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Synthetic studies of phosphonate derivatives

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SYNTHETIC STUDIES OF PHOSPHONATE DERIVATIVES

by

Gregorio Valentin Sanchez, Jr.

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 2006

Copyright 2006 Gregorio Valentin Sanchez, Jr.

ii
DEDICATION

This work is dedicated to those in my life cut down in their prime because of a
terminal illness. Their examples of strength, courage and love continue to
strengthen my resolve to positively impact human health.

In loving memory of:

Linda Prado Founds

Evangeline Mungaray Gutierrez

Robert M. C. Synder

Joshua E. “Sasha” Young

iii
ACKNOWLEDGEMENTS

I would like to start off by thanking God Almighty and my guardian angels for
blessing me here at USC and beyond with an awesome support network and lots
and lots of patience! To my mother Betty and my grandmothers Dolores and
Juanita thank you for your unconditional love and support. Even if you didn’t
understand what I was talking about, you always provided me with a kind word
and good food! To my father Greg Sr. and grandfather Frank I thank you from
the bottom of my heart for teaching me the importance of hard-work and
determination. These traits have come in handy everyday and I am truly grateful.
To my friend and companion Carter, thank you for your love and support, you
have truly been an answer to my prayers. To my cousin Sylvia, my catalyst for
success, thank you for introducing me to the right people and getting me out of
hospital administration and into science. To my brothers Chris, Joseph, and
Steven thank you for always keeping me grounded and reminding me of my roots.
I love you all. To my nephews Chris Jr. and Sammy never stop reaching for the
stars and know that if you work hard enough one day your dreams can become
your reality.

iv
To Professor Charles E. McKenna and Dr. Boris Kashemirov both skilled
chemist, knowledgeable educators and mentors. Thank you for taking a chance
on me five years ago. You took a raw undergraduate with a weak foundation in
chemistry and gave him the tools and guidance to strengthen that base and build
upward. It is because of you both that I find myself more confident of my
abilities and ready for the next phase of my scientific endeavors. I will always be
grateful for my time working diligently with you both in the lab.

To the McKenna group: Ulrika, Mong, JJ, Christian, Noi, James, Tom, Larryn,
and Joy, thank you all for some great times. I’ve learned so much from all of
you…and not just chemistry. From international relations to World Cup Soccer
to online gaming…you all made the time fly by!

In every place I have worked previously, there has been a special group of people
that really made the day in and day out routine more enjoyable. The same can be
said of the USC Department of Chemistry, home to so many great staff and
faculty members. I especially have to thank Michele Dea, Marie de la Torre, Judy
Hom, Jim Merritt, Cory Schultz, Heather Connor, Jamie Avila, Bruno Herreros,
the VWR staff (Marie and Darryl), Dr. Marie McKenna, Dr. Hanna Reisler, Dr.
Curt Wittig, Dr. Mark Thompson, Dr. James Haw, Dr. William Weber, Dr. James
Ellern, Dr. Robert Bau, Dr. Amy Barrios, Dr. John Tower and Dr. Surya Prakash.

v
Through your generosity, kindness and dedication to the students of the
department you have all made my tenure here at USC the most memorable time of
my life.

To my friends and mentors at Cal Poly Pomona, Dr. Barbara Burke and Dr. Paul
Hiemenz, thank you so much for encouraging me to go to graduate school. I
couldn’t have made it without your support!

Finally I must thank my friends, past and present, for their unwavering support.
To Adam, Denise, Leslie, Renee, Ricky, Rose, Daniel, Janette, Shellie, Cathy,
Roger, Alma, Laura, Cydne, Richard, Audrey, Beckie, Claudia, Ulrika, Crystal,
Kristin, Nichelle, Jessie, Larryn, Patrick, Marco, Iris, Miranda, Simona, Paulin,
Tom, and anyone else I may have forgotten, God bless you all. It has been a hard
five years, but through it all you have all been there for me in one way or another.

vi

TABLE OF CONTENTS

Dedication ii
Acknowledgements iii
List of Tables xiii
List of Figures xiv
List of Schemes xv
Abstract xviii
Chapter 1:
A Novel Route to α-Hydroxy, α-Substituted Phosphonates via Reaction
of α-Carbonylphosphonates with Organometallic Reagents
1
Introduction 1
Results and Discussion 4
Conclusion 13
Acknowledgments 14
Experimental 14
Tetraisopropyl (1-hydroxyethane-1,1-diyl)bis(phosphonate)
(2a)
16
Tetraisopropyl
[Hydroxy(phenyl)methylene]bis(phosphonate) (2b)
17
Tetraisopropyl (1-Hydroxy-2-phenylethane-1,1-
diyl)bis(phosphonate) (2c)
18
Tetraisopropyl [(2-chloropyridine-4-yl)[(1-
trimethylsilyl)oxy]methylene]bis(phosphonate) (4a)
18
Ethyl (2-chloropyridin-4-yl)(diethoxyphosphoryl)[(1-
trimethylsilyl)oxy] methylene]acetate (7a)
19
Tetraisopropyl [1-[(trimethylsilyl)oxy]-2-iodoethane-1,1-
diyl]bis(phosphonate) (4b)

20

vii
[Hydroxy(phenyl)methylene]bis(phosphonic acid)
(triethylammonium salt) (3b)
20
(1-Hydroxy-2-phenylethane-1,1-diyl)bis(phosphonic acid)
(triethylammonium salt) (3c)
21
[(2-chloropyridine-4-yl)-1-hydroxy-
methylene]bis(phosphonic acid) (triethyl ammonium salt)
(6a)
22
[1-(2-chloropyridin-4-yl)-2-ethoxy-1-hydroxy-methylene]-2-
oxoethyl]phosphonic acid, ethyl ester (triethylammonium
salt) (8a)
23
Tetraisopropyl (4-hydroxybut-1-ene-4,4-
diyl)bis(phosphonate) (9a)
23
Tetraisopropyl [(2)-5-hydroxypent-2-ene-5,5-
diyl)bis(phosphonate) (9b)
24
Tetraisopropyl [(1)-4-hydroxy-1-phenylbut-1-ene-4,4-
diyl]bis(phosphonate) (9c)
25
Tetraisopropyl (2-Bromo-4-hydroxybut-1-ene-4,4-
diyl)bis(phosphonate) (9d)
25
Tetraisopropyl [2-(ethoxycarbonyl)-4-hydroxybut-1-ene-4,4-
diyl]bis(phosphonate) (9f)
25
Ethyl 2-(diethoxyphosphoryl)-2-hydroxypent-4-enoate (11a) 26
Diethyl 2-(diethoxyphosphoryl)-2-hydroxy-4-
methylenepentanedioate (11b)
26
4-(ethoxycarbonyl)-2-hydroxy-2-phosphonopent-4-enoic acid
(11c)
26
[(1)-4-hydroxy-1-phenylbut-1-ene-4,4-diyl]bis(phosphonic
acid) (triethylammonium salt) (10a)
27
(2-Bromo-4-hydroxybut-1-ene-4,4-diyl)bis(phosphonic acid)
(triethylammonium salt) (10b)
28
(4-methylene-5-oxotetrahydrofuran-2,2-diyl)bis(phosphonic
acid) (triethylammonium salt) (10c)
28
[(3)-1-(ethoxycarbonyl)-1-hydroxy-4-phenylbut-3-en-1-
yl]phosphonic acid, (triethylammonium salt) (12a)
29
[1,3-bis(ethoxycarbonyl)-1-hydroxybut-3-en-1-yl]phosphonic
acid (triethylammonium salt) (12b)
29
5-ethoxy-4-hydroxy-2-methylene-5-oxo-4-
phosphonopentanoic acid (triethylammonium salt) (12c)
30
Chapter 1 References 31

viii
Chapter 2:
The Synthesis of Branched Troika Acid Resins for Chelation of
Divalent Metal Cations
36
Introduction 36
Results and Discussion 40
Conclusion 57
Acknowledgements 58
Experimental 58
Synthesis of Troika acid bound macroporous resin (4) 60
Qualitative chelation study of macro resin (4) 61
Synthesis of the phosphonate branched micro resin (6) 62
Diethyl (2- {[(3-hydroxypropyl)amino]oxy}2-
oxoethyl)phosphonate (Ligand A)
62
[(Amino)oxy-2-oxoethyl]phosphonic acid bound micro resin 63
Synthesis of the “daisy-chained” micro resin (8) 63
Ligand B 63
Troika precursor “daisy-chain” resin (7) 66
Troika “daisy-chain” micro resin (8) 67
Semi-qualitative chelation study for micro resins 7 and 8 68
Synthesis of MicB and MacB 69
MicB 69
MacB 69
Synthesis of amino-bound dipropanol to resin surface (MicB or
MacB) containing phosphonate linked Troika diethyl
carbmoylmethylphosphonate (CMP) (11)
69
Diethyl CMP 69
Sodium monosalt of CMP 71
MicB Troika CMP precursor resin (9) 71
MicB Troika CMP resin (11) 72
MacB Troika CMP precursor resin (MacB-9) 72
MacB Troika CMP resin (MacB-11) 73
Semi-qualitative chelation study for micro resins 9 and 11 73
Synthesis of resin bound 3,3’-iminodipropan-1-ol (MicB) containing
phosphonate linked Troika diethyl phosphonoacetonitrile (PAN)
75
Diethyl PAN 75
Lithium monosalt of PAN 76
MicB Troika PAN precursor resin (10) 76
MicB Troika PAN resin (12) 76
Semi-qualitative chelation study for micro resins 10 and 12 77
Synthesis of amide linked Troika acid to PEG-1000 micro resin 78

ix
Amide-linked Troika precursor bound to PEG-1000 micro
resin (13)
78
Amide-linked Troika acid bound to PEG-1000 micro resin
(14)
78
Chapter 2 References 79
Chapter 3:
Oxidative Pathways of α-Diazo Bisphosphonates: A Continuation
82
Introduction 82
Results and Discussion 84
Conclusion 95
Experimental 96
Protected Uridine Conjugates 99
[Chloro-(dimethoxy-phosphoryl)-(2-nitro-benzyloxy)-
methyl]-phosphonic acid 6-(2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d]-1,3-
dioxol-4-ylmethyl ester methyl ester (5)
99
[6-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-2,2-dimethyl-
tetrahydro-furo[3,4-d]-1,3-dioxol-4-ylmethoxy]-hydroxy-
phosphoranecarbonyl)-phosphoric acid (9)
100
Unprotected Thymidine Conjugates 101
(Diazo-{[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-tetrahydro-furan-2-ylmethoxy]-methoxy-
phosphoryl}-methyl)-phosphonic acid dimethyl ester (2)
101
(Chloro-{[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-tetrahydro-furan-2-ylmethoxy]-methoxy-
phosphoryl}-methoxymethyl)-phosphonic acid dimethyl ester
(6)
102
{Hydroxy-[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-tetrahydro-furan-2-ylmethoxy]-hydroxy-
phosphoranecarbonyl}-phosphoric acid (10)
103
AZT Conjugates 104
({[3-Azido-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-
1-yl)-tetrahydro-furan-2-ylmethoxy]-methoxy-phosphoryl}-
chloro-methoxy-methyl)-phosphonic acid dimethyl ester (7)

104
[3-Azido-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-
yl)-tetrahydro-furan-2-ylmethoxy]-hydroxy-
phosphoranecarbonyl)-phosphonic acid (11)
104
Synthesis of 11 via t-butyl alcohol substitution for water in
the “moisture modification”
105
Protected Adenosine Conjugates 105

x
[Diazo-(dimethoxy-phosphoryl)-methyl]-phosphonic acid 6-
(6-dibenzoylamino-purin-9-yl)-2,2-dimethyl-tetrahydro-
furo[3,4-d]-1,3-dioxol-4-ylmethyl ester methyl ester (4)
105
{Chloro-(dimethoxy-phosphoryl)-[(2-nitrobenzyl)oxy]-
methyl}-phosphonic acid 6-(6-dibenzoylamino-purin-9-yl)-
2,2-dimethyl-tetrahydro-furo[3,4-d]-1,3-dioxol-4-ylmethyl
ester methyl ester (8)
106
[6-(6-Dibenzoylamino-purin-9-yl)-2,2-dimethyl-tetrahydro-
furo[3,4-d]-1,3-dioxol-4-ylmethoxy]-hydroxy-
phosphoranecarbonyl)-phosphonic acid (12)
107
Chapter 3 References 108
Alphabetized Bibliography 111
Appendix A:
NMR SPECTRA FOR CHAPTER 1
121
2a 121
2b 123
3b 124
3c 126
4a 128
7a 131
4b 133
6a 136
8a 139
9a 141
9b 143
9c 144
9d 145
9f 146
10a 147
10b 150
10c 152
11a 155
11b 156
11c 158
12a 160
12b 162
12c 164

xi
Appendix B:
NMR SPECTRA FOR CHAPTER 2
167
Ligand A 167
L-B-b 169
Ligand B 170
7 172
8 173
CMP 174
Sodium monosalt CMP 176
MicB 9 178
Mic B 11 179
PAN 180
Lithium monosalt of PAN 182
MicB 10 183
MicB 12 184
13 185
14 186
FT-IR Spectra for Chapter 2 187
8 187
Mic-B 188
Mac-B 189
CMP 190
9 191
11 192
MacB-9 193
MacB-11 194
PAN 195
12 196
13 197
Chelation Studies for Chapter 2
Key for Chelation Data Chart on page 199
198
Aqueous and Organic Chelation Data for Tables 1 to 3

199

xii
Appendix C:
NMR SPECTRA FOR CHAPTER 3
200
5 200
9 201
2 202
6 204
10 205
7 206
11 207
4 208
8 210
12 212

xiii
LIST OF TABLES
Table

2.1

Change in Copper Ion Concentration Using Resins 7 and 8 48
2.2

Changes in Metal Ion Concentration Using Resin 11 52
2.3

Changes in Metal Ion Concentration Using Resin 12 54
3.1

Nucleoside/α-diazoBP Conjugates and
31
P NMR Spectra 89
3.2 Nucleoside/α-chloro, α-alkoxyBP Conjugates and
31
P NMR
Spectra

90
3.3 Nucleoside/α-COBP Conjugates (as triethylammonium salts)
and
31
P NMR Spectra

93

xiv
LIST OF FIGURES

Figure

2.1

Z-isomer of Troika Acid 37
2.2

Coupling Methods for the Troika Ligand 39
2.3

Various Resin-Ligand Motifs 40
2.4

P-O Stretch as Observed in MacA bound PAA FT-IR Spectra 42
2.5

Effects of Multiple DCC Coupling Steps 43
2.6

Effects of Solvents on the Nitrosation Reaction via FT-IR 44
2.7

Branched and “Daisy-chained” Synthetic Targets 45
3.1 HPLC Chromatogram of the “Moisture Modified” and 2-Step
Anhydrous Method Generated Protected Uridine/α-COBP
Conjugate

91
3.2 HPLC Chromatogram of a Mixture of “Moisture Modified”
and 2-Step Anhydrous Method Generated Protected Uridine/α-
COBP Conjugate

92

xv
LIST OF SCHEMES
Scheme

1.1

P-C-O-P Rearrangement Mechanism 2
1.2

Oxidation Methodologies 3
1.3

Nucleophilic Addition to iPr
4
COBP 3
1.4

Formation of Dimer and Trimer via Addition of Phosphite 4
1.5 Alkyl and Aryl Grignard Reagent Addition to iPr
4
COBP and
Subsequent Generation of the Bisphosphonic Acid

5
1.6 Generation of Heteroatom-containing Grignard Reagents via
Metal:Halogen Exchange

6
1.7 Heteroatom-containing Grignard Reagent Addition to
iPr
4
COBP and Subsequent Generation of the Bisphosphonic
Acid

7
1.8 Heteroatom-containing Grignard Reagent Addition to
COTEPA and Subsequent Generation of the Bisphosphonic
Acid

8
1.9 Decomposition Pathways of 1.4b

9
1.10 Allyl Indium Reagent Additions to iPr
4
COBP and Subsequent
Generation of the Bisphosphonic Acid

11
1.11 Allyl Indium Reagent Additions to COTEPA and Subsequent
Generation of the Bisphosphonic Acid

12
1.12 Possible Cyclization Mechanism for 1.9f

13
2.1 Nirtosation Reactions

38
2.2 Conversion of Chloromethylated Resins to Aminomethylated
Resins
41

xvi
2.3 DCC Coupling of PAA to Aminomethylated Macro Resin

42
2.4 Synthesis of the Troika Ligand

44
2.5 Synthesis of Ligand A

45
2.6 BTMS Delkylation of MicA Bound PAA

46
2.7 Synthesis of Ligand B

47
2.8 Coupling of Ligand B to Aminomethylated Micro Resin

48
2.9 Synthesis of Diethylamine Platform

49
2.10 Arbuzov Synthesis of CMP

49
2.11 Synthesis of Solution-Phase Troika Derivative

50
2.12 Synthesis of Resin 2.11

51
2.13 Synthesis of Resin 2.12

53
2.14 Troika Acid Attached via PEG to Micro Resin

55
2.15 Synthesis of Troika Acid Attached via PEG to Micro Resin

56
3.1 McKenna
2
Formic Acid Promoted Oxidation of α-
Diazobisphosphonates

83
3.2 McKenna “Moisture Modification” Method

83
3.3 Nucleophilic Activation of Oxidation Mechanism

85
3.4 Hydrolytic Cleavage of o-Nitrobenzyl Alcohol

86
3.5 Alcohol Promoted Oxidation of α-Diazobisphosphonate

86
3.6 Replacement of Water in the “Moisture Modification” with a
Hindered Alcohol

87

xvii
3.7 Kashemirov et al.
9
Procedure

88
3.8 Ketone Reactivity Trend at pH 7

95

xviii
ABSTRACT

Pyrophosphonate analogs such as methylenebisphosphonates and
phosphonoacetates have applications in pharmaceutical chemistry and also in
material sciences. Herein, a series of synthetic studies focusing on the preparation
of potentially useful new organophosphorus compounds are presented. In
Chapter 1, the addition of organometallic reagents to carbonylbisphosphonate and
oxophosphonoacetate alkyl esters to generate α-hydroxy, α-substituted
methylenebisphosphonate or phosphonoacetate alkyl esters is investigated.
Chapter 2 discusses synthetic approaches to branched (E)-
(hydroxyimino)(dihydroxyphosphinyl)acetic acid ligands and their possible
application as metal chelating agents. Finally, Chapter 3 presents the
hypochlorite oxidation of diazomethylenebisphosphonate alkyl esters from
various alcohols for use in synthesis of nucleoside 5’-triphosphate
carbonylbisphosphonate analogs.

