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Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications
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Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications
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Content
FLUORESCENT IMAGING PROBES OF NITROGEN-CONTAINING BONE
ACTIVE DRUGS: DESIGN, SYNTHESIS AND APPLICATIONS
by
Shuting Sun
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
December 2013
Copyright 2013 Shuting Sun
ii
DEDICATION
To my beloved grandma, who will be in my heart, always!
iii
ACKNOWLEDGMENTS
“Rome was not built in one day”; this is the first English phase I learnt when I just
started to learn English. It is such a good metaphor and so appropriate expression to use
here after I finished the five chaptered PhD dissertation. Besides, there is another
sentence I learnt and created from my experiences during the last few years of PhD study:
Rome was also not built by one person.
To build Rome, one could imagine that the first and foremost thing is to have an
excellent commander in chief, who could point out a general direction, make a
construction plan and provide significant suggestions when obstacles appear; and this
title has to go to my mentor Prof. Charles E. McKenna. Although a simple “thank you” is
not enough since the dissertation (“Rome”) would not be possible without him, I still
want to say sincerely: thank you, Prof. McKenna, for all your advice and support during
my PhD study. Prof. McKenna not only brought me the great opportunity to work in the
fluorescent imaging research field, but also provided me numerous chances in other areas,
such as teaching me to prepare grant proposals, introducing me to be the judge of the
USC Annual Undergraduate Symposium, encouraging me to be the mentor of REU
program of Chemistry Department, training me to prepare patent applications, as well as
supporting me to attend international and national conferences nationwide and abroad; all
these experiences made my stay at USC fruitful and colorful and also shaped me from a
novice to a young professional with confidence and courage.
I would like to acknowledge Dr. Boris Kashemirov for his kind help and useful
suggestions provided to my research as well as his sharing of perspectives on life. It is he
who helped to create a great laboratory enviroment for my research. Many thanks should
also go to Dr. Katarzyna Blazewska and Dr. Feng Ni, for all their help in the lab both at
USC and XMU (Xiamen University). For Katarzyna, the happy time we spent together
both in the lab and in everyday life is so memorable and the inspiring discussions about
science, ideals, and life will always remind me how lucky I am to have this “acquired”
sister. For Feng, your keen instinct in science and rigorous standards for research are so
amazing and I have to admit that I learnt a lot from you through plenty of chemistry
discussions and sometimes heated debates.
As I said, Rome was not built by one person; and it is also reasonable to deduce
that Rome was not built on one brick. I am indebted to our biological collaborators
worldwide, who tested the imaging probes I made and proved the various applications of
my work, making the “Rome” more steady and further furnished and decorated pretty.
Many thanks go to but not limited to the following experts: Dr. F. Hal Ebetino for his
biological research contribution and previous funding support; Dr. Xuchen (Aimee) Duan,
iv
Prof. James Triffitt and Prof. Graham Russell at Oxford University, UK for the
hydroxyapatite binding assays; Dr. Anke Roelofs and Dr. Fraser Coxon at University of
Aberdeen, UK for the prenylation and cell viability assays as well as the skeletal
distribution studies; Dr. Akishige Hokugo and Prof. Ichiro Nishimura at University of
California Los Angeles, USA for the applications of fluorescent imaging probe in
mechanism study of osteonecrosis of the jaw; Dr. Woo Seok Kang, Dr. David Jung, Dr.
Alicia Quesnel, Dr. William Sewell, and Prof. Michael McKenna at Harvard Medical
School for the fluorescent probe distribution study in cochlear. My other numerous
biological collaborators are recognized as co-authors on joint publications cited in this
dissertation. In addition, I would like to acknowledge Dr. Joy Nemirow for her previous
work on carboxyfluorescein-risedronate conjugate and related analogues, Dr. Katarzyna
Blazewska for her contributions on the rhodamine red-X and X-rhodamine-RISPC
conjugates; Anastasia Kadina for her contributions on preliminary study of reaction
between the epichlorohydrin linker and pyridine; and the undergraduate REU students I
have trained: Adam Subhas, Alma Castaneda, Hamid Barkhordar, for their help on
preparation of useful synthetic precursors during their undergraduate research program.
The great input from outside are especially important in the process of building
“Rome”; thus I am so grateful to my screening, qualifying and defense committee
members: Prof. Wei-Chiang Shen, Prof. Peter Z. Qin, Prof. G. K. Surya Prakash, Prof.
Kyung W. Jung and Prof. Richard L. Brutchey for their insightful advice and
encouragement. In addition, I owe thanks to Prof. Travis J. William for his kind help on
NMR studies and Prof. Matthew R. Pratt for kindly lending me the reagents for click
chemistry studies.
I appreciate the USC Chemistry staff a lot for their great work on instrument
maintenance and administration: Allan Kershaw, Michele Dea, Katie McKissick, Marie
de la Torre, Inah Kang, and VWR staff Darryl and Leo, thank you all for your help so
that I can concentrate on my research and was not distracted by other matters.
While everyone is building his or her own “Rome” diligently, people in McKenna
group are always open to help each other. Thank you to all McKenna group members, in
the past and present, especially to Dr. Michaela Serpi, Dr. Valeriya Zakharova, Dr. Yue
Wu for the girls’ happy shopping and zumba time; to Dr. Brian Chamberlain for the
accompany in Poland while attending ICPC; to Dr. Ivan Krylov for the always interesting
conversations; to Dr. Jorge Osuna for always helping me lifting the 5 gallon water tank
and opening the liquid nitrogen trap; to Kim Nguyen for the great suggestions on piano
selection; to Amirsoheil Negahbani and Maryam Nakhjiri for the tasty Persian food they
always offer me to try; and also to Feng, Anastasia, Amirsoheil and Kim for their help to
proofread my dissertation. You guys made my stay in McKenna group so sweet and
delightful.
My family is always a strong support and the source where I could gain unlimited
energy and strength when I feel exhausted. The unconditional love and support from my
parents Mr. Rongming Sun and Mrs Gairong Qin and my young sister Shujing are always
there and I share the whole credit of my dissertation with them. With them around, I have
v
always felt more fearless and energetic to pursue my dreams. A lot of thanks also go to
my dear friends worldwide, who are always supportive and eager to share their own
experiences; I feel so blessed to have you in my life.
“Life is a string of diamonds, while each diamond is an important occasion or
period that plays an important role in one’s development and destination of striving”, this
is the sentence I have written in my personal statement when I applied the USC
Chemistry graduate program years ago; and now I could give myself a satisfied answer
that I have already obtained this shining diamond called “USC graduate study time”,
which will be always cherished and glittering in the future.
vi
TABLE OF CONTENTS
DEDICATION .................................................................................................................. ii
ACKNOWLEDGMENTS ...............................................................................................iii
LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES .......................................................................................................... x
LIST OF SCHEMES ..................................................................................................... xvi
LIST OF ABBREVIATIONS ...................................................................................... xvii
ABSTRACT.................................................................................................................. xviii
Chapter 1 ........................................................................................................................... 1
Introduction – Bisphosphonates as “Magic Bullets” in Clinic and Research ............. 1
1.1
Bisphosphonates and bone diseases ................................................................................1
1.2
Side effects of bisphosphonate therapy...........................................................................5
1.3
Bisphosphonates as bone targeting moiety in drug delivery, imaging and other
related studies .............................................................................................................................9
1.3.1 Bisphosphonates as bone targeting moiety in small molecule drug delivery .................9
1.3.2 Bisphosphonates as bone targeting moieties in imaging studies ..................................12
1.3.3 Bisphosphonates as a bone targeting moiety in other related studies...........................16
1.4
References........................................................................................................................20
Chapter 2 ......................................................................................................................... 27
Design and Synthesis of Fluorescent N-Heterocyclic Bisphosphonate Probes.......... 27
2.1
Background .....................................................................................................................27
2.2
Results and discussion ....................................................................................................31
2.2.1
Synthesis of BP-linker intermediates via Route A ....................................................32
2.2.2
Synthesis of BP-linker intermediates via Route B ....................................................36
2.2.3
Syntheses of fluorescent BP imaging probes ............................................................37
2.2.4
Spectroscopic properties............................................................................................39
2.3
Conclusion .......................................................................................................................40
2.4
Experimental ...................................................................................................................41
2.4.1
General ......................................................................................................................41
2.4.2
Synthesis of drug-linker intermediates 4a-4c via route A.........................................43
2.4.2.1 Synthesis of epoxide linker 5 (tert-butyl (oxiran-2-ylmethyl)carbamate) .......................... 43
2.4.2.2 Synthesis of drug-linker intermediates 4a-4c...................................................................... 44
2.4.3
Synthesis of drug-linker intermediate 4d (3-(3-amino-2-hydroxypropyl)-1-(2-
hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium)...........................................................47
2.4.4
Synthesis of drug-linker intermediates 4e (1-(3-amino-2-hydroxypropyl)-3-(2-
hydroxy-2,2-diphosphonoethyl)imidazo[1,2-a]pyridin-1-ium, Route B)..............................49
2.4.5
Synthesis of drug-linker intermediates 4f (1-(3-amino-2-hydroxypropyl)-3-(2-
carboxy-2-hydroxy-2-phosphonoethyl)imidazo[1,2-a]pyridin-1-ium, Route B) ..................50
2.4.6
General method for preparation of compounds 7a-7f ...............................................51
2.4.7
UV-VIS absorption and fluorescence emission spectra ............................................68
2.5
References........................................................................................................................69
vii
Chapter 3 ......................................................................................................................... 74
Activity Characterization / Evaluation of Fluorescent N-Heterocyclic
Bisphosphonates as Imaging Probes ............................................................................. 74
3.1
Background .....................................................................................................................74
3.2
Results and discussion ....................................................................................................76
3.2.1
Hydroxyapatite binding affinity assays.....................................................................76
3.2.1.1 HAP column chromatography assay ................................................................................... 76
3.2.1.2 Quantitative measurement of BP-HAP interaction by using Langmuir adsorption isotherms
......................................................................................................................................................... 80
3.2.2
Inhibition of protein prenylation and cell viability assays ........................................83
3.3
Conclusion .......................................................................................................................85
3.4
Experimental ...................................................................................................................86
3.4.1 Hydroxyapatite column chromatography assay............................................................86
3.4.2 Quantitative measurement of BP-HAP interaction by using Langmuir adsorption
isotherms................................................................................................................................87
3.4.3
Inhibition of protein prenylation and cell viability assays ........................................88
3.5
References........................................................................................................................89
Chapter 4 ......................................................................................................................... 93
Applications of Fluorescent N-heterocyclic Bisphosphonate Probes ......................... 93
4.1
General.............................................................................................................................93
4.2
Study of bisphosphonate distribution pattern in bone skeleton by simultaneous
imaging approach.....................................................................................................................93
4.2.1
Binding of fluorescent bisphosphonate analogues to mineral surfaces.....................94
4.2.2
Distribution of high- and low-affinity BP analogues on cortical bone surfaces in vivo
96
4.2.4
Mineral surface penetration of high- and low-affinity BP analogues .....................100
4.2.5
Penetration of the osteocyte canalicular network by high- and low-affinity BP
analogues .............................................................................................................................103
4.2.6
Localization of high- and low-affinity BP analogues 7 days after administration..106
4.2.7
Conclusion...............................................................................................................109
4.3
Mechanism study of osteonecrosis of the jaw by fluorescent bisphosphonate........109
4.3.1
FAM-ZOL retained the pharmacological effect of ZOL in vivo.............................110
4.3.2
BP adsorption kinetics to CaP disc in vitro.............................................................111
4.3.3
Bio-distribution pattern of FAM-ZOL with different administration protocols in vivo
115
4.3.4
Effect of different ZOL administration protocols on anti-catabolic bone remodeling
116
4.3.5
Effect of different ZOL administration protocols on the development of ONJ-like
lesions in the rat maxilla ......................................................................................................118
4.3.6
Conclusion...............................................................................................................119
4.4
Study of zoledronate distribution in cochlea by systemic and local delivery..........120
4.5
Conclusion .....................................................................................................................125
4.6
Experimental .................................................................................................................125
4.6.1 Study of bisphosphonate distribution pattern in bone skeleton by simultaneous
imaging approach.................................................................................................................125
4.6.2 Mechanism study of osteonecrosis of the jaw by fluorescent bisphosphonate...........125
4.6.3 Study of zoledronate distribution in cochlear by systemic and local delivery ...........126
4.7
References......................................................................................................................127
viii
Chapter 5 ....................................................................................................................... 133
Design and Synthesis of Dual Functional Clickable Bisphosphonate for Preparation
of Fluorescent Imaging Probes .................................................................................... 133
5.1
Background ...................................................................................................................133
5.2
Results and discussion ..................................................................................................137
5.2.1
Synthesis of bifunctional azido-containing N-heterocyclic bisphosphonate (amino-
azido-para-dRIS, 1-(3-amino-2-azidopropyl)-4-(2,2-diphosphonoethyl)pyridin-1-ium)....137
5.2.2
Clickable reactivity studies of amino-azido-para-dRIS (7).....................................140
5.3
Conclusion .....................................................................................................................142
5.4
Experimental .................................................................................................................143
5.4.1
General ....................................................................................................................143
5.4.2
Synthesis of bifunctional azido-containing N-heterocyclic bisphosphonate (amino-
azido-para-dRIS, 7, 1-(3-amino-2-azidopropyl)-4-(2,2-diphosphonoethyl)pyridin-1-ium) 143
5.4.3
Clickable reactivity studies of amino-azido-para-dRIS (7).....................................147
5.5
References......................................................................................................................149
Chapter 6 ....................................................................................................................... 152
Conclusions and Perspectives ...................................................................................... 152
6.1
Summary........................................................................................................................152
6.2
References......................................................................................................................155
BIBLIOGRAPHY......................................................................................................... 157
APPENDIX A. Chapter 2 Supporting Data ............................................................... 173
APPENDIX B. Chapter 5 Supporting Data ............................................................... 245
ix
LIST OF TABLES
Table 1.1 Selected safety concerns of interest relating to BP therapy ............................... 6
Table 2.1 Spectroscopic properties of fluorescent bisphosphonate probes...................... 39
Table 3.1 Dissociation constants (K
d
) and maximum capacities (Bmax) of fluorescent
BP/PC probes for HAP ............................................................................................. 82
Table 4.1 Serum bone remodeling markers of vitamin D deficient rats with different ZOL
injection protocols................................................................................................... 117
x
LIST OF FIGURES
Figure 1.1 Examples of bisphosphonate drugs. ................................................................. 3
Figure 1.2 FPPS participation in isoprenoid biosynthetic pathways. ................................ 4
Figure 1.3 The appearance of osteonecrosis of the jaw. .................................................... 9
Figure 1.4 Bisphosphonates as “magic bullet” in drug delivery...................................... 10
Figure 1.5 The perspective views of structure of
99m
Tc-MDP (1:1 ratio)........................ 13
Figure 1.6 Examples of DOTA-BP conjugates................................................................ 14
Figure 1.7 “Magic linker” synthesis of fluorescent risedronate conjugates and related
analogues................................................................................................................... 16
Figure 1.8 Examples of bisphosphonates for protein conjugation................................... 18
Figure 2.1 Heterocyclic N-BPs and related analogues in literature................................. 29
Figure 2.2 Heteroatom reactivity in aromatic heterocyclic rings..................................... 32
Figure 2.3 Reaction study of ZOL (1d) and 5.................................................................. 34
Figure 2.4
31
P NMR of reaction mixture of MinPC (1f) and 5 via Route A.................... 35
Figure 2.5 Reactions of pyridine/imidazole/N-methylimidazole with epichlorohydrin.. 37
Figure 2.6 Preparative reverse phase HPLC separation of 5(6)-FAM-ZOL mixture
through a semi-preparative C18 column................................................................... 38
Figure 3.1 Comparison of peak retention time of RIS, dRIS, RISPC and their related
fluorescent conjugates............................................................................................... 78
Figure 3.2 Comparison of peak retention time of ZOL and its related fluorescent
conjugates. ................................................................................................................ 79
Figure 3.3 Comparison of peak retention time of MIN, MINPC and their related
fluorescent conjugates............................................................................................... 80
Figure 3.4 The Langmuir adsorption isotherm for the binding of BPs to HAP............... 81
Figure 3.5 Adsorption isotherms for the binding of four fluorescent BP/PC probes....... 81
Figure 3.6 Prenylation assay and J774.2 cell viability assay of some fluorescent BP
imaging probes.......................................................................................................... 83
Figure 4.1 Relative mineral affinities of fluorescent BP analogues. ............................... 95
Figure 4.2 Distribution at forming endocortical and resorbing periosteal surfaces......... 97
Figure 4.3 Distribution at resorbing and quiescent periosteal surfaces. .......................... 99
xi
Figure 4.4 Penetration at mineral surfaces. Mineral surface penetration of fluorescent BP
analogues was compared in tibiae 1 day after administration. ............................... 102
Figure 4.5 Penetration of the osteocyte network............................................................ 105
Figure 4.6 Distribution of fluorescent BP analogues one week after administration. ... 108
Figure 4.7 FAM-ZOL anti-resorptive activity assay. .................................................... 111
Figure 4.8 Study of FAM-ZOL absorption on HAP...................................................... 113
Figure 4.9 Fluorescent BP “displacement” experiment................................................. 114
Figure 4.10 In vivo absorption pattern of FAM-ZOL in rat bone tissue. ...................... 115
Figure 4.11 Different biological consequences to the modulated administration of ZOL.
................................................................................................................................. 117
Figure 4.12 Different dosing protocal results in different prevalence of ONJ-like lesions.
................................................................................................................................. 119
Figure 4.13 FAM-ZOL and otosclerosis........................................................................ 121
Figure 4.14 Systemic delivery and ototoxicity studies of 6-FAM-ZOL........................ 122
Figure 4.15 Local delivery studies of 6-FAM-ZOL. ..................................................... 123
Figure 4.16 Ototoxicity and quantification of fluorescence by local 6-FAM-ZOL
treatment. ................................................................................................................ 124
Figure 5.1 Alkynyl bisphosphonates and azido bisphosphonates.................................. 135
Figure 5.2 Examples of some alkynyl/azido containing reagents.................................. 136
Figure 5.3 Bifunctional azido-containing N-heterocyclic bisphosphonate (amino-azido-
para-dRIS)............................................................................................................... 136
Figure 5.4 Linkers tested for para-dRIS-linker synthesis. ............................................. 138
Figure 5.5
31
P NMR trace of reaction 13→14. .............................................................. 139
Figure 5.6 HPLC separation of (A) 5- and 6-isomers of triazole product (24) and (B)
FAM-alkyne (23). ................................................................................................... 142
Figure A1.
1
H NMR (CDCl
3
) spectrum of tert-butyl-allylcarbamate............................ 173
Figure A2.
1
H NMR (CDCl
3
) spectrum of epoxide linker 5.......................................... 173
Figure A3.
1
H NMR (D
2
O) spectrum of RIS-linker 4a. ................................................ 174
Figure A4.
31
P NMR (D
2
O) spectrum of RIS-linker 4a................................................. 174
Figure A5.
1
H NMR (D
2
O) spectrum of RISPC-linker 4b. ........................................... 175
Figure A6.
31
P NMR (D
2
O) spectrum of RISPC-linker 4b............................................ 175
xii
Figure A7.
1
H NMR (D
2
O) spectrum of dRIS-linker 4c................................................ 176
Figure A8.
31
P NMR (D
2
O) spectrum of dRIS-linker 4c............................................... 176
Figure A9.
1
H NMR (D
2
O) spectrum of ZOL-linker 4d................................................ 177
Figure A10.
31
P NMR (D
2
O) spectrum of ZOL-linker 4d............................................. 177
Figure A11.
1
H NMR (D
2
O) spectrum of ZOL-N,O-dilinker P1. ................................. 178
Figure A12.
31
P NMR (D
2
O) spectrum of ZOL-N,O-dilinker P1. ................................ 178
Figure A13. Mass spectrum (+) of ZOL-N,O-dilinker P1............................................. 179
Figure A14.
1
H NMR (D
2
O) spectrum of P3 of HPLC trace (ZOL)............................. 180
Figure A15.
31
P NMR (D
2
O) spectrum of P3 of HPLC trace (ZOL). ........................... 180
Figure A16.
1
H NMR (D
2
O) spectrum of MIN-linker 4e.............................................. 181
Figure A17.
31
P NMR (D
2
O) spectrum of MIN-linker 4e. ............................................ 181
Figure A18.
1
H NMR (D
2
O) spectrum of MINPC-linker 4f.......................................... 182
Figure A19.
31
P NMR (D
2
O) spectrum of MINPC-linker 4f......................................... 182
Figure A20. N, C(P)-O-dilinker-MINPC side products................................................. 183
Figure A21.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-RIS, 7a1..................................... 183
Figure A22.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-RIS, 7a1. ................................... 184
Figure A23.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-RISPC, 7b1................................ 184
Figure A24.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-RISPC, 7b1. .............................. 185
Figure A25. HPLC trace of 5(6)-FAM-RISPC, 7b1...................................................... 185
Figure A26.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-dRIS, 7c1. .................................. 186
Figure A27.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-dRIS, 7c1................................... 186
Figure A28.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-RIS, 7a4. ..................................... 187
Figure A29.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-RIS, 7a4...................................... 187
Figure A30. Mass spectrum of 5(6)-RhR-RIS, 7a4....................................................... 188
Figure A31.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-RISPC, 7b2. ................................ 189
Figure A32.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-RISPC, 7b2................................. 189
Figure A33.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-dRIS, 7c2..................................... 190
Figure A34.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-dRIS, 7c2.................................... 190
Figure A35. Mass spectrum of 5(6)-RhR-dRIS, 7c2. .................................................... 191
Figure A36.
1
H NMR (D
2
O) spectrum of 5-ROX-RIS, 7a5.......................................... 192
Figure A37.
31
P NMR (D
2
O) spectrum of 5-ROX-RIS, 7a5. ........................................ 192
xiii
Figure A38. Mass spectrum of 5-ROX-RIS, 7a5........................................................... 193
Figure A39. HPLC trace of 5-ROX-RIS, 7a5................................................................ 194
Figure A40.
1
H NMR (D
2
O) spectrum of 5-ROX-RISPC, 7b3..................................... 194
Figure A41.
31
P NMR (D
2
O) spectrum of 5-ROX-RISPC, 7b3. ................................... 195
Figure A42. Mass spectrum of 5-ROX-RISPC, 7b3. .................................................... 196
Figure A43. HPLC trace of 5-ROX-RISPC, 7b3........................................................... 197
Figure A44.
1
H NMR (D
2
O) spectrum of AF647-RIS, 7a6........................................... 197
Figure A45.
31
P NMR (D
2
O) spectrum of AF647-RIS, 7a6.......................................... 198
Figure A46. Mass spectrum of AF647-RIS, 7a6. .......................................................... 199
Figure A47. HPLC trace of AF647-RIS, 7a6. ............................................................... 200
Figure A48.
1
H NMR (D
2
O) spectrum of AF647-RISPC, 7b4. .................................... 201
Figure A49.
31
P NMR (D
2
O) spectrum of AF647-RISPC, 7b4..................................... 201
Figure A50. Mass spectrum of AF647-RISPC, 7b4. ..................................................... 202
Figure A51. HPLC trace of AF647-RISPC, 7b4. .......................................................... 203
Figure A52.
1
H NMR (D
2
O) spectrum of 5-FAM-ZOL, 7d1........................................ 203
Figure A53.
31
P NMR (D
2
O) spectrum of 5-FAM-ZOL, 7d1. ...................................... 204
Figure A54. Mass spectrum of 5-FAM-ZOL, 7d1......................................................... 205
Figure A55.
1
H NMR (D
2
O) spectrum of 6-FAM-ZOL, 7d2........................................ 206
Figure A56.
31
P NMR (D
2
O) spectrum of 6-FAM-ZOL, 7d2. ...................................... 206
Figure A57. Mass spectrum of 6-FAM-ZOL, 7d2......................................................... 207
Figure A58.
1
H NMR (D
2
O) spectrum of AF647-ZOL, 7d3......................................... 208
Figure A59.
31
P NMR (D
2
O) spectrum of AF647-ZOL, 7d3. ....................................... 208
Figure A60. Mass spectrum of AF647-ZOL, 7d3. ........................................................ 209
Figure A61. HPLC trace of AF647-ZOL, 7d3............................................................... 210
Figure A62.
1
H NMR (D
2
O) spectrum of 800CW-ZOL, 7d4. ...................................... 210
Figure A63.
31
P NMR (D
2
O) spectrum of 800CW-ZOL, 7d4....................................... 211
Figure A64. Mass spectrum of 800CW-ZOL, 7d4. ....................................................... 212
Figure A65. HPLC trace of 800CW-ZOL, 7d4. ............................................................ 213
Figure A66.
1
H NMR (D
2
O) spectrum of Sulfo-Cy5-ZOL, 7d5. .................................. 213
Figure A67.
31
P NMR (D
2
O) spectrum of Sulfo-Cy5-ZOL, 7d5................................... 214
Figure A68. Mass spectrum (+) of Sulfo-Cy5-ZOL, 7d5.............................................. 215
xiv
Figure A69. HPLC trace of Sulfo-Cy5-ZOL, 7d5......................................................... 216
Figure A70.
1
H NMR (D
2
O) spectrum of 5-FAM-MIN, 7e1. ....................................... 216
Figure A71.
31
P NMR (D
2
O) spectrum of 5-FAM-MIN, 7e1........................................ 217
Figure A72. Mass spectrum of 5-FAM-MIN, 7e1......................................................... 218
Figure A73.
1
H NMR (D
2
O) spectrum of 6-FAM-MIN, 7e2. ....................................... 219
Figure A74.
31
P NMR (D
2
O) spectrum of 6-FAM-MIN, 7e2........................................ 219
Figure A75. Mass spectrum of 6-FAM-MIN, 7e2......................................................... 220
Figure A76. HPLC separation of 5-FAM-MIN (7e1) and 6-FAM-MIN (7e2). ............ 221
Figure A77.
1
H NMR (D
2
O) spectrum of 5-FAM-MINPC, 7f1.................................... 221
Figure A78.
31
P NMR (D
2
O) spectrum of 5-FAM-MINPC, 7f1. .................................. 222
Figure A79. Mass spectrum of 5-FAM-MINPC, 7f1..................................................... 223
Figure A80.
1
H NMR (D
2
O) spectrum of 6-FAM-MINPC, 7f2.................................... 224
Figure A81.
31
P NMR (D
2
O) spectrum of 6-FAM-MINPC, 7f2. .................................. 224
Figure A82. Mass spectrum of 6-FAM-MINPC, 7f2..................................................... 225
Figure A83. HPLC separation of 5-FAM-MINPC (7f1) and 6-FAM-MINPC (7f2)..... 226
Figure A84. UV-VIS, fluorescence emission spectra of compounds 7a1 – 7f2............ 226
Figure B1.
1
H NMR (400 MHz, D
2
O) spectrum of para-dRIS, 10................................ 245
Figure B2.
31
P NMR (162 MHz, D
2
O) spectrum of para-dRIS, 10. .............................. 246
Figure B3. MS(+) spectrum of para-dRIS, 10. .............................................................. 247
Figure B4.
1
H NMR (400 MHz, CDCl
3
) spectrum of para-dRIS-linker-OH, 12. ......... 248
Figure B5.
31
P NMR (162 MHz, CDCl
3
) spectrum of para-dRIS-linker-OH, 12.......... 248
Figure B6. MS(+) spectrum of para-dRIS-linker-OH, 12.............................................. 249
Figure B7a.
31
P NMR (162 MHz, CDCl
3
) spectrum of intermediate 13. ...................... 250
Figure B7b.
MS(+) spectrum of intermediate 13........................................................... 251
Figure B8.
31
P NMR (162 MHz, CDCl
3
) monitoring of reaction intermediate 13→14.252
Figure B9.
1
H NMR (400 MHz, CDCl
3
) spectrum of para-dRIS-linker-N3-ester, 14. . 252
Figure B10.
31
P NMR (162 MHz, CDCl
3
) spectrum of para-dRIS-linker-N3-ester, 14.253
Figure B11. MS spectrum of para-dRIS-linker-N3-ester, 14......................................... 254
Figure B12a.
1
H NMR (500 MHz, D
2
O) spectrum of amino-azido-para-dRIS, 7. ....... 255
Figure B12b. gCOSY spectrum of amino-azido-para-dRIS, 7...................................... 256
Figure B13.
31
P NMR (203 MHz, D
2
O) spectrum of amino-azido-para-dRIS, 7.......... 257
xv
Figure B14a. MS(+) spectrum of amino-azido-para-dRIS, 7. ....................................... 258
Figure B14b. MS(-) spectrum of amino-azido-para-dRIS, 7......................................... 259
Figure B15.
1
H NMR (500 MHz, Methanol-d
4
) spectrum of 5(6)-FAM-alkyne, 23..... 260
Figure B16a. MS(+)spectrum of 5(6)-FAM-alkyne, 23. ............................................... 261
Figure B16b. MS(-)spectrum of 5(6)-FAM-alkyne, 23................................................. 262
Figure B17.
1
H NMR (400 MHz, D
2
O) spectrum of 6-FAM-triazole-para-dRIS, 24a. 263
Figure B18.
31
P NMR (162 MHz, D
2
O) spectrum of 6-FAM-triazole-para-dRIS, 24a. 263
Figure B19.
1
H NMR (400 MHz, D
2
O) spectrum of 5-FAM-triazole-para-dRIS, 24b. 264
Figure B20.
31
P NMR (162 MHz, D
2
O) spectrum of 5-FAM-triazole-para-dRIS, 24b. 264
Figure B21a. MS(+) spectrum of 5(6)-FAM-triazole-para-dRIS, 24. ........................... 265
Figure B21b. MS(-) spectrum of 5(6)-FAM-triazole-para-dRIS, 24............................. 266
Figure B22. UV-VIS and fluorescence emission spectra of 5(6)-FAM-triazole-para-dRIS,
24............................................................................................................................. 267
xvi
LIST OF SCHEMES
Scheme 2.1 Synthesis of fluorescent bisphosphonate ‘toolkit’........................................ 31
Scheme 2.2 Synthetic route of epoxide linker 5 (tert-butyl (oxiran-2-ylmethyl)carbamate)
................................................................................................................................... 43
Scheme 5.1 Synthesis of bifunctional azido-containing N-heterocyclic bisphosphonate.
................................................................................................................................. 138
Scheme 5.2 Synthesis of 5(6)-FAM-alkyne (23). .......................................................... 141
Scheme 5.3 Click reaction of amino-azido-para-dRIS (7) and 5(6)-FAM-alkyne (23). 141
xvii
LIST OF ABBREVIATIONS
AF647 Alexa Fluor 647
ALN Alendronate
ALP Alkaline phosphatase
BP Bisphosphonate
CaP Calcium phosphonate
CTX Type I cross-linked C-telopeptide
DMAPP Dimethylallyl pyrophosphate
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
FAM Carboxyfluorescein
FGPP/GFPP Farnesylgeranyl pyrophosphate/Geranylfarnesyl pyrophosphate
FGPPS Farnesylgeranyl pyrophosphate synthase
FPP Farnesyl pyrophosphate
FPPS/FDPS Farnesyl pyrophosphate synthase/Farnesyl diphosphonate synthase
FT Farnesyl transferase
GGPPS Geranylgeranyl pyrophosphate synthase
GGT Geranylgeranyl transferase
GPP Geranyl pyrophosphate
HAP Hydroxyapatite
IPP Isoprenyl pyrophosphate
IV Intravenous
MBP/MDP Methylene bisphosphonate/diphosphonate
MIN Minodronate
N-BP Nitrogen-containing bisphosphonate
ONJ Osteonecrosis of the Jaw
PAM Pamidronate
PC Phosphonocarboxylate
r.t. Room Temperature
RCT Randomized, placebo-controlled clinical trial
RGGT Rab geranylgeranyl transferase
RhR Rhodamine Red-X
RIS Risedronate
ROX X-Rhodamine
TRACP-5b Tartrate-resistant Acid Phosphatase 5b
ZOL Zoledronate
xviii
ABSTRACT
Bisphosphonates (BPs) are therapeutic agents for treatment of bone disorders such
as osteoporosis and Paget’s disease. Several nitrogen-containing bisphosphonates (N-BPs)
and phosphonocarboxylate (PC) analogues also have potential as anti-cancer agents.
However, details of the skeletal distribution, cellular uptake and mechanisms of these
drugs remain to be elucidated, stimulating the creation of imaging probes which mimic
some or all of their pharmacological properties.
A fluorescent imaging “toolkit” with more than 20 probes derived from all three
heterocyclic N-BP drugs (risedronate, zoledronate and minodronate) and related
analogues have been successfully synthesized. A linking strategy with two routes was
applied under exceedingly mild conditions and introduced a terminal amino group in
drug-linker intermediates capable to conjugate with the commercially available activated
ester of fluorescent dyes. All the fluorescent probes were prepared in good yield (50-77%)
and high purity (> 95%), and are fully characterized by HPLC, UV-VIS and fluorescence
emission spectroscopy,
1
H and
31
P NMR and high-resolution MS.
The ‘toolkit’ contains a series of fluorescent probes ranging from visible to near
infrared optical window and the probes generally retain substantial affinity for bone
mineral reflecting the varying affinities of their parent drug components, depending on
the structure of the conjugating fluorescent dye. In addition, we have obtained evidence
that certain probes (e.g. the FAM- and ROX- conjugates) have anti-prenylation and anti-
resorptive effects in vitro and in vivo, demonstrating that the probes could retain
biological activities of the parent BP drugs.
Due to their diverse spectroscopic and pharmacological properties, the fluorescent
xix
imaging probe “toolkit” has been successfully utilized in various biological researches
including osteoclast imaging, drug distribution at bone skeleton and cellular level,
mechanism studies of osteonecrosis of the jaw (ONJ), cancer imaging, et al., and several
papers have been published based on the obtained results, demonstrating the versatility of
the imaging probe “toolkit”.
The first example of a bifunctional amino/azido-containing N-heterocyclic
bisphosphonate (amino-azido-para-dRIS, compound 7 in Chapter 5) has been synthesized;
and it has been successfully applied for the preparation of fluorescent probes via Cu(I)
catalyzed alkyne-azide coupling (CuAAC) click reaction. The synthetic strategy
developed based on para-dRIS, should be adaptable to other N-heterocyclic deoxy-
bisphosphonates and related analogues, such as dRIS, dRISPC, et al. In addition, two
functionalities, the azido and amino groups, were introduced sequentially to make dual-
conjugation of the molecule possible, providing the opportunity for novel cleavable
imaging agents and drug delivery system design.
1
Chapter 1
Introduction – Bisphosphonates as “Magic Bullets” in Clinic
and Research
1.1 Bisphosphonates and bone diseases
Methylenebisphosphonates are pyrophosphate analogues in which a carbon atom
replaces the bridging oxygen atom between the two phosphate groups. Various
substitutions on the bridging carbon with different side chains (R
1
, R
2
; Figure 1.1)
produce novel compounds, making possible the generation of many compounds derived
from this simple scaffold.
1, 2
For convenience, as a class these compounds are commonly
referred to as ‘bisphosphonates’ (BPs), although strictly this term applies to any
compound containing two phosphonate groups, and certain clinical drugs are actually
formulated as bisphosphonic acids (e.g., zoledronate and minodronate in Figure 1.1). In
vivo, they will be ionized to an extent determined by their physiological environment and
particular structure. BPs are currently the major class of drugs used for the treatment of
osteoporosis and other diseases characterized by excessive bone resorption.
2, 3
BPs were first used clinically in the late 1960s and early 1970s
2
; some of the early
examples of BPs developed for the treatment of diseases characterized by abnormal
calcium metabolism include etidronate and clodronate (Figure 1.1); these early
generation of BPs do not have a nitrogen-containing side chain, which are categorized as
non nitrogen-containing BPs (non N-BPs). These non N-BPs were found to be
incorporated into the corresponding non-hydrolyzable, β,γ-methylene analogues of
2
adenosine triphosphate (ATP)
4, 5
by the reversal of aminoacyl-tRNA synthetase reactions
which are normally involved in activating amino acids during protein synthesis.
6
The
non N-BPs are metabolized to AppCp-type nucleotides in this way, perhaps because of
their resemblance to naturally occurring pyrophosphate. Induction of osteoclast apoptosis
following the intracellular accumulation of such metabolites appears to be the major
mode of action of these non N-BPs.
4
The non-hydrolyzable ATP analogue of clodronate
has been shown to inhibit the adenine nucleotide translocase in the mitochondrial
membrane.
7
This presumably affects mitochondrial permeability and initiates caspase
activation, which is an irreversible step toward apoptotic cell death.
8
For the later generation of BPs such as alendronate, risedronate, and zoledronate,
their side chains usually contain a nitrogen atom, by which they are categorized as
nitrogen-containing BPs (N-BPs, Figure 1.1). N-BPs generally have higher potency than
non N-BPs and are more widely used clinically nowadays, but it was only in the early
2000s that their mechanism of action began to emerge.
1, 9, 10
During this period, it
became clear that these N-BP drugs inhibit an important enzyme in mevalonate pathway -
--- farnesyl pyrophosphate synthase (FPPS).
11, 12
3
Figure 1.1 Examples of bisphosphonate drugs.
For simplicity of illustration, all are depicted in bisphosphonic acid form.
Farnesyl pyrophosphate synthase (FPPS, also known as farnesyl diphosphate
synthase (FDPS)) is one of the key enzymes involved in the mevalonate pathway (Figure
1.2).
13-15
This enzyme catalyzes the two-step synthesis of the C15 isoprenoid farnesyl
pyrophosphate (FPP): isoprenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate
(DMAPP) are coupled to produce geranyl pyrophosphate (GPP), which is then condensed
with an additional IPP to produce FPP. This sesquiterpene derivative is a crucial
precursor in the synthesis of several classes of essential metabolites, including sterols,
dolichols, ubiquinones and carotenoids. It is also used by farnesyl transferase (FT) to
prenylate biomolecules that function as hormones, visual pigments, constituents of
membranes and components of signal transduction.
16, 17
N
P
P
HO
O
OH
OH
O
OH
OH
N
N
P
P
HO
O
OH
OH
O
OH
OH
P
P
HO
H
2
N
O
OH
OH
O
OH
OH
P
P
HO
H
2
N
O
OH
OH
O
OH
OH
Alendronate Pamidronate
Risedronate Zoledronate
P
P
HO
N
O
OH
OH
O
OH
OH
Ibandronate
R
1
C
P
P
R
2
O
OH
OH
O
OH
OH
Bisphosphonate
N
N
P
P
HO
O
OH
OH
O
OH
OH
Minodronate
Cl P
P
Cl
O
OH
OH
O
OH
OH
Clodronate
H
3
C P
P
HO
O
OH
OH
O
OH
OH
Etidronate
O
P
P
O
OH
OH
O
OH
OH
Pyrophosphate
H P
P
S
O
OH
OH
O
OH
OH
Tiludronate
Cl
P
P
HO
O
OH
OH
O
OH
OH
Neridronate
P
P
HO
O
OH
OH
O
OH
OH
Olpadronate
N
H
2
N
Non nitrogen-containing BPs (non N-BPs)
Nitrogen-containing BPs (N-BPs)
4
FPPS belongs to the prenyltransferase group and FPPS proteins purified from
different organisms have all proved to be homodimeric with subunit sizes from 32 to 44
kDa, typically displaying two aspartate-rich motifs with two substrate binding sites per
monomer, one for an allylic diphosphate and one for IPP.
13, 15
Since the discovery of
FPPS in 1959
18
, knowledge about the prenyltransferases has advanced significantly,
providing an understanding of their reaction kinetics, catalytic mechanisms and
conformational changes.
14, 19
Due to its importance in the isoprenoid biosynthesis
pathway, many studies have evaluated the potential of FPPS as a drug target.
20-23
Figure 1.2 FPPS participation in isoprenoid biosynthetic pathways.
CO
2
-
OH
HO
O P O
O
O
-
P
O
O
-
O
-
O P O
O
O
-
P
O
O
-
O
-
O P O
O
O
-
P
O
O
-
O
-
Mevalonate
IPP DMAPP
IPP
FPPS
GPP
IPP
FPPS
O P O
O
O
-
P
O
O
-
O
-
O P O
O
O
-
P
O
O
-
O
-
2
FPP
IPP
GGPPS
GGPP
Farnesylated proteins
Geranylgeranylated proteins
Farnesyl
transferase (FT)
Geranylgeranyl transferase
(GGT), e.g. RGGT
Steroids
Phytosterols
Cholesterols
Ubiquinones
Dolichols
Carotenoids
Retinoids
Diterpenes
Chlorophylls
Tocopherols
O P O
O
-
P O
-
O
-
3
GFPP
Sesquiterpenes
Heme a
Vitamin K2
FGPPS
IPP
Archaeal ether linked
lipids
O O
5
FPPS was recently identified as the apparent main biochemical target of nitrogen-
containing bisphosphonate (N-BP) drugs (Figure 1.1)
11, 12
that are used to treat bone
resorption diseases such as osteoporosis. Inhibition of FPPS by these N-BPs prevents the
biosynthesis of FPP and downstream products, for example, geranylgeranyl
pyrophosphate (GGPP) (Figure 1.2). Besides being an intermediate for biosynthesis of
important biomolecules ranging from retinoids and chlorophylls, GGPP is required for
post-translational prenylation of small GTPases, such as the Ras, Rho and Rab family
proteins in osteoclasts.
24
Disruption of protein prenylation leads to loss of essential
signaling processes that are necessary for osteoclast function and survival.
12, 25-27
Although these pathways are crucial in all cell types, the extraordinary ability of
N-BPs to bind to bone mineral selectively results in the exposure of osteoclasts to high
concentrations of the drug when they mediate resorption of surface bone in vivo.
1
The
successful application of some N-BPs as FPPS inhibitors for treatment of bone diseases
has underscored the importance of FPPS as a drug target, stimulating research groups in
both industry and academia to focus on design of new FPPS inhibitors as well as new
applications of existing drugs.
28
1.2 Side effects of bisphosphonate therapy
Due to their strong binding affinity to bone minerals, BPs are rapidly absorbed to
bone once they enter circulation; apart from a negligible amount of BPs transiently
exposed to other tissues, most of the remainder BPs is excreted unchanged shortly in
urine via filtration and proximal tubular secretion.
1
Thus, it is accepted that BPs have a
6
strong safety and tolerability profile
3
, although people start to think and question the
long-term safety of BP therapy recently.
29-31
The major safety concerns relating to BP therapy include two categories
31
: 1)
Non-skeletal safety issues, including gastrointestinal intolerance, hypocalcaemia, acute-
phase (i.e., postdose) reactions, renal toxicity, hepatotoxicity and cardiovascular safety
(specifically, atrial fibrillation). 2) Skeletal safety issues, including atypical fractures,
delayed healing, chronic musculoskeletal pain and a rare but severe phenomenon called
osteonecrosis of the jaw (ONJ). Among the above safety concerns, it is considered that
some are well documented and have biological plausibility, while others are suspected
but there is still controversy on whether they are associated with BP therapy.
31
Table 1.1
summarizes these safety concerns, and it is noteworthy that the determination of
biological plausibility and causality is partly subjective in nature, based on the author’s
assessment of the medical evidence and the findings of expert panels reviewing the
evidence, when available. In addition, it is very important to understand that the so-called
“adverse effects” are not observed for all the clinical BP agents.
Table 1.1 Selected safety concerns of interest relating to BP therapy
(Adapted from ref. 30, 31)
Adverse event Biological
plausibility/Possible
mechanism
Causality
demonstrated
Main types of
supporting evidence,
comments
Gastrointestinal
intolerance
Yes (may through the
toxicity to the soft
tissues, such as mucosa
cells)
Yes Preclinical studies,
case reports,
postmarketing
database reviews
Esophageal
cancer
Uncertain No Case reports,
database reviews
Atrial
fibrillation
No No One RCT
7
Renal toxicity Yes (very high systemic
drug concentrations on
renal tubular cells)
Yes Preclinical studies,
case reports, few
clinical trials;
primarily seen with
IV BP with excessive
dose or rate of
infusion
Acute phase
reaction/Post-
dose symptoms
Yes (release of pro-
inflammatory cytokines)
Yes RCTs
Hypocalcaemia Yes (BPs reduce calcium
efflux from bone,
resulting a small decrease
in serum calcium levels,
and a compensatory rise
in serum parathyroid
hormone levels)
Yes Preclinical studies,
RCTs
Hepatotoxicity No No Case reports
Ocular
inflammation
Uncertain No Case reports, RCTs
Osteonecrosis
of the jaw
Uncertain No Case reports,
database reviews
Atypical femur
fractures
Uncertain No Case reports,
database reviews
Chronic
musculoskeletal
pain
No No Case reports
Impaired
healing of acute
fractures
No No Hypothetical concern
Impaired
healing of stress
fractures
Yes (may through
inhibition of osteoclast
activity)
No Speculated to play a
role in pathogenesis
of BP-associated
atypical femur
fractures
IV = intravenous; RCT = randomized, placebo-controlled clinical trial.
8
The detailed pathophysiology studies of most of the above mentioned adverse
effects are beyond the scope of the dissertation, thus are simply summarized in Table 1,
and will not be discussed further. The mechanism of ONJ will be discussed in Chapter
4.3. In general, these safety concerns of long-term use of BPs do remind people to bear in
mind the positive benefit-to-risk ratio when making treatment recommendations for
patients.
Since 2003, phenomena collectively referred to as osteonecrosis of the jaw (ONJ,
Figure 1.3) have been reported as a rare but potentially severe adverse event in patients
receiving high dose of certain BPs, such as zoledronate, alendronate, and pamidronate.
32-
34
ONJ is clinically characterized as unresolved exposure of partially necrotic jawbone to
the oral cavity and is frequently associated with dentoalveolar procedures in cancer
patients receiving high doses of intravenously administered BPs.
33, 35-38
The etiology and
pathogenesis of ONJ remain poorly characterized, and there are still controversies on
whether ONJ is associated with BPs from drug research community and dental research
community.
38-40
Numerous hypotheses are proposed to explain the mechanism of ONJ,
however, none is widely accepted yet. Thus, more solid experimental evidence are highly
desirable in the mechanism study of ONJ.
9
Figure 1.3 The appearance of osteonecrosis of the jaw.
Picture is courtesy of Dr. Ichiro Nishimura, UCLA.
1.3 Bisphosphonates as bone targeting moiety in drug delivery, imaging and
other related studies
Since BPs have strong and unique binding affinity to bone minerals, this specific
targeting property makes them a magic bone seeking “bullet”, which enables their
applications in various studies, such as drug delivery and imaging research.
1.3.1 Bisphosphonates as bone targeting moiety in small molecule drug delivery
According to Paul Ehrlich’s idea of “magic bullet”, the drug which can be
considered as “magic bullet” should ideally act specifically on the targeted organs or
tissues, without affecting other organs or tissues; clinically used BPs themselves can be
considered as “magic bullets” for bone related diseases from this sense. Due to this
special targeting property, researchers are investigating their potential use as the targeting
moiety for other small molecule drug delivery.
41
The bone-specific delivery of several classes of therapeutic agents and other small
molecules has already been studied using BPs as targeting moiety/carrier; these agents
10
include
41
: 1) anti-neoplastic agents (e.g. 5-fluorouracil, radionuclides, cisplatin,
doxorubicin, et al.); 2) anti-bacterial agents (e.g. ciprofloxacin); and 3) anti-osteoporosis
agents (Src kinase inhibitors, raloxifene, nitric-oxide, et al.) (Figure 1.4A). Four
approaches are utilized to incorporate BPs as targeting moiety: a) covalently modify the
agents by adding the P-C-P functionality; b) covalently modify the agents by conjugating
the agents and bisphosphonates with a linker/tether; c) deliver the agents by BP-drug
non-covalent interactions; d) deliver the agents by BP-containing generic drug carriers,
which utilize BPs as building blocks and complex/conjugate with drugs through non-
covalent/covalent interactions (Figure 1.4B).
Figure 1.4 Bisphosphonates as “magic bullet” in drug delivery.
(A) The type of drugs that use BPs as targeting moiety in drug delivery. (B) The approaches to
incorporate BPs as targeting moiety in drug delivery.
The approaches B(a) and B(b) generally yield brand new agents which may retain
the activity of the parent drugs to certain extend depending on the different drugs
themselves; it is also reasonable to observe that some new agents have the same or even
A
B
Bisphosphonate Drug Drug Carrier
Linkage types between
bisphosphonate and drugs
non-covalent linker covalent
c
d
b
a
"Magic bullet"
Bisphosphonate
Anti-neoplastic
agents
Anti-bacterial
agents
N
NH
O
O
F
N
N F
N
COOH
O
Anti-resorptive
agents
OH
NH
P
P
O
OH
O
OH HO
HO
O
P
P
O
OH
O
OH HO
HO
P
P
O
OH
O
HO
HO
H
N
O
O
S
O
OH
O
N
11
higher activity compared with the parent drugs, indicating the new agents may function
through different pathways.
41
Meanwhile, when a linker is used in the modification, the
different selecting options of linkers provide a possibility to release the parent drugs, thus
this approach can be considered as a “prodrug” strategy.
42, 43
Notably, the nitric oxide
local delivery has been achieved by conjugating the BP moiety with organic nitric oxide
donor.
44
In addition, some anti-metabolites (e.g. 2’-deoxy-5-fluorouridine (5-FdU), 1-β-
D-arabinofuranosyluracil (araU), 3’-azido-2’,3’-dideoxythymidine (AZT)) were also
covalently modified by adding a BP moiety, and the underlying mechanisms of these new
agents remain to be further evaluated.
45
The approach c has successfully been achieved with some radiopharmaceuticals
46
and platinum agents
47, 48
by chelating BPs (e.g. β, γ-emitting
186/188
Re); and it is
noteworthy that BPs in approach c could also be regarded as a specific “drug carrier” in a
general sense.
46
However, as in approach d, the design of “generic” carriers of other
therapeutic agents is usually difficult since the drugs are larger in size, and designing a
generic molecular host will be not as straightforward as simple metal-chelators.
49-51
Nevertheless, people have started to look for possibilities of utilizing endogenous
molecules such as heparins or antibodies as hosts for larger therapeutic agents;
52, 53
in
addition, nanocarriers (polymeric or micellar)
54-56
have been explored to encapsulate
small molecule drugs. These drug carriers incorporated BPs as building blocks to achieve
bone-specific delivery. These carrier systems under development should be able to
deliver a range of therapeutic agents, rather than a specific one, which are likely to meet
the requirement of “generic” drug carrier for specific bone delivery.
12
In conclusion, BPs have already been demonstrated as an effective targeting
moiety and enhanced the small molecule drug delivery to osseous tissues. The full
potential of BPs in site-specific drug delivery should be further explored by researchers
from both academia and industry. Besides retaining/enhancing the desired bone binding
affinity of the therapeutic agent-BP conjugates/complexes, one should also take it into
consideration of devising better means to release the therapeutic agents from the BP
conjugates or complexes.
2
1.3.2 Bisphosphonates as bone targeting moieties in imaging studies
Although it is becoming prevalent in the last couple of years to investigate the
applications of BPs as a targeting moiety in drug delivery studies as discussed in Section
1.3.1, the idea was introduced as early as 1970s in skeletal imaging agent studies, when
99m
Tc-MDP (methylenebisphosphonate, where R
1
and R
2
are –H, Figure 1.1) was
successfully introduced and is still widely utilized in bone scintigraphy nowadays.
57
99m
Tc-labeled skeletal imaging agents first utilized long chain polyphosphates as a
targeting moiety, which were superseded by pyrophosphate and subsequently the BPs,
such as MDP and etidronate (HEDP) (Figure 1.1).
99m
Tc-pyrophosphates and
99m
Tc-BP
are known as the second and third generation diagnostic agents; and
186/188
Re-BPs were
developed afterwards, which are not only β-emitters for diagnosis, but also γ-emitters for
therapeutic application.
46
Despite the proven success of Tc/Re-BPs as the imaging probes in abnormal
calcification or osseous tissues related disease diagnoses, such as tumor burden at the
osseous sites, meningiomas, et al., these radio-labeled probes are far from optimal from a
13
chemical and imaging point of view. For example, despite decades of applications, their
exact structures and compositions remain unknown. The chromatographic analysis
reveals that commercially available
99m
Tc-BPs (e.g.
99m
Tc-MDP) are composed of a
mixture of multiple
99m
Tc species with different properties, indicating that
99m
Tc does not
form a single complex with BPs.
46, 58
The only reported
99m
Tc-MDP structure shows a
1:1 ration of
99m
Tc and MDP in the complex, where each
99m
Tc atom chelates with two
MDPs, and each MDP chelates with two
99m
Tc atoms
59
(Figure 1.5). Thus BPs act both
as chelator and targeting group; each role will compromise the other, and will eventually
affect their performances in imaging studies. In addition, the
186/188
Re-BPs are not stable
enough in vivo, and tend to degrade to perrhenate within 24 hrs, leading to reduced bone
uptake and increased soft-tissue doses, which will bring low S/N ratio for imaging studies.
60
Figure 1.5 Perspective views of structure of
99m
Tc-MDP (1:1 ratio).
(A) One MDP ligand bridging two
99m
Tc centers. (B) One
99m
Tc atom bridging two MDP ligands.
(Adapted from ref. 59)
To overcome the drawbacks of the Tc/Re-BPs probes, the targeted bifunctional
ligands are proposed, in which the targeting moiety (BPs) and metal-chelating group are
separated within the molecule, so that they can function independently and will not
compromise each other.
49
It is noteworthy that this approach is the same as the drug
Communications to the Editor
Table I. Summary of Crystal Datao
2477
molecular formulab
formula weight, amu
obsd density, g
cald density, g cm-3
space group
Z
unit cell constants, A
unit cell volume, A3
data collection
specifics
2.12 (2)
2.1 1
RT, No. 148, hexagonal setting
18
a = 21.771 (8),c = 12.208 (3)
501 1.3
Cu Ka radiation, X = 1.541 8 8,
8/28 technique, 28,,, = 1 12O
1108 reflections with I > 2a(I)
Estimated standard deviations of the last significant figure are
given in parentheses.
Table 11. Selected Bond Parameterso
MDP = methylenediphosphonate.
Tc Distances
Tc-O(l) 2.000 (14) Tc-0(3) 1.983 (14)
Tc-0(6) 2.036 (1 6) Tc-0(5) 2.029 (1 2)
Tc-OH 1.968 (1 5) Tc-OH' 1.917 (12)
Diphosphonate Distances
P( 1)-O( 1) 1.534 (I 5) P(2)-0(4) 1 SO2 (1 8)
P(1)-0(2) 1.460 (16) P(2)-0(5) 1.552 (14)
P(1)-0(3) 1.551 (14) P(2)-0(6) 1.552 (15)
P( I)-c 1.83 (2) P(2)-C 1.78 (2)
Diphosphonate Angles
O(l)-P(l)-O(2) 114.4 (9) 0(4)-P(2)-0(5) 108.9 (9)
O(l)-P(l)-O(3) 110.1 (8) 0(4)-P(2)-0(6) 109.9 (9)
O(1)-P(l)-C 106.7 (9) 0(4)-P(2)-C 111.9 (10)
0(2)-P(I)-0(3) 11 1.1 (9) 0(5)-P(2)-0(6) 111.1 (8)
0(2)-P(I)-C 110.4 (IO) 0(5)-P(2)-C 106.6 (10)
0(3)-P(l)-C 103.4 (9) 0(6)-P(2)-C 108.4 (10)
P(I)-C-P(2) 110.7 (12)
Distances are in angstroms, angles are in degrees, and atom
identifications are as shown in Figures I and 2. Estimated standard
deviations of the last significant figure(s) are given in parentheses.
In a typical preparation 0.2 mmol of (NH4)2TcBr6 is dis-
solved in -10 mL of N,N-dimethylformamide and a ninefold
molar excess of H4MDP is added to the stirred solution. Within
a few minutes the solution changes from an orange-red to a
brown-gold color, and it is then heated at 80 OC with stirring
for -20 min. After dilution with -300 mL of aqueous chlo-
roacetate buffer solution, the pH is adjusted to 3.2. This so-
lution is loaded onto a column (1.25-cm-i.d. X 5.0 cm) of
anion-exchange resin (Bio-Rad, AG MP-1, 100-200 mesh,
chloride form) and the column rinsed with 0.05 M aqueous
chloroacetate buffer at pH 3.2. At this stage the brown product
is sorbed at the top of the column; passage of 0.5 M LiC104
through the column removes most of this material by what
appears to be a displacement phenomenon. Upon standing for
several weeks, small brown crystals of quality suitable for the
X-ray diffraction experiment appear in the acidified eluant
solution. A well-formed, but rather small (0.08 X 0.08 X 0.05
mm), cryst$ was subjected to X-ray diffraction analysis and
the structure was solved using standard Patterson and least-
squares analysis. Table I lists data pertinent to this experiment.
Full-matrix least-squares refinement using statistical weights
and varying (I) the overall scale factor, (2) all independent
positional parameters, (3) the lithium atom isotropic tem-
perature factors, and (4) anisotropic temperature factors for
all remaining atoms, converged at R = 0.077 (0.067 weighted).
The final difference map shows no features other than a ran-
dom background below 1.2 e/A3 and no indications of disorder.
Portions of the structure are shown in Figures 1 and 2, while
selected bond parameters are listed in Table 11.
The solid state structure of the Tc-MDP complex consists
of infinite polymeric chains. Each MDP ligand bridges two
symmetry related technetium atoms (Figure l), and each
Figure 1. Perspective view of a portion of the ([Li(H20)3][Tc(OH)-
(MDP)].'/3H20), structure showing one MDP ligand bridging two
technetium centers.
04"
A
Figure 2. Perspective view of a portion of the {[Li(H20)3][Tc(OH)-
(MDP)].'/3H20)n structure showing one Tc center bridging two MDP
ligands.
technetium atom is bound to two symmetry related MDP li-
gands (Figure 2)-the MDP/Tc ratio within the polymer is
therefore 1:l. The polymeric repeat unit is completed by an
oxygen atom (presumably in the form of an hydroxyl ion) that
bridges two symmetry-related technetium atoms (Figure 1)
and by an hydrated lithium cation which neutralizes the charge
associated with each repeat unit. In addition, there is a single
oxygen atom (presumably in the form of a disordered water
molecule) on the threefold axis of the space group. The mo-
lecular formula of the polymeric Tc-MDP complex may thus
be represented as { [Li(H20)3] [TC'~(OH)(MDP)].'/~H~O}~
where the indicated protonation states of the bridging and
noncoordinated oxygen atoms are chemically reasonable and
consistent with an assumed Tc(IV) oxidation state, but are not
definitively established by the X-ray diffraction data. Alter-
nate, equivalently reasonable, representations may be con-
structed about other technetium oxidation states and other
degrees of protonation of the bridging and noncoordinated
oxygen atoms, e.g. { [Li(H20)3] [TC'~(O)MDP)]-]/~H~O}~ or
{[Li(H20)31 [TC~(O)(MDP)I.'/~H~O~,.
Each technetium center has approximately octahedral
coordination geometry, the two bridging oxygen atoms oc-
cupying cis coordination sites. The bridging oxygen to tech-
netium bond distance (1.94 (2) A average) is generally in line
with observed Tc-0 single bond lengths (2.00-2.03 A),12
Tc=O double bond lengths being considerably shorter
(1.65-1.70 A).12 Bond lengths and bond angles within the
MDP ligand are generally as expected from structural studies
on the free acid H4MDPI6 and on related diphosphonate
One of the most important structural features of the
Communications to the Editor
Table I. Summary of Crystal Datao
2477
molecular formulab
formula weight, amu
obsd density, g
cald density, g cm-3
space group
Z
unit cell constants, A
unit cell volume, A3
data collection
specifics
2.12 (2)
2.1 1
RT, No. 148, hexagonal setting
18
a = 21.771 (8),c = 12.208 (3)
501 1.3
Cu Ka radiation, X = 1.541 8 8,
8/28 technique, 28,,, = 1 12O
1108 reflections with I > 2a(I)
Estimated standard deviations of the last significant figure are
given in parentheses.
Table 11. Selected Bond Parameterso
MDP = methylenediphosphonate.
Tc Distances
Tc-O(l) 2.000 (14) Tc-0(3) 1.983 (14)
Tc-0(6) 2.036 (1 6) Tc-0(5) 2.029 (1 2)
Tc-OH 1.968 (1 5) Tc-OH' 1.917 (12)
Diphosphonate Distances
P( 1)-O( 1) 1.534 (I 5) P(2)-0(4) 1 SO2 (1 8)
P(1)-0(2) 1.460 (16) P(2)-0(5) 1.552 (14)
P(1)-0(3) 1.551 (14) P(2)-0(6) 1.552 (15)
P( I)-c 1.83 (2) P(2)-C 1.78 (2)
Diphosphonate Angles
O(l)-P(l)-O(2) 114.4 (9) 0(4)-P(2)-0(5) 108.9 (9)
O(l)-P(l)-O(3) 110.1 (8) 0(4)-P(2)-0(6) 109.9 (9)
O(1)-P(l)-C 106.7 (9) 0(4)-P(2)-C 111.9 (10)
0(2)-P(I)-0(3) 11 1.1 (9) 0(5)-P(2)-0(6) 111.1 (8)
0(2)-P(I)-C 110.4 (IO) 0(5)-P(2)-C 106.6 (10)
0(3)-P(l)-C 103.4 (9) 0(6)-P(2)-C 108.4 (10)
P(I)-C-P(2) 110.7 (12)
Distances are in angstroms, angles are in degrees, and atom
identifications are as shown in Figures I and 2. Estimated standard
deviations of the last significant figure(s) are given in parentheses.
In a typical preparation 0.2 mmol of (NH4)2TcBr6 is dis-
solved in -10 mL of N,N-dimethylformamide and a ninefold
molar excess of H4MDP is added to the stirred solution. Within
a few minutes the solution changes from an orange-red to a
brown-gold color, and it is then heated at 80 OC with stirring
for -20 min. After dilution with -300 mL of aqueous chlo-
roacetate buffer solution, the pH is adjusted to 3.2. This so-
lution is loaded onto a column (1.25-cm-i.d. X 5.0 cm) of
anion-exchange resin (Bio-Rad, AG MP-1, 100-200 mesh,
chloride form) and the column rinsed with 0.05 M aqueous
chloroacetate buffer at pH 3.2. At this stage the brown product
is sorbed at the top of the column; passage of 0.5 M LiC104
through the column removes most of this material by what
appears to be a displacement phenomenon. Upon standing for
several weeks, small brown crystals of quality suitable for the
X-ray diffraction experiment appear in the acidified eluant
solution. A well-formed, but rather small (0.08 X 0.08 X 0.05
mm), cryst$ was subjected to X-ray diffraction analysis and
the structure was solved using standard Patterson and least-
squares analysis. Table I lists data pertinent to this experiment.
Full-matrix least-squares refinement using statistical weights
and varying (I) the overall scale factor, (2) all independent
positional parameters, (3) the lithium atom isotropic tem-
perature factors, and (4) anisotropic temperature factors for
all remaining atoms, converged at R = 0.077 (0.067 weighted).
The final difference map shows no features other than a ran-
dom background below 1.2 e/A3 and no indications of disorder.
Portions of the structure are shown in Figures 1 and 2, while
selected bond parameters are listed in Table 11.
The solid state structure of the Tc-MDP complex consists
of infinite polymeric chains. Each MDP ligand bridges two
symmetry related technetium atoms (Figure l), and each
Figure 1. Perspective view of a portion of the ([Li(H20)3][Tc(OH)-
(MDP)].'/3H20), structure showing one MDP ligand bridging two
technetium centers.
04"
A
Figure 2. Perspective view of a portion of the {[Li(H20)3][Tc(OH)-
(MDP)].'/3H20)n structure showing one Tc center bridging two MDP
ligands.
technetium atom is bound to two symmetry related MDP li-
gands (Figure 2)-the MDP/Tc ratio within the polymer is
therefore 1:l. The polymeric repeat unit is completed by an
oxygen atom (presumably in the form of an hydroxyl ion) that
bridges two symmetry-related technetium atoms (Figure 1)
and by an hydrated lithium cation which neutralizes the charge
associated with each repeat unit. In addition, there is a single
oxygen atom (presumably in the form of a disordered water
molecule) on the threefold axis of the space group. The mo-
lecular formula of the polymeric Tc-MDP complex may thus
be represented as { [Li(H20)3] [TC'~(OH)(MDP)].'/~H~O}~
where the indicated protonation states of the bridging and
noncoordinated oxygen atoms are chemically reasonable and
consistent with an assumed Tc(IV) oxidation state, but are not
definitively established by the X-ray diffraction data. Alter-
nate, equivalently reasonable, representations may be con-
structed about other technetium oxidation states and other
degrees of protonation of the bridging and noncoordinated
oxygen atoms, e.g. { [Li(H20)3] [TC'~(O)MDP)]-]/~H~O}~ or
{[Li(H20)31 [TC~(O)(MDP)I.'/~H~O~,.
Each technetium center has approximately octahedral
coordination geometry, the two bridging oxygen atoms oc-
cupying cis coordination sites. The bridging oxygen to tech-
netium bond distance (1.94 (2) A average) is generally in line
with observed Tc-0 single bond lengths (2.00-2.03 A),12
Tc=O double bond lengths being considerably shorter
(1.65-1.70 A).12 Bond lengths and bond angles within the
MDP ligand are generally as expected from structural studies
on the free acid H4MDPI6 and on related diphosphonate
One of the most important structural features of the
A B
14
carrier strategy that has been discussed as a way for targeted drug delivery in Section
1.3.1 (Figure 1.4B), only replacing the small molecule drugs with radio-imaging agents.
A lot of studies have successfully been done, and this approach is extended to a range of
imaging agents design, such as the DOTA-BP conjugates
51, 61
(DOTA represents
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Figure 1.6), in which the
DOTA group can complex with an appropriate metal ion or radiotracer (e.g. Gd(III),
Ln(III),
160
Tb,
90
Y,
67/68
Ga, et al.), and the complexes can function as imaging probes for
variety imaging technique applications, such as MRI, PET, SPECT, and SPECT-CT, as
well as possible therapeutic agents.
50
A recent paper reviews nicely the various
radionuclides as well as the different chelating groups capable for metal/radiotracer
complexation which can be conjugated with BP for targeted imaging or drug delivery
studies.
46
Figure 1.6 Examples of DOTA-BP conjugates
Considering the heath hazards of exposure to the above mentioned radionuclide
probes, fluorescence-based imaging agents were investigated, which is also a major topic
N N
N N
COOH
COOH HOOC
HOOC
DOTA
N N
N N
COOH HOOC
HOOC
HN
O
PO
3
H
2
PO
3
H
2
N N
N N
COOH HOOC
HOOC
O PO
3
H
2
PO
3
H
2
HN
BPAMD BPAPD
N N
N N
P
COOH HOOC
HOOC PO
3
H
2
PO
3
H
2
BPPED/DO3AP
BP
OH O
N N
N N
COOH HOOC
HOOC
O
PO
3
H
2
PO
3
H
2
HN
DOTA-HBP
HO
N N
N N
Linker
COOH HOOC
HOOC BP
BP-DOTA conjugates
... ...
15
in this dissertation. A short summary will be given here and detailed discussion will be
offered in Chapter 2.
Frangioni, et al. first introduced the pamidronate-based fluorescent probe, in
which pamidronate (Figure 1.1) was conjugated to IRDye78 (PAM78)
62
; they introduced
a similar probe (PAM800, pamidronate is conjugated with IRDye800CW) several years
later, which was synthesized from different chemistry.
63
These conjugates were observed
to bind to HAP and were applied in osteoclast imaging, breast cancer microcalcification
mapping, and atherosclerosis imaging.
62-65
It is noteworthy that their published chemistry
of synthesizing PAM800 is not reproducible by our hands.
Rogers et al. reported the synthesis of a fluorescently labeled analogue of
alendronate, AF-ALN, by coupling the primary aminoalkyl N-BP alendronate (Figure
1.1) to Alexa Fluor 488 via the γ-amino group.
66
Utilizing AF-ALN, they detected the
internalization route of bisphosphonate into cells and measured bisphosphonate
internalization by osteoclasts and non-resorbing cells.
67
However, the conjugated labeled
drug (AF-ALN) was obtained in only 7% purity.
In all of these cases, the imaging dyes were attached via an amide linkage at the
terminal amino group of the molecule, resulting in a large decrease in nitrogen basicity,
which would be expected to greatly lower the inhibitory potency of the resulting
conjugate. Notably, this linking approach is not applicable to the clinically utilized
heterocyclic N-BPs such as risedronate (Figure 1.1), which lack a primary amino group
susceptible to facile acylation by activated esters of fluorescent labels.
16
Our group at USC introduced the ‘magic linker’ methodology
68
which permits
attaching a fluorescent label to one of the modern, more potent, heterocyclic N-BPs,
risedronate (Figure 1.1). The synthesis is based on the use of a functionalized epoxide to
attach a universal linker group to the drug under mild reaction conditions (aqueous, near
neutral pH, 21–40 °C). The drug-linker is facilely conjugated to a suitable ester of a
fluorescent dye, to yield a fluorescent bisphosphonate imaging probe (Figure 1.7). The
method typically affords probes of high purity (greater than 98%) which are fully
characterized by UV–VIS absorption, fluorescent emission, NMR, HRMS, and HPLC. In
this dissertation, the method is successfully further developed for other N-heterocyclic
BPs, such as zoledronate, minodronate and their related analogues; a probe “toolkit” with
varieties of bone binding affinity, pharmacological activity, as well as spectroscopic
property is developed and has been successfully applied in a wide range of bone related
studies; the detailed discussion will be covered in Chapter 2-4.
Figure 1.7 “Magic linker” synthesis of fluorescent risedronate conjugates and related
analogues
1.3.3 Bisphosphonates as a bone targeting moiety in other related studies
Besides the targeted small molecule delivery and imaging, BPs are also applied in
N
PO
3
H
2
R
2 R
1
O
NHBoc
N
PO
3
H
2
R
2 R
1
5(6)-carboxyfluorescein
rt, in
darkness
O O HO
CO
2
H
O
HN
N
HO
PO
3
H
2
R
1
R
2
5(6)-FAM-RIS, R
1
= OH, R
2
= P(O)(OH)
5-FAM-RIS, R
1
= OH, R
2
= P(O)(OH)
2
6-FAM-RIS, R
1
= OH, R
2
= P(O)(OH)
2
5(6)-FAM-RISPC, R
1
= OH, R
2
= CO
2
H
5-FAM-RISPC, R
1
= OH, R
2
= CO
2
H
6-FAM-RISPC, R
1
= OH, R
2
= CO
2
H
5(6)-FAM-dRIS, R
1
= H, R
2
= P(O)(OH)
2
OH
NH
2
17
other studies which require specificity, such as protein delivery to osseous tissues.
41
While several endogenous proteins exhibit a significant binding affinity towards
biological apatite, the clinically useful recombinant proteins do not own this targeting
property; thus BP substitution of these proteins are proposed for protein targeting to the
bone.
41
Considering the possible denaturation of proteins under extreme conditions, such
as high temperature, pH extremes, or exposure to organic solvents, the ideal BPs for
protein conjugation should be water-soluble and with readily-reactive functional groups.
Three most common reacting moieties for protein chemistry are –NH
2
, -SH, and –COOH.
41
Interestingly, Uludağ, et al. found that direct modification of the –NH
2
group of amino-
bisphosphonates (pamidronate, alendronate) was not readily achieved in their hands,
although some groups reported that they could utilize this –NH
2
group of amino-
bisphosphonates for direct coupling to targeted molecules under aqueous conditions.
62, 69
Thus, Uludağ, et al. explored the bisphosphonates with active –SH, –COOH, and –NH-
NH
2
groups for protein coupling and the structures are summarizes in Figure 1.8.
41, 42, 54,
70, 71
18
Figure 1.8 Examples of bisphosphonates for protein conjugation.
The reactive sites for protein coupling are highlighted by orange (-SH group), pink (-
NHS group), blue (-NH-NH
2
group) and red (-COOH).
Compared with the molecular weight of BPs (<1 kDa), bioactive proteins have a
much bigger MW ranging from 5 kDa to 150 kDa; thus, to achieve effective
enhancement of protein targeting to the bone, multiple BP substitution of proteins is
desired, although Bachas, et al. recently reported a single hydrazine-BP conjugation of
PTH (1-34, a 34 amino acids peptide with MW of 4118) significantly enhanced the bone
affinity compared with unmodified PTH (1-34).
72
Depending on the BPs utilized and
protein sizes, different numbers of BPs are coupled to certain proteins; for example, a
maximum of 3-4 molecules of Di-BP (Figure 1.8) was attached to albumin (~ 66 kDa)
while maximum 6-7 molecules of Di-BP per IgG (~ 150 kDa); for the BP molecules such
as BP-NHS (Figure 1.8), a maximum number of 15-20 BP molecules are usually used for
albumin or IgG labeling.
41
The number of BP substitution is undoubtedly an important
factor for the bone mineral binding affinity, however, the correlation between the bone
H
2
O
3
P
H
2
O
3
P
NH
+
H
2
N
SH
H
2
O
3
P
H
2
O
3
P
NH
+
H
2
N
S S
O
O
N
O
O
Cl
-
Cl
-
H
2
O
3
P
H
2
O
3
P
NH
+
H
2
N
S
N
O
O
O
O
N
O
O
H
2
O
3
P
H
2
O
3
P
NH
+
H
2
N
S
N
O
O
NH
O
H
2
N
Cl
-
Cl
-
H
2
O
3
P
H
2
O
3
P
HN
SH
H
2
O
3
P
H
2
O
3
P
S
COOH
HN NH
COOH
HN NH
H
2
O
3
P
PO
3
H
2
PO
3
H
2
PO
3
H
2
O O
NH
H
N
H
2
O
3
P
H
2
O
3
P
PO
3
H
2
H
2
O
3
P
H
N
HN
H
2
O
3
P PO
3
H
2
PO
3
H
2
PO
3
H
2
H
2
O
3
P
H
2
O
3
P
S S S
SH
O
O
N
O
O
Di-BP Tetra-BP
BP-SH BP-NHS
BP-NH-NH
2
Dendritic BP-COOH
19
targeting efficiency after systemic delivery and the extent of BP substitution remains to
be fully elucidated.
41
Besides, the effect of R
1
and R
2
chains (Figure 1.1) of applied BP
molecule on the bone affinity of the final protein-BP conjugates is profound and needs to
be studied further.
41
It is worth to mention that in addition to the above direct conjugation with BP
approach, different strategies of targeted protein delivery to bone by BPs are also
explored. The “drug carrier” concept in Section 1.3.1 was also adopted for protein
delivery; for example, Uludağ, et al. introduced the drug delivery system composed with
collagen/hydroxyapatite composition scaffold (Col/HA) and BP-derivatized liposomes;
the system successfully encapsulated two small molecules as well as lysozyme (MW. ~
14 kDa).
52
It should be pointed out that this system could also function as the sustained
drug release platform. Bachas, et al. reported a modified amino-bisphosphonate in which
a biotin molecule was attached; this biotin-BP molecule generally retains the mineral
binding affinity, and the biotin functionality is still capable to be recognized by antibiotin
antibody.
73
Other studies using BPs’ specific mineral targeting property include the protein
immobilization on hydroxyapatite surface
74
, and the isolation of calcium phosphate
crystals from complex biological fluids by using BP-modified superparamagnetic beads.
75
In conclusion, the use of BPs as a specific targeting moiety in a wide range of
studies has been recognized at very early stage of BP development, and the interest in this
field has never faded, becoming even keener in the last few years. The strong and specific
20
mineral affinity of BPs makes them a “magic bullet” targeting the osseous tissues and
calcification related studies, which is also the stimulus and major topic of this dissertation.
1.4 References
1. Russell, R. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J., Mechanisms of action
of bisphosphonates: similarities and differences and their potential influence on clinical
efficacy. Osteoporos Int 2008, 19 (6), 733-59.
2. Russell, R. G. G., Bisphosphonates: The first 40 years. Bone 2011, 49 (1), 2-19.
3. Greenspan, S. L.; Harris, S. T.; Bone, H.; Miller, P. D.; Orwoll, E. S.; Watts, N.
B.; Rosen, C. J., Bisphosphonates: Safety and efficacy in the treatment and prevention of
osteoporosis. Am Fam Physician 2000, 61 (9), 2731-2736.
4. Frith, J. C.; Monkkonen, J.; Blackburn, G. M.; Russell, R. G. G.; Rogers, M. J.,
Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog,
adenosine 5'-(beta,gamma-dichloromethylene) triphosphate, by mammalian cells in vitro.
J Bone Miner Res 1997, 12 (9), 1358-1367.
5. Frith, J. C.; Monkkonen, J.; Auriola, S.; Monkkonen, H.; Rogers, M. J., The
molecular mechanism of action of the antiresorptive and antiinflammatory drug
clodronate - Evidence for the formation in vivo of a metabolite that inhibits bone
resorption and causes osteoclast and macrophage apoptosis. Arthritis Rheum 2001, 44 (9),
2201-2210.
6. Rogers, M. J.; Ji, X. H.; Russell, R. G. G.; Blackburn, G. M.; Williamson, M. P.;
Bayless, A. V.; Ebetino, F. H.; Watts, D. J., Incorporation of Bisphosphonates into
Adenine-Nucleotides by Amebas of the Cellular Slime-Mold Dictyostelium-Discoideum.
Biochem J 1994, 303, 303-311.
7. Lehenkari, P. P.; Kellinsalmi, M.; Napankangas, J. P.; Ylitalo, K. V.; Monkkonen,
J.; Rogers, M. J.; Azhayev, A.; Vaananen, H. K.; Hassinen, I. E., Further insight into
mechanism of action of clodronate: Inhibition of mitochondrial ADP/ATP translocase by
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21
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27
Chapter 2
Design and Synthesis of Fluorescent N-Heterocyclic
Bisphosphonate Probes
2.1 Background
Bisphosphonates (BPs, Figure 2.1) are therapeutic agents for treatment of bone
disorders such as osteoporosis and Paget’s disease and have also found use in the cancer
clinic.
1-3
Several nitrogen-containing bisphosphonates (N-BPs) and
phosphonocarboxylate
4, 5
(PC, Figure 2.1, 1b) analogues demonstrate antitumor effects
in vitro and in vivo. Although farnesyl diphosphate synthase (FPPS) has been clearly
established as the enzymatic target for N-BPs, details of the skeletal distribution, cellular
uptake and mechanisms of these drugs remain to be elucidated.
3
Furthermore, it is
reported that high-dose usage of some N-BPs in patients causes some side effects, such as
osteonecrosis of the jaw (ONJ)
6, 7
; several models have been proposed to explain the
mechanism of ONJ, however, none of them provide strong experimental support.
8-10
All
of these unsolved problems point to the need for special imaging probes which mimic
some or all of BPs or PCs’ pharmacological properties. Of these, the most important is
the affinity of the parent drugs for bone mineral; however, it is also of interest to retain at
least some of the cellular effects of the drug as well.
The P-C-P bond of BPs mimics the P-O-P bond of naturally-occurring inorganic
pyrophosphate, thus retaining strong binding affinity to hydroxyapatite (HAP), an
inorganic material found in bone, as well as giving the exceptional stability of BP
28
molecules against both chemical and biological degradation.
2
This specific bone-
targeting property of BPs makes them an ideal carrier to introduce desired drugs or
macromolecules to the bones in drug delivery studies.
11
In addition, due to their strong
and specific affinity to HAP, modified BPs with an appropriate imaging label can be used
as molecular indicator for calcium enrichment site, thus can find utilization of mapping
breast cancer microcalcification, calcium urolithiasis, and atherosclerosis.
12-14, 23
Compared with radioactive isotope-labeled imaging probes, fluorescent probes
are highly sensitive and don’t have potential long-term toxic effects
15
; especially the near
infrared (NIR) imaging probes with emission wavelength between 600-1000 nm are ideal
tools for in vivo imaging because the autofluorescence from the tissues will be minimized
in this optical window
16
; a refined Fluorescence-Assisted Resection and Exploration
(FLARE
TM
) imaging system was recently introduced and utilized in a first-in-human
testing in women undergoing sentinel lymph node mapping for breast cancer
17
; the
successful clinical translation of this system shows the advantages of NIR imaging for
image-guided oncologic surgery
18
, and also indicates the great potential of NIR imaging
in disease prognosis and monitoring treatment effects in time.
19
Thus, fluorescent probes
of bisphosphonates are of increasing interest as biological probes in bone diseases related
imaging studies.
In the literature, both alendronate
20, 21
and pamidronate
22, 23
based fluorescent
probes were developed; unpurified AF-ALN and F-ALN (alendronate-based imaging
probes labeled with Alexa Fluor 488
TM
and carboxyfluorescein, respectively) were
previously employed for cellular uptake studies but were unable to inhibit protein
prenylation, a key function of N-BP drugs.
20, 21
Similarly, near-IR analogues of the
29
alkylamino BP pamidronate, including Pam78, Pam800, and commercialized
OsteoSense
TM
680EX and 750EX (Perkin Elmer products), have also been visualized in
vitro and in vivo, but their pharmacological activity was not reported, and their synthetic
chemistry suffers from either low yields or a complicated purification procedure
22, 23
; the
chemistry of large scale synthesis of Pam800 was not reproducible in my hand. In general,
the imaging moieties of these examples are connected to the N-BPs by converting the
drug’s pharmacologically important terminal amino group into an amide linkage, thereby
altering the inhibitory effects of the resulting fluorescent BP. Furthermore, this type of
direct acylation to the label by the BP is not applicable to the more potent heterocyclic N-
BPs, such as risedronate (RIS, 1a), zoledronate (ZOL, 1d) and minodronate (MIN, 1e),
which lack a primary amino group necessary for facile acylation to activated esters of
fluorescent labels.
Figure 2.1 Heterocyclic N-BPs and related analogues in literature
Recently, our laboratory introduced a so-called ‘magic linker’ synthesis of
FAMRIS and related analogues
24
(Scheme 2.1, route A), the first example of
fluorescently labeled heterocyclic N-BP, formed from RIS (1a) and related
phosphonocarboxyl (PC, 1b) analogues with 5(6)-carboxyfluorescein (8). The synthesis
, R
1
= OH, R
3
= P(O)(OH)
2
1a: RIS, R
2
=
N
1b: RISPC R
2
= , R
1
= OH, R
3
= CO
2
H
N
N
, R
1
= H, R
3
= P(O)(OH)
2
1c: dRIS R
2
=
1d: ZOL, R
2
=
1e: MIN, R
2
=
1f: MINPC, R
2
=
N
N
N
N
N
N
, R
1
= OH, R
3
= P(O)(OH)
2
, R
1
= OH, R
3
= P(O)(OH)
2
, R
1
= OH, R
3
= CO
2
H
R
2
P
R
1
R
3
O
OH
OH
1a-1f
30
centered on an important linking step, using a synthesized epoxide (5) to introduce a
universal linker group to drug in an exceptionally mild (pH near neutral, aqueous
conditions, 40 ̊C) and regioselective reaction condition. After typical deprotection of
compounds 2a-2c, the resulting drug-linker complexes 4a-4c advantageously included a
primary amine for facile conjugation to any activated esters of any imaging agent, a
positively charged pyridinium nitrogen that may mimic the carbocation intermediate
25
in
enzymatic catalysis, and an additional hydroxyl group that may counteract the addition of
the the hydrophobic alkyl chain.
It would be desirable to extend the family of fluorescent bisphosphonates to other
N-BPs and their analogues with different chemical functionalities and those capable to
emit different wavelength fluorescence, thus generating a fluorescent bisphosphonate
‘toolkit’ which will open the doors to biological experiments where different BPs and PC
compounds may be visualized within the same assay and may also find new fields to use
as imaging tools. Herein, in this dissertation, the simplified, adaptable and high-yield
synthesis of a fluorescent bisphosphonate probe ‘toolkit’ is described which is based on
all the heterocyclic N-BPs and their important analogues in current literature. Our ‘toolkit’
has already found versatile applications in various bone imaging studies, including ONJ
mechanism and otoscleorosis studies, which will be discussed in detail in Chapter 4.
31
Scheme 2.1 Synthesis of fluorescent bisphosphonate ‘toolkit’.
I(i): ~5% MeOH/H
2
O, 40-50 ̊C; I(ii): 1:1 TFA/H
2
O, r.t.; II(i): H
2
O, r.t.; II(ii): NH
3
⋅H
2
O, r.t.; (iii):
FAM, SE (8), RhR-X, SE (9), ROX, SE (10), AF647, SE (11) or IRDye 800CW, SE (12), Sulfo-
Cy5, SE (13), NaHCO
3
/DMF, pH 8.3 - 9.0, r.t., in darkness (SE: succinimidyl ester).
2.2 Results and discussion
The overall synthetic route for fluorescent bisphosphonate “toolkit” is depicted in
Scheme 2.1. Bisphosphonates (1a-1f) were conjugated with an epoxide (5 or 6) via two
different routes (A and B), yielding the BP-linker intermediates, which were then reacted
with the activated ester of a range of fluorescent dyes (commercially available
O
NHBOC
R
2
P
R
1
R
3
O
OH
OH
I(i)
R
2
P
R
1
R
3
O
OH
OH
OH
NHBOC
I(ii)
R
2
P
R
1
R
3
O
OH
OH
OH
H
2
N
R
2
P
R
1
R
3
O
OH
OH
O
Cl
II(ii)
R
2
P
R
1
R
3
O
OH
OH
OH
Cl
N
+
P
R
3
R
2
O
OH
OH
OH
NH
N
+
N
P
R
3
R
2
O
OH
OH
OH
NH
N
N
+
P
R
3
R
2
O
OH
OH
OH
NH
R
4 R
4 R
4
N
O
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
HO
3
S SO
3
H
O N
CO
2
H
N
O
O O HO
CO
2
H
O
O N N
SO
3
H
SO
2
NH
N
HO
3
S
N
+
SO
3
H
O
SO
3
H
SO
3
H O
Carboxyfluorescin
(FAM)
Carboxy-X-Rhodamine
(ROX)
Rhodamine Red-X
(RhR-X)
Alexa Fluor 647
(AF647)
IRDye 800CW
(800CW)
7a1: 5(6)-FAM-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= FAM
7a2: 5-FAM-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 5-FAM
7a3: 6-FAM-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 6-FAM
7a4: 5(6)-RhR-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= RhR-X
7a5: 5(6)-ROX-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= ROX
7a6: AF647-RIS. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= AF647
7b1: 5(6)-FAM-RISPC. R
2
= OH, R
3
= CO
2
H, R
4
= FAM
7b2: 5(6)-RhR-RISPC. R
2
= OH, R
3
= CO
2
H, R
4
= RhR-X
7b3: 5(6)-ROX-RISPC. R
2
= OH, R
3
= CO
2
H, R
4
= ROX
7b4: AF647-RISPC. R
2
= OH, R
3
= CO
2
H, R
4
= AF647
7c1: 5(6)-FAM-dRIS. R
2
= H, R
3
= P(O)(OH)
2
, R
4
= FAM
7c2: 5(6)-RhR-dRIS. R
2
= H, R
3
= P(O)(OH)
2
, R
4
= RhR-X
7d1: 5-FAM-ZOL. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 5-FAM
7d2: 6-FAM-ZOL. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 6-FAM
7d3: AF647-ZOL. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= AF647
7d4: 800CW-ZOL. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 800CW
7d5: Sulfo-Cy5-ZOL. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= Sulfo-Cy5
7e1: 5-FAM-MIN. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 5-FAM
7e2: 6-FAM-MIN. R
2
= OH, R
3
= P(O)(OH)
2
, R
4
= 6-FAM
7f1: 5-FAM-MINPC. R
2
= OH, R
3
= CO
2
H, R
4
= 5-FAM
7f2: 6-FAM-MINPC. R
2
= OH, R
3
= CO
2
H, R
4
= 6-FAM
R
1
=
N
R
1
= R
1
=
N
N
N
N
, R
1
= OH, R
3
= P(O)(OH)
2
a R
2
=
N
b R
2
= , R
1
= OH, R
3
= CO
2
H
N
N
, R
1
= H, R
3
= P(O)(OH)
2
c R
2
=
d R
2
=
e R
2
=
f R
2
=
N
N
N
N
N
N
, R
1
= OH, R
3
= P(O)(OH)
2
, R
1
= OH, R
3
= P(O)(OH)
2
, R
1
= OH, R
3
= CO
2
H
1a-1f
2a-2f
3a-3f 4a-4f
1a-1f
5 6
7a-7c 7d
7e-7f
Route A
Route B
II(i)
iii
N
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
O
Sulfo-Cy5
75-95% Quant.
Quant. 75-85%
50-80%
O
32
succinimidyl ester form was applied), to afford the final fluorescent bisphosphonate
imaging probes.
2.2.1 Synthesis of BP-linker intermediates via Route A
The basicity and nucleophilicity of the nitrogen atom in heterocyclic N-BPs varies
between each other
26
, thus the ‘magic linker’ methodology for RIS requires some
adaptation to ZOL (1d), MIN (1e) and their related analogues (1f). When the heteroatom
of aromatic heterocyclic rings is substituted (e.g. the nitrogen of pyridine; and the N3
atom of imidazole (the lone electron pair of N1 is part of the sextet of π-electrons for
aromaticity, thus N1 is non-basic nitrogen), see Figure 2.2), there is almost no reactivity
on the heteroatom towards electrophiles, due to lack of lone electron pair. Although
basicity and nucleophilicity are different concept, and pKa is often used to evaluate
basicity, it is also a useful quantitative indication of the tendency to undergo electrophilic
attack on heteroatom such as nitrogen. Thus one could imagine that the protonation status
of the heteroatom at different pH conditions is essential to the reactivity.
Figure 2.2 Heteroatom reactivity in aromatic heterocyclic rings
For example, the pKa of heterocyclic nitrogen of RIS, ZOL and MIN are 5.65,
N
N
NH
pyridine
N
pyridinium
cation
imidazole
R
1
2
3
4
5
reacts
fairly readily
no reaction
(no lone pair)
nitrogen atoms
are different
14
15 16
33
6.67, 6.54
*
; in previous ‘magic linker’ synthesis of RIS
24
, the pH of reaction mixture for
linking step was adjusted to 6.0, under which more than 50% (~ 71.3% calculated) of the
nitrogen of RIS will be deprotonated thus have better reactivity towards the opening of
the epoxide ring. However, at pH 6.0, only 17.7% of the nitrogen of ZOL is deprotonated,
while 22.4% for MIN and its related analogues
**
; the concentration of reactive species
(deprotonated) is not enough for the reaction, and our results showed that the linking
reaction was very slow for ZOL-linker (4d) synthesis at pH 6.0. Thus we tried pH 7.2-7.8,
and reaction went faster as expected; however, compared with RIS under same reaction
temperature and reactant ratio, the reaction for ZOL is still slower; in addition,
regioselectivity (N-alkylation or O-alkylation) of reactions between the epoxide 5 and
ZOL, MIN or related analogues (1d, 1e, 1f) is not as good as RIS and its analogues (1a,
1b, 1c); 10-20% of side products could be observed (Figure 2.3), which were confirmed
later by HPLC, NMR and MS to be the products of N,O(P)-dialkylation compounds (O-
alkylation occurs between phosphonate/carboxylate group of the drug and the epoxide)
(Figure A11-13.). The reaction rate of MinPC (1f)-epoxide (5) coupling is even slower
than ZOL at the same reaction condition (same reactant ratio, pH 7.5, 40-45 ̊C), which
might be due to the steric effect; in addition, the percent of side products was higher and
O(P)-monoalkylation, O(C)-monoalkylation, N, O(C)-dialkylation product were also
observed in the reaction mixture (Figure 2.4).
*
pKa of ZOL and MIN were calculated by MarvinSketch and the error is ±0.5.
**
The protonation percentage of each compound is calculated based on their estimated pKa values by
MarvinSketch.
34
Figure 2.3 Reaction study of ZOL (1d) and 5.
(A)
31
P NMR of reaction mixture of ZOL (1d) and 5 via Route A (in D
2
O, pH 7.5) (Heating at 50
̊C for 41 hrs then deprotect t-Boc group in TFA:H
2
O (1:1)). (B) HPLC separation of reaction
mixture of ZOL (1d) and 5 through a strong anion exchange (SAX) column (Macherey-Nagel
21.4 mm x 250 mm SP15/25 Nucleogel column), eluted with A: H
2
O, B: 0.5 M TEAB pH 7.5
using a gradient that was increased from 0-30% over 10 min, maintained at 30% from 10-15 min,
and then increased to 100% of buffer B from 15-35 min using a 9 mL/min flow rate; UV-VIS
detection set at 230 nm.
N
N
P
P
HO
O
OH
OH
O
OH
OH
P2
P3
P1
P2
P1
P3
A
B
N
N
P
P
HO
O
OH
OH
O
OH
OH
OH
NH
2
N
N
P
P
HO
O
O
OH
O
OH
OH
OH
NH
2
HO
NH
2
35
Figure 2.4
31
P NMR of reaction mixture of MinPC (1f) and 5 via Route A.
(1f : 5 (eq.) = 1 : 3, in D
2
O, pH 7.5, heating at 45 ̊C for 48 hrs).
In order to accelerate the reaction and reduce side products for Route A (Scheme
1), the effects of various factors, such as reactant ratio, pH and temperature, were
investigated; however, no optimal reaction condition was found to achieve >95%
regioselectivity and fast reaction rate; generally, more equivalences of epoxide linker (5)
result in higher reaction rate as well as higher side product percentage; higher
temperature can speed up the reaction, but also increase the side product percentage; at
pH below the pKa value, reactions are very slow and the optimal pH value is between
7.4-8.0, but there is still 10-15% of side products. Finally, the condition with a reactant
ratio (drug : linker (5) = 1 : 2.5), at 50 ̊C was applied, and after 41 hrs, ~85% of desired
drug-linker intermediate (4d) yielded with 15% of side products.
N
N
P
C
HO
O
OH
OH
O
OH
N
N
P
C
HO
O
OH
OH
O
OH
OH
NHBoc
N
N
P
C
HO
O
OH
OH
O
O
OH
NHBoc
NHBoc
OH
N
N
P
C
HO
O
OH
OH
O
O
NHBoc
OH
N
N
P
C
HO
O
O
OH
O
OH
OH
NHBoc
OH
NHBoc
N
N
P
C
HO
O
O
OH
O
OH
OH
NHBoc
36
2.2.2 Synthesis of BP-linker intermediates via Route B
In addition, a new epoxide linker, the commercially available epichlorohydrin (6),
was also explored (Route B, Scheme 2.1). Compared with the previous epoxide linker (5),
epichlorohydrin (6) is more water-soluble and more reactive. In this route (Scheme 2.1,
route B), epichlorohydrin was reacted with drugs (1a-1f), yielding intermediates 3a-3f,
which were then ammonolyzed to give final drug-linker compounds (4a-4f). The results
showed that the linking reaction (Scheme 1, 1a-1f→3a-3f) went faster compared with the
previous route (Scheme 1, 1a-1f→2a-2f) under the same reactant ratio and pH even at r.t.;
however, the percentage of side products for 1d-1f was similar as in route A; compared
with the high regioselectivity of route A for 1a-1c, route B for 1a-1c results in more side
products. Therefore, we decided to keep route A for syntheses of 4a-4c, while use route B
for syntheses of 4d-4f. Finally, a reactant ratio of 1:5 (drug : linker (6)) with appropriate
pH (pH 7.4-8.0) was adopted; in around 20 hrs, 85-90% of 4d-4f formed at r.t..
Microwave heating was also applied to optimize the linking reaction, however, no
improvement on regioselectivity was observed.
It is interesting that Demberelnyamba, et al. reported the reaction of
pyridine/imidazole/N-methylimidazole and epichlorohydrin in MeCN to make ionic
liquids
27
; instead of the epoxide ring-opening, the heterocyclic nitrogen displaced the
chlorine in epichlorohydrin, generating product 17-19 (Figure 2.5 A-C). Our experiment
of pyridine with epichlorohydrin reaction confirmed the product 17 by MS, although the
NMR data is different from a literature report that may be questionable (Figure 2.5D).
37
Figure 2.5 Reactions of pyridine/imidazole/N-methylimidazole with epichlorohydrin.
A-C: reaction scheme. D: Characterization data of 17 from literature and our experiment.
Different methods were tried to purify 4d-4f. The most straightforward one is to
convert corresponding N(O)-dialkylation side products to 4d-4f, however, hydrolysis of
the N(O)-dialkylation side products did not succeed under both acidic (concentrated HCl,
HBr) and basic conditions (NaOH), indicating the phosphate monoester is resistant to
hydrolysis as reported in literature, and harsh conditions or catalysts are usually needed
for hydrolytic process
28-32
; hydrolysis by BTMS did not succeed either, due to the poor
solubility of 4d-4f in BTMS and organic solvents. Hence the anion exchange HPLC was
applied to obtain pure 4d-4f for conjugation with fluorescent dyes in the following step
(Figure 2.3B).
2.2.3 Syntheses of fluorescent BP imaging probes
A series of fluorescent dyes with distinguishable emission spectra, including three
near infrared dyes, Alexa Fluor
®
647 (AF647), Sulfo-Cy5 and IRDye
®
800CW (800CW),
was selected to generate the fluorescent bisphosphonate probe ‘toolkit’. The conjugation
occurs between drug-linker intermediates (4a-4f) and succinimidyl ester (SE) of
fluorescent dyes; reaction conditions (Scheme 2.1, step iii) for different fluorescent dyes
17: literature data 17: our data
MS: m/e = 136 m/e = 136
NMR (
1
H, in D2O):
8.92-8.85 (m, 2H, ring);
8.66-8.59 (m, 1H, ring);
8.16-8.10 (m, 2H, ring);
5.11-4.66 (m, 1H, CHOCH2);
3.80-3.63 (m, 2H, CHOCH2);
1.2-1.17 (d, 2H, CH2-N).
8.73-8.78 (m, 2H, ring);
8.45-8.53 (m, 1H, ring);
7.96-8.02 (m, 2H, ring);
5.00-6.06 (dd, 1H, CH-N);
4.40-4.47 (dd, 1H, CH-N);
3.50-3.57 (m, 1H, CHOCH2);
2.95-2.98 (dd, 1H, CHOCH2);
2.66-2.68 (dd, 1H, CHOCH2).
D
N
O
Cl N
O
+
MeCN
r.t. 24 hrs
A
B
C
17
N
NH
O
Cl
MeCN
r.t. 24 hrs
+
N
N
O
N
N
O
Cl
MeCN
reflux, 12 hrs
+
N
N
18
O
19
Cl
-
Cl
-
38
are similar with minor modification of reactant ratio and reaction pH; the reaction is very
fast and can be monitored by TLC (100% MeOH as eluent). To obtain pure fluorescent
bisphosphonate probes (7a-7f), chromatography methods were used. Except
carboxyfluorescein (FAM, 8) and Rhodamine X-Red (RhR-X, 9) conjugates which need
TLC (100% MeOH as eluent) to remove free dye labels from reaction mixture, all the
other fluorescent conjugates could be purified by a single reverse phase HPLC (see
details in experimental section), with both unreacted drug/drug-linker (4a-4f) and free
dye labels removed. All the final fluorescent conjugates are fully characterized by HPLC,
UV-VIS and fluorescence emission spectroscopy,
1
H and
31
P NMR and high-resolution
MS (see details in experimental section). It is noteworthy that 5- and 6- isomers of
carboxyfluorescein (FAM, 8) conjugates can either be directly synthesized from their
respective isomerically pure FAM, SE starting materials, or be separated from mixed
isomers of FAM conjugates (Figure 2.6), and the latter is obviously more cost efficient.
Figure 2.6 Preparative reverse phase HPLC separation of 5(6)-FAM-ZOL mixture through
a semi-preparative C18 column.
(Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size), eluted with A: 0.1 M
TEAC, 10% MeOH, pH 7.0, B: 0.1 M TEAC, 75% MeOH, pH 7.8 using a gradient that was
increased from 0-40% of buffer B over 25 min, and then increased to 70% of buffer B from
25-100 min using a 4 mL/min flow rate; UV-VIS detection set at 230 nm and then 492 nm
after ZOL/ZOL-Linker (1d/4d) was eluted.
Chrom. 1 0.0 mins. 60.2 mins.
1
6-FAM-ZOL (7d2) 5-FAM-ZOL (7d1)
ZOL/ZOL-Linker(1d/4d)
39
2.2.4 Spectroscopic properties
The above probes fluoresce across a wide optical wavelength window (Table 2.1),
and their spectroscopic properties are summarized in Table 2.1. The diverse
spectroscopic properties make possible the simultaneous detection of individual low and
high affinity BPs and PCs in bone tissues and cells.
Table 2.1 Spectroscopic properties of fluorescent bisphosphonate probes
Probes
Maximum
absorption
wavelength
(λmax (abs),
nm)
[a]
Maximum
emission
wavelength
(λmax (em),
nm)
[a]
Extinction
coefficient
(M
-1
cm-
1
)
[b], [c]
5(6)-FAM-RIS
(7a1)
493 518
73,000 (pH 7.2)
5-FAM-RIS
(7a2)
493 521 73,000 (pH 7.2)
6-FAM-RIS
(7a3)
493 517 73,000 (pH 7.2)
5(6)-RhR-RIS
(7a4)
567.5 589 114,850 (pH 7.5)
5(6)-ROX-RIS
(7a5)
580 606 72,000 (pH 8.0)
AF647-RIS
(7a6)
648 666 240,000 (pH 7.0)
5(6)-FAM-
RISPC (5(6)-
FAM-3-PEHPC,
7b1)
493 518 73,000 (pH 7.2)
5(6)-RhR-RISPC
(5(6)-RhR-3-
PEHPC, 7b2)
568 589 114,850 (pH 7.5)
5(6)-ROX-
RISPC (5(6)-
ROX-3-PEHPC,
7b3)
579 606 72,000 (pH 8.0)
AF647-RISPC
(AF647-3-
PEHPC, 7b4)
648 666 240,000 (pH 7.0)
5(6)-FAM-dRIS
(7c1)
493 518 73,000 (pH 7.2)
40
5(6)-RhR-dRIS
(7c2)
567.5 589 114,850 (pH 7.5)
5-FAM-ZOL
(7d1)
493 521 73,000 (pH 7.2)
6-FAM-ZOL
(7d2)
493 516 73,000 (pH 7.2)
AF647-ZOL
(7d3)
648.5 666 240,000 (pH 7.0)
800CW-ZOL
(7d4)
774 789 240,000 (pH 7.0)
Sulfo-Cy5-ZOL
(7d5)
644 663 271,000 (1×
PBS, pH 7.4)
5-FAM-MIN
(7e1)
493 522 73,000 (pH 7.2)
6-FAM-MIN
(7e2)
493 518 73,000 (pH 7.2)
5-FAM-MINPC
(5-FAM-3-
IPEHPC, 7f1)
493 522 73,000 (pH 7.2)
6-FAM-MINPC
(6-FAM-3-
IPEHPC, 7f2)
493 517 73,000 (pH 7.2)
[a] There is ± 1nm error of λmax (abs) and λmax (em).
[b] The extinction coefficient of each probe is assumed the same as its
corresponding fluorescent dye.
[c] Unless specified, 0.1 M phosphate buffer is used for all the
measurements.
2.3 Conclusion
In conclusion, we have successfully synthesized a fluorescent bisphosphonate
probe ‘toolkit’. A linking strategy with two routes was applied to all the heterocyclic N-
BPs and related analogues in literature under exceedingly mild condition. Route A was
previously demonstrated for the synthesis of carboxyfluorescein-risedronate and related
conjugates (FAM-RIS), and further developed for preparation of new near-infrared
risedronate conjugates, fluorescent zoledronate, minodronate and related analogues.
Route B adopted a commercially available epoxide linker, epichlorohydrin, which
41
enhanced the reaction rate significantly. The linking strategy via both routes introduced a
terminal amino group in drug-linker intermediates capable to conjugate with the
commercially available activated ester of fluorescent dyes; in addition, the permanent
positive charge on heterocyclic nitrogen may mimic a putative carbocation-like transition
state analogue in the active site of FPPS, providing the possibility to retain the
pharmacological activities of parent BP drugs (see details in Chapter 3); furthermore, the
hydroxyl group from epoxide ring-opening could balance the hydrophobic alkyl chain as
well as offer the potential to convert to other functionalities (see details in Chapter 5). All
the fluorescent probes were prepared in good yield (50-77%) and high purity (> 95%),
and fully characterized by HPLC, UV-VIS and fluorescence emission spectroscopy,
1
H
and
31
P NMR and high-resolution MS (See characterization data and spectra in
Experimental section and Appendix A). The ‘toolkit’ contains a series of fluorescent
probes with diverse spectroscopic and pharmacological properties, providing versatile
tools for different purposed bone related imaging studies.
2.4 Experimental
2.4.1 General
Reagents and Spectral Measurements: 5(6)-, 5-, and 6-carboxyfluorescein,
succinimidyl ester (FAM, SE) were purchased from Sigma Aldrich or Invitrogen, US.
5(6)-Rhodamine Red-X, SE (5(6)-RhR, SE), 5(6)-carboxy-X-Rhodamine, SE (5(6)-ROX,
SE) and Alexa Fluor 647, SE (AF647, SE) were purchased from Invitrogen, US; sulfo-
Cy5, SE was purchased from Lumiprobe, US, and IRDye
®
800CW, SE was purchased
from LI-COR Biosciences, US. Compounds 1a-1c were kind gifts from Warner Chilcott
42
Pharmaceuticals (former P&G Pharmaceuticals). Compound 1d (zoledronic acid) was
purchased from Molekula, UK. Compound 1e (minodronic acid) was purchased from
Shanghai Hengrui International Trading Co. LTD, PRC. Compound 1f (3-IPEHPC) was
synthesized in our lab according to a published procedure.
33
All other compounds were
purchased from Aldrich or Alfa Aesar. Triethylamine (TEA) was distilled from KOH;
CH
2
Cl
2
was distilled from P
2
O
5
; and allylamine was distilled under N
2
. All other
compounds were used as supplied by the manufacturer. Thin layer chromatography was
performed on Merck Silica Gel 60 F
254
plates, and the developed plates were visualized
under a UV lamp at 354 nm. HPLC separations were performed on a Rainan Dynamax
Model SD-200 system with a Rainan Dynamax absorbance detector Model UV-DII.
NMR spectra were recorded on either 400 MHz Varian, 500 MHz Varian, 600 MHz
Varian or 500 MHz Bruker spectrometers. UV spectra were recorded on a DU 800
spectrometer, and fluorescence emission spectra were taken on either a Jobin Yvon
Horiba FluoroMax-3 fluorimeter equipped with a DataMax Software version 2.20 (Jobin
Yvon Inc), Jobin Yvon Nanolog fluorimeter (Jobin Yvon Inc), SHIMADZU
spectrofluorophotometer RF-5301PC, or PTI QuantaMaster model C-60SE Spectrometer
equipped with a 928 PMT detector. High resolution mass spectra were performed by Dr.
Ron New at UC Riverside High Resolution Mass Spectrometry Facility on a PE
Biosystems DE-STR MALDI TOF spectrometer with a WinNT (2000) Data System.
Other mass spectra were taken on ESI Thermo-Finnigan LCQ DECA XPmax Ion Trap
LC/MS/MS spectrometer.
43
2.4.2 Synthesis of drug-linker intermediates 4a-4c via route A
24
2.4.2.1 Synthesis of epoxide linker 5 (tert-butyl (oxiran-2-ylmethyl)carbamate)
34
Scheme 2.2 Synthetic route of epoxide linker 5 (tert-butyl (oxiran-2-
ylmethyl)carbamate)
Freshly distilled allylamine (2.3 mL, 30 mmol, 1.0 eq) in 10 mL dry CH
2
Cl
2
was
cooled in an ice bath (0 °C). To this cold solution, 6.54 g di-tert-butyl dicarbonate (30.0
mmol, 1.00 eq) in 20 mL dry CH
2
Cl
2
was added. The solution was brought to r.t. and
stirred for 4 h. The reaction mixture was then diluted with additional CH
2
Cl
2
(25 mL) and
washed with 5% citric acid solution, followed by brine. The organic layer was dried over
Na
2
SO
4
and concentrated in vacuo, yielding 3.39 g (73%) of the tert-butyl allylcarbamate.
1
H NMR (CDCl
3
): δ 5.72- 5.84 (m, 1H), 5.02-5.20 (m, 2H), 4.70 (brs, 1H), 3.75 (br, 2H),
1.28 (s, 9H).
The tert-butyl allylcarbamate (1.0 g, 6.4 mmol, 1.0 eq) was dissolved in 50 mL
dry CH
2
Cl
2
. The solution was cooled to 0 °C and kept cold during addition of 2.8 g of 3-
chlorobenzenecarboperoxoic acid (MCPBA, commercially available as 77% pure; 12
mmol, 1.9 eq). The solution was then brought to r.t. and stirred overnight. The reaction
mixture was then diluted with additional CH
2
Cl
2
(80 mL). The solution was washed with
10% Na
2
SO
3
, followed by washing with saturated NaHCO
3
(3×), and finally by washing
with water. The organic layer was dried over Na
2
SO
4
and concentrated in vacuo, yielding
crude epoxide 5. According to the
1
H NMR spectrum, the yield was 88%.
1
H NMR
NH
2
H
N
O
O
H
N
O
O
O
(Boc)
2
O
CH
2
Cl
2 CH
2
Cl
2
MCPBA
44
(CDCl
3
): δ 4.65 (brs, 1H), 3.53 - 3.42 (m, 1H), 3.21 - 3.09 (m, 1H), 3.07 – 2.98 (m, 1H),
2.72 (dd, J = 4.7, 4.0 Hz, 1H), 2.53 (dd, J = 4.7, 2.6 Hz, 1H), 1.38 (s, 9H).
2.4.2.2 Synthesis of drug-linker intermediates 4a-4c
General procedure: the parent drug (1a-1c) is dissolved in water and the pH
adjusted to 5.7-6.0 with 1 M NaOH. Epoxide 5 is dissolved in minimal methanol (MeOH)
and added to the water solution, causing a slight precipitation to occur. The precipitate
disappears on heating (40-50 °C) and as the reaction progresses. The reaction is
monitored by
31
P NMR. After 90-95% of the desired product is obtained (
31
P NMR), the
solvent is removed in vacuo, and the resulting white powder is washed with diethyl ether,
filtered, and dried in a dessicator. Standard deprotection is performed with 1:1
trifluoroacetic acid (TFA): H
2
O. After the reaction mixture is stirred for 3-4 h at r.t., the
solvent is removed in vacuo, and the resulting crystals are washed with diethyl ether and
MeOH to yield the drug-linker intermediates.
Synthesis of 1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-
diphosphonoethyl)pyridinium (4a):
The monosodium salt of (1-hydroxy-2-pyridin-3-ylethane-1,1-
diyl)bis(phosphonic acid), 1a (288 mg, 0.94 mmol, 1.00 eq), was dissolved in 4 mL water,
and the pH adjusted to 6.2 with 1 M NaOH. To this solution, 164 mg of 5 (0.94 mmol,
1.00 eq) in minimal MeOH was added. The reaction mixture was stirred at 40 °C for 18.5
h, yielding 90% of 2a by
31
P NMR. The solvent was removed in vacuo, and the residue
was washed with diethyl ether, filtered, and dried in a desiccator. 2a, a white solid, was
then used without further purification.
1
H NMR (D
2
O): δ 8.68 (s, 1H), 8.46 (d, J = 6.3 Hz,
1H), 8.42 (d, J = 8.1 Hz, 1H), 7.78 (dd, J = 8.2, 5.8 Hz, 1H), 4.67 – 4.62 (part. obscured
45
by HDO, about 1H), 4.27 (dd, J = 13.6, 9.6 Hz, 1H), 4.13 – 3.92 (m, 1H), 3.41 – 3.10 (m,
4H), 1.31 (s, 9H).
31
P NMR (D
2
O) δ 16.55 (d, J = 21.7s Hz, 1P), 16.33 (d, J = 21.9 Hz,
1P).
The entire sample of 2a was dissolved in 50:50 water:TFA (v/v). After the
solution was stirred at r.t. for 3 h, a 100% yield of 4a was achieved according to
1
H NMR.
The solvent was then removed in vacuo, and the resulting solids were washed with ether,
filtered, and dried, yielding 4a as white crystals, which were used without further
purification.
1
H NMR (D
2
O): δ 8.71 (s, 1H), 8.54 (d, J = 6.0 Hz, 1H), 8.44 (d, J = 8.1 Hz,
1H), 7.84 (dd, J = 8.1, 6.0 Hz, 1H), 4.74 (part. obscured by HDO, about 1H), 4.41 – 4.21
(m, 2H), 3.39 – 3.21 (m, 3H), 2.96 (dd, J = 13.0, 9.9 Hz, 1H).
31
P NMR (D
2
O): δ 16.35 (d,
J = 26.4 Hz, 1P), 16.04 (d, J = 27.7 Hz, 1P).
Synthesis of 1-(3-amino-2-hydroxypropyl)-3-(2-carboxy-2-hydroxy-2-
phosphonoethyl)pyridinium (4b):
Compound 1b (0.52 g, 2.10 mmol, 1.00 eq), 2-hydroxy-2-phosphono-3-pyridin-3-
ylpropanoic acid, was dissolved in 10 mL water, and the pH adjusted to 5.9 with 1 M
NaOH. To this solution, 0.45 g of 5 (2.57 mmol, 1.22 eq) in minimal MeOH was added.
The reaction mixture was stirred at 50 °C for 6 h and then stirred at r.t. overnight,
yielding 90% of 2b (
31
P NMR). The solvent was removed in vacuo, and the residue was
washed with diethyl ether, filtered, and dried in a desiccator, leaving 2b
(diastereoisomeric mixture), which was then used without further purification.
1
H NMR
(D
2
O): δ 8.53 - 8.49 (brd, 1H), 8.47 (d, J = 6.0 Hz, 1H), 8.29 - 8.24 (m, 1H), 7.78 (dd, J =
8.3 Hz, 6.2 Hz, 1H), 4.64 – 4.58 (brd, 1H), 4.27 – 4.19 (m, 1H), 4.00 – 3.91 (m, 1H), 3.49
– 3.43 (m, 1H), 3.23 – 3.00 (m, 3H), 1.27 (s, 9H).
31
P NMR (D
2
O): δ 14.97 (s, 1P).
46
The entire sample of 2b was dissolved in 50:50 water:TFA (v/v). After stirring at
r.t. for 4 h, a 100% yield of 4b was obtained according to
1
H NMR. The solvent was then
removed in vacuo, and the residue was washed with diethyl ether, filtered, and dried,
yielding 4b (a diastereoisomeric mixture) as white crystals, which were used without
further purification.
1
H NMR (D
2
O): δ 8.68 – 8.64 (m, 1H), 8.63 – 8.59 (m, 1H), 8.42 –
8.39 (m, 1H), 7.91 (dd, J = 8.0 Hz, 6.4 Hz, 1H), 4.78 – 4.72 (m, 1H), 4.46 – 4.33 (m, 1H),
4.24 – 4.14 (m, 1H), 3.59 – 3.49 (m, 1H), 3.33 – 3.21 (m, 2H), 2.95 (ddd, J = 13.3, 10.0,
3.6 Hz, 1H).
31
P NMR (D
2
O): δ 12.73 – 12.51 (m, 1P).
Synthesis of 1-(3-amino-2-hydroxypropyl)-3-(2,2-diphosphonoethyl)pyridinium
(4c):
Compound 1c (38.0 mg, 0.14 mmol, 1.00 eq), (2-pyridin-3-ylethane-1,1-
diyl)bis(phosphonic acid), was dissolved in 1 mL water and the pH adjusted to 5.4 with 1
M NaOH. To this solution was added 25.5 mg of 5 (0.15 mmol, 1.07 eq) in minimal
MeOH. The reaction mixture was stirred at 40 °C overnight, and the reaction was
monitored by
31
P NMR. After 19 h, 80% of 2c yielded. Thus, an additional 5.30 mg (0.03
mmol, 0.21 eq) of 5 in MeOH was added to the reaction mixture. After 42 h, 90% of the
desired product was obtained. The solvent was removed in vacuo, and the resulting white
powder was washed with diethyl ether, filtered, and dried, giving 2c, which was used
without further purification.
1
H NMR (D
2
O): δ 8.69 (s, 1H), 8.49 (d, J = 6.1 Hz, 1H),
8.42 (d, J = 8.3 Hz, 1H), 7.84 (dd, J = 8.1 Hz, 6.1 Hz, 1H), 4.66 - 4.61 (m, 1H), 4.27 (dd,
J = 13.5 Hz, 9.6 Hz, 1H), 4.00 – 3.94 (m, 1H), 3.30 – 3.10 (m, 4H), 2.15 (tt, J = 21.0 Hz,
7.2 Hz, 1H), 1.26 (s, 9H).
31
P NMR (D
2
O): δ 17.25 (s, 2P).
The entire sample of 2c was dissolved in 50:50 water:TFA (v/v). After stirring at
47
r.t. for 4 h, a 100% yield of 4c was obtained according to
1
H NMR. The solvent was
removed in vacuo, and the residue was washed with diethyl ether and methanol, filtered,
and dried, yielding 4c as white crystals, which was then used without further purification.
1
H NMR (D
2
O): δ 8.73 (s, 1H), 8.54 (d, J = 6.1 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H), 7.89
(dd, J = 8.1 Hz, 6.1 Hz, 1H), 4.76 – 4.70 (m, 1H), 4.37 (dd, J = 13.4 Hz, 9.3 Hz, 1H),
4.26 (t, J = 9.6 Hz, 1H), 3.37 – 3.13 (m, 3H), 2.98 (dd, J = 13.1, 9.8 Hz, 1H), 2.28 (tt, J =
21.4, 7.2 Hz, 1H).
31
P NMR (D
2
O): δ 17.35 (s, 2P).
2.4.3 Synthesis of drug-linker intermediate 4d (3-(3-amino-2-hydroxypropyl)-1-(2-
hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium)
Route A: Compound 1d (40.0 mg, 0.15 mmol, 1.00 eq.), [1-hydroxy-2-(1H-
imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), was dissolved in 3 mL water and the
pH adjusted to 7.4 with Na
2
CO
3
(s). To this solution was added 51 mg of 5 (0.29 mmol, 2
eq) in minimal MeOH. The reaction mixture was stirred at 50 °C overnight, and the
reaction was monitored by
31
P NMR. After 19 h, 76% of 2d yielded. Thus, an additional
10.9 mg (0.06 mmol, 0.42 eq) of 5 in MeOH was added to the reaction mixture. After 41
h, less than 10% of starting materials was left and 81% of desired compound 2d yielded
with ~15% of side products. The solvent was removed in vacuo, and the resulting white
powder was washed with diethyl ether, filtered, and dried, giving 2d, which was used
without further purification. The entire sample of 2d was dissolved in 50:50 water:TFA
(v/v). After stirring at r.t. for overnight, Boc group was fully deprotected, and desired
compound 4d was obtained according to
1
H NMR. The solvent was removed in vacuo,
and the residue was washed with diethyl ether and methanol, filtered, and dried, which
was then subjected to SAX HPLC purification. SAX column (Macherey-Nagel 21.4 mm
48
x 250 mm SP15/25 Nucleogel column), flow rate: 9 mL/min, UV-VIS detection at 230
nm. Sample was eluted with A: H
2
O, B: 0.5 M TEAB pH 7.5 using a gradient that was
increased from 0-30% over 10 min, maintained at 30% from 10-15 min, and then
increased to 100% of buffer B from 15-35 min. The biggest peak eluting from 12 – 14
min was collected (the retention time has ±1.5 min error between different runs), and
solvents were evaporated, yielding compound 4d for next step reaction.
1
H NMR (D
2
O):
δ 8.76 (s, 1H), 7.45 (s, 1H), 7.33 (s, 1H), 4.53 (dd, J = 7.4, 6.8 Hz, 2H), 4.33 (d, J = 13.3
Hz, 1H), 4.11 - 4.07 (m, 2H), 3.21 – 3.17 (m, 1H), 2.89 (brd, 1H).
31
P NMR (D
2
O): δ
14.02 (s, 2P).
Route B: Compound 1d (50.0 mg, 0.18 mmol, 1.00 eq.), [1-hydroxy-2-(1H-
imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), was dissolved in 4 mL water and the
pH adjusted to 7.4 - 7.8 with Na
2
CO
3
(s). To this solution was added 72.8 µL of 6 (0.93
mmol, 5 eq). The reaction mixture was stirred at r.t. overnight, and the reaction was
monitored by
31
P NMR. After 19 h, 79% of 3d yielded with ~15% of side products and
less than 10% of starting materials was left. The solution of reaction mixture was washed
with diethyl ether (3×), and the solvent of aqueous phase was removed in vacuo, giving
3d, which was used without further purification. The entire sample of 3d was dissolved in
2 mL of NH
3
•H
2
O. After stirring at r.t. for 30 hrs, chlorine was displaced and desired
compound 4d was obtained according to MS. The solvent was removed in vacuo, and the
residue was washed with diethyl ether and methanol, filtered, and dried, which was then
subjected to SAX HPLC purification. SAX column (Macherey-Nagel 21.4 mm x 250 mm
SP15/25 Nucleogel column), flow rate: 9 mL/min, UV-VIS detection at 230 nm. Sample
was eluted with A: H
2
O, B: 0.5 M TEAB pH 7.5 using a gradient that was increased from
49
0-30% over 10 min, maintained at 30% from 10-15 min, and then increased to 100% of
buffer B from 15-35 min. The biggest peak eluting from 13.5 – 15.0 min was collected
(the retention time has ±1.5 min error between different runs), and solvents were
evaporated, yielding compound 4d for next step reaction.
1
H NMR (D
2
O): δ 8.76 (s, 1H),
7.45 (s, 1H), 7.33 (s, 1H), 4.53 (dd, J = 7.4, 6.8 Hz, 2H), 4.33 (d, J = 13.3 Hz, 1H), 4.11 -
4.07 (m, 2H), 3.21 – 3.17 (m, 1H), 2.89 (brd, 1H).
31
P NMR (D
2
O): δ 13.9 (s, 2P).
2.4.4 Synthesis of drug-linker intermediates 4e (1-(3-amino-2-hydroxypropyl)-3-
(2-hydroxy-2,2-diphosphonoethyl)imidazo[1,2-a]pyridin-1-ium, Route B)
Compound 1e (140.4 mg, 0.44 mmol, 1.00 eq), (1-Hydroxy-2-imidazo[1,2-
a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, was dissolved in 4 mL water and the
pH adjusted to 7.4 - 7.8 with 10 M NaOH. To this solution was added 170.9 µL of 6 (2.18
mmol, 5 eq). The reaction mixture was stirred at r.t. overnight, and the reaction was
monitored by
31
P NMR. After 19 h, 70% of 3e yielded with ~15% of side products and ~
15% of starting materials was left. The solution of reaction mixture was washed with
diethyl ether (3 -5 times), and the solvent of aqueous phase was removed in vacuo, giving
3e, which was used without further purification. The entire sample of 3e was dissolved in
8 mL of NH
3
•H
2
O. After stirring at r.t. for 44 hrs, chlorine was displaced and desired
compound 4e was obtained according to MS. The solvent was removed in vacuo, and the
residue was washed with diethyl ether and methanol, filtered, and dried, which was then
subjected to SAX HPLC purification. SAX column (Macherey-Nagel 21.4 mm x 250 mm
SP15/25 Nucleogel column), flow rate: 9 mL/min, UV-VIS detection at 280 nm. Sample
was eluted with A: H
2
O, B: 0.5 M TEAB pH 7.6 using a gradient that was increased from
0-30% over 10 min, maintained at 30% from 10-18 min, and then increased to 100% of
50
buffer B from 18-35 min. The biggest peak eluting from 9.4 – 11.5 min was collected
(the retention time has ±1.5 min error between different runs), and solvents were
evaporated, yielding compound 4e for next step reaction.
1
H NMR (D
2
O): δ 8.77 (d, J =
7.0 Hz, 1H), 7.87 – 7.70 (m, 3H), 7.32 (dt, J = 7.8, 4.2 Hz, 1H), 4.47 (d, J = 13.5 Hz, 1H),
4.40 – 4.13 (m, 2H), 3.53 (t, J = 11.3 Hz, 2H), 3.29 – 3.20 (m, 1H), 2.99 – 2.90 (part.
obscured by triethylamine, about 1H).
31
P NMR (D
2
O): δ 16.6 (s, 2P).
2.4.5 Synthesis of drug-linker intermediates 4f (1-(3-amino-2-hydroxypropyl)-3-(2-
carboxy-2-hydroxy-2-phosphonoethyl)imidazo[1,2-a]pyridin-1-ium, Route B)
Compound 1f (100 mg, 0.35 mmol, 1.00 eq), 2-hydroxy-3-imidazo[1,2-a]pyridin-
3-yl-2-phosphonopropionic acid, was dissolved in 2.85 mL water and the pH adjusted to
7.4 - 7.8 with 10 M NaOH. To this solution was added 137 µL of 6 (1.75 mmol, 5 eq).
The reaction mixture was stirred at r.t. overnight, and the reaction was monitored by
31
P
NMR. After 19 h, 61% of 3f yielded with ~24% of side products and ~ 15% of starting
materials was left. The solution of reaction mixture was washed with diethyl ether (3 -5
times), and the solvent of aqueous phase was removed in vacuo, giving 3f, which was
used without further purification. The entire sample of 3f was dissolved in 5 mL of
NH
3
•H
2
O. After stirring at r.t. for 44 hrs, chlorine was displaced and desired compound
4f was obtained according to MS. The solvent was removed in vacuo, and the residue
was washed with diethyl ether and methanol, filtered, and dried, which was then
subjected to SAX HPLC purification. SAX column (Macherey-Nagel 21.4 mm x 250 mm
SP15/25 Nucleogel column), flow rate: 9 mL/min, UV-VIS detection at 280 nm. Sample
was eluted with A: H
2
O, B: 0.5 M TEAB pH 7.6 using a gradient that was increased from
0-30% over 10 min, maintained at 30% from 10-18 min, and then increased to 100% of
51
buffer B from 18-35 min. The biggest peak eluting from 9.3 – 11.5 min was collected
(the retention time has ±1.5 min error between different runs), and solvents were
evaporated, yielding compound 4f for next step reaction.
1
H NMR (D
2
O): δ 8.64 (d, J =
7.1 Hz, 1H), 7.91 – 7.71 (m, 2H), 7.62 (s, 1H), 7.29 (ddd, J = 7.0, 5.9, 2.2 Hz, 1H), 4.51 –
4.39 (m, 1H), 4.35 – 4.14 (m, 2H), 3.66 (dd, J = 15.8, 3.6 Hz, 1H), 3.35 (dd, J = 15.7, 7.4
Hz, 1H), 3.26 – 3.15 (m, 1H), 2.98 – 2.79 (m, 1H).
31
P NMR (D
2
O): δ 14.8.
2.4.6 General method for preparation of compounds 7a-7f
The following synthesis and purification steps were performed under minimal
lighting. 4a, 4b, ... or 4f (3-5 eq) is dissolved in H
2
O. The pH is adjusted to 8.3 with solid
Na
2
CO
3
. 5(6)-FAM, SE (1 eq), 5(6)-RhR-X, SE (1 eq), 5(6)-ROX, SE
***
(1 eq), AF647,
SE (1 eq, the structure of AF647 is determined by MS and
1
H NMR of its corresponding
BP/PC conjugates, which is different from the proposed structure in literature
35
), sulfo-
Cy5, SE (1 eq) or IRDye 800CW, SE (1 eq) is dissolved in anhydrous DMF and
combined with the water solution. The pH can be re-adjusted to 8.2 - 8.5 with Na
2
CO
3
,
dissolving any precipitate, and the reaction mixture is stirred for 3 h - overnight under r.t.
in darkness. Crude products of FAM and 5(6)-RhR-X conjugates (7a1-7a4, 7b1-7b2,
7c1-7c2, 7d1-7d2, 7e1-7e2, 7f1-7f2) are purified by TLC on plates 20 x 20 cm or 7 x 20
cm (the size of TLC plates chosen depends on the total crude amount) eluted with 100%
MeOH. Crude reaction mixtures of other dye conjugates are not purified by TLC. The
phosphonate-containing compounds remaining at the origin (R
f
= 0) are extracted with
water; the combined aqueous extracts may be treated with Chelex (sodium form) to aid
***
There is only 5-isomer existing in the commercially available 5(6)-ROX, SE (mixed isomer); thus the
final ROX-RIS is 5-ROX-RIS; and the ROX-RISPC is 5-ROX-RISPC.
52
the extraction process. The solution is centrifuged, and then concentrated in vacuo. The
resulting solids are then dissolved in either water, 20% MeOH in 0.1 M
triethylammonium acetate buffer (TEAAc, pH 5.0 - 5.5) or triethylammonium carbonate
buffer (TEAC, pH 7.0 - 7.8) and filtered through Nanosep 30K Omega filters. The
solution is then purified by preparative/semi-preparative reverse-phase HPLC according
to the appropriate method. The final amount of labeled product is calculated from the
UV-VIS absorption spectrum, and the isolated eluent is concentrated in vacuo and
lyophilized.
Method A: Dynamax C18 (21.4 mm x 25 cm, 5 µm, 100 Å pore size) column, flow
rate 8.0 mL/min, UV detection at 260 nm, gradient as follows: linearly increasing
from 10% MeOH 0.1 M TEAAc (pH 5.0 – 5.5) or TEAC (pH 7.0 – 7.8, buffer A) to
40% of 75% MeOH 0.1 M TEAAc (pH 5.0 – 5.5) or TEAC (pH 7.0 – 7.8, buffer B)
in 12 min, then increasing to 70% of buffer B from 12 - 100 min;
Method B: Dynamax C18 (21.4 mm x 25 cm, 5 µm, 100 Å pore size) column, flow
rate 8.0 mL/min, UV detection at 260 nm, isocratic elution with 20% MeOH 0.1 M
TEAC (pH 7.0 – 7.8, buffer A) for 12 min, linearly increasing to 100% of 70%
MeOH 0.1 M TEAC (pH 7.0 – 7.8, buffer B) from 12 - 22 min;
Method C: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 6.0 mL/min, UV detection at 260 nm and 568 nm, isocratic elution of 20%
MeOH in 0.1 M TEAC (pH 7.0 – 7.8, buffer A) for 5 min, linearly increasing to
100% of 75% MeOH in 0.1 M TEAC (pH 7.0 – 7.8, buffer B) in 1 min;
Method D: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 260 nm and 568 nm, isocratic elution of 20%
53
MeOH in 0.1 M TEAAc (pH 5.0 – 5.5, buffer A) for 5 min, linearly increasing to
100% of 75% MeOH in 0.1 M TEAAc (pH 5.0 – 5.5, buffer B) in 1 min;
Method E: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4 mL/min, UV detection at 260 nm and 568 nm, isocratic elution of 20 %
MeOH in 0.1 M TEAC (pH 7.0 - 7.8, buffer A) for 5 min, linearly increasing to 100%
of 70% MeOH in 0.1 M TEAC (pH 7.0 - 7.8, buffer B) in 1 min;
Method F: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 260 nm and 576 nm, isocratic elution of 20%
MeOH in 0.1 M TEAAc buffer (pH 5.0 - 5.5, buffer A) for 5 min, linearly increasing
to 100% of 70% MeOH in 0.1 M TEAAc buffer (pH 5.0 - 5.5 buffer B) in 5 min;
Method G: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 260 nm and 576 nm, isocratic elution of 10%
MeOH in 0.1 M TEAC buffer (pH 7.0 - 7.8, buffer A) for 5 min, linearly increasing
to 100% of 70% MeOH in 0.1 M TEAC buffer (pH 7.0 - 7.8, buffer B) in 5 min;
Method H: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 260 nm (7a6, 7b4) or 230 nm (7d3) and 598
nm, isocratic elution of 20% MeOH in 0.1 M TEAAc buffer (pH 5.0 – 5.5, buffer A)
for 5 min, linearly increasing to 40% of 70% MeOH in 0.1 M TEAAc buffer (pH 5.0
– 5.5, buffer B) in 20 min;
Method I: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 230 nm (7d1 - 7d2) or 280 nm (7e1, 7e2, 7f1,
7f2) and 492 nm, gradient as follows: linearly increasing from 10% MeOH in 0.1 M
TEAC (pH 7.0 - 7.8, buffer A) to 40% of 75% MeOH in 0.1 M TEAC (pH 7.0 – 7.8,
54
buffer B) in 25 min, then increasing to 70% of buffer B from 25 - 100 min;
Method J: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection at 230 nm and 598 nm, isocratic elution of 20%
MeOH in 0.1 M TEAAc buffer (pH 5.0 – 5.5, buffer A) for 7 min, linearly increasing
to 100% of 70% MeOH in 0.1 M TEAAc buffer (pH 5.0 – 5.5, buffer B) from 7 - 25
min;
Method K: Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size),
flow rate 4.0 mL/min, UV detection 230 nm and 598 nm, isocratic elution of 20%
MeOH in 0.1 M TEAAc buffer (pH 5.0 – 5.5, buffer A) for 5 min, linearly increasing
to 40% of 70% MeOH in 0.1 M TEAAc buffer (pH 5.0 – 5.5, buffer B) from 5 - 15
min; maintained at 40% of buffer B from 15 – 30min, finally increase to 100% of
buffer B from 30 – 35 min.
5(6)-FAM-RIS (7a1), 5-FAM-RIS (7a2): 1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-
3H-xanthen-9-yl)benzamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-
diphosphonoethyl)pyridin-1-ium; 6-FAM-RIS (7a3): 1-(3-(4-carboxy-3-(6-hydroxy-3-
oxo-3H-xanthen-9-yl)benzamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-
diphosphonoethyl)pyridin-1-ium):
Synthesized according to the method above with 86.5 mg of 4a (as TFA
-
, Na
+
salt,
0.18 mmol, 1.7 eq) in 2 mL of H
2
O and pH adjusted to 8.3 with Na
2
CO
3
(s), to which
added in 50.0 mg of 5(6)-FAM, SE (0.11 mmol, 1.00 eq) in 600 µL anhydrous DMF; the
pH of reaction solution was further adjusted to pH 8.3 to dissolve precipitates, and the
reaction mixture was then stirred at r.t. for overnight. After TLC purification (100%
MeOH as eluent), the product was purified by HPLC according to Method A with
55
TEAAc buffers. Peaks eluting from 25-75 min were collected. During evaporation of the
buffer solution, product precipitated from the solution. Consequently, a second HPLC
purification was performed according to Method A but eluting with TEAC buffers (pH
7.5). Obtained 29.0 mg, 46.8% yield (triethylammonium bicarbonate salt).
1
H NMR
(D
2
O): δ 8.76 – 8.62 (m, 1H), 8.54 – 8.48 (m, 1H), 8.42 (dt, J = 17.6, 7.8 Hz, 1H), 8.04 (s
0.6 H), 7.92 – 7.64 (m, 2H), 7.45 (s, 0.4 H), 7.13 (s, 1H), 6.93 (ddd, J = 9.1, 4.5, 1.9 Hz,
2H), 6.45 – 6.38 (m, 4H), 4.82 – 4.70 (m, 1H), 4.44 – 4.28 (m, 1H), 4.28 – 4.12 (m, 1H),
3.65 – 3.55 (m, 1H), 3.56 – 3.44 (m, 1H), 3.44 – 3.19 (m, 2H).
31
P NMR (D
2
O): δ 16.36
(s, 2P).
HPLC Separation of 5- and 6-FAM-RIS (7a2 and 7a3): Synthesized according
to method described for 7a1. Under HPLC conditions described as Method A, 6-
FAMRIS and 5-FAMRIS elute at very different retention times, 27 and 44 min (the
retention time has ±1.5 min error between different runs), respectively. Each isomer was
collected separately and then concentrated in vacuo to remove buffer. Compound 7a2 and
7a3 were also directly synthesized from 5-FAM, SE and 6-FAM, SE according to the
method described above. Detailed NMR descriptions of 7a2 and 7a3 can be found from
ref.
24
5(6)-FAM-RISPC (7b1, also know as 5(6)-FAM-3-PEHPC. 5-FAM-RISPC: 3-
(2-carboxy-2-hydroxy-2-phosphonoethyl)-1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)benzamido)-2-hydroxypropyl)pyridin-1-ium; 6-FAM-RISPC: 3-(2-
carboxy-2-hydroxy-2-phosphonoethyl)-1-(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)benzamido)-2-hydroxypropyl)pyridin-1-ium):
Synthesized according to method described above with 94.8 mg of intermediate
56
4b (0.21 mmol, 3.5 eq) in 1 mL of water and pH adjusted to 8.3 with Na
2
CO
3
(s), to
which added in 30 mg of 5(6)-FAM, SE (0.06 mmol, 1.0 eq) in 200 µL anhydrous DMF.
The pH of reaction solution was further adjusted to pH 8.4 to dissolve precipitates, and
the reaction mixture was then stirred at r.t. for overnight. After TLC purification (100%
MeOH as eluent), the mixture was purified by HPLC according to Method B. Peaks
eluting at 21-25 min were collected together as 7b1. Obtained 23.2 mg, 53.9% yield
(triethylammonium bicarbonate salt).
1
H NMR (D
2
O): δ 8.66 – 8.44 (m, 2H), 8.29 (brd,
1H), 8.05 (s, 0.6 H), 7.89 – 7.68 (m, 2H), 7.45 (s, 0.4 H), 7.13 (d, J = 8.0 Hz, 1H), 6.93 (d,
J = 9.2 Hz, 2H), 6.50 – 6.35 (m, 4H), 4.43 – 4.25 (m, 2H), 3.78 – 3.55 (m, 1H), 3.54 –
3.41 (m, 2H), 3.41 – 3.23 (m, 1H), 2.87 (part. obscured by triethylamine, about 1H).
31
P
NMR (D
2
O): δ 15.15 (brs, 1P). HRMS (positive ion MALDI): calcd 679.1324 m/z; found
[M]
+
= 679.1321 m/z.
5(6)-FAM-dRIS (7c1, 5-FAM-dRIS: 1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)benzamido)-2-hydroxypropyl)-3-(2,2-diphosphonoethyl)pyridin-1-ium; 6-
FAM-dRIS: 1-(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-
hydroxypropyl)-3-(2,2-diphosphonoethyl)pyridin-1-ium):
Synthesized according to method described above with 53 mg of intermediate 4c
(0.1 mmol, 2.5 eq) in 1 mL HPLC water and pH adjusted to 8.3 with Na
2
CO
3
(s), to
which added in 18.0 mg of 5(6)-FAM, SE (0.04 mmol, 1.00 eq) in 100 µL anhydrous
DMF. The pH of reaction solution was further adjusted to pH 8.4 to dissolve precipitates,
and the reaction mixture was then stirred at r.t. for overnight. After TLC purification, the
mixture was purified according to Method B. Peaks eluting from 27-45 min were
collected as 7c1. Obtained 9.4 mg, 36% yield (triethylammonium acetate salt).
1
H NMR
57
(D
2
O): δ 8.73 – 8.69 (m, 1H), 8.50 (d, J = 6.1 Hz, 1H), 8.47 – 8.36 (m, 1H), 8.06 (d, J =
1.9 Hz, 0.6 H), 7.94 – 7.68 (m, 2H), 7.53 (s, 0.4 H), 7.23 (d, J = 8.0 Hz, 1H), 6.99 (dd, J
= 9.7, 2.4 Hz, 2H), 6.50 – 6.40 (m, 4H), 4.82 – 4.72 (m, 1H), 4.42 – 4.29 (m, 1H), 4.29 –
4.09 (m, 1H), 3.62 (dd, J = 14.1, 4.5 Hz, 1H), 3.23 – 3.11 (obscured by solvent peak,
about 1H), 3.58 – 3.32 (m, 2H), 2.14 – 1.87 (m, 1H).
31
P NMR (D
2
O): δ 17.17 (brs).
HRMS (positive ion MALDI): calcd 699.1139 m/z; found [M]
+
= 699.1137 m/z.
5(6)-RhR-RIS (7a4, 1-{3-[6-({4-[6-(diethylamino)-3-(diethylimino)-3H-xanthen-
9-yl]-3-sulfobenzene}sulfonamido)hexanamido]-2-hydroxypropyl}-3-(2-hydroxy-2,2-
diphosphonoethyl)pyridinium):
Synthesized according to method described above with 11.2 mg of compound 4a
(0.032 mmol, 4.9 eq) in 0.5 mL H
2
O and pH adjusted to 9.0 with Na
2
CO
3
(s), to which
added in 5 mg of RhR-X, SE (0.0065 mmol, 1 eq.) in 250 µL DMF. After TLC
purification, the mixture was purified by HPLC according to Method C. Peak eluting
between 12.8 – 18 minutes (the retention time has ±1.0 min error between different runs)
were collected. Obtained 0.2 mg, 3% yield (as a triethylammonium bicarbonate salt).
1
H
NMR (D
2
O): δ 8.66 (s, 1H), 8.49 – 8.31 (m, 3H), 8.09 (s, 1H), 7.70 (s, 1H), 7.59 – 7.33
(m, 1H), 7.01 – 6.57 (m, 7H), 4.22 – 3.89 (m, 3H), 3.55 – 3.23 (m, obscured by solvent
peak and TEA peak, around 12H), 3.02 – 2.96 (m, obscured by TEA peak, around 3H),
2.19 – 2.01 (m, 2H), 1.47 – 1.24 (m, obscured by TEA peak, around 5H), 1.10 (obscured
by TEA peak, about 12H).
31
P NMR (D
2
O): δ 16.76 (s, 2P). HRMS (positive ion
MALDI): calcd 1011.2913 m/z, found [M-H]
+
= 1010.2866 m/z.
5(6)-RhR-RISPC (7b2, 3-(2-carboxy-2-hydroxy-2-phosphonoethyl)-1-{3-[6-({4-
[6-(diethylamino)-3-(diethylimino)-3H-xanthen-9-yl]-3-
58
sulfobenzene}sulfonamido)hexanamido]-2-hydroxypropyl}pyridinium, also known as
5(6)-RhR-3-PEHPC):
Synthesized according to method described above with 10.9 mg of compound 4b
(0.04 mmol, 3 eq) in 0.5 mL of H
2
O and pH adjusted to 8.3 with Na
2
CO
3
(s), to which
added in 5 mg of 5(6)-RhR-X, SE in 500 µL DMF. After TLC purification, the solution
was then purified by HPLC according to Method D. Peak eluting at 13 min (the retention
time has ±1.0 min error between different runs) is collected as 7b2. Obtained 2.1 mg,
33% yield (triethylammonium bicarbonate salt).
1
H NMR (400 MHz, D
2
O): δ 8.51 (s,
1H), 8.43 (d, J = 10.1 Hz, 2H), 8.29 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.83 – 7.66 (m, 1H),
7.45 (d, J = 8.0 Hz, 1H), 6.87 – 6.70 (m, 4H), 6.65 (s, 2H), 4.56 – 4.43 (m, 1H), 4.16 (d, J
= 14.4 Hz, 1H), 3.95 (s, 1H), 3.48 (dd, J = 23.6, 8.0 Hz, 8H), 3.28 – 3.12 (m, obscured by
solvent, about 4H), 2.93 (td, J = 17.6, 16.8, 8.9 Hz, 3H), 2.10 (t, J = 7.6 Hz, 2H), 1.47 –
1.21 (m, 5H), 1.09 (obscured by TEA peak, about 12H).
31
P NMR (D
2
O): 15.2 (s).
HRMS (positive ion MALDI): calcd 975.3148 m/z, found [M-H]
+
= 974.3118 m/z.
5(6)-RhR-dRIS (7c2, 1-{3-[6-({4-[6-(diethylamino)-3-(diethylimino)-3H-
xanthen-9-yl]-3-sulfobenzene}sulfonamido)hexanamido]-2-hydroxypropyl}-3-(2,2-
diphosphonoethyl)pyridin-1-ium):
Synthesized according to method described above with 9.4 mg of compound 4c
(0.02 mmol, 3.3 eq) in 0.6 mL H
2
O and pH adjusted to 8.3 with Na
2
CO
3
(s), to which
added in 5 mg of 5(6)-RhR-X, SE (0.0065 mmol, 1 eq) in 0.45 mL of DMF.
Precipitation was observed. The reaction mixture was stirred for 2 h, then evaporated to
dryness. The resulting solids were extracted with acetone (3 x 1 mL, in order to remove
partially unconjugated dye. The remaining precipitate was dissolved in ~2 mL H
2
O and
59
purified by TLC as described above, followed by HPLC purification (Method E). A
broad peak eluting between 13 – 17.3 min (the retention time has ±1.0 min error between
different runs) was collected. Obtained 2.75 mg, 42.5% yield (triethylammonium
bicarbonate salt).
1
H NMR (D
2
O): δ 8.67 (d, J = 2.2 Hz, 1H), 8.50 – 8.36 (m, 3H), 8.10 (t,
J = 6.9 Hz, 1H), 7.79 (dd, J = 8.4, 6.4 Hz, 1H), 7.42 (t, J = 8.6 Hz, 1H), 6.86 – 6.68 (m,
4H), 6.68 – 6.57 (m, 2H), 4.62 – 4.50 (m, 1H), 4.19 (dd, J = 13.3, 9.7 Hz, 1H), 4.10 –
3.94 (m, 1H), 3.45 (p, J = 7.0 Hz, 8H), 3.34 – 3.13 (m, 4H), 2.97 (q, J = 6.7 Hz, 3H), 2.36
– 2.01 (m, 3H), 1.53 – 1.23 (m, 5H), 1.10 (td, J = 7.0, 3.2 Hz, 12H).
31
P NMR (D
2
O):
17.29 (s). HRMS (positive ion MALDI): calcd 995.2695 m/z, found [M-H]
+
= 994.2872
m/z.
5(6)-ROX-RIS (7a5, 5-ROX-RIS: 16-[2-carboxy-4-({2-hydroxy-3-[4-(2-
hydroxy-2,2-diphosphonoethyl)pyridin-1-ium-1-yl]propyl}carbamoyl)phenyl]-3-oxa-
9λ⁵,23-diazaheptacyclo[17.7.1.1⁵,⁹.0²,¹⁷.0⁴,¹⁵.0²³,²⁷.0¹³,²⁸]octacosa-
1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium):
Synthesized according to method described above with 23.6 mg of compound 4a
(0.047 mmol, 3 eq.) in 0.8 mL of H
2
O/NaHCO
3
(pH 9.0), to which added in 10 mg of
5(6)-ROX, SE (0.016 mmol, 1 eq.) in 200 µL anhydrous DMF, and the solution was
stirred overnight. The solvent was concentrated under vacuo, and the resulting purple
residue was dissolved in 20% MeOH in 0.1 M TEAAc buffer (pH 5.3) and purified by
HPLC (Method F). Peaks eluting at 17.0 min (the retention time has ±1.0 min error
between different runs) were collected as 7a5. Obtained 7.4 mg, 54.0% yield
(triethylammonium acetate salt).
1
H NMR (D
2
O): δ 8.74 (s, 1H), 8.55 (d, J = 6.0 Hz, 1H),
8.43 (d, J = 8.1 Hz, 1H), 8.07 (s, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H),
60
6.77 (d, J = 7.9 Hz, 1H), 6.52 (s, 2H), 4.35 – 4.21 (m, 2H), 3.57 – 3.48 (m, 2H), 3.37 –
3.16 (m, 13H), 2.70 – 2.62 (m, 2H), 2.47 – 2.27 (m, 5H), 1.79 – 1.53 (m, 7H).
31
P NMR
(D
2
O): 16.36 (s). HRMS (positive ion MALDI): calcd 873.2660 m/z, found [M-H]
+
=
873.2647 m/z.
5(6)-ROX-RISPC (7b3, also known as 5(6)-ROX-3-PEHPC, 5-ROX-RISPC:
16-[2-carboxy-4-({3-[4-(2-carboxy-2-hydroxy-2-phosphonoethyl)pyridin-1-ium-1-yl]-2-
hydroxypropyl}carbamoyl)phenyl]-3-oxa-9λ⁵,23-
diazaheptacyclo[17.7.1.1⁵,⁹.0²,¹⁷.0⁴,¹⁵.0²³,²⁷.0¹³,²⁸]octacosa-1(27),2(17),4,9(28),13,15,18-
heptaen-9-ylium):
Synthesized according to method described above with 54.3 mg of 4b (0.119
mmol, 3 eq.) in 1.6 mL of H
2
O/NaHCO
3
(pH 9.0) and 25 mg of 5(6)-ROX, SE (0.04
mmol, 1 eq.) in 1 mL anhydrous DMF, and the solution was stirred overnight. The
solvent was concentrated under vacuo, and the resulting purple residue was dissolved in
10% MeOH in 0.1 M TEAC buffer (pH 7.0) and purified by HPLC (Method G). Peaks
eluting at 21.9 min (the retention time has ±1.0 min error between different runs) were
collected as 7b3. Obtained 9.4 mg, 35.0% yield (triethylammonium bicarbonate salt).
1
H
NMR (D
2
O): δ 8.63 (s, 1H), 8.54 (d, J = 6.2 Hz, 1H), 8.31 (s, 1H), 8.03 (d, J = 1.9 Hz,
1H), 7.86 – 7.78 (m, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H), 6.59 (s, 2H), 4.45 – 4.33
(m, 1H), 4.31 – 4.18 (m, 1H), 3.67 – 3.48 (m, 2H), 3.36 – 3.14 (m, 13H), 2.83 – 2.72 (m,
2H), 2.51 – 2.35 (m, 5H), 1.81 – 1.62 (m, 7H).
31
P NMR (D
2
O): 14.34 (s). HRMS
(negative ion MALDI): calcd 835.2750 m/z, found [M-3H]
-
= 835.2733 m/z.
AF647-RIS (7a6, 2-(5-(3-(6-((2-hydroxy-3-(3-(2-hydroxy-2,2-
diphosphonoethyl)pyridin-1-ium-1-yl)propyl)amino)-6-oxohexyl)-3-methyl-5-sulfo-1-(3-
61
sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-1-(3-
sulfopropyl)-3H-indol-1-ium):
Synthesized according to method described above with 25.9 mg of compound 4a
(0.05 mmol, 10 eq.) in 1 mL of H
2
O/NaHCO
3
(pH 8.3) and 5 mg of AF647, SE (0.005
mmol, 1 eq.) in 250 µL anhydrous DMF, and the solution was stirred at r.t. overnight.
The solvent was concentrated under vacuo, and the resulting blue residue was dissolved
in 20% MeOH in 0.1 M TEAAc buffer (pH 5.3) and purified by HPLC (Method H).
Peaks eluting at 19.8 min (the retention time has ±1.0 min error between different runs)
were collected as 7a6. Obtained 4.8 mg, 76.7% yield (triethylammonium acetate salt).
1
H
NMR (D
2
O): δ 8.61 (s, 1H), 8.45 (d, J = 6.2 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.00 (t, J =
13 Hz, 2H), 7.80 – 7.67 (m, 5H), 7.31 – 7.20 (m, 2H), 6.55 (t, J = 12.6 Hz, 1H), 6.37 –
6.23 (m, 2H), 4.70 – 4.60 (obscured by HDO, about 1H), 4.14 – 3.98 (m, 4H), 2.92 – 2.80
(m, 6H), 2.14 – 2.10 (m, 5H), 1.95 – 1.92 (m, 2H), 1.57 – 1.53 (m, 9H), 1.48 – 1.45 (m,
1H), 1.29 – 1.27 (m, 2H), 1.00 – 0.95 (m, 3H), 0.82 – 0.79 (m, 3H), 0.45 – 0.43 (m, 2H).
31
P NMR (D
2
O): δ 16.50 (d, J = 26.9 Hz, 1P), 16.30 (d, J = 29.0 Hz, 1P). HRMS
(positive ion MALDI): calcd 1198.2410 m/z, found [M-H]
+
= 1197.2358 m/z.
AF647-RISPC (7b4, 2-(5-(3-(6-((3-(3-(2-carboxy-2-hydroxy-2-
phosphonoethyl)pyridin-1-ium-1-yl)-2-hydroxypropyl)amino)-6-oxohexyl)-3-methyl-5-
sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-1-(3-
sulfopropyl)-3H-indol-1-ium, also known as AF647-3-PEHPC):
Synthesized according to method described above with 22.5 mg of compound 4b
(0.05 mmol, 10 eq.) in 1 mL of H
2
O and pH adjusted to 8.3 with Na
2
CO
3
(s), to which
added in 5 mg of AF647, SE (0.005 mmol, 1 eq.) in 300 µL anhydrous DMF. The
62
solution was stirred at r.t. overnight. The solvent was concentrated under vacuo, and the
resulting blue residue was dissolved in 20% MeOH in 0.1 M TEAAc buffer (pH 5.3) and
purified by HPLC (Method H). Peaks eluting at 18.8 min (the retention time has ±1.0 min
error between different runs) were collected as 7b4. Obtained 5.3 mg, 87.2% yield
(triethylammonium acetate salt).
1
H NMR (D
2
O): δ 8.47 (m, 2H), 8.25 (d, J = 7.7 Hz,
1H), 7.94 (t, J = 13.1 Hz, 2H), 7.81 – 7.73 (m, 1H), 7.73 – 7.63 (m, 4H), 7.26 (t, J = 8.0
Hz, 2H), 6.52 (t, J = 12.4 Hz, 1H), 6.25 (dd, J = 13.6, 9.8 Hz, 2H), 4.70 – 4.60 (obscured
by HDO, about 1H), 4.21 – 4.11 (m, 4H), 3.42 – 3.40 (m, 1H), 2.91 – 2.83 (m, 5H), 2.20
– 2.01 (m, 6H), 1.95 – 1.92 (m, 2H), 1.51 – 1.50 (m, 9H), 1.25 – 1.20 (obscured by
triethylamine peak, about 4H), 0.99 – 0.95 (obscured by triethylamine peak, 4H), 0.69 –
0.42 (m, about 2H).
31
P NMR (D
2
O): δ 15.21 (s). HRMS (positive ion MALDI): calcd
1162.2645 m/z, found [M-H]
+
= 1161.2572 m/z.
5-FAM-ZOL (7d1, 3-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)benzamido)-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-
ium) and 6-FAM-ZOL (7d2, 3-(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)benzamido)-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-
ium):
Synthesized according to method described above with 60.2 mg of compound 4d
(as TEA
+
salt (4 eq. of TEA), 0.08 mmol, 2.7 eq.) in 0.5 mL of H
2
O and pH adjusted to
8.4 with Na
2
CO
3
(s), to which added in 15.2 mg of 5(6)-FAM, SE (0.03 mmol, 1 eq.) in
100 µL anhydrous DMF. Re-adjust pH to 8.3 to dissolve precipitates and the solution was
stirred at r.t. overnight. After TLC purification, the product was purified by HPLC
(Method I). 6-FAM-ZOL (7d2) and 5-FAM-ZOL (7d1) were eluted at very different
63
retention times, 20 and 30 min (the retention time has ±1.5 min error between different
runs), respectively. Each isomer was collected separately and then concentrated in vacuo
to remove buffer. Compound 7d1 and 7d2 could also be directly synthesized from 5-
FAM, SE and 6-FAM, SE according to the method described above. Detailed NMR
descriptions given below correspond to the HPLC-separated products. Total amount of
7d1 and 7d2 is 15.4 mg, 68.3% yield. 5-FAM-ZOL (7d1, triethylammonium bicarbonate
salt): obtained 9.2 mg (triethylammonium bicarbonate salt).
1
H NMR (D
2
O): δ 8.74 (s,
1H), 8.11 – 8.03 (m, 1H), 7.84 (dd, J = 8.0, 1.9 Hz, 1H), 7.45 (t, J = 1.7 Hz, 1H), 7.34 (t,
J = 1.8 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 6.50 – 6.43 (m, 4H),
4.57 – 4.44 (m, 2H), 4.36 (d, J = 12 Hz, 1H), 4.22 – 4.03 (m, 2H), 3.57 (dd, J = 14.0, 4.5
Hz, 1H), 3.43 (dd, J = 14.0, 6.7 Hz, 1H).
31
P NMR (D
2
O): δ 14.02 (s). HRMS (positive
ion MALDI): calcd 704.1041 m/z, found M
+
= 704.1013 m/z. 6-FAM-ZOL (7d2,
triethylammonium bicarbonate salt): obtained 6.2 mg (triethylammonium bicarbonate
salt).
1
H NMR (D
2
O): δ 8.70 (s, 1H), 7.90 (dd, J = 8.1, 1.8 Hz, 1H), 7.78 (d, J = 8.1, 1H),
7.57 (d, J = 1.7 Hz, 1H), 7.43 (t, J = 1.7 Hz, 1H), 7.30 (t, J = 1.8 Hz, 1H), 7.03 (d, J = 8.8
Hz, 2H), 6.56 – 6.42 (m, 4H), 4.56 – 4.42 (m, 2H), 4.32 (d, J = 12.5 Hz, 1H), 4.17 – 3.99
(m, 2H), 3.51 (dd, J = 14.1, 4.2 Hz, 1H), 3.40 – 3.33 (m, 1H).
31
P NMR (D
2
O): δ 14.03
(s). HRMS (positive ion MALDI): calcd 704.1041 m/z, found M
+
= 704.1027 m/z.
AF647-ZOL (7d3, 2-(5-(3-(6-((2-hydroxy-3-(1-(2-hydroxy-2,2-
diphosphonoethyl)-1H-imidazol-3-ium-3-yl)propyl)amino)-6-oxohexyl)-3-methyl-5-
sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-1-(3-
sulfopropyl)-3H-indol-1-ium):
Synthesized according to method described above with 18.9 mg of compound 4d
64
(0.05 mmol, 5 eq.) in 500 µL of H
2
O and pH adjusted to 8.4 with Na
2
CO
3
(s), to which
added in and 10 mg of AF647, SE (0.0105 mmol, 1 eq.) in 300 µL anhydrous DMF. The
solution was stirred at r.t. overnight and then was concentrated under vacuo, and the
resulting blue residue was dissolved in 20% MeOH in 0.1 M TEAAc buffer (pH 5.3) and
purified by HPLC (Method H). Peaks eluting at 16.5 min were collected as 7d3 (the
retention time has ±1.5 min error between different runs). Obtained 6.6 mg, 53.1% yield
(triethylammonium acetate salt).
1
H NMR (D
2
O): δ 8.63 (s, 1H), 7.99 (t, J = 13.2 Hz, 2H),
7.78 – 7.65 (m, 4H), 7.39 (s, 1H), 7.34 – 7.20 (m, 3H), 6.55 (t, J = 12.5 Hz, 1H), 6.29 (dd,
J = 13.6, 9.7 Hz, 2H), 4.52 – 4.48 (m, 2H), 4.26 – 4.07 (m, 5H), 3.98 – 3.81 (m, 2H), 2.95
– 2.85 (m, 5H), 2.13 – 2.08 (m, 6H), 1.93 – 1.89 (m, 2H), 1.58 – 1.54 (m, 9H), 1.34 –
1.21 (m, 3H), 1.04 – 0.94 (m, 2H), 0.81 – 0.66 (m, 1H), 0.52 – 0.36 (m, 1H).
31
P NMR
(D
2
O): δ 13.52 (s). HRMS (positive ion MALDI): calcd 1186.2290 m/z, found [M-H]
+
=
1186.2337 m/z.
800CW-ZOL (7d4, (E)-2-((E)-2-(3-((E)-2-(1-(6-((2-hydroxy-3-(1-(2-hydroxy-
2,2-diphosphonoethyl)-1H-imidazol-3-ium-3-yl)propyl)amino)-6-oxohexyl)-3,3-
dimethyl-5-sulfo-3H-indol-1-ium-2-yl)vinyl)-2-(4-sulfophenoxy)cyclohex-2-en-1-
ylidene)ethylidene)-3,3-dimethyl-1-(4-sulfonatobutyl)indoline-5-sulfonate, sodium salt):
Synthesized according to method described above with 7.4 mg of compound 4d
(0.021 mmol, 5.3 eq.) in 1 mL of H
2
O and pH adjusted to 8.4 with Na
2
CO
3
(s), to which
added in and 5 mg of IRDye 800CW, SE (0.004 mmol, 1 eq.) in 100 µL anhydrous DMF.
The solution was stirred at 4 ̊C overnight and then was concentrated under vacuo, and the
resulting greenish black residue was dissolved in 20% MeOH in 0.1 M TEAAc buffer
(pH 5.3) and purified by HPLC (Method J). Peaks eluting at 23.5 min were collected as
65
7d4 (the retention time has ±1.5 min error between different runs). Obtained 4.8 mg,
83.2% yield (triethylammonium acetate salt).
1
H NMR (D
2
O): δ 8.65 (s, 1H), 7.67 (d, J =
8.6 Hz, 2H), 7.63 – 7.50 (m, 6H), 7.39 (s, 1H), 7.28 (t, J = 1.8 Hz, 1H), 7.14 – 6.96 (m,
4H), 5.99 – 5.84 (dd, J = 14.2, 9.4 Hz, 2H), 4.56 – 4.46 (m, 2H), 4.18 (d, J = 12.6 Hz,
1H), 4.02 – 3.66 (m, 6H), 3.15 – 3.09 (m, 2H), 2.81 – 2.73 (m, 3H), 2.45 (brd, 5H), 2.09
– 2.06 (m, 2H), 1.83 (obscured by solvent peak, around 12H), 1.80 – 1.59 (obscured by
solvent peak, around 6H), 1.53 – 1.38 (m, 4H).
31
P NMR (D
2
O): δ 13.65 (s). HRMS
(positive ion MALDI): calcd 1330.2865 m/z, found [M-H]
+
= 1330.2885 m/z.
Sulfo-Cy5-ZOL (7d5, 1-(6-((2-hydroxy-3-(1-(2-hydroxy-2,2-diphosphonoethyl)-
1H-imidazol-3-ium-3-yl)propyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E,5E)-5-
(1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-
sulfonate, sodium salt):
Synthesized according to method described above with 22.71 mg of compound 4d
(0.066 mmol, 5.1 eq.) in 0.95 mL of H
2
O and pH adjusted to 8.34 with Na
2
CO
3
(s), to
which added in and 10 mg of Sulfo-Cy5, SE (0.013 mmol, 1 eq.) in 450 µL anhydrous
DMF. Precipitates could be seen. The solution was stirred at r.t. overnight and then was
concentrated under vacuo, and the resulting blue residue was dissolved in 20% MeOH in
0.1 M TEAAc buffer (pH 5.3) and purified by HPLC (Method K). Peaks eluting at 18
min were collected as 7d5 (the retention time has ±1.5 min error between different runs).
Obtained 5.3 mg, 41.2% yield (triethylammonium acetate salt).
1
H NMR (D
2
O): δ 8.65 (s,
1H), 7.81 (td, J = 13.1, 4.3 Hz, 2H), 7.74 – 7.56 (m, 4H), 7.39 (s, 1H), 7.26 (s, 1H), 7.15
(dd, J = 8.4, 2.2 Hz, 2H), 6.32 (t, J = 12.5 Hz, 1H), 6.00 (dd, J = 19.6, 13.7 Hz, 2H), 4.57
– 4.44 (m, 2H), 4.17 (d, J = 13.0 Hz, 1H), 3.92 (m, 4H), 3.42 (s, 3H), 2.16 – 2.09 (m, 2H),
66
1.68 – 1.61 (m, 2H), 1.53 – 1.41 (m, 15H), 1.26 – 1.17 (obscured by triethylamine peak,
around 3H).
31
P NMR (D
2
O): δ 13.51 (s). MS (negative ion ESI): calcd 483.6 m/z, found
[M-4H]
2-
= 484.0 m/z.
5-FAM-MIN (7e1, 1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)benzamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)imidazo[1,2-
a]pyridin-1-ium) and 6-FAM-MIN (7e2, 1-(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)benzamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-
diphosphonoethyl)imidazo[1,2-a]pyridin-1-ium):
Synthesized according to method described above with 22.4 mg of compound 4e
(0.057 mmol, 2.5 eq.) in 1 mL of H
2
O and pH adjusted to 8.58 with Na
2
CO
3
(s), to which
added in 10.8 mg of 5(6)-FAM, SE (0.023 mmol, 1 eq.) in 300 µL anhydrous DMF. Re-
adjust pH to 8.4 to dissolve precipitates and the solution was stirred at r.t. overnight.
After TLC purification, the product was purified by HPLC (Method I). 6-FAM-MIN
(7e2) and 5-FAM-MIN (7e1) were eluted at very different retention times, 21.5 and 31.5
min (the retention time has ±3 min error between different runs), respectively. Each
isomer was collected separately and then concentrated in vacuo to remove buffer.
Compound 7e1 and 7e2 could also be directly synthesized from 5-FAM, SE and 6-FAM,
SE according to the method described above. Detailed NMR descriptions given below
correspond to the HPLC-separated products. Total amount of 7e1 and 7e2 is 11.6 mg,
67.2% yield. 5-FAM-MIN (7e1, triethylammonium bicarbonate salt): obtained 6.3 mg
(triethylammonium bicarbonate salt).
1
H NMR (D
2
O): δ 8.77 (d, J = 7.0 Hz, 1H), 8.09 (d,
J = 1.6 Hz, 1H), 7.94 – 7.67 (m, 4H), 7.31 (td, J = 6.4, 1.6 Hz, 1H), 7.26 (d, J = 8.0 Hz,
1H), 7.06 (s, 1H), 7.03 (s, 1H), 6.55 (dq, J = 5.0, 2.3 Hz, 4H), 4.57 – 4.46 (m, 1H), 4.42 –
67
4.21 (m, 2H), 3.72 – 3.42 (m, 4H).
31
P NMR (D
2
O): δ 16.52 (s). HRMS (positive ion
MALDI): calcd 754.1198 m/z, found M
+
= 754.1178 m/z. 6-FAM-MIN (7e2,
triethylammonium bicarbonate salt): obtained 5.3 mg (triethylammonium bicarbonate
salt).
1
H NMR (D
2
O): δ 8.75 (d, J = 7.0 Hz, 1H), 7.89 (dd, J = 8.1, 1.8 Hz, 1H), 7.82 –
7.67 (m, 4H), 7.55 (d, J = 1.6 Hz, 1H), 7.27 (td, J = 6.7, 1.6 Hz, 1H), 7.07 (s, 1H), 7.05 (s,
1H), 6.63 – 6.51 (m, 4H), 4.50 – 4.42 (m, 1H), 4.34 – 4.19 (m, 2H), 3.63 – 3.48 (m, 3H),
3.42 (dd, J = 14.1, 6.9 Hz, 1H).
31
P NMR (D
2
O): δ 16.52 (s). HRMS (positive ion
MALDI): calcd 754.1198 m/z, found M
+
= 754.1187 m/z.
5-FAM-MINPC (7f1, also known as 5-FAM-3-IPEHPC, 3-(2-carboxy-2-
hydroxy-2-phosphonoethyl)-1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)benzamido)-2-hydroxypropyl)imidazo[1,2-a]pyridin-1-ium) and 6-FAM-MINPC
(7f2, also known as 6-FAM-3-IPEHPC, 3-(2-carboxy-2-hydroxy-2-phosphonoethyl)-1-
(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-
hydroxypropyl)imidazo[1,2-a]pyridin-1-ium):
Synthesized according to method described above with 20 mg of compound 4f
(0.056 mmol, 2.5 eq.) in 1 mL of H
2
O and pH adjusted to 8.57 with Na
2
CO
3
(s), to which
added in 10.5 mg of 5(6)-FAM, SE (0.022 mmol, 1 eq.) in 300 µL anhydrous DMF. Re-
adjust pH to 8.4 to dissolve precipitates and the solution was stirred at r.t. overnight.
After TLC purification, the product was purified by HPLC (Method I). 6-FAM-MINPC
(7f2) and 5-FAM-MINPC (7f1) were eluted at very different retention times, 19 and 28
min (the retention time has ±1.5 min error between different runs), respectively. Each
isomer was collected separately and then concentrated in vacuo to remove buffer.
Compound 7f1 and 7f2 could also be directly synthesized from 5-FAM, SE and 6-FAM,
68
SE according to the method described above. Detailed NMR descriptions given below
correspond to the HPLC-separated products. Total amount of 7f1 and 7f2 is 11.7 mg,
73.2% yield. 5-FAM-MINPC (7f1, triethylammonium bicarbonate salt): obtained 6.3 mg
(triethylammonium bicarbonate salt).
1
H NMR (D
2
O): δ 8.69 (d, J = 6.9 Hz, 1H), 8.08
(dq, J = 1.6, 0.8 Hz, 1H), 7.88 – 7.75 (m, 3H), 7.68 (d, J = 4.4 Hz, 1H), 7.33 (ddd, J = 6.9,
5.8, 2.1 Hz, 1H), 7.27 (dt, J = 8.0, 0.7 Hz, 1H), 7.01 (dt, J = 9.2, 0.9 Hz, 2H), 6.53 – 6.46
(m, 3H), 4.50 (dt, J = 14.6, 2.9 Hz, 1H), 4.32 (dd, J = 14.6, 9.0 Hz, 1H), 4.26 (dq, J = 9.2,
5.1, 4.1 Hz, 1H), 3.72 (dd, J = 15.9, 3.2 Hz, 1H), 3.62 (ddd, J = 14.1, 4.8, 1.9 Hz, 1H),
3.48 (ddd, J = 14.0, 7.0, 4.3 Hz, 1H), 3.40 (dd, J = 15.7, 7.4 Hz, 1H).
31
P NMR (D
2
O): δ
14.82 (s). HRMS (positive ion MALDI): calcd 718.1433 m/z, found M
+
= 718.1399 m/z.
6-FAM-MINPC (7f2, triethylammonium bicarbonate salt): obtained 5.4 mg
(triethylammonium bicarbonate salt).
1
H NMR (D
2
O): δ 8.64 (d, J = 7.0 Hz, 1H), 7.85
(ddd, J = 8.1, 1.8, 0.9 Hz, 1H), 7.78 – 7.62 (m, 4H), 7.50 (dt, J = 1.7, 0.8 Hz, 1H), 7.28
(td, J = 6.8, 1.6 Hz, 1H), 7.04 – 6.95 (m, 2H), 6.53 – 6.45 (m, 4H), 4.46 – 4.37 (m, 1H),
4.29 – 4.12 (m, 2H), 3.68 (dd, J = 15.7, 3.6 Hz, 1H), 3.52 (dd, J = 14.0, 4.0 Hz, 1H), 3.43
– 3.33 (m, 2H).
31
P NMR (D
2
O): δ 15.03 (s). HRMS (positive ion MALDI): calcd
718.1433 m/z, found M
+
= 718.1416 m/z.
2.4.7 UV-VIS absorption and fluorescence emission spectra
All labeled samples were dissolved in water and diluted with 0.1 M phosphate
buffer or 1×PBS (for 7d5). Assuming that the labeled bisphosphonates have the same
extinction coefficient (ε) as the carboxylic acid of the free fluorescent label, the final
concentrations for all labeled products are calculated from UV-VIS absorption spectra at
λ = 493 nm (ε = 73000 M
-1
cm
-1
at pH 7.2) for FAM conjugates (7a1-7a3, 7b1, 7c1, 7d1-
69
7d2, 7e1-7e2, 7f1-7f2), λ = 567.5 nm (ε = 114850 M
-1
cm
-1
at pH 7.5) for RhR-X
conjugates (7a4, 7b2, 7c2), λ = 580 nm (ε = 72000 M
-1
cm
-1
at pH 8.0) for ROX
conjugates (7a5, 7b3), λ = 648 nm (ε = 240000 M
-1
cm
-1
at pH 7.2) for AF647 conjugates
(7a6, 7b4, 7d3), λ = 774 nm (ε = 240000 M
-1
cm
-1
at pH 7.4) for 800CW conjugates (7d4),
λ = 644 nm (ε = 271000 M
-1
cm
-1
at pH 7.4, 1×PBS) for Sulfo-Cy5 conjugates (7d5).
36-42
Emission spectra for FAM and RhR-X conjugates were recorded using an
excitation wavelength of 490 nm or 520 nm, respectively. Emission spectra for AF647
conjugates and Sulfo-Cy5 conjugates were recorded using an excitation wavelength of
600 nm. Emission spectra of 5(6)-ROX conjugates were recorded using an excitation
wavelength of 575 nm. Emission spectra of IRDye CW800 conjugate was recorded using
an excitation wavelength of 750 nm. The set up of excitation slit, emission slit,
integration time and increment were determined to get optimal spectra for each
compound respectively, depending on the sample concentration and spectrometer used
every time.
2.5 References
1. Russell, R. G. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J., Mechanisms of
action of bisphosphonates: similarities and differences and their potential influence on
clinical efficacy. Osteoporos Int 2008, 19 (6), 733-59.
2. Russell, R. G. G., Bisphosphonates: The first 40 years. Bone 2011, 49 (1), 2-19.
3. Ebetino, F.; Hogan, A.; Sun, S.; Tsoumpra, M.; Duan, X.; Triffitt, J.; Kwaasi, A.;
Dunford, J.; Barnett, B.; Oppermann, U.; Lundy, M.; Boyde, A.; Kashemirov, B.;
McKenna, C.; Russell, R., The relationship between the chemistry and biological activity
of the bisphosphonates. Bone 2011, 49 20-33.
4. Coxon, F.; Helfrich, M.; Larijani, B.; Muzylak, M.; Dunford, J.; Marshall, D.;
McKinnon, A.; Nesbitt, S.; Horton, M.; Seabra, M.; Ebetino, F.; Rogers, M.,
Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl
70
transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J
Biol Chem 2001, 276 (51), 48213-22.
5. Coxon, F.; Ebetino, F.; Mules, E.; Seabra, M.; McKenna, C.; Rogers, M.,
Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the
prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo.
Bone 2005, 37 (3), 349-58.
6. Reid, I.; Bolland, M.; Grey, A., Is bisphosphonate-associated osteonecrosis of the
jaw caused by soft tissue toxicity? Bone 2007, 41 (3), 318-20.
7. Siddiqi, A.; Payne, A.; Zafar, S., Bisphosphonate-induced osteonecrosis of the
jaw: a medical enigma? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009, 108
(3), e1-8.
8. Allen, M.; Burr, D., The pathogenesis of bisphosphonate-related osteonecrosis of
the jaw: so many hypotheses, so few data. J Oral Maxillofac Surg 2009, 67 (5 Suppl), 61-
70.
9. Reid, I., Osteonecrosis of the jaw: who gets it, and why? Bone 2009, 44 (1), 4-10.
10. Reid, I. R.; Cornish, J., Epidemiology and pathogenesis of osteonecrosis of the
jaw. Nature reviews Rheumatology 2012, 8 (2), 90-6.
11. Zhang, S.; Gangal, G.; Uludag, H., 'Magic bullets' for bone diseases: progress in
rational design of bone-seeking medicinal agents. Chem Soc Rev 2007, 36 (3), 507-31.
12. Figueiredo, J.; Passerotti, C.; Sponholtz, T.; Nguyen, H.; Weissleder, R., A novel
method of imaging calcium urolithiasis using fluorescence. J Urol 2008, 179 (4), 1610-4.
13. Jaffer, F.; Libby, P.; Weissleder, R., Optical and multimodality molecular
imaging: insights into atherosclerosis. Arterioscler Thromb Vasc Biol 2009, 29 (7), 1017-
24.
14. Subramanian, S.; Jaffer, F.; Tawakol, A., Optical molecular imaging in
atherosclerosis. J Nucl Cardiol 2010, 17 (1), 135-44.
15. Kubicek, V.; Lukes, I., Bone-seeking probes for optical and magnetic resonance
imaging. Future Med Chem 2010, 2 (3), 521-31.
16. Hilderbrand, S.; Weissleder, R., Near-infrared fluorescence: application to in vivo
molecular imaging. Curr Opin Chem Biol 2010, 14 (1), 71-9.
17. Troyan, S.; Kianzad, V.; Gibbs-Strauss, S.; Gioux, S.; Matsui, A.; Oketokoun, R.;
Ngo, L.; Khamene, A.; Azar, F.; Frangioni, J., The FLARE intraoperative near-infrared
fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel
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lymph node mapping. Ann Surg Oncol 2009, 16 (10), 2943-52.
18. Gioux, S.; Choi, H.; Frangioni, J., Image-guided surgery using invisible near-
infrared light: fundamentals of clinical translation. Mol Imaging 2010, 9 (5), 237-55.
19. Leblond, F.; Davis, S.; Valdes, P.; Pogue, B., Pre-clinical whole-body
fluorescence imaging: Review of instruments, methods and applications. J Photochem
Photobiol B 2010, 98 (1), 77-94.
20. Thompson, K.; Rogers, M.; Coxon, F.; Crockett, J., Cytosolic entry of
bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. Mol
Pharmacol 2006, 69 (5), 1624-32.
21. Coxon, F. P.; Thompson, K.; Roelofs, A. J.; Ebetino, F. H.; Rogers, M. J.,
Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-
resorbing cells. Bone 2008, 42 (5), 848-60.
22. Zaheer, A.; Lenkinski, R.; Mahmood, A.; Jones, A.; Cantley, L.; Frangioni, J., In
vivo near-infrared fluorescence imaging of osteoblastic activity. Nat Biotechnol 2001, 19
(12), 1148-54.
23. Bhushan, K.; Tanaka, E.; Frangioni, J., Synthesis of conjugatable bisphosphonates
for molecular imaging of large animals. Angew Chem Int Ed Engl 2007, 46 (42), 7969-71.
24. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.
G.; Coxon, F. P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled
risedronate and related analogues: "magic linker" synthesis. Bioconjug Chem 2008, 19
(12), 2308-10.
25. Martin, M.; Arnold, W.; Heath, H. r.; Urbina, J.; Oldfield, E., Nitrogen-containing
bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis.
Biochem Biophys Res Commun 1999, 263 (3), 754-8.
26. Hounslow, A.; Carran, J.; Brown, R.; Rejman, D.; Blackburn, G.; Watts, D.,
Determination of the microscopic equilibrium dissociation constants for risedronate and
its analogues reveals two distinct roles for the nitrogen atom in nitrogen-containing
bisphosphonate drugs. J Med Chem 2008, 51 (14), 4170-8.
27. Demberelnyamba, D.; Yoon, S. J.; Lee, H., New epoxide molten salts: Key
intermediates for designing novel ionic liquids. Chem Lett 2004, 33 (5), 560-561.
28. Brown, D. M.; Higson, H. M., Phospholipids .1. The Hydrolysis of Some Esters
of Cyclohexanediol Phosphates. J Chem Soc 1957, (May), 2034-2041.
29. Cox, J. R.; Ramsay, O. B., Mechanisms of Nucleophilic Substitution in Phosphate
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30. Kirby, A. J.; Varvoglis A. G., Reactivity of Phosphate Esters. Monoester
Hydrolysis. J Am Chem Soc 1967, 89 (2), 415-&.
31. Guthrie, J. P., Hydration and Dehydration of Phosphoric-Acid Derivatives - Free-
Energies of Formation of Pentacoordinate Intermediates for Phosphate Ester Hydrolysis
and of Monomeric Metaphosphate. J Am Chem Soc 1977, 99 (12), 3991-4001.
32. Sprecher, M.; Oppenheimer, R.; Nov, E., Phosphonate and Phosphate Monoester
Hydrolysis. Synth Commun 1993, 23 (1), 115-120.
33. McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M.; Mallard-Favier, I.;
Stewart, C. A.; Rojas, J.; Lundy, M. W.; Ebetino, F. H.; Baron, R. A.; Dunford, J. E.;
Kirsten, M. L.; Seabra, M. C.; Bala, J. L.; Marma, M. S.; Rogers, M. J.; Coxon, F. P.,
Synthesis, chiral high performance liquid chromatographic resolution and enantiospecific
activity of a potent new geranylgeranyl transferase inhibitor, 2-hydroxy-3-imidazo[1,2-
a]pyridin-3-yl-2-phosphonopropionic acid. J Med Chem 2010, 53 (9), 3454-64.
34. Rocheblave, L.; Bihel, F.; De Michelis, C.; Priem, G.; Courcambeck, J.; Bonnet,
B.; Chermann, J. C.; Kraus, J. L., Synthesis and antiviral activity of new anti-HIV
amprenavir bioisosteres. J Med Chem 2002, 45 (15), 3321-3324.
35. White, S. S.; Li, H. T.; Marsh, R. J.; Piper, J. D.; Leonczek, N. D.; Nicolaou, N.;
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switch in free solution. J Am Chem Soc 2006, 128 (35), 11423-11432.
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V.; Lukhtanov, E.; Metcalf, M.; Mills, A.; Reed, M. W.; Sanders, S.; Shishkina, I.;
Vermeulen, N. M. J., Reduced aggregation and improved specificity of G-rich
oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases. Nucleic
Acids Res 2002, 30 (22), 4952-4959.
37. Mende, I.; Hoffmann, P.; Wolf, A.; Lutterbuse, R.; Kopp, E.; Baeuerle, P. A.; de
Baey, A.; Kufer, P., Highly efficient antigen targeting to M-DC8(+) dendritic cells via Fc
gamma RIII/CD16-specific antibody conjugates. Int Immunol 2005, 17 (5), 539-547.
38. Lefevre, C.; Kang, H. C.; Haugland, R. P.; Malekzadeh, N.; Arttamangkul, S.;
Haugland, R. P., Texas Red-X and rhodamine Red-X, new derivatives of sulforhodamine
101 and lissamine rhodamine B with improved labeling and fluorescence properties.
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73
40. Balaz, M.; Sundberg, M.; Persson, M.; Kvassman, J.; Monsson, A., Effects of
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41. Li-Cor IRDye 800CW, SE spectroscopic property.
http://www.licor.com/bio/products/reagents/irdye_800cw_nhs_ester/irdye_800cw_nhs_es
ter.jsp (accessed August, 2013).
42. Lumiprobe Sulfo-Cy5, SE spectroscopic property.
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74
Chapter 3
Activity Characterization / Evaluation of Fluorescent N-
Heterocyclic Bisphosphonates as Imaging Probes
3.1 Background
An excellent probe is the one that can meet the aim of proposed studies. As for
bone imaging, one important requirement is to retain the bone-specific targeting property
1
;
in addition, some studies require that the probes are good mimic of the native drugs,
which means that the probes would retain at least partially the pharmacological activity,
e.g. the inhibitory activity of protein prenylation/bone resorption as for bisphosphonate
probes.
2
The alendronate
3, 4
and pamidronate
5, 6
based fluorescent probes in literature
generally have a certain degree of mineral binding affinity which is not thoroughly
quantified though; however, their inhibitory activity of protein prenylation is either
lacking or not reported. 5(6)-FAMRIS reported earlier by our group demonstrated anti-
prenylation effect in vitro
7
; thus it is of great importance to systematically investigate the
binding affinity and pharmacological activity of the fluorescent bisphosphonate imaging
probe “toolkit” which is discussed in Chapter 2.
Different approaches have been utilized to study the mineral binding affinity of
bisphosphonate drugs over the years. Nancollas, et al.,
8
measured the kinetic binding
affinity of 6 clinically used bisphosphonates by a constant composition method at 37 °C
and at physiological ionic strength (0.15 M), with the affinity constants (K
L
) for the
adsorption of bisphosphonates calculated from kinetic studies on hydroxyapatite (HAP)
75
crystal growth. In addition, they also suggested that BPs’ effects on zeta potential and
interfacial tension have relevance with their interaction with the bone matrix. The same
methodology was also applied with carbonated apatite (CAP), and similar results were
obtained in terms of binding affinity rank order of the 6 bisphosphonates.
9
Jahnke, et
al.,
10
reported in 2010 an NMR-based in vitro assay for measurement of binding affinities
of small molecules to HAP or bone powder; and the assay can be done in either a direct
binding format or in competition mode. A few clinically relevant bisphosphonates and
some common bone-staining agents such as alizarin and its derivatives were tested by
this method, confirming the binding of these molecules in the new assay. Van Beek, et
al.,
11
adopted a radioactive competition assay using
14
C-dimethyl APD (dimethyl-APD
also known as olpadronate, see Chapter 1) for the binding affinity assay of several
heterocycle-containing bisphosphonate analogues; and Reszka, et al.,
12
utilized the
similar method later with another radio-labeled BP,
14
C-alendronate, to investigate
binding affinities of several clinically used BPs.
Russell, et al., developed a novel approach using HAP column chromatography in
recent years.
13-16
In this approach, BP molecules initially adsorbed at the top of HAP
column, and were then driven out of the crystal surface with elution by sodium or
potassium phosphate buffer (pH ~ 6.8); due to different affinities of BPs for HAP, the
time taken for BPs to be eluted from HAP column varies, which can reflect their binding
affinities. This methodology has already been successfully applied in binding affinity
assays of BPs and related analogues.
7, 13, 15, 16
It should be mentioned that each method
for binding affinity assay has its advantages and drawbacks, and the affinity rank order of
bisphosphonates is not identical across these assays thus there is still controversy whether
76
and what extend the R
2
group of bisphosphonates would affect their mineral binding
affinities
16, 17
; however, most of these methods are capable enough as a way to investigate
the mineral binding affinities of novel agents. Therefore, the binding affinity assay of
above mentioned fluorescent bisphosphonate probe “toolkit” was performed by both
HAP column chromatography and adsorption isotherms, in collaboration with Dr. R.
Graham Russell.
The mechanism of anti-resorptive action of N-BPs/PCs is through the inhibition
of key enzymes in isoprenoid biosynthetic pathways [FPPS as the target for N-BPs
2
while
rab geranylgeranyl transferase (RGGT) as the target for PC analogues
18-21
], finally
resulting in dysfunction and reduced cell viability of osteoclasts as discussed in Chapter 1.
It is found that N-BPs also reduce cell viability and induce apoptosis in the macrophage-
like cell line J774 by preventing protein prenylation
22
, indicating that N-BPs inhibit
osteoclast-mediated bone resorption and reduce J774 macrophage viability by the same
molecular mechanism. Thus, the effects of fluorescent bisphosphonate imaging probes on
protein prenylation and cell viability of J774.2 mouse macrophages were investigated to
determine their pharmacological activities, in collaboration with Drs. Anke Roelofs,
Fraser P. Coxon and Michael J. Rogers.
3.2 Results and discussion
3.2.1 Hydroxyapatite binding affinity assays
3.2.1.1 HAP column chromatography assay
It is generally accepted that the two phosphonate groups as well as the R
1
-OH
group of bisphosphonates are essential for their strong bone binding affinity
17
; thus the
77
affinities of RIS and its two analogues [RISPC, in which one phosphonate was replaced
by a carboxylate; and deoxy-RIS (dRIS), in which R
1
is H] were tested and compared by
the HAP column chromatography method (Figure 3.1). As expected, the data shows that
RISPC and dRIS have statistically reduced retention time compared to RIS (P < 0.001),
indicating their weaker binding affinity to bone mineral. In addition, RISPC has the
shortest retention time, suggesting that the mineral binding affinity of BPs is
predominantly determined by the phosphonate groups; while the R
1
-OH group also plays
a clear role in mineral affinity, which is hypothesized to interact with bone mineral
through tridentate binding together with two phosphonates.
11
To investigate whether the attached fluorophore could influence the mineral
binding affinity or not, the retention time of each fluorescent BP conjugate on HAP
column was measured and normalized to retention time of its counterpart BP molecule
(Figure 3.1 – 3.3). As shown in Figure 3.1, each fluorescent conjugate generally retains
most of the HAP affinity compared to its counterpart molecule, with 5(6)-ROX
conjugates displaying even higher affinity than their counterparts. 5(6)-FAM-RIS has a
slightly decrease of HAP affinity (~ 6%) while 5(6)-RhR-RIS has almost the same HAP
affinity as RIS. AF647-RIS exhibits the largest decrease of HAP affinity compared to
other fluorescent RIS conjugates, but still retains 74%. These findings have further been
confirmed using an in vitro HAP crystal growth assay as a model to determine the
relative affinity for bone mineral, which demonstrated that both 5(6)-FAM-RIS and
AF647-RIS significantly inhibited HAP crystal growth.
23
RISPC and dRIS fluorescent conjugates generally have negligible affinity
reduction compared to RISPC and dRIS themselves, and 5(6)-ROX and 5(6)-RhR
78
conjugates show even higher affinity than their counterparts. Notably, the affinity rank
order of RIS, dRIS and RISPC conjugates with the same fluorophore remains the same as
RIS > dRIS/RISPC, but the difference between dRIS and RISPC conjugates is not
statistically significant as their counterparts [the retention time of 5(6)-FAM-dRIS (0.50)
on HAP column is similar as 5(6)-FAM-RISPC (0.52)]. In addition, it is observed that
AF647-RIS has even slightly weaker HAP affinity than 5(6)-ROX-RISPC (0.74 vs. 0.78),
indicating the fluorophores as part of R
2
group have non-negligible impacts on HAP
affinity. These results further support the notion that phosphonates are the major
determinant of mineral binding affinity, and R
2
group also has important influences
(generally slight reduction but in some cases enhancement) on binding affinity, and may
diminish the effects caused by R
1
and even phosphonate groups.
Figure 3.1 Comparison of peak retention time of RIS, dRIS, RISPC and their related
fluorescent conjugates.
Data are shown as mean ± SD from three individual studies, as relative retention times
normalized to RIS.
1.00
0.58
0.47
0.94
0.50 0.52
0.74
0.46
1.46
0.78
1.02
0.68
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Relative retention time normalized to RIS
RIS, dRIS, RISPC and their fluorescent probes
79
The results for ZOL and its fluorescent conjugates are similar as for RIS (Figure
3.2), with the fluorescent ZOL conjugates have ~ 23 – 38% affinity reduction compared
to ZOL. It is noted that 6-FAMZOL has a little longer retention time on HAP column
than its 5-isomer, which is in good accordance with the results for 6-FAM-RIS and 5-
FAM-RIS isomer.
7
Same comparison results were also observed for 6-FAM-MIN and 5-
FAM-MIN (Figure 3.3); however, the retention time rank order for 6-FAM-MINPC and
5-FAM-MINPC reverses (Figure 3.3), indicating the profound effects introduced by
attached fluorophores on the R
2
group. In addition, the different results obtained for 5-
FAM and 6-FAM conjugate isomers also suggest that HAP column chromatography
could differentiate even subtle structural differences.
Figure 3.2 Comparison of peak retention time of ZOL and its related fluorescent
conjugates.
Data are shown as mean ± SD from three individual studies, as relative retention times
normalized to ZOL.
1.00
0.66
0.68
0.62
0.77
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Zoledronate 5-FAM-ZOL 6-FAM-ZOL AF647-ZOL 800CW-ZOL
Relative retention time normalized to ZOL
ZOL and its fluorescent probes
80
Figure 3.3 Comparison of peak retention time of MIN, MINPC and their related fluorescent
conjugates.
Data are shown as mean ± SD from three individual studies, as relative retention times
normalized to MIN.
3.2.1.2 Quantitative measurement of BP-HAP interaction by using Langmuir adsorption
isotherms
The above HAP column chromatography method has proved to be capable for
relative binding affinity comparison of different fluorescent BP probes; however, it does
not allow quantitative calculation of affinity constants that represent binding capacities
and affinities of these probes. Thus a binding assay that could be used for quantitative
measurement of BP-HAP interaction is of great interest.
It is considered that BP binds to HAP crystals via the Langmuir adsorption
isotherm model (Figure 3.4).
14
With the increasing concentration of BP in solution, the
amount bound to HAP will depend on its affinity and increase up to a saturation level that
indicates the capacity of HAP to adsorb a given BP onto their surfaces. The equilibrium
1.00
0.55
0.74
0.79
0.53
0.49
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Minodronate MINPC 5-FAM-MIN 6-FAM-MIN 5-FAM-MINPC 6-FAM-MINPC
Relative retention time normalized to MIN
BPs and their fluorescent probes
81
dissociation constant (K
d
) of BP-HAP complex as well as the maximal binding capacity
of HAP for a certain BP (Bmax) can be calculated from this non-linear adsorption
isotherm curve.
Figure 3.4 The Langmuir adsorption isotherm for the binding of BPs to HAP.
Adapted from ref. 14
Figure 3.5 Adsorption isotherms for the binding of four fluorescent BP/PC probes.
A: 5(6)-ROX-RIS, B: 5(6)-ROX-RISPC, C: AF647-RIS, D: AF647-RISPC to HAP at pH 6.8
Binding of ROXRIS to HAP
0 20 40 60 80 100
0.0
0.5
1.0
1.5
2.0
Kd= 7.681 ± 0.7848 µM
Bmax= 1.664 ± 0.05405 µmol/m
2
Equilibrium BP concentration (µM)
BP HAP surface conc. (µmol/m
2
)
Scatchard plot
0.0 0.5 1.0 1.5 2.0
0.00
0.05
0.10
0.15
0.20
0.25
Bmax/Kd
Bmax
Bound, µmol/m
2
Bound/Free
Binding of ROXRISPC to HAP
0 50 100 150
0.0
0.5
1.0
1.5
Kd= 8.442 ± 0.8492 µM
Bmax= 1.261 ± 0.03434 µmol/m
2
Equilibrium BP concentration (µM)
BP HAP surface conc. (µmol/m
2
)
Scatchard plot
0.0 0.5 1.0 1.5
0.00
0.05
0.10
0.15
0.20 Bmax/Kd
Bmax
Bound, µmol/m
2
Bound/Free
Binding of AF647RIS to HAP
0 50 100 150 200 250
0.0
0.2
0.4
0.6
0.8
1.0
Kd= 8.754 ± 1.251 µM
Bmax= 0.8437 ± 0.02790 µmol/m
2
Equilibrium BP concentration (µM)
BP HAP surface conc. (µmol/m
2
)
Scatchard plot
0.0 0.2 0.4 0.6 0.8 1.0
0.00
0.05
0.10
0.15
Bmax/Kd
Bmax
Bound, µmol/m
2
Bound/Free
Binding of AF647RISPC to HAP
0 100 200 300
0.0
0.2
0.4
0.6
Kd= 53.20 ± 28.90 µM
Bmax= 0.4467 ± 0.08379 µmol/m
2
Equilibrium BP concentration (µM)
BP HAP surface conc. (µmol/m
2
)
Scatchard plot
0.0 0.1 0.2 0.3 0.4 0.5
0.000
0.002
0.004
0.006
0.008
0.010
Bmax/Kd
Bmax
Bound, µmol/m
2
Bound/Free
A"
B"
C" D"
Binding&of&5(6)-ROX-RIS&to&HAP& Binding&of&5(6)-ROX-RISPC&to&HAP&
Binding&of&AF647-RIS&to&HAP& Binding&of&AF647-RISPC&to&HAP&
82
with Scatchard plots of the same data as inset, data are mean ± SD (n=3).
Four fluorescent BP probes were analyzed by this method, and their adsorption
isotherms were depicted in Figure 3.5. The K
d
and Bmax values were derived from the
data as illustrated in Table 3.1. A small K
d
reflects higher affinity of BP to HAP while a
large K
d
reflects lower affinity. Furthermore, Bmax is also an important parameter for
affinity measurement, with the larger Bmax indicating higher binding affinity. The results
show that 5(6)-ROX conjugates have higher affinity than their corresponding AF647
conjugates, while the BP conjugates have higher affinity than the PC conjugates with the
same fluorescent dye, which are in keeping with the results obtained from HAP column
chromatography. Notably, the Langmuir adsorption isotherm has also been used in
fluorescence competitive binding assay with the aid of 5(6)-FAM-RIS for the
dissociation constants measurement of nine clinically relevant BP drugs with high
sensitivity and accuracy.
16
Table 3.1 Dissociation constants (K
d
) and maximum capacities (Bmax) of fluorescent
BP/PC probes for HAP
Fluorescent probe K
d
(µM) Bmax (µmol/m
2
)
5(6)-ROX-RIS 7.681±0.7848 1.664±0.05405
5(6)-ROX-RISPC 8.442±0.8492 1.261±0.03434
AF647-RIS 8.754±1.251 0.8437±0.02790
AF647-RISPC 53.20±28.90 0.4467±0.08379
Data are shown as mean ± SD (n=3)
83
3.2.2 Inhibition of protein prenylation and cell viability assays
The detection of unprenylated Rap1A (uRap1A) and cell viability of J774.2
macrophages has been used widely as an in vitro screening of pharmacological activity of
novel BP and related analogues.
7, 13, 24
Since the native PC compounds (RISPC, MINPC)
are much less active than the BPs in these assays, fluorescent PC analogues could not be
adequately tested for such activity as too high concentrations were needed; thus only
fluorescent BP and deoxy-BP (dRIS) conjugates were analyzed (Figure 3.6).
Figure 3.6 Prenylation assay and J774.2 cell viability assay of some fluorescent BP imaging
probes.
A-C: Western blot assays for unprenylated Rap1A (uRap1A). J774.2 macrophages were treated
for 24 h with 10 or 100 µM of fluorescent analogues of RIS (A), dRIS (B), and ZOL(C), the
ß"ac%n'
uRap1A'
10' 10' 100'
dRIS' 5(6)"FAM"dRIS'
0' 100'
ß"ac%n'
uRap1A'
0' 10' 100' 10' 100' 10' 100' 10' 100'
ZOL' 5"FAM"ZOL' 6"FAM"ZOL' 800CW"ZOL'
ß"ac%n'
uRap1A'
10' 10' 100' 10' 100' 10' 100' 10' 100'
RIS' 5"FAM"RIS' 6"FAM"RIS'5(6)"ROX"RIS' AF647"RIS'
0' 100'
A"
B"
C"
D"
E"
F"
Conc'(µM)'
Conc'(µM)'
Conc'(µM)'
1.6$ 1.8$ 4.1$ 1.3$ 4.8$ 0.1$ 0.8$ 0.1$ 0.0$ 0.0$ 3.2$ Ra,o$
0.5$ 0.1$ 3.0$ 0.1$ 4.0$ Ra,o$
0.0$ 1.8$ 2.1$ 0.1$ 0.1$ 0.1$ 0.7$ 0.1$ 0.0$ Ra,o$
84
respective native BP, or vehicle, for 24 h. Detection of β-actin served as loading control. The
ratio between abundance of unprenylated Rap1A and β-actin is indicated for each sample below
the blots. D-F: Cell viability assays of J774.2 macrophages. Cells were then treated with 10, 100
or 500 µM of fluorescent analogues of RIS (D), dRIS (E), and ZOL (F), the respective native BP,
or vehicle, for 48 h. Results are shown as mean ± SD of ≥ 2 independent experiments, performed
at least in duplicate.
It was found that unprenylated Rap1A was clearly detectable in cell lysate of both
5-FAM-RIS and 6-FAM-RIS treated J774.2 mouse macrophages (Figure 3.6A),
suggesting they are active towards inhibition of protein prenylation as previously
reported. 5(6)-FAM-dRIS is also active, with potency comparable to its parent BP dRIS
(Figure 3.6B). Both 5-FAM-ZOL and 6-FAM-ZOL retain some activity, although their
activities are weaker than native ZOL (Figure 3.6C). It is interesting that the activity is
more apparent for 6-FAM-ZOL than 5-FAM-ZOL at the concentrations used, which
represents the same rank order of their above mentioned HAP binding affinity. The
cellular activities of 5-FAM-MIN and 6-FAM-MIN were not obtained due to unspecific
cell death of control experiment, thus will not be reported here.
5(6)-ROX-RIS also shows some activity, and its activity is weaker than native
RIS or 5,6-FAM-RIS (Figure 3.6A). The cell lysate treated with two near infrared BP
conjugates, AF647-RIS and 800CW-ZOL did not show accumulation of unprenylated
Rap1A at the concentrations used, suggesting they are inactive. The results of cell
viability assay are in good accordance with the prenylation assay, with the exception that
AF647-RIS and 800CW-ZOL treated J774.2 macrophages exhibit modest decrease in
viable cell number at high concentrations, which may be due to a non-specific effect, e.g.
calcium chelation. The near infrared fluorophores generally have larger size than FAM
and ROX fluorophores, which may interfere the interactions of fluorescent BP and its
85
enzymatic target, resulting the loss of activity. An extended length of linker may be used
in the fluorescent BP conjugates to exclude the fluorophores outside the enzymatic
activity site, which may help retaining some activity.
3.3 Conclusion
The mineral binding affinity and pharmacological cellular activity were tested for
the fluorescent imaging probe “toolkit”, which provide the first fully characterization of
the probes. The imaging probes generally retain substantial affinity for bone mineral,
reflecting the varying affinities of their parent drug components. The conjugated
fluorophores also have important influences (generally slight reduction but in some cases
enhancement) on mineral affinity of the probes.
Both the FAM and ROX conjugates display cellular activity including inhibition
of protein prenylation and reduction of cell viability of J774.2 macrophages, albeit
weaker than their parent drug compounds. The near infrared conjugates (AF647-RIS and
800CW-ZOL) do not show clear activity, which may be due to the interference to their
interactions with the enzymatic target caused by the larger sized near infrared
fluorophores. It will be of great interest to measure the IC
50
values of these probes
towards specific enzyme target, e.g. FPPS, to elucidate the exact mechanism of their
cellular activity. The current in vitro anti-prenylation assays have provided a general
screening of cellular activity of these probes for the first time, which is the basis for
further pharmacological activity measurement as well as selecting the appropriate
imaging probes from the versatile probe “toolkit”, for different purpose-designed
biological studies.
86
3.4 Experimental
3.4.1 Hydroxyapatite column chromatography assay
The fast performance liquid chromatography (FPLC) system consisted of a
Waters 650E advanced protein purification system (Millipore Corp., Waters
chromatography division, Milford, MA), a 600E system controller and a 484 tunable
absorbance detector for UV absorbance assessment. Ceramic hydroxyapatite [HAP,
Ca
10
(PO
4
)
6
(OH)
2
, Macro-Prep
®
Ceramic Hydroxyapatite Type II 20 µm 100 g, Bio-Rad
Laboratories, Inc. Hercules, CA] was equilibrated with 1 mM phosphate buffer (pH 6.8)
and packed in a 0.66 cm (diameter) x 6.5 cm (length) glass column (Omnifit, Bio-chem
valve™ inc., Cambridge, U.K.), which was attached to the Waters 650E advanced protein
purification system. Each sample was prepared in 1 mM potassium phosphate buffer, and
100 µL of 1 mM (0.1 µmol) sample was injected into the FPLC system. As a
consequence, the compounds were absorbed and subsequently eluted at a flow rate of 2
ml/min by using a linear concentration gradient of phosphate from 1 to 1000 mM at pH
6.8. Fractions of each sample were collected in 80 tubes using an automated fraction
collector (Gilson, France) and then used for subsequent ultraviolet (SPECTRAmax PLUS
384, Molecular Devices, CA) or fluorescence spectrometry detection (WALLAC
VICTOR
TM
, 1420 MULTILABEL COUNTER, Perkin Elmer, USA). Each fraction
contained eluent in 0.3 min. The elution profile for each sample was determined in
triplicate for statistical analysis (Prism, GraphPad Software, USA). Using the native BP
(RIS, ZOL and MIN) as a retention time control/comparator, the chromatographic
profiles of each fluorescent probe were normalized to its BP counterpart to allow relative
comparisons of separations performed at different times. Data are presented as mean
87
retention times normalized to native BP ± standard deviation (SD).
3.4.2 Quantitative measurement of BP-HAP interaction by using Langmuir
adsorption isotherms
1 mM phosphate-buffered saline (PBS) with 0.15 M NaCl (pH 6.8) was prepared
freshly. Stock solutions of 5(6)-ROX-RIS, 5(6)-ROX-RISPC, AF647-RIS and AF647-
RISPC were made by dissolving the compounds in 1mM PBS to yield a 10 mM solution.
Hydroxyapatite (Macro-Prep® Ceramic hydroxyapatite Type II 20 µM 100 g) was
obtained from Bio-Rad Laboratories, Inc. Hercules, CA.
To measure and compare the bone mineral affinities of fluorescent BP/PCs,
adsorption isotherm studies were carried out under identical experimental conditions.
Accurately weighed HAP powder (1.4-1.6 mg) was suspended in 4 mL clear vial
containing the appropriate volume of 1 mM PBS with 0.15 M NaCl (pH 6.8) for 3 hours.
After premixing, 10 mM fluorescent BP stock solutions were added, resulting in
concentrations of the fluorescent BP/PC additives ranging as 25, 50, 100, 200 and 300
µM. Equilibrium with the HAP was performed by rotating the vials end-over-end on a
shaker at room temperature for 16 hours. Each sample was prepared in triplicate.
Subsequent to the equilibrium period, the vials were centrifuged at 10,000 rpm for 5 min
to separate the solids and the supernatant. 0.3 mL of the supernatant was collected and
the equilibrium solution concentration was measured by using Nanodrop UV
spectrometer. For the calibration series, fluorescent BP/PC standards were prepared by
serial dilution from the stock solution with the same isotherm buffer to give the range
from 0 to 400 µM. Calibration curves were constructed using standard solutions of the
88
target fluorescent BP.
The amount of fluorescent BP/PC bound to the HAP (µmol/m
2
) was calculated by
comparing the end point concentration of fluorescent BP/PC detected after equilibrium to
the initial fluorescent BP/PC additive concentration (µM) using the following equation:
Fluorescent BP/PC HAP surface concentration = (Initial fluorescent BP/PC
concentration – end point concentration) / HAP surface area of the sample
Where HAP surface area of the sample was 6700 m
2
/L in our case. A plot of
fluorescent BP/PC HAP surface concentration versus BP end point concentration
provided the adsorption isotherm.
To describe the equilibrium binding of fluorescent BP/PC to HAP as a function of
increasing fluorescent BP/PC concentration, the experimental data were fitted to a
saturation binding equation: Y (specific binding) = Bmax * X/(K
d
+X) by using a non-
linear curve-fitting algorithm, implemented in the Prism program (Graphpad, USA).
Where X is the concentration of the fluorescent BP/PC, Y is the specific binding, and
Bmax is the maximum number of binding sites, expressed in the same units as the Y-axis.
K
d
is the equilibrium dissociation constant, expressed in the same units as the X-axis
(concentration). When the drug concentration equals K
d
, half of the binding sites are
occupied at equilibrium.
3.4.3 Inhibition of protein prenylation and cell viability assays
To determine the effect of fluorescent BP probes on protein prenylation, J774.2
mouse macrophages were plated out at 2x10
5
cells/mL in 24-well plates and left to adhere
overnight. Cells were then treated with 10 or 100 µM of fluorescent analogues of RIS,
89
dRIS, and ZOL, the respective native BP, or vehicle, for 24 h. Cells were lysed in
radioimmunoprecipitation buffer, and proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membranes by
western blotting. Membranes were then incubated with antibodies to the unprenylated
form of Rap1A (uRap1A) and the housekeeping protein β-actin, which were detected by
incubation with fluorescently-labeled secondary antibodies and scanning of membranes
on a LI-COR Infrared Imager. Results shown are representative of 2 independent
experiments. The ratio between abundance of unprenylated Rap1A and β-actin is
indicated for each sample below the blots in Figure 3.6.
To determine the effect of fluorescent BP analogues on cell viability, J774.2
mouse macrophages were plated at 2x10
5
cells/mL in 96-well plates and left to adhere
overnight. Cells were then treated with 10, 100 or 500 µM of fluorescent analogues of
RIS, dRIS, and ZOL, the respective native BP, or vehicle, for 48 h. At the end of the
incubation period, the medium was removed, and cells were washed twice with PBS.
Medium with AlamarBlue reagent was then added and cells were incubated for a further
3 h. Fluorescence was detected on a plate reader, and expressed as percent of vehicle
control. Results are shown as mean ± SD of ≥ 2 independent experiments, performed at
least in duplicate.
3.5 References
1. Russell, R. G. G., Bisphosphonates: The first 40 years. Bone 2011, 49 (1), 2-19.
2. Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.;
Rogers, M. J.; Russell, R. G. G.; Oppermann, U., The molecular mechanism of nitrogen-
containing bisphosphonates as antiosteoporosis drugs. Proc Natl Acad Sci U S A 2006,
103 (20), 7829-34.
90
3. Thompson, K.; Rogers, M.; Coxon, F.; Crockett, J., Cytosolic entry of
bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. Mol
Pharmacol 2006, 69 (5), 1624-32.
4. Coxon, F. P.; Thompson, K.; Roelofs, A. J.; Ebetino, F. H.; Rogers, M. J.,
Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-
resorbing cells. Bone 2008, 42 (5), 848-60.
5. Zaheer, A.; Lenkinski, R.; Mahmood, A.; Jones, A.; Cantley, L.; Frangioni, J., In
vivo near-infrared fluorescence imaging of osteoblastic activity. Nat Biotechnol 2001, 19
(12), 1148-54.
6. Bhushan, K.; Tanaka, E.; Frangioni, J., Synthesis of conjugatable bisphosphonates
for molecular imaging of large animals. Angew Chem Int Ed Engl 2007, 46 (42), 7969-71.
7. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.
G.; Coxon, F. P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled
risedronate and related analogues: "magic linker" synthesis. Bioconjug Chem 2008, 19
(12), 2308-10.
8. Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.;
Mangood, A.; Russell, R. G. G.; Ebetino, F. H., Novel insights into actions of
bisphosphonates on bone: Differences in interactions with hydroxyapatite. Bone 2006, 38
(5), 617-627.
9. Henneman, Z. J.; Nancollas, G. H.; Ebetino, F. H.; Russell, R. G. G.; Phipps, R. J.,
Bisphosphonate binding affinity as assessed by inhibition of carbonated apatite
dissolution in vitro. J Biomed Mater Res A 2008, 85A (4), 993-1000.
10. Jahnke, W.; Henry, C., An in vitro Assay to Measure Targeted Drug Delivery to
Bone Mineral. Chemmedchem 2010, 5 (5), 770-776.
11. van Beek, E. R.; Lowik, C. W. G. M.; Ebetino, F. H.; Papapoulos, S. E., Binding
and antiresorptive properties of heterocycle-containing bisphosphonate analogs:
Structure-activity relationships. Bone 1998, 23 (5), 437-442.
12. Leu, C. T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reszka, A. A., Relative
binding affinities of bisphosphonates for human bone and relationship to antiresorptive
efficacy. Bone 2006, 38 (5), 628-636.
13. Marma, M. S.; Xia, Z. D.; Stewart, C.; Coxon, F.; Dunford, J. E.; Baron, R.;
Kashemirovli, B. A.; Ebetino, F. H.; Triffitt, J. T.; Russell, R. G. G.; McKenna, C. E.,
Synthesis and biological evaluation of alpha-halogenated bisphosphonate and
phosphonocarboxylate analogues of risedronate. J Med Chem 2007, 50 (24), 5967-5975.
91
14. Russell, R. G. G.; Xia, Z.; Dunford, J. E.; Oppermann, U.; Kwaasi, A.; Hulley, P.
A.; Kavanagh, K. L.; Triffitt, J. T.; Lundy, M. W.; Phipps, R. J.; Barnett, B. L.; Coxon, F.
P.; Rogers, M. J.; Watts, N. B.; Ebetino, F. H., Bisphosphonates - An update on
mechanisms of action and how these relate to clinical efficacy. Ann NY Acad Sci 2007,
1117, 209-257.
15. Lawson, M. A.; Xia, Z.; Barnett, B. L.; Triffitt, J. T.; Phipps, R. J.; Dunford, J. E.;
Locklin, R. M.; Ebetino, F. H.; Russell, R. G. G., Differences Between Bisphosphonates
in Binding Affinities for Hydroxyapatite. J Biomed Mater Res B 2010, 92B (1), 149-155.
16. Duan, X. Physiological and biological mechanisms of bisphosphonate action.
Medical Sciences Division, University of Oxford, 2010.
17. Russell, R. G. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J., Mechanisms of
action of bisphosphonates: similarities and differences and their potential influence on
clinical efficacy. Osteoporos Int 2008, 19 (6), 733-59.
18. Coxon, F.; Helfrich, M.; Larijani, B.; Muzylak, M.; Dunford, J.; Marshall, D.;
McKinnon, A.; Nesbitt, S.; Horton, M.; Seabra, M.; Ebetino, F.; Rogers, M.,
Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl
transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J
Biol Chem 2001, 276 (51), 48213-22.
19. Coxon, F.; Ebetino, F.; Mules, E.; Seabra, M.; McKenna, C.; Rogers, M.,
Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the
prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo.
Bone 2005, 37 (3), 349-58.
20. Roelofs, A.; Hulley, P.; Meijer, A.; Ebetino, F.; Russell, R.; Shipman, C.,
Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of
risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells. Int J
Cancer 2006, 119 (6), 1254-61.
21. Baron, R.; Tavare, R.; Figueiredo, A.; Blazewska, K.; Kashemirov, B.; McKenna,
C.; Ebetino, F.; Taylor, A.; Rogers, M.; Coxon, F.; Seabra, M., Phosphonocarboxylates
inhibit the second geranylgeranyl addition by Rab geranylgeranyl transferase. J Biol
Chem 2009, 284 (11), 6861-8.
22. Luckman, S. P.; Coxon, F. P.; Ebetino, F. H.; Russell, R. G. G.; Rogers, M. J.,
Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by
preventing protein prenylation: Evidence from structure-activity relationships in J774
macrophages. J Bone Miner Res 1998, 13 (11), 1668-1678.
23. Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.;
Nancollas, G. H.; Sun, S.; Blazewska, K. M.; Bala, J. L.; Kashemirov, B. A.; Khalid, A.
B.; McKenna, C. E.; Rogers, M. J., Fluorescent risedronate analogues reveal
92
bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in
vivo. J Bone Miner Res 2010, 25 (3), 606-16.
24. McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M.; Mallard-Favier, I.;
Stewart, C. A.; Rojas, J.; Lundy, M. W.; Ebetino, F. H.; Baron, R. A.; Dunford, J. E.;
Kirsten, M. L.; Seabra, M. C.; Bala, J. L.; Marma, M. S.; Rogers, M. J.; Coxon, F. P.,
Synthesis, chiral high performance liquid chromatographic resolution and enantiospecific
activity of a potent new geranylgeranyl transferase inhibitor, 2-hydroxy-3-imidazo[1,2-
a]pyridin-3-yl-2-phosphonopropionic acid. J Med Chem 2010, 53 (9), 3454-64.
93
Chapter 4
Applications of Fluorescent N-heterocyclic Bisphosphonate
Probes
4.1 General
The fluorescent imaging probe “toolkit” has been applied in various biological
research studies including osteoclast imaging,
1
drug distribution at bone skeleton and
cellular levels,
2-4
mechanism of osteonecrosis of the jaw (ONJ) studies,
5
cancer imaging,
et al., and several papers have been published based on the obtained results; as space is
limited in the dissertation, only a few examples of these collaborative investigations will
be discussed.
4.2 Study of bisphosphonate distribution pattern in bone skeleton by
simultaneous imaging approach
As discussed in Chapter 3, it has become evident that bisphosphonate drugs have
differing affinities for bone mineral; although their rank order given by different assays
was not identical, overall findings were similar, with alendronate and pamidronate
consistently identified as having relatively high affinity, whereas risedronate (RIS) has
intermediate to low affinity among the clinically used BPs, which were proposed as a
result of differences in charge on the nitrogen atom of their R
2
side chain.
6, 7
However, it
is still unclear whether such differences affect their distribution in bone and how this may
impact on their clinical effects. Previous studies using radiolabeled alendronate or
etidronate demonstrated preferential localization to areas of bone turnover, binding to
94
newly formed mineral and mineral exposed by resorption.
8-10
Furthermore, it was
recently demonstrated, using fluorescent analogues of RIS
2
, that these compounds
penetrate the osteocyte network and bind to osteocyte lacunar walls in vivo. However,
whether differences exist between high- and low-affinity compounds in their preferential
binding to formation and resorption sites, and their degree of penetration of the osteocyte
network, is unclear. The fluorescent imaging probe “toolkit” including a variety of
fluorescent BPs and related analogues with different mineral binding affinities makes it
possible to investigate the relation between mineral affinity and distribution pattern. As a
“proof-of-concept” study, fluorescent conjugates of risedronate (high affinity), and its
analogues deoxy-risedronate (dRIS, medium affinity) and 3-PEHPC (RISPC, low
affinity), were used to compare the localization of compounds with differing mineral
affinities in vivo
*
.
4.2.1 Binding of fluorescent bisphosphonate analogues to mineral surfaces
Binding to dentine in vitro confirmed differences in mineral binding between
different fluorescent BP/PC compounds, which was influenced predominantly by the
characteristics of the parent compound but also by the choice of fluorescent tag (Figure
4.1A-C), and the results are in good accordance with the HAP column chromatography
assays in Chapter 3. In addition, similar differences in bone mineral binding in vivo was
also observed; compared with AF647-RIS, AF647-3-PEHPC (AF647-RISPC) showed a
significantly lower degree of binding to tibias from rats treated with equimolar amounts
of each compound respectively and the tibias were scanned either 1 or 7 days after
*
All FAM-, ROX- and RhR- conjugates used in this study are as their 5- and 6- isomer mixtures,
synthesized directly from the corresponding 5- and 6- isomer mixture of fluorescent dyes.
95
intravenous administration (Figure 4.1D). The results are consistent with a study in
humans comparing urinary excretion of
14
C-alendronate and
14
C-risedronate as an inverse
measure of their skeletal binding, which shows significantly higher urinary excretion of
risedronate (55%) compared with the high-affinity alendronate (48%; p < 0.02) within the
first 72 hours after administration.
11
For both compounds, fluorescence at day 7 was
similar to day 1, which is consistent with pharmacokinetic studies of BPs in humans,
showing the vast majority of BPs is cleared from the circulation by either binding to bone
or being excreted via the urine within 24 hours.
12
In addition, the data also show that the
lower affinity AF647-3-PEHPC was efficiently retained in the skeleton once bound to
bone, similar to AF674-RIS.
Figure 4.1 Relative mineral affinities of fluorescent BP analogues.
(A-C) Percentage of each of the fluorescent BP analogues that was bound to dentine discs in vitro
following incubation with 1 or 10 µM at pH 7.4 for 1 hour. ** P<0.01; *** p<0.001, as analyzed
by two-way ANOVA. (D) Fluorescence intensities of rat tibiae 1 or 7 days after intravenous
administration of either AF647-RIS (0.29 mg/kg) or AF647-3-PEHPC (0.28 mg/kg), determined
using an Infrared Imager. Fluorescence intensity for each tibia was corrected for background
A
D
AF647-RIS
AF647-3-PEHPC
Vehicle
***
B
C
***
***
***
***
***
**
***
96
fluorescence of the vehicle and expressed as mean ± SD (n=2) relative to the AF647-RIS at 1-day
group. *** p<0.001 (combined data of 1 and 7 day animals), as analyzed by unpaired t-test.
Examples of scan results are shown on the right, with fluorescence shown in black (composite
image). Adapted from ref. 4.
4.2.2 Distribution of high- and low-affinity BP analogues on cortical bone surfaces
in vivo
To assess potential differences in binding to resorption and formation surfaces,
cross sections through the tibias of the growing rats were analyzed, focusing on specific
areas that exhibited extensive endocortical bone formation and periosteal bone resorption
and areas that were quiescent on both surfaces. At the doses used, all compounds
preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces
in cortical bone, 1 day after administration (Figure 4.2A-F). In contrast, these differences
were not observed between quiescent endocortical and periosteal surfaces (Figure 4.2G),
suggesting that the differential labeling relates specifically to the type of surface (i.e.,
forming or resorbing) rather than the anatomical site. The relatively high degree of
labeling at forming surfaces is probably because of the large surface area provided by the
newly formed mineral crystals available for binding; and Kozloff, et al.,
13
also found
intense labeling at forming surfaces, although no quantitative comparison was made
between labeling at forming and resorbing surfaces. In contrast, previous studies with
radiolabeled BP have shown the preferential binding to resorbing surfaces.
8-10
The
different results obtained from radiolabeled BP and fluorescent BP could be a function of
the specific activity of the labeled materals, regarding that the purity of fluorescent BPs is
> 95% whereas only approximately 0.014% of the BP molecules are labeled with tritium.
In addition, it is worth mentioning that preferential labeling of resorbing surfaces were
97
observed for both radiolabeled alendronate and etidornate at 1.3 mmol/kg dose; however,
even higher degree of preferential labeling of resorbing surfaces was observed when the
dose of radiolabeled alendronate decreased from 1.3 mmol/kg to its pharmacologically
effective dose 0.12 mmol/kg, whereas etidronate showed similar labeling of resorbing
and forming surfaces when the dose increased from 1.3 mmol/kg to 73 mmol/kg (its
pharmacologically effective dose), suggesting the differences are also dose dependent.
10
Figure 4.2 Distribution at forming endocortical and resorbing periosteal surfaces.
Optical sections acquired by confocal microscopy of cortical bone surfaces in tibiae of 9-week
old rats given a single dose of AF647-RIS, FAM-dRIS and ROX-3-PEHPC (n=3; A, D), ROX-
RIS, FAM-dRIS and AF647-3-PEHPC (n=3; B, E, G), or ROX-RIS, FAM-RIS and AF647-RIS
(n=3; C, F) one day prior to sacrifice. (A-C) Labeling of areas of cortical bone with a forming
endocortical (left side of images) and resorbing periosteal surface (right side). For each image,
fluorescence of the different labels (AF647, FAM, ROX) are shown separately for clarity. Scale
bars = 100 µm. (D-F) Quantification of labeling of forming endocortical and resorbing periosteal
FAM-dRIS AF647-RIS ROX-3-PEHPC
FAM-dRIS ROX-RIS AF647-3-PEHPC
B
A
G FAM-dRIS ROX-RIS AF647-3-PEHPC
FAM-RIS ROX-RIS AF647-RIS C F
D
E
*
***
***
**
**
**
**
***
**
98
surfaces by densitometry using ImageJ software (≥ 3 images/rat). Data was corrected for
background fluorescence and expressed as mean ± SD (n=3) of endocortical surface labeling
intensity relative to periosteal surface labeling intensity (dotted lines) for each compound. *
p<0.05; ** p<0.01; *** p<0.001, relative to periosteal surface. (G) Labeling of quiescent
endocortical (left side) and periosteal surfaces (right side) in an area of cortical bone, showing a
similar degree of labeling on both surfaces. Scale bars = 100 µm. Adapted from ref. 4.
4.2.3 Distribution of high- and low-affinity BP analogues at resorbing surfaces
When analyzing the labeling at resorbing surfaces in more detail, it was observed
that the lower-affinity compound had a preferential binding to distinct areas that
resembled Howