMR of 1a (D 2O, 202 MHz) .................................................................. 142
Figure A.32 Preparative SAX HPLC purification of 1b .................................................... 143
Figure A.33
1
H NMR of 1b (D 2O, 500 MHz) ................................................................... 144
Figure A.34
31
P NMR of 1b (D 2O, 202 MHz) .................................................................. 144
Figure B.1
1
H NMR of 2a (CDCl 3, 500 MHz) ................................................................... 146
Figure B.2
31
P NMR of 2a (CDCl 3, 202 MHz) .................................................................. 146
xi

Figure B.3
1
H NMR of 2b (CDCl 3, 500 MHz) .................................................................. 147
Figure B.4
31
P NMR of 2b (CDCl 3, 202 MHz).................................................................. 147
Figure B.5
1
H NMR of 3a (CDCl 3, 500 MHz) ................................................................... 148
Figure B.6
31
P NMR of 3a (CDCl 3, 202 MHz) .................................................................. 148
Figure B.7
13
C NMR of 3a (CDCl 3, 126 MHz) .................................................................. 149
Figure B.8
1
H NMR of 3b (CDCl 3, 500 MHz) .................................................................. 150
Figure B.9
31
P NMR of 3b (CDCl 3, 202 MHz).................................................................. 150
Figure B.10
13
C NMR of 3b (CDCl 3, 126 MHz)................................................................ 151
Figure B.11
1
H NMR of 4a (CDCl 3, 500 MHz) ................................................................. 152
Figure B.12
31
P NMR of 4a (CDCl 3, 202 MHz) ................................................................ 152
Figure B.13
13
C NMR of 4a (CDCl 3, 126 MHz) ................................................................ 153
Figure B.14
1
H -
31
P gHMBC NMR of 4a (CDCl 3, 500 MHz) ............................................. 153
Figure B.15
1
H NMR of 4b (CDCl 3, 500 MHz)................................................................. 154
Figure B.16
31
P NMR of 4b (CDCl 3, 202 MHz) ................................................................ 154
Figure B.17
13
C NMR of 4b (CDCl 3, 126 MHz)................................................................ 155
Figure B.18
1
H –
31
P gHMBC NMR of 4b (CDCl 3, 500 MHz) ............................................ 155
Figure B.19
31
P –
1
H NMR of 4b (CDCl 3, 202 MHz) ........................................................ 156
Figure B.20
1
H NMR of 6a (mixture of 4 isomers) (CDCl 3, 500 MHz) ............................. 157
Figure B.21
31
P NMR of 6a (mixture of 4 isomers) (CDCl 3, 202 MHz) ............................ 157
Figure B.22
1
H NMR of 6b (mixture of 4 isomers) (CDCl 3, 400 MHz) ............................. 158
Figure B.23
31
P NMR of 6b (mixture of 4 isomers) (CDCl 3, 162 MHz) ............................ 158
Figure B.24
1
H NMR of 7a (mixture of 4 isomers) (CD 3OD, 500 MHz) ........................... 159
Figure B.25
31
P NMR of 7a (mixture of 4 isomers) (CD 3OD, 202 MHz) .......................... 159
xii

Figure B.26
1
H NMR of 7b (mixture of 4 isomers) (CD 3OD, 400 MHz) ........................... 160
Figure B.27
31
P NMR of 7b (mixture of 4 isomers) (CD 3OD, 162 MHz) .......................... 160
Figure B.28
1
H NMR of α-1a (D 2O, 500 MHz) ................................................................ 161
Figure B.29
31
P NMR of α-1a (D 2O, 202 MHz) ............................................................... 161
Figure B.30
1
H NMR of 1a (D 2O, 500 MHz) ................................................................... 162
Figure B.31
31
P NMR of 1a (D 2O, 202 MHz) .................................................................. 162
Figure B.32
1
H NMR of 1b (D 2O, 400 MHz) ................................................................... 163
Figure B.33
31
P NMR of 1b (D 2O, 162 MHz) .................................................................. 163
Figure C.1
31
P NMR of the r.m. of synthesis of 3a (CDCl 3, 243 MHz) ............................. 167
Figure C.2
1
H NMR of 3a (CDCl 3, 600 MHz) ................................................................... 167
Figure C.3
31
P NMR of 3a (CDCl 3, 243 MHz) .................................................................. 168
Figure C.4
13
C NMR of 3a (CDCl 3, 151 MHz) .................................................................. 168
Figure C.5
1
H NMR of 4a (CDCl 3, 500 MHz) ................................................................... 169
Figure C.6
31
P NMR of 4a (CDCl 3, 202 MHz) .................................................................. 169
Figure C.7
13
C NMR of 4a (CDCl 3, 126 MHz) .................................................................. 170
Figure C.8
1
H NMR of 5a (CDCl 3, 500 MHz) ................................................................... 171
Figure C.9
31
P NMR of 5a (CDCl 3, 202 MHz) .................................................................. 171
Figure C.10
1
H NMR of 6a (CD 3OD, 500 MHz) ............................................................... 172
Figure C.11
31
P NMR of 6a (CD 3OD, 202 MHz) .............................................................. 172
Figure C.12
1
H NMR of benzyl 2-iodoethyl ether (CDCl 3, 400 MHz) .............................. 173
Figure C.13 Purification trace of compound 3b. Fractions 10-26
contained isomer 1 ...................................................................................................... 173
Figure C.14
1
H NMR of 3b (isomer 1) (CDCl 3, 500 MHz) ................................................ 174
xiii

Figure C.15
31
P NMR of 3b (isomer 1) (CDCl 3, 202 MHz) ............................................... 174
Figure C.16
1
H NMR of 4b (mixture of 2 isomers) (CDCl 3, 500 MHz) ............................. 175
Figure C.17
31
P NMR of 4b (mixture of 2 isomers) (CD 3OD, 202 MHz) .......................... 175
Figure C.18
1
H NMR of 5b (mixture of 8 isomers) (CDCl 3, 500 MHz) ............................. 176
Figure C.19
31
P NMR of 5b (mixture of 8 isomers) (CDCl 3, 202 MHz) ............................ 176
Figure C.20
1
H NMR of 6b (mixture of 8 isomers) (CD 3OD, 500 MHz) ........................... 177
Figure C.21
31
P NMR of 6b (mixture of 8 isomers) (CD 3OD, 202 MHz) .......................... 177
Figure C.22
1
H NMR of 1b (mixture of 2 isomers) (D 2O, pH 9, 500 MHz) ....................... 178
Figure C.23
31
P NMR of 1b (mixture of 2 isomers) (D 2O, pH 9, 202 MHz) ...................... 178
Figure C.24 HPLC trace of purification of compound 1b ............................................... 179
Figure C.25 Trace of compound 1b .............................................................................. 179
Figure C.26 Purification trace of compound 3c. Fractions 13-39
were collected ............................................................................................................. 180
Figure C.27
1
H NMR of 3c (mixture of 2 isomers) (CDCl 3, 500 MHz) .............................. 181
Figure C.28
31
P NMR of 3c (mixture of 2 isomers) (CDCl 3, 202 MHz) ............................. 181
Figure C.29
19
F NMR of 3c (mixture of 2 isomers) (CDCl 3, 470 MHz) ............................. 182
Figure C.30
13
C NMR of 3c (mixture of 2 isomers) (CDCl 3, 126 MHz) ............................. 182
Figure C.31
1
H NMR of 4c (mixture of 2 isomers) (CD 3OD, 500 MHz) ............................ 183
Figure C.32
31
P NMR of 4c (mixture of 2 isomers) (CD 3OD, 202 MHz) ........................... 183
Figure C.33
19
F NMR of 4c (mixture of 2 isomers) (CD 3OD, 470 MHz) ........................... 184
Figure C.34
1
H NMR of 5c (mixture of 8 isomers) (CDCl 3, 500 MHz) .............................. 185
Figure C.35
31
P NMR of 5c (mixture of 8 isomers) (CDCl 3, 202 MHz) ............................. 185
Figure C.36
19
F NMR of 5c (mixture of 8 isomers) (CDCl 3, 470 MHz) ............................. 186
xiv

Figure C.37
1
H NMR of 6c (mixture of 8 isomers) (CD 3OD, 500 MHz) ............................ 187
Figure C.38
31
P NMR of 6c (mixture of 8 isomers) (CD 3OD, 202 MHz) ........................... 187
Figure C.39
19
F NMR of 6c (mixture of 8 isomers) (CD 3OD, 470 MHz) ........................... 188
Figure C.40
1
H NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 500 MHz) ....................... 189
Figure C.41
31
P NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 202 MHz) ...................... 189
Figure C.42
19
F NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 470 MHz) ...................... 190
Figure D.1
1
H NMR of 5 (CDCl 3, 500 MHz) .................................................................... 192
Figure D.2
31
P NMR of 5 (CDCl 3, 202 MHz) ................................................................... 192
Figure D.3
13
C NMR of 5 (CDCl 3, 126 MHz) ................................................................... 193
Figure D.4
1
H NMR of 6 (CDCl 3, 500 MHz) .................................................................... 194
Figure D.5
31
P NMR of 6 (CDCl 3, 202 MHz) ................................................................... 194
Figure D.6
13
C NMR of 6 (CDCl 3, 126 MHz) ................................................................... 195
Figure D.7 HRMS (ESI/APCI) of 6 .................................................................................. 196
Figure D.8
1
H NMR of 7 (D 2O, 500 MHz) ....................................................................... 197
Figure D.9
31
P NMR of 7 (D 2O, 202 MHz) ...................................................................... 197
Figure D.10
13
C NMR spectrum of 7 (D 2O, 126 MHz) .................................................... 198
Figure D.11 HRMS (ESI/APCI) of 7 ................................................................................ 199
Figure D.12 Part of the
1
H NMR of α- and β- anomers of 1
(after SAX purification) (D 2O, 500 MHz) ....................................................................... 200
Figure D.13
1
H NMR of 1 (D 2O, 500 MHz) ..................................................................... 201
Figure D.14
31
P NMR of 1 (D 2O, 202 MHz) .................................................................... 201
Figure D.15
13
C NMR of 1 (D 2O, 126 MHz) .................................................................... 202

xv

Figure D.16 HRMS (ESI/APCI) of 1 ................................................................................ 203
Figure D.17 Inhibition of SAMHD1 by 1 ........................................................................ 204

xvi

List of Schemes
Scheme 1.1 Synthesis of chiral synthons 10a/b for synthesis of
(β,γ)-CHCl-dTTP individual diastereomers ......................................................................12
Scheme 1.2. Synthesis of individual diastereomers of (β,γ)-CHCl-dTTP (1a, 1b) .............13
Scheme 2.1. Flip Synthesis of 4a/b ................................................................................37
Scheme 2.2. [1,3] N C Migration Mechanism ..............................................................38
Scheme 2.3. Synthesis of “Met-Im” Nucleotides 1a/b ....................................................40
Scheme 2.4. Microwave irradiation reduces BTMS-promoted anomerization
of 1a ..............................................................................................................................42
Scheme 3.1 Synthesis of intermediate 3a ......................................................................68
Scheme 3.2. Synthesis of intermediate 3b .....................................................................69
Scheme 3.3. Synthesis of intermediate 3c and its double fluorination
byproduct ......................................................................................................................70
Scheme 3.4. Synthesis of 6a-c ........................................................................................71
Scheme 3.5.Full deprotection of 6a-c.............................................................................71
Scheme 3.6 Products of decomposition of 1b ................................................................75
Scheme 4.1. General method of synthesis of 5’-methylene nucleotide
analogues ......................................................................................................................97
Scheme 4.2. Synthesis of pppCH 2dU (1) .........................................................................99

xvii

Abstract
Deoxyribonucleoside triphosphates (dNTPs) are ubiquitous substrates for a variety of
cellular enzymes, such as polymerases and triphosphohydrolases. The triphosphate
moiety of dNTPs represents a versatile site for a rational design of probes of dNTP-utilizing
enzymes. Substituting bridging oxygens in dNTP with non-hydrolysable moieties, such as
CXY or NH, has provided a number of probes and inhibitors of the enzymes which break
P–O bonds in dNTPs. In an effort to expand the scope of dNTP non-hydrolysable analogues
as enzyme inhibitors, we focused on the design and synthesis of (α,β)-, (β,γ)-, or 5’-non-
hydrolysable dNTP analogues as inhibitors of two important therapeutic target enzymes
– DNA polymerase β and SAMHD1.
Working within the framework of a collaboration with Prof. M. Goodman (USC) and
S. Wilson, M.D. (NIEHS), we created a novel class of “Met-Im” inhibitors of DNA pol β:
dNTP analogues in which the P α–O–P β and P β–O–P γ bridging oxygens are replaced by
hydrolytically-resistant CXY and NH in alternation. The first two representatives of this
class were synthesized via an unusual [1,3]-N –> C rearrangement (or “flip”) of
diphosphoryl amine precursors. Conventional dealkylation with BTMS incurred
anomerization, however this could be largely prevented by microwave radiative heating,
which may be a useful approach to this problem more generally. Enzymatic studies
confirmed that both compounds inhibit DNA pol β, but the inhibitor having the NH group
in the (β,γ)-position was 9-fold more potent. This difference was associated with the aid
xviii

of X-ray crystallographic analysis with a water-mediated H-bond of the (β,γ)-NH group
with the R183 residue in the active site of DNA pol β.
The H-bond identified in the crystal structure of parent “Met-Im” inhibitors inspired
an attempt to design “next generation” higher affinity inhibitors of this class. In a
preliminary study, two H-bonding functionalities were installed into the parent “Met-Im”
dNTP analogues to optimize their interaction with DNA pol β. The synthesis of the novel
inhibitors was accomplished analogously to the parent “Met-Im” dNTPs.
In collaboration with Prof. J. Stivers (John Hopkins School of Medicine) we also
designed and synthesized a 5’-non-hydrolysable dNTP analogue (pppCH 2dU), the first
non-reactive inhibitor of SAMHD1, to explore potential modes of inhibition of SAMHD1.
pppCH 2dU, initially viewed as a simple competitive inhibitor, was determined to act by an
unusual mechanism of disrupting the formation of SAMHD1 oligomers. Our results
demonstrated that SAMHD1, an important link in the immune response pathways, is a
druggable enzyme, subject to rational inhibitor design.
1

Chapter 1 - Synthesis of Individual Diastereomers of (β,γ)-CHCl-dTTP
1.1 DNA polymerase β and base excision repair (BER)
DNA polymerases synthesize DNA by adding nucleotides to the 3’-end of a DNA
primer, using template DNA to insert the correct nucleotide. They participate in all
processes in which DNA is extended such as DNA replication and repair. All known DNA
polymerases have been divided into seven families based on their primary structure.
1
In
mammalian cells, 15 DNA polymerases have been identified, belonging to five families (A-
, B-, RT-, X-, Y-).
2
DNA polymerase β (pol β),
3
identified in 1971,
4
belongs to the X-family
of DNA polymerases. Pol β fills short gaps in DNA during base excision repair (BER).
5
BER
may occur when DNA damage results from modifications of the bases, caused, for
example, by antitumor agents, such as bleomycin
6
or cisplatin;
7
however, BER is mostly
limited to the modifications that do not significantly modify size or shape of the base.
Such modifications are commonly caused by alkylating agents
8
or reactive oxygen species
(ROS).
9

BER (Figure 1.1) is initiated when DNA glycosylases recognize and remove damaged
bases via hydrolytic glycosyl cleavage. The resulting abasic sugar is often referred to as
the apurinic/apyrimidinic (AP) site. The number of AP sites formed in a mammalian cell
per day is estimated to be as many as 10
4
.
10
While monofunctional glycosylases only
create an AP site by cleaving the base from the sugar ring, bifunctional glycosylases
additionally incise the strand at the AP site forming free 3’-phosphate (Figure 1.1). AP
2

endonuclease further processes the AP site by incising the DNA strand at the 5’-position,
leading to formation of a one-nucleotide gap. This gap is filled by pol β. In addition to its
ability to fill single-nucleotide gaps, DNA pol β also possesses lyase activity. If DNA
glycosylase has not initially incised the AP site at the 3’-phosphate, the lyase activity of
pol β removes the residual deoxyribophosphate (dRP), and DNA is ligated by DNA ligase
to restore a healthy strand.
Pol β possesses lower fidelity compared to replicative polymerases.
11
Furthermore, it
lacks proofreading activity, depending upon other enzymes to correct
misincorporations,
12
and incorrect insertion sites have the potential to become
mutagenic.

Figure 1.1. Base excision repair pathways
3a

In normal cells, pol β gene (POLB) expression levels demand tight regulation since
overexpression of pol β interferes with normal replication, resulting in mutagenesis.
13

3

High levels of pol β have been observed in gastric, uterine, prostate, ovarian and thyroid
carcinomas.
2
Overexpression of pol β may interfere with some anti-cancer drugs, such as
alkylating agents, which damage DNA bases to trigger cell apoptosis. Instead, pol β
involves the damaged site in BER preventing cell death. Mutant forms of pol β, which are
often found in cancer cells,
11, 13b, 14
have a higher chance to incorporate the wrong base,
resulting in enhanced mutagenesis.
15
Therefore, steady regulation of pol β forms and
levels in a cell is necessary for preventing carcinogenesis.
1.2 Structure and mechanism of DNA polymerase β
Pol β (Figure 1.2) is a small (39kDa, 335 residues) eukaryotic DNA polymerase,
belonging to the X-family of DNA polymerases.
16
It consists of an N-terminal lyase domain
(8 kDa, ≈ 90 residues),
17
connected via a short protease-sensitive segment to C-terminal
polymerase domain (31 kDa, ≈ 250 residues). The polymerase domain, in turn, consists of
three distinct subdomains: C- (catalytic subdomain), coordinates two divalent metal
cations, facilitating DNA synthesis; N- (nascent base pair binding subdomain); and D-
(duplex DNA binding subdomain).
4

Figure 1.2. Organization of DNA pol β. Full length enzyme consists of lyase domain
(white), and polymerase domain, subdivided into three subdomains (D-, duplex DNA
binding (purple), C-, catalytic (yellow), and N-, nascent base pair binding (green)).
Incoming dTTP, shown in ball-and-stick (blue), is paired to A on the template DNA (red)
3a

Pol β apoenzyme has an extended conformation,
18
but upon binding to a single-
stranded DNA template and two divalent metal ion cofactors (usually magnesium) the
enzyme assumes a doughnut-like conformation. Pol β utilizes two substrates: a single-
nucleotide gapped DNA primer annealed to template and dNTP complementary to the
corresponding template base. DNA extension occurs as a result of primer’s 3’-OH
nucleophilic attack onto the P α of the incoming dNTP, leading to a pentacoordinated
bipyramidal P α in the transition state (Figure 1.3). One of the magnesium ions (catalytic)
activates the OH-nucleophile by lowering the pK a of the hydroxyl group, while the second
Mg
2+
(templating) optimizes dNTP conformation for the nucleophilic attack. The
transition state is resolved upon release of pyrophosphate (PP i) leaving group (Figure 1.3).
5

Figure 1.3. Scheme of the pre-transition state structure of DNA pol β. 3’-OH group of
DNA primer (red) attacks the P α of the incoming dNTP or dNTP analogue (blue). Two Mg
2+

cofactors and 3 Asp residues in the enzyme active site (black) lower the activation barrier
of the transformation by increasing the nucleophilicity of 3’-oxygen and keeping the
substrates in the optimal conformation
19

DNA polymerase fidelity, an important characteristic of polymerases, is the
polymerase’s ability to choose a correct dNTP from a pool of structurally similar incorrect
nucleotides. Quantitatively, fidelity is the ratio of specificity constants for correct and
incorrect nucleotides: [(k cat/K m) corr/(k cat/K m) incorr]. Importantly, the efficiency of the
insertion of incorrect nucleotides is similar for low- and high-fidelity polymerases.
20

Therefore, fidelity is mostly influenced by the efficiency of correct nucleotide insertion.
Pol β has low to moderate fidelity, with the error rate for single-gap nucleotide filling
being about 1 error in 3000 nucleotides.
21
Error rate tends to increase for larger gaps.
22

1.3 Probing the active site of pol β
Better understanding of the activity and fidelity of pol β is the driving force of
exploring its active site and mechanism, and, particularly, establishing the rate-
6

determining step (RDS) in the DNA synthesis.
23
RDS can be a chemical transformation or
conformational change, taking place before or after transformation. Our group has
previously developed a library of dNTP analogues,
24
modified at P β–O–P γ bridging oxygen,
(β,γ)-CXY-dNTP (Figure 1.4a). These analogues are substrates for pol β, however, upon
transformation they release the corresponding bisphosphonate instead of
pyrophosphate, the product of turnover of natural dNTPs. It has been suggested that in
the absence of significant steric effects or interactions specific to X/Y in the active site
induced by leaving group (LG) modification, reaction rate constants would correlate with
the pK a of the LG. Since it was found that (β,γ)-CXY-dGTP analogues assume a native
conformation in the active site of pol β, and no steric interactions as well as solvent- or
Mg-induced effects were identified in the active site by analysis of the X-ray structures of
the number of analogues,
19, 24
analysis of the kinetics of a series of (β,γ)-CXY-dNTPs offers
an unambiguous approach for determining RDS.
(β,γ)-CXY-dNTP “tool kit”
19, 24-25
has been shown to accommodate a wide range of
substituted methylene groups with various stereoelectronic properties. Varying
electronegativity of X and Y changes the pK a4 of the corresponding bisphosphonic acid,
modifying the leaving group aptitude. A wide range of pK a4 can be achieved using H, CH 3,
and Hal as X and Y.
19, 24, 26
Stabilization of the products leads to the lowering of the
preceding activation barrier. If the RDS of the transformation is chemistry (which
inherently depends on the X and Y properties), the plot of the log(k pol) vs pK a4 (Brønsted
plot
27
) is predicted to be linear (linear free energy relationship, LFER)
28
with a negative
7

slope. The slope magnitude determines the sensitivity of the transformation rate to the
leaving group aptitude. The LFER is unambiguously observed in the studies of the (β,γ)-
CXY-dNTP series (N = G, T) (Figure 1.4b). Interestingly, dihalogenated (β,γ)-CXY-dNTPs (X,
Y = Hal) were found to have lower transformation rates for similar pK a values compared
to non- or monohalogenated dNTPs. The slopes of -0.54 (for both normal and dihalo lines
in G-C correct pairing) to -0.71 and -1.1 (normal and dihalo lines in G-T incorrect pairing,
respectively) are relatively steep,
19, 24-25
supporting the hypothesis of chemical
transformation, and, particularly, LG elimination, being RDS.

Figure 1.4. (A) (β,γ)-CXY-dNTP analogues (X,Y = H, Me, Hal, N 3); (B) LFERs correlating
log(k pol) and pK a4 of the leaving group for a library of (β,γ)-CXY-dGTP analogues comparing
right (G-C) and wrong (G-T) pairing modes
25

1.4 (β,γ)-CXY-dGTPs individual diastereomers as stereochemical probe of DNA pol β
The (β,γ)-CXY-dNTPs where X ≠ Y are obtained as mixtures of two diastereomers
because a new chiral center is generated at the (β,γ)-bridging carbon. X-ray
crystallographic studies of ternary complexes formed from (β,γ)-CXF-dGTP
(A)
(B)
8

diastereomeric mixtures (≈1:1) incubated with binary DNA-pol β complex have revealed
the presence of a single (R)-diastereomer in the active site for monofluorinated analogues
(X = H, Cl, Me; Y = F).
26b, 26c
Other β,γ-monohalogenated (X = H; Y = Cl, Br),
monomethylated (X = H, Y = Me), and heterodihalogenated (X = Cl, Y = Br) analogues
populated the active site evenly in the crystal complex.
26b, 26c
To explain the apparent
stereoselectivity of pol β towards monofluorinated dNTP analogues, individual
diastereomers of (β,γ)-CHCl-dNTPs and (β,γ)-CHF-dGTPs have been synthesized. The
insights into behavior of individual diastereomers can also provide explanation for the
positioning of dihalogenated (β,γ)-CXY-dNTPs analogues on a separate LFER line.
The first synthesis of individual diastereomers of (β,γ)-CHX-dGTP (X = F, Cl) was
accomplished by Y. Wu et al.
29
The approach centered on the preparation of synthetic
intermediates bearing a chiral auxiliary (mandelic acid morpholidate), followed by their
separation by reverse phase (RP) HPLC. The diastereomerically pure synthons were
demorpholidated, the resulting intermediates coupled to dGTP, and the chiral auxiliary
was removed by catalytic hydrogenolysis. The absolute configurations of (β,γ)-CHX-dGTP
(X = F, Cl) were assigned using X-ray crystallographic analysis.
Both individual diastereomers of (β,γ)-CHX-dGTPs (X = F, Cl) were found to populate
the active site of DNA pol β, ruling out the possibility that one of the diastereomers could
not be accommodated due to the steric clashes of (S)-halogen with the active site
residues.
30
Since both diastereomers could be accommodated, possible reasons of pol β
stereospecificity may include differences in analogues binding affinities (K d), reaction
9

rates (k pol), or combination of both. To determine if the enzyme was stereospecific to (R)-
(β,γ)-CHF-dGTP in solution as well as in the crystal, kinetic experiments were performed.
30

The 1:1 mixture of (R/S)-(β,γ)-CHF-dGTP diastereomers was incubated with the protein
and DNA, and the relative consumption of isomers was determined to occur at a 3:1 ratio
(R):(S) by
19
F NMR. Therefore, (S)-isomer was also found to be a substrate of pol β,
however slower than (R)-isomer.
Pre-steady state kinetic experiments were also conducted for individual
diastereomers of (R)- and (S)-(β,γ)-CHF-dGTP.
30
The (R)-isomer was incorporated with a
2-fold larger k pol, and had a 1.5 fold higher affinity to pol β than the (S)-isomer, resulting
in a stereospecificity of 3.8. When paired to the “wrong” T, a greater difference between
(R)- and (S)-isomers’ k pol was observed, while the binding constants were similar, resulting
in a stereospecificity of 11.
A similar result was observed for (R)- and (S)-(β,γ)-CHCl-dGTP analogues. In the
enzymatic DNA synthesis, the diastereomers were consumed during the reaction in a 7:1
ratio (R:S) as determined by
31
P NMR. In a pre-steady-state experiment, (R)-(β,γ)-CHCl-
dGTP was utilized by pol β 1.6 times faster than (S)-isomer, and possessed an affinity 3
times higher, resulting in stereospecificity of 6.3 for the correct pairing. For incorrect
pairing, stereospecificity was determined to be 7.8. Interestingly, for diastereomeric
mixtures (R/S)-(β,γ)-CHF-dGTP and (R/S)-(β,γ)-CHCl-dGTP k pol value is a weighted mean of
contributions from individual diastereomers.
10

Figure 1.5. (R)-(β,γ)-CHCl-dTTP (1a) and (S)-(β,γ)-CHCl-dTTP (1b)
The differences in the stereospecificities of the (R)- and (S)-(β,γ)-CHX-dGTPs (X = F, Cl)
diastereomers suggested that placement of CHF/CHCl atom in the active site of pol β,
determined by the configuration of the chiral (β,γ)-carbon, can influence the energy of
the transition state during turnover, particularly for mispair.
1.5 Synthesis of individual diastereomers of (β,γ)-CHCl-dTTP
Influence of H-bonding between the incoming dNTP and the template, and base-
stacking effects on the transition state may be of great importance for the reaction
kinetics as well.
25
H-bonding energy stabilization in A-T pairs is lower than in G-C pairs (in
fact, A-T pairing in DNA can be destabilizing).
31
Base-stacking effect has a larger
contribution for purines than for pyrimidine bases due to their larger surface area and
greater polarizability.
31-32
To obtain sufficient data to account for base stacking and H-
bonding influence on the reaction kinetics, we are working towards constructing the
library of (β,γ)-CXY-dTTP (X, Y = H, Hal, Me) analogues. Synthesis of the full “tool-kit”
would allow the systematic assessment of the base pairing and stacking effect differences
in G-C and G-T vs T-A and T-G pairs. Synthesis of individual diastereomers of (β,γ)-CHCl-
dTTP (1a/b) (Figure 1.5) completes the construction of the T-based “tool-kit”.
11

We utilized the Wu et al. procedure with minor modifications to synthesize individual
diastereomers of (β,γ)-CHCl-dTTP (1a and 1b) (Figure 1.5).
29
Tetramethyl
(chloromethylene)bis(phosphonate) 5 was synthesized according to the previously
published method from readily available materials in 4 steps (Scheme 1.1).
33
Single
methyl group could be easily removed by refluxing 5 with TEA in acetonitrile,
34
followed
by ion exchange with DOWEX H
+
to convert triethylmethylammonium salt of 6 into the
acid form. The above method has advantages over the conventional method using NaI
since it largely avoids double demethylation of symmetrical tetramethyl
(methylene)bis(phosphonates), reduces the reaction time (20 min vs overnight reaction
with NaI), and significantly improves the yield of the demethylation.
Acid 6 was coupled to (R)-(-)-methyl mandelate under Mitsunobu conditions, resulting
in the formation of 7. The S N2 Mitsunobu reaction inverts the chiral carbon of methyl
mandelate to the (S)-configuration. Thus, 7 has 3 chiral centers, one of which (methyl
mandelate) has a definite (S)-configuration, resulting in 4 possible diastereomers. Indeed,
all 4 isomers of 7 are formed in the coupling reaction as can be determined by the
31
P
NMR spectrum. Each diastereomer has two phosphorus atoms, resulting in 8 signals in
31
P NMR. Each phosphorus signal is coupled to the neighboring phosphorus, resulting in
the
31
P NMR of 7 showing 15 lines (2 lines coincidently overlap).
Silyldealkylation of 7 was effected using bromotrimethylsilane (BTMS),
35
and the
trimethylsilyl ester was subjected to methanolysis to afford 8 (Scheme 1.1). BTMS can
selectively remove methyl groups over methyl mandelate as the limiting step in
12

silyldealkylation is an S N2 reaction, which favors smaller methyl groups over bulky
secondary alkyls.
Scheme 1.1 Synthesis of chiral synthons 10a/b for synthesis of (β,γ)-CHCl-dTTP
individual diastereomers

Following the Wu procedure, methyl ester 8 was hydrolyzed using Na 2CO 3 to obtain
9a/b. Compound 9 has two chiral centers, one of which (methyl mandelate) has a definite
(S)-configuration; thus, 9 exists as a mixture of 2 diastereomers, with 2 distinct sets of
phosphorus doublet signals in
31
P NMR spectrum.
Upon DCC-mediated coupling to morpholine, 9a/b formed 2 isomers of P,C-
dimorpholinamides 10a/b,
29
which have been separated using RP HPLC, giving 2
diastereomerically pure chiral synthons 10a and 10b (Scheme 1.1).
13

Scheme 1.2. Synthesis of individual diastereomers of (β,γ)-CHCl-dTTP (1a, 1b)

After separation, individual diastereomers 10a and 10b were hydrolyzed to form 11a
and 11b using HCl, and the resulting monomorpholidates were coupled to morpholidate-
activated dTMP.
36
The chiral auxiliary in 12
26b, 26c
was removed by catalytic
hydrogenolysis, yielding the individual diastereomers of (β,γ)-CHCl-dTTP 1a and 1b, which
were purified using RP HPLC.
26c

Results and Discussion
The Wu et al. approach was successfully extended to the synthesis of individual
diastereomers of (β,γ)-CHCl-dTTP, characterized by NMR, HPLC, and mass-spectrometry.
The key intermediates 10a/b, having a chiral auxiliary, were separated by RP HPLC, and
their separation was confirmed by
31
P NMR of individual diastereomers 10a and 10b.
Inidividual diastereomers of 10 were hydrolyzed to monomorpholidates 11a and 11b. The
14

final compounds were successfully synthesized via coupling of tetrabutylammonium salts
of 11 to morpholidate-activated dTMP, followed by the removal of the mandelic acid
morpholidate auxiliary using catalytic hydrogenolysis. The final products 1a and 1b were
purified by RP HPLC, and obtained from chiral synthons 10a and 10b with 16% and 13%
yield, respectively.
We have determined k pol and K d values for (β,γ)-CHCl-dTTPs to examine the effect of
stereochemistry of substitution on DNA synthesis kinetics (k pol) and substrate binding (K d),
and the data were added to the dataset of the (β,γ)-CXY-dTTPs (X, Y = H or Hal) library.
The results are summarized in Table 1.1, with the data for the novel individual
diastereomers of (β,γ)-CHCl-dTTP highlighted in bold.
Table 1.1. k pol and K d of a (β,γ)-CXY-dTTPs (X, Y = H or Hal) library for correct (T-A) and
incorrect (T-G) pairing. The data for the individual diastereomers of (β,γ)-CHCl-dTTP is
highlighted in bold
25

-X- pK a4 M-N k pol (s
-1
) K d (μM) M-N k pol (s
-1
) K d (µM)
CF 2 7.8 T-A 18.1 ± 1.0 15.2 ± 2.0 T-G 0.088 ± 0.018 370 ± 190
CFCl (mix) 8.4 T-A 5.6 ± 0.6 33.0 ± 0.6 T-G 0.024 ± 0.003 1300 ± 230
CCl 2 8.8 T-A 2.0 ± 0.3 98 ± 15 T-G
0.00082 ±
0.00002
170 ± 50
O 8.9 T-A 20.2 ± 1.1 8.2 ± 2.6 T-G 0.55 ± 0.05 910 ± 130
CHF (mix) 9.0 T-A 23.8 ± 1.8 19.1 ± 3.7 T-G 0.27 ± 0.05 360 ± 90
CBr 2 9.3 T-A 0.42 ± 0.09 180 ± 60 T-G n.d. n.d.
CHBr 9.9 T-A 7.1 ± 0.9 19.2 ± 2.0 T-G 0.028 ± 0.005 500 ± 170
CH 2 10.5 T-A 2.0 ± 0.0 8.3 ± 1.9 T-G 0.014 ± 0.001 310 ± 30
CHCl (mix) 9.5 T-A 9.1 ± 1.6 31.7 ± 5.0 T-G 0.092 ± 0.009 1100 ± 100
CHCl (R) 9.5 T-A 13.3 ± 1.0 21.8 ± 1.3 T-G 0.19 ± 0.02 1500 ± 200
CHCl (S) 9.5 T-A 3.9 ± 0.3 57.4 ± 6.9 T-G 0.022 ± 0.005 1600 ± 300
15

LFER plots of log(k pol) vs pK a4 were constructed from the full set of data in Table 1.1
for both correct (T-A) and incorrect (T-G) pairing (Figure 1.6). Similar to what was
observed for dGTP analogues, dihalo lines (blue for correct T-A and grey for incorrect T-G
pairs) are clearly distinct from monohalo, CH 2, and O lines (red for correct T-A and yellow
for incorrect T-G pairs). Interestingly, the slopes of mono- and dihalo- lines are different
for both correct and incorrect pairing, while for the dGTP-based library, the lines were
parallel for correct G-C pairing. For both correct and incorrect pairings, (R)-(β,γ)-CHCl-
dTTP isomer is found to be above the line, showing the higher reaction rate compared to
that of (S)-(β,γ)-CHCl-dTTP, which is found below, similar to dGTP series.
30

To justify the difference in the reaction rate for the two isomers, several explanations
can be suggested. The first possibility is that steric hindrance in the active site inhibits the
primer reaction with the (S)-isomer compared to (R)-isomer. However, the crystal
structure analysis shows that the active site has enough space to accommodate the Cl
atom in (S)-(β,γ)-CHCl-dGTP. Besides, (β,γ)-CCl 2-dGTP and even (β,γ)-CBr 2-dGTP can be
utilized by pol β, making the possibility that steric effects in the active site are fully
responsible for the rate difference unlikely.
Another possibility is that the (R)-isomer can favorably interact with R183 in the
enzyme active site, lowering the energy of the corresponding transition state (TS). In
previous experiments k pol of (R)-(β,γ)-CHCl-dGTP have been shown to be greater
compared to its (S)-counterpart. Supporting this hypothesis, the R183A mutant form of
16

pol β did not show a rate difference for individual diastereomers of (β,γ)-CHCl-dGTP.
However, this is not supported by the dissociation constants K d for (β,γ)-CHX-dGTPs (X =
F, Cl). Although K d is a characteristic of ground state (GS) and not the TS of the
transformation, some of the interactions in the GS can be expected to contribute into the
energy of all states along the reaction coordinate, including the TS. A more
electronegative F can be expected to have a higher affinity to arginine residue; however,
K d for (R)-isomers is generally lower for (β,γ)-CHCl-dGTP analogues (3.2 µM for correct
and 290 µM for incorrect pairs) than for (β,γ)-CHF-dGTPs (6.2 µM for correct and 740 µM
for incorrect pairs). Interestingly, affinities for (S)-(β,γ)-CHX-dGTPs are very similar (within
error) when X = F or Cl.

Figure 1.6. Brønsted plots correlating log(k pol) and the leaving group pK a4 for (β,γ)-
CHX-dTTP. Correlations represent “right” A-T (red and blue) and “wrong” G-T (yellow and
grey) pairing modes. Also plotted are individual diastereomers of (β,γ)-CHCl-dTTP (red
(“right”) and yellow (“wrong”) boxes)
25

-4
-3
-2
-1
0
1
2
7 8 9 10 11
T opp. A / T opp. G
CH
2
O
CHF
CHF
CF
2
CFCl
CCl
2
CF
2
CFCl
CCl
2
CBr
2
O
R-CHCl
CHBr
S-CHCl
CHCl (mix)
CHCl (mix)
S-CHCl
R-CHCl
CHBr
pK a
log(k pol)
17

Yet another possibility is that the difference in the reaction rate between the isomers
can be attributed to the repulsion between accumulating negative charge on the newly
formed non-bridging oxygen (P α–O–P β) the halogen atom in the (S)-position of the leaving
bisphosphonate. As the crystal structures of pol β in complex with individual
diastereomers of (β,γ)-CHCl-dGTPs reveal, the halogen atom in the (S)-isomer is located
close (2.8 Å) to the oxygen in P α–O–P β, while this distance for (R)-isomer is much longer
(4.0 Å) (Figure 1.7).
29
This distance corresponds to the GS, however, the spatial
arrangement of the halogen and the developing negative charge on the bridging oxygen
can be expected to be similar during the reaction course even though the distances will
be increasing. Thus, this repulsive interaction may contribute into the energy of all states
along the reaction coordinates, including TS, leading to the significant destabilization of
TS for (S)-isomer compared to its (R)-counterpart.
This suggests a possible contributing factor to the “downward shift” of the dihalo line.
In (β,γ)-CXY-dNTP (X, Y = Hal) either X or Y would inevitably have an (S)-configuration, and
would be located within close proximity to the anion-to-be-formed. The halogen atom in
(S)-isomer, not surprisingly, will be located closer to the accumulating negative charge
compared to halogen in (R)-position, resulting in the placement of (S)-isomers on the LFER
plot closer to the dihalo line compared to (R)-(β,γ)-CHF-dGTP or (R)-(β,γ)-CHCl-dTTP.
18

Figure 1.7. Active site of DNA pol β incorporating (A) (R)-isomer and (B) (S)-isomer of
(β,γ)-CHCl-dGTP
Experimental Section
Materials and Methods
Tetraisopropyl methylenebisphosphonate (TiPMBP) was generously provided by
Albright and Wilson Americas, Inc. All other reagents and solvents were obtained from
Sigma-Aldrich, VWR, or Alfa Aesar. Preparative HPLC was performed using a Varian
ProStar equipped with a Shimadzu SPD-10A UV detector (0.5 mm path length) with
detection at 260 nm. Strong Anion Exchange (SAX) HPLC was performed using a Macherey
Nagel 21.4 mm  250 mm SP15/25 Nucleogel column, C-18 HPLC was performed using a
Phenomenex Luna 5u C18 (21.20 mm  250 mm). Mass spectrometry was performed on
a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the
negative ion mode. LC-MS utilized Finnigan Survey or PDA 158 Plus detector (1 cm path
length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7. The final
products, obtained as triethylammonium salts, were quantitated by UV absorbance using
(A)
(B)
19

the extinction coefficient (λ = 260 nm, ε= 9600 M
-1
*cm
-1
) of the natural dTTP in phosphate
buffer at pH 7.0.
1
H,
13
C and
31
P NMR spectra were obtained on Varian 400-MR or VNMRS-500
spectrometers.
13
C and
31
P NMR spectra were proton-decoupled. All chemical shifts (δ)
are reported in parts per million (ppm) relative to internal CH 3OH in CD 3OD (δ 3.34,
1
H
NMR), CHCl 3 in CDCl 3 (δ 7.26,
1
H NMR), HDO in D 2O (δ 4.80,
1
H NMR), external 85% H 3PO 4
(δ 0.00,
31
P NMR) or internal CDCl 3 (δ 77.00,
13
C NMR). NMR spectra processing was
performed with MestReNova 9.0.0. Multiplicities are reported using the following
abbreviations: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad resonance.
Tetraisopropyl (dichloromethylene)bis(phosphonate) (2)
33a

An aqueous solution of NaOCl (60 g, 5.25%, 42 mmol) was cooled in an ice bath, and
tetraisopropyl (methylene)bis(phosphonate) (2g, 5.8 mmol) was added dropwise under
vigorous stirring. The ice bath was removed, and r.m. was stirred for 20 min at r.t. The
cloudy r.m. was extracted with hexanes (5  20 mL). Combined organic layers were dried
over MgSO 4, filtered, and volatiles were evaporated under reduced pressure. Product 2
was obtained as a white crystalline solid (2.19 g, 91%).
1
H NMR (400 MHz, CDCl 3): δ 5.01-
4.93 (m, 4H, 4  CH(CH 3) 2), 1.42 (d, J = 6.2 Hz, 24H, 4  CH(CH 3) 2)).
31
P NMR (162 MHz,
CDCl 3): δ 6.70 (s, 2P). Lit. data:
33a

1
H NMR (270 MHz, CDCl 3): δ 4.95 (m, 4H), 1.38 (d, J = 6
Hz).
31
P NMR (109 MHz, CDCl 3): δ 7.3 (s, 2P).
20

Tetraisopropyl (chloromethylene)bis(phosphonate) (3)
33a

A solution of Na 2SO 3 (2.48 g, 19.7 mmol) in water (65 mL) was added to an ice cold
solution of 2 (2.19 g, 5.3 mmol) in EtOH (16 mL). The reaction mixture was stirred at r.t.
for 1 h. The product was extracted with CHCl 3 (5  10 mL), and the combined organic
layers were dried over Na 2SO 4. After evaporation of solvent, product 3 was collected as a
clear colorless oil (2.0 g, >99%), which was used without further purification.
1
H NMR (400
MHz, CDCl 3): δ 4.93-4.80 (m, 4H, 4  CH(CH 3) 2), 3.89 (t, J = 17.7 Hz, 1H, CHCl), 1.39 (m,
24H, 4  CH(CH 3) 2)).
31
P NMR (162 MHz, CDCl 3): δ 11.56 (s, 2P). Lit. data:
33a

1
H NMR (270
MHz, CDCl 3): δ 4.83 (m, 4H), 3.85 (t, J = 17.5 Hz), 1.35 (d, J = 6.5 Hz, 24H).
31
P NMR (109
MHz, CDCl 3): δ 12.2 (s, 2P).
(Chloromethylene)bis(phosphonic acid) (4)
Solution of compound 3 (2.0 g, 5.28 mmol) in HCl (30 mL, conc.) was refluxed for 5 h.
After removal of solvent the product was thoroughly dried under high vacuum at 60 ⁰C,
yielding 4 as a white crystalline solid (1.11 g, >99%), which was used without further
purification.
1
H NMR (400 MHz, D 2O): δ 4.02 (t, J = 16.2 Hz, 1H, CHCl).
Tetramethyl (chloromethylene)bis(phosphonate) (5)
37

Trimethyl orthoformate (8 mL, 73 mmol) was added to 4 (650 mg, 3.1 mmol), and the
r.m. was set to reflux. After the solution became clear, the r.m. was cooled down, and
volatiles were evaporated at reduced pressure. The product was dried under reduced
pressure, yielding 5 as a yellowish viscous oil (753 mg, 91%).
1
H NMR (400 MHz, CDCl 3): δ
4.09 (t, J = 17.7 Hz, 1H, CHCl), 3.95-3.90 (m, 12H, 4  CH 3).
31
P NMR (162 MHz, CDCl 3): δ
21

15.39 (s, 2P). Lit. data:
33b

1
H (400 MHz, CDCl 3): δ 4.0 (t, J = 18 Hz, 1H), 3.7 (m, 12H);
31
P
(101 MHz, CDCl 3): δ 15.5.
Methyl hydrogen (chloro(dimethoxyphosphoryl)methyl)phosphonate (6)
34

To a solution of 5 (360 mg, 1.35 mmol) in acetonitrile (2 mL) triethylamine (2 mL) was
added, and r.m. was refluxed for 15 min. After cooling down, all volatiles were
evaporated, yielding 6 as a triethylmethylammonium salt (494 mg, 99%).
1
H NMR (400
MHz, D 2O) δ 4.41 (dd, J = 17.2, 15.9 Hz, 1H, CHCl), 3.92 (d, J = 11.1 Hz, 3H, OCH 3), 3.91 (d,
J = 11.1 Hz, 3H, OCH 3), 3.71 (d, J = 10.8 Hz, 3H, OCH 3), 3.33 (q, J = 7.3 Hz, 6H, 3  CH 2CH 3),
2.95 (s, 3H, NCH 3), 1.35 – 1.30 (3  t, J = 7.3 Hz, 12H, 3  CH 2CH 3).
31
P NMR (162 MHz, D 2O)
δ 21.89 (d, J = 3.6 Hz, 1P), 8.78 (d, J = 3.6 Hz, 1P).
Methyl (2S)-2-(((chloro(dimethoxyphosphoryl)methyl)(methoxy)phosphoryl)oxy)-2-
phenylacetate (7)
29

A solution of the triethylmethylammonium salt of 6 (735 mg, 2.0 mmol) in methanol
was loaded onto a DOWEX H
+
column and eluted with methanol. Acidic fractions were
collected, combined, and evaporated to dryness. The resulting acid (510 mg, 2.0 mmol)
was dissolved in anhydrous dioxane (2 mL). To this solution, triphenylphosphine (802 mg,
3.1 mmol) and (R)-(-)-methylmandelate (507 mg, 3.1 mmol) was added, followed by DIAD
(600 µL, 3.1 mmol). The reaction mixture was stirred under N 2 overnight. All volatiles were
removed under vacuum. R.m. was partitioned between EtOAc and 5% aqueous solution
of NaHCO 3. The aqueous phase was extracted with EtOAc (3  15 mL), the combined
organic layers were dried over Na 2SO 4 and concentrated under vacuum. The residue was
22

taken up in a 1:5 mixture of hexanes – EtOAc (15 mL). Precipitated PPh 3O was removed
by filtration, the solution was concentrated again, and the product was purified by column
chromatography on silica gel (hexanes : EtOAc = 1 : 5), giving 407 mg of 7 (51%) as a
colorless oil as a mixture of 2 diastereomers.
1
H NMR (500 MHz, CDCl 3): δ 7.51-7.38 (m,
5H, C 6H 5), 5.98-5.94 (m, 1H, CHPh), 4.48-4.34 (m, 1H, CHCl), 4.05-3.68 (m, 12H, 4  OCH 3).
31
P NMR (202 MHz, CDCl 3): δ 15.77-14.53 (m, 2P). Lit. data:
29

31
P NMR (202 MHz; CDCl 3):
δ 16-15 (m).
(Chloro(hydroxy((S)-2-methoxy-2-oxo-1-phenylethoxy)phosphoryl)methyl)phosphonic
acid (8)
To a solution of 7 (72 mg, 0.18 mmol) in acetonitrile (5 mL) BTMS (80 µL, 0.6 mmol)
was added. The reaction mixture was stirred at r.t. and monitored by MS. After 2 h,
volatiles were removed under vacuum, and the residue was taken up in methanol. The
solution was stirred at r.t. for 15 min. The solvent was evaporated to dryness, and the
residue was thoroughly dried to yield 8a/b as a clear viscous oil (mixture of 2
diastereomers). The product was identified by MS (m/z): calcd for C 10H 12ClO 8P 2
-
357.0 [M-
H]
-
, found 357.0 [M-H]
-
, and was not further characterized. It was used in the next step
without further purification.
Sodium (2S)-2-(((chloro(phosphono)methyl)(hydroxy)phosphoryl)oxy)-2-phenylacetate
(9)
29

Crude 8a/b was dissolved in H 2O (10 mL) and the pH was adjusted to 8 using Na 2CO 3
(solid). The r.m. was washed with DCM to remove residual PPh 3O, and the aqueous phase
23

was collected and stirred overnight. The solvent was evaporated to dryness, yielding 9a/b
(mixture of 2 diastereomers) as a white solid (46 mg, 70% over 2 steps).
1
H NMR (500
MHz, D 2O): δ 7.57-7.39 (m, 5H, C 6H 5), 5.60-5.57 (m, 1H, CHPh), 3.76-3.69 (m, 1H, CHCl).
31
P NMR (202 MHz, D 2O): δ 13.86 (app. s., 1P, major isomer), 13.48 (d, J = 4.2 Hz, 1P, minor
isomer), 9.41 (d, J = 3.9 Hz, 1P, major isomer), 9.31 (d, J = 4.9 Hz, 1P, minor isomer).
((1R)-chloro(hydroxy((S)-2-morpholino-2-oxo-1-
henylethoxy)phosphoryl)methyl)(morpholino)phosphinic acid (10a)
29

To the solution of 9a/b (0.1 mmol) in 1:1 mixture of t-BuOH and H 2O (10 mL)
morpholine (70 µL, 0.8 mmol) was added. pH was adjusted to 2 using 10% HCl. The r.m.
was stirred at r.t. for 15 min, then set to reflux. DCC (412 mg, 2 mmol) was dissolved in 3
mL of t-BuOH and added to the reaction mixture in aliquots over 2 h. After cooling down
to r.t. all volatiles were evaporated, and residue was re-suspended in H 2O (2 mL). The
precipitate was filtered out, the resulting solution was concentrated again, and subjected
to separation by RP HPLC (0.1 M TEAB:15% MeCN, pH = 7.4). The two isomers were
collected separately. The first eluting (fast) isomer 10a eluted at 19.2 min and was
obtained as a triethylammonium salt (0.021 mmol, 21%).
1
H NMR (400 MHz, D 2O): δ 7.57-
7.46 (m, 5H, C 6H 5), 6.22 (d, J = 8.5 Hz, 1H, CHPh), 3.82 (dd, J = 16.1 Hz, J = 14.8 Hz, 1H,
CHCl), 3.75-3.12 (m, 16H, morpholine).
31
P NMR (162 MHz, D 2O) (chemical shifts and
coupling constants determined via simulation): δ 11.03 (d, J = 14.0 Hz, 1P), 10.93 (d, J =
14.0 Hz, 1P). MS (m/z): calcd for C 17H 24ClN 2O 8P 2
-
481.1 [M-H]
-
, found 481.0 [M-H]
-
. Lit.
data:
29

1
H NMR (500 MHz; D 2O): δ 7.55-7.46 (m, 5H), 6.20 (d, J = 8.7 Hz, 1H), 3.82-3.76 (m,
24

5H), 3.63 (m, 8H), 3.16-3.14 (m, 4H).
31
P NMR (202 MHz; D 2O): δ 11.79-11.63 (d, J = 5.3
Hz, 2P).
((1S)-chloro(hydroxy((S)-2-morpholino-2-oxo-1-
henylethoxy)phosphoryl)methyl)(morpholino)phosphinic acid (10b)
29

Following the procedure for synthesis and separation of 10a, HPLC separation
provided second eluting (slow) isomer 10b eluting at 21.7 min. It was obtained as
triethylammonium salt (0.033 mmol, 33%).
1
H NMR (400 MHz, D 2O): δ 7.58-7.45 (m, 5H,
C 6H 5), 6.18 (d, J = 8.6 Hz, 1H, CHPh), 3.83 (dd, J = 15.9 Hz, J = 14.7 Hz, 1H, CHCl), 3.74-3.14
(m, 16H, morpholine).
31
P NMR (162 MHz, D 2O): δ 11.70 (d, J = 4.5 Hz, 1P), 10.93 (d, J =
4.5 Hz, 1P). MS (m/z): calcd for C 17H 24ClN 2O 8P 2
-
481.1 [M-H]
-
, found 481.1 [M-H]
-
. Lit.
data:
29

1
H NMR (500 MHz; D 2O): δ 7.56-7.45 (m, 5H), 6.16 (d, J = 8.6 Hz, 1H), 3.84-3.78 (m,
5H), 3.65-3.63 (m, 8H), 3.16-3.13 (m, 4H).
31
P NMR (202 MHz; D 2O): δ 12.46 (d, J = 4.5 Hz,
1P), 11.67 (d, J = 4.6 Hz, 1P).
((1R)-chloro(hydroxy((S)-2-morpholino-2-oxo-1-
phenylethoxy)phosphoryl)methyl)phosphonic acid (11a)
29

A solution of 10a (0.021 mmol) in H 2O (8 mL) was stirred with DOWEX H
+
(2 mL) for
30 min at r.t. After DOWEX was removed by filtration, 4 drops of 1M HCl were added and
the r.m. was stirred for another 30 min. All solvents were removed by evaporation,
yielding 11a, which was used without further purification.
1
H NMR (500 MHz, CD 3OD): δ
7.57-7.41 (m, 5H, C 6H 5), 6.35 (d, J = 7.9 Hz, 1H, CHPh), 4.24 (app. t, J = 17.1 Hz, 1H, CHCl),
25

3.70-3.24 (m, 8H, morpholine).
31
P NMR (202 MHz, CD 3OD): δ 12.43 (app. s, 1P), 11.25
(app. s, 1P). MS (m/z): calcd for C 13H 17ClNO 8P 2
-
412.0 [M-H]
-
, found 412.1 [M-H]
-
.
((1S)-chloro(hydroxy((S)-2-morpholino-2-oxo-1-
phenylethoxy)phosphoryl)methyl)phosphonic acid (11b)
29

Following the procedure for synthesis of 11a, 10b (0.033 mmol) was hydrolyzed to
form 11b, which was used without further purification.
1
H NMR (500 MHz, CD 3OD): δ 7.56-
7.42 (m, 5H, C 6H 5), 6.37 (d, J = 7.8 Hz, 1H, CHPh), 4.29 (app. t, J = 17.2 Hz, 1H, CHCl), 3.70-
3.25 (m, 8H, morpholine).
31
P NMR (202 MHz, CD 3OD): δ 12.67 (d, J = 4.6 Hz, 1P), 11.11
(d, J = 4.4 Hz, 1P). MS (m/z): calcd for C 13H 17ClNO 8P 2
-
412.0 [M-H]
-
, found 412.3 [M-H]
-
.
((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methyl morpholinophosphonate
36

Sodium salt of dTMP monohydrate (175 mg, 0.455 mmol) was dissolved in a 1:1
mixture of t-BuOH – H 2O (12 mL). The pH was adjusted to 2 with 0.1 HCl, and distilled
morpholine (118 μL, 1.365 mmol) was added dropwise. R.m. was stirred at r.t. for 15 min,
then set to reflux. A solution of DCC (281 mg, 1.365 mmol) in t-BuOH (3 mL) was added
over the period of 2 h. After cooling down to r.t. all volatiles were evaporated under
reduced pressure, and the residue was re-suspended in H 2O (2 mL). The precipitate was
filtered out, and the solution was evaporated to dryness. The resulting product was used
in the following steps without further purification.
26

5'-O-[({[(S)-chloro{hydroxy[(1S)-2-(morpholin-4-yl)-2-oxo-1-
phenylethoxy]phosphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-
deoxythymidine (12a)
29

11a was dissolved in EtOH (2 mL). A solution of Bu 3N in EtOH (1:10) was added to
adjust pH to 4.5. The mixture was stirred at r.t. for 30 min, evaporated to dryness and co-
evaporated with DMF 3 times before being dissolved in dry DMSO (1 mL). To this solution,
solution of dTMP in dry DMSO (1 mL) was added, and the reaction mixture was stirred at
r.t. for 72 h. Products have been purified using SAX HPLC (0-0.5 M TEAB gradient in 18
min, pH = 7.4) giving 12a (eluting at 14.8 min) as a triethylammonium salt.
1
H NMR (500
MHz, D 2O): δ 7.67 (d, J = 1.1 Hz, 1H, CH-T), 7.56-7.43 (m, 5H, C 6H 5), 6.39 (t, J = 7.0 Hz, 1H,
CH-T), 6.24 (d, J = 8.7 Hz, 1H, CHPh), 4.64-4.62 (m, 1H, H-3’), 4.19-4.17 (m, 3H, H-4’, H-5’),
4.10 (dd, J = 17.2 Hz, J = 16.2 Hz, 1H, CHCl), 3.74-3.26 (m, 8H, morpholine), 2.34-2.31 (m,
2H, H-2’), 1.91 (d, J = 1.0 Hz, 1H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ 10.14 (d, J = 9.3 Hz,
P γ), 1.83 (dd, J = 26.7, J = 9.4 Hz, P β), -11.36 (d, J = 26.6 Hz, P α). MS (m/z): calcd for
C 23H 30ClN 3O 15P 3
-
716.1 [M-H]
-
, found 715.8 [M-H]
-
.
5'-O-[({[(R)-chloro{hydroxy[(1S)-2-(morpholin-4-yl)-2-oxo-1-
phenylethoxy]phosphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-
deoxythymidine (12b)
29

Following the procedure for synthesis of 12a, 12b have been prepared from 11b
(eluting at 15.1 min) as triethylammonium salt.
1
H NMR (500 MHz, D 2O): δ 7.63 (s, 1H,
CH-T), 7.56-7.44 (m, 5H, C 6H 5), 6.39 (t, J = 7.0 Hz, 1H, CH-T), 6.22 (d, J = 8.2 Hz, 1H, CHPh),
27

4.64-4.62 (m, 1H, H-3’), 4.23-4.08 (m, 4H, CHCl, H-4’, H-5’), 3.78-3.39 (m, 8H, morpholine),
2.33-2.31 (m, 2H, H-2’), 1.89 (s, 1H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ 10.59 (d, J = 8.9
Hz, P γ), 2.04 (dd, J = 26.3, J = 8.8 Hz, P β), -11.27 (d, J = 26.4 Hz, P α). MS (m/z): calcd for
C 23H 30ClN 3O 15P 3
-
716.1 [M-H]
-
, found 716.0 [M-H]
-
.
5'-O-[({[(S)-
chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-
deoxythymidine (1a)
29

12a was dissolved in 3 mL of 0.1 M TEAB:MeOH (1:1). The pH was adjusted to 8 with
CO 2 (gas). Pd/C (2 mg) was added, and the r.m. was stirred under 1 atm H 2 for 3 h. The
catalyst was filtered out, and product was purified by RP HPLC (0.1 M TEAB:4.5% MeCN,
pH = 7.4) to obtain 1.76 mg of 13a as a colorless film (16% over 4 steps).
1
H NMR (500
MHz, D 2O): δ 7.70 (d, J = 1.1 Hz, 1H, CH-T), 6.40 (t, J = 7.0 Hz, 1H, CH-T), 4.69-4.66 (m, 1H,
H-3’), 4.26-4.19 (m, 3H, H-4’, H-5’), 3.92 (dd, J = 16.1 Hz, J = 15.7 Hz, 1H, CHCl), 2.41-2.32
(m, 2H, H-2’), 1.93 (s, 1H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ 8.59 (s, P γ), 6.79 (d, J = 27.2
Hz, P β), -11.12 (d, J = 27.2 Hz, P α). MS (m/z): calcd for C 11H 17ClN 2O 13P 3
-
513.0 [M-H]
-
, found
513.0 [M-H]
-
.
5'-O-[({[(R)-
chloro(phosphono)methyl](hydroxy)phosphoryl}soxy)(hydroxy)phosphoryl]-2'-
deoxythymidine (1b)
29

Following the procedure for synthesis of 1a, 2.09 mg of 1b has been prepared as a
triethylammonium salt (13% over 4 steps).
1
H NMR (500 MHz, D 2O): δ 7.68 (d, J = 1.1 Hz,
28

1H, CH-T), 6.40 (t, J = 7.0 Hz, 1H, H-1’), 4.69-4.67 (m, 1H, H-3’), 4.28-4.18 (m, 3H, H-4’, H-
5’), 3.91 (dd, J = 16.6, J = 15.2 Hz, 1H, CHCl), 2.41-2.31 (m, 2H, H-2’), 1.93 (s, 3H, CH 3-T).

31
P NMR (202 MHz, D 2O) δ 8.58 (d, J = 6.3 Hz, P γ), 6.76 (dd, J = 27.4, J = 6.3 Hz, P β), -11.10
(d, J = 27.2 Hz, P α). MS (m/z): calcd for C 11H 17ClN 2O 13P 3
-
513.0 [M-H]
-
, found 513.0 [M-H]
-
.

29

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(24) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martinek, V.; Xiang, Y.; Beard, W.
A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F.,
Biochemistry 2007, 46, 461-471.
(25) Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.;
McKenna, C. E.; Goodman, M. F., Biochemistry 2014, 53, 1842-1848.
(26) (a) Batra, V. K.; Shock, D. D.; Beard, W. A.; McKenna, C. E.; Wilson, S. H., Proc. Natl. Acad. Sci.
U. S. A. 2012, 109, 113-118, S113/111-S113/111; (b) Batra, V. K.; Pedersen, L. C.; Beard, W. A.;
Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E., J. Am. Chem. Soc.
2010, 132, 7617-7625; (c) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman,
M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H., J. Am. Chem. Soc. 2007, 129, 15412-15413; (d)
Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E., J. Org. Chem. 2011, 76, 5132-
5136.
(27) Braun-Sand, S.; Olsson, M. H. M.; Warshel, A., Adv. Phys. Org. Chem. 2005, 40, 201-245.
(28) Schweins, T.; Geyer, M.; Kalbitzer, H. R.; Wittinghofer, A.; Warshel, A., Biochemistry 1996,
35, 14225-14231.
31

(29) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.; Wilson, S. H.;
McKenna, C. E., J. Am. Chem. Soc. 2012, 134, 8734-8737.
(30) Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard, W. A.; Wilson, S.
H.; McKenna, C. E.; Goodman, M. F., Biochemistry 2012, 51, 8491-8501.
(31) (a) Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D., Nucleic Acids Res. 2006, 34,
564-574; (b) Kool, E. T., Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 1-22.
(32) Bommarito, S.; Peyret, N.; SantaLucia, J., Jr., Nucleic Acids Res. 2000, 28, 1929-1934.
(33) (a) McKenna, C. E.; Khawli, L. A.; Ahmad, W. Y.; Pham, P.; Bongartz, J. P., Phosphorus Sulfur
1988, 37, 1-12; (b) Hutchinson, D. W.; Semple, G., J. Organomet. Chem. 1986, 309, C7-C10.
(34) Ahlmark, M. J.; Vepsalainen, J. J., Tetrahedron 1997, 53, 16153-16160.
(35) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C., Tetrahedron Lett. 1977, 155-158.
(36) Moffatt, J. G.; Khorana, H. G., J. Am. Chem. Soc. 1961, 83, 649-658.
(37) Nicholson, D. A.; Cilley, W. A.; Quimby, O. T., J. Org. Chem. 1970, 35, 3149-3150.

32

Chapter 2 - Synthesis of (α,β)/(β,γ)-CH
2
/NH “Met-Im” Analogues of dTTP
(The research discussed in this chapter has been substantially published
1
)
2.1 Inhibitors of DNA pol β
As DNA pol β helps cancer cells tolerate DNA damage through BER,
2
its selective
inhibition is considered a promising approach towards increasing the efficacy of anti-
cancer drugs.
3
In the past two decades, many inhibitors of pol β have been identified,
including fatty acids, polar lipids, triterpenoids, pamoic acid and ribonucleoside analogues
(Figure 2.1).
4
Indeed, some of the known inhibitors have been shown to enhance
cytotoxicity of bleomycin in cultured mammalian cells.
5

Figure 2.1. Structures of selected inhibitors of DNA pol β
The majority of known inhibitors bind to the active site of the lyase domain of pol β
instead of polymerase domain (Table 2.1). In addition, most of the known inhibitors lack
33

the desired activity and selectivity.
4
New potent and selective inhibitors of the
polymerase domain of pol β may inhibit BER more efficiently, significantly improving the
efficacy and reducing the dosage of anti-cancer drugs.
Table 2.1. Selected inhibitors of DNA pol β
Inhibitor Binding Site Affinity Reference
Linoleic acid Lyase domain IC 50 = 38 µM
6

Nervonic acid Lyase domain IC 50 = 5.8 µM
6

Sulfolipid 1 Not identified IC 50 = 3 µg / mL
7

KN-208 Not identified K i = 0.05 µM
8

Oleanolic acid Not identified IC 50 = 7.5 µM
9

Pamoic acid Lyase domain K D = 9 µM
10

Bredinine 5’-MP Polymerase domain IC 50 = 20 µM
11

2.2 Design of novel DNA pol β substrate mimic inhibitors
Our purpose was to synthesize dNTP mimics, which would inhibit the polymerase
domain of DNA pol β. Substrate mimics or transition state analogues often possess good
activity due to the pre-designed optimal interactions with residues in the active site of an
enzyme.
12
Such mimics should not be substrates themselves; for that purpose, (α,β)-non-
hydrolyzable dNTP analogues
13
are good candidates. If non-hydrolysable CXY (X,Y = H, Hal,
CH 3), or NH group replaces the P α–O–P β bridging oxygen, the turnover is blocked, and
thus, the dNTP analogue is not a substrate but an inhibitor of pol β (Figure 2.2A). Activities
34

K i of these inhibitors depend on the stereoelectronic properties of P α–Z–P β group (Z = CXY
or NH).
13-14

In contrast, if CXY (X,Y = H, Hal, CH 3) or NH replaces bridging oxygen in P β–O–P γ of
dNTPs (Figure 2.2B), the resulting analogues are substrates of pol β. Turnover results in
elimination of bisphosphonate instead of pyrophosphate, the byproduct of incorporation
of natural dNTPs. As was discussed in Chapter 1, the stereoelectronic properties of the
bisphosphonate leaving group affecting the rate of the DNA synthesis suggests that
chemical transformation is the RDS in DNA extension,
15
such that the reaction rates (k pol)
of (β,γ)-CXY-dNTP analogues depend on the stereoelectronic properties of the X and Y
substituents.
15-16

O
OH
O P
O
-
O
B
Z
P
O
O
-
O
P
O
O
-
-
O
(B)
Z = NH or CXY (X, Y = H, halo, CH
3
)
O
OH
O P
O
-
O
B
O
P
O
O
-
Z
P
O
O
-
-
O
(A)

Figure 2.2. Singly bridge-modified dNTP analogues as (A) inhibitors and (B) substrates
of pol β
13-16

Due to the location of the (α,β)-bridging atom of dNTP near the transformation locus,
sterically demanding groups are generally not well tolerated in this position, resulting in
large K i values for corresponding dNTP analogues.
13
Substituents at the (β,γ)-position are
better tolerated, and can potentially be used to tune K i values of dNTP mimics. One
possibility to improve the affinity of dNTP mimic inhibitors to pol β is to enforce its
interaction with the R183 residue, located close to the P β–O–P γ bridging oxygen in the
35

active site of pol β.
17
To achieve this favorable interaction, the modifier capable of H-
bonding can be introduced to the (β,γ)-position to “anchor” to R183.
16a
The oxygen
linkage in P β–O–P γ of the (α,β)-non-hydrolysable dNTPs (Figure 2.2A), however, cannot be
modified. Replacing this oxygen with CXY or NH creates a versatile site potentially capable
of incorporation of an H-bond acceptor “anchoring” R183. Thus, we chose to target
potential inhibitors that have non-hydrolysable NH and CHX moieties replacing P α–O–P β
and P β–O–P γ oxygen linkages.
Bond lengths and angles of the P–C–P moiety differ substantially from those of the
natural P–O–P linkages.
18
Using CH 2 to replace both the (α,β)- and (β,γ)-linkage may
deviate the geometry of its ternary complex with pol β from that normally adopted with
the natural dNTPs.
19
Yet another concern with a doubled CH 2 linkage replacing (α,β)- and
(β,γ)-linkages is the difference in polarity between CH 2 and O.
20
A double CF 2 linkage had
been previously considered to address this problem, however, when a CF 2 group replaced
both oxygen bridges in dTTP, the pol β affinity was drastically decreased.
21
A similar
approach can be considered with a double replacement of P–O–P by a CHF linkage,
however, such modification introduces complicated stereochemical effects as new chiral
centers are generated at the (α,β)- and (β,γ)-CHF carbon atoms. Besides, this limits the
number of potential modifications to a single (α,β),(β,γ)-bisCHF-dNTP. Analysis of the
crystal structures of the ternary complexes of (α,β)- or (β,γ)-CH 2-dNTP with pol β and
DNA
22
suggests that the structural perturbations introduced by a single CH 2 linkage can
be accommodated.
13
On the other hand, P–N–P geometrical parameters closely resemble
36

those of the natural P–O–P linkage.
18
Therefore, substituting both the (α,β)- and (β,γ)-
linking oxygens with imido linkages does not significantly perturb the geometry of the
ternary complex with the enzyme. However, substitution of both P–O–P with P–N–P
linkage limits options for modifications relative to the more synthetically versatile
methylene group. We therefore considered two new scaffolds to explore the inhibition of
pol β, in which the P α–O–P β and P β–O–P γ oxygens are replaced by either a methylene (CH 2)
or an imido (NH) group in alternation: “Met-Im” nucleotides (Figure 2.3).

Figure 2.3. (A) (α,β)-CH 2-(β,γ)-NH-dTTP (1a) and (B) (α,β)-NH-(β,γ)-CH 2-dTTP (1b)
“Met-Im” nucleotides
2.3 Synthesis of “Met-Im” Nucleotides
Our approach to the synthesis of “Met-Im” nucleotides centered on the construction
of the pentaalkyl phosphorimido-bisphosphonate synthons 4a and 4b (Scheme 2.1).
Regioisomers 4a and 4b have terminal (EtO) 2P(O) or (MeO) 2P(O) groups connected via a
NBn or CH 2 bridge to a central EtOP(O). Benzyl (Bn) protection was chosen as it can be
removed by catalytic hydrogenolysis without affecting other sensitive functional groups.
Regioselective removal of one terminal methyl with amines or NaI would expose a free
P(O)OH, which would become the P α in the final dNTP analogue product (1a or 1b) after
coupling to thymidine and deprotection.
37

Scheme 2.1. Flip Synthesis of 4a/b

To synthesize 4b, we initially prepared precursor 3b from dimethyl
benzylphosphoramidate 2b and ethyl methylphosphonochloridate
23
in the presence of t-
BuOK (Scheme 2.1). Phosphoramidate 2b
24
was conveniently synthesized from readily
available dimethyl phosphite and benzylamine in a presence of CCl 4. Next, we envisioned
converting the 3b into 4b by reacting its lithium salt with diethyl chlorophosphate.
Unexpectedly, the LDA-promoted reaction of 3b with diethyl chlorophosphate proceeded
with the formation of the regioisomer 4a (Scheme 2.1). We hypothesized that the
transformation occurred via an anionic 1,3-rearrangement
25
of the carbanion, formed
upon the reaction of 3b with LDA. The driving force of this N C rearrangement
25a-e
is
likely the formation of the more stable N-anion via a rapid migration of the
dimethoxyphosphoryl group to the anionic carbon, and the following reaction of the
phosphoramide anion with diethyl chlorophosphate results in the formation of the
“flipped” product 4a (Scheme 2.2).
1

This result suggested that the original intermediate target, 4b, could be obtained via
the same “flip” chemistry by swapping the ethyl groups in (EtO) 2P(O)Cl with the methyl
38

groups in 3b. Indeed, 3a has reacted with (MeO) 2P(O)Cl under similar conditions, yielding
4b (Scheme 2.1). It should be noted that no non-flipped product was detected in
syntheses of 4a and 4b, suggesting that the rearrangement occurs very fast.
Scheme 2.2. [1,3] N C Migration Mechanism

The isomers 4a and 4b were characterized by
1
H,
31
P, and
13
C NMR spectra and by MS.
The
31
P resonance at δ 22.7 ppm corresponds to a (MeO) 2P(O)CH 2 in 4a (Δδ +3 ppm
relative to (EtO) 2P(O)CH 2 in 4b), both coupled (J PP = 4 Hz) to the same phosphorus at δ
≈23 ppm, assigned to the central P(O)OEt. The most upfield resonance (δ 3 ppm) is then
(EtO) 2P(O)NBn in 4a, with the (MeO) 2P(O)NBn resonance in 4b observed 3 ppm downfield
(both with J PP = 20 Hz to the central phosphorus nucleus).
The structures of 4a and 4b were unambiguously determined by
1
H–
31
P gHMBC NMR
(Figure 2.4). For 4a (Figure 2.4A) a strong cross-peak is detected for the protons of two
OCH 3 groups (3.84 and 3.79 ppm) and the phosphonate
31
P resonance (22.7 ppm), while
no cross-peak of these methyl esters is observed with the terminal phosphorimide
31
P
resonance (2.79 ppm). In addition, for phosphorimide
31
P resonance cross-peaks are
observed with methylene protons of ethyl esters (4.2, 4.0, and 3.8 ppm (the last signal
O
P
N
Bn
EtO
H
3
C
P
O
OMe
OMe
LDA, (EtO)
2
POCl
THF
O
P
N
Bn
P
O
OEt
OEt
EtO
P
O
MeO
MeO
O
P
N
H
P
O
-
O
-
O
O
-
P
O
O
-
O O
OH
T
O
P
N
Bn
EtO
-
H
2
C
P
O
OMe
OMe
O
P
NBn
OEt
LDA (EtO)
2
POCl
P
O
MeO
MeO
O
P
N
Bn
P
O
OEt
OEt
EtO
P
O
HO
MeO
TEA
MeCN
3b 4a
39

overlaps with OCH 3 group resonance)). In contrast, the
1
H–
31
P gHMBC NMR of 4b (Figure
2.4B) reveals a strong cross-peak for the OCH 3 protons (3.70 and 3.54 ppm) and
phosphorimide
31
P resonance (6 ppm), while no cross-peak is observed for these protons
and the terminal phosphonate
31
P resonance (19.65 ppm). Phosphonate
31
P resonance
shows a cross-peak with the methylene protons of ethyl groups (4.3-4.2 and 4.0 ppm).

Figure 2.4.
1
H –
31
P gHMBC NMR (CDCl 3, 500 MHz) of (A) 4a and (B) 4b
The following procedure was utilized for synthesis of both 1a and 1b (Scheme 2.3). A
single methyl group from 4a or 4b was removed by TEA under reflux in acetonitrile.
26

Reaction progress was monitored by
31
P NMR. The resulting salts were converted to the
corresponding acids (5a or 5b) by DOWEX H
+
. Acids 5a and 5b were coupled with
thymidine under Mitsunobu conditions
27
to form 6a or 6b. We used DMF as a solvent
since thymidine is not soluble in conventional Mitsunobu solvents such as THF or dioxane.
Debenzylation of 6a and 6b by catalytic hydrogenolysis resulted in the formation of
immediate precursors 7a or 7b (Scheme 2.3).
16b
It is interesting to note that the range of
the chemical shifts for the phosphorus signals in
31
P NMR gets narrower for diastereomers
(A)
(B)
40

of compounds 7a/b compared to 6a/b due to absence of the shielding/deshielding effects
of an adjacent benzyl group present in 6a/b. This effect is similar to the one used by
Mosher to deduce the absolute configuration of chiral amines and alcohols,
28
and was
recently shown by Blazewska et al. to have a similar application for assigning the absolute
configuration chiral carbon in hydroxyl- and aminophosphonates.
29

Scheme 2.3. Synthesis of “Met-Im” Nucleotides 1a/b

Simultaneous removal of the methyl and three ethyl groups was achieved by
silyldealkylation with BTMS followed by neutral hydrolysis (Scheme 2.4).
30
The major
product had the anticipated MS (m/z [M-H]
-
= 478), but preliminary pol β dTTP
incorporation assay demonstrated its low (K i > 1 mM) activity. Analysis of its
1
H NMR
(Figure B.28) spectrum suggested that the major isolated product has been sufficiently
anomerized. It was previously observed that BTMS, when utilized for deprotection of
phosphorus ester groups in deoxyribonucleotides, may induce significant
anomerization.
31
A plausible explanation is that electron-withdrawing group in a 2’-
41

position of the nucleotides slows down the process of anomerization, and undesired α-
anomers are generally not observed when BTMS is applied to ribonucleotides.
32

In search for the optimal conditions for BTMS-mediated deprotection we discovered
that microwave (MW) acceleration
33
significantly improved the yield and reduced
anomerization: treatment of 7a with microwave irradiation for 30 min at 40 ⁰C followed
by hydrolysis gave 80% of the desired β-anomer 1a (Scheme 2.4), which was subsequently
purified using two-stage preparative (strong anion exchange (SAX) followed by RP)
HPLC.
16a

To unambiguously prove that conventional BTMS reaction results in formation of α-
1a, while microwave acceleration suppresses anomerization, and β-anomer is
predominantly formed,
1
H NMR spectra of the products obtained via conventional and
MW-assisted BTMS reaction were compared to those of α- and β-dTMP. Table 2.2
summarizes the main differences in the
1
H NMR spectra of α- and β-1a, and α- and β-
dTMP. H-1’ resonance is located 0.15 ppm upfield for α-anomer compared to that of β-
anomer. Particularly, the splitting pattern of H-1’ resonance is different for α- (dd, J ≈ 7
Hz, J ≈ 3 Hz) and β-anomers (app. t, J ≈ 7 Hz) of 1a. These data are consistent with the
splitting patterns of α- and β-dTMP (Table 2.2).
34
H-4’ signal is located 0.45 ppm upfield
in β-1a compared to α-1a. Another distinction is the difference between chemical shifts
of H-2’ protons in β- and α-anomers: in
1
H NMR of β-1a two signals from H-2’ protons are
clustered (2.39-2.30 ppm), while in
1
H NMR of α-1a H-2’ protons have chemical shifts
differing by ≈0.65 ppm. A similar pattern is observed for β- and α-dTMP, respectively.
42

Based on this analysis, we can confidently assign the α-configuration to product 1a
obtained by conventional BTMS reaction, and β-configuration – to product obtained with
the microwave assistance.
Scheme 2.4. Microwave irradiation reduces BTMS-promoted anomerization of 1a

Similar to 7a, deprotection of 7b was achieved in 7 min at 40 ⁰C, with minor (10%) α-
anomer formation, and the pure β-product 1b was isolated by preparative two-stage
HPLC.
Table 2.2. Main differences in
1
H NMR spectra of α- and β-anomers of dTMP and their
α- and β-1a analogues
Chemical shift, ppm
β-1a α-1a β-dTMP
34a
α-dTMP
34b

H-1’ 6.39 (t, J = 7.0 Hz) 6.24 (dd, J = 7.2, 3.1 Hz) 6.30 (t, J = 7.0 Hz) 6.12 (dd, J = 7.2, 3.0 Hz)
H-4’ 4.17-4.07 4.59-4.54 4.11-4.10 4.43
H-2’ 2.39-2.30 2.86-2.80, 2.20-2.16 2.39-2.26 2.66-2.76, 2.02-2.09

43

Results and Discussion
Novel compounds 1a and 1b were synthesized with 12% and 6% overall yields,
respectively. The structures of final products have been verified by
1
H and
31
P NMR
spectroscopy, and MS. The regiochemistry of important “flipped” intermediates 4a and
4b have been confirmed by
1
H-
31
P gHMBC NMR.
In order to estimate the ability of 1a or 1b to inhibit the incorporation of unmodified
dTTP into DNA by pol β, reactions with varying amounts of inhibitor and a constant
amount of dTTP were performed and aliquots quenched at various time points (Figure
2.5A). For each reaction time, the samples were loaded on the gel in increasing
concentration of 1a/1b. The first lane for each time, labeled –I, corresponds to the
reaction mixture without the inhibitor. The final lane for each time corresponds to the
reaction with only 1a/1b and no dTTP, showing that inhibitors cannot be incorporated
into the DNA. As expected, as the amount of inhibitors is increased in the reaction, the
rate and amount of primer extension is decreased (Figure 2.5A). For each inhibitor
concentration, an observed rate was determined and then plotted against the
corresponding concentration and fit as a hyperbolic decay curve from which the K i was
calculated (Figure 2.5B).
44

Figure 2.5. Inhibition of pol β by “Met-Im” dTTP analogues. (A) Representative gel for
inhibition by 1a. Aliquots from the reaction mixture were quenched at 5, 15, and 30 min
and the DNA run on denaturing polyacrylamide gels to separate the unextended and
extended primers. In each set, the first lane is the control (reaction without inhibitor),
followed by a series of increasing amounts. The final lane in each set is the reaction
without dTTP, showing that 1a is not incorporated by pol β into the DNA. (B) Hyperbolic
decay fits of data from inhibition reactions. Filled circles represent 1a and open circles 1b.
For each concentration of scaffold, the observed rate constant for the incorporation of
dTTP is plotted opposite the corresponding scaffold concentration. Error bars represent
the standard deviation of three independent experiments. The data are then fit to a
hyperbolic decay yielding the K i values of 2.7 µM for 1a and 23 µM for 1b. (Data obtained
by Dr. Keri Oertell in Prof. Myron Goodman’s laboratory)
The K i values for the two inhibitors differed by 9-fold (1a, 2.7 µM, 1b, 23 µM). To
explain this difference in K i, X-ray crystal structures of 1a and 1b in ternary complexes
with pol β and DNA have been solved. Analysis of the crystal structures reveals that both
1a and 1b assume conformations similar to those of the natural nucleotide (Figure 2.6).
However, 1a, having its NH group at the (β,γ)-bridging position is capable of a water-
45

mediated H-bonding interaction with R183 in the active site. The H-bond distances N 1a…O
and N Arg183…O are close to 3 Å, indicating that the H-bonding may make a large
contribution into the overall stabilization of the complex. This interaction is unavailable
for 1b as it has a methylene group at the (β,γ)-bridge, and the α,β-NH in 1b has no
apparent interactions with the proximal active site residues, suggesting, that some or all
of the observed K i difference could be attributed to this water-mediated H-bond.

Figure 2.6. 1a (left) and 1b (right) in ternary complex with pol β and DNA. R183 makes
a water-mediated H-bond with the (β,γ)-NH in 1a. A similar interaction between R183 and
the (α,β)-NH of 1b is not available. (Data obtained by Dr. Vinod Batra and Samuel Wilson,
M.D. at NIEHS)
The synthetic strategy devised to prepare 1a and 1b could be applied to the synthesis
of other derivatives of “Met-Im” nucleotides by coupling the appropriate nucleoside with
modified analogues of synthons 4a and 4b. Their synthesis would take advantage of the
[1,3] N C P(O)(OR 2) migration, which provides both desired compounds by a simple
46

swap of terminal alkyl ester groups. The observed suppression of unwanted
anomerization by microwave acceleration in the BTMS-mediated deprotection of 7a and
7b may be of general use in the removal of phosphorus ester groups in preparation of
phosphonic acids incorporating nucleoside groups.
Experimental Section
Materials and Methods
All the reagents and solvents were purchased from VWR, Sigma-Aldrich, or Alfa Aesar
and used without further purification, unless noted otherwise.
1
H,
13
C and
31
P NMR
spectra were obtained on Varian 400-MR or VNMRS-500 spectrometers.
13
C and
31
P NMR
spectra were proton-decoupled unless stated otherwise. All chemical shifts (δ) are
reported in parts per million (ppm) relative to residual CH 3OH in CD 3OD (δ 3.34,
1
H NMR),
CHCl 3 in CDCl 3 (δ 7.26,
1
H NMR), HDO in D 2O (δ 4.80,
1
H NMR), external 85% H 3PO 4 (δ
0.00,
31
P NMR) or internal CDCl 3 (δ 77.00,
13
C NMR). The concentration of the NMR
samples was 2 – 15 mg/ml. 1D and 2D NMR spectra processing was performed with
MestReNova 9.0.0. Multiplicities are reported using the following abbreviations: s =
singlet, d = doublet, t = triplet, m = multiplet, br = broad resonance.
Preparative HPLC was performed using a Varian ProStar equipped with a Shimadzu
SPD-10A UV detector (0.5 mm path length) with detection at 260 nm. Strong Anion
Exchange (SAX) HPLC was performed using a Macherey Nagel 21.4 mm  250 mm SP15/25
Nucleogel column, C-18 HPLC was performed using a Phenomenex Luna 5u C18 (21.20
47

mm  250 mm). Mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass
spectrometer equipped with an ESI source in the negative ion mode. Microwave‐assisted
synthesis was performed using a Milestone Ethos Synth Microwave Labstation.
Compound IUPAC names were assigned with the assistance of MarvinSketch 6.1.5. The
molar yields of the final products were determined by UV absorbance using the extinction
coefficient of dTTP in phosphate buffer at pH 7.0 at 260 nm, ε = 9600 M
-1
*cm
-1
.
Diethyl benzylphosphoramidate (2a)
Compound 2a was previously made by the reaction of diethyl phosphite and
benzylamine in the presence of hydrogen peroxide and a catalytic amount of I 2,
35

however, we found that it could be conveniently obtained by adapting the procedure of
Glidewell.
24

Benzylamine (2.14 g, 0.02 mol) was added to a solution of diethylphosphite (1.38 g,
0.01 mol) in CCl 4 (20 mL). A white precipitate appeared immediately, and the reaction
mixture was stirred overnight at r.t. The white precipitate was filtered, washed with
CH 2Cl 2, and volatiles were removed from the filtrate by rotary evaporation (water pump).
The residue was dried under vacuum at r.t. (<1 mm), giving 2.4 g of the product 2a as a
colorless oil (99%).
1
H NMR (500 MHz, CDCl 3): δ 7.36-7.27 (m, 5H, C 6H 5), 4.13-3.99 (m, 6H,
CH 2C 6H 5 and CH 2CH 3), 2.89 (br, 1H, NH), 1.31 (t, J = 7.1 Hz, 6H, 2 × OCH 2CH 3).
31
P NMR
(202 MHz, CDCl 3): δ 8.36. Lit. data:
35

1
H NMR (400 MHz, CDCl 3): δ 7.33-7.25 (m, 5H, C 6H 5),
4.11-3.98 (m, 6H, CH 2C 6H 5 and CH 2CH 3), 3.22 (br, 1H, NH), 1.29 (t, J = 7.1 Hz, 6H, 2 ×
OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 8.50.
48

Dimethyl benzylphosphoramidate (2b)
24

Benzylamine (3.3 g, 0.03 mol) was added to a solution of dimethyl phosphite (1.65 g,
0.015 mol) in CCl 4 (20 mL). A white precipitate appeared immediately, and the reaction
mixture was stirred overnight at r.t. The white precipitate was filtered, washed with
CH 2Cl 2, and volatiles were removed from the combined filtrate by rotary evaporation
(water pump). The residue was dried under vacuum at r.t. (<1 mm), giving 2.98 g of the
product 2b as a colorless oil that crystallized in the refrigerator (≈-20°C) but remelted on
rewarming to r.t. (92%).
1
H NMR (500 MHz, CDCl 3): δ 7.36-7.28 (m, 5H, C 6H 5), 4.10 (dd, J
= 9.7 Hz, J = 7.0 Hz, 2H, CH 2C 6H 5), 3.72 (d, J = 10.7 Hz, 6H, 2 × OCH 3), 2.88 (br, 1H, NH).
31
P
NMR (202 MHz, CDCl 3): δ 10.93. Lit. data:
24

1
H NMR (60 MHz, CCl 4) δ 7.30 (s, 5H), 5.38 (dt,
J = 11 Hz, J = 6 Hz, lH), 3.98 (dd, J = 11 Hz, J = 6 Hz, 2H), 3.58 (d, J = 11 Hz, 6H).
31
P NMR
(32 MHz, CCl 4): δ 11.7.
Ethyl methylphosphonochloridate
23

Oxalyl chloride (1.27 g, 0.01 mol) was added in one portion to a solution of diethyl
methylphosphonate (1.52 g, 0.01 mol) in 15 mL of dry CH 2Cl 2. The reaction mixture was
stirred for 16 h at r.t. The solvent was removed by evaporation, and the residue was
distilled in vacuum, yielding 1.03 g of the product as a colorless liquid (72%): bp 75-76
⁰C/14 mmHg (lit. bp 62-64 ⁰C/8 mmHg).
23

1
H NMR (500 MHz, CDCl 3): δ 4.32 (m, 1H,
CH 2CH 3), 4.23 (m, 1H, CH 2CH 3), 1.97 (d, J = 17.5 Hz, 3H, P-CH 3), 1.40 (t, J = 7.1 Hz, 3H,
CH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 40.06. Lit. data:
23

1
H NMR (300/500 MHz, CDCl 3): δ
4.41-4.17 (m, 2H, OCH 2), 1.98 (d, J = 18 Hz, 3H, P-CH 3), 1.41 (t, J = 7 Hz, 3H, CH 3).
49

Benzyl(diethoxyphosphoryl)[ethoxy(methyl)phosphoryl]amine (3a)
To a suspension of t-BuOK (800 mg, 7.16 mmol) in 15 mL of dry toluene was slowly
added a solution of compound 2a (1.16 g, 4.77 mmol) in 5 mL of dry toluene. The reaction
mixture was stirred for 5 min, and a solution of ethyl methylphosphonochloridate (1.02
g, 7.16 mmol) in 2 mL of dry toluene was added. The reaction mixture was stirred
overnight, and then quenched by addition of a saturated aqueous solution of NH 4Cl. The
product was extracted into EtOAc (3  15 mL), and the combined organic layers were
washed with brine, then dried over Na 2SO 4. After evaporation of the solvent, the residual
product was purified by column chromatography on silica gel (EtOAc:Hexanes (3:1) 
EtOAc), giving 1.176 g of 3a as a colorless oil (71%).
1
H NMR (500 MHz, CDCl 3): δ 7.49-7.21
(m, 5H, CH 2C 6H 5), 4.67-4.42 (m, 2H, CH 2C 6H 5), 4.11-3.67 (m, 6H, 3 × OCH 2CH 3), 1.71 (d, J HP
= 17.5 Hz, 3H, PCH 3), 1.27 (t, J = 7.1 Hz, 3H, OCH 2CH 3), 1.22 (t, J = 7.1 Hz, 3H, OCH 2CH 3),
1.16 (t, J = 7.1 Hz, 3H, OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 32.99 (d, J = 18.1 Hz, 1P,
CH 3(EtO)P(O)), 3.55 (d, J = 18.1 Hz, 1P, P(O)(OEt) 2).
13
C NMR (126 MHz, CDCl 3): δ 138.6,
128.9, 128.1, 127.4, 63.02 (d, J CP = 5.9 Hz), 62.98 (d, J CP = 5.1 Hz), 60.9 (d, J CP = 6.7 Hz),
49.0, 16.1 (d, J CP = 7.1 Hz), 15.9 (d, J CP = 7.2 Hz), 15.8 (d, J CP = 7.5 Hz), 15.2 (d, J CP = 134 Hz).
MS (m/z): calcd for C 14H 25NNaO 5P 2
+
[M+Na]
+
: 372.1, found: 372.4 [M+Na]
+
.
Benzyl(dimethoxyphosphoryl)[ethoxy(methyl)phosphoryl]amine (3b)
To a suspension of t-BuOK (400 mg, 3.6 mmol) in 7 mL of dry toluene was slowly added
a solution of compound 2b (645 mg, 3 mmol) in 5 mL of dry toluene. The reaction mixture
was stirred for 5 min, and a solution of ethyl methylphosphonochloridate (442 mg, 3.1
50

mmol) in 3 mL of dry toluene was added. The mixture was stirred overnight, and then
quenched by addition of a saturated aqueous solution of NH 4Cl. The product was
extracted into EtOAc (3  15 mL), and the combined organic layers were washed with
brine, then dried over Na 2SO 4. After evaporation of the solvent, the residual product was
purified by column chromatography on silica gel (EtOAc  3% MeOH in EtOAc), giving
0.88 g of 3b as a colorless oil (91%).
1
H NMR (500 MHz, CDCl 3): δ 7.52-7.25 (m, 5H,
CH 2C 6H 5), 4.69-4.43 (m, 2H, CH 2C 6H 5), 4.15-4.07 (m, 1H, OCH 2CH 3), 3.97-3.89 (m, 1H,
OCH 2CH 3), 3.64 (d, J = 11.4 Hz, 3H, OCH 3), 3.53 (d, J = 11.4 Hz, 3H, OCH 3), 1.71 (d, J = 17.4
Hz, 3H, CH 3P(O)), 1.30 (t, J = 7.1 Hz, 3H, OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 32.89 (d,
J = 18.2 Hz, 1P, H 3C(EtO)P(O)), 6.54 (d, J = 18.2 Hz, 1P, (MeO) 2P(O)).
13
C NMR (101 MHz,
CDCl 3): δ 138.4, 128.9, 128.2, 127.6, 61.1 (d, J CP = 6.5 Hz), 53.5 (d, J CP = 5.4 Hz), 53.4 (d, J CP
= 5.4 Hz), 49.1, 16.1 (d, J CP = 7.6 Hz), 15.1 (d, J CP = 134 Hz). MS (m/z): calcd for
C 12H 22NO 5P 2
+
: 322.1 [M+H]
+
, found: 322.4 [M+H]
+
.
Dimethyl
({[benzyl(diethoxyphosphoryl)amino](ethoxy)phosphoryl}methyl)phosphonate (4a)
A solution of BuLi in hexane (2.8 mL, 4.4 mmol) was added to a dry flask under N 2 and
cooled to -20 ⁰C. Then freshly distilled diisopropylamine (b.p. = 82-84 ⁰C) (655 L, 4.68
mmol) in 3 mL of dry THF was added. After stirring for 5 min, the reaction mixture was
cooled to -78 ⁰C, and a solution of 3b (680 mg, 2.12 mmol) in 3 mL of dry THF was added.
51

The reaction mixture was stirred for 1.5 min at -78 ⁰C, and a solution of diethyl
chlorophosphate (439 mg, 2.54 mmol) was slowly added, after which the reaction mixture
was allowed to warm up gradually to r.t. and left to stand overnight.
After cooling to 0 ⁰C, the reaction mixture was quenched with saturated NH 4Cl, and
the product extracted into EtOAc (3  15 mL). The combined organic fractions were
washed with brine and dried with Na 2SO 4. The product 4a was purified by column
chromatography on silica gel (2%  5% MeOH in EtOAc) and isolated as a colorless oil
(800 mg, 83%).
1
H NMR (500 MHz, CDCl 3): δ 7.52-7.23 (m, 5H, CH 2C 6H 5), 4.76-4.39 (m, 2H,
CH 2C 6H 5), 4.18-4.09 (m, 2H, OCH 2CH 3), 4.02-3.95 (m, 3H, OCH 2CH 3), 3.88-3.82 (m, 1H,
OCH 2CH 3), 3.84 (d, J = 11.3 Hz, 3H, OCH 3), 3.79 (d, J = 11.3 Hz, 3H, OCH 3), 3.06-2.77 (m,
2H, PCH 2P), 1.28 (t, J = 7.0 Hz, 6H, OCH 2CH 3), 1.15 (t, J = 7.1 Hz, 3H, OCH 2CH 3).
31
P NMR
(202 MHz, CDCl 3): δ 23.22 (dd, J = 19.8, 4.3 Hz, 1P, PCH 2PNP), 22.70 (d, J = 4.3 Hz, 1P,
PCH 2PNP), 2.79 (d, J = 19.8 Hz, 1P, PCH 2PNP).
13
C NMR (126 MHz, CDCl 3): δ 138.3, 129.3,
128.1, 127.6, 63.5 (d, J CP = 5.2 Hz), 63.1 (d, J CP = 5.7 Hz), 62.0 (d, J CP = 6.8 Hz), 53.2 (d, J CP =
6.1 Hz), 52.9 (d, J CP = 6.1 Hz), 49.9, 27.2 (dd, J CP = 135.1 Hz, J CP = 127.3 Hz), 16.1 (d, J CP =
7.3 Hz), 16.0 (d, J CP =7.6 Hz), 15.8 (J CP = 7.6 Hz).
Diethyl
({[benzyl(dimethoxyphosphoryl)amino](ethoxy)phosphoryl}methyl)phosphonate (4b)
A solution of butyllithium in hexane (3.75 mL, 6 mmol) was added to a dry flask under
N 2 and cooled to -20 C. Then freshly distilled diisopropylamine (924 L, 6.6 mmol) in 3 mL
52

of dry THF was added. After stirring for 5 min, the reaction mixture was cooled to -78 ⁰C,
and a solution of 3a (1.047 g, 3 mmol) in 6 mL of dry THF was added.
The resulting solution was stirred for 1.5 min at -78 ⁰C, and a solution of dimethyl
chlorophosphate (520 mg, 3.6 mmol) in dry THF (2 mL) was slowly added. The reaction
mixture was allowed to warm up gradually to r.t. and left to stand overnight.
After cooling to 0 ⁰C, the reaction mixture was quenched with a saturated aqueous
solution of NH 4Cl and extracted into EtOAc (3  15 mL). The combined organic fractions
were washed with brine and dried with Na 2SO 4. The product 4b was purified by column
chromatography on silica gel (EtOAc  2% MeOH in EtOAc) and isolated as a colorless oil
(805 mg, 59%).
1
H NMR (500 MHz, CDCl 3): δ 7.52-7.25 (m, 5H, CH 2C 6H 5), 4.76-4.37 (m, 2H,
CH 2C 6H 5), 4.26-3.93 (m, 6H, 3 × OCH 2CH 3), 3.70 (d, J = 11.4 Hz, 3H, OCH 3), 3.54 (d, J = 11.5
Hz, 3H, OCH 3), 3.02-2.72 (m, 2H, PCH 2P), 1.35 (t, J = 7.2 Hz, 3H, OCH 2CH 3), 1.34 (t, J = 7.1
Hz, 3H, OCH 2CH 3), 1.27 (t, J = 7.1 Hz, 3H, OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 23.38
(dd, J = 19.8, 4.0 Hz, 1P, PCH 2PNP), 19.65 (d, J = 4.0 Hz, 1P, PCH 2PNP), 5.76 (d, J = 19.8 Hz,
1P, PCH 2PNP).
13
C NMR (126 MHz, CDCl 3): δ 138.2, 129.3, 128.2, 127.6, 62.7 (d, J = 6.3 Hz),
62.3 (d, J = 6.2 Hz), 62.0 (d, J = 6.7 Hz), 53.8 (d, J = 5.3 Hz), 53.4 (d, J = 5.7 Hz), 49.9, 28.0
(dd, J CP = 134.7 Hz, J CP = 127.0 Hz), 16.4 (d, J CP = 6.4 Hz), 16.3 (d, J CP =6.4 Hz), 16.1 (J CP = 7.1
Hz). MS (m/z): calcd for C 16H 31NO 8P 3
+
458.1 [M+H]
+
, found: 458.5 [M+H]
+
.
53

[(2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-
2-yl]methyl methyl
({[benzyl(diethoxyphosphoryl)amino](ethoxy)phosphoryl}methyl)phosphonate (6a)
A solution of 4a (320 mg, 0.7 mmol) and 3 mL of triethylamine in 3 mL of acetonitrile
was refluxed for 12 h. The reaction progress was monitored by
31
P NMR. After completion,
solvents were removed under reduced pressure and the residue dried in vacuum. The
resulting oil was passed through a column of DOWEX H
+
, the acidic fractions were
collected and the solvent removed under reduced pressure, yielding 279 mg (90%) of the
corresponding acid 5a as a colorless oil, which was used without further purification.
Acid 5a (279 mg, 0.63 mmol), thymidine (153 mg, 0.63 mmol), and PPh 3 (247 mg,
0.945 mmol) were co-evaporated with 5 mL of dry DMF 3 times, and then dissolved in 2
mL of dry DMF. DIAD (186 μL, 0.945 mmol) was added dropwise and the resulting mixture
allowed to stand at r.t.
After 3 d, the solvent was removed under reduced pressure and the residue dissolved
in EtOAc and washed with 10 mL of 10% NaHCO 3. The aqueous layer was extracted with
EtOAc (3  15 mL), and the combined organic layers were washed with brine and dried
over Na 2SO 4. Product 6a was isolated by column chromatography (EtOAc  10% MeOH
in EtOAc) as a mixture of 4 isomers (247 mg, 59%).
1
H NMR (500 MHz, CDCl 3): δ 8.73-8.71
(1H, NH), 7.52-7.24 (6H, CH 2C 6H 5, CH-T), 6.43-6.16 (1H, H-1'), 4.72-3.78 (15H, CH 2C 6H 5, 3
× OCH 2CH 3, H-5', H-4', H-3', OCH 3), 3.43-2.68 (2H, PCH 2P), 2.49-2.19 (2H, H-2'), 1.94-1.90
54

(3H, CH 3-T), 1.41-1.15 (9H, 3 × OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 23.80-19.84 (2P,
P β and P α), 2.64-2.26 (1P, P γ).
Diethyl ({[benzyl({[(2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino](ethoxy)phosphoryl}methyl)phosphonate
(6b)
A solution of 4b (220 mg, 0.48 mmol) in 2 mL of MeCN and 2 mL of TEA was refluxed
for 3 h. The reaction progress was monitored by
31
P NMR. After completion, solvents were
removed and the residue was dissolved in water and extracted with EtOAc (10 ml). The
aqueous layer was evaporated at reduced pressure and the residue dried in vacuum. It
was then dissolved in MeOH (5mL) and stirred with DOWEX H
+
(300 mg) overnight. After
removal of the solvent at reduced pressure, 155 mg (73%) of the acid 5b was obtained as
a colorless oil, which was used without further purification.
Thymidine (90 mg, 0.35 mmol), acid 5b (155 mg, 0.35 mmol), and PPh 3 (137 mg, 0.525
mmol) were co-evaporated with 3 mL of dry DMF 3 times, and then dissolved in 2 mL of
dry DMF. To this solution, DIAD (103 μL, 0.525 mmol) was slowly added and the reaction
mixture was allowed to stand at r.t.
After 4 days, the solvent was evaporated and the residue was dissolved in EtOAc and
washed with 10 mL of 10% NaHCO 3. The aqueous layer was extracted with EtOAc (3 15
mL), and the combined organic layers were washed with brine, then dried over Na 2SO 4.
Product 6b was isolated by column chromatography (EtOAc  6% MeOH in EtOAc) as a
55

mixture of 4 isomers (72 mg, 31%).
1
H NMR (400 MHz, CDCl 3): δ 9.32-9.28 (1H, NH), 7.50-
7.24 (6H, CH 2C 6H 5, CH-T), 6.29-6.15 (1H, H-1'), 4.82-3.71 (12H, CH 2C 6H 5, 3 × OCH 2CH 3, H-
5', H-4', H-3'), 3.54-3.47 (3H, OCH 3), 3.26-2.54 (2H, PCH 2P), 2.40-2.18 (2H, H-2'), 1.92-1.87
(3H, CH 3-T), 1.41-1.23 (9H, 3 × OCH 2CH 3).
31
P NMR (162 MHz, CDCl 3): δ 23.28-22.83 (1P,
P β), 19.53-19.27 (1P, P γ), 5.65-4.79 (1P, P α).
[(2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-
2-yl]methyl methyl
({[(diethoxyphosphoryl)amino](ethoxy)phosphoryl}methyl)phosphonate (7a)
Compound 6a (247 mg, 0.37 mmol) was dissolved in methanol (7 mL), and 15 mg (6
wt. %) of Pd(OH) 2/C was added. The reaction mixture was stirred under 1 atm H 2 for 20
h, then filtered through a celite pad, and the volatiles evaporated to yield 206 mg of 7a
as a mixture of 4 isomers as a colorless oil (97%).
1
H NMR (500 MHz, CD 3OD): δ 7.59-7.56
(1H, CH-T), 6.29-6.26 (1H, H-1'), 4.47-4.04 (10H, 3 × OCH 2CH 3, H-5', H-4', H-3'), 3.83-3.79
(3H, OCH 3), 3.04-2.84 (2H, PCH 2P), 2.29-2.25 (2H, H-2'), 1.91-1.90 (3H, CH 3-T), 1.37-1.33
(9H, 3  OCH 2CH 3).
31
P NMR (202 MHz, CD 3OD): δ 22.89-21.95 (1P, P α), 18.76-18.34 (1P,
P β), -0.22-(-0.31) (1P, P γ). MS (m/z): calcd for C 18H 33N 3O 12P 3
-
576.1 [M-H]
-
, found: 576.1
[M-H]
-
.
56

Diethyl ({ethoxy[({[(2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino]phosphoryl}methyl)phosphonate (7b)
Compound 6b (72 mg, 0.108 mmol) was dissolved in methanol (3 mL), and 7 mg (10
wt. %) of Pd(OH) 2/C was added. The reaction mixture was stirred under 1 atm H 2 for 4 h,
then filtered through a celite pad. Volatiles were evaporated from the filtrate, yielding 60
mg of 7b as a mixture of 4 isomers as a colorless oil (96%).
1
H NMR (400 MHz, CD 3OD): δ
7.58-7.56 (1H, CH-T), 6.30-6.26 (1H, H-1'), 4.48-4.03 (10H, 3 × OCH 2CH 3, H-5', H-4', H-3'),
3.86-3.81 (3H, OCH 3), 2.95-2.74 (2H, PCH 2P), 2.27-2.24 (2H, H-2'), 1.91-1.90 (3H, CH 3-T),
1.38-1.32 (9H, 3  OCH 2CH 3).
31
P NMR (162 MHz, CD 3OD): δ 19.75-19.66 (1P, P β), 18.70-
18.55 (1P, P γ), 1.91-1.59 (1P, P α).
{[({[(2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-
yl)oxolan-2-yl]methyl phosphonato}methyl)phosphinato]amino}phosphonate (TEA/Na
salt) [(α,β)-CH 2-(β,γ)-NH-dTTP (1a)
A mixture of 7a (10 mg, 17 μmol), dry DMF (20 μL) and BTMS (200 μL, 1.52 mmol) was
microwaved at 40 ⁰C for 30 min. The reaction mixture was allowed to cool to r.t., volatiles
were removed in vacuum, and the residue was treated with 1 mL of Et 3N-MeOH (1:5). The
product was purified by two-stage preparative HPLC: first, on a SAX column (0-0.5 M TEAB
gradient in 20 min); then, on a C-18 column (0.1 M TEAB:5% MeCN, pH = 6.9). The
fractions containing 1a (identified by MS) were collected and combined, and evaporated
yielding the TEA salt of compound 1a. The solution of 1a in water was treated with
57

DOWEX Na
+
(20 mg) and lyophilized again to yield Na-TEA salt of compound 1a (2.2 mg,
27%).
1
H NMR (500 MHz, D 2O): δ 7.62 (s, 1H, CH-T), 6.39 (t, J = 7.0 Hz, 1H, H-1'), 4.64-4.62
(m, 1H, H-3'), 4.17-4.07 (m, 3H, H-4', H-5'), 2.40 (t, J = 19.5 Hz, 2H, PCH 2P), 2.39-2.30 (m,
2H, H-2'), 1.92 (s, 3H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ 19.89 (d, J = 9.8 Hz, 1P, P α), 12.28
(d, J = 9.8 Hz, 1P, P β), -0.31 (s, 1P, P γ). MS (m/z): calcd for C 11H 19N 3O 12P 3
-
478.0 [M-H]
-
,
found: 478.1 [M-H]
-
.
({[({[(2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-
yl)oxolan-2-yl]methoxy}phosphinato)amino]phosphinato}methyl)phosphonate (TEA
salt) [(α,β)-NH-(β,γ)-CH 2-dTTP] (1b)
A mixture of 7b (5.0 mg, 8.7 μmol), dry DMF (100 μL) and BTMS (100 μL, 760 µmol)
was microwaved at 40 ⁰C for 8 min. The reaction mixture was cooled to r.t., all volatiles
removed in vacuum, and 1 mL of Et 3N-MeOH (1:5) added. The crude product was purified
by two-stage preparative HPLC: first, on a SAX column (0-0.5 M TEAB gradient over 20
min); second, on a C-18 (0.1 M TEAB:5% MeCN, pH = 7.0). The fractions containing 1b
(identified by MS) were collected and combined, and then lyophilized to dryness to
provide a TEA salt of compound 1b (1.9 mg, 46%).
1
H NMR (400 MHz, D 2O): δ 7.71 (s, 1H,
CH-T), 6.38 (t, J = 6.9 Hz, 1H, H-1'), 4.68-4.65 (m, 1H, H-3'), 4.19-4.08 (m, 3H, H-4', H-5'),
2.44-2.32 (m, 2H, H-2'), 2.08 (t, J = 19.2 Hz, 2H, PCH 2P), 1.93 (d, J = 1.1 Hz, 3H, CH 3-T).
31
P
NMR (162 MHz, D 2O): δ 15.32 (dd, J = 8.3, J = 3.9 Hz, 1P, P β), 12.30 (d, J = 8.3 Hz, 1P, P γ),
0.68 (d, J = 3.9, 1P, P α). MS (m/z): calcd for C 11H 19N 3O 12P 3
-
478.0 [M-H]
-
, found: 478.1 [M-
H]
-
.
58

DNA synthesis, purification, radiolabeling, and annealing
Kinetic experiments were performed by Dr. Keriann Oertell in Prof. Myron Goodman’s
lab.
Primer (5´-TAT TAC CGC GCT GAT GCG C), template, (5´-GCG TTG TTC CGA CAG CGC
ATC AGC GCG GTA ATA), and 5´-phosphorylated downstream (5´-GTC GGA ACA ACG C)
oligomers were synthesized on a solid phase DNA synthesizer and purified by 16%
denaturing polyacrylamide gel electrophoresis, followed by desalting using
oligonucleotide purification cartridges. 1 mol equiv primer was 5´-end labeled with 0.4
U/µL T4 polynucleotide kinase and 0.7 mol equiv [γ-
32
P]ATP with the supplied buffer at
37 ⁰C for 30 min, followed by heat inactivation at 95 ⁰C for 10 min. The primer was
annealed by mixing with 1.2 mol equiv template and 1.5 mol equiv downstream
oligomers. The mixture was heated to 95 ⁰C and cooled slowly to room temperature. The
reaction buffer consisted of 50 mM Tris-Cl, 20 mM KCl, 20 mM NaCl, 10 mM MgCl 2, 1 mM
DTT, and 6% glycerol at pH 8.0, 37 ⁰C.
DNA synthesis kinetics
Radiolabeled 1 nt gapped DNA (100 nM) was incubated with pol β (600 nM) in reaction
buffer (2x mixture) for 3 minutes at 37 ⁰C. Equal volumes of the DNA/pol β mixture and a
2x solution of the 1a/1b at different concentrations and dTTP at 0.2 µM in reaction buffer
were combined and incubated at 37 ⁰C. After the appropriate reaction time, an aliquot of
the reaction mixture was quenched with 0.5 M EDTA at pH 8.0. Reaction products were
separated by 20% denaturing polyacrylamide gel electrophoresis (39 cm x 33 cm x 0.4
59

mm). Dehydrated gels were exposed to a phosphor screen and detected by
phosphorescence emission.
For each set of reactions, the percentage of primer extended is plotted versus time,
and the data for each concentration of analog is fit to the first order exponential y = a(1-
e
-kt
), where a is the maximum percent of primer extension and k is the observed rate
constant. The observed rate constant (k obs) is then plotted versus the corresponding
inhibitor concentration and the data fit to the hyperbolic decay equation k obs = k pol×K i/(K i
+ [inhibitor]) to give the K i parameter.
Crystallization of the Pol  Substrate Complex
Crystal structures were obtained by Dr. Vinod Batra and Samuel Wilson, M.D. at
NIEHS.
Binary complex crystals of human pol  with a dideoxy-terminated primer in a 1-
nucleotide gapped DNA were grown as described previously.
14
Briefly, the sequence of
the template strand (16-mer) was 5′-CCGACAGCGCATCAGC-3′. The primer strand (9-mer)
sequence was 5′-GCTGATGCG-3′. The downstream oligonucleotide (5-mer) was 5′-
phosphorylated, and the sequence was 5′-GTCGG-3′. These oligonuleotides were
annealed in a ratio of 1:1:1 by heating at 90 ⁰C for 10 min and cooling to 4 ⁰C (1 ⁰C/min)
using a PCR thermocycler, resulting in a 2-nucleotide gapped duplex DNA. These annealed
oligonucleotides were further incubated with pol β in a solution containing ddCTP to
create a ddCMP-terminated primer. This pol β-DNA binary complex was crystallized by
sitting-drop vapor diffusion at 18 ⁰C by mixing 2 µl of the complex with 2 µl of
60

crystallization buffer. The crystallization buffer consisted of 16% PEG-3350, 350 mM
sodium acetate, and 50 mM imidazole, pH 7.5. Crystals grew in approximately 2-4 days
after seeding. The ternary complexes were obtained by soaking crystals of the binary (one
nucleotide gapped DNA) complex in artificial mother liquor containing 50 mM MgCl 2, 20%
PEG-3350, 12% ethylene glycol, and 4 mM (α,β)-CH 2-(β,γ)-NH-dTTP or (α,β)-NH-(β,γ)-CH 2-
dTTP for 1-2 hours. The crystals were flash-frozen in a liquid nitrogen stream. Diffraction
quality data were then collected for the ternary complex crystals as described below.
Data collection and structure determination
Data were collected at 100 ⁰K on a CCD detector system mounted on a MiraMax
®
-
007HF (Rigaku Corporation) rotating anode generator. Data were integrated and reduced
with HKL2000 software.
36
All crystals belong to the space group P2 1. The ternary complex
structures were solved by molecular replacement using 2FMP
14
as a reference model. The
structure was refined using PHENIX and manual model building using Coot. The
crystallographic statistics are reported in Table B.1.
Structure factors and the coordinates for the pol β complexes with (α,β)-CH 2-(β,γ)-
NH-dTTP (1a) and (α,β)-NH-(β,γ)-CH 2-dTTP (1b) have been deposited with Protein Data
Bank with accession codes 4RT2 and 4RT3, respectively.

61

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64

Chapter 3 - Design and Synthesis of Modified “Met-Im” Analogues of dTTP
– Potential Inhibitors of DNA pol β
3.1 Design of the H-bonding “Met-Im” analogue inhibitors of DNA pol β
As discussed in chapter 2, two non-hydrolysable dTTP analogues, (α,β)-CH 2-(β,γ)-NH-
dTTP and (α,β)-NH-(β,γ)-CH 2-dTTP, demonstrated promising affinity to DNA pol β.
1

Interestingly, the former dTTP analogue having an NH group in (β,γ)-position displayed
affinity to pol β 9 times higher than the latter analogue. Analysis of X-ray structures of
ternary complexes of (α,β)/(β,γ) CH 2/NH-dTTPs with DNA pol β and DNA revealed that a
water-mediated favorable interaction between (β,γ)-NH and R183
2
may be responsible
for this significant difference in activity. This result supported the idea that H-bonding
between dNTP analogue and R183 can be used to improve the affinity of the inhibitor to
DNA pol β, and inspired us to design new inhibitors of pol β having H-bonding functional
groups at the (β,γ)-position. According to the multiple X-ray structures of ternary pol β
complexes with DNA and dNTP analogues, the (β,γ)-bridging atom is located spatially
close to R183.
2-3
We speculated that introduction of H-bond acceptors into the (β,γ)-
methylene group of (α,β)-NH-(β,γ)-CH 2-dTTP may “anchor” new inhibitors to R183, thus
improving their affinity to the enzyme.
Carboxyl and hydroxyl groups are commonly used as H-bond acceptors. At pH 7, a
negatively charged carboxylate group can be expected to be a better H-bond acceptor
due to its higher basicity compared to hydroxyl group. Regardless of the H-bond acceptor
type, high energy bonding is achieved when the distance between oxygen acceptor and
65

nitrogen is close to 3 Å.
4
The distance between R183 and an H-bond acceptor, and the
resulting interaction, can be optimized by an appropriate spatial arrangement of the
arginine and the carboxyl or hydroxyl group. This arrangement can be modified using a
spacer of the appropriate length to adjust the position of an oxygen atom in the H-bond
acceptor. Analysis of the crystal structures of ternary complexes of DNA pol β with DNA
and substrate analogues using docking software (Autodock Vina 1.1.2)
5
suggested that a
hydroxyl group separated from the (β,γ)-bridging carbon with a 2-carbon spacer may be
located within close proximity to R183, while for the carboxylate group a shorter 1-carbon
spacer may be sufficient to place it in the optimal position.
Another option, avoiding the introduction of chemically non-inert H-bonding groups,
is to incorporate a fluorine atom into the (β,γ)-position of (α,β)-NH-(β,γ)-CH 2-dTTP. It has
previously been suggested that the CHF bridge may introduce a weak yet favorable
interaction between F and R183,
2
providing additional binding energy. In addition,
geometrical parameters and the polarity of the P–CHF–P linkage are expected not to
significantly deviate from those of the P–O–P moiety.
2-3

To estimate the beneficial effect of the interaction between R183 and H-bond
acceptors or fluorine to the binding affinity of dNTP analogues, we proposed to synthesize
the analogues of (α,β)-NH-(β,γ)-CH 2-dTTP with a CH 2CH 2OH, CH 2COO
-
, and F group in the
(β,γ)-position, (α,β)-NH-(β,γ)-CH(CH 2COO
-
)-dTTP (1a), (α,β)-NH-(β,γ)-CH(CH 2CH 2OH)-dTTP
(1b) and (α,β)-NH-(β,γ)-CHF-dTTP (1c), respectively (Figure 3.1).
66

Figure 3.1. (α,β)-NH-(β,γ)-CH(CH 2COO-)-dTTP (1a), (α,β)-NH-(β,γ)-CH(CH 2CH 2OH)-dTTP
(1b), (α,β)-NH-(β,γ)-CHF-dTTP (1c)
Introduction of a single substituent into the (β,γ)-position of (α,β)-NH-(β,γ)-CH 2-dTTP
results in generation of a new chiral center at (β,γ)-carbon atom.
6
Thus, each 1a-c can
exist in the form of two diastereomers. Different diastereomers can display different
affinity to pol β,
3b
and to explore the inhibition of pol β by 1a-c it would be highly desirable
to obtain 1a-c as individual diastereomers.
3b
However, no established procedure for
synthesis and/or separation of individual diastereomers of 1a-c exist, thus, our initial
purpose was to generate 1a-c as a mixture of diastereomers and determine their activity
regardless of stereochemical considerations.
3.2 Synthesis of “Met-Im” analogues 1a-c
To synthesize 1a-c we utilized the same synthetic approach that we have previously
reported for the synthesis of (α,β)-NH-(β,γ)-CH 2-dTTP.
1
Triphosphate analogue 2 was
synthesized according to the previously reported procedure,
1
and the modifying H-bond
acceptors were attached to the “triphosphate” moiety by a spacer of an appropriate
length (except fluorine) attached to the phosphonate bridging carbon atom of 2.
Upon introduction of modifiers into compound 2, a new chiral center is generated at
the (β,γ)-methylene carbon atom in addition to the chiral center at the central
67

phosphorus atom, resulting in the possible formation of 2 diastereomers –isomer A and
isomer B (Figure 3.2, black boxes). Each diastereomer represents a pair of enantiomers:
SS/RR for isomer A and SR/RS for isomer B. It is clear that the phosphonate carbon in a
single diastereomer (either black box) is not stereochemically uniform. Diastereomers of
compounds 3a-c, in which the phosphonate carbons have alike absolute configurations,
are shown in red boxes on Figure 3.2.

Figure 3.2. Stereochemistry of compounds 3a-c. Black boxes correspond to individual
diastereomers of 3 (isomer A and isomer B), while red boxes show two diastereomers
having same absolute configuration at the phosphonate carbon.

Triphosphate analogue 3a, the key intermediate in the synthesis of 1a, was obtained
via alkylation of 2 with benzyl bromoacetate. Protection of the carboxyl group as a benzyl
ester was chosen to eliminate an extra deprotection step since Bn can be removed
simultaneously with deprotection of the benzylimido group. The electron-withdrawing
properties of the ester function improve the electrophilicity of the α-carbon of benzyl
bromoacetate, and the alkylation of 2 was easily achieved using t-BuOK to generate the
potassium salt of 2, which was reacted with benzyl bromoacetate to yield 3a.
68

Scheme 3.1 Synthesis of intermediate 3a

With 2 chiral centers (Figure 3.2), 3a was formed as two diastereomers in a 2.7:1 ratio
with 80% conversion of the starting material, as determined by
31
P NMR of the reaction
mixture. Using column chromatography, 3a was isolated as a mixture of diastereomers in
a 4:1 ratio.
Triphosphate analogue 3b, the key intermediate in the synthesis of 1b, was obtained
via alkylation of 2 with benzyl 2-iodoethyl ether.
7
Benzyl protection of hydroxyl group was
utilized for similar reasons as in the synthesis of 3a. In benzyl 2-haloethyl ether, the β-
carbon is less electrophilic than the α-carbon in haloacetate due to the electron-
withdrawing properties of the ester functionality. Thus, harsher conditions were needed
to alkylate 2 with a good yield. A brief screening of alkylation conditions was performed
(Table 3.1), with various combinations of solvent, base, and temperature. The reaction
conversion was estimated by
31
P NMR of the crude reaction mixture. The optimal results
were obtained when sodium salt of 2 was generated by reaction of 2 and NaH in DMF,
and then reacted with benzyl 2-iodoethyl ether at 50 ⁰C (Scheme 3.2).
Table 3.1. Screening of conditions of alkylation of 2 with benzyl 2-iodoethyl ether
69

Entry Solvent Base Temperature Yield of 3b (
31
P NMR), % Isomers ratio
1 DMF NaH r.t. 20 1:1
2 Tol t-BuOK r.t. 0 -
3 THF NaH r.t. 0 -
4 THF NaH 50 ⁰C 0 -
5 DMF NaH 50 ⁰C 75 2:1

Intermediate 3b has two chiral centers as well, and two diastereomers were formed
in 2:1 ratio with 75% conversion of the starting material as determined by
31
P NMR of the
alkylation reaction mixture. The products were purified by flash chromatography, and the
first (fast eluting) isomer was used in further transformations.
Scheme 3.2. Synthesis of intermediate 3b

To synthesize 3c, the key intermediate in the synthesis of 1c, we fluorinated the
sodium salt of 2 using Selectfluor® in DMF (Scheme 3.3).
2, 8
Two isomers of compound 3c
were initially formed in a 1:1 ratio as determined by
31
P NMR. However, chromatographic
purification of 3c significantly enriched the second (slow-eluting) isomer due to the similar
R f values of the first (fast-eluting) isomer of 3c and the product of double fluorination of
2 (Scheme 3.3).

70

Scheme 3.3. Synthesis of intermediate 3c and its double fluorination byproduct

Compounds 1a-c were synthesized from modified triphosphate analogues 3a-c
according to the previously reported procedure.
1
Selective removal of a single methyl
group by triethylamine (TEA)
9
afforded compounds 4a-c. Reaction progress was
monitored by
31
P NMR. It is interesting to note that TEA induced isomerization of the
chiral carbon in 3a-c. After the reaction completion, two isomers of
triethylmethylammonium salts of 4a-c were obtained in a roughly 1:1 ratio even though
different ratios of diastereomers 3a-c were subjected to demethylation by TEA. This
isomerization can be explained by a relatively low pK a of the PCH(R)P group (R =
CH 2CO 2Bn, CH 2CH 2OBn, F): TEA is capable of deprotonating the phosphonate carbon with
the formation of a planar anion, which, upon addition of a proton, can result in formation
of both diastereomers (Figure 3.2).
The resulting triethylmethylammonium salts were converted to the corresponding
acids (4a-c) using DOWEX H
+
. The acids were coupled to thymidine via Mitsunobu
reaction
10
in DMF to form 5a-c, correspondingly. Full debenzylation of 5a-c was effected
by hydrogenolysis in presence of Pd(OH) 2/C (Scheme 3.4) to give 6a-c. Similar to the effect
observed for (α,β)-CH 2-(β,γ)-NH-dTTP and (α,β)-NH-(β,γ)-CH 2-dTTP, removal of the benzyl
groups eliminated their shielding/deshielding effects, resulting in a smaller range of
chemical shifts in the
31
P NMR, and, for 3c, in the
19
F NMR.
71

Scheme 3.4. Synthesis of 6a-c
Similar to the procedure for deprotection of parent dNTP analogues, full deprotection of
compounds 6a-c was achieved using bromotrimethylsilane (BTMS)
11
under microwave
irradiation (Scheme 3.5). Reaction progress was monitored by MS. Significantly longer
times (≈20 min) were required to completely deprotect compounds 6a and 6b,
presumably due to the steric hindrance inherent to the bulky substituents at the (β,γ)-
carbon. Fluorine atom is less sterically demanding, and 6c was successfully deprotected
in 10 min, as determined by MS.
Scheme 3.5.Full deprotection of 6a-c

Results and Discussion
Formation of compound 1a under MW conditions by reaction with BTMS was
confirmed by mass spectrometry. However, quenching the reaction mixture with various
72

quenching systems (H 2O, triethylammonium bicarbonate (TEAB) buffer, MeOH, or
MeOH:TEA) initiated rapid decomposition of 1 at r.t. (23-25 ⁰C).
Products 1b and 1c did not show significant decomposition upon quenching with TEAB
buffer, and were successfully purified by two-stage preparative (strong anion exchange
(SAX) followed by reversed-phase (RP)) HPLC.
2
RP HPLC successfully separated β-anomer
from the traces of α-anomer, and 1b and 1c were isolated as mixtures of two
diastereomers.
In the
31
P NMR spectrum of 1b, two diastereomers have discrete doublet (J = 7.6 Hz)
P α signals at 1.05 and 1.02 ppm (Figure 3.3). This finding confirms that 1b was isolated as
a 1:1 mixture of diastereomers as determined by integration of the outermost lines of
two P α signals.

Figure 3.3. Portion of
31
P NMR of 1b. Integration of the outermost lines of the P α
signals corresponding to two diastereomers demonstrates that the isomers are present
in equal amounts.
73

Despite being significantly more stable than carboxyl derivative 1a, 1b has been
shown to slowly decompose in aqueous solutions at r.t. (23-25 ⁰C). The rate and the
products of decomposition of 1b have been analyzed by LC-MS (Figure 3.4). The reaction
mixture composition was monitored for 120 h after the reaction quench with TEAB buffer.
We determined that the rate of decomposition is pH-dependent: in acidic conditions
(pH 5) the decomposition rate of 1b was significantly higher compared to conditions close
to neutral (pH 8). The decomposition products have been identified by LC-MS (Figure 3.4).
Of the three injections shown, the first two correspond to the reaction quenched with
TEAB buffer at pH 8, while the third injection corresponds to reaction quench at pH 5. At
pH 5 (injection 3) decomposition proceeds at the higher rate, which is demonstrated by
the fact that the peak, corresponding to 1 (m/z = 522.0 and m/z = 544.0) (Figure 3.4B and
C) is present in lower amount compared to the other two injections (pH 8), with
simultaneous accumulation of the peaks, which, by MS analysis, were assigned to 7 (m/z
= 320.0) (Figure 3.4D) and dTMP (8) (m/z = 321.0) (Figure 3.4E). The last trace, which was
assigned to 9 (m/z = 201.0) based on the MS, is larger in injection 3 (pH 5) compared to
pH 8, but still present in injections 1 and 2 (pH 8). It is likely that this peak (m/z = 201.0),
besides being the product of decomposition of 1b, is also formed upon fragmentation of
1b under mass spectrometry conditions.
74

Figure 3.4. Representative LC-MS trace of decomposition of 1b at pH 8 and pH 5 at r.t.
The trace corresponds to the reaction time of 120 h. Three injections shown. Injections 1
and 2 correspond to the r.m. at pH = 8, injection 3 – to the r. m. at pH = 5. Traces, from
top to bottom: (A) Total UV absorption, λ = 260 nm; (B) MS (m/z): 522, corresponding to
[1b – H]
-
; (C) MS (m/z): 544, corresponding to [1b + Na]
-
; (D) MS (m/z): 320, 7; (E) MS
(m/z): 321, [8 – H]
-
; (F) MS (m/z): 201, [9 – H]
-

Analysis of the products of decomposition suggests that the reaction occurs via an
intramolecular attack of the hydroxyl group onto P β of 1b with the elimination of dTMP-
NH 2 (7). Product 7 is subsequently partially hydrolyzed by water to dTMP 8 (Scheme 3.6).
This plausible mechanism suggests an explanation for the pH-dependence of the rate of
decomposition of 1b. At lower pH, a larger proportion of the P β non-bridging oxygen in
1b is protonated. The protonation makes P β more susceptible to the nucleophilic attack
by an internal hydroxyl nucleophile, resulting in an increase of the intramolecular
nucleophilic attack rate.
75

Scheme 3.6 Products of decomposition of 1b

In contrast to 1a and 1b, (α,β)-NH-(β,γ)-CHF-dTTP (1с), which does not have
nucleophilic groups incorporated into the (β,γ)-position, did not display any
decomposition signs at pH ranging from 5 to 8 at r.t. (23-25 ⁰C).
In summary, the strategy devised for synthesis of (α,β)-NH-(β,γ)-CH 2-dTTP proved
applicable to the synthesis of its derivatives. Potential inhibitors (α,β)-NH-(β,γ)-
CH(CH 2CH 2OH)-dTTP (1b) and (α,β)-NH-(β,γ)-CHF-dTTP (1c) were synthesized from
triphosphate analogue 2 with overall yields of 2%. Carboxyl derivative 1a, (α,β)-NH-(β,γ)-
CH(CH 2CO 2OH)-dTTP, decomposed quickly in protic solvents. (α,β)-NH-(β,γ)-
CH(CH 2CH 2OH)-dTTP (1b) was found to slowly decompose in aqueous solutions, but was
stable at pH above neutral. Thus, the rate of decomposition of 1b increases at lower pH,
plausibly by shifting the equilibrium towards protonation of non-brydging oxygen on P β
and thus making it more susceptible to the intramolecular nucleophilic attack by the
hydroxyl group. Compound 1c did not decompose in protic solvents, presumably due to
the absence of nucleophilic groups in (β,γ)-position.
76

Experimental Section
Materials and Methods
All the reagents and solvents were purchased from VWR, Sigma-Aldrich, or Alfa Aesar
and used without further purification, unless noted otherwise. Compound 2 was
synthesized according to the previously reported procedure.
1
Compounds 3b-c have been
purified using ISCO CombiFlash® column chromatography equipped with ELSD detector,
compound 3a was purified by column chromatography.
1
H,
13
C and
31
P NMR spectra were
obtained on Varian 400-MR or VNMRS-500 spectrometers.
13
C and
31
P NMR spectra were
proton-decoupled. All chemical shifts (δ) are reported in parts per million (ppm) relative
to residual CH 3OH in CD 3OD (δ 3.34,
1
H NMR), CHCl 3 in CDCl 3 (δ 7.26,
1
H NMR), HDO in
D 2O (δ 4.80,
1
H NMR), external 85% H 3PO 4 (δ 0.00,
31
P NMR) or internal CDCl 3 (δ 77.00,
13
C NMR). NMR spectra processing was performed with MestReNova 9.0.0. Multiplicities
are reported using the following abbreviations: s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad resonance.
Preparative HPLC was performed using a Varian ProStar equipped with a Shimadzu
SPD-10A UV detector (0.5 mm path length) with detection at 260 nm. Strong Anion
Exchange (SAX) HPLC was performed using a Macherey Nagel 21.4 mm  250 mm SP15/25
Nucleogel column, C-18 HPLC was performed using a Phenomenex Luna 5u C18 (21.20
mm  250 mm). Mass spectrometry and LC-MS experiments were performed on a
Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the
negative ion mode. LC-MS utilized Finnigan Survey or PDA 158 Plus detector (1 cm path
77

length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7.
Microwave‐assisted synthesis was performed using a Milestone Ethos Synth Microwave
Labstation. Compound IUPAC names were assigned with the assistance of MarvinSketch
15.5. The molar yields of the final products were determined by UV absorbance using the
extinction coefficient of dTTP in phosphate buffer at pH 7.0 at 260 nm, ε = 9600 M
-1
*cm
-
1
.
Synthesis of benzyl 3-{[benzyl(dimethoxyphosphoryl)amino](ethoxy)phosphoryl}-3-
(diethoxyphosphoryl)propanoate (3a)
To a suspension of t-BuOK (143 mg, 1.28 mmol) in dry toluene (3 mL) was added
solution of 2 (390 mg, 0.85 mmol) in dry toluene (3 mL) at 0 ⁰C. After 5 min, solution of
benzyl bromoacetate (292 mg, 1.28 mmol) in dry toluene (2 mL) was added dropwise.
R.m. was stirred at r.t. for 4 h before being quenched with saturated NH 4Cl. Organic layer
was separated, and aqueous layer extracted with EtOAc (3  15 mL). Combined organic
layers were dried over Na 2SO 4, solvent evaporated, and residue was purified by column
chromatography (EtOAc) to give 321 mg of 3a as a mixture of 2 isomers as a colorless oil
(62%).
1
H NMR (500 MHz, CDCl 3): δ 7.53-7.21 (m, 10H, 2  CH 2C 6H 5), 5.15-5.11 (m, 2H,
OCH 2C 6H 5), 4.89-4.82 (m, 0.8H, NCH 2C 6H 5), 4.65-4.38 (m, 0.4H, NCH 2C 6H 5), 4.33-3.76 (m,
0.8H, NCH 2C 6H 5, 6H, OCH 2CH 3, and 1H, PCHP), 3.71-3.53 (m, 6H, OCH 3), 3.01-2.85 (m, 2H,
CH 2CO 2Bn), 1.34-1.12 (9H, OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 27.24 (dd, J = 18.3, 8.2
Hz, PCHPNP, major isomer), 26.51 (dd, J = 16.8, 3.0 Hz, PCHPNP, minor isomer), 22.03 (d,
J = 3.0 Hz, PCHPNP, minor isomer), 21.97 (d, J = 8.2 Hz, PCHPNP, major isomer), 6.11 (d, J
78

= 16.9 Hz, PCHPNP, minor isomer), 5.64 (d, J = 18.3 Hz, PCHPNP, major isomer).
13
C (151
Hz, CDCl 3) δ: major isomer: 170.4 (d, J = 9.2 Hz), 138.6, 135.7, 129.5, 128.4, 128.3, 128.0,
127.5, 66.8, 63.4 (d, J = 6.0 Hz), 62.3 (d, J = 6.0 Hz), 61.6 (d, J = 6.0 Hz), 54.0 (d, J = 5.4 Hz),
53.6 (d, 5.9 Hz), 51.0, 34.5 (dd, J = 133.8 Hz, 127.5 Hz), 30.65 (t, J = 4.9 Hz), 16.4 (d, J = 6.1
Hz), 16.23 (d, J = 6.2 Hz), 15.8 (d, J = 6.9). Minor isomer δ: 170.4 (d, J = 9.2 Hz), 138.6,
138.2, 129.2, 128.23, 128.21, 128.19, 66.8, 62.9 (d, J = 7.0 Hz,), 62.7 (d, J = 5.9 Hz), 62.6
(d, J = 6.3 Hz), 53.9 (d, J = 5.6 Hz), 53.7 (d, J = 5.8 Hz), 50.1, 30.1 (d, J = 4.5 Hz), 16.3 (d, J =
7.6 Hz), 16.0 (d, J = 6.7 Hz). MS (m/z): calcd for C 25H 40NNaO 9P 3
+
614.2 [M+Na]
+
, found:
614.2 [M+Na]
+
.
Synthesis of [benzyl({[3-(benzyloxy)-1-(diethoxyphosphoryl)-3-
oxopropyl](ethoxy)phosphoryl})amino](methoxy)phosphinic acid (4a)
3a (320 mg, 0.53 mmol) was refluxed in a 1:1 mixture of TEA and acetonitrile (4 mL)
for 3 h. Reaction progress was monitored by
31
P. Upon completion, all volatiles were
evaporated under reduced pressure, residue was re-dissolved in MeOH, and the resulting
solution was stirred with DOWEX H
+
for 1 h. The solvent was evaporated, and the residue
was thoroughly dried under reduced pressure to give acid 4a (286 mg) as a mixture of 2
isomers in 3:2 ratio (91%).
1
H NMR (500 MHz, CDCl 3): δ 9.48 (br s, 1H, P(O)OH), 7.60-7.22
(m, 10H, 2  CH 2C 6H 5), 5.17-5.10 (m, 2H, OCH 2C 6H 5), 4.80-4.39 (m, 2H, NCH 2C 6H 5), 4.34-
3.77 (m, 6H, OCH 2CH 3, and 0.4H, PCHP, minor isomer), 3.55 (d, J = 11.7 Hz, 1.2H, OCH 3,
minor isomer), 3.51 (d, J = 11.7 Hz, 1.8H, OCH 3, major isomer), 3.29 (tt, J = 23.5, 6.3 Hz,
0.6H, PCHP, major isomer), 3.09-2.72 (m, 2H, CH 2CO 2Bn), 1.37-1.08 (9H, OCH 2CH 3).
31
P
79

NMR (202 MHz, CDCl 3): δ 25.16 (dd, J = 19.0, 11.7 Hz, PCHPNP, minor isomer), 24.48 (d, J
= 15.3 Hz, PCHPNP, major isomer), 23.64 (s, PCHPNP, major isomer), 23.28 (d, J = 11.6 Hz,
PCHPNP, minor isomer), 4.06 (d, J = 19.0 Hz, PCHPNP, major isomer), 4.02 (d, J = 15.4 Hz,
PCHPNP, minor isomer).
Synthesis of benzyl 3-{[benzyl({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino](ethoxy)phosphoryl}-3-
(diethoxyphosphoryl)propanoate (5a)
Acid 4a (285 mg, 0.48 mmol), thymidine (117 mg, 0.48 mmol), and PPh 3 (189 mg, 0.72
mmol) were co-evaporated with 3 mL of dry DMF 3 times, and then dissolved in 3 mL of
dry DMF. DIAD (142 μL, 0.72 mmol) was added dropwise and the resulting mixture
allowed to stand at r.t.
After 3 d, the solvent was removed under reduced pressure and the residue dissolved
in EtOAc and washed with 10 mL of 10% NaHCO 3. The aqueous layer was extracted with
EtOAc (3  15 mL), and the combined organic layers were washed with water and dried
over Na 2SO 4. Product 5a was isolated by column chromatography (EtOAc  10% MeOH
in EtOAc) as a mixture of 8 isomers (191 mg, 49%).
1
H NMR (500 MHz, CDCl 3): δ 9.38-9.31
(1H, NH), 7.55-7.20 (11H, OCH 2C 6H 5, NCH 2C 6H 5, CH-T), 6.31-6.20 (1H, H-1'), 5.18-5.08 (2H,
OCH 2C 6H 5), 4.91-3.50 (16H, NCH 2C 6H 5, 3 × OCH 2CH 3, H-5', H-4', H-3', PCHP, OCH 3), 3.00-
2.80 (2H, CH 2CO 2Bn), 2.47-2.08 (2H, H-2'), 1.96-1.86 (3H, CH 3-T), 1.36-1.12 (9H, 3 ×
80

OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 26.90-25.64 (1P, P β), 21.81-21.58 (1P, P γ), 6.38-
4.87 (1P, P α).
Synthesis of 3-(diethoxyphosphoryl)-3-{ethoxy[({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino]phosphoryl}propanoate (6a)
Compound 5a (132 mg, 0.16 mmol) was dissolved in methanol (4 mL), and 15 mg (10
wt. %) of Pd(OH) 2/C was added. The reaction mixture was stirred under 1 atm H 2 for 6 h,
then filtered through a celite pad, and the volatiles evaporated to yield 99 mg of 6a as a
mixture of 8 isomers as a colorless oil (96%).
1
H NMR (500 MHz, CD 3OD): δ 7.59-7.56 (1H,
CH-T), 6.29-6.26 (1H, H-1'), 4.48-4.05 (10H, 3 × OCH 2CH 3, H-5', H-4', H-3'), 3.86-3.81 (3H,
OCH 3), 3.50-3.30 (1H, PCHP), 3.03-2.71 (2H, CH 2CO 2H), 2.27-2.24 (2H, H-2'), 1.95-1.90 (3H,
CH 3-T), 1.39-1.31 (9H, 3  OCH 2CH 3).
31
P NMR (202 MHz, CD 3OD): δ 22.42-21.37 (2P, P β,
P γ), 2.42-1.84 (1P, P α).
Synthesis of benzyl 2-iodoethyl ether
12

To a vigorously stirred solution of benzyl 2-hydroxyethyl ether (2.28 g, 15 mmol) in
DCM (50 mL) PPh 3 (11.4 g, 44 mmol) and imidazole (6 g, 88 mmol) were added. The
resulting solution was cooled down to 0⁰ C, and iodine (11 g, 44 mmol) was slowly added.
R.m. was stirred at r.t.
In 24 hours, r.m. was quenched with saturated solution of Na 2S 2O 3. Product was
extracted with DCM (3  30 mL), and the combined organic layers were dried over Na 2SO 4.
After evaporation of the solvent, the residual product was purified by column
81

chromatography (EtOAc : Hex, 1:10), giving 3.78 g of benzyl 2-iodoethyl ether as a
colorless oil (97%).
1
H NMR (400 MHz, CDCl 3): δ 7.36-7.28 (m, 5H, CH 2C 6H 5), 4.58 (s, 2H,
CH 2C 6H 5), 3.74 (t, J = 6.8 Hz, 2H, CH 2OBn), 3.29 (t, J = 6.8 Hz, 2H, CH 2I). Lit. data:
12

1
H NMR
(CDCl 3, 300 MHz) δ 7.41-7.35 (m, 5H), 4.62 (s, 2H), 3.78 (t, J = 6.8 Hz, 2H), 3.32 (t, J = 6.6
Hz, 2H).
Synthesis of diethyl (1-{[benzyl(dimethoxyphosphoryl)amino](ethoxy)phosphoryl}-3-
(benzyloxy)propyl)phosphonate (3b)
NaH (60% dispersion in oil) (52 mg, 1.3 mmol) was suspended in dry DMF (2 mL) and
cooled down to 0 ⁰C, and slowly added to a cooled solution of compound 2 (457 mg, 1
mmol) in 2 mL of dry DMF. The resulting solution was stirred at r.t. In 1 h, r.m. was cooled
down to 0 ⁰C, and the solution of benzyl 2-iodoethyl ether (917 mg, 3.5 mmol) was added
dropwise. R.m. was heated for 5 h to 40 ⁰C. In 5 h, r.m. was quenched with saturated
NH 4Cl, and the products were extracted with EtOAc. Products were purified using
Combiflash® (0-20% EtOAc in hexanes in 20 min, to give 178 mg of major (fast eluting)
isomer 3b as a colorless oil (30%).
1
H NMR (500 MHz, CDCl 3): δ 7.53-7.21 (m, 10H, 2 
CH 2C 6H 5), 4.96-4.87 (m, 1H, NCH 2C 6H 5), 4.54-4.48 (m, 2H, OCH 2C 6H 5), 4.32-4.18 (m, 1H,
NCH 2C 6H 5 and 2H, OCH 2CH 3), 4.11-4.04 (m, 3H, 1.5  OCH 2CH 3), 3.85-3.79 (m, 1H, 0.5 
OCH 2CH 3), 3.80 (d, J = 11.4 Hz, 3H, OCH 3), 3.70-3.65 (m, 2H, CH 2OBn), 3.47 (d, J = 11.5 Hz,
3H, OCH 3), 3.53-3.41 (1H, tt, J = 24 Hz, 5.9 Hz, PCHP), 2.34-2.18 (m, 2H, CHCH 2CH 2), 1.35
(t, 3H, J = 7.1 Hz, OCH 2CH 3), 1.27 (t, 3H, J = 7.1 Hz, OCH 2CH 3), 1.19 (t, J = 7.1 Hz, 3H,
OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 29.25 (dd, J = 20.7 Hz, 10.6 Hz, 1P, PCHPNP), 23.53
82

(d, J = 10.6 Hz, 1P, PCHPNP), 5.93 (d, J = 20.7 Hz, 1P, PCHPNP). MS (m/z): calcd for
C 25H 40NNaO 9P 3
+
614.2 [M+Na]
+
, found: 614.2 [M+Na]
+
.
Synthesis of [benzyl({[3-(benzyloxy)-1-
(diethoxyphosphoryl)propyl](ethoxy)phosphoryl})amino](methoxy)phosphinic acid
(4b)
A solution of 3b (178 mg, 0.3 mmol) and 2 mL of triethylamine in 2 mL of acetonitrile
was refluxed for 4 h. The reaction progress was monitored by
31
P NMR. After completion,
solvents were removed under reduced pressure and the residue dried in vacuo. The
resulting oil was then dissolved in MeOH (5mL) and stirred with DOWEX H
+
(200 mg) for
two hours. After filtering out DOWEX H
+
, the solvent was evaporated yielding 172 mg
(99%) of the corresponding acid 4b as a mixture of 2 isomers in a ratio of 10:8.
1
H NMR
(500 MHz, CD 3OD): δ 7.53-7.24 (m, 10H, 2  CH 2C 6H 5), 4.82-3.86 (m, 10H, 3  OCH 2CH 3,
NCH 2C 6H 5, OCH 2C 6H 5), 3.69-3.58 (m, 2H, CH 2OBn), 3.48 (d, J = 11.6 Hz, 3H, OCH 3, major
isomer), 3.47 (d, J = 11.7 Hz, 3H, OCH 3, minor isomer), 3.49-3.22 (1H, m, PCHP), 2.32-2.05
(m, 2H, CH 2CH 2OH), 1.36-1.20 (6 t, J = 7.1 Hz, 9H, OCH 2CH 3).
31
P NMR (202 MHz, CD 3OD):
δ 28.87 (dd, J = 20.7 Hz, 10.6 Hz, 1P, PCHPNP, minor isomer), 28.87 (dd, J = 20.7 Hz, 7.6
Hz, 0.8P, PCHPNP, minor isomer), 28.45 (d, J = 19.8 Hz, 2.2 Hz, 1P, PCHPNP, major isomer),
24.11 (d, J = 7.6 Hz, 1P, PCHPNP, minor isomer), 24.01 (d, J = 2.2 Hz, 1P, PCHPNP, major
isomer), 3.41 (d, J = 19.8 Hz, 1P, PCHPNP, major isomer), 3.40 (d, J = 20.7 Hz, 1P, PCHPNP,
minor isomer).
83

Synthesis of diethyl (1-{[benzyl({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino](ethoxy)phosphoryl}-3-
(benzyloxy)propyl)phosphonate (5b)
Acid 4b (172 mg, 0.3 mmol), thymidine (80 mg, 0.33 mmol), and PPh 3 (118 mg, 0.45
mmol) were co-evaporated with 2 mL of dry DMF 3 times, and then dissolved in 2 mL of
dry DMF. DIAD (87 μL, 0.45 mmol) was added dropwise and the resulting mixture allowed
to stand at r.t.
After 3 d, the solvent was removed under reduced pressure and the residue dissolved
in EtOAc and washed with 10 mL of 10% NaHCO 3. The aqueous layer was extracted with
EtOAc (3  15 mL), and the combined organic layers were washed with brine and dried
over Na 2SO 4. Product 5b was isolated by column chromatography (EtOAc  10% MeOH
in EtOAc) as a mixture of 8 isomers (114 mg, 47%).
1
H NMR (500 MHz, CDCl 3): δ 7.87-7.12
(11H, OCH 2C 6H 5, NCH 2C 6H 5, CH-T), 6.19-6.14 (1H, H-1'), 4.90-3.09 (20H, NCH 2C 6H 5,
OCH 2C 6H 5, 3 × OCH 2CH 3, H-5', H-4', H-3', PCHP, CH 2OBn, OCH 3), 2.43-1.99 (4H, CHCH 2CH 2,
H-2'), 1.88-1.80 (3H, CH 3-T), 1.35-1.10 (9H, 3 × OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ
28.86-27.87 (1P, P β), 23.73-23.22 (1P, P γ), 6.42-5.07 (1P, P α). MS (m/z): calcd for
C 34H 50N 3NaO 13P 3
+
824.2 [M+H]
+
, found: 824.3 [M+H]
-
.
84

Synthesis of diethyl (1-{ethoxy[({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino]phosphoryl}-3-hydroxypropyl)phosphonate
(6b)
Compound 5b (113 mg, 0.141 mmol) was dissolved in methanol (3 mL), and 7 mg (6
wt. %) of Pd(OH) 2/C was added. The r.m. was stirred under 1 atm H 2 for 20 h, then filtered
through a celite pad, and the volatiles evaporated to yield 93 mg of 6b as a mixture of 8
isomers as a colorless oil (99%).
1
H NMR (500 MHz, CD 3OD): δ 7.62-7.58 (1H, CH-T), 6.34-
6.23 (1H, H-1'), 4.47-4.06 (10H, 3 × OCH 2CH 3, H-5', H-4', H-3'), 3.86-3.68 (5H, CH 2OH,
OCH 3), 2.99-2.78 (1H, PCHP), 2.27-2.03 (4H, CHCH 2CH 2, H-2'), 1.98-1.91 (3H, CH 3-T), 1.38-
1.33 (9H, 3  OCH 2CH 3).
31
P NMR (202 MHz, CD 3OD): δ 23.98-23.73 (1P, P γ), 22.99-22.57
(1P, P β), 2.41-1.92 (1P, P α). MS (m/z): calcd for C 20H 38N 3NaO 13P 3
+
644.2 [M+Na]
+
, found:
644.4 [M+Na]
+
.
Synthesis of (3-hydroxy-1-{[({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}phosphinato)amino]phosphinato}propyl)phosphonate (1b)
A mixture of 6b (12 mg, 19 μmol), dry DMF (200 μL) and BTMS (200 μL, 1.52 mmol)
was microwaved at 40 C for 18 min. The reaction mixture was allowed to cool to r. t.,
volatiles were removed in vacuo, and the residue was treated 0.1 M TEAB (pH = 7), then
pH was adjusted to 10 using TEA. The product was purified by two-stage preparative
HPLC: first, on a SAX column (0-0.5 M TEAB gradient in 23 min); then, on a C-18 column
85

(0.1 M TEAB:5% MeCN, pH = 7.7). The fractions containing 2 (identified by MS) were
collected and combined, and evaporated yielding the TEA salt of compound 2 (1.25 mg,
13%).
1
H NMR (500 MHz, D 2O): δ 7.80 (s, 1H, CH-T), 6.37 (t, J = 6.9 Hz, 1H, H-1'), 4.69-4.66
(m, 1H, H-3'), 4.20-4.10 (m, 3H, H-4', H-5'), 3.84-3.77 (m, 2H, CH 2OH), 2.46-2.34 (m, 2H,
H-2'), 2.28-2.05 (m, 3H, PCHP, CH 2CH 2OH), 1.96 (s, 3H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ
19.11-19.05 (1P, P β), 16.04 (s, 1P, P γ), 1.05 (d, J = 7.6 Hz, isomer 1, P α), 1.02 (d, J = 7.6 Hz,
isomer 2, P α). MS (m/z): calcd for C 13H 23N 3O 13P 3
-
: 522.0 [M-H]
-
, found: 522.0 [M-H]
-
.
Synthesis of diethyl
({[benzyl(dimethoxyphosphoryl)amino](ethoxy)phosphoryl}(fluoro)methyl)phosphona
te (3c)
NaH (60% dispersion in oil) (52 mg, 1.3 mmol) was suspended in dry DMF (4 mL) and
cooled down to 0 ⁰C, and slowly added to a cooled solution of compound 2 (471 mg, 1.03
mmol) in 3 mL of dry DMF. The resulting solution was stirred at r.t. In 1 h, r. m. was cooled
down to 0 ⁰C, and the solution of Selectfluor® (440 mg, 1.24 mmol) in 3 mL of dry DMF
was added dropwise. R.m. was stirred for 5 h to r.t., then quenched with saturated NH 4Cl,
and the products was extracted with EtOAc. Products formed in the reaction were
purified using Combiflash® (0-30% EtOAc in hexanes in 26 min, fractions 13-39 were
collected), to give 241 mg of compound 3c (49%) as a mixture of 2 isomers.
1
H NMR (500
MHz, CDCl 3): δ 7.51-7.25 (m, 5H, NCH 2C 6H 5), 5.86 (ddd, 0.3H, J = 45.1 Hz, 13.5 Hz, 12.3 Hz,
CHF, minor isomer), 5.73 (ddd, 0.7H, J = 44.6 Hz, 14.0 Hz, 11.8 Hz, CHF, major isomer),
4.88-4.18 (m, 8H, NCH 2C 6H 5, 3  OCH 2CH 3), 3.80 (d, 0.9H, J = 11.5 Hz, OCH 3, minor isomer),
86

3.54 (d, 2H, J = 11.6 Hz, OCH 3, major isomer), 3.53 (d, 2H, J = 11.5 Hz, OCH 3, major isomer),
3.45 (d, J = 11.7 Hz, 3H, OCH 3, minor isomer), 1.41-1.27 (9H, 3  OCH 2CH 3).
31
P NMR (202
MHz, CDCl 3): δ 18.40 (dt, 1P, J = 75.0 Hz, 20.8 Hz, PCHPNP, major isomer), 17.42 (dt, 1P, J
= 64.1 Hz, 24.2 Hz, PCHPNP, minor isomer), 11.50 (dd, 1P, J = 60.7 Hz, 20.8 Hz, PCHPNP,
major isomer), 11.28 (dd, 1P, J = 60.3 Hz, 24.2 Hz, PCHPNP, minor isomer), 5.09 (d, 1P, J =
23.7 Hz, PCHPNP, minor isomer), 4.59 (d, 1P, J = 23.7 Hz, PCHPNP, major isomer).
19
F NMR
(470 MHz, CDCl 3): δ -224.8 (ddd, J = 75.0, 60.5, 44.5 Hz, major isomer), -226.0 (ddd, J =
64.1, 60.3, 45.1 Hz, minor isomer).
13
C NMR (126 MHz, CDCl 3) δ 137.7 (minor isomer),
137.7 (major isomer), 129.2 (major isomer), 128.7 (minor isomer), 128.13 (major isomer),
128.10 (major isomer), 127.7 (minor isomer), 127.6 (minor isomer), 85.5 (ddd, J = 193.2,
152.7, 143.4 Hz, minor isomer), 84.7 (ddd, J = 189.6, 152.4, 149.6 Hz, major isomer), 64.5
(d, J = 6.4 Hz, minor isomer), 64.4 (d, J = 6.2 Hz, major isomer), 64.1 (d, J = 6.6 Hz, minor
isomer), 63.9 (d, J = 7.1 Hz, major isomer), 63.5 (d, J = 6.6 Hz, minor isomer), 63.30 (d, J =
6.6 Hz, major isomer), 54.06 (d, J = 5.2 Hz), 53.56 (dd, J = 4.9, 2.7 Hz), 53.30 (d, J = 5.2 Hz),
50.3 (minor isomer), 49.33 (major isomer), 16.4 (d, J = 5.9 Hz, major isomer), 16.3 – 16.2
(5  d, major and minor isomers). MS (m/z): calcd for C 16H 29FNNaO 8P 3
+
498.1 [M+Na]
+
,
found: 498.4 [M+Na]
+
.
87

Synthesis of
[benzyl({[(diethoxyphosphoryl)(fluoro)methyl](ethoxy)phosphoryl})amino](methoxy)p
hosphinic acid (4c)
A solution of 3c (240 mg, 0.5 mmol) and 2 mL of triethylamine in 2 mL of acetonitrile
was refluxed for 3 h. The reaction progress was monitored by
31
P NMR. After completion,
solvents were removed under reduced pressure and the residue dried in vacuo. The
resulting oil was then dissolved in MeOH (5mL) and stirred with DOWEX H
+
(200 mg) for
two hours. After filtering out DOWEX H
+
, the solvent was evaporated yielding 230 mg
(99%) of the corresponding acid 4c as a mixture of 2 isomers in a ratio of 10:8.
1
H NMR
(500 MHz, CD 3OD): δ 7.51-7.25 (m, 5H, CH 2C 6H 5), 5.84-5.64 (m, 1H, CHF), 4.79-4.70 (m,
0.5H, NCH 2Ph), 4.60-4.06 (m, 6.5H, 3  OCH 2CH 3, 0.5  NCH 2C 6H 5), 3.49 (d, J = 11.8 Hz,
1.5H, OCH 3), 3.41 (d, J = 11.8 Hz, 1.5H, OCH 3), 1.40-1.24 (6  t, 9H, OCH 2CH 3).
31
P NMR
(202 MHz, CD 3OD) δ 17.84 (ddd, J PF = 76.0 Hz, J PP = 21.8 Hz, J PP = 18.0 Hz, PCHPNP, isomer
1), 17.27 (ddd, J PF = 62.9 Hz, J PP = 24.0 Hz, J PP = 22.3 Hz, PCHPNP, isomer 2), 11.65 (dd, J PF
= 64.4 Hz, J PP = 22.3 Hz, PCHPNP, isomer 2), 11.64 (d, J PF = 63.9 Hz, J PP = 18.0 Hz, isomer
1), 2.22 (d, J = 24.0 Hz, isomer 2), 1.90 (d, J = 21.8 Hz, isomer 1).
19
F NMR (470 MHz, CD 3OD)
δ -227.67 (app td, J FP = 62.9 Hz, J FP = 64.4 Hz, J FH =45.2 Hz, isomer 2), -228.58 (ddd, J FP =
76.0 Hz, J FP = 63.8 Hz, J FH = 44.7 Hz, isomer 1). MS (m/z): calcd for C 15H 26FNO 8P 3
-
: 460.1
[M-H]
-
, found: 460.0 [M-H]
-
.
88

Synthesis of diethyl ({[benzyl({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino](ethoxy)phosphoryl}(fluoro)methyl)phospho
nate (5c)
Acid 4c (230 mg, 0.5 mmol), thymidine (145 mg, 0.6 mmol), and PPh 3 (200 mg, 0. 75
mmol) were co-evaporated with 3 mL of dry DMF 3 times, and then dissolved in 3 mL of
dry DMF. DIAD (1487 μL, 0.75 mmol) was added dropwise and the resulting mixture
allowed to stand at r.t.
After 3 d, the solvent was removed under reduced pressure and the residue dissolved
in EtOAc and washed with 10 mL of 10% NaHCO 3. The aqueous layer was extracted with
EtOAc (3  15 mL), and the combined organic layers were washed with brine and dried
over Na 2SO 4. Product 5c was isolated by column chromatography (EtOAc  10% MeOH
in EtOAc) as a mixture of 8 isomers (101 mg, 30%).
1
H NMR (500 MHz, CDCl 3): δ 9.09-8.98
(1H, NH), 7.56-7.22 (5H, NCH 2C 6H 5, NCH 2C 6H 5, CH-T), 6.29-6.16 (1H, H-1'), 6.01-5.45 (CHF),
4.93-3.36 (15H, NCH 2C 6H 5, 3 × OCH 2CH 3, H-5', H-4', H-3', OCH 3), 2.44-1.86 (5H, H-2', CH 3-
T), 1.41-1.23 (9H, 3 × OCH 2CH 3).
31
P NMR (202 MHz, CDCl 3): δ 18.36-16.65 (1P, P β), 11.45-
10.35 (1P, P γ), 5.06-4.31 (1P, P α).
19
F NMR (470 MHz, CDCl 3): δ -223.42-(-224.37), -225.27-
(-226.23). MS (m/z): calcd for C 25H 39FN 3NaO 12P 3
+
708.2 [M+Na]
+
, found: 708.3 [M+Na]
+
.
89

Synthesis of diethyl ({ethoxy[({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy}(methoxy)phosphoryl)amino]phosphoryl}(fluoro)methyl)phosphonate (6c)
Compound 5c (100 mg, 0.146 mmol) was dissolved in methanol (4 mL), and 6 mg (6
wt. %) of Pd(OH) 2/C was added. The reaction mixture was stirred under 1 atm H 2 for 5 h,
then filtered through a celite pad, and the volatiles evaporated to yield 82 mg of 6c as a
mixture of 8 isomers as a colorless oil (94%).
1
H NMR (500 MHz, CD 3OD): δ 7.58 (1H, CH-
T), 6.34-6.25 (1H, H-1'), 5.54-5.37 (CHF), 4.45-3.82 (13H, 3 × OCH 2CH 3, H-5', H-4', H-3',
OCH 3), 2.29-2.19 (2H, H-2'), 1.96-1.90 (3H, CH 3-T), 1.38-1.37 (9H, 3  OCH 2CH 3).
31
P NMR
(202 MHz, CD 3OD): δ 12.61-11.82 (1P, P α), 11.55-10.97 (1P, P β), 1.71-0.96 (1P, P γ).
19
F NMR
(470 MHz, CD 3OD): δ -226.79-(-227.32), -228.15-(-228.64) MS (m/z): calcd for
C 18H 33N 3O 12P 3
-
576.1 [M-H]
-
, found: 576.1 [M-H]
-
. MS (m/z): calcd for C 18H 33FN 3NaO 12P 3
+

618.1 [M+Na]
+
, found: 618.2 [M+Na]
+
.
Synthesis of hydrogen {fluoro[hydroxy({[hydroxy({[(2R,5R)-3-hydroxy-5-(5-methyl-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-
yl]methoxy})phosphoryl]amino})phosphoryl]methyl}phosphonate (1c)
A mixture of 6c (5 mg, 8.4 μmol), dry DMF (100 μL) and BTMS (100 μL, 0.76 mmol) was
microwaved at 40 C for 10 min. The reaction mixture was allowed to cool to r.t., volatiles
were removed in vacuo, and the residue was treated 0.1 M TEAB (pH = 7), then pH was
adjusted to 10.2 using TEA. The product was purified by two-stage preparative HPLC: first,
on a SAX column (0-0.5 M TEAB gradient in 20 min); then, on a C-18 column (0.1 M
90

TEAB:5% MeCN, pH = 7.5). The fractions containing 1c (identified by MS) were collected
and combined, and evaporated yielding the compound 1c as a mixture of 2 diastereomers
as a TEA salt (1.5 mg, 18%).
1
H NMR (500 MHz, D 2O): δ 7.66 (s, 0.5H, CH-T), 7.64 (s, 0.5H,
CH-T), 6.22 (t, J = 7.0 Hz, 1H, H-1'), 4.56-4.50 (m, 1H, H-3'), 4.06-3.96 (m, 3H, H-4', H-5'),
2.32-2.19 (m, 2H, H-2'), 1.81 (s, 3H, CH 3-T).
31
P NMR (202 MHz, D 2O): δ 9.06-8.69 (1P, P β),
7.94-7.60 (1P, P γ), 0.08 (s, 1P, P α).
19
F NMR (470 MHz, D 2O): δ -215.6-(-216.02) (m). MS
(m/z): calcd for C 11H 18FN 3O 12P 3
-
: 496.0 [M-H]
-
, found: 496.0 [M-H]
-
.

91

Chapter 3 references
(1) Kadina, A. P.; Kashemirov, B. A.; Oertell, K.; Batra, V. K.; Wilson, S. H.; Goodman, M. F.;
McKenna, C. E., Org. Lett. 2015, 17, 2586-2589.
(2) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.;
Beard, W. A.; Wilson, S. H., J. Am. Chem. Soc. 2007, 129, 15412-15413.
(3) (a) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.;
Goodman, M. F.; McKenna, C. E., Journal of the American Chemical Society 2010, 132, 7617-7625;
(b) Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard, W. A.; Wilson, S.
H.; McKenna, C. E.; Goodman, M. F., Biochemistry 2012, 51, 8491-8501; (c) 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 2008, 47, 870-879.
(4) Jeffrey, G. A.; Editor, An Introduction to Hydrogen Bonding. Oxford Univ Press1997; p 272 pp.
(5) Trott, O.; Olson, A. J., Journal of Computational Chemistry 2010, 31, 455-461.
(6) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.; Wilson, S. H.;
McKenna, C. E., J. Am. Chem. Soc. 2012, 134, 8734-8737.
(7) King, B. W. Preparation of quinolinylmethoxyphenyl-substituted lactam derivatives as
inhibitors of matrix metalloproteinases and/or TNF-alpha converting enzyme. US20040266751A1,
2004.
(8) Banks, R. E.; Mohialdin-Khaffaf, S. N.; Lal, G. S.; Sharif, I.; Syvret, R. G., Journal of the Chemical
Society, Chemical Communications 1992, 595-596.
(9) Ahlmark, M. J.; Vepsalainen, J. J., Tetrahedron 1997, 53, 16153-16160.
(10) Mitsunobu, O.; Yamada, M., Bull. Chem. Soc. Jpn. 1967, 40, 2380-2382.
(11) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C., Tetrahedron Lett. 1977, 155-158.
(12) Zhao, Y.; Chen, G., Organic Letters 2011, 13, 4850-4853.

92

Chapter 4 - Synthesis and Inhibition Mechanism of “pppCH2dU” –
Inhibitor Probe of SAMHD1
(The research discussed in this chapter has been substantially published
1
)
4.1 SAMHD1 structure and function
SAM domain and HD domain-containing protein (SAMHD1) is the only known human
enzyme having an unusual non-specific dNTP triphosphohydrolase activity (Figure 4.1),
which helps the cells to maintain optimal dNTP levels for DNA replication.
2
SAMHD1 is
expressed at basal levels in a large number of tissues,
3
but is found in high concentrations
in dendritic and other myeloid cells.
4

Figure 4.1. GTP-dependent SAMHD1-catalyzed hydrolysis of dNTP to PPP i and
nucleoside
SAMHD1 consists of an N-terminal Sterile Alpha Motif (SAM) domain and a C-terminal
HD domain (Figure 4.2). The HD domain is often found in the classes of enzymes having
phosphohydrolase activity, such as phosphodiesterases, phosphatases, and nucleases.
Tripolyphosphate product likely degrades to 3 P i (phosphate anions) by cellular
pyrophosphatases or tripolyphosphatases.
5
Degradation to the level of nucleoside (dN)
rather than dNDP or dNMP is possibly needed to make the process energetically or
93

kinetically difficult to reverse, with the additional possibility that the neutral nucleoside
can be irreversibly transported out of the cell through the cell wall.
6

Figure 4.2. Crystal structure of SAMHD1 monomer unit catalytic core (residues 109-
626) from the GTP-bound SAMHD1 dimer showing the major lobe, minor lobe and C-
terminal region. N-terminal SAM domain (pink) and C-terminal HD domain (lilac) are
shown
7

SAMHD1 is an important HIV-1 and HSV-1 restriction factor.
2, 8
It is generally believed
that the mechanism of viral restriction is based on the protein’s ability to deplete the pool
of dNTP substrates of viral reverse transcriptase in myeloid lineage target cells of HIV-1,
such as resting T cells, macrophages and dendritic cells.
2, 9
However, recent findings
suggested that another possible mechanism for viral restriction is SAMHD1-catalyzed
hydrolysis of viral RNA or the newly synthesized DNA. In support to this hypothesis,
SAMHD1 has been shown to have RNA and ssDNA exonuclease activity.
10
Mutations in
SAMHD1 have been linked to Aicardi-Goutières syndrome (AGS), a rare genetic
autoimmune disease.
11
Unsurprisingly, CD4
+
T cells in AGS patients are permissive
towards HIV-1 infection.
12
Paradoxically, SAMHD1-related suppression of HIV infection in
94

dendritic cells may prevent activation of CD4
+
T cells through the IFN1-toll receptor
pathway, thus preventing the stimulation of a strong adaptive immune response by the
host.
13

4.2 SAMHD1 activation mechanism
SAMHD1 displays a complex sequential activation (Figure 4.3). In the absence of
nucleotides, SAMHD1 is in equilibrium between monomer and dimer forms and does not
display any activity. Binding of GTP or dGTP to a guanine nucleotide-specific activator site
(A1) on each monomer induces dimerization.
14
The cellular level of GTP is 1000-fold
higher than that of dGTP and, therefore, it is the primary source of enzyme activation.
14-
15
The A1 site is highly specific for guanine due to a unique interaction between the
guanine base and Asp137 in the A1 site.
7
The possible structural basis for the equilibrium
shift towards enzyme dimer is the electrostatic interaction between (d)GTP triphosphate
group and the second monomer unit.
16
Each monomer subunit of the resulting dimer can
bind any dNTP molecule to a second nonspecific activator site (A2).
16
Presumably, sterical
hindrance between 2’-OH group in ribonucleotides and Phe157 prevents the binding of
NTPs to the second A2 site,
7
so GTP cannot activate SAMHD1 in the absence of dNTPs. In
the dimer, the catalytic site is blocked by the C-terminal, preventing the substrate from
entry to the catalytic site. dNTP binding to the A2 site induces tetramerization, which
results in a large conformational shift of the SAMHD1 C-terminal region, and thus allows
the substrate to enter the catalytic site. The combined effect of dNTP binding to the A2
95

and, eventually, to the catalytic sites shifts the entire equilibrium to an active, long-lived
tetrameric state, which can persist long after activator molecules are removed from the
environment.
14
Thus, the fully activated tetramer contains four occupied pairs of activator
sites (A1A2) 4 and four catalytic sites (S) 4.
16-17
This mechanism of activation presents
multiple opportunities for the design of small molecules modulating the enzyme activity.

Figure 4.3. Model for sequential activation of SAMHD1 by GTP activators and dNTP
coactivators and substrates by stepwise formation of a long-lived activated tetramer. The
(A1A2) 4S 4 complex is shown as isomerizing (K iso) to a long-lived tetramer that performs
steady-state turnover. Activator sites of the long-lived tetramer are not in equilibrium
with free nucleotides.
14
Crystal structures of singly (GTP) and doubly (GTP + dNTP)
activated SAMHD1 are shown on top of their schematic counterparts.
7

96

4.3 Design of the inhibitor probe of SAMHD1
Inhibitors of SAMHD1 could serve as stimulators of an adaptive immune response by
restoring the host’s innate immune activity that is otherwise deficient.
18
Our purpose was
to design, synthesize and characterize an inhibitor of SAMHD1, which may in addition
prove helpful in studying the possible sites and mechanisms of enzyme inhibition.
The dNTP hydrolysis occurs via a SAMHD1-catalyzed attack of a water molecule onto
the P α of the dNTP in the catalytic site, followed by the elimination of the nucleoside
leaving group. This mechanism was confirmed by mass-spectrometry studies of the
products of SAMHD1-catalyzed reaction between dGTP and H 2
18
O.
1
Subsequently, if the
5’-oxygen atom in substrate dNTP is replaced by a non-hydrolyzable moiety (such as a
methylene group),
19
enzymatic cleavage of dNTP will be prevented. Therefore, we
designed 5’-methylene dUTP (pppCH 2dU, 1) (Figure 4.4) as a potential competitive
inhibitor of SAMHD1.

Figure 4.4. pppCH2dU (1) – inhibitor probe of SAMHD1
4.4 General method of synthesis of 5’-methylene (d)NTPs
Nucleoside phosphonates have been previously utilized as chain terminators of RNA
and DNA synthesis, such as in the synthesis of DNA by HIV reverse transcriptase for anti-
97

HIV chemotherapy.
20
Furthermore, 5’-methylene dNTP analogues have been considered
as HCV RNA-polymerase inhibitors and chain terminators.
21
Nucleoside phosphonates
offer an advantage over their phosphate analogues for their higher metabolic stability
since they are not susceptible to hydrolysis by phosphatases.
Nucleoside 5’-monophosphonate cores are commonly synthesized by the following
general approach (Scheme 4.1).
21-22
First, 3’ and 2’-OH groups are protected, and the free
5’-OH group is oxidized to aldehyde at 5’-carbon. The oxidizing agents commonly used for
this transformation include 2-iodoxybenzoic acid (IBX),
23
Dess-Martin periodinane
(DMP),
24
and DCC/DMSO (Pfitzner-Moffatt oxidation).
25
The aldehyde, without further
purification, is converted to alkene using either sodium salt of tetraalkyl
methylenebisphosphonate (Horner-Wadsworth-Emmons reaction) or diaryl
triphenylphosphoranylidene methylphosphonate.
26
The double bond of the alkene is
then hydrogenated under catalytic conditions, and the nucleoside phosphonate is
obtained after removal of protecting groups from 2’- and 3’-hydroxyls.
Scheme 4.1. General method of synthesis of 5’-methylene nucleotide analogues

98

4.5 Synthesis of pppCH 2dU – inhibitor probe of SAMHD1
We have initiated the synthesis of pppCH 2dU (Scheme 4.2) with protection of the 3’-
and 5’-hydroxyl groups of deoxyuridine (dU) with tert-butyldimethylsilyl chloride
(TBDMSCl).
27
5’-silyl group was selectively removed from 2 with trichloroacetic acid to
form free 5’-OH group.
22a, 28
Adapting the procedure of Schinazi,
22a
alcohol 3 was then
oxidized with IBX,
23
and the resulting aldehyde 4 was reacted with sodium salt of
tetramethyl (methylene)bisphosphonate (TMMBP) carbanion. Since methyl groups on
the phosphonate are the easiest to be removed on the following steps, TMMBP was
preferred over more readily available tetraisopropyl (methylene)bisphosphonate.
Formation of 5 was confirmed by
1
H NMR spectrum of the reaction product. Two new
signals were identified at 5.9 ppm and 6.8 ppm, corresponding to alkene 5’- and 6’-
protons, respectively. Alkene 5 was predominantly formed as the trans-isomer, as
determined by the coupling constant (J HH = 17.1 Hz) between 5’ and 6’ protons, which is
larger than common cis-coupling constants for vicinal alkene protons. Hydrogenation of
the double bond in compound 5 was effected by H 2 (1atm) in the presence of Pd/C,
affording the 3'-TBS dimethyl ester of pCH 2dU, 6.
99

O
OH
HO
U
TBDMSCl
N
N
H
O
OTBS
TBSO
U
O
OTBS
HO
U CCl
3
COOH
THF/H
2
O
O
OTBS
U
IBX
MeCN
dU
O
O
OTBS
U
P
O
MeO
MeO
O
OTBS
U
P
O
MeO
MeO
TMMBP
NaH, THF
H
2
, Pd/C
MeOH
1) BTMS, 7 min
MW, MeCN
2) H
2
O
O
OH
U
P
O
HO
HO
O
H
N
DCC
t-BuOH/H
2
O
O
OH
U
P
O
N
HO
O
DMSO
O
OH
U
P
O
P
O
O O
P
O
-
O
-
O
O
-
O
-
2 (97% ) 3 (58% )
4 5
(79% over 2 steps)
6 (94% )
7
8 1
(25% over 3 steps)
pppCH
2
dU
TBA/PP
i
Scheme 4.2. Synthesis of pppCH 2dU (1)
The methyl and t-butyldimethylsilyl protecting groups were removed by
bromotrimethylsilane (BTMS)
29
providing a facile hydrolysis resulting in the formation of
pCH 2dU (7). It was previously reported
22b
that conventional BTMS deprotection of the
diethyl ester of 7 resulted in significant anomerization of the nucleotide (β:α-anomers,
2:3). In contrast, we found that microwave-assisted BTMS silyldealkylation
30
(MeCN,
60 C, 7 min, MW), followed by hydrolysis with H 2O, provided phosphonic acid 7 with
minimal anomerization ( : -anomers, 8:1) (Figure 4.5). The ratios are based on values
obtained after the conversion to 1 and after the first round of purification (SAX), which
eliminates the majority of impurities.
100

Figure 4.5. Part of the 500 MHz
1
H NMR spectrum of α- and β- anomers of 1 in D 2O
(after SAX purification)
Although it was possible to prepare 7 free of -anomer by HPLC purification, 7 was
more conveniently first converted to the corresponding dUTP analogue 1 via coupling of
morpholidate 8 with tributylammonium pyrophosphate (TBA/PP i) in DMSO,
31
followed by
the isolation of the pure -anomer of 1 using two-step HPLC purification (SAX followed by
RP). The final product, pppCH 2dU (1) was characterization by
1
H,
31
P and
13
C NMR and by
high resolution mass spectrometry (HRMS).
Results and Discussion
Enzymatic studies were performed by the research group of Prof. James T. Stivers at
John Hopkins School of Medicine in Baltimore, Maryland.
Biological data revealed that 1 was completely nonreactive after 24 h incubation with
SAMHD1 regardless of whether GTP activator was present. Thus, to the best of our
101

knowledge, compound 1 is the first completely nonreactive substrate analogue of
SAMHD1. The inhibition mechanism of 1 was investigated using dUTP as the substrate. 1
appeared to follow a simple competitive inhibition with K i = 80 ± 6 μM with respect to
dUTP, which is surprisingly 20-fold lower than the K m for the substrate dUTP (Figure
D.17A).
14

Since the K i of 1 was much lower than the K m for dUTP and the A2 site can bind any
dNTP, including dUTP,
14
it is possible that 1 targets the A2 site in addition to the catalytic
site, but fails to activate the protein (the possibility that 1 binds to the A1 site is excluded
because this site is highly specific for guanine nucleotides).
14
It was recently determined
that A1 and A2 sites become inaccessible to free ligands after formation of the active
tetramer, but the catalytic site remains accessible.
14
Thus, order-of-addition experiments
in which 1 was added before or after dUTP and GTP can differentiate between the
inhibition caused by the binding of 1 to either A2 or catalytic site. To test for binding of 1
to the catalytic site, a reaction of SAMHD1 with GTP and dUTP was initiated and allowed
to proceed for 2.5 min to ensure the occupation of the A1 and A2 sites by GTP and dUTP,
respectively, and the sequestration of these sites from free ligand. Subsequently, various
concentrations of 1 were added to the reaction, and the rate of dUTP consumption was
determined at different time points. The K i (670 ± 110 μM) obtained under these
conditions was similar to the K m = 1.5 mM for dUTP. This suggests, that when the A2 site
is unavailable, 1 targets the catalytic site. In contrast, when dUTP was added after
SAMHD1 was pre-incubated with GTP and 1, K i = 52 ± 5 μM was obtained, which is
102

significantly lower than the K m for dUTP. Thus, order-of-addition experiments strongly
suggest that 1 targets both the A2 site and the catalytic site, but its activity largely results
from the binding to the A2 site (Figure D.17B).
Since 1 was initially presumed to behave as a dUTP analogue, it was not clear why its
binding to the A2 site resulted in inhibition rather than activation. The initial hypothesis
that 1 prevents the formation of active tetramer was confirmed by glutaraldehyde
crosslinking in the presence and absence of 1 under conditions of the inhibition
experiments. Separation of the cross-linked forms by denaturing gel electrophoresis
showed that 2 mM of 1 completely prevented the tetramer formation in the presence of
1 mM dUTP and 5 mM GTP.
1

To further explore this unforeseen mechanism, a recently developed dilution-jump
(DJ) method was used. DJ allows assessment of the activity and stability of oligomeric
forms of SAMHD1.
14
This approach involves the formation of complexes of SAMHD1 with
activators and/or dNTPs at high concentration (pre-jump condition) followed by a rapid
100-fold dilution into a solution with low activator and/or substrate dNTP concentration
(post-jump condition). The post-jump assay took two forms: kinetic and structural (Figure
D.17C). In the structural mode (DJXL), oligomeric states are identified that were present
at the time of dilution, as well as their rate of decay in a given post-jump condition. In the
dilution-jump kinetic (DJK) mode, chronological changes in the catalytic activity of the
enzyme after dilution into a post-jump solution containing a radiolabeled substrate ([5-
3
H] dUTP) are determined. The DJXL experiments assessed the effect of 1 on the
103

oligomeric states of SAMHD1. When the pre-jump solution contained [dUTP + GTP] but
not 1, the SAMHD1 tetramer was efficiently formed, while in the absence of these
nucleotides, only the monomer and dimer forms of SAMHD1 were found.
14
The tetramer
generated in the pre-jump persisted for hours, even in the absence of GTP activator in the
post-jump, confirming the previous findings that the tetramer is long-lived in the absence
of activators. Including 1 into the pre-jump solution prevented the formation of the
tetramer, as determined by glutaraldehyde cross-linking.
1

The DJK method evaluated the effect of 1 on the post-jump enzymatic activity of
SAMHD1. The pre-jump solution consisted of SAMHD1, GTP activator, and dUTP substrate
in the absence or presence of 1, while the post-jump solution contained [5-
3
H] dUTP
substrate but no GTP activator. In the absence of 1 in the pre-jump, burst-phase for dUTP
hydrolysis in the post-jump was detected. The burst-phase decayed exponentially and
eventually yielded a linear steady-state rate lasting for hours. The burst-decay period (t 1/2
∼ 10 min) was attributed to the decay of SAMHD1 from a highly active form, generated
in the pre-jump solution, to a less active however long-lived form that persists even in the
absence of GTP activator. With 1 (5 mM) in the pre-jump solution, the post-jump burst-
decay difference was almost abolished, and the linear steady-state rate was reduced
significantly even though the concentration of pppCH 2dU in the post-jump was much
lower than that of the dUTP substrate.
1

104

Figure 4.6. Double hit inhibition mechanism of SAMHD1 by 1. 1 prevents the ordered-
essential activation mechanism of SAMHD1 by binding to the A2 activator site abolishing
the tetramer formation (K i = 52 µM). In addition, 1 binds to the catalytic site and
competitively inhibits substrate binding (K i = 670 µM).
1
(Data obtained by Kyle Seamon
and Eric Hansen in Prof. James Stivers laboratory)
In conclusion, although 1 was initially viewed as a substrate mimic and a competitive
inhibitor, the above data strongly suggest that 1 binds to SAMHD1 in a manner that is
disruptive not only to catalytic activity but also to tetramer formation (Figure 4.6). This
unusual inhibition mechanism takes advantage of the sequential activation of SAMHD1.
Binding of 1 to the A2 sites provides a reasonable explanation for the long-term inhibition
of tetramer formation caused by 1 in the DJXL experiment. When 1 binds to the A2 sites
in the pre-jump solution, it disrupts the ordered binding of GTP and dUTP, and in the
absence of GTP in the post-jump, tetramer reassembly is not possible.
These findings reveal a wide range of possible inhibition mechanisms of SAMHD1 by
dNTP analogues. By binding to two active sites within the enzyme, 1 interferes with the
105

sequential activation mechanism and prevents tetramerization. This, along with the
multiple ligand binding pockets on SAMHD1, suggests that rational design of inhibitors
and activators, modulating its activity, is possible. Modulator ligands could have research
and therapeutic use in preventing or treatment of autoimmune and chronic inflammatory
diseases
32
as well as viral infection.
33

Experimental Section
Materials and methods
All reagents for chemical synthesis were purchased from Alfa Aesar or Sigma-Aldrich.
Dry dimethylformamide (DMF), tetrahydrofuran (THF), and acetonitrile were purchased
from EMD Chemicals and used without further purification. Methanol and t-butyl alcohol
were purchased from Macron Fine Chemicals and TCI, respectively. DOWEX 50WX8-200
ion exchange resin was purchased from Sigma-Aldrich. Sodium hydride was used as a 60%
dispersion in oil. Iodoxybenzoic acid (IBX) was prepared by the method of Sputore
1
. Thin
layer chromatography (TLC) plastic-back sheets (20  20; silica gel 60 F 254) were purchased
from EMD Chemicals. Silica gel, Grade 62 (60-200 mesh) used for column chromatography
was purchased from Macron. HPLC experiments were carried out using a Varian ProStar
210, equipped with a semi-preparative column (21.4  150 mm SP 15/20 Nucleogel SAX,
Macherey-Nagel or 21.20  250 mm Phenomenex Luna 5u C18 columns) and a Shimadzu
SPD-10A VP UV-Vis detector. Eluates were detected at λ = 260 nm.
1
H,
13
C, and
31
P NMR
spectra were measured on a Varian VNMRS-500 NMR spectrometer. Chemical shifts ( )
106

are reported in parts per million (ppm) relative to internal residual CHCl 3 in CDCl 3 (  7.26,
1
H) or HDO in D 2O (  4.80,
1
H), internal CDCl 3 (δ 77.0,
13
C) or external 85% H 3PO 4 (  0.00,
31
P). NMR FIDs were processed using MestReNova v9.0.0. Low resolution mass
spectrometry for chemical synthesis was performed on a Finnigan LCQ Deca XP Max
spectrometer equipped with an ESI source operated in negative ion mode. HRMS data
were obtained at the UC Riverside (Dr. Ron New) high resolution mass spectrometry
facility. Microwave-assisted synthesis was performed using a Milestone Ethos Synth
Microwave Labstation. Compound IUPAC names were assigned using MarvinSketch 6.1.5.
1‐[(2R,5R)‐4‐[(tert‐butyldimethylsilyl)oxy]‐5‐{[(tert‐
butyldimethylsilyl)oxy]methyl}oxolan‐2‐yl]‐1,2,3,4‐tetrahydropyrimidine‐2,4‐dione
(2)
22a, 27

To a solution of 2’-deoxyuridine (0.4 g, 1.75 mmol) and imidazole (0.6 g, 8.75 mmol)
in 4 mL of dry DMF was added t-butyldimethylsilyl chloride (660 mg, 4.4 mmol). The
reaction mixture was stirred for 4 h at r.t. The reaction mixture was quenched by addition
of saturated NaHCO 3, and the product was extracted with CH 2Cl 2 (3 15 ml). The organic
layers were washed with brine, dried over Na 2SO 4, filtered, and the solvent was
evaporated. The white crystalline product (774 mg, 97%) was used in the next step
without further purification.
1
H NMR (500 MHz, CDCl 3):  7.97 (1H, br, NH), 7.90 (1H, d, J
= 8.2 Hz, H-6), 6.28 (1H, t, J = 6.2 Hz, H-1’), 5.67 (1H, d, J = 8.2 Hz, H-5), 4.41 (1H, dt, J =
6.5 Hz, J = 4.0 Hz, H-3’), 3.93-3.91 (1H, m, H-4’), 3.91-3.75 (2H, m, H-5’), 2.32 (1H, ddd, J =
13.5 Hz, J = 6.2 Hz, J = 4.2 Hz, H-2’), 2.06 (1H, dt, J = 13.5 Hz, J = 6.3 Hz, H-2’), 0.92 (9H, s,
107

C(CH 3) 3), 0.89 (9H, s, C(CH 3) 3), 0.10 (3H, s, Si(CH 3)), 0.01 (3H, s, Si(CH 3)), 0.08 (3H, s,
Si(CH 3)), 0.07 (3H, s, Si(CH 3)).
1‐[(2R,5R)‐4‐[(tert‐butyldimethylsilyl)oxy]‐5‐(hydroxymethyl)oxolan‐2‐yl]‐1,2,3,4-
tetrahydropyrimidine‐2,4‐dione (3)
22a, 28

A solution of 2 (0.775 g, 1.7 mmol) in 8 mL of THF was cooled to 0 ⁰C, and a solution
of trichloroacetic acid (5.56 g, 34 mmol) in 10 mL of H 2O was added. The r.m. was stirred
for 1 h at 0 ⁰C. Reaction progress was monitored by TLC (EtOAc:Hexanes = 2:1).
After 1 h the reaction mixture was quenched with solid Na 2CO 3, and 20 mL of water
was added. The product was extracted with CH 2Cl 2 (3 10 mL). The combined organic
layers were washed with brine and dried over Na 2SO 4. After filtration, the solvent was
removed, and the residue was purified by silica gel column chromatography
(EtOAc:Hexanes, 1:1  3:1). The product was isolated as a white crystalline solid (344 mg,
60%).
1
H NMR (400 MHz, CDCl 3):  8.05 (1H, br, NH), 7.62 (1H, d, J = 8.1 Hz, H-6), 6.16 (1H,
t, J = 6.6 Hz, H-1’), 5.73 (1H, d, J = 8.1 Hz, H-5), 4.49 (1H, dt, J = 6.2 Hz, J = 4.0 Hz, H-3’),
3.96-3.75 (3H, m, H-4’, H-5’), 2.35-2.24 (2H, m, H-2’), 2.12 (1H, br, OH), 0.90 (9H, s,
C(CH 3) 3), 0.09 (6H, s, Si(CH 3) 2).
Dimethyl [(E)‐2‐[(2R,5R)‐3‐[(tert‐butyldimethylsilyl)oxy]‐5‐(2,4‐dioxo‐1,2,3,4‐
tetrahydropyrimidin‐1‐yl)oxolan‐2‐yl]ethenyl]phosphonate (5)
22a

Alcohol 3 (120 mg, 0.35 mmol) was dissolved in 4 mL of CH 3CN, and freshly prepared
IBX (108 mg, 0.385 mmol) was added. The reaction mixture was refluxed for 1 h. Product
formation was monitored by TLC (EtOAc:Hexanes = 2:1). Upon completion of the reaction,
108

the mixture was cooled to 0 ⁰C, filtered and washed with cold acetonitrile. The solvent
was removed, and the residue was co-evaporated with dry THF and then dried under high
vacuum. Aldehyde 4 was used in the following reaction without further purification.
To a suspension of NaH (26 mg, 0.65 mmol) in dry THF (2 mL) at 0 ⁰C was added a
solution of tetramethyl methylenebisphosphonate in 4 mL of dry THF. The reaction
mixture was stirred at 0 ⁰C for 10 min. The resulting suspension was then added dropwise
to a solution of aldehyde 4 in 4 mL of dry THF at 0 ⁰C. The reaction mixture was stirred for
3 h and allowed to slowly warm up to r.t. before being quenched with saturated NH 4Cl
and extracted with EtOAc (3 10 mL). The combined organic layers were washed with
brine and dried over Na 2SO 4. The residue was purified by silica gel column
chromatography (1% MeOH in EtOAc), giving 124 mg of 5 as a colorless oil (79% over two
steps).
31
P NMR (202 MHz, CDCl 3):  19.68 (s);
1
H NMR (500 MHz, CDCl 3):  9.66 (1H, bs,
NH), 7.31 (1H, d, J = 8.1 Hz, H-6), 6.83 (1H, ddd, J = 22.2 Hz, 17.1 Hz, 5 Hz, H-6’), 6.27 (1H,
t, J = 6.5 Hz, H-1’), 5.94 (1H, ddd, J = 19.4 Hz, 17.1 Hz, 1.7 Hz, H-5’), 5.76 (1H, dd, J = 8.1
Hz, 1.7 Hz, H-5), 4.36-4.33 (1H, m, H-3’), 4.24-4.21 (1H, m, H-4’), 3.73 (3H, d, J = 11.1 Hz,
P(O)(OCH 3)), 3.73 (3H, d, J = 11.2 Hz, P(O)(OCH 3)), 2.31 (1H, ddd, J = 13.4 Hz, 6.3 Hz, 4.7
Hz, H-2’), 2.12 (1H, dt, J = 13.4 Hz, 6.6 Hz, H-2’), 0.87 (9H, s, C(CH 3) 3), 0.06 (6H, s, Si(CH 3) 2);
13
C NMR (126 MHz, CDCl 3):  163.1 (C-4), 150.2 (C-2), 148.5 (d, J = 5.7 Hz, C-5’), 139.3 (C-
6), 117.7 (d, J = 189.3 Hz, C-6’), 103.0 (C-5), 86.1 (d, J = 22.4 Hz, C-4’), 85.3 (C-1’), 74.7 (d,
J = 1.9 Hz, C-3’), 52.5 (d, J = 9.0 Hz, P(O)(OCH 3)), 52.5 (d, J = 8.9 Hz, P(O)(OCH 3)), 40.1 (C-
2’), 25.6 (C(CH 3) 3), 17.8 (C(CH 3) 3), -4.8 (Si(CH 3)), -4.9 (Si(CH 3)).
109

Dimethyl {2‐[(2R,5R)‐3‐[(tert‐butyldimethylsilyl)oxy]‐5‐(2,4‐dioxo‐1,2,3,4‐
tetrahydropyrimidin‐1‐yl)oxolan‐2‐yl]ethyl}phosphonate (6)
22a

To a solution of compound 5 (40 mg, 0.09 mmol) in 4 mL of methanol was added Pd/C
(4 mg, 10 wt %), and the reaction mixture stirred under H 2 (1 atm) for 1 h. It was then
filtered through a celite pad, and the methanol was evaporated. The residue was dried
under vacuum, yielding 38 mg of compound 5 (94%) as a colorless oil, which was used
without further purification.

HRMS (ESI/APCI): calcd for C 18H 34N 2O 7PSi, [M-H]
-
449.1867;
found 449.1871 m/z.
31
P NMR (202 MHz, CDCl 3):  33.70 (s);
1
H NMR (500 MHz, CDCl 3): 
9.59 (1H, br, NH), 7.31 (1H, d, J = 8.1 Hz, H-6), 6.15 (1H, t, J = 6.5 Hz, H-1’), 5.74 (1H, d, J =
8.1 Hz, H-5), 4.05 (1H, dt, J = 6.7 Hz, 4.4 Hz, H-3’), 3.79-3.73 (1H, m, H-4’), 3.73 (6H, d, J =
10.8 Hz, P(O)(OCH 3) 2), 2.28 (1H, ddd, J = 13.6 Hz, 6.5 Hz, 4.4 Hz, H-2’), 2.06 (1H, dt, J = 13.6
Hz, 6.6 Hz, H-2’), 2.00-1.74 (4H, m, H-5’, H-6’), 0.86 (9H, s, C(CH 3) 3), 0.05 (3H, s, Si(CH 3)),
0.05 (3H, s, Si(CH 3)).
13
C NMR (126 MHz, CDCl 3):  163.3 (C-4), 150.2 (C-2), 139.4 (C-6),
102.7 (C-5), 86.0 (d, J = 16.7 Hz, C-4’), 85.0 (C-1’), 74.5 (C-3’), 52.5 (d, J = 6.3 Hz,
P(O)(OCH 3)), 52.4 (d, J = 6.3 Hz, P(O)(OCH 3)), 40.6 (C-2’), 26.4 (d, J = 4.7 Hz, C-5’), 25.6
(C(CH 3) 3), 21.2 (d, J = 143.4 Hz, C-6’)), 17.8 (C(CH 3) 3), -4.6 (Si(CH 3)), -4.9 (Si(CH 3)).
{2‐[(2R,5R)‐5‐(2,4‐dioxo‐1,2,3,4‐tetrahydropyrimidin‐1‐yl)‐3‐hydroxyoxolan‐2‐
yl]ethyl}phosphonate (TEA salt) (7)
22b

Compound 6 (17 mg, 0.038 mmol) was dissolved in 500 L of dry acetonitrile, and
BTMS (11 L, 0.08 mmol) was added. The reaction mixture was microwaved at 60 ⁰C for
7 min. Volatiles were removed under reduced pressure, and the residue treated with
110

water. After stirring for 30 min, water was evaporated, the reaction mixture dried under
vacuum and the resulting residue used in the following step without further purification.
A sample of compound 7 was isolated using two-step HPLC (SAX (0 50% linear
gradient, A = water, B = 0.5 M TEAB, pH 7.5, 20 min, 8 mL/min) followed by RP HPLC
(isocratic, 0.1 M TEAB, 5% CH 3CN, pH 7.0, 8 mL/min)). HRMS (ESI/APCI): calcd for
C 10H 14N 2O 7P
-
, [M-H]
-
305.0533; found 305.0548 m/z.
31
P NMR (202 MHz, D 2O):  20.92;
1
H
NMR (500 MHz, D 2O):  7.71 (1H, d, J = 7.8 Hz, H-6), 6.30 (1H, t, J = 6.9 Hz, H-1’), 5.87 (1H,
d, J = 7.8 Hz, H-5), 4.35 (1H, dt, J = 6.9 Hz, J = 3.7 Hz, H-3’), 3.97 (1H, td, J = 6.9 Hz, J = 3.7
Hz, H-4’), 2.39-2.28 (2H, m, H-2’), 1.93-1.84 (2H, m, H-5’/6’), 1.61-1.40 (2H, m, H-5’/6’).
13
C NMR (126 MHz, D 2O):  163.6 (C-4), 157.6 (C-2), 141.4 (C-6), 103.4 (C-5), 88.4 (d, J =
17.5 Hz, C-4’), 85.6 (C-1’), 73.9 (C-3’), 39.0 (C-2’), 29.1 (d, J = 3.4 Hz, C-5’), 26.0 (d, J = 130.5
Hz, C-6’).
({[({2‐[(2R,5R)‐5‐(2,4‐dioxo‐1,2,3,4‐tetrahydropyrimidin‐1‐yl)‐3‐hydroxyoxolan‐2‐
yl]ethyl}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}oxy)phosphonate (TEA salt) (1)
Compound 7 was dissolved in 1 mL of a 1:1 mixture of t-BuOH and H 2O. To this solution
was added distilled morpholine (10 L, 0.114 mmol), and the reaction mixture was stirred
at r.t. for 10 min, then set to reflux. A solution of DCC (25 mg, 0.114 mmol) in 0.5 mL of t-
BuOH was added over a period of 1.5 h, and the reaction mixture was then refluxed for 5
h. Volatiles were removed under reduced pressure, the residue taken up in water, and
the resulting suspension filtered. The aqueous solution was extracted twice with ether,
111

and then re-evaporated under reduced pressure. The resulting residue (8) was dried using
the oil pump and used in the next step without further purification.
A solution of tetrasodium pyrophosphate (30 mg, 0.114 mmol) in 1 mL water was
passed through DOWEX H
+
. The solvent was removed and the residue re-dissolved in 1
mL 50% aqueous ethanol. Tributylamine (34 L, 0.171 mmol, 1.5 eq) was added and the
resulting solution stirred at r.t. for 1 h. The solvent was removed, and the residue was co-
evaporated with ethanol, and the resulting glassy solid was dried using an oil pump. The
product was dissolved in 1 mL of dry DMSO.
The solution of tributylammonium pyrophosphate in DMSO was added dropwise to a
solution of morpholidate 8 in 1 mL dry DMSO dropwise. The reaction was stirred for 24 h
at r.t. The product 1 was isolated using two-step preparative HPLC: SAX (0  100% linear
gradient, A = water, B = 0.5 M TEAB, pH = 7.5, 25 min, 8 mL/min) followed by RP HPLC
(isocratic, 0.1 M TEAB, 5% CH 3CN, pH = 7, 8 mL/min). The fractions containing 1 were
combined and lyophilized, giving the product as a white crystalline solid (4.1 mg, 25% over
three steps). HRMS (ESI/APCI): calcd for C 10H 16N 2O 13P 3
-
, [M-H]
-
464.9871; found 464.9872
m/z.
31
P NMR (202 MHz, D 2O):  18.63 (1P, d, J = 25.8 Hz, P α), -10.67 (1P, d, J = 16.4 Hz,
P γ), -23.32 (1P, dd, J = 25.8, 16.4, P β);
1
H NMR (500 MHz, D 2O):  7.79 (1H, d, J = 8.1 Hz, H-
6), 6.29 (1H, t, J = 6.9 Hz, H-1’), 5.95 (1H, d, J = 8.1 Hz, H-5), 4.42-4.39 (1H, m, H-3’), 4.06-
4.03 (1H, m, H-4’), 2.43-2.34 (2H, m, H-2’), 2.06-1.83 (4H, m, H-5’, H-6’).
13
C NMR (126
MHz, D 2O):  166.7 (C-4), 152.1 (C-2), 142.3 (C-6), 102.9 (C-5), 87.6 (d, J = 19.3 Hz, C-4’),
85.6 (C-1’), 73.6 (C-3’), 38.4 (C-2’), 27.4 (d, J = 4.2 Hz, C-5’), 24.5 (d, J = 138.1 Hz, C-6’).
112

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124

Appendix A: Chapter 1 Supporting Data
125

Figure A.1
1
H NMR of 2 (CDCl 3, 400 MHz)

Figure A.2
31
P NMR of 2 (CDCl 3, 162 MHz)

CHCl3
126

Figure A.3
1
H NMR of 3 (CDCl 3, 400 MHz)
Figure A.4
31
P NMR of 3 (CDCl 3, 162 MHz)

CHCl3
X
127

Figure A.5
1
H NMR of 5 (CDCl 3, 400 MHz)

Figure A.6
31
P NMR of 5 (CDCl 3, 162 MHz)

CHCl3
X
128

Figure A.7
1
H NMR of TEA salt of 6 (D 2O, 400 MHz)
Figure A.8
31
Р NMR of TEA salt of 6 (D 2O, 162 MHz)

HDO
129

Figure A.9
1
H NMR of 7 (CDCl 3, 500 MHz)

Figure A.10
31
P NMR of 7 (CDCl 3, 202 MHz)

P P
O O
O
OMe
MeO
MeO
Cl
CO
2
Me
Ph
7
CHCl3
130

Figure A.11
1
H NMR of 9 (D 2O, 500 MHz)
Figure A.12
31
P NMR of 9 (D 2O, 202 MHz)

HDO
X
131

Figure A.13 Preparative HPLC separation of diastereomers 10a and 10b

10a
10b
P P
O O
O
OH
HO
Cl
Ph
O
N
O
N
O
10a/b
132

Figure A.14
1
H NMR of 10a (D 2O, 400 MHz)

Figure A.15
31
P NMR of 10a (D 2O, 162 MHz)

TEA
MeCN
HDO
TEA
133

Figure A.16 Simulation of
31
P NMR of 10a (D 2O, 162 MHz) (blue) superimposed with
31
P NMR
(brown)

134

Figure A.17
1
H NMR of 10b (D 2O, 400 MHz)
Figure A.18
31
P NMR of 10b (D 2O, 162 MHz)

HDO
P P
O O
O
OH
HO
Cl
Ph
O
N
O
N
O
10b
X
135

Figure A.19
1
H NMR of 11a (CD 3OD, 500 MHz)
Figure A.20
31
P NMR of 11a (CD 3OD, 202 MHz)

P P
O O
O
OH
HO
Cl
Ph
O
N
O
HO
11a
HDO
CH3OH
CD2HOD
136

Figure A.21
1
H NMR of 11b (CD 3OD, 500 MHz)
Figure A.22
31
P NMR of 11a (CD 3OD, 202 MHz)

P P
O O
O
OH
HO
Cl
Ph
O
N
O
HO
11b
HDO
CD2HOD
P P
O O
O
OH
HO
Cl
Ph
O
N
O
HO
11b
X
137

Figure A.23 Preparative SAX HPLC purification of 12a

12a
138

Figure A.24
1
H NMR of 12a (D 2O, 500 MHz)
Figure A.25
31
P NMR of 12a (D 2O, 202 MHz)

HDO
TEA
TEA
X
139

Figure A.26 Preparative SAX HPLC purification of 12b

12b
140

Figure A.27
1
H NMR of 12b (D 2O, 500 MHz)
Figure A.28
31
P NMR of 12b (D 2O, 202 MHz)

NH
O
O N
O
OH
O P
O
-
O
O
P P
O O
O
-
O
-
O
O
N
O Ph
Cl
12b
TEA
TEA
HDO
X
141

Figure A.29 Preparative SAX HPLC purification of 1a

1a
142

Figure A.30
1
H NMR of 1a (D 2O, 500 MHz)
Figure A.31
31
P NMR of 1a (D 2O, 202 MHz)

TEA
TEA
HDO
143

Figure A.32 Preparative SAX HPLC purification of 1b

1b
144

Figure A.33
1
H NMR of 1b (D 2O, 500 MHz)
Figure A.34
31
P NMR of 1b (D 2O, 202 MHz)

TEA
TEA
HDO
145

Appendix B: Chapter 2 Supporting Data
146

Figure B.1
1
H NMR of 2a (CDCl 3, 500 MHz)

Figure B.2
31
P NMR of 2a (CDCl 3, 202 MHz)

CHCl 3
147

Figure B.3
1
H NMR of 2b (CDCl 3, 500 MHz)

Figure B.4
31
P NMR of 2b (CDCl 3, 202 MHz)

H 2O
CHCl 3
148

Figure B.5
1
H NMR of 3a (CDCl 3, 500 MHz)
Figure B.6
31
P NMR of 3a (CDCl 3, 202 MHz)

149

Figure B.7
13
C NMR of 3a (CDCl 3, 126 MHz)

CDCl 3
150

Figure B.8
1
H NMR of 3b (CDCl 3, 500 MHz)

Figure B.9
31
P NMR of 3b (CDCl 3, 202 MHz)

CHCl 3
×
151

Figure B.10
13
C NMR of 3b (CDCl 3, 126 MHz)

CDCl 3
152

Figure B.11
1
H NMR of 4a (CDCl 3, 500 MHz)
Figure B.12
31
P NMR of 4a (CDCl 3, 202 MHz)

153

Figure B.13
13
C NMR of 4a (CDCl 3, 126 MHz)

Figure B.14
1
H -
31
P gHMBC NMR of 4a (CDCl 3, 500 MHz)

CDCl 3
154

Figure B.15
1
H NMR of 4b (CDCl 3, 500 MHz)
Figure B.16
31
P NMR of 4b (CDCl 3, 202 MHz)
×
×
155

Figure B.17
13
C NMR of 4b (CDCl 3, 126 MHz)

Figure B.18
1
H –
31
P gHMBC NMR of 4b (CDCl 3, 500 MHz)

×
CDCl 3
156

Figure B.19
31
P –
1
H NMR of 4b (CDCl 3, 202 MHz)

157

Figure B.20
1
H NMR of 6a (mixture of 4 isomers) (CDCl 3, 500 MHz)

Figure B.21
31
P NMR of 6a (mixture of 4 isomers) (CDCl 3, 202 MHz)

CHCl 3
158

Figure B.22
1
H NMR of 6b (mixture of 4 isomers) (CDCl 3, 400 MHz)
Figure B.23
31
P NMR of 6b (mixture of 4 isomers) (CDCl 3, 162 MHz)

CHCl 3
159

Figure B.24
1
H NMR of 7a (mixture of 4 isomers) (CD 3OD, 500 MHz)
Figure B.25
31
P NMR of 7a (mixture of 4 isomers) (CD 3OD, 202 MHz)

H 2O
CD 2HOD,
MeOH
160

Figure B.26
1
H NMR of 7b (mixture of 4 isomers) (CD 3OD, 400 MHz)

Figure B.27
31
P NMR of 7b (mixture of 4 isomers) (CD 3OD, 162 MHz)

CD 2HOD,
MeOH
H 2O
161

Figure B.28
1
H NMR of α-1a (D 2O, 500 MHz)

Figure B.29
31
P NMR of α-1a (D 2O, 202 MHz)

HDO
TEA
TEA
×
162

Figure B.30
1
H NMR of 1a (D 2O, 500 MHz)

Figure B.31
31
P NMR of 1a (D 2O, 202 MHz)

TEA
TEA
HDO
163

Figure B.32
1
H NMR of 1b (D 2O, 400 MHz)
Figure B.33
31
P NMR of 1b (D 2O, 162 MHz)

TEA
TEA
HDO
164

Table B.1 Crystallographic Statistics

( α , β)-CH 2- ( β, γ )-NH-dTTP ( α , β)-NH- ( β, γ )-CH 2-dTTP
PDB Code 4RT2 4RT3
Data Collection
Space Group P2 1 P2 1
a (Å) 50.7 50.6
b (Å) 79.9 79.4
c (Å) 55.3 55.3
 (°) 107.5 107.3
d min (Å) 1.92 1.92
R merge (%)
a, b
0.084 (0.493) 0.079 (0.479)
Completeness (%) 94.0 (67.4) 89.8 (48)
Unique Reflections 30067 (2142) 28973 (1547)
Total Reflections 106757 96986
I/σ 13.8 (2.0) 16.6 (1.9)
Refinement
r.m.s. deviations
Bond lengths (Å) 0.007 0.008
Bond angles (°) 1.020 1.040
R work (%)
c
17.6 18.0
R free (%) 21.1 22.6
Average B Factors (Å)
Protein 27.4 33.6
DNA 34.4 40.5
Analog 18.7 21.8
Ramachandaran Analysis
Favored 98.5 98.5
Allowed 100 100

a
R merge=100 x Σ hΣ i |I h,i-I h| Σ h Σ i I h,j, where I h is the mean intensity of symmetry related reflections I h,j.

165

b
Numbers in the parentheses refer to the highest resolution shell of data (10%).

c
R work = 100 x Σ |F obs|-|F calc|/Σ|F obs|.

166

Appendix C: Chapter 3 Supporting Data
167

Figure C.1
31
P NMR of the r.m. of synthesis of 3a (CDCl 3, 243 MHz)

Figure C.2
1
H NMR of 3a (CDCl 3, 600 MHz)

O
P P
O
EtO
EtO
N
Bn
OEt
P
O
OMe
OMe
CO
2
Bn
3a
168

Figure C.3
31
P NMR of 3a (CDCl 3, 243 MHz)

Figure C.4
13
C NMR of 3a (CDCl 3, 151 MHz)

CDCl3
169

Figure C.5
1
H NMR of 4a (CDCl 3, 500 MHz)

Figure C.6
31
P NMR of 4a (CDCl 3, 202 MHz)

O
P P
O
EtO
EtO
N
Bn
OEt
P
O
OH
OMe
CO
2
Bn
4a
170

Figure C.7
13
C NMR of 4a (CDCl 3, 126 MHz)

CDCl3
171

Figure C.8
1
H NMR of 5a (CDCl 3, 500 MHz)

Figure C.9
31
P NMR of 5a (CDCl 3, 202 MHz)

O
P P
O
EtO
EtO
N
Bn
OEt
P
O
OMe
O O
OH
T
CO
2
Bn
5a
CHCl3
172

Figure C.10
1
H NMR of 6a (CD 3OD, 500 MHz)

Figure C.11
31
P NMR of 6a (CD 3OD, 202 MHz)

H2O
CHD2OD
CH3OH
173

Figure C.12
1
H NMR of benzyl 2-iodoethyl ether (CDCl 3, 400 MHz)
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
1.97
2.00
2.01
4.42
3.27
3.29
3.30
3.73
3.74
3.76
4.58
7.26
7.28
7.29
7.30
7.31
7.32
7.33
7.35
7.36

Figure C.13 Purification trace of compound 3b. Fractions 10-26 contained isomer 1

CHCl 3
H 2O
174

Figure C.14
1
H NMR of 3b (isomer 1) (CDCl 3, 500 MHz)

Figure C.15
31
P NMR of 3b (isomer 1) (CDCl 3, 202 MHz)

175

Figure C.16
1
H NMR of 4b (mixture of 2 isomers) (CDCl 3, 500 MHz)

Figure C.17
31
P NMR of 4b (mixture of 2 isomers) (CD 3OD, 202 MHz)

CD 2HOD
CH 3OH
H 2O
176

Figure C.18
1
H NMR of 5b (mixture of 8 isomers) (CDCl 3, 500 MHz)

Figure C.19
31
P NMR of 5b (mixture of 8 isomers) (CDCl 3, 202 MHz)

O
P
N
Bn
P
O
O
OMe
OEt
P
O
EtO
EtO
O
OH
T
OBn
5b
CHCl 3
O
P
N
Bn
P
O
O
OMe
OEt
P
O
EtO
EtO
O
OH
T
OBn
5b
177

Figure C.20
1
H NMR of 6b (mixture of 8 isomers) (CD 3OD, 500 MHz)

Figure C.21
31
P NMR of 6b (mixture of 8 isomers) (CD 3OD, 202 MHz)

CD 2HOD
CH 3OH
H 2O
178

Figure C.22
1
H NMR of 1b (mixture of 2 isomers) (D 2O, pH 9, 500 MHz)

Figure C.23
31
P NMR of 1b (mixture of 2 isomers) (D 2O, pH 9, 202 MHz)

HDO
TEA TEA
179

Figure C.24 HPLC trace of purification of compound 1b

Figure C.25 Trace of compound 1b

1b
1b
180

Figure C.26 Purification trace of compound 3c. Fractions 13-39 were collected

181

Figure C.27
1
H NMR of 3c (mixture of 2 isomers) (CDCl 3, 500 MHz)

Figure C.28
31
P NMR of 3c (mixture of 2 isomers) (CDCl 3, 202 MHz)

182

Figure C.29
19
F NMR of 3c (mixture of 2 isomers) (CDCl 3, 470 MHz)

Figure C.30
13
C NMR of 3c (mixture of 2 isomers) (CDCl 3, 126 MHz)

CDCl 3
183

Figure C.31
1
H NMR of 4c (mixture of 2 isomers) (CD 3OD, 500 MHz)

Figure C.32
31
P NMR of 4c (mixture of 2 isomers) (CD 3OD, 202 MHz)

CD 2HOD
CH 3OH
H 2O
184

Figure C.33
19
F NMR of 4c (mixture of 2 isomers) (CD 3OD, 470 MHz)

185

Figure C.34
1
H NMR of 5c (mixture of 8 isomers) (CDCl 3, 500 MHz)

Figure C.35
31
P NMR of 5c (mixture of 8 isomers) (CDCl 3, 202 MHz)

O
P
N
Bn
P
O
O
OMe
OEt
P
O
EtO
EtO
F
O
OH
T
5c
CHCl 3
O
P
N
Bn
P
O
O
OMe
OEt
P
O
EtO
EtO
F
O
OH
T
5c
186

Figure C.36
19
F NMR of 5c (mixture of 8 isomers) (CDCl 3, 470 MHz)

O
P
N
Bn
P
O
O
OMe
OEt
P
O
EtO
EtO
F
O
OH
T
5c
187

Figure C.37
1
H NMR of 6c (mixture of 8 isomers) (CD 3OD, 500 MHz)

Figure C.38
31
P NMR of 6c (mixture of 8 isomers) (CD 3OD, 202 MHz)

H 2O
CD 2HOD
CH 3OH
188

Figure C.39
19
F NMR of 6c (mixture of 8 isomers) (CD 3OD, 470 MHz)

189

Figure C.40
1
H NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 500 MHz)

Figure C.41
31
P NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 202 MHz)

HDO
TEA
TEA
190

Figure C.42
19
F NMR of 1c (mixture of 2 isomers) (D 2O, pH 9, 470 MHz)

191

Appendix D: Chapter 4 Supporting Data
192

Figure D.1
1
H NMR of 5 (CDCl 3, 500 MHz)

Figure D.2
31
P NMR of 5 (CDCl 3, 202 MHz)

CHCl 3
193

Figure D.3
13
C NMR of 5 (CDCl 3, 126 MHz)

CDCl 3
194

Figure D.4
1
H NMR of 6 (CDCl 3, 500 MHz)

Figure D.5
31
P NMR of 6 (CDCl 3, 202 MHz)

CHCl 3
195

Figure D.6
13
C NMR of 6 (CDCl 3, 126 MHz)

CDCl 3
196

Figure D.7 HRMS (ESI/APCI) of 6

197

Figure D.8
1
H NMR of 7 (D 2O, 500 MHz)

Figure D.9
31
P NMR of 7 (D 2O, 202 MHz)

HDO
TEA
TEA
198

Figure D.10
13
C NMR spectrum of 7 (D 2O, 126 MHz)

199

Figure D.11 HRMS (ESI/APCI) of 7

200

Figure D.12 Part of the
1
H NMR of α- and β- anomers of 1 (after SAX purification) (D 2O, 500 MHz)

201

Figure D.13
1
H NMR of 1 (D 2O, 500 MHz)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
4.45
2.15
1.04
1.06
0.95
1.00
1.04
1.29
1.30
1.32
1.83
1.84
1.87
1.88
1.89
1.92
1.93
1.95
1.96
1.99
2.01
2.05
2.06
2.34
2.36
2.37
2.39
2.40
2.41
2.43
3.20
3.22
3.23
3.25
4.03
4.03
4.04
4.05
4.05
4.06
4.39
4.40
4.41
4.41
4.42
4.80
5.94
5.95
6.27
6.29
6.30
7.78
7.79

Figure D.14
31
P NMR of 1 (D 2O, 202 MHz)
-28 -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
f1 (ppm)
0.97
1.00
1.04
-23.43
-23.33
-23.22
-10.71
-10.63
18.57
18.70

TEA
TEA
HDO
202

Figure D.15
13
C NMR of 1 (D 2O, 126 MHz)

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
f1 (ppm)
9.30
24.53
25.63
28.02
28.05
39.05
47.74
74.17
86.23
88.13
88.29
103.54
142.95
152.67
167.35

203

Figure D.16 HRMS (ESI/APCI) of 1

204

Figure D.17 Inhibition of SAMHD1 by 1. (a) dUTP hydrolysis in the presence of various fixed con-
centrations of 1 as indicated. The reactions contain a saturating concentration of GTP activator (5
mM). The entire data were fitted globally to a competitive inhibition model to obtain the inhibi-
tion constant (K i = 80 ± 6 M). The two left panels show silver stained polyacrylamide gel images
of reactions (with and without 2 mM 1) that were quenched with the cross linking agent glutara-
dehyde. The monomer (M), dimer (D) and tetramer (T) cross-linked forms of SAMHD1 are indi-
cated. (b) Order of addition experiments in which 1 is added before or after substrate. (c) Dilution-
jump kinetic (DJK) and corresponding cross-linking (DJXL) experiment to evaluate the effects of 1
on the activity and oligomeric state of SAMHD1. The various pre-jump conditions are listed above
the dotted lines and the post-jump reactions contained 1 mM dUTP in all cases. The post-jump
delay times at which 50 mM glutaraldehyde was added are indicated above the gel images and
the monomer (M), dimer (D) and tetramer (T) cross-linked forms are indicated.

0 60 120 180 240 300 360
0
250
500
750
1000
Time (min)
dU ( M)
Post-Jump
Time (min)
0 60
Pre-jump
GTP + 1
GTP + dUTP + 1
GTP + dUTP
GTP
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
[pppCH
2
dU] (mM)
V
i
/V
o
Pre-Incubation
0 1 2 3 4 5 6
0.0
0.5
1.0
1.5
2.0
[dUTP] mM
dUTP hydrolysis ( M/s)
[1] (mM)
0
0.05
0.1
0.2
0.4
1.0
2.0
a b
c
Initiation
S
E + GTP + S
E + GTP + 1
1
T
T
T
T
T
T
D
M
D
M
D
M
D
M
D
M
D
M

Abstract (if available)

Abstract Deoxyribonucleoside triphosphates (dNTPs) are ubiquitous substrates for a variety of cellular enzymes, such as polymerases and triphosphohydrolases. The triphosphate moiety of dNTPs represents a versatile site for a rational design of probes of dNTP‐utilizing enzymes. Substituting bridging oxygens in dNTP with non‐hydrolysable moieties, such as CXY or NH, has provided a number of probes and inhibitors of the enzymes which break P–O bonds in dNTPs. In an effort to expand the scope of dNTP non‐hydrolysable analogues as enzyme inhibitors, we focused on the design and synthesis of (α,β)‐, (β,γ)‐, or 5’‐non‐hydrolysable dNTP analogues as inhibitors of two important therapeutic target enzymes—DNA polymerase β and SAMHD1. ❧ Working within the framework of a collaboration with Prof. M. Goodman (USC) and S. Wilson, M.D. (NIEHS), we created a novel class of ""Met‐Im"" inhibitors of DNA pol β: dNTP analogues in which the Pα–O–Pᵦ and Pᵦ–O–Pᵧ bridging oxygens are replaced by hydrolytically‐resistant CXY and NH in alternation. The first two representatives of this class were synthesized via an unusual [1,3]‐N → C rearrangement (or ""flip"") of diphosphoryl amine precursors. Conventional dealkylation with BTMS incurred anomerization, however this could be largely prevented by microwave radiative heating, which may be a useful approach to this problem more generally. Enzymatic studies confirmed that both compounds inhibit DNA pol β, but the inhibitor having the NH group in the (β,γ)‐position was 9‐fold more potent. This difference was associated with the aid of X‐ray crystallographic analysis with a water‐mediated H‐bond of the (β,γ)‐NH group with the R183 residue in the active site of DNA pol β. ❧ The H‐bond identified in the crystal structure of parent ""Met‐Im"" inhibitors inspired an attempt to design “next generation” higher affinity inhibitors of this class. In a preliminary study, two H‐bonding functionalities were installed into the parent ""Met‐Im"" dNTP analogues to optimize their interaction with DNA pol β. The synthesis of the novel inhibitors was accomplished analogously to the parent ""Met‐Im"" dNTPs. ❧ In collaboration with Prof. J. Stivers (John Hopkins School of Medicine) we also designed and synthesized a 5’‐non‐hydrolysable dNTP analogue (pppCH₂dU), the first non-reactive inhibitor of SAMHD1, to explore potential modes of inhibition of SAMHD1. pppCH₂dU, initially viewed as a simple competitive inhibitor, was determined to act by an unusual mechanism of disrupting the formation of SAMHD1 oligomers. Our results demonstrated that SAMHD1, an important link in the immune response pathways, is a druggable enzyme, subject to rational inhibitor design.

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Creator Kadina, Anastasia (author)

Core Title Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 08/26/2015

Defense Date 07/31/2015

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

Tag cancer,OAI-PMH Harvest,polymerase,SAMHD1,triphosphate analogues

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Advisor McKenna, Charles E. (committee chair), Armani, Andrea (committee member), Brutchey, Richard L. (committee member)

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