1
Chapter 1
A NOVEL ROUTE TO α-HYDROXY, α-
SUBSTITUTED PHOSPHONATES VIA REACTION
OF α-CARBONYLPHOSPHONATES WITH
ORGANOMETALLIC REAGENTS
Introduction
Biological phosphonate analogs have been of interest as pharmaceutical targets
for sinces the 1970’s. Bisphosphonate (BP) and phosphonoacetate (PA)
derivatives have been used in the treatment of bone diseases
1, 2
, cancer
3, 4
, and viral
therapies
5-7
. Inherent in both analogs is the bridging methylene carbon (α-
carbon), which can be functionalized to thereby fine-tune biological activity. It
has been well established that incorporating a methylene-bridged nitrogen-
containing heterocycle at the α-carbon into a α−hydroxy
methylenebisphosphonate (α-HRMBP) increases binding affinity to the farnesyl
diphosphate synthase (FPPS) active site
8-10
. In addition studies have also
revealed that the same bridged heterocyclic derivative of α-HRMBP and the
α−hydroxy, methylenephosphonoacetate (α-HRMPA) analog inhibit Rab
geranylgeranyl transferase, disrupting the prenylation of Rab proteins in
osteoclasts
11
.
2
Conventional synthetic methodologies for both analogs are accomplished by the
reaction of a carboxylic acid (RCO
2
H) with various phosphorylating reagents
under harsh conditions
12-17
. With respect to the BP, milder routes through ester
intermediates have also been explored including the synthesis of tetrasilyl esters
18,
19
and tetraalkyl methylenebisphosphonates
20
. Unlike the predominately stable
α−HRMBP salts
21
, ester derivatives have been observed to rearrange to the
corresponding phosphonophosphate (P-O-C-P) (Scheme 1.1) or fragment into a
phosphite and ketone (Scheme II), both of which are acid, base, and thermally
catalyzed
21-26
. Furthermore, strongly basic nucleophiles, when reacted with α-
keto acylphosphonates, were also shown to promote dephosphorylation
24, 27
. This
presents a major obstacle in the utilization of BP esters as synthons to novel and
currently administered BP.
RO
P
RO
O
C P
O
OR
OR
OH
R
RO
P
RO
O
C O
R
P
O
OR
OR
RO
P
RO
O
C P
O
OR
OR
O
-
R
H
RO
P
RO
O
H
C O
R
P
O
OR
OR
Fragmentation
Scheme 1.1: P-C-O-P Rearrangement Mechanism
In spite of these problems, our laboratory reported the synthesis of the alkyl esters
of carbonylphosphonoacetate and carbonylbisphosphonate (Scheme 1.2)
28-31
which, in principle, makes possible a mild approach to α-HRMBP and α-
3
HRMPA esters via addition of nucleophilic species to the reactive α-ketone group
(Scheme 1.3). Preliminary nucleophilic addition studies with water and methanol
confirmed that triethyl α-keto phosphonoacetate (COTEPA) was more reactive
with respect to α-keto tetraisopropyl methylenebisphosphonate (iPr
4
COBP)
28, 32
.
Decomposition to phosphite and ketone in both COTEPA and iPr
4
COBP produces
pentaalkyl phosphonoacetate (dimer) and hexaalkyl bisphosphonophosphate
(trimer), respectively (Scheme 1.4)
32
, indicating that the additions to the α-ketone
was not selective.
RO
P
RO
O
C P
O
OR
OR
O
RO
P
RO
O
C P
O
OR
OR
N
2
oxidation
O
P C
N
2
C
O
RO
RO
OR
O
P C
O
C
O
RO
RO
OR
Oxidation for milligram scale reactions completed in-situ by 'moisture modification'
t-butyl hypochlorite is added to diazo- intermediate dissolved in ethyl acetate with a catalytic amount of water present
!
a
in-situ yields by
31
P-NMR
( 95%
a
)
( 70%
a
)
1a
1b
Scheme 1.2: Oxidation Methodologies
O
P
O
O
C P
O
O
O
O
1. Nu
2. acidic work-up
O
P
O
O
C P
O
O
O
OH
Nu
Scheme 1.3: Nucleophilic Addition to iPr
4
COBP
4
RO
P
RO
O
C R'
O
RO
P
RO
O
H
RO
P
RO
OH
P
H
C O
R'
P
O
OR
OR
R' C P
P
RO OR
O
O
OR
OR
O
-
O
RO
RO
O
P
RO
RO
C
O
OR
R = alkyl; R' = or
Scheme 1.4: Formation of Dimer and Trimer via Addition of Phosphite
NMR-scale reactions of alkyl and aryl Grignard reagents with iPr
4
COBP
28, 32
established precedence for the continued study and possible creation of novel
libraries employing Grignard and other organometallic reagents. Herein, we
report our advancements to date in the generation of α-HRMBP and α-HRMPA
acids from various organometallic reagents employing unique adaptations limiting
common rearrangement/decomposition pathways.
Results and Discussion
Early findings by Maeda et al. demonstrated that acylphosphonates could react
with alkyl Grignard reagents to generate the stable dialkyl α-hydroxy, α-alkyl
phosphonate, by adjusting solvents to limit fractionation
27
. In 1998, we
introduced our preliminary NMR-scale synthesis of 2a-c via nucleophilic addition
of the respective Grignard reagents with iPr
4
COBP
28, 32
. Recently, our group has
5
optimized these reactions for large-scale (0.5-1.0 g) synthesis, improving the
yields of 2b and 2c by 20% and 30% (by
31
P NMR) respectively, via adjusting
Et
2
O:THF ratios in the Grignard reagents before adding iPr
4
COBP (Scheme 1.5).
During this time, we discovered that the tetraisopropyl esters of 2b and 2c could
not be efficiently purified on a silica gel column due to an increase in
rearrangement and fragmentation on the column. Furthermore Ruel et al.
observed that bulkier substituents were more sensitive to rearrangement
22
. I
believe this sensitivity stems from the electronic effect provided by the ring
systems present in 2b and 2c, which facilitate the generation of the oxygen anion
and subsequent rearrangement. Chromatography was circumvented by direct
dealkylation of the reaction mixture by typical bromotrimethylsilane (BTMS)
conditions
33, 34
, purified by HPLC and characterized as the triethylammonium salt.
O
P
O
O
C P
O
O
O
O
O
P
O
O
C P
O
O
O
OH
R
HO
P
HO
O
C P
O
OH
OH
OH
R
I II
I: i: CO-TIPMBP in toluene added to RMX in Et
2
O:THF @ RT ii: acidic work-up; II i: BTMS in DCM ii: aqueous work-up
2 3
1a
2a R = methyl ( 65%
a
)
2b R = phenyl ( 41%
a
)
2c R = benzyl ( 18%
a
)
3b R = phenyl ( 22%
b
)
3c R = benzyl (20%
b
)
!
a
Purified by silica column chromatography.
b
Purified by HPLC as triethyl ammonium salts.
Scheme 1.5: Alkyl and Aryl Grignard Reagent Addition to iPr
4
COBP and
Subsequent Generation of the Bisphosphonic Acid
6
Understanding the critical role nitrogen-containing heterocyclic α-HRMBP play
in the regulation of bone metabolism and the high risk of base-catalyzed
rearrangement, we set out on the daunting task of generating a heterocyclic
Grignard reagent for reaction with iPr
4
COBP. There are several literature
examples of how to synthesize functionalized heterocyclic Grignard reagents
35-37
.
Knochel et al. described a method to heterocyclic Grignard reagents via a low
temperature halogen-magnesium exchange
36
. 2-Chloro-4-iodopyridine was
chosen for facile generation of the corresponding Grignard reagent
16
(Scheme
1.6).
MgBr
I R
THF @-78
o
C
R MgBr + I
R = C N
Cl
H
2
C I R =
Scheme 1.6: Generation of Heteroatom-containing Grignard Reagents via
Metal:Halogen Exchange
Our first attempt at the synthesis of the 2-chloropyridine
methylenebisphosphonate derivative produced P-O-C-P as the major product.
Considering the mechanism of rearrangement, we pondered the idea of
intercepting the oxygen anion with a silylating agent such as chlorotrimethylsilane
(TMSCl). After several attempts, we discovered that co-addition of the iPr
4
7
COBP with excess TMSCl (20 eq.) at low temperatures (-78°C) generated both 4a
and 5a in a 3:2 ratio according to
31
P NMR integration (Scheme 1.7). We can only
speculate on the outcome of this reaction but consider that excess TMSCl
immediately silates the oxygen anion producing HCl, which in turn protonates
pyridine, blocking further base-catalyzed rearrangement. As observed with the
aryl products, rearrangement and fragmentation during large-scale
chromatography prompted the direct transfer of ester to acid. Typical BTMS
dealkylation
33, 34
was found to be ineffective for complete dealkylation of this
product. Use of dried and distilled acetonitrile provided the dealkylated form of
6a and the 2-chloropyridine P-O-C-P derivative.
III i: CO-TIPMBP in toluene and 20 eq. TMSCl are added to Grignard reagent @ -78
o
C ii: acidic work-up;
IV i: BTMS in acetonitrile ii: aqueous work-up
O
P
O
O
C P
O
O
O
O
O
P
O
O
C P
O
O
O
O
R
HO
P
HO
O
C P
O
OH
OH
OH
R
III IV
4 & 5
6
1a
4a R' = SiMe
3
, R = C N
Cl
H
2
C I 4b R' = SiMe
3
, R =
6a R = C N
Cl
( 31%
a
)
( 30%
a
)
( 43%
b
)
!
a
Purified by preparative thin layer chromatography.
b
Purified by HPLC as triethyl ammonium salts.
c
By
31
P NMR
R'
5a R' = H, R = C N
Cl
( 40%
c
)
Scheme 1.7: Heteroatom-containing Grignard Reagent Addition to iPr
4
COBP and
Subsequent Generation of the Bisphosphonic Acid
8
Employing the modification with the 2-chloropyridine Grignard addition to
COTEPA successfully provides the silyl-protected α-HRMPA intermediate (10a),
which proved to be less labile on silica gel in comparison to 4a. Dealkylation to
the phosphonic acid was accomplished by BTMS affording the ethyl ester (11a)
(Scheme 1.8).
O
P C
O
C
O
EtO
EtO
OEt
O
P C
O
C
O
EtO
EtO
OEt
R
O
P C
OH
C
O
HO
HO
OEt
R
C N
7a R =
Cl
C N 8a R =
Cl
!
a
Purified by preparative thin layer chromatography.
b
Yield overall of triethylammonium salt.
III i: CO-TIPMBP in toluene and 20 eq. TMSCl are added to Grignard reagent @ -78
o
C ii: acidic work-up;
IV i: BTMS in acetonitrile ii: aqueous work-up
III IV
SiMe
3
( 27%
a
)
22%
b
1b
Scheme 1.8: Heteroatom-containing Grignard Reagent Addition to COTEPA and
Subsequent Generation of the Bisphosphonic Acid
Directing our attention to the synthesis of the methylene-bridged heterocyclic
derivatives, we found the generation and use of heteroatom-containing ‘benzylic-
like’ Grignard reagents challenging
38, 39
. Exploration commenced on α-hydroxy-
protected methylenephosphonates containing an α-methyl halide substitutent,
which could be later functionalized to the desired bridged heterocycle. Braun et
al. demonstrated that diiodomethane undergoes halogen-magnesium exchange at
low temperature, generating the corresponding Grignard reagent
40
(Scheme 1.7).
Following the above procedure using the ‘TMSCl-modification’, 4b was
9
generated and found to be stable as a yellow crystal if properly stored after silica
gel chromatography (Scheme 1.7). Synthesis of the methyl iodide α-HRMPA
derivative was more elusive, decomposing on silica gel and during dealkylation.
Preliminary results from addition chemistries to 4b, were limited by P-C-O-P
rearrangement of the 4b starting material under current reaction conditions.
Further examination revealed that even under conditions that should favor
elimination of iodide for intramolecular cyclization to the oxiranylidene-BP (Path
B), the methyl iodide P-C-O-P derivative predominates (Path A) (Scheme 1.9).
P C
O
P
O O
CH
2
OR
OR
RO
RO
I
SiMe
3
P C
O
P
O O
CH
2
OR
OR
RO
RO
I
P
C
P
O CH
2
O O
OR
OR
RO
RO
I
P
C
P
O CH
2
O O
OR
OR
RO
RO
P C
O
P
O O
CH
2
OR
OR
RO
RO
I
P
H
C O
O
CH
2
RO
RO
P
O
OR
OR
I
Path A
Path B
deprotection
Scheme 1.9: Decomposition Pathways of 4b
Our work with Grignard reagents provided motivation to further study other
organometallic reagents with iPr
4
COBP and COTEPA. Alkyl lithium, samarium
and magnesium-lithium reagents in addition to aryl zinc reagents propagated
rearrangement and decomposition according to
31
P NMR studies. Wiemer et al.
demonstrated that allyl InBr
2
reagents react well with acyl phosphonates to yield
the corresponding α-hydroxy alkylphoshonate in the presence acetic acid
41
. With
10
respect to the other organometallic reagents studied, other then zinc, indium
reagents were considered much milder organometallic reagents, exhibiting a
higher tolerance for the presence of heteroatoms and were relatively unaffected by
oxygen and water
42
. Encouraged by these findings and the possibility of
generating α-unsaturated, α-hydroxy methylenephosphonate derivatives, a
practice currently inaccessible by conventional means, we initiated a series of
allylation studies with COTEPA and iPr
4
COBP.
Following Wiemer’s protocol
41
, we attempted the allyllation of iPr
4
COBP via the
in situ generated allyl InBr
2
reagent in the presence of acetic acid.
31
P NMR
spectral analysis of the reaction mixture confirmed no P-O-C-P formed; however,
we observed only a fair amount of the desired product, with trimer as the major
component.
31
P NMR experiments uncovered that indium metal and acetic acid
promote the decomposition of carbonylbisphosphonates to phosphite,
consequently generating trimer. To better understand this phenomenon, we
examined the role of acetic acid in the reaction by monitoring the effects of acids
with varying pKa. Formic acid (pKa= 3.75) and p-nitrophenol (pKa= 7.2) were
compared against acetic acid (pKa= 4.7) with no enhancement in product yield.
11
P C
O
P
O
O
O
O
O
O
HO
P
HO
O
C P
O
OH
OH
OH
P C
OH
P
O
O
O
O
O
O
R
R
9a R =
9b R =
H
2
C
H
2
C
CH
3
H
2
C
H
2
C
Br
H
2
C
9c R =
9d R =
9e R =
( 90%
a
)
( 63%
a
)
( 67%
a
)
( 67%
a
)
( 60%
a
)
10a R =
H
2
C
H
2
C
Br
10b R =
( 32%
b
)
( 34%
b
)
V i: CO-TIPMBP in toluene is added to indium reagent followed by the addition of 1 eq. TMSCl, ))) ii:
acidic work-up; VI i: BTMS in acetonitrile ii: aqueous work-up
V VI
a
By
31
P NMR .
b
Purified by HPLC as triethyl ammonium salts.
H
2
C
OEt
O
9f R =
10c
( 67%
a
)
( 37%
b
)
O
C
P P
O O
O
OH
OH
HO
HO
1a
Scheme 1.10: Allyl Indium Reagent Additions to iPr
4
COBP and Subsequent
Generation of the Bisphosphonic Acid
Lewis acids, such as TMSCl or BF
3
• etherate, have been shown to enhance or
catalyze organometallic reactions. Augé et al. observed that allylation of
aldehydes and ketones is enhanced by stoichiometric amounts of indium (s) and
TMSCl
43
. Varying amounts of TMSCl were added in place of acetic acid to the
reaction mixture. A major increase in the allylation product was observed when 1
12
eq. of TMSCl was added to the reaction mixture immediately following the
addition of CO-TIPMBP and sonication. Direct deakylation of the ester was
completed using BTMS and acetonitrile to access the corresponding acid in good
yield. The optimized protocol quickly enabled us to synthesize a handful of novel
α-hydroxy, α- unsaturated BP and PA derivatives (Schemes 1.10 and 1.11).
H
2
C
O
P C
O
C
O
EtO
EtO
OEt
O
P C
OH
C
O
EtO
EtO
OEt
R
O
P C
OH
C
O
HO
HO
OEt
R
H
2
C
H
2
C
EtO
O
11a R =
11b R =
11c R =
H
2
C
H
2
C
EtO
O
12a R =
12b R =
a
By
31
P NMR.
b
Purified using preparative thin layer chromatography.
c
Purified by HPLC as
triethyl ammonium salts.
V i: CO-TIPMBP in toluene is added to indium reagent followed by the addition of 1 eq. TMSCl, ))) ii:
acidic work-up; VI i: BTMS in acetonitrile ii: aqueous work-up
V
VI
( 55%
a
)
( 34%
c
)
(39%
c
)
( 42%
b
)
( 30%
b
)
12c R =
H
2
C
HO
O
1b
(40%
a
)
Scheme 1.11: Allyl Indium Reagent Additions to COTEPA and Subsequent
Generation of the Bisphosphonic Acid
Further examination of the α-methylene ester derivative 9f uncovered a
propensity toward intramolecular cyclization. Lactone formation in compounds
containing α-hydroxy, α-methylene esters under acidic conditions is not
13
uncommon, especially when flanked by strong electron withdrawing groups
(Scheme 1.12)
44, 45
. Our findings indicate that the acidic conditions generated
upon aqueous work-up of the BTMS reaction mixture promote cyclization of the
bisphosphonate (9f) but not the phosphonoacetate derivative, which provides the
ester (12b) as the predominant product. Generation of 12c is attained via
hydrolytic cleavage after prolonged exposure to the HPLC mobile phase.
P C
OH
CH
2
O
P
RO
RO
C H
2
C
C
O
O
P C
O
HO
HO
P
H
2
C
C
C
O
O
H
2
C
O
C
O
C
CH
2
C
O
P
P
O
RO
OR
H
-EtOH
O
OR
OR
O
RO
RO
O
OH
OH
R = Si
H
3
O
+
Scheme 1.12: Possible Cyclization Mechanism for 9f
Conclusion
In summary, we have found that simple alkyl and aryl Grignard reagents in
addition to indium mediated Barbier allyl derivatives react with iPr
4
COBP and
COTEPA with minimal rearrangement or decomposition, providing the
corresponding esters. In cases where iPr
4
COBP and COTEPA are added to
heteroatom-containing Grignard reagents generated at low temperatures,
14
rearrangement can be eliminated by employing our ‘TMSCl-modification’
forming the protected α-hydroxy. This methodology has provided a route to the
first examples of heteroatom-containing silyl-protected α-HRMBP and α-
HRMPA esters via nucleophilic addition. Facile dealkylation via BTMS in
acetonitrile provide the α-HRMBP and α-HRMPA acids in good to fair yields.
Acknowledgements
This research was financially supported by the University of Southern California
department of Chemistry. We also acknowledge the assistance of Dr. Ron New,
University of California at Riverside High Resolution Mass Spectroscopy
Facility, for his effort in collecting the HR-MS data.
Experimental
Materials and Methods:
All reactions were carried out under a nitrogen atmosphere, unless otherwise
indicated. Toluene and tetrahydrofuran (THF) (both reagent grade purchased
from Mallinckrodt Chemicals) were dried and distilled over
sodium/benzophenone. Ethyl acetate and acetonitrile (both HPLC grade
purchased from Mallinckrodt Chemicals) were dried and distilled over P
2
O
5
.
15
Anhydrous diethyl ether (Et
2
O) was purchased from EMD Chemicals, Inc.
Acetone (HPLC grade) was purchased from Mallinckrodt Chemicals and hexanes
(reagent grade) from EM Science. Isopropylmagnesium bromide (15% in THF,
ca. 1 mol/L) was purchased from TCI. Indium metal (100 mesh, 99.99%),
magnesium turnings (≥ 99.5%), chlorotrimethylsilane (≥ 99%),
bromotrimethylsilane (97%), and all halogenated starting materials were
purchased from Sigma Aldrich. Tetraisopropyl methylenebisphosphonate and
trimethyl phosphonoacetate were graciously donated by Albright & Wilson
Americas, Inc. iPr
4
COBP and COTEPA were synthesized in situ via “the
moisture modification” (McKenna et al.
28, 31, 32
) and co-evaporated from ethyl
acetate with dry toluene. Thin layer chromatography plastic-back sheets (20 X
20; silica gel 60 F
254
) were purchased from EMD Chemicals, Inc. Preparative thin
layer chromatography glass-back sheets (20 X 20; 1000 microns) were purchased
from Analtech. Silica gel 150 (60-200 mesh) used for column chromatography
(column width 1-2 in) of bisphosphonate esters was purchased from Mallinckrodt
Chemicals; the esters were eluted using either a gradient from 100% toluene to
1:1 acetone:toluene or from 100% hexanes to 2:3 acetone:hexanes. Preparative
HPLC was accomplished using the Waters 600E Multisolvent Delivery System
with Waters 486 Tunable Absorbance Detector, equipped with a Varian Dynamax
column (Microsorb 100-5, C
18
; 250 X 21.4 mm). The mobile phase was 0.1 M
16
triethylamine/acetic acid (0.1 M TEA:AA) at pH 7.0 using a gradient of 1% to
20% acetonitrile (HPLC grade) at a flow rate of 8 mL/min, detected at λ = 254
nm.
Proton (
1
H), carbon (
13
C), and phosphorus (
31
P) NMR spectra were measured
either on a Bruker AM-360 MHz, Varian Mercury-400 MHz, or Bruker AMX-
500 MHz spectrometer. Chemical shifts are reported relative to external TMS
(
1
H), internal CDCl
3
[δ = 77.0] (
13
C) or external 85% H
3
PO
4
(
31
P). NMR samples
of BP and PA esters were dissolved in CDCl
3
, while BP and PA
triethylammonium salts were dissolved in D
2
O. Triethylammonium salt peaks are
on average 1.08 (t,
2
J
HH
= 7, 3H); 3.00 (q,
2
J
HH
= 7, 2H) for (
1
H) and 7.8 (s, CH
3
);
46.2 (s, CH
2
) for (
13
C) and are omitted from the reported NMR spectral data.
High-resolution mass spectrometry was performed at the University of California
at Riverside High Resolution Mass Spectrometry Facility using a VG-ZAB mass
spectrometry instrument, operated in the negative ion mode.
Preparation of substituted 1-hydroxymethylene-1-phosphonic acid, alkyl ester
derivatives via Grignard Chemistry
Tetraisopropyl (1-hydroxyethane-1,1-diyl)bis(phosphonate) (2a)
The methyl Grignard reagent
46
(5 eq.) was obtained from magnesium turnings and
methyliodide in 10 mL of a 1:1 dry Et
2
O:THF solution at 5°C. iPr
4
COBP (0.200
17
g, 0.54 mmol) was generated in situ and co-evaporated from ethyl acetate using 2
mL dry toluene and added via a glass syringe to a magnetically stirred solution of
the Grignard reagent at 5°C. After 10-25 min, 5 mL of 10% acetic acid was
added at 5°C and stirred magnetically for 10 min. The aqueous phase was
extracted twice with 5 mL portions of Et
2
O. The organic layer and Et
2
O extracts
were combined and dried over Na
2
SO
4
, filtered and the solvent removed by rotary
evaporation under reduced pressure at 50°C. The residue was purified by column
chromatography eluted using a gradient from 100% toluene to 1:1
acetone:toluene. Solvent was removed by rotary evaporation under reduced
pressure (~1 mm Hg) at room temperature to constant weight, leaving 2a as a
viscous oil (0.135 g, 65% yield overall).
δ
P
19.4
47
δ
H
1.3-1.4 (m, 24H), 1.6 (t,
3
J
HP
= 16 Hz, 3H), 4.6-4.8 (m, 4H)
47
Tetraisopropyl [Hydroxy(phenyl)methylene]bis(phosphonate) (2b)
Prepared as for 2a. 2b was obtained as a viscous yellow oil (0.10 g, 41% yield
overall).
δ
P
15.4 (s)
δ
H
1.2 (m, 24H), 4.6 (m, 4H), 7.2-7.9 (m, 5H)
18
Tetraisopropyl (1-Hydroxy-2-phenylethane-1,1-diyl)bis(phosphonate) (2c)
Prepared as for 2a. 2c was obtained as a light yellow viscous oil (0.045 g, 18%
yield)
δ
P
19.6 (s)
δ
H
1.3 (m, 24H), 3.5 (t,
3
J
HP
= 14 Hz, 2H), 4.6 (m, 4H), 7.2-7.9 (m, 5H)
Tetraisopropyl [(2-chloropyridine-4-
yl)[(1trimethylsilyl)oxy]methylene]bis(phosphonate) (4a)
The Grignard reagent of 2-chloro-4-iodopyridine was synthesized according to
Abarbri et al.
16
In a 25 mL pear-shape flask, iPr
4
COBP (0.200 g, 0.54 mmol) was
generated in situ and co-evaporated from ethyl acetate using 5 mL toluene.
TMSCl (1 mL, ~10 eq.) was added by a glass syringe under N
2
(g) and
subsequently taken back up into the glass syringe. The ketone/TMSCl solution
was added to a magnetically stirred solution of the pyridinyl Grignard reagent (1.5
eq. in THF) at -60°C (dry ice/acetone bath). The solution was first stirred at -
60°C for 20 min, then at room temperature for 10 min. The reaction mixture was
worked up by adding 5 mL of 10% acetic acid at 5°C stirring magnetically for 10
min. The aqueous phase was extracted twice with 5 mL portions of Et
2
O. The
organic layer and Et
2
O extracts were combined and dried over Na
2
SO
4
, filtered
and the solvent removed by rotary evaporation under reduced pressure at 50°C.
The residue was purified by preparative thin layer chromatography (silica gel)
19
using a 1:1 acetone:toluene mobile phase (R
f
= 0.68). Solvent was removed by
rotary evaporation under reduced pressure (~1 mm Hg) at room temperature to a
constant weight, leaving 4a as a light yellow viscous oil (0.091 g, 31% yield
overall).
δ
P
13.1 (s)
δ
H
0.3 (s, 9H), 1.2-1.3 (m, 24H), 4.6 (m, 2H), 4.8 (m, 2H), 7.6 (broad m, 1H), 7.7
(broad s, 1H), 8.3 (broad d,
3
J
HH
= 5, 1H)
δ
C
2.7 (s), 23.8 (m), 73.1 (d,
2
J
PC
= 30 Hz), 121.1 (s), 122.8 (s), 148.3 (s), 150.8 (s)
Ethyl (2-chloropyridin-4-yl)(diethoxyphosphoryl)[(1-trimethylsilyl)oxy]
methylene]acetate (7a)
Prepared as for 4a using 0.135 g (0.54 mmol) of COTEPA. 7a was purified by
preparative thin layer chromatography using a 2:3 acetone:hexane mobile phase,
providing a light yellow viscous oil (0.062 g, 27% yield overall).
δ
P
13.2 (s)
δ
H
0.23 (s, 9H), 1.2 (m, 6H), 1.3 (t,
3
J
HH
= 7, 3H), 4.1 (m, 4H), 4.3 (m, 2H), 7.5
(broad m, 1H), 7.6 (broad s, 1H), 8.3 (broad d,
3
J
HH
= 5 Hz, 1H)
20
Tetraisopropyl [1-[(trimethylsilyl)oxy]-2-iodoethane-1,1-diyl]bis(phosphonate)
(4b)
The Grignard reagent of diiodomethane was synthesized according to Braun
40
4b
was prepared for as 4a. Purification was accomplished by preparative thin layer
chromatography using a 1:1 acetone:toluene mobile phase (R
f
= 0.86) providing
yellow crystals (0.091 g, 30% yield overall)
δ
P
12.3 (s)
δ
H
0.3 (s, 9H), 1.3 (m, 24H), 3.7 (t,
3
J
HP
= 14, 2H), 4.8 (m, 4H)
δ
C
-0.1 (s), 5.8 (s), 21.5 (m), 69.1 (s), 70.3 (s)
HR-MS (FAB
+
; MH
+
): calcd for 573.1063, found 573.1061
Preparation of substituted 1-hydroxymethylene-1-phosphonic acids
(triethylammonium salts) derivatives
[Hydroxy(phenyl)methylene]bis(phosphonic acid) (triethylammonium salt) (3b)
from 2b
Direct dealkylation of the 2b reaction mixture (70% by
31
P NMR) was performed
by first drying the residue by rotary evaporation under reduced pressure (~1 mm
Hg) at room temperature to a constant weight. Neat BTMS (0.5 mL, ~10 eq.) was
added under N
2
(g) and the mixture was stirred magnetically for 12 hrs.
Unreacted BTMS was removed by rotary evaporation under reduced pressure at
40°C. To the residue, 5 mL of water was added. After being stirred magnetically
21
for 30 min at room temperature, the mixture was extracted twice with 5 mL
portions of Et
2
O. The aqueous phase was collected and water removed by rotary
evaporation under reduced pressure (~1 mm Hg) at 40°C leaving a light yellow
viscous oil. The crude product was dissolved in 0.5 mL of 0.1 M TEA:AA at pH
7.0 and purified by HPLC using a linear gradient from 1% to 10% acetonitrile
(t
R
= 16.8 min). Solvent was removed by rotary evaporation under reduced
pressure (~1 mm Hg) at ~5°C. Excess triethylamine and acetic acid was removed
by adding 0.5 mL of water and freeze-drying (~0.7 mm Hg). This process was
repeated twice, providing the pure salt of 3b as a white foam (0.054 g, 22% yield
overall as a di-salt).
δ
P
15.5 (s)
δ
H
7.1-7.7 (m, 5H)
HR-MS (FAB
-
; [M
2-
+H
+
]
-
) calcd for 266.9823, found 266.9816
(1-Hydroxy-2-phenylethane-1,1-diyl)bis(phosphonic acid) (triethylammonium
salt) (3c) from 2c
Prepared as for 3b. 3c was purified by HPLC using a 1% to 10% acetonitrile
linear gradient (t
R
= 24.8 min), providing a white foam (0.051 g, 20% yield
overall).
δ
P
17.5 (s)
22
δ
H
3.1 (t, J
HP
= 14 Hz, 2H), 7.1-7.3 (m, 5H)
HR-MS (FAB
-
; [M
2-
+H
+
]
-
): calcd 280.9980, found 280.9986
[(2-chloropyridine-4-yl)-1-hydroxy-methylene]bis(phosphonic acid) (triethyl
ammonium salt) (6a) from 4a
Direct dealkylation of the 4a reaction mixture (60% by
31
P NMR) was employed.
The crude product was dried under reduced pressure (~1 mm Hg) at room
temperature to a constant weight in a 25 mL pear-shape flask. Under N
2
(g) 4a
was first dissolved with dry acetonitrile, followed by the addition of neat BTMS
(0.5 mL, ~10 eq.) and allowed to stir magnetically for 12 hours. Work up was
similar to the procedure followed for 3b. 6a was purified by HPLC using a 1% to
10% acetonitrile linear gradient (t
R
= 19.3 min), providing a light yellow foam
(0.116 g, 43% yield overall)
δ
P
12.9 (s)
δ
H
7.5 (broad m, 1H), 7.6 (broad s, 1H), 8.0 (d,
3
J
HH
= 6, 1H)
δ
C
120.2 (s), 121.5 (s), 147.5 (s), 149.5 (s), 153.8 (s)
HR-MS (FAB
-
; [M
2-
+H
+
]
-
): calcd for 301.9383, found 301.9384
23
[1-(2-chloropyridin-4-yl)-2-ethoxy-1-hydroxy-methylene]-2-oxoethyl]phosphonic
acid, ethyl ester (triethylammonium salt) (8a) from 7a
Prepared as for 6a (7a in the reaction mixture was 58% by
31
P NMR). 8a was
purified by HPLC using a 1% to 20% acetonitrile linear gradient (t
R
= 18.5 min),
providing a light yellow foam (0.058 g, 22% yield overall as a mono-salt)
δ
P
13.1 (s)
δ
H
4.1 (m, 2H), 7.6 (broad m, 1H), 7.7 (broad s, 1H), 8.1 (broad d,
3
J
HH
= 5 Hz,
1H)
HR-MS (FAB
-
; [M
2-
+H
+
]
-
): calcd. for 293.9937, found 293.9941
Preparation of unsaturated 1-hydroxymethylene-1-phosphonic acids, alkyl ester
derivatives via allyl InBr
2
reagents
Tetraisopropyl (4-hydroxybut-1-ene-4,4-diyl)bis(phosphonate) (9a)
Indium (s) (0.075 g, 0.65 mmol) and 5 mL THF were added to a 25 mL pear-
shape flask. The flask was placed in an ultrasonicator (Bransonic 2510; 40 kHz)
for 60 min at 30°C. Allyl bromide (0.055 mL, 0.65 mmol) was injected via a
glass micro-syringe, and the flask was returned to the sonicator for an additional
60 min at 30-40°C. iPr
4
COBP (0.200 g, 0.54 mmol) was generated in situ, co-
evaporated from ethyl acetate with 3 mL of toluene and injected via a glass
syringe into the indium reagent at 5°C under N
2
(g). Immediately following the
24
addition of ketone, 0.08 mL TMSCl (0.634 mmol) was injected via a micro-glass
syringe to the reaction mixture and further sonicated at 30-40°C for 60-90 min or
until the yellow color of the ketone was no longer observed. The reaction mixture
was worked up by adding 5 mL of 10% acetic acid at 5°C stirring magnetically
for 5 min. The aqueous phase was extracted twice with 5 mL portions of Et
2
O.
The organic layer and Et
2
O extracts were combined and dried over Na
2
SO
4
,
filtered and the solvent removed by rotary evaporation under reduced pressure at
50°C. A small amount (0.020 g) of the crude product was purified by thin layer
chromatography (using an iodine chamber to follow product movement) using a
1:1 acetone:toluene mobile phase. Some product decomposed on silica,
decreasing the overall yield. Solvent was removed by rotary evaporation under
reduced pressure (~1 mm Hg) at room temperature to constant weight providing a
colorless oil (90% yield by
31
P NMR).
δ
P
18.6 (s)
δ
H
1.4-1.5 (m, 24H), 2.7 (dt,
3
J
HH
=4,
3
J
PH
= 15, 2H), 4.7 (m, 4H), 5.0-5.1 (m, 2H),
6.1 (m, 1H)
Tetraisopropyl [(2)-5-hydroxypent-2-ene-5,5-diyl)bis(phosphonate) (9b)
Prepared as for 9a. The product was not purified by chromatography (63% yield
by
31
P NMR), but was dried under reduced pressure (~1 mm Hg) at room
25
temperature to constant weight for direct dealkylation.
δ
P
18.1 (dd,
2
J
PP
= 40 Hz)
Tetraisopropyl [(1)-4-hydroxy-1-phenylbut-1-ene-4,4-diyl]bis(phosphonate) (9c)
Prepared as for 9a. The product was not purified by chromatography (67% yield
by
31
P NMR), but was dried under reduced pressure (~1 mm Hg) at room
temperature to constant weight for direct dealkylation.
δ
P1
16.2 (d,
2
J
PP
= 37 Hz)
δ
P2
18.1 (d,
2
J
PP
= 34 Hz)
Tetraisopropyl (2-Bromo-4-hydroxybut-1-ene-4,4-diyl)bis(phosphonate) (9d)
Prepared as for 9a. The product was not purified by chromatography (67% yield
by
31
P NMR), but was dried under reduced pressure (~1 mm Hg) at room
temperature to constant weight for direct dealkylation.
δ
P
17.2 (s)
Tetraisopropyl [2-(ethoxycarbonyl)-4-hydroxybut-1-ene-4,4-
diyl]bis(phosphonate) (9f)
Prepared as for 9a. The product was not purified by chromatography (67% yield
by
31
P NMR), but was dried under reduced pressure (~1 mm Hg) at room
26
temperature to constant weight for direct dealkylation.
δ
P
16.9 (s)
Ethyl 2-(diethoxyphosphoryl)-2-hydroxypent-4-enoate (11a)
Prepared as for 9a. The product was not purified by chromatography (55% yield
by
31
P NMR).
δ
P
14.7 (s)
Diethyl 2-(diethoxyphosphoryl)-2-hydroxy-4-methylenepentanedioate (11b)
Prepared as for 9a using 0.162 g of COTEPA generated in situ and co-evaporated
from ethyl acetate using 1.5 mL toluene. Product was purified by preparative thin
layer chromatography using a 2:3 acetone:hexane mobile phase providing a light
yellow oil (0.096 g, 42% yield overall).
δ
P
15.6 (s)
δ
H
1.1 (broad m, 6H), 1.4 (broad t,
3
J
HH
= 7, 3H), 3.5 (m, 1H), 3.8 (m, 1H), 3.9 (m,
2H), 4.1 (d,
3
J
PH
= 10, 1H), 4.3 (m, 2H), 5.1 (m, 2H), 6.1 (dt,
3
J
HH
= 18,
3
J
PH
= 10,
1H), 7.2-7.5 (m, 5H)
4-(ethoxycarbonyl)-2-hydroxy-2-phosphonopent-4-enoic acid (11c)
Prepared as for 9a using 0.21 g of COTEPA generated in situ and co-evaporated
from ethyl acetate using 1.5 mL toluene. The product was purified by preparative
27
thin layer chromatography using a 2:3 acetone:hexane mobile phase providing a
light yellow oil (0.089 g, 30% yield overall).
δ
P
14.5 (s)
δ
H
1.3 (m, 12H), 3.0 (dd,
2
J
HH
= 14,
3
J
PH
= 6, 1H), 3.2 (dd,
2
J
HH
= 14,
3
J
PH
= 9, 1H),
3.9 (d,
3
J
PH
= 7, 1H), 4.2 (m, 6H), 4.3 (d,
3
J
PH
= 7, 1H), 5.7 (s, 1H), 6.2 (s, 1H)
Preparation of unsaturated 1-hydroxymethylene-1-phosphonic acid (triethyl
ammonium salts) derivatives
[(1)-4-hydroxy-1-phenylbut-1-ene-4,4-diyl]bis(phosphonic acid)
(triethylammonium salt) (10a) from 9c
Prepared as for 6a. 10a was purified by HPLC using a 1% to 10% acetonitrile
linear gradient (t
R
= 26.3 min), providing a colorless oil (0.088 g, 32% yield
overall).
δ
P
16.7 (s)
δ
H
3.8 (broad s, 1H), 4.7-4.9 (m, 2H), 6.3 (m, 1H), 7.0 (t,
3
J
HH
= 7, 1H), 7.1 (t,
3
J
HH
= 7, 2H), 7.2(d,
3
J
HH
= 7, 2H)
δ
C
52.2 (s), 114.8 (s), 125.5 (s), 127.0 (s), 128.8 (s), 137.8 (s), 140.5 (s)
HR-MS (FAB
-
; M
-
): calcd for 307.0136, found 307.0139
28
(2-Bromo-4-hydroxybut-1-ene-4,4-diyl)bis(phosphonic acid) (triethylammonium
salt) (10b) from 9d
Prepared as for 6a. 10b was purified by HPLC using a 1% to 10% acetonitrile
linear gradient (t
R
= 24.8 min), providing a white foam (0.093 g, 34% yield
overall).
δ
P
16.4 (s)
δ
H
δ 5.5 (s, 1H), 5.7 (s, 1H)
HR-MS (FAB
-
; [M
2-
+H
+
]
-
): calcd for 308.8928, found 308.8932
(4-methylene-5-oxotetrahydrofuran-2,2-diyl)bis(phosphonic acid)
(triethylammonium salt) (10c) from 9f
Prepared according to the procedure for 6a. 10c was purified by HPLC using a
1% to 10% acetonitrile linear gradient (t
R
= 18.2 min), providing a colorless oil (
0.091 g, 37% yield overall).
δ
P
12.9 (s)
δ
H
3.2 (t,
3
J
PH
= 15, 2H), 5.7 (s, 1H), 6.0 (s, 1H)
δ
C
22.3 (s), 80.1 (t,
1
J
PC
= 108 Hz), 123.1 (s), 133.4 (s), 173.0 (s)
HR-MS (FAB
-
; M
-
): calcd for 256.9616, found 256.9619
29
[(3)-1-(ethoxycarbonyl)-1-hydroxy-4-phenylbut-3-en-1-yl]phosphonic acid,
(triethylammonium salt) (12a) from 11b
Prepared as for 6a. 12a was purified by HPLC using a 1% to 20% acetonitrile
linear gradient (t
R
= 18.4 min), providing a white foam (0.09 g, 34% yield overall).
δ
P
12.2 (s)
δ
H
1.2 (t,
3
J
HH
= 6, 3H), 4.0 (d,
3
J
PH
= 9, 1H), 4.1 (m, 2H), 5.1 (m, 2H), 6.1 (dt,
3
J
HH
=
18,
3
J
PH
= 9, 1H), 7.1 (t,
3
J
HH
= 7, 1H), 7.2 (t,
3
J
HH
= 7, 2H), 7.3 (d,
3
J
HH
= 7, 2H)
δ
C
14.1 (s), 55.6 (s), 62.3 (s), 80.9 (d,
1
J
PC
= 120 Hz), 117.5 (s), 126.5 (s), 127.2
(s), 128.4 (s), 136.1 (s), 138.9 (s), 174.3 (s)
HR-MS (FAB
-
; [M
—
H
+
]
-
): calcd for 299.0684, found 299.0678
[1,3-bis(ethoxycarbonyl)-1-hydroxybut-3-en-1-yl]phosphonic acid
(triethylammonium salt) (12b) from 11c
Prepared as for 6a. 12b was purified by HPLC using a 1% to 20% acetonitrile
linear gradient (t
R
= 25.8 min), providing a colorless oil (0.130 g, 39% yield
overall).
δ
P
14.0 (s)
δ
H
2.8 (dd,
3
J
PH
= 10, 1H), 4.0 (m, 4H), 5.6 (s, 1H), 6.0 (s, 1H)
HR-MS (FAB
-
; [M
—
H
+
]
-
): calcd for 295.0582, found 295.0580
30
5-ethoxy-4-hydroxy-2-methylene-5-oxo-4-phosphonopentanoic acid
(triethylammonium salt) (12c) from 11c
Prepared as for 6a. 12c was purified by HPLC using a 1% to 20% acetonitrile
linear gradient (t
R
= 18.5 min), providing a colorless oil.
δ
P
10.1 (broad)
δ
H
3.3 (t,
3
J
PH
= 14, 2H), 4.2 (m, 2H), 5.8 (s, 1H), 6.1 (s, 1H)
δ
C
13.0 (s), 34.6 (s), 63.2 (s), 124.3 (s), 133.8 (s), 171.3 (d), 173.2 (s)
HR-MS (FAB
-
; M
-
): calcd for 249.0164, found 249.0159
31
Chapter 1 References
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Patient, Fourth Edition. 2000; p 350 pp.
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Tsurumi, T., Architecture of replication compartments formed during Epstein-
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Lushington, G. H.; Schoenbrunn, E., Interaction of Phosphonate Analogues of the
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Synthase in Atomic Detail. Biochemistry 2005, 44, (9), 3241-3248.
7. Li, L.; Murphy, K. M.; Kanevets, U.; Reha-Krantz, L. J., Sensitivity to
phosphonoacetic acid: A new phenotype to probe DNA polymerase d in
Saccharomyces cerevisiae. Genetics 2005, 170, (2), 569-580.
8. Reszka, A. A.; Rodan, G. A., Nitrogen-containing bisphosphonate
mechanism of action. Mini-Reviews in Medicinal Chemistry 2004, 4, (7), 711-
719.
9. Reszka, A. A.; Rodan, G. A., The mechanism of action of nitrogen-
containing bisphosphonates. Osteoporosis 2003, 447-457.
10. Ebetino, F. H.; Roze, C. N.; McKenna, C. E.; Barnett, B. L.; Dunford, J.
E.; Russell, R. G. G.; Mieling, G. E.; Rogers, M. J., Molecular interactions of
nitrogen-containing bisphosphonates within farnesyl diphosphate synthase.
Journal of Organometallic Chemistry 2005, 690, (10), 2679-2687.
32
11. Coxon, F. P.; Ebetino, F. H.; Mules, E. H.; Seabra, M. C.; McKenna, C.
E.; Rogers, M. J., Phosphonocarboxylate inhibitors of Rab geranylgeranyl
transferase disrupt the prenylation and membrane localization of Rab proteins in
osteoclasts in vitro and in vivo. Bone (San Diego, CA, United States) 2005, 37,
(3), 349-358.
12. Mikroyannidis, J. A., Hydroxy- and/or carboxy-substituted phosphonic
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Sulfur and the Related Elements 1987, 32, (3-4), 113-18.
13. Lazzarato, L.; Rolando, B.; Lolli, M. L.; Tron, G. C.; Fruttero, R.; Gasco,
A.; Deleide, G.; Guenther, H. L., Synthesis of NO-Donor Bisphosphonates and
Their in-Vitro Action on Bone Resorption. Journal of Medicinal Chemistry 2005,
48, (5), 1322-1329.
14. Xie, Y.; Ding, H.; Qian, L.; Yan, X.; Yang, C.; Xie, Y., Synthesis and
biological evaluation of novel bisphosphonates with dual activities on bone in
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15. Neves, M.; Gano, L.; Pereira, N.; Costa, M. C.; Costa, M. R.; Chandia,
M.; Rosado, M.; Fausto, R., Synthesis, characterization and biodistribution of
bisphosphonates Sm-153 complexes: correlation with molecular modeling
interaction studies. Nuclear Medicine and Biology 2002, 29, (3), 329-338.
16. Abarbri, M.; Thibonnet, J.; Berillon, L.; Dehmel, F.; Rottlaender, M.;
Knochel, P., Preparation of New Polyfunctional Magnesiated Heterocycles Using
a Chlorine-, Bromine-, or Iodine-Magnesium Exchange. Journal of Organic
Chemistry 2000, 65, (15), 4618-4634.
17. Wei, C.; Lu, H.; Liang, B.; Wang, S.; Wang, S.; Shi, X.; Zhang, C.; Ma,
G. Process for preparing 1-hydroxyl-1-carboxyalkylphosphonic acids. 2000-
1289171309128, 20000915., 2001.
18. Lecouvey, M.; Leroux, Y., Synthesis of 1-hydroxy-1,1-bisphosphonates.
Heteroatom Chemistry 2000, 11, (7), 556-561.
19. Lecouvey, M.; Mallard, I.; Bailly, T.; Burgada, R.; Leroux, Y., A mild and
efficient one-pot synthesis of 1-hydroxymethylene-1,1-bisphosphonic acids.
Preparation of new tripod ligands. Tetrahedron Letters 2001, 42, (48), 8475-8478.
33
20. Arstad, E.; Hoff, P.; Skattebol, L.; Skretting, A.; Breistol, K., Studies on
the synthesis and biological properties of non-carrier-added [(125)I and (131)I]-
labeled arylalkylidenebisphosphonates: potent bone-seekers for diagnosis and
therapy of malignant osseous lesions. Journal of medicinal chemistry 2003, 46,
(14), 3021-32.
21. Fitch, S. J.; Moedritzer, K., Nuclear magnetic resonance study of the P-
C(OH)-P to P-CO-P rearrangement: Tetraethyl-I-
hydroxyalkylidenediphosphonates. Journal of the American Chemical Society
1962, 84, 1876-9.
22. Ruel, R.; Bouvier, J.-P.; Young, R. N., Single-Step Preparation of 1-
Hydroxybisphosphonates via Addition of Dialkyl Phosphite Potassium Anions to
Acid Chlorides. Journal of Organic Chemistry 1995, 60, (16), 5209-13.
23. Burgos-Lepley, C. E.; Mizsak, S. A.; Nugent, R. A.; Johnson, R. A.,
Tetraalkyl oxiranylidenebis(phosphonates). Synthesis and reactions with
nucleophiles. Journal of Organic Chemistry 1993, 58, (15), 4159-61.
24. Katzhendler, J.; Ringel, I.; Karaman, R.; Zaher, H.; Breuer, E.,
Acylphosphonate hemiketals - formation rate and equilibrium. The electron-
withdrawing effect of dimethoxyphosphinyl group. Journal of the Chemical
Society, Perkin Transactions 2: Physical Organic Chemistry 1997, (2), 341-349.
25. Pudovik, A. N.; Zimin, M. G., Addition reactions of partially-esterified
phosphorus acids. Rearrangements of a-hydroxyalkyl phosphorus esters and their
a-mercapto and a-amino-analogs. Pure and Applied Chemistry 1980, 52, (4), 989-
1011.
26. Arstad, E.; Skattebol, L., Reactions of diethyl mesyl- or
tosyloxyphosphonates with diethyl phosphite and base: a method claimed to yield
bisphosphonates. Tetrahedron Letters 2002, 43, (48), 8711-8712.
27. Maeda, H.; Takahashi, K.; Ohmori, H., Reactions of acyl
tributylphosphonium chlorides and dialkyl acylphosphonates with Grignard and
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28. McKenna, C. E.; Kashemirov, B. A. Preparation and use of a-keto
bisphosphonates. 99-US1577 2000002889, 19990713., 2000.
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29. McKenna, C. E.; Kashemirov, B. A.; Roze, C. N.,
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diphosphate. Bioorganic Chemistry 2002, 30, (6), 383-395.
30. Bonaz-Krause, P. I.; Kashemirov, B. A.; McKenna, C. E., Oxidative
pathways of a-diazo phosphonates. Phosphorus, Sulfur and Silicon and the
Related Elements 2002, 177, (10), 2271.
31. Kashemirov, B. A.; Roze, C. N.; McKenna, C. E.,
Carbonylbisphosphonate analogues of nucleoside 5'-diphosphates. Phosphorus,
Sulfur and Silicon and the Related Elements 2002, 177, (10), 2275.
32. McKenna, C. E.; Kashemirov, B. A., Recent progress in
carbonylphosphonate chemistry. Topics in Current Chemistry 2002, 220, (New
Aspects in Phosphorus Chemistry I), 201-238.
33. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C., The facile
dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane.
Tetrahedron Letters 1977, (2), 155-8.
34. McKenna, C. E.; Schmidhauser, J., Functional selectivity in phosphonate
ester dealkylation with bromotrimethylsilane. Journal of the Chemical Society,
Chemical Communications 1979, (17), 739.
35. Trecourt, F.; Breton, G.; Bonnet, V.; Mongin, F.; Marsais, F.; Queguiner,
G., New syntheses of substituted pyridines via bromine-magnesium exchange.
Tetrahedron 2000, 56, (10), 1349-1360.
36. Knochel, P.; Dohle, W.; Gommermann, N.; Kneisel, F. F.; Kopp, F.; Korn,
T.; Sapountzis, I.; Vu, V. A., Highly functionalized organomagnesium reagents
prepared through halogen-metal exchange. Angewandte Chemie, International
Edition 2003, 42, (36), 4302-4320.
37. Rottlander, M.; Boymond, L.; Berillon, L.; Lepretre, A.; Varchi, G.;
Avolio, S.; Laaziri, H.; Queguiner, G.; Ricci, A.; Cahiez, G.; Knochel, P., New
polyfunctional magnesium reagents for organic synthesis. Chemistry--A European
Journal 2000, 6, (5), 767-770.
38. Beumel, O. F., Jr.; Smith, W. N.; Rybalka, B., Preparation of 2- and 4-
picolyllithium. Synthesis 1974, (1), 43-5.
35
39. Sanchez-Sancho, F.; Herradon, B., Stereoselective conjugate addition of
metalated 2-methylpyridine to functionalized a,b-unsaturated carbonyl
compounds. Heterocycles 2003, 60, (8), 1843-1854.
40. Braun, H. A.; Meusinger, R.; Schmidt, B., 2-Iodoethanols from aldehydes,
diiodomethane, and isopropylmagnesium chloride. Tetrahedron Letters 2005, 46,
(15), 2551-2554.
41. Kim, D. Y.; Wiemer, D. F., Addition of allylindium reagents to acyl
phosphonates: synthesis of tertiary a-hydroxy alkylphosphonates. Tetrahedron
Letters 2003, 44, (14), 2803-2805.
42. Ranu, B. C., Indium metal and its halides in organic synthesis. European
Journal of Organic Chemistry 2000, (13), 2347-2356.
43. Auge, J.; Lubin-Germain, N.; Marque, S.; Seghrouchni, L., Indium-
catalyzed Barbier allylation reaction. Journal of Organometallic Chemistry 2003,
679, (1), 79-83.
44. Guenin, E.; Degache, E.; Liquier, J.; Lecouvey, M., Synthesis of 1-
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of a new cyclic 1-acyloxymethylene-1,1-bis(phosphonic acid). European Journal
of Organic Chemistry 2004, (14), 2983-2987.
45. Steurer, S.; Podlech, J., Aminoalkyl-substituted a-methylene-g-
butyrolactones from a-amino acids using an indium-mediated Barbier allyl
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46. Vogel, A. I., Vogel's Elementary Practical Organic Chemistry. 3rd Ed.
1979; p 350 pp.
47. Turhanen, P. A.; Ahlgren, M. J.; Jarvinen, T.; Vepsalainen, J. J.,
Bisphosphonate prodrugs. Synthesis and identification of (1-hydroxyethylidene)-
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2001, 170, 115-133.
36
Chapter 2
THE SYNTHESIS OF BRANCHED TROIKA ACID
RESINS FOR CHELATION OF DIVALENT METAL
CATIONS
Introduction
As global industries and the world’s population continue to grow, so does the
threat of environmental pollutants entering our water supplies. Exposure to toxic
heavy metals, such as arsenic, mercury, lead, and chromium found in industrial
wastewaters, have been shown to promote cancer in humans and animals
1
.
Furthermore, plant models have also been observed to absorb and store heavy
metals, providing an entryway into the food chain and further contact
2
. Several
methodologies have been employed in remediation of heavy metals from
wastewaters
3
. Currently, ion-exchange resins are the preferred means of chelation
due to efficient and selective removal of impurities from solutions
3
. These resins
function by replacing an undesirable metal ion by another one, which is neutral to
the environment. Modifications to functional groups (i.e. increased concentration
or variation in groups) on the chelating ligands can alter exchange capacity, ion-
selectivity, and pH dependence.
37
Previous work in our laboratory in the field of chelation chemistry provided
Troika acid, (α-hydroxyimino)phosphonoacetic acid, a novel compound
containing phosphonate, oxime, and carboxylate moieties all linked by a common
α-carbon
4-7
. The Troika precursor diethyl phosphonoacetic acid (PAA) and other
derivatives, such as diethyl phosphonoacetonitrile (PAN) and diethyl
carbmoylmethylphosphonate (CMP), can either be purchased or synthesized by
Arbuzov chemistry
8, 9
and subsequently, converted to Troika derivatives by
nitrosation reactions (Scheme 2.1)
4
. In liquid-liquid studies, these tri-functional
ligands, particularly the Z-isomer of Troika acid (Figure 2.1), exhibit features
consistent with other strong chelating ligands, such as the iminodiacetic acid
ligand found in the commercial chelating resin Chelex-100. These features
include multiple ionizable sites over a wide range of pH and facile regeneration
under mild acidic conditions
4
.
RO
P
RO
O
C C
O
OH
N
HO
Z-Troika Acid
Figure 2.1: Z-isomer of Troika Acid
38
RO
P
RO
O
C C
O
OH
N
OH
t-butyl ONO, HCl (g)
phosphonoacetic acid (PAA)
Troika Acid (1)
RO
P
RO
O
C
H
H
C
O
NH
2
RO
P
RO
O
C
N
C
O
NH
2
OH
RO
P
RO
O
C C
O
OH
H
H
carbmoylmethylphosphonate (CMP)
Troika Derivative (2)
RO
P
RO
O
C
H
H
C N
RO
P
RO
O
C
N
C N
OH
propyl ONO, potassium
t-butoxide
phosphonoacetonitrile (PAN)
Troika Derivative (3)
Troika Derivatives
Scheme 2.1: Nitrosation Reactions
Liquid-liquid data prompted coupling of the active Troika derivatives (1 to 3) to a
microporous polystyrene resin (1% crosslinking) (micro resin), either through the
phosphonate or the carboxylic acid (as in Troika acid 1, Figure 2.2) by various
coupling reactions. Once bound and characterized, chelation studies using Cu
(II), Ni (II) and Co (II) were preformed in both aqueous (0.6 M acetate buffer)
and organic (1:1 dioxane:methanol) solutions
4
. It was observed that the resin
bound Troika derivatives had no affinity for metal in aqueous solution, but
strongly bound metals from organic solutions, suggesting a deficiency in
39
hydrophilic character hinders interactions between the resin and metal ions in
aqueous solutions.
RO
P
RO
O
C C
O N
polystyrene resin bead
RO
P
O
O
C C
O N
OH
OH
OH
A
B
A: Mitsunobu Coupling; B: Dicyclocarbodiimide Coupling
Coupling Methodologies
Figure 2.2: Coupling Methods for the Troika Ligand
To increase the hydrophilic character of the solid-supported Troika derivatives,
two strategies were proposed. The first method required exchanging the micro
polystyrene resin, which is not water soluble, for a support more capable of
interacting with the aqueous solution. Macroporous polystyrene resins (8%
crosslinking, macro resin) were considered 1) for increased resin pore-size
enhancing aqueous solution penetration into the resin beads, and 2) increased
surface area providing a higher molar concentration of bound Troika derivatives.
The second approach explores incorporating branched Troika ligands on the resin
surface. It was proposed that attaching Troika derivatives to the micro resin via
branched “spacer-units” or a “daisy-chain” motif (Figure 2.3), would allow the
ligand to move independently of the support and therefore, enhance binding.
40
These motifs could also provide higher concentrations of polar functional groups,
introduced from either the Troika ligand and/or the “spacer-units”, improving
solubility in aqueous solutions. Herein, we present the synthesis, characterization,
and preliminary chelation studies of novel branched Troika derivatives bound to
micro and macro resin supports.
Branched
"Spacer-unit"
Troika Ligands
Resin Motif
polystyrene resin bead
"Daisy-chain"
Troika Ligands
Resin Motif
polystyrene resin bead
Figure 2.3: Various Resin-Ligand Motifs
Results and Discussion
Polystyrene-divinylbenzene (DVB) macro resins contain increased amounts of
crosslinked DVB, which increases pore-size (in Å) and enhances overall stability
versus the micro resins. The larger pores allow for greater penetration of aqueous
41
solutions and interaction with the inner surface of the resin, providing a greater
chance of supported Troika derivatives capable of aqueous interactions. The
conversion (and calculation of milliequivalents, meq.) of chloromethylated micro
resin to aminomethylated micro resin (MicA), described by Carrick
4
, was found
to be applicable to the macro resin (MacA) via the Gabriel reaction (Scheme 2.2).
PS
N
O
O
PS
NH
2
NH
2
NH
2
PS
NH
DMF
Cl
O
O
Micro or Macro resin
MicA or MacA
Scheme 2.2: Conversion of Chloromethylated Resins to Aminomethylated Resins
Unlike micro resins, which can be analyzed by a number of techniques, macro
resin analysis is complicated by increased crosslinking. Gel
31
P NMR is difficult
to attain even after grinding macro resin into a fine powder. Convoluted FT-IR
spectra, due to decreased signal:noise ratios, are also common in highly
crosslinked resins
10, 11
. FT-IR analysis using a diamond compression cell
equipped with a ZnSe lens beam condensing unit has been observed to increase
signal:noise ratios, providing an efficient method for the analysis of macro
resins
12
. Utilizing this FT-IR technique during the addition of PAA via
dicyclocarbodiimide (DCC) coupling (Scheme 2.3) proved to be advantageous.
42
Monitoring changes in the P-O stretch (1310 cm
-1
and 1100 cm
-1
) intensities in the
spectrum with respect to a constant peak (CH
2
stretch 2850 cm
-1
to 2920 cm
-1
)
allowed for the optimal amount of PAA to be loaded on the resin (Figure 2.4 and
2.5).
PS
NH
2
PS
HN C
O
C P
O
OEt
OEt
H
H
RO
P
RO
O
C C
O
OH
H
H
DCC
Scheme 2.3: DCC Coupling of PAA to Aminomethylated Macro Resin
0
20
40
60
80
100
120
350 850 1350 1850 2350 2850 3350 3850 4350
Wavelength (nm)
Absorbance
Phosphonoacetic acid
Resin after DCC
coupling
-NH2 resin
Figure 2.4: P-O Stretch as Observed in MacA bound PAA FT-IR Spectra
P-O signals
43
0
10
20
30
40
50
60
70
80
90
100
350 850 1350 1850 2350 2850 3350 3850
Wavelength (nm)
Absorbance
1st DCC coupling
2nd DCC couupling
3rd DCC coupling
Figure 2.5: Effects of Multiple DCC Coupling Steps
The nitrosation reaction yielding the Troika acid macro resin 4 (Scheme 2.4) was
attempted in various solvents. As the hydroxyimino functional group does not
produce an IR signal, it was anticipated that the spectrum resulting from the
hydroxyimino compound (Figure 2.6) would exhibit no major signal changes. A
qualitative determination (i.e. color change) using aqueous copper acetate
revealed that dioxane was the optimal solvent for this experiment yielding the
most intense green color. A chelation study using known amounts of Cu (II) ion
in organic (1:1 dioxane:methanol) and aqueous (0.6 M acetate buffer, pH = 5.4)
solutions revealed that 4 had higher metal affinity in the organic versus aqueous
solution.
44
PS
HN C
O
C P
O
OEt
OEt
H
H
PS
HN C
O
C P
O
OEt
OEt
N
OH
t-butyl ONO, HCl (g)
4
Scheme 2.4: Synthesis of the Troika Ligand
0
20
40
60
80
100
120
140
160
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Wavelength (nm)
Absorbance
Tetrahydrofuran
Dioxane
Toluene
Figure 2.6: Effects of Solvents on the Nitrosation Reaction via FT-IR
Following the synthesis and study of 4, our efforts focused on synthesizing the
micro resin containing either a “spacer-unit” or “daisy-chain” motif (Figure 2.7).
The construction of micro resin 6 began with the synthesis of the amide-linked
aminopropanol to PAA (ligand A). Coupling was first attempted required the
synthesis of the acid chloride of PAA via thionylchloride and subsequent addition
of the amine
13, 14
. This route proved to be problematic as it generated the desired
product in addition to the formation of ester coupling and the dimer product. A
selective synthesis of ligand A was attained by first activating PAA with p-
nitrophenol, followed by addition of aminopropanol (Scheme 2.5)
15
, which was
purified by column chromatography and verified by
31
P and
1
H NMR.
45
N
O
O
P
EtO
O
C
P
EtO
O
C
N
PS
R
R
N
O
P
OEt
C C
O O
N
H
N
OH
C
H
H
H
N
n
C
O
C
N
P
O
OEt
OEt
PS
N
H
C C
O
P
O H
H
H
N C
O
C P
O
OEt
OEt
O C
H
H
n
PS
N
H
N C
O
C P
O
OEt
OEt
O C
H
H
n
N
OH
OH
OH
OH
OH
O
C
NH
2
C N
12
11
R, R =
R, R =
6
8
Figure 2.7: Branched and “Daisy-chained” Synthetic Targets
EtO
P
EtO
O
C C
O
OH
EtO
P
EtO
O
C C
O
N
H
1) p-nitrophenol,
4-pyrolidinopyridine,
DCC, DCM
OH
2) aminopropanol
H
H
H
H
Ligand A
Scheme 2.5: Synthesis of Ligand A
Dealkylation of diethyl PAA bound to the micro resin was required for the
addition of ligand A. Phosphonate dealkylation using bromotrimethylsilane
(BTMS) is accomplished by generating the phosphonate silyl esters, followed by
hydrolysis during aqueous work up yielding the phosphonic acid. The PAA
bound micro resin was treated with BTMS, followed by aqueous work-up
(Scheme 2.6).
31
P NMR and FT-IR spectral data was inconclusive, providing no
46
evidence that phosphonic acid had been generated. Subsequent Mitsunobu and
DCC coupling experiments using the presumed “phosphonic acid micro resin”
failed to produce the Troika precursor micro resin. It was speculated that the
rigidity of the solid-support and limited rotation of resin bound PAA sterically
hinder silylation and generation of the final acid bound resin, confirming similar
findings observed by Carrick
4
. Furthermore modifications to the synthesis of
micro resin 6 were suspended to focus on the synthesis of resins 8, 11, and 12.
EtO
P
EtO
C
O
C OH
O
DCC, DMF
H
2
N
OEt
P
OEt
C C
O O
HN
H
PS
H
H
H
Diethyl PAA Microporous Resin
OH
P
OH
C C
O O
HN
H
PS
H
No Reaction
BTMS,
DCC
PS
Scheme 2.6: BTMS Delkylation of MicA Bound PAA
Ligand B used in the ‘daisy-chained’ micro resin 8 required a multi-step
synthesis
16
(Scheme 2.7). Steps L-B-a to L-B-c were verified by NMR, while
final purification by column chromatography was completed at step L-B-d. DCC
coupling of ligand B to the aminomethylated micro resin was accomplished
providing micro resin 7. Nitrosation of micro resin 7 was monitored using a new
gel
31
P NMR technique, the micro resin is swollen with a mixture of DMF and d-
methanol providing a higher quality spectrum, confirms the incorporation of
oxime to 40% of ligand B (Scheme 2.8). A second nitrosation attempt was made
47
decreasing the amount of oxime bound to the resin and increasing the amount of
an unknown phosphonate.
Both micro resins 7 and 8 (after 2
nd
nitrosation) were converted to the Troika
potassium salt by rinsing with ethanolic potassium t-butoxide and semi-
quantitatively tested. Changes in Cu (II) ion levels in a 18.7 mM copper acetate
solution (0.6 M acetate buffer, pH = 6.5) were monitoring by UV-Vis (748 nm).
Measurements were taken before and after (24 hours) addition of the resin to the
solution. It was discovered that micro resin 7 bound Cu (II) 5-times more
efficiently then 8; however, binding was modest with respect to the standard
Chelex-100 (Table 2.1). These findings confirm the loss of the Troika ligand
upon the second nitrosation as observed by
31
P NMR, suggesting that full
nitrosation of micro resin 7 is difficult.
H
2
N N
H
C O
O
EtO
P
O
C
O
C OEt
O
Li
O
N
H
C O
O
P
OEt
C C
O O
EtO
1) Acidic deprotection
DCC, Et
3
N, DCM
EtO
P
EtO
C
O
C OH
O
O
NH
P
OEt
C C
O O
HO
C O
C
P
EtO
EtO
O
t-BOC protection
L-B-a
Mitsunobu coupling of L-B-a
L-B-b
L-B-b
Ligand B
H
H
H
H
H
H
H
H
H
H
2
OH
2
HO
2
2
2)
3) KOH, EtOH / Water
L-B-d
Scheme 2.7: Synthesis of Ligand B
48
O NH
P
OEt
C C
O O
N
H
C O
P
EtO
EtO
O
N
N
Propylnitrite, HCl (g), Dioxane
OH
OH
PS
8
Ligand B
PS
NH
2
DCC, HOBt, DCM
O NH
P
OEt
C C
O O
HN
C O
C
P
EtO
EtO
O
H
PS
H
H
H
7
7
Scheme 2.8: Coupling of Ligand B to Aminomethylated Micro Resin
Table 2.1: Change in Copper Ion Concentration Using Resins 7 and 8
The limitations discovered for the synthesis of target micro resins 5 and 8,
prompted the examination of coupling diethanolamine to the chloromethylated
resin. Preliminary solution-phase studies of the reaction conditions used in
coupling diethanolamine to benzylchloride
17
were found to be applicable to either
18.7 mM Cu(OAc)
2
in
acetate buffer (pH=
6.5) Micro resin 7 Mirco resin 8
Chelex-100
O hours 18.7 18.7 18.7
24 hours 17.2 18.4 0.7
Change in [Cu (II)]* 1.5 0.6 18
*Changes in [Cu (II)] were monitored by UV-VIS (Initial absorbance at 1.18 @ 748 nm) and referenced against a calibration curve
49
microporous or macroporous resins (Scheme 2.9) providing the desired
diethanolamine platform (MicB and MacB).
PS
HN
OH
OH
Na
2
CO
3
/ acetonitrile
PS
Cl
N
OH
OH
MacB or MicB
Scheme 2.9: Synthesis of Diethylamine Platform
Several coupling strategies to the dipropanolamine moiety were explored in the
solution-phase before transferring to the resins. CMP was used as the Troika
precursor for the first set of experiments and was synthesized using the Arbuzov
reaction
8
followed by the formation of the sodium salt
18
(Scheme 2.10). DCC
coupling reactions in DMF of the CMP ligand to the benzyl dipropanolamine
were found to yield the desired product by
1
H and
31
P NMR, however the rate of
reaction is very slow (50% coupled product after 72 hours). Heating the reaction
mixture was found to increase product formation in addition to side products not
seen in the un-heated reaction mixture.
P OEt
OEt
OEt
Cl C C NH
2
O
Reflux
P C C NH
2
O O
EtO
EtO
NaOH, EtOH
P C C NH
2
O O
EtO
O
Na
H
H
H
H
H
H
Scheme 2.10: Arbuzov Synthesis of CMP
50
The second approach employed the Mitsunobu coupling reaction in DMF at 55°C
while swirling on a shaker bath. The McKenna group has found that under these
conditions the Mitsunobu reaction can be highly selective when coupling
monoacidic phosphonates to multifunctional 1° alcohols (unpublished results).
The coupling was completed in 4 hours. The crude product was then purified by
column chromatography and characterized by
1
H,
13
C, and
31
P NMR and FT-IR.
Nitrosation was completed providing the Troika derivative and characterized
(Scheme 2.11). The reaction mixture contained ~80% of the Troika CMP ligand
(in the EE, ZZ, ZE and EZ geometries) and was found to be contaminated with an
unknown phosphonate. The mixture was found to be miscible in water and
chelated Cu (II) from a copper acetate solution, turning the solution from blue to a
dark olive green color.
N
O
O
P
EtO
O
C C
P
EtO
O
C C NH
2
O
O
NH
2
N
O
O
P
EtO
O
C C
P
EtO
O
C C NH
2
O
O
NH
2
N
N
Propylnitrite, HCl (g), Dioxane
H
H
H
H
OH
OH
Mistunobu Product
Solution-Phase Troika Derivative
Scheme 2.11: Synthesis of Solution-Phase Troika Derivative
The elaborated Mitsunobu method for CMP was then transferred to the micro and
macro resin supports (Scheme 2.12). This method provided resin 9 at an
increased rate, with limited side products generated. Nitrosation to micro and
51
macro resin 11 were followed by gel
31
P NMR and FT-IR, respectively. Micro
resin 11 was found to only contain ~50% oxime contaminated with an unknown
phosphonate. This mixed-surface resin was found to qualitatively chelate Cu (II)
from an aqueous solution only after treatment with ethanolic potassium t-
butoxide. Nitrosation of the macro resin 11 was also attempted with similar
results. Semi-quantitative testing (by UV-Vis) with various metal solutions at
pH= 6.5 (0.6 M acetate buffer) demonstrated that micro resin 11 was able to
chelate Cu (II) in aqueous solution; however, the binding was modest with respect
to the commercial standard Chelex-100 (Table 2.2). It was also observed that
micro resin 11 had comparable Cu (II) binding with respect to the Troika
precursor resin 9, limiting our ability to discern a mechanism of metal chelation.
PS
N
OH
OH
P C C R
O O
EtO
HO
H
H
Mitsunobu Coupling in DMF
@ 55
o
C
MicB and MacB
N
O
O
P
EtO
O
C
P
EtO
O
C
H
PS
R
R
O
C
NH
2 9
H
H
H
R, R =
PS
N
O
O
P
EtO
O
C C
P
EtO
O
C C NH
2
O
O
NH
2
PS
N
O
O
P
EtO
O
C C
P
EtO
O
C C NH
2
O
O
NH
2
N
N
Propylnitrite, HCl (g), Dioxane
H
H
H
H
OH
OH
11
9
Scheme 2.12: Synthesis of Resin 11
52
Table 2.2: Changes in Metal Ion Concentration Using Resin 11
The PAN derivative, synthesized via Arbuzov chemistry
9
and converted to the
lithium salt
19
, was also coupled to Mic-A by the Mitsunobu method described
above (Scheme 2.13). Three peaks were observed in the
31
P NMR corresponding
to the product (80%) and a pair of unknown phosphonates. Adjustments to
reaction conditions, particularly temperature (< 25°C), were found to only reduce
18.7 mM Cu(OAc)
2
in
acetate buffer (pH=
6.5) Micro resin 9 Mirco resin 11
Chelex-100
O hours 18.7 18.7 18.7
24 hours 17.8 17.9 0.7
Change in [Cu (II)]* 0.8 0.8 17.9
*Changes in [Cu (II)] were monitored by UV-VIS (Initial absorbance at 1.18 @ 748 nm) and referenced against a calibration curve
15.8 mM CoCl
2
in
acetate buffer (pH=
6.5) Micro resin 9 Mirco resin 11
Chelex-100
O hours 15.8 15.8 15.8
24 hours 15.6 15.8 3.9
Change in [Co (II)]* 0.2 0 11.9
*Changes in [Co (II)] were monitored by UV-VIS (Initial absorbance at 0.20 @ 514 nm) and refernced againist a calibration curve
17.8 mM NiCl
2
in
acetate buffer (pH=
6.5) Micro resin 9 Mirco resin 11
Chelex-100
O hours 17.8 17.8 17.8
24 hours 16.6 17.1 2
Change in [Ni (II)]* 1.2 0.7 15.8
*Changes in [Ni (II)] were monitored by UV-VIS (Initial absorbance at 0.18 @398 nm) and referenced against a calibration curve
53
and remove these side products. Amine-catalyzed phosphonate dealkylation, a
phenomenon observed when a six-member ring configuration can be induced
between an amine and phosphonate ester, generating the phosphonate anion, in
turn promotes fragmentation
20
. It is believed that this fragmentation pathway
may also explain the presense of phosphonate in micro resin 11. Nitrosation using
this resin provided ~10% oxime insertion into the existing bound PAN ligands
and were semi-quantitatively tested for Cu (II) and Ni (II) chelation (Table 2.3).
EtO
P
EtO
C
O
C N
P OEt
OEt
OEt
Cl C C N
Reflux
LiBr
Methylethylketone
EtO
P
O
C
O
C N
Li
H
H
H
H
H
H
PS
N
OH
OH
P C C R
O O
EtO
HO
H
H
Mitsunobu Coupling in DMF
@ 55
o
C
MicB
N
O
O
P
EtO
O
C
P
EtO
O
C
H
PS
R
R
R, R = C N 10
H
H
H
K t-butoxide, propylnitrite,
EtOH / THF
PS
N
O
O
P
EtO
O
C R
P
EtO
O
C R
N
N
OH
OH
R, R = C N 12
10
Scheme 2.13: Synthesis of Resin 12
54
Table 2.3: Changes in Metal Ion Concentration Using Resin 12
DCC coupling (72 hours) at room temperature was also examined providing the
micro resin devoid of side product; however, attempts at nitrosation using either
potassium t-butoxide or sodium hydride with propyl nitrite did not generate the
Troika derivative. The Troika derivative of the PAN ester is generated via
electrophilic attack of a carbanion generated under basic conditions at the α-
carbon. Carbanion formation on resin-bound PAN presents a pathway to several
unpredictable side reactions. A broad peak between 2200 cm
-1
to 2600 cm
-1
is
observed in the FT-IR spectrum of the DCC coupled micro resin suggesting
formation of various amine salts. A contributing factor to salt formation is the
six-atom distance between the α-carbon and the tertiary amine. These salts and
18.7 mM Cu(OAc)
2
in
acetate buffer (pH=
6.5) Micro resin 10 Mirco resin 12
Chelex-100
O hours 18.7 18.7 18.7
24 hours 18 17.9 0.7
Change in [Cu (II)]* 0.7 0.7 17.9
*Changes in [Cu (II)] were monitored by UV-VIS (Initial absorbance at 1.18 @ 748 nm) and referenced against a calibration curve
17.8 mM NiCl
2
in
acetate buffer (pH=
6.5) Micro resin 10 Mirco resin 12
Chelex-100
O hours 17.8 17.8 17.8
24 hours 17 17.7 2
Change in [Ni (II)]* 0.8 0.05 15.8
*Changes in [Ni (II)] were monitored by UV-VIS (Initial absorbance at 0.18 @398 nm) and referenced against a calibration curve
55
other nucleophilic species (i.e. un-reacted hydroxy ligands) can intercept the
oxime electrophile, abating nitrosation. The FT-IR spectrum of the Mitsunobu
coupled PAN resin did not exhibit a strong amine salt stretch; however, the same
rationale is applicable, explaining the decreased amount of oxime inserted in PAN
and CMP coupled micro resins.
The addition of the amine moiety was found to perpetuate side reactions in the
coupling and generation of micro resins 11 and 12 with little to no effect on
increasing the hydrophilic character of the resin. According to Lezzi et al., the
incorporation of polyethylene glycol (PEG) to polystyrene/DVB microporous
resins was found to substantially increase the resin’s hydrophilicity
21-23
. Using the
cited procedure, we attempted the synthesis of an amino-terminated PEG
microporous resin for DCC coupling to PAA and subsequent conversion to Troika
acid (Scheme 2.14).
O
O
n
N
H
C
O
C
H
P
O
OEt
OEt
O
O
n
N
H
C
O
C
N
P
O
OEt
OEt
OH
H
PS PS
nitrosation
13 14
Scheme 2.14: Troika Acid Attached via PEG to Micro Resin
56
Synthesis of micro resin 13 began by converting the hydroxy-terminated PEG
micro resin to the chloro-terminated
24
resin followed by Gabriel chemistry
providing the amino-terminated micro resin
4
. DCC coupling of PAA to the
amino-terminated resin was accomplished and found to reach maximum load after
two consecutive coupling reactions, according to FT-IR comparision of the amide
and P-O stretch with respect to the alkyl stretch. Nitrosation of resin 13 provide a
micro resin 14 with ~80% oxime insertion according to
31
P NMR (Scheme 2.15).
Qualitative studies where micro resin 14 was pre-treated with aqueous sodium
hydroxide, rinsed with water, and suspended in a Cu (II) aqueous solution
provided the first examples of aqueous chelation with resin-supported Troika
derivatives. Further study of the effectiveness of these micro resins is highly
recommended.
O
O
n
N
H
C
O
C
H
P
O
OEt
OEt
O
O
n
N
H
C
O
C
N
P
O
OEt
OEt
OH
H
PS
PS
ONOCl, Dioxane
13
14
13
Cl
PS
O
O
1000
PS
H
O
O
1000
PS
NH
2
1) PEG-1000, KOH,
THF
2) tert-n-butylammonium
hydrogen sulfate
1) Thionylchloride, DCM
2) Potassium phthalimide,
DCM
3) Hydrazine hydrate,
EtOH
O
O
1000
PS
NH
2
P C C OH
O O
EtO
EtO
H
H
DCC, DMF
Scheme 2.15: Synthesis of Troika Acid Attached via PEG to Micro Resin
57
Conclusion
A series of novel micro and macro resins containing various Troika derivatives
were synthesized and studied for the purpose of metal chelation from aqueous
solutions. The coupling of Troika acid to macro resins was found to have
marginal chelation abilities in an aqueous solution of copper acetate. The
proposed branching and “daisy-chained” micro resins were also found to have
limited chelating capacity in aqueous solution. These limitations can be attributed
to a number of decomposition and competing reactions, which either hinder
oxime insertion into the Troika precursor derivatives or facilitate the loss of the
Troika precursor ligand.
It was also discovered that the addition of polar “spacer-units” such as
dipropanolamine and other amino-alcohols only slightly influence hydrophilicity.
Larger polar “spacer-units”, such as polyethylene glycol, did show promise in
increasing the hydrophilic character of micro resins. Coupling of PAA to the
peglated resin and subsequent conversion to Troika acid provided the first
example of a Troika acid containing micro resin capable of aqueous metal
chelation without organic (ethanolic potassium t-butoxide) regeneration. Further
studies of peglated micro resin supports, in addition to stable Troika ligands
58
devoid of phosphonate ester alkyl chains, may provide a metal chelating resin
comparable to the commercial standard Chelex-100.
Acknowledgments
I would like to thanks EPRI project manager Paul Chu for his continued support
and helpful suggestions in the exploration of resin-bound Troika derivatives and
for financial support.
Experimental
Materials and Methods:
All reactions were carried out under a nitrogen atmosphere, unless otherwise
indicated. Toluene and tetrahydrofuran (THF), dioxane (reagent grade purchased
from Mallinckrodt Chemicals) were dried and distilled over
sodium/benzophenone. Chloroform and dichloromethane (DCM) (both reagent
grade purchased from Mallinckrodt Chemicals) were dried and distilled over
P
2
O
5
. Anhydrous diethyl ether (Et
2
O) was purchased from EMD Chemicals, Inc.
Anhydrous N, N-dimethylformimide (DMF) was purchased from EMD
Chemicals, Inc as a DriSolv™. Acetone and methanol (both HPLC grade) were
purchased from Mallinckrodt Chemicals, and ethanol (200 proof) was purchased
59
from EM Science. Chloromethylated macroporous resin (8% DVB crosslinked
with polystyrene) was purchased from ResinTech. Merrifield microporous resin
(1% crosslinked), triethyl phosphonoacetate (98%), triethyl phosphite(98%),
dipropanolamine (95%), aminopropanol (≥99%), thionylchloride (≥98%), N, N’-
dicyclohexylcarbodiimide (DCC) (99%), triphenylphosphine (≥95%), diisopropyl
azodicarboxylate (DIAD) (95%) and all other reagents were purchased from
Sigma Aldrich. Thin layer chromatography plastic-back sheets (20 X 20; silica
gel 60 F
254
) were purchased from EMD Chemicals, Inc. Preparative thin layer
chromatography glass-back sheets (20 X 20; 1000 microns) were purchased from
Analtech. Silica gel 150 (60-200 mesh) used for column chromatography
(column width 1-2 in) was purchased from Mallinckrodt Chemicals. Proton (
1
H),
carbon (
13
C), and phosphorus (
31
P) NMR spectra were measured either on a
Bruker AM-360 MHz, Varian Mercury-400 MHz, or Bruker AMX-500 MHz
spectrometer. Chemical shifts are reported relative to external TMS (
1
H), internal
CDCl
3
[δ = 77.0] (
13
C) or external 85% H
3
PO
4
(
31
P). NMR samples of
microporous resins were swelled forming a gel using either CDCl
3
or a mixture of
DMF:d-methanol. FT-IR spectra where taken on a Perkins-Elmer Spectrum 2000
spectrometer. Resins samples for FT-IR were 1) ground into a fine power using a
mortar and pestle, 2) mixed with KBr (spectral grade, Sigma Aldrich) then
compressed into a disk and 3) place on a diamond compression cell equipped with
a ZnSe beam condensing unit (Gatsby-Specac, Inc.) for analysis. UV-visible
60
detection was completed on a Agilent 8457 Spectrometer (equipped with HP UV-
visible ChemStation Software).
Synthesis of Troika acid bound macroporous resin (4)
PAA bound macro resin
Chloromethylated macro resin (5.16 meq of Cl per gram resin) was converted to
aminomethylated macro resin and titrated to determine NH
2
concentration
according to the procedure described by Carrick
4
. FT-IR analysis confirms
primary amine incorporation on to the resin (N-H stretch @ 3300 cm
-1
, N-H bend
@1600 cm
-1
). Triethyl phosphonoacetate is dealkylated using KOH in 100 mL
ethanol according to Carrick providing PAA
4
. Aminomethylated macro resin (0.5
g, 2.1 mmol) was added to a 100 mL round bottom flask equipped with a
magnetic stir bar under N
2
(g). DMF (10 mL) was added by glass syringe and
allowed to swell for 60 min while magnetically stirred. To the stirring solution,
2.4 g (0.012 mol) PAA was added drop-wise by pipette. DCC (2.5 g, 0.012 mol)
was dissolved in 10 mL DMF, and then added by glass syringe to the PAA/resin
solution and allowed to stir magnetically for 24 hours at 55°C. The resin was
filtered and rinsed with 200 mL ethanol and 200 mL water, and repeated till all
urea crystals were removed. Finally, it was rinsed with 200 mL saturated sodium
bicarbonate solution, 200 mL ethanol and followed by 200 mL methanol. The
resin was dried in a vacuum oven under reduced pressure at 30°C for 24 hours.
61
FT-IR analysis was completed and the coupling reaction was repeated until the P-
O stretch (1310 cm
-1
and 1100 cm
-1
) and the amide stretch (1690 cm
-1
) reached a
maximum with respect to the aliphatic C-H stretch (2850 cm
-1
to 2920 cm
-1
).
Troika acid bound macro resin (4)
Approximately 0.05 g (meq undetermined) of PAA bound macro resin was added
to a 50 mL round bottom flask equipped with a magnetic stir bar under N
2
(g).
Dry dioxane (10 mL) was added via glass syringe to PAA bound macro resin and
allowed to swell for 60 min. Excess nitrosyl chloride was added directly from the
storage ample and quickly capped (solution goes dark red), then stirred
magnetically for 12 hours. Resin 2-4 was filtered and rinsed with 200 mL
ethanol, followed by 100 mL 5% KOH solution (resin turns light orange).
Qualitative chelation study of macro resin (4)
Two 0.01 g samples of macro resin 4 were prepared by first rinsing each sample
with 100 mL of ethanolic potassium t-butoxide and 200 mL of water, and then
allowing it to air dried on the filter for 10 min. One sample was added to 2 mL of
organic solution (1:1 dioxane:methanol) and the other to 2 mL of aqueous (0.6 M
acetate buffer, pH = 5.4) containing an undetermined amount of copper acetate
and allowed to swirl for 10 min. Immediately upon addition, resin 2-4 in organic
62
solution turned dark green, while the aqueous sample turned only a very light
green color.
Synthesis of the phosphonate branched micro resin (6)
Diethyl (2- {[(3-hydroxypropyl)amino]oxy}2-oxoethyl)phosphonate (Ligand A)
In a 100 mL round bottom three-neck flask equipped with a magnetic stir bar,
3.92 g (0.02 mol) PAA, 2.78 (0.02 mol) p-nitrophenol, 0.3 g (0.002 mol) 4-
pyrolidinopyridine were added and dissolved using 50 mL of DCM. An addition
funnel was charged with 4.16 g (0.02 mol) DCC in DCM and added drop-wise to
the stirring solution at 20°C. The reaction intermediate was allowed to stir at
room temperature and was monitored by
31
P NMR. Once all the PAA was gone,
the intermediate was filtered, retaining the filtrate. A DCM solution of
aminopropanol (1.5 g, 0.02 mol) was added by pipette to the filtrate and allowed
to stir till completion. Solvent was removed by rotary evaporation at room
temperature under reduced pressure. The residue was purified by column
chromatography (2 in. diameter) using a gradient from 100% chloroform to 4:1
chloroform:acetone mobile phase. Ligand A was collected in fractions 180-224
and solvent removed by rotary evaporation at room temperature under reduced
pressure, providing pure Ligand A as a colorless oil.
31
P NMR: (CDCl
3
) δ
P
23.3 (s)
63
1
H NMR: (CDCl
3
) δ
H
1.25 (t,
4
J
HH
= 7 Hz, 3H), 1.62 (quint,
4
J
HH
= 12 Hz, 2H), 2.81
(d,
2
J
PH
= 22 Hz, 2H), 3.31 (quart, J
HH
= 12 Hz, 2H), 3.56 (t,
3
J
HH
= 6 Hz, 2H), 4.06
(quint,
3
J
HH
= 15 Hz, 4H)
[(Amino)oxy-2-oxoethyl]phosphonic acid bound micro resin
The PAA bound micro resin is synthesized according to procedure described by
Carrick
4
. PAA bound micro resin (0.5 g) was added to a 50 mL round bottom
flack equipped with a magnetic stir bar under N
2
(g). DCM (20 ml) was added to
the resin and allowed to swell for 30 min. Excess BTMS was added to the stirring
resin solution via pipette and quickly capped and allowed to stir for 12 hours. The
resin was filtered and rinsed with 100 mL methanol, 100 mL ethanol, and a final
rinse with 50 mL diethyl ether. It was then dried at room temperature under
reduced pressure for 12 hours.
31
P NMR did not show a chemical shift associated
with a phosphonic acid.
Synthesis of the “daisy-chained” micro resin (8)
Ligand B
L-B-a: In a graduated cylinder, a solution of 2.58 g (0.025 mol) aminopentanol in
10 mL of water was made. In another graduated cylinder, 6 g (0.027 mol) t-BOC
was dissolved with 20 mL dioxane. The two solutions were mixed together in a
100 mL round bottom flask equipped with a magnetic stir bar at 0°C. Sodium
64
carbonate (3.2 g, mol) was added slowly to rapid evolution of CO
2
(g), and stirred
magnetically until the reaction was completed (monitored by
1
H NMR). L-B-a
was extracted twice with 5 mL portions of DCM from the reaction mixture. The
DCM extracts were combined and dried with anhydrous Na
2
SO
4
, filtered, and the
solvent was removed by rotary evaporation under reduced pressure providing a
colorless residue (80% pure by
1
H NMR).
L-B-b: In a 50 mL round bottom flask equipped with a magnetic stir bar, the
phosphonic lithium salt of diethylphosphonoacetate (1.0 g, 0.005 mol) was
transferred to the phosphonic acid form using 3.0 g Dowex ion-exchange resin in
20 mL methanol. The solvent was removed using a rotary evaporator under
reduced pressure at room temperature and allowed to come to a constant weight.
The residue was then dissolved with 10 mL of dry dioxane under N
2
(g). L-B-a
(1.53 g, 0.0075 mol) and 1.94 g (0.0075 mol) were weighed out in test tubes and
dissolved with 5 mL dioxane and then added to the magnetically stirred acid
solution under N
2
(g). DIAD (1.5 g, 0.0075 mol) in 5 mL of dioxane was added
drop-wise using a glass syringe. The reaction was monitored by
31
P NMR until
completed, followed by removal of the solvent using a rotary evaporator under
reduced pressure at 30°C. The residue was dissolved with 15% diethyl ether in
hexanes, generating a yellowish precipitate. The reaction mixture was filtered,
retaining the filtrate, then solvent removed by rotary evaporation under reduced
65
pressure at 30°C.
31
P NMR analysis indicates a minor contamination of L-B-b
(product weight 1.96 g) with triphenylphosphine oxide.
31
P NMR: (CDCl
3
) δ
P
19.9 (s)
Direct deprotection of tritylated L-B-b was accomplished using 10 mL of 4 M
HCl in dioxane at 0°C while stirring magnetically for 30 min. Solvent was
removed using rotary evaporation at 30°C until a constant weight was achieved.
The crude residue was taken forward for coupling to PAA via DCC coupling.
To a 100 mL round bottom flask equipped with a magnetic stir bar, 0.74 g (0.0037
mol) PAA and 1.3 g (~0.004 mol) of the crude L-B-b residue were dissolved with
10 mL DCM under N
2
(g). DCC (0.82 g, 0.004 mol) was dissolved with 10 mL
DCM in a test tube and subsequently added to the magnetically stirred solution of
PAA/residue solution at 10°C. The reaction mixture was monitored by
31
P NMR
until completed, followed by removal of the solvent by rotary evaporation under
reduced pressure at room temperature providing a cloudy residue. Purification of
the acid protected Ligand B was achieved by column chromatography using a
gradient from 100% chloroform to 4:1 chloroform:acetone mobile phase. The
solvent was removed by rotary evaporation under reduced pressure at 30°C.
providing 0.56 g (31% yield). Direct dealkylation of the acetate ester was
accomplished using 1 eq. of KOH in a 75% ethanol:water solution. The final
66
product was extracted twice from the aqueous phase with 5 mL portions of DCM.
The DCM extracts were combined and dried over Na
2
SO
4
(s) and filtered. The
filtrate was collected and solvent was removed by rotary evaporation under
reduced pressure at room temperature providing Ligand B as a light yellow oil
(0.48 g, 98% yield).
31
P NMR: (CDCl
3
) δ
P
20.5 (s), 23.6 (s)
1
H NMR: (CDCl
3
) δ
H
1.26 (m, 9H), 1.45 (m, 4H), 1.61 (m, 2H), 2.85 (dd,
2
J
PH
=26
Hz, 2H), 2.90 (d,
2
J
PH
= 22 Hz, 2H), 3.19 (m, 2H), 4.08 (m, 8H), 7.08 (broad s, 1H)
Troika precursor “daisy-chain” resin (7)
Ligand B (0.223 g, 0.54 mmol) was dried to a constant weight under vacuum (1
mm Hg) at room temperature in a 100 mL round bottom flask equipped with a
magnetic stir bar. DCM (20 mL) was added by glass syringe to dissolve ligand B
under N
2
(g). In separate test tubes, 0.167 g (0.81 mmol) of DCC and 0.072 g
(0.53 mmol) of HOBt were weighed out and dissolved with 5 mL of DCM under
N
2
(g). The solution of ligand B was allowed to cool to 0°C, then equipped with
an addition funnel charged with the DCC solution. While magnetically stirring,
the DCC solution was added drop-wise to ligand B, followed by the addition of
the HOBt solution. The ligand B/HOBt intermediate was monitored by
31
P NMR
until the reaction was completed, followed by the addition of dry
aminomethylated micro resin (0.54 g, 0.54 mmol) and allowed to stir
67
magnetically for 12 hours at room temperature under N
2
(g). The resin was
filtered and rinsed with 100 mL DCM and twice with a 200 mL portion of ethanol
and 200 mL portion of water. Micro resin 7 was finally rinsed with diethyl ether
and dried in a vacuum oven under reduced pressure at 25°C, providing a yellow
resin.
31
P NMR: (DMF: d-methanol) δ
P
21.4 (broad s)
Troika “daisy-chain” micro resin (8)
Micro resin 7 (0.250 g) was weighed in a 100 mL round bottom flask equipped
with a magnetic stir bar. Dioxane (10 mL) was added by glass syringe to the
resin; the resin was allowed to swell for 1 hour. Under N
2
(g), 0.5 mL (4.6 mmol)
of t-butylnitrite was added via glass syringe to the resin followed by bubbling HCl
(g) (generated in situ by mixing NaCl and H
2
SO
4
in a round bottom flask) through
the resin until the solution turned a dark red color. The solution was then stirred
for 4 hours, filtered, and rinsed with 200 mL ethanol, and 100 mL 5% KOH
solution (resin turns light orange).
31
P NMR: (DMF: d-methanol) δ
P
0.7 (broad s), 7.19 (broad s)
FT-IR: (as KBr pellet)
68
Semi-qualitative chelation study for micro resins 7 and 8
Calibration curve for Cu(OAc)
2
in acetate buffer
A stock solution of 18.7 mM Cu(OAc)
2
was prepared in acetate buffer (0.6 M, pH
6.5). Serial dilutions of the stock solution were done using the same buffer
resulted in 0, 2, 4, 8, and 16.6 mM solutions. The absorbance of each solution
was measured (using quartz cuvettes) at λ
max
748 nm (ε
max
29.6) and plotted versus
the known concentrations to produce the calibration curve (y = 0.063x + 0.116; r
2
= 0.989). Measurements were taken at room temperature.
Copper chelation study using resins 7 and 8 at initial pH=6.5
A 0.05 g sample of micro resin 7 and 8, in addition to the commercial standard
Chelex-100, were prepared by first rinsing each sample with 100 mL of ethanolic
potassium t-butoxide, 200 mL of water, and then allowing them to air dry on the
filter for 10 min. Each sample was transferred to a test tube containing 10 mL of
the stock Cu(OAc)
2
solution (18.7 mM) and swirled using a shaker bath. The
initial absorbance value (1.18 at λ
max
748 nm) was taken of the solution with no
resin added, while the final absorbance values were taken 24 hours after the
addition of resin at room temperature. These values were referenced against the
calibration curve providing the change in Cu (II) concentrations.
69
Synthesis of MicB and MacB
MicB resin
Merrifield resin (1.99 g, 2 mmol Cl/g resin), 0.81 g (7.6 mmol) diethanolamine,
and 0.42 g (0.40 mmol) anhydrous sodium carbonate was added to 50 mL round
bottom flask equipped with a magnetic stir bar under N
2
(g). Acetonitrile (20 mL)
was added via glass syringe and the solution was allowed to reflux at 90°C for 24
hours. The solution was cooled to room temperature, filtered, and rinsed with 100
mL acetonitrile, 200 mL ethanol, 200 mL acetone and 100 mL diethyl ether, and
then placed in a vacuum oven and dried under reduced pressure at 25°C providing
an off-white resin (2.16 g).
FT-IR: (as KBr pellet)
MacB resin
Prepared as for MicB using 1.11 g (5.0 mmol Cl/g resin) of chloromethylated
macro resin.
FT-IR: (as KBr pellet)
Synthesis of amino-bound dipropanol to resin surface (MicB or MacB)
containing phosphonate linked Troika CMP (11)
Diethyl carbmoylmethylphosphonate (CMP)
Triethyl phosphite (16.6 g, 0.099 mol) was weighted out in a 100 mL round
70
bottom flask equipped with a condenser and stirred magnetically while heating to
120°C in a sand bath. Into three test tubes, a total of 9.35 g (0.37 mmol) 2-
chloroacetamide was weighed out then roughly added one half of the contents of a
test tube to the stirring solution and allowed to stir for 20 min. This process is
repeated until all the 2-chloroacetamide is added, turning the solution yellow. At
this point the temperature is increase to 165°C for 20 min or until the solution
turns a dark carmel color to avoid the formation of side products. The solution is
allowed to cool to 80°C and is then placed on a rotary evaporator under reduced
pressure (1 mmHg) and lightly heated to drive off un-reacted phosphite and the
ethylchloride by-product providing a viscous solution. Upon the addition of 30
mL of ethylacetate to the product mixture a crystalline precipitate forms. The
solution is then filtered and the crystals were washed with an additional amount of
ethyl acetate and dried providing white crystals (11.20 g, 60% yield after
purification).
31
P NMR: (D
2
O) δ
P

24.3 (s)
1
H NMR: (D
2
O) δ
H
1.21 (t,
4
J
HH
= 7 Hz, 6H), 2.95 (d,
3
J
PH
= 22 Hz, 2H), 4.07
(quint,
4
J
HH
= 15Hz, 4H)
FT-IR: (as KBr pellet)
71
Sodium monosalt of CMP
Diethyl CMP (11.2 g, 0.057 mol) was added to a 100 mL round botton flask and
dissolved with 50 mL of ethanol while magnetically stirred at room temperature.
An ethanolic (5 mL) NaOH (2.30 g. 0.057 mol) solution was added to the stirring
solution and heated to 65°C for 3.5 hours providing a white precipitate. The
solution was allowed to cool to room temperature, then filtered and rinsed with
ethanol providing a white crystalline product (6.77 g, 64% yield). The product
was found to be hygroscopic and wash dried in a vacuum oven under reduced
pressure (1 mm Hg) at room temperature.
31
P NMR: (D
2
O) δ
P

16.6 (s)
1
H NMR: (D
2
O) δ
H
1.02 (t,
4
J
HH
= 7 Hz, 3H), 1.10 (t,
4
J
HH
= 7 Hz, 3H), 2.58 (d,
3
J
PH
= 22 Hz, 2H), 3.49 (quart,
4
J
HH
= 7 Hz, 2H), 3.79 (t,
4
J
HH
= 7 Hz, 2H)
MicB Troika CMP precursor resin (9)
The sodium salt of CMP was converted to the monoacid (0.230 g, 1.16 mmol)
using Dowex 50WX8-200 and concentrated under reduced pressure (1 mm Hg) to
a constant weight. MicB resin (0.5 g, 2 mmol hydroxyl) was dried in the vacuum
oven under reduced pressure (1 mm Hg) at 30°C for 4 hours. The resin was then
added to a 25 mL pear-shape flask and allowed to swell in 10 mL DMF while
swirling on a shaker bath. To the solution, 0.36 g (1.4 mmol) of triphenyl
phosphine was added under N
2
(g) followed by the addition of the monoacid. A 5
72
mL DMF solution of DIAD (0.28 g, 1.4 mmol) was added via a glass syringe
while swirling the solution by hand. The reaction mixture was returned to the
shaker bath and heated to 55°C for 24 hours.
31
P NMR was used to monitor the
amount of monoacid in the reaction solution to determine completion. The
reaction mixture was filtered and rinsed with 50 mL DMF, 200 mL methanol, 100
mL 10% HCl, and 100 mL water until filtrate was neutral by pH paper. The resin
was then dried in a vacuum oven under reduced pressure (1 mm Hg) at room
temperature for 12 hours providing a beige colored resin.
31
P NMR: (DMF:d-methanol) δ
P
25.1 (broad s)
FT-IR: (as KBr pellet)
MicB Troika CMP resin (11)
Prepared for as Troika resin 8 using 0.15 g micro resin 9 providing 50% oxime
insertion by
31
P NMR. Insertion of oxime was confirmed qualitatively by color
change (light yellow after acidic wash, orange after basic wash).
31
P NMR: (DMF:d-methanol) δ
P
0.4 (broad s), 8.4 (broad s), 22.3 (broad s)
FT-IR: (as KBr pellet)
MacB Troika CMP precursor resin (MacB-9)
Prepared for as MicB resin 9 using 0.250 g (~10 mmol hydroxyl/g resin) MacB
73
resin providing MacB-9 as a yellow resin.
FT-IR: (as KBr pellet)
MacB Troika CMP resin (MacB-11)
Prepared for as MicB resin 11 using 0.15 g MacB-9 providing undetermined
amount of oxime insertion. Oximation confirmed qualitatively by color change
(light yellow after acidic wash, orange after basic wash).
Semi-qualitative chelation study for micro resins 9 and 11
Calibration curve for Cu (OAc)
2
in acetate buffer
Described above for micro resins 7 and 8.
Calibration curve for CoCl
2
in acetate buffer
A stock solution of 15.8 mM CoCl
2
was prepared in acetate buffer (0.6 M, pH
6.5). Serial dilutions of the stock solution were done using the same buffer
resulted in 0, 2, 4, 8, and 16.6 mM solutions. The absorbance of each solution
was measured (using quartz cuvettes) at λ
max
514 nm (ε
max
5.26) and plotted versus
the known concentrations to produce the calibration curve (y = 0.013x – 0.0103;
r
2
= 1). Measurements were taken at room temperature.
74
Calibration curve for NiCl
2
in acetate buffer
A stock solution of 17.8 mM NiCl
2
was prepared in acetate buffer (0.6 M, pH
6.5). Serial dilutions of the stock solution were done using the same buffer
resulted in 0, 2, 4, 8, 16.6 mM solutions. The absorbance of each solution was
measured (using quartz cuvettes) at λ
max
398 nm (ε
max
5.75) and plotted versus the
known concentrations to produce the calibration curve (y = 0.0106x – 0.0115;
r
2
=0.99). Measurements were taken at room temperature.
Copper chelation study using resins 9 and 11 at initial pH = 6.5
Prepared as for micro resins 7 and 8.
Cobalt chelation study using resins 9 and 11 at initial pH = 6.5
A 0.05 g sample of micro resin 9 and 11, in addition to the commercial standard
Chelex-100, were prepared by first rinsing each sample with 100 mL of ethanolic
potassium t-butoxide, 200 mL of water, and then allowing each to air dry on the
filter for 10 min. Each sample was transferred to a test tube containing 10 mL of
the stock CoCl
2
solution (15.8 mM) and swirled using a shaker bath. The initial
absorbance value (0.206 at λ
max
514 nm) was taken of the solution with no resin
added, while the final absorbance values were taken 24 hours after the addition of
resin at room temperature. These values were referenced against the calibration
curve providing the change in Co (II) concentrations.
75
Nickel chelation study using resins 9 and 11 at initial pH = 6.5
A 0.05 g sample of micro resin 9 and 11, in addition to the commercial standard
Chelex-100, were prepared by first rinsing each sample with 100 mL of ethanolic
potassium t-butoxide, 200 mL of water, and then allowing each to air dry on the
filter for 10 min. Each sample was transferred to a test tube containing 10 mL of
the stock NiCl
2
solution (17.8 mM) and swirled using a shaker bath. The initial
absorbance value (0.188 at λ
max
398 nm) was taken of the solution with no resin
added, while the final absorbance values were taken 24 hours after the addition of
resin at room temperature. These values were referenced against the calibration
curve providing the change in Ni (II) concentrations.
Synthesis of resin bound 3,3’-iminodipropan-1-ol (MicB) containing phosphonate
linked Troika diethyl phosphonoacetonitrile (PAN)
Diethyl phosphonoacetonitrile (PAN)
Prepared as for diethyl CMP using 7.55 g (0.10 mol) chloroacetonitrile and 16.6 g
(0.10 mol) triethyl phosphite providing white crystals (12.39 g, 70% yield).
31
P NMR: (D
2
O) δ
P
18.9 (s)
1
H NMR: (D
2
O) δ
H
1.41 (t,
3
J
HH
= 7 Hz, 6H), 2.89 (d,
3
J
PH
= 22 Hz, 2H), 4.27
(quint,
3
J
HH
= 16 Hz, 4H)
76
Lithium monosalt of PAN
Diethyl PAN (8.86 g, 0.050 mol) and 4.34 g LiBr (0.050 mol) were added to a 50
mL pear-shape flask and dissolved with 5 mL of methylethyl ketone while
magnetically stirred at 90°C. White precipitate formed within 2 hours and then
monitored by
31
P NMR until completed. The reaction mixture was filtered and
the white crystals were rinsed with 200 mL of diethyl ether and dried under
reduced pressure (1 mm Hg) at room temperature (6.97 g, 90% yield).
31
P NMR: (D
2
O) δ
P
11.1 (s)
FT-IR: (as KBr pellet)
MicB Troika PAN precursor resin (10)
Prepared as for micro resin 9 using 0.76 g (5.10 mmol) PAN monoacid and 0.56 g
(2 mmol hydroxyl) MicB resin providing a beige colored resin.
31
P NMR: (d-DMF) δ
P
9.7 (broad s), 14.0 (broad s), 18.5 (broad s)
FT-IR: (as KBr pellet)
MicB Troika PAN resin (12)
Micro resin 10 (0.318 g) was weighted out in a 50 mL round bottom flask
equipped with a magnetic stir bar. Under N
2
(g), the resin was swelled with 20
mL dioxane added via a glass syringe. In a separate 25 mL pear-shape flask, 0.15
g (1.31 mmol) of potassium t-butoxide was dissolved with the minimum amount
77
of ethanol (0.4 mL). To the magnetically stirred solution of resin, 0.12 g (1.31
mmol) propylnitrite was added followed by the drop-wise addition of the t-
butoxide solution, which turns the solution a dark orange color. The reaction is
allowed to proceed for 4 hours at room temperature. The reaction mixture was
filtered and the resin, a bright yellow, was rinsed with 200 mL of ethanol and 100
mL of 10% HCl. The resin was rinsed with water until the filtrate was neutral by
pH paper providing 12 as a light yellow color with ~14% oxime insertion by
31
P
NMR.
31
P NMR: (CDCl
3
) δ
P
–1.1 (broad s), 9.1 (s)
FT-IR: (as KBr pellet)
Semi-qualitative chelation study for micro resins 10 and 12
Calibration curve for Cu (OAc)
2
in acetate buffer
Described above for micro resins 7 and 8.
Calibration curve for NiCl
2
in acetate buffer
Described above for micro resins 9 and 11.
Copper chelation study using resins 10 and 12 at initial pH=6.5
Prepared for as micro resins 7 and 8.
78
Cobalt chelation study using resins 9 and 11 at initial pH=6.5
Prepared for as micro resins 7 and 8.
Synthesis of amide linked Troika acid to PEG-1000 micro resin
Amino-terminated PEG-1000 micro resin
The peglation of the Merrifield resin was accomplished according to Lezzi et al.
24
and converted to the amino-terminated micro resin examined by according to the
procedure described by Carrick
4
followed by FT-IR.
Amide-linked Troika precursor bound to PEG-1000 micro resin (13)
Prepared for as macro resin 4 using 0.27 g (0.27 mmol) of the amino-terminated
PEG-1000 micro resin and 0.32 g (1.63 mmol) PAA providing, after drying,
micro resin 13 as a beige resin in large clumps.
31
P NMR: (DMF:d-methanol) δ
P
20.8, 23.5 (PAA: δ
P
21.9)
FT-IR: (as KBr pellet)
Amide-linked Troika acid bound to PEG-1000 micro resin (14)
Prepared for as macro resin 5 using 0.30 g (0.30 mmol) micro resin 13 and
allowed to react for 4 hours. Oximation was confirmed by
31
P NMR and
qualitatively by color change (yellow after acidic wash, orange after basic wash).
31
P NMR: (DMF:d-methanol) δ
P
0.1 (s), 6.9 (broad s), 10.8 (broad s)
79
Chapter 2 References
1. Leonard, S. S.; Bower, J. J.; Shi, X., Metal-induced toxicity,
carcinogenesis, mechanisms and cellular responses. Molecular and Cellular
Biochemistry 2004, 255, (1&2), 3-10.
2. Murzaeva, S. V., Effect of Heavy Metals on Wheat Seedlings: Activation
of Antioxidant Enzymes. Applied Biochemistry and Microbiology (Translation of
Prikladnaya Biokhimiya i Mikrobiologiya) 2004, 40, (1), 98-103.
3. Dabrowski, A.; Hubicki, Z.; Podkoscielny, P.; Robens, E., Selective
removal of the heavy metal ions from waters and industrial wastewaters by ion-
exchange method. Chemosphere 2004, 56, (2), 91-106.
4. Carrick, J. M. Novel troika acid derivatives: photochemistry and metal
chelation. 2000.
5. Kashemirov, B. A.; Fujimoto, M.; McKenna, C. E., (E)-
(Hydroxyimino)(hydroxymethoxyphosphinyl)acetic acid: synthesis and pH-
dependent fragmentation. Tetrahedron Letters 1995, 36, (52), 9437-40.
6. Kashemirov, B. A.; Ju, J.-Y.; Bau, R.; McKenna, C. E., \"Troika Acids\":
Synthesis, Structure, and Fragmentation Pathways of Novel a-
(Hydroxyimino)phosphonoacetic Acids. Journal of the American Chemical
Society 1995, 117, (27), 7285-6.
7. McKenna, C. E.; Kashemirov, B. A.; Ju, J. Y., E/Z stereoisomer
assignment by 13C NMR in trifunctional phosphonate a-oximes and a-
arylhydrazones. Journal of the Chemical Society, Chemical Communications
1994, (10), 1211-12.
8. Garner, A. Y.; Chapin, E. C.; Scanlon, P. M., Mechanism of the
Michaelis-Arbuzov reaction: olefin formation. Journal of Organic Chemistry
1959, 24, 532-6.
9. Harizi, A.; Hajjem, B.; Zantour, H.; Baccar, B., Reaction of organozinc
compounds with cyanoalkylphosphonates: method for general synthesis of b-, g-
and d-ketophosphonates. Phosphorus, Sulfur and Silicon and the Related
Elements 2000, 159, 37-46.
80
10. Ayres, N.; Haddleton, D. M.; Shooter, A. J.; Pears, D. A., Synthesis of
Hydrophilic Polar Supports Based on Poly(dimethylacrylamide) via Copper-
Mediated Radical Polymerization from a Cross-Linked Polystyrene Surface:
Potential Resins for Oligopeptide Solid-Phase Synthesis. Macromolecules 2002,
35, (10), 3849-3855.
11. Yan, B.; Fell, J. B.; Kumaravel, G., Progression of Organic Reactions on
Resin Supports Monitored by Single Bead FTIR Microspectroscopy. Journal of
Organic Chemistry 1996, 61, (21), 7467-7472.
12. Liao, J. C.; Beaird, J.; McCartney, M.; DuPriest, M. T., An improved
FTIR method for polymer resin bead analysis to support combinatorial solid-
phase synthesis. American Laboratory (Shelton, Connecticut) 2000, 32, (14), 16,
18-20.
13. Muller, C.; Even, P.; Viriot, M.-L.; Carre, M.-C., Protection and labelling
of thymidine by a fluorescent photolabile group. Helvetica Chimica Acta 2001,
84, (12), 3735-3741.
14. Vandevoorde, S.; Jonsson, K.-O.; Fowler Christopher, J.; Lambert Didier,
M., Modifications of the ethanolamine head in N-palmitoylethanolamine:
synthesis and evaluation of new agents interfering with the metabolism of
anandamide. Journal of medicinal chemistry 2003, 46, (8), 1440-8.
15. Granier, C.; Guilard, R., First unequivocal synthesis of 1- or 8-N-
monosubstituted 1,4,8,12-tetraazacyclopentadecane. Tetrahedron 1995, 51, (4),
1197-208.
16. Ho, T.-L.; Gorobets, E., Synthesis of tacamonine. Tetrahedron 2002, 58,
(24), 4969-4973.
17. Sonoda, A.; Takagi, N.; Ooi, K.; Hirotsu, T., Complex Formation between
Boric Acid and Triethanolamine in Aqueous Solutions. Bulletin of the Chemical
Society of Japan 1998, 71, (1), 161-166.
18. Bartels, G. a-Aminoalkylphosphonic and a-aminoalkylphosphinic acids.
84-3445300 3445300, 19841212., 1986.
19. Krawczyk, H., A convenient route for monodealkylation of diethyl
phosphonates. Synthetic Communications 1997, 27, (18), 3151-3161.
81
20. Katzhendler, J.; Schneider, H.; Ta-Shma, R.; Breuer, E., Fragmentation of
methyl hydrogen a-hydroxyiminobenzylphosphonates - kinetics, mechanism and
the question of metaphosphate formation. Perkin 2 2000, (9), 1961-1968.
21. Lezzi, A.; Cobianco, S., Chelating resins supporting dithiocarbamate and
methylthiourea groups in adsorption of heavy metal ions. Journal of Applied
Polymer Science 1994, 54, (7), 889-94.
22. Buchardt, J.; Meldal, M., A chemically inert hydrophilic resin for solid
phase organic synthesis. Tetrahedron Letters 1998, 39, (47), 8695-8698.
23. Cobianco, S.; Lezzi, A.; Scotti, R., A spectroscopic study of Cu(II)-
complexes of chelating resins containing nitrogen and sulfur atoms in the
chelating groups. Reactive & Functional Polymers 2000, 43, (1,2), 7-16.
24. Lezzi, A.; Cobianco, S.; Roggero, A., Synthesis of thiol chelating resins
and their adsorption properties toward heavy metal ions. Journal of Polymer
Science, Part A: Polymer Chemistry 1994, 32, (10), 1877-83.
82
Chapter 3
OXIDATIVE PATHWAYS OF α-DIAZO
BISPHOSPHONATES: A CONTINUATION
Introduction
Nucleoside analogs are well-established drug targets in fields ranging from anti-
cancer therapeutics to anti-retroviral agents
1
. Modifications to the nucleic acid or
ribose ring of the nucleoside, has demonstrated inhibitory effects on enzymatic
functions related to DNA/RNA processes. Enzymatic inhibition has also been
observed by various phosphonate analogs
2
. Over the years, several research
groups have studied the inhibitory effects of combining these analogs generating
novel nucleotide triphosphates
3
. Variation in the triphosphate structure stem from
substituting the bridging oxygen at the α, β, or γ positions either with a sulfur
4
,
nitrogen
5-8
, or carbon
9-12
providing a point for additional functional groups to be
incorporated into the triphosphate tail, modulating inhibition.
Previous studies in the McKenna group uncovered the inhibitory effects of α-
ketobisphosphonate (α-COBP) and α-ketophosphonoacetic acid (α-COPAA)
derivatives
2
. These pyrophosphate analogues containing a ketone group at the α-
position have been generated from the α-diazobisphosphonate ester (α-diazoBP)
83
utilizing the hypochlorite-promoted oxidation of diazo intermediates as described
by Regitz
13
(Scheme 3.1). Adjustments to the synthesis provided the McKenna
“moisture modification” procedure, substituting a catalytic amount of water for
formic acid, generating the α-CO moiety (Scheme 3.2)
14
. Water promotes
decomposition to the phosphite, but can be quickly trapped with excess
trimethylsilylchloride (TMSCl) limiting phosphite formation. This procedure
became the first one-pot synthesis for the quantitative generation of α-COBP
esters.
O
RO
P
OR
C
P
RO OR
O
O
N
2
RO
P
OR
C
P
RO OR
O
O
N
2
RO
P
OR
C
P
RO OR
O
O
O Cl Cl CH
O
HCOOH
Cl
OH
OOCH
N
2
Scheme 3.1: McKenna
2
Formic Acid Promoted Oxidation of α-
Diazobisphosphonates
RO
P
OR
C
P
RO OR
O
O
N
2
RO
P
OR
C
P
RO OR
O
O
O Cl Cl
OH/H
2
O
N
2
H
HCl
RO
P
OR
C
P
RO OR
O
O
O
Excess H
2
O
RO
P
OR
C
P
RO OR
O
O
O O H H
CO
2
O
P
OR
OR
2 H
Scheme 3.2: McKenna “Moisture Modification” Method
84
Coupling the α-COBP to the nucleoside was elaborated by Kashemirov et al.
using 3'-azido-2'-deoxythymidine (AZT)
9
. The successful synthesis of the
conjugate facilitated continued studies of 1) other nucleoside/α-COBP conjugates,
and 2) a method of phosphorylation converting these diphosphate analogs to the
desired nucleotide triphosphate analogs. Preliminary efforts in the synthesis of
novel conjugates revealed two novel pathways to diazo oxidation, providing
alternatives to the “moisture modification” procedure. Herein, the conception
and application to nucleoside analog synthesis of both methods will be discussed.
Furthermore, enzymatic phosphorylation trials using the generated analogs will be
reviewed.
Results and Discussion
As noted in the above scheme, the α-COBP ester (alkyl or silyl) is generated in
situ via the loss of HCl from the α-chloro, α-hydroxy BP intermediate.
Formation of the intermediate occurs via nucleophilic addition to the reactive α-
carbon promoting the loss of N
2
(g)
10
. When a nucleophile, such as water, is not
added to drive the reaction to the desired product, several reaction pathways can
occur. One such pathway, which can predominate under modified reaction
conditions, is the addition of in situ-generated t-butanol providing a stable α-
chloro, α-(t-butoxy) BP intermediate (Scheme 3.3).
85
O
RO
P
OR
C
P
RO OR
O
O
N
2
RO
P
OR
C
P
RO OR
O
O
N
2
Cl
Cl
OH
N
2
Nu
H
Nu = nucleophile, including in situ-generated t-butyl alcohol
Nu
RO
P
OR
C
P
RO OR
O
O
Nu Cl
Scheme 3.3: Nucleophilic Activation of Oxidation Mechanism
This serendipitous formation of the α-chloro, α-alkoxy BP intermediate prompted
a study using o-nitrobenzyl alcohol as the nucleophile employed in forming the
intermediate. It was believed that the UV-promoted loss of the o-nitrobenzyl
group in aqueous solution would generate the α-hydroxy, providing the α-COBP
ester via the loss of HCl. Preliminary reaction conditions provided the α-chloro,
α-(o-methoxy-nitrobenzene) intermediate, which was isolated on silica gel using
a 1:1 heptane:ethyl acetate mobile phase, followed by NMR characterization.
Following the procedure described by Carrick
15
, the intermediate was dealkylated
using bromotrimethylsilane (BTMS) in preparation for photolytic cleavage. Upon
aqueous work-up, the α-COBP acid was discovered without exposure to UV
radiation. Separation by TLC of the organic phase extracts from the reaction
mixture revealed the presence of o-nitrobenzyl alcohol. This led us to suspect that
acid catalyzed hydrolytic cleavage of the o-nitrobenzyl group during aqueous
BTMS work-up had occurred, yielding the desired product (Scheme 3.4).
86
O
P
O
O
C
P
O
O
O
O
Si
Si Si
Si
Cl
O
2
N
1) H
2
O
NO
2
OH
+ Si HO
HO
P
O
O
C
P
O
O
OH
O
2) Aqueous Na
2
CO
3
, pH= 7.5
Na
Na
Scheme 3.4: Hydrolytic Cleavage of o-Nitrobenzyl Alcohol
α-COBP acid formation absent of UV-radiation prompted the study of non-UV
active protic reagents for the synthesis of α-chloro, α-alkoxy BP esters (Scheme
3.5). Methanol and t-butanol generated their respective intermediate products
according to
31
P NMR, but isolation proved to be challenging. Use of formic or
acetic acids provided no major advantage over the alcohols tested, alleviating the
influence of reagent pKa on intermediate formation.
MeO
P
MeO
O
C
P
O
OMe
OMe
N
2
MeO
P
MeO
O
C
P
O
OMe
OMe Cl
Nu
Nu
t-butyl hypochlorite,
DCM @ -10
o
C
Purified by
column chromatography
BTMS
DCM
HO
P
HO
O
C
P
O
OH
OH
O
Scheme 3.5: Alcohol Promoted Oxidation of α-Diazobisphosphonate
Generation of the α-chloro, α-alkoxy BP tetrasilyl ester was attempted using t-
butanol in place of water in the “moisture modification” procedure. According to
87
Kashemirov
9
RMe 3 N 2BP ester (R = nucleoside) was first dealkylated using
BTMS in DCM providing the trisilyl ester (R(Me 3Si) 3N 2BP). Co-evaporation
using anhydrous ethyl acetate eliminates un-reacted BTMS and DCM affording
the R(Me 3Si) 3N 2BP. Co-administration of t-BuOCl and dry t-butanol in anhydrous
ethyl acetate versus the “moisture modification”, limited premature deprotection
of the silyl ester, providing α-COBP acid and an uncharacterized decomposition
product after aqueous work-up (Scheme 3.6). Decreasing the reaction
temperature to -10°C, limited the decomposition product formation, generating a
method comparable to the original “moisture modification”.
MeO
P
MeO
O
C
P
O
OMe
OMe
N
2
1) BTMS, DCM
2) t-butyl hypochlorite/
t-butylalcohol in ethyl acetate
@ -10
o
C
HO
P
HO
O
C
P
O
OH
OH
O
Scheme 3.6: Replacement of Water in the “Moisture Modification” with a
Hindered Alcohol
The synthesis of novel nucleoside/α-COBP conjugates provided a practical
application for the newly elaborated diazo oxidation methods. As described by
Kashemirov et al.
9
, utilization of Mitsunobu coupling provided the desired
AZT/α-diazoBP trimethyl ester in good yield (Scheme 3.7). The coupling
88
procedure was applicable in the synthesis of 2’, 3’-O-isopropylideneuridine
(protected uridine) (1) and N, N-dibenzoyl-2',3'-O-(1-methylethylidene)
adenosine (protected adenosine) (4); however, under the same conditions,
according to
31
P NMR, both 1° and 2° alcohols of thymidine (2) coupled the α-
diazoBP ester. To combat this problem without protecting thymidine, Mitsunobu
conditions (dry DMF at 55°C) were found to promote selectivity towards the 1°
alcohol.
O
R' R
H H
H H
HO
Base
MeO
P
O
O
C
P
O
OMe
OMe
N
2
Na
Dowex-500 IE resin
MeOH
MeO
P
HO
O
C
P
O
OMe
OMe
N
2
Triphenylphosphine, DIAD,
Dioxane, 0-55
o
C
Nucleoside-
diazobisphosphonate
conjugate
Scheme 3.7: Kashemirov et al.
9
Procedure
Conversion of the silica column-purified α-diazo conjugates (1 to 4, Table 3.1) to
the α-chloro intermediates (5 to 8, Table 3.2) were monitored by
31
P NMR for an
increase in splitting due to the incorporation of a chiral center. Each sample was
found to react with either the o-nitrobenzyl alcohol or methanol nucleophiles
generating the desired intermediate. Reaction mixtures could be directly
dealkylated and oxidized to the α-COBP using BTMS; however, analysis of the
crude product provided convoluted
31
P NMR spectra with reduced ketone
formation. The solubility of the starting material, as in the case of 2, dictated
which nucleophile was used to generate the α-chloro intermediates.
89
Nucleoside-DiazoBP
Conjugates
(Trimethyl esters)
31
P-NMR Spectral Data
1
NH
O
O N
O
O O
H H
H H
O
C
P
OR
O
C
P
O
RO
RO
N
2
2
NH
O
O N
O
H OH
H H
H H
O
P
OR
O
C
P
O
RO
RO
O
3
NH
O
O N
O
H N
3
H H
H H
O
P
OR
O
C
P
O
RO
RO
N
2
4
N
N
N
N
N
O
O O
H H
H H
O
O
O
P
OR
O
C
P
O
RO
RO
N2
C
Table 3.1: Nucleoside/α-diazoBP Conjugates and
1
H decoupled
31
P NMR Spectra
90
Nucleoside-α-chloro, α-
alkoxyBP Conjugates
(Trimethyl esters)
31
P-NMR Spectral Data
5
NH
O
O N
O
O O
H H
H H
O
C
P
OR
O
C
P
O
RO
RO
O
Cl
O
2
N
6
NH
O
O N
O
H OH
H H
H H
O
P
OH
O
C
P
O
HO
HO
O
Cl
7
NH
O
O N
O
H N
3
H H
H H
O
P
OR
O
C
P
O
RO
RO
O
Cl
8
N
N
N
N
N
O
O O
H H
H H
O
O
O
P
OR
O
C
P
O
RO
RO
C
Cl
O
NO
2
Table 3.2: Nucleoside/α-chloro, α-alkoxyBP Conjugates and
1
H decoupled
31
P
NMR Spectra
91
The dealkylation and subsequent formation of α-COBP conjugates was first
tested using sample 5. After aqueous BTMS work-up, the crude product was
found to be 90% pure according to
31
P NMR integration. A comparison study of
sample 9 and the same compound generated via the “moisture modification” was
completed using analytical HPLC. Both samples were found to have the same
retention times (12.8 min; flow rate 1.5 mL/min; absorbance 280 nm, Figure 3.1)
using a 0.1 N phosphate buffer (15% acetonitrile) and overlapped retention (13.1
min, Figure 3.2) when mixed together. This data, in addition to
31
P NMR,
confirmed the formation of 9 via the purified α-chloro method.
Figure 3.1: HPLC Chromatogram of the “Moisture Modified” and 2-Step
Method Generated Protected Uridine/α-COBP Conjugate

Chrom. 1 0.0
mins.
42.9
mins.
1
2-Step Anhydrous
Method
Absorbance
@260 nm
NH
O
O N
O
O O
H H
H H
O
C
P
OH
O
C
P
O
HO
HO
O
“Moisture
Modification”
Time (min) 12.8 12.8
92
Figure 3.2: HPLC Chromatogram of a Mixture of “Moisture Modified” and 2-
Step Method Generated Protected Uridine/α-COBP Conjugate
Preliminary studies replacing water in the “moisture modification” procedure with
a hindered alcohol (t-butyl alcohol) to generate α-COBP conjugates was explored
with sample 3. The alcohol was meticulously dried and distilled over solid
sodium and used the same day to avoid the presence of water in the reaction
mixture. Co-administration of t-butyl hypochlorite in a solvent amount of t-butyl
alcohol (0.4 mL) was found to generate 80% pure 11 (according to
31
P NMR). As
described for sample 9, analytical HPLC comparison studies using “moisture
modified” generated sample 11 validated the formation of the product. The
tautomerization of t-butyl alcohol to 2-methyl, 2-propene and water may possibly
explain the generation of α-COBP and has not been ruled out. Further study of
this methodology is necessary to determine the correct mechanism.
Absorbance
@260 nm
NH
O
O N
O
O O
H H
H H
O
C
P
OH
O
C
P
O
HO
HO
O
Time (min) 12.8
Chrom. 1 0.0 mins. 23.7 mins.
1
1
3
.
1
93
Nucleoside-α-COBP
Conjugates
(Triethylammonium salts)
31
P-NMR Spectral Data
9
NH
O
O N
O
O O
H H
H H
O
C
P
OH
O
C
P
O
HO
HO
O
10
NH
O
O N
O
H OH
H H
H H
O
P
OH
O
C
P
O
HO
HO
O
11
NH
O
O N
O
H N
3
H H
H H
O
P
OH
O
C
P
O
HO
HO
O
12 N
N
N
N
N
O
O O
H H
H H
O
O
O
P
OH
O
C
P
O
HO
HO
O
C
Table 3.3: Nucleoside/α-COBP Conjugates (as triethylammonium salts) and
31
P
NMR Spectra
94
With the completion of samples 9 to 12 (Table 3.3), the search for a means of
phosphorylation of these samples to the desired nucleoside triphosphates
commenced. After a careful review of the literature, enzymatic phosphorylation
was deemed the most efficient manner for the conversion to the triphosphate.
Following the procedure demonstrated by the Kenyon group
5, 6
for
phosphorylation of imido BP (P-N-P) nucleoside conjugates, an attempt was
made to phosphorylate sample 11 using creatine phosphokinase and creatine
phosphate in HEPES buffer at pH 7.4. The reaction mixture was allowed to
incubate for 4 and 24 hours at 30°C. Samples were micro-filtered and monitored
by analytical HPLC using a SAX (strong anion exchange) column. The
generation of triphosphate was never observed. A second attempt using a dual
enzyme phosphorylation technique
16, 17
currently employed by the McKenna
group was also attempted with similar results.
Understanding the reactivity of the α-COBP may provide an explanation for the
above results. The tetra ester of this ketone is known to be much more susceptible
to nucleophilic addition then the tetra anion form. Reactivity at the α-carbon can
be limited by the delocalization of the negatively charged oxygen present at pH =
7 or by steric hinderance, as discussed by Bonaz-Krause
18
. In the case of the
AZT/α-COBP conjugate, at pH = 7, the negative charge at the terminal
phosphonate is delocalized, impeding nucleophilic addition to the α-carbon.
95
Upon phosphorylation to the triphosphate analog, the adjacent delocalization
provided by the phosphonate is lost, leaving the α-carbon vulnerable to attack and
subsequent decomposition. As this is only a speculation, further investigation is
required to understand the factors functioning in the reactivity of the α-carbon.
O
P
O
O
C P
O
O
OH
O
O
P
O
O
C P
O
OR
O
O
RO
P
RO
O
C P
O
OR
OR
O
< < < <
Scheme 3.8: Ketone Reactivity Trend at pH 7
Conclusion
The synthesis of nucleoside/α-diazoBP trimethyl ester conjugates 1,3 and 4 were
elaborated using the established Mitsunobu procedure described by Kashemirov
et al. Modifications to the conditions of the Mitsunobu reaction provided
selective 1° alcohol coupling of the α-diazoBP monoacid to unprotected
thymidine. Exploiting a reaction pathway hindered by the conditions of the
“moisture modification”, a method was generated using various alcohols
providing the α-chloro, α-alkoxy intermediates 5 thru 8. Dealkylation of these
esters was accomplished using BTMS, and upon aqueous work-up provided the
nucleoside/α-COBP conjugates 9 thru 12. A second method, which requires
further study, believed to limit premature desilylation of the protected α-diazoBP
ester conjugates was also discovered, wherein t-butyl alcohol is substituted for
96
water in the “moisture modification” procedure. Finally, a thorough investigation
of enzymatic or chemical phosphorylation of these compounds is necessary to
determine if nucleoside/α-COBP conjugates can form stable nucleotide
triphosphates.
Experimental
Materials and Methods:
All reactions were carried out under a nitrogen atmosphere, unless otherwise
indicated. Toluene, tetrahydrofuran (THF), dioxane, methanol, t-butyl alcohol
(all reagent grade purchased from Mallinckrodt Chemicals) were dried and
distilled over sodium/benzophenone. Ethyl acetate (HPLC grade purchased from
Mallinckrodt Chemicals), acetonitrile (HPLC grade purchased from Mallinckrodt
Chemicals), chloroform, and dichloromethane (DCM) (both reagent grade
purchased from Mallinckrodt Chemicals) were dried and distilled over P
2
O
5
.
Anhydrous diethyl ether (Et
2
O) was purchased from EMD Chemicals, Inc.
Acetone (HPLC grade) was purchased from Mallinckrodt Chemicals and heptanes
(reagent grade) from EM Science. o-Nitrobenzyl alcohol (97%), 2’, 3’-O-
isopropylideneuridine (99%), 2’, 3’-O-isopropylideneadenosine (98%), thymidine
(≥ 99%), 3’-azido-3’-deoxythymidine (98%), chlorotrimethylsilane (≥ 99%),
bromotrimethylsilane (97%), and Dowex 50WX8-200 ion exchange resin were
97
purchased from Sigma Aldrich. MgCl
2
•6H
2
O, KCl, Na
2
CO
3
were all reagent-
grade purchased from Mallinckrodt Chemicals. HEPES (≥99.5%), creatine
phosphokinase from rabbit muscle, sodium creatine phosphate di-basic
tetrahydrate (≥98%), pyruvate kinase from rabbit muscle, phospho(enol) pyruvic
acid mono potassium salt (≥97%, enzymatic), nucleoside 5’-diphosphate kinase
from baker’s yeast, and adenosine 5’-triphosphate disodium salt were purchased
from Sigma (Fluka). t-Butyl hypochlorite and α-diazo BP esters in toluene,
which was subsequently converted by the “moisture modification” to the α-COBP
salts, were synthesized according to Bonaz-Krause
18
. The synthesis of protected
uridine)/α-diazoBP trimethyl ester (1) was elaborated by Roze (dissertation in
preparation). AZT/α-diazoBP trimethyl ester (3) was synthesized according to
Kashemirov et al
9
. N, N-Dibenzoyl-2',3'-O-(1-methylethylidene) adenosine was
synthesized according to Vrudhula et al
19
. Enzymatic reactions were
accomplished according to the procedure described by Kenyon
5, 6
and Upton
(paper in preparation). Thin layer chromatography plastic-back sheets (20 X 20;
silica gel 60 F
254
) were purchased from EMD Chemicals, Inc. Preparative thin
layer chromatography glass-back sheets (20 X 20; 1000 microns) were purchased
from Analtech. Silica gel 150 (60-200 mesh) used for column chromatography
(column width 1-2 in) of bisphosphonate esters was purchased from Mallinckrodt
Chemicals; the esters were eluted using a gradient from 100% heptane to 1:1 ethyl
acetate:heptane.
98
HPLC studies and purification was accomplished using the Waters 600E
Multisolvent Delivery System with Waters 486 Tunable Absorbance Detector,
equipped with either an analytical column (Varian analytical C
18
column; 250 x
4.6 mm; Microsorb-MV, 100-5 C
18
) or semi-preparative column (Varian
Dynamax column; Microsorb 100-5, C
18
; 250 X 21.4 mm). The mobile phase was
either a 0.1 M triethylamine/acetic acid (0.1 M TEA:AA) or 0.1 M
triethylamine/carbonate at pH 7.2 using a gradient of either acetonitrile (HPLC
grade) or methanol (HPLC grade) at various flow rates, detected at λ = 260 nm.
Proton (
1
H), carbon (
13
C), and phosphorus (
31
P) NMR spectra were measured
either on a Bruker AM-360 MHz, Varian Mercury-400 MHz, or Bruker AMX-
500 MHz spectrometer. Chemical shifts are reported relative to external TMS
(
1
H), internal CDCl
3
[δ = 77.0] (
13
C) or external 85% H
3
PO
4
(
31
P). NMR samples
of BP and BP conjugate intermediate esters were dissolved in CDCl
3
, while
COBP conjugate triethylammonium salts were dissolved in D
2
O. Reaction
mixtures were monitored using
31
P NMR by adding a D
2
O capillary to the NMR
tube enabling the instrument to lock. High-resolution mass spectrometry was
performed at the University of California at Riverside High Resolution Mass
Spectrometry Facility using a +MALDI mass spectrometry instrument, operated
in the negative ion mode.
99
Protected Uridine Conjugates
[Chloro-(dimethoxy-phosphoryl)-(2-nitro-benzyloxy)-methyl]-phosphonic acid 6-
(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d]-
1,3-dioxol-4-ylmethyl ester methyl ester (5)
Conjugate 1 (0.05 g, 1.0 mmol) was added to a 25 mL round bottom flask
equipped with a magnetic stir bar and dried under reduced pressure (~1 mm Hg)
to a constant weight. A solution of o-nitrobenzyl alcohol (0.14 g, 10 mmol) in 3
mL of DCM was added to the conjugate via a glass syringe and allowed to chill to
-10°C while magnetically stirred under N
2
(g). In a 10 mL pear-shaped flask, 0.01
g (1 mmol) of t-butyl hypochlorite was weighed out and dissolved with 1 mL of
DCM. The hypochlorite solution was added via glass syringe to the chilled
conjugate/alcohol mixture, immediately evolving N
2
(g). The reaction was
monitored by
31
P NMR for completion, followed by solvent removal using a
rotary evaporator at room temperature under reduced pressure. Preparative TLC
using a 3:2 ethyl acetate:heptane mobile phase purified the crude product.
Solvent was removed under reduced pressure providing a colorless oil (78% yield
according to
31
P NMR integration).
31
P NMR: (CDCl
3
) δ
P
8.154 (m)
100
[6-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-2,2-dimethyl-tetrahydro-furo[3,4-
d]-1,3-dioxol-4-ylmethoxy]-hydroxy-phosphoranecarbonyl)-phosphoric acid (9)
Intermediate 5 (0.03 g, 0.04 mmol) was added to a 25 mL round bottom flask
equipped with a magnetic stir bar and was dried to a constant weight under
reduced pressure (~ 1 mm Hg) at room temperature. The intermediate was
dissolved with 5 mL of DCM and and chilled to 5°C in an ice bath. Under N
2
(g),
BTMS (30 µL, 0.2 mmol) was added via a glass syringe and allowed to stir for 3
hours or until the reaction was completed. After 10 min, 5 mL of water was
added and allowed to stir for 20 min followed by adjustment of the pH with
saturated Na
2
CO
3
solution to above 7.5. The two layers were separated, and the
aqueous layer was extracted twice with 5 mL portions of diethyl ether. The
aqueous layer was retained and water removed by rotary evaporation at 30°C
under reduced pressure (~1 mm Hg) providing 9 as crude sodium salt.
31
P NMR: (D
2
O) δ
P
-1.29 (dd,
2
J
PP
= 222 Hz)
Comparison studies with the “moisture modified” generated 9, were detected at λ
= 260 nm, flowrate 1.5 mL/min using a 0.1 N phosphate buffer (15% acetonitrile,
2 µL TBAP ion pairing agent) at pH 7.0. Both samples of 9 were dissolved in the
mobile phase for injection.
101
Unprotected Thymidine Conjugates
(Diazo-{[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
tetrahydro-furan-2-ylmethoxy]-methoxy-phosphoryl}-methyl)-phosphonic acid
dimethyl ester (2)
The mono-sodium salt of α-diazo BP (0.152 g, 0.5 mmol) was added to a 25 mL
round bottom flask equipped with a magnetic stir bar and dissolved, while
stirring, with 10 mL of methanol. Dowex 50WX8-200 ion exchange resin in the
acidic form (2.5 g) was added to the solution and allowed to stir for 30 min. The
resin was then removed by filtration and the filtrate collected in a 50 mL round
bottom flask which was then concentrated by rotary evaporation under reduced
pressure (~1 mm Hg) at 30°C to a constant weight providing the monoacid (0.132
g, 0.54 mmol).
The residue was dissolved with 5 mL of anhydrous DMF, followed by the
addition of triphenyl phosphine (0.226 g, 0.86 mmol), thymidine (0.214 g, 0.86
mmol), and a magnetic stir bar and placed under N
2
(g) while stirring. An
addition funnel was charged with a 5 mL of DMF, 0.170 g (0.86 mmol) DIAD
and added drop-wise over a 20 min period to the stirring solution. The reaction
mixture was capped and stirred at 50°C until the reaction was completed
according to
31
P NMR. The product was purified using a preparative TLC plate
102
with a 4:1 chloroform:methanol mobile phase (R
f
= 0.66) providing a viscous
light yellow oil upon removal of the solvent by rotary evaporation under reduced
pressure at room temperature.
31
P NMR: (d-methanol) δ
P
20.37 (m)
1
H NMR: (d-methanol) δ
H
1.17 (m, 3H), 1.53 (m, 2H), 2.13 (s, 1H), 2.27 (s, 1H),
2.62 (s, 1H), 3.15 (m, 6H), 3.36 (m, 1H), 3.64 (m, 3H), 5.57 (t,
3
J
HH
= 7, 1H), 6.83
(dd,
4
J
HH
= 7, 1H)
(Chloro-{[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
tetrahydro-furan-2-ylmethoxy]-methoxy-phosphoryl}-methoxymethyl)-phosphonic
acid dimethyl ester (6)
In a 25 mL round bottom flask equipped with a magnetic stir bar, 0.10 g (0.2
mmol) 2 was added and dissolved with 200 µL of dried and distilled methanol.
While stirring under N
2
(g), 2 mL of DCM was added via a glass syringe followed
by 0.04 g (0.3 mmol) of t-butyl hypochlorite. The evolution of gas signaled the
formation of the intermediate, which was monitored by
31
P NMR. The solvent
and unreacted methanol was removed by rotary evaporation under reduced
pressure at room temperature. The residue was purified on a preparative TLC
plate using a 4:1 chloroform:methanol mobile phase (R
f
= 0.64), providing 6 as a
light yellow viscous oil.
31
P NMR: (d-methanol) δ
P
11.1 (m)
103
{Hydroxy-[3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
tetrahydro-furan-2-ylmethoxy]-hydroxy-phosphoranecarbonyl}-phosphoric acid
(10)
In a 25 mL round bottom flask equipped with a magnetic stir bar, 0.03 g (0.05
mmol) of 6 was dried under reduced pressure (~1 mm Hg) until a constant weight
was reached. Under N
2
(g), 2 mL of DCM was added by a glass syringe to 6 and
allowed to stir. BTMS (0.5 mL) was added to the solution using a pasteur pipette,
capped and followed by
31
P NMR until completed. Solvent and un-reacted BTMS
were removed by rotary evaporation under reduced pressure at 30°C. A saturated
Na
2
CO
3
solution (5 mL) was added to the silylate product (pH above 7.5) and was
allowed to stir for an additional 20 min. The two layers were separated and the
aqueous layer was extracted twice with 5 mL portions of diethyl ether. The
aqueous layer was retained and water removed by rotary evaporation at 30°C
under reduced pressure (~1 mm Hg), providing a crude sodium salt 10. Semi-
preparative HPLC using a 0.1 N triethylamine/carbonate buffer (containing 3%
methanol at pH 7.2) mobile phase eluted 10 at 20.4 min.
31
P NMR: (D
2
O) δ
P
-0.97 (d,
2
J
PP
= 32)
104
AZT Conjugates
({[3-Azido-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-
furan-2-ylmethoxy]-methoxy-phosphoryl}-chloro-methoxy-methyl)-phosphonic
acid dimethyl ester (7)
Prepared as for 6. Preparative TLC using a 4:1 DCM:methanol mobile phase
purified the crude product. Solvent was removed under reduced pressure
providing a slightly yellow colored oil (60% yield).
31
P NMR: (CDCl
3
) δ
P
10.8 (m)
[3-Azido-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-
furan-2-ylmethoxy]-hydroxy-phosphoranecarbonyl)-phosphonic acid (11)
Prepared as for 6. Semi-preparative HPLC using a 0.1 N triethylamine/acetic acid
buffer (gradient from 2% to 15% acetonitrile at pH 7.2) mobile phase eluted 11 at
19.3 min.
31
P NMR: (D
2
O) δ
P
–1.29 (dd,
2
J
PP
= 190)
Synthesis of 11 via t-butyl alcohol substitution for water in the “moisture
modification”
To a 25 mL round bottom flask equipped with a magnetic stir bar, 0.07 g (0.15
mmol) 11 was dried under reduced pressure (~1 mm Hg) until a constant weight
was attained. Under N
2
(g), while stirring, 3 mL of DCM followed by 0.5 mL of
105
BTMS was added to 11 and allowed to react for 3 hours. Ethyl acetate (5 mL)
was added to the reaction mixture for co-evaporation under N
2
(g). The total
volume was reduced by half, and the process was repeated twice. After the final
ethyl acetate wash, the solution remained yellowish with no precipitate. The
reaction mixture was then chilled in an ice bath to ~5°C. A chilled (~5°C)
anhydrous solution of t-butyl alcohol (0.4 mL) was co-administered with 0.04 g
(0.35 mmol) t-butyl hypochlorite via a glass syringe to the mixture. After 10 min,
5 mL of water was added and allowed to stir for 20 min followed by adjustment
of the pH with saturated Na
2
CO
3
solution to above 7.5. The reaction mixture was
extracted twice with 5 mL portions of diethyl ether. The aqueous layer was
retained and water removed by rotary evaporation at 30°C under reduced pressure
(~1 mm Hg), providing a crude sodium salt 11.
Protected Adenosine Conjugates
N, N-Dibenzoyl-2',3'-O-(1-methylethylidene) adenosine was synthesized
according to Vrudhula et al
19
.
[Diazo-(dimethoxy-phosphoryl)-methyl]-phosphonic acid 6-(6-dibenzoylamino-
purin-9-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d]-1,3-dioxol-4-ylmethyl ester
methyl ester (4)
Prepared according to the procedure described by Kashemirov et al
9
.
106
Preparative TLC using a 9:1 Et
2
O:methanol mobile phase purified the crude
product (Rf = 0.27). Solvent was removed under reduced pressure providing a
slightly yellow oil (60% yield).
31
P NMR: (CDCl
3
) δ
P
16.36 (m)
1
H NMR: (CDCl
3
) δ
H
1.25 (t,
3
J
HH
= 7, 2H), 1.38 (s, 1H), 1.45 (s, 2H), 1.66 (m,
3H), 4.12 (quart,
3
J
HH
= 7, 2H), 4.49 (dd,
3
J
HH
= 5, 2H), 4.71 (m, 2H), 5.19 (m,
3H), 5.58 (dd,
3
J
HH
= 7, 1H), 5.94 (d,
3
J
HH
= 6, 1H), 6.24 (d,
3
J
HH
= 3, 1H), 7.36
(quart,
3
J
HH
= 7, 3H), 7.52 (m, 7H), 7.84 (m, 3H)
HR-MS (+MALDI): calcd 764.1587, found 764.1589
{Chloro-(dimethoxy-phosphoryl)-[(2-nitrobenzyl)oxy]-methyl}-phosphonic acid
6-(6-dibenzoylamino-purin-9-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d]-1,3-dioxol-
4-ylmethyl ester methyl ester (8)
Prepared as for 3-5. Preparative TLC using a 9:1 Et
2
O:methanol mobile phase (R
f
= 0.46) purified the crude product. Solvent was removed using rotary evaporation
under reduced pressure providing a slightly yellow colored oil (80% yield).
31
P NMR: (CDCl
3
) δ
P
15.9 (m)
1
H NMR: (CDCl
3
) δ
H
1.20 (quart,
3
J
HH
= 14, 2H), 1.36 (m, 3H), 1.61 (m, 3H),
3.48 (m, 2H), 3.89 (m, 6H), 4.06 (m, 1H), 4.48 (m, 3H), 5.08 (ddd,
3
J
HH
= 9 Hz,
1H), 5.19 (dt,
3
J
HH
= 7 Hz, 1H), 5.33 (m, 1H), 5.40 (d,
3
J
HH
= 7 Hz, 1H), 6.22
107
(ddd, 9, 13 Hz, 1H), 7.33 (t,
3
J
HH
= 8 Hz, 3H), 7.45 (m, 3H), 7.60 (m 1H), 7.71
(m, 1H), 7.82 (m, 3H), 8.03 (m, 1H), 8.39 (m, 1H)
[6-(6-Dibenzoylamino-purin-9-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d]-1,3-
dioxol-4-ylmethoxy]-hydroxy-phosphoranecarbonyl)-phosphonic acid (12)
Prepared for as 9 providing a yellowish “flaky” salt.
31
P NMR: (D
2
O) δ
P
-0.81 (d,
2
J
PP
= 15.3)
108
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121
Appendix A
NMR SPECTRA FOR CHAPTER 1
2a: (CDCl
3
, 400 mHz) δ
P
19.4
47

122

2a: (CDCl
3
, 400 MHz) δ
H
1.3-1.4 (m, 24H), 1.6 (t,
3
J
HP
= 16 Hz, 3H), 4.6-4.8 (m,
4H)
47

123

2b: (CDCl
3
, 400 MHz) δ
P
15.4 (s)

124

3b: (D
2
O, 400 MHz) δ
P
15.5 (s)
125

3b: (D
2
O, 400 MHz) δ
H
7.1-7.7 (m, 5H)
126

3c: (D
2
O, 400 MHz) δ
P
17.5 (s)
127

3c: (D2O, 400 MHz) δ
H
3.1 (t, J
HP
= 14 Hz, 2H), 7.1-7.3 (m, 5H)
128

4a: (CDCl
3
, 360 MHz) δ
P
13.1 (s)
129

4a: (CDCl
3
, 360 HMz) δ
H
0.3 (s, 9H), 1.2-1.3 (m, 24H), 4.6 (m, 2H), 4.8 (m, 2H),
7.6 (broad m, 1H), 7.7 (broad s, 1H), 8.3 (broad d,
3
J
HH
= 5, 1H)
130

4a: (CDCl
3
, 400 MHz) δ
C
2.7 (s), 23.8 (m), 73.1 (d,
2
J
PC
= 30 Hz), 121.1 (s), 122.8
(s), 148.3 (s), 150.8 (s)
131

7a: (CDCl
3
, 500 MHz) δ
P
13.2 (s)

132

7a: (CDCl
3
, 500 HMz) δ
H
0.23 (s, 9H), 1.2 (m, 6H), 1.3 (t,
3
J
HH
= 7, 3H), 4.1 (m,
4H), 4.3 (m, 2H), 7.5 (broad m, 1H), 7.6 (broad s, 1H), 8.3 (broad d,
3
J
HH
= 5 Hz,
1H)

133

4b: (CDCl
3
, 400 MHz) δ
P
12.3 (s)
134

4b: (CDCl
3
, 500 MHz) δ
H
0.3 (s, 9H), 1.3 (m, 24H), 3.7 (t,
3
J
HP
= 14, 2H), 4.8 (m,
4H)
135

4b: (CDCl
3
, 500 MHz) δ
C
-0.1 (s), 5.8 (s), 21.5 (m), 69.1 (s), 70.3 (s)
136

6a: (D
2
O, 400 MHz) δ
P
12.9 (s)
137

6a: (D
2
O, 400 MHz) δ
H
7.5 (broad m, 1H), 7.6 (broad s, 1H), 8.0 (d,
3
J
HH
= 6, 1H)

138

6a: (D
2
O, 500 MHz) δ
C
120.2 (s), 121.5 (s), 147.5 (s), 149.5 (s), 153.8 (s)

139

8a: (D2O, 500 MHz) δ
P
13.1 (s)

140

8a: (D
2
O, 500 MHz) δ
H
4.1 (m, 2H), 7.6 (broad m, 1H), 7.7 (broad s, 1H), 8.1
(broad d,
3
J
HH
= 5 Hz, 1H)

141

9a: (CDCl
3
, 400 MHz) δ
P
18.6 (s)

142

9a: (CDCl
3
, 400 MHz) δ
H
1.4-1.5 (m, 24H), 2.7 (dt,
3
J
HH
=4,
3
J
PH
= 15, 2H), 4.7
(m, 4H), 5.0-5.1 (m, 2H), 6.1 (m, 1H)
143

9b: (CDCl
3
, 400 MHz) δ
P
18.1 (dd,
2
J
PP
= 40 Hz)

144

9c: (CDCl3, 400 MHz) δ
P1
16.2 (d,
2
J
PP
= 37 Hz); δ
P2
18.1 (d,
2
J
PP
= 34 Hz)
145

9d: (CDCl
3
, 400 MHz) δ
P
17.2 (s)
146

9f: (CDCl3, 400 MHz) δ
P
16.9 (s)
147

10a: (D2O, 500 MHz) δ
P
16.7 (s)
148

10a: (D
2
O, 500 MHz) δ
H
3.8 (broad s, 1H), 4.7-4.9 (m, 2H), 6.3 (m, 1H), 7.0 (t,
3
J
HH
= 7, 1H), 7.1 (t,
3
J
HH
= 7, 2H), 7.2(d,
3
J
HH
= 7, 2H)
149

10a: (D
2
O, 500 MHz) δ
C
52.2 (s), 114.8 (s), 125.5 (s), 127.0 (s), 128.8 (s), 137.8
(s), 140.5 (s)
150

10b: (D2O, 400 MHz) δ
P
16.4 (s)

151

10b: (D
2
O, 400 MHz) δ
H
δ 5.5 (s, 1H), 5.7 (s, 1H)
152

10c: (D
2
O, 400 MHz) δ
P
12.9 (s)

153

10c: (D2O, 400 MHz) δ
H
3.2 (t,
3
J
PH
= 15, 2H), 5.7 (s, 1H), 6.0 (s, 1H)
154

10c: (D
2
O, 360 MHz) δ
C
22.3 (s), 80.1 (t,
1
J
PC
= 108 Hz), 123.1 (s), 133.4 (s),
173.0 (s)
155

11a: (CDCl3, 500 MHz) δ
P
14.7 (s)

156

11b: (CDCl
3
, 500 MHz) δ
P
15.6 (s)

157

11b: (CDCl3, 500 MHz) δ
H
1.1 (broad m, 6H), 1.4 (broad t,
3
J
HH
= 7, 3H), 3.5 (m,
1H), 3.8 (m, 1H), 3.9 (m, 2H), 4.1 (d,
3
J
PH
= 10, 1H), 4.3 (m, 2H), 5.1 (m, 2H), 6.1
(dt,
3
J
HH
= 18,
3
J
PH
= 10, 1H), 7.2-7.5 (m, 5H)
158

11c: (CDCl
3
, 500 MHz) δ
P
14.5 (s)
159

11c: (CDCl
3
, 500 MHz) δ
H
1.3 (m, 12H), 3.0 (dd,
2
J
HH
= 14,
3
J
PH
= 6, 1H), 3.2 (dd,
2
J
HH
= 14,
3
J
PH
= 9, 1H), 3.9 (d,
3
J
PH
= 7, 1H), 4.2 (m, 6H), 4.3 (d,
3
J
PH
= 7, 1H), 5.7
(s, 1H), 6.2 (s, 1H)
160

12a: (D
2
O, 500 MHz) δ
P
12.2 (s)

161

12a: (D2O, 500 MHz) δ
H
1.2 (t,
3
J
HH
= 6, 3H), 4.0 (d,
3
J
PH
= 9, 1H), 4.1 (m, 2H),
5.1 (m, 2H), 6.1 (dt,
3
J
HH
= 18,
3
J
PH
= 9, 1H), 7.1 (t,
3
J
HH
= 7, 1H), 7.2 (t,
3
J
HH
= 7,
2H), 7.3 (d,
3
J
HH
= 7, 2H)
162

12b: (D
2
O, 500 MHz) δ
P
14.0 (s)
163

12b: (D
2
O, 500 MHz) δ
H
2.8 (dd,
3
J
PH
= 10, 1H), 4.0 (m, 4H), 5.6 (s, 1H), 6.0 (s,
1H)
164

12c: (D
2
O, 500 MHz) δ
P
10.1 (broad)

165

12c: (D2O, 500 MHz) δ
H
3.3 (t,
3
J
PH
= 14, 2H), 4.2 (m, 2H), 5.8 (s, 1H), 6.1 (s,
1H)
166

12c: (D2O, MHz) δ
C
13.0 (s), 34.6 (s), 63.2 (s), 124.3 (s), 133.8 (s), 171.3 (d),
173.2 (s)
167
Appendix B
NMR SPECTRA FOR CHAPTER 2
Ligand A: (CDCl
3
, 360 MHz) δ
P
23.3 (s)
168

Ligand A: (CDCl
3
, 360 MHz) δ
H
1.25 (t,
4
J
HH
= 7 Hz, 3H), 1.62 (quint,
4
J
HH
= 12
Hz, 2H), 2.81 (d,
2
J
PH
= 22 Hz, 2H), 3.31 (quart, J
HH
= 12 Hz, 2H), 3.56 (t,
3
J
HH
= 6
Hz, 2H), 4.06 (quint,
3
J
HH
= 15 Hz, 4H)
169

L-B-b: (CDCl
3
, 360 MHz) δ
P
19.9 (s)
170

Ligand B: (CDCl
3
, 360 MHz) δ
P
20.5 (s), 23.6 (s)
171

Ligand B: (CDCl3, 360 MHz) δ
H
1.26 (m, 9H), 1.45 (m, 4H), 1.61 (m, 2H), 2.85
(dd,
2
J
PH
=26 Hz, 2H), 2.90 (d,
2
J
PH
= 22 Hz, 2H), 3.19 (m, 2H), 4.08 (m, 8H), 7.08
(broad s, 1H)
172

7: (DMF: d-methanol, 360 MHz) δ
P
21.4 (broad s)
173

8 (after 2
nd
nitrosation): (DMF: d-methanol, 360 MHz) δ
P
0.7 (broad s), 7.19
(broad s)

174

CMP: (D
2
O, 360MHz) δ
P

24.3 (s)
175

CMP: (D2O, 360 MHz)
1
H NMR: (D
2
O) δ
H
1.21 (t,
4
J
HH
= 7 Hz, 6H), 2.95 (d,
3
J
PH
= 22 Hz, 2H), 4.07 (quint,
4
J
HH
= 15Hz, 4H)
176

Sodium monosalt CMP: (D
2
O, 360 MHz) δ
P

16.6 (s)
177

Sodium monosalt CMP: (D
2
O, 360 MHz) δ
H
1.02 (t,
4
J
HH
= 7 Hz, 3H), 1.10 (t,
4
J
HH
= 7 Hz, 3H), 2.58 (d,
3
J
PH
= 22 Hz, 2H), 3.49 (quart,
4
J
HH
= 7 Hz, 2H), 3.79 (t,
4
J
HH
= 7 Hz, 2H)
178

MicB 9: (DMF:d-methanol, 360 MHz) δ
P
25.1 (broad s)
179

Mic B 11: (DMF:d-methanol, 360 MHz) δ
P
0.4 (broad s), 8.4 (broad s), 22.3
(broad s)
180

PAN: (D
2
O, 400 MHz) δ
P
18.9 (s)
181

PAN:
1
H NMR: (D
2
O, 400 MHz) δ
H
1.41 (t,
3
J
HH
= 7 Hz, 6H), 2.89 (d,
3
J
PH
= 22
Hz, 2H), 4.27 (quint,
3
J
HH
= 16 Hz, 4H)
182

Lithium monosalt of PAN: (D
2
O, 400 MHz) δ
P
11.1 (s)
183

MicB 10: (d-DMF, 360 MHz) δ
P
9.7 (broad s), 14.0 (broad s), 18.5 (broad s)
184

MicB 12:
31
P NMR: (CDCl
3
) δ
P
–1.1 (broad s), 9.1 (s)
185

13:
31
P NMR: (DMF:d-methanol, 500 MHz) δ
P
20.8, 23.5 (PAA: δ
P
21.9)
186

14: (DMF:d-methanol, 500 MHz) δ
P
0.1 (s), 6.9 (broad s), 10.8 (broad s)
187
FT-IR Spectra for Chapter 2

FT-IR Spectrum for Resin 8
"Daisy-chain" Troika Ligand on Micro Resin
10
20
30
40
50
60
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700
Wavelength (nm)
Absorbance
\
188

FT-IR Spectrum for Resin Mic-B

MicB resin
75
95
115
135
155
175
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
189

FT-IR Spectrum for Resin Mac-B
MacB
50
70
90
110
130
150
170
190
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
190

FT-IR Spectrum for CMP
Carbmoylmethylphosphonate (CMP)
0
10
20
30
40
50
60
70
80
90
100
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
191

FT-IR Spectrum for Resin 9

MicB Troika CMP Precursor Resin (9)
5
15
25
35
45
55
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
192

FT-IR Spectrum for Resin 11
MicB Troika CMP Resin (11)
25
30
35
40
45
50
55
60
65
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
193

FT-IR Spectrum for Resin MacB-9

MacB Troika CMP Precursor Resin (MacB-9)
45
55
65
75
85
95
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
194

FT-IR Spectrum for Resin MacB-11
MaB Troika CMP Resin (MacB-11)
0
5
10
15
20
25
30
35
40
45
50
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
195

FT-IR Spectrum for Resin PAN

Lithium Monosalt of Phosphonoacetonitrile (PAN)
50
70
90
110
130
150
170
190
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
196

FT-IR Spectrum for Resin 12
MicB Troika PAN Resin (12)
10
20
30
40
50
60
70
80
90
500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
197

FT-IR Spectrum for Resin 13
PEG-PAA Micro Resin (13)
10
20
30
40
50
60
70
80
90
100
700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 3100 3300 3500 3700 3900
Wavelength (nm)
Absorbance
198

Chelation Studies for Chapter 2

Key for Chelation Data Chart on page 199
Key:
Solutions A
B
C
D
E
F
Micro-resins Chelex
2-7
2-8
2-9
2-11
2-10
2-12
Chelation Data for Studies Completed 2-24-05 to 2-26-05
Cu
2+
from Cu(OAc)
2
.
H
2
O in 0.6 M Acetate Buffer (pH 6.5)
Co
2+
from CoCl
2
.
6H
2
O in 0.6 M Acetate Buffer (pH 6.5)
Ni
2+
from NiCl
2
.
H
2
O in 0.6 M Acetate Buffer (pH 6.5)
Cu
2+
from Cu(OAc)
2
.
H
2
O in 1:1 Dioxane:Methanol
Co
2+
from CoCl
2
.
6H
2
O in 1:1 Dioxane:Methanol
Ni
2+
from NiCl
2
.
H
2
O in 1:1 Dioxane:Methanol
Commercial Chelex-100 resin
MicB Troika PAN Precursor Resin
MicB Troika PAN Resin
Troika Precursor "Daisy-Chain" Resin
Troika "Daisy-Chain" Resin
MicB Troika CMP Precusor Resin
MicB Troika CMP Resin
199

Aqueous and Organic Chelation Data for Tables 1 to 3
Micro-resins Solutions
Absorbance at 0
hr
Initial
[Metal]
(mM)
Absorbance
after 24 hr
Final
[Metal]
(mM)
Change in
[Metal]
(mM)
Chelex A 1.19 @ 748 nm 18.71 0.05 @ 748 nm 17.83 17.92
2-7 A 1.19 @ 748 nm 18.71 1.09 @ 748 nm 17.19 1.51
2-8 A 1.19 @ 748 nm 18.71 1.16 @ 748 nm 18.41 0.29
2-9 A 1.19 @ 748 nm 18.71 1.13@ 748 nm 17.83 0.87
2-11 A 1.19 @ 748 nm 18.71 1.13 @ 748 nm 17.9 0.8
2-10 A 1.19 @ 748 nm 18.71 1.14 @ 748 nm 18.08 0.62
2-12 A 1.19 @ 748 nm 18.71 1.14 @ 748 nm 17.99 0.7
Chelex B 0.21 @ 514 nm 15.86 0.05 @ 512 nm 3.95 11.91
2-9 B 0.21 @ 514 nm 15.86 0.20 @ 512 nm 15.62 0.23
2-11 B 0.21 @ 514 nm 15.86 0.21 @ 512 nm 15.85 0
Chelex C 0.18 @ 398 nm 17.83 0.02 @ 398 nm 2.02 15.81
2-9 C 0.18 @ 398 nm 17.83 0.17 @ 398 nm 16.62 1.21
2-11 C 0.18 @ 398 nm 17.83 0.18 @ 398 nm 17.12 0.7
2-10 C 0.18 @ 398 nm 17.83 0.18 @ 398 nm 17.04 0.8
2-12 C 0.18 @ 398 nm 17.83 0.19 @ 398 nm 17.77 0.05
2-9 D 3.96 @ 685 nm 16.94 3.66 @ 685 nm 15.61 1.33
2-11 D 3.96 @ 685 nm 16.94 2.87 @ 685 nm 12.91 4.75
2-10 D 3.96 @ 685 nm 16.94 4.00 @ 685 nm 16.94 0
2-12 D 3.96 @ 685 nm 16.94 3.10 @ 685 nm 13.16 3.77
2-8 E 1.86 @ 672 nm 14.7 1.79 @ 673 nm 14.21 0.48
2-9 E 1.86 @ 672 nm 14.7 0.98 @ 673 nm 7.88 6.82
2-11 E 1.86 @ 672 nm 14.7 0.86 @ 672 nm 6.88 7.82
2-10 E 1.86 @ 672 nm 14.22 0.86 @ 672 nm 6.91 7.31
2-12 E 1.86 @ 672 nm 14.22 1.08 @ 673 nm 8.64 5.58
2-7 F 0.34 @ 417 nm 16.76 0.26 @ 417 nm 12.73 4.01
2-8 F 0.34 @ 417 nm 16.76 0.25 @ 417 nm 12.66 4.09
2-9 F 0.34 @ 417 nm 16.76 0.34 @ 417 nm 16.75 0
2-11 F 0.34 @ 417 nm 16.76 0.22 @ 417 nm 11.11 5.64
2-10 F 0.34 @ 417 nm 17.63 0.31 @ 417 nm 15.31 2.32
2-12 F 0.34 @ 417 nm 17.63 0.36 @ 417 nm 17.63 0
200
Appendix C
NMR SPECTRA FOR CHAPTER 3
5: (CDCl
3
, 360 MHz) δ
P
8.154 (m)
201

9: (D
2
O, 360 MHz) δ
P
-1.29 (dd,
2
J
PP
= 222 Hz)
202

2: (d-methanol, 360 MHz) δ
P
20.37 (m)

203

2: (d-methanol, 360 MHz) δ
H
1.17 (m, 3H), 1.53 (m, 2H), 2.13 (s, 1H), 2.27 (s,
1H), 2.62 (s, 1H), 3.15 (m, 6H), 3.36 (m, 1H), 3.64 (m, 3H), 5.57 (t,
3
J
HH
= 7, 1H),
6.83 (dd,
4
J
HH
= 7, 1H)
204

6: (CDCl
3
, 360 MHz) δ
P
11.1 (m)

205

10: (D
2
O, 360 MHz) δ
P
-0.97 (d,
2
J
PP
= 32)

206

7: (CDCl
3
, 400 MHz) δ
P
10.8 (m)

207

11: (D
2
O, 500 MHz) δ
P
-1.29 (dd,
2
J
PP
= 190)

208

4: (CDCl
3
, 400 MHz) δ
P
16.36 (m)
209

4: (CDCl
3
, 360 MHz) δ
H
1.25 (t,
3
J
HH
= 7, 2H), 1.38 (s, 1H), 1.45 (s, 2H), 1.66
(m, 3H), 4.12 (quart,
3
J
HH
= 7, 2H), 4.49 (dd,
3
J
HH
= 5, 2H), 4.71 (m, 2H), 5.19
(m, 3H), 5.58 (dd,
3
J
HH
= 7, 1H), 5.94 (d,
3
J
HH
= 6, 1H), 6.24 (d,
3
J
HH
= 3, 1H),
7.36 (quart,
3
J
HH
= 7, 3H), 7.52 (m, 7H), 7.84 (m, 3H)
210

8: (CDCl
3
, 360 MHz) δ
P
15.9 (m)

211

8: (CDCl
3
, 360 MHz) δ
H
1.20 (quart,
3
J
HH
= 14, 2H), 1.36 (m, 3H), 1.61 (m, 3H),
3.48 (m, 2H), 3.89 (m, 6H), 4.06 (m, 1H), 4.48 (m, 3H), 5.08 (ddd,
3
J
HH
= 9 Hz,
1H), 5.19 (dt,
3
J
HH
= 7 Hz, 1H), 5.33 (m, 1H), 5.40 (d,
3
J
HH
= 7 Hz, 1H), 6.22
(ddd, 9, 13 Hz, 1H), 7.33 (t,
3
J
HH
= 8 Hz, 3H), 7.45 (m, 3H), 7.60 (m 1H), 7.71
(m, 1H), 7.82 (m, 3H), 8.03 (m, 1H), 8.39 (m, 1H)

212

12: (D
2
O, 360 MHz) δ
P
-0.81 (d,
2
J
PP
= 15.3)

Abstract (if available)

Abstract Pyrophosphonate analogs such as methylenebisphosphonates and phosphonoacetates have applications in pharmaceutical chemistry and also in material sciences. Herein, a series of synthetic studies focusing on the preparation of potentially useful new organophosphorus compounds are presented. In Chapter 1, the addition of organometallic reagents to carbonylbisphosphonate and oxophosphonoacetate alkyl esters to generate a-hydroxy, a-substituted methylenebisphosphonate or phosphonoacetate alkyl esters is investigated. Chapter 2 discusses synthetic approaches to branched (E)-(hydroxyimino)(dihydroxyphosphinyl)acetic acid ligands and their possible application as metal chelating agents. Finally, Chapter 3 presents the hypochlorite oxidation of diazomethylenebisphosphonate alkyl esters with various alcohols for use in synthesis of nucleoside 5'-triphosphate carbonylbisphosphonate analogs.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Sanchez, Gregorio Valentin, Jr. (author)

Core Title Synthetic studies of phosphonate derivatives

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 10/19/2006

Defense Date 01/01/2006

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

Tag anti-viral agents,bisphosphonates,metal chelating agents,nucleoside analogs,OAI-PMH Harvest,organometallic chemistry,osteoporosis,phosphonates

Language English

Advisor McKenna, Charles E. (committee chair), Barrios, Amy (committee member), Tower, John G. (committee member)

Creator Email gregoris@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m99

Unique identifier UC1194015

Identifier etd-Sanchez-20061019 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-25923 (legacy record id),usctheses-m99 (legacy record id)

Legacy Identifier etd-Sanchez-20061019.pdf

Dmrecord 25923

Document Type Dissertation

Rights Sanchez, Gregorio Valentin, Jr.

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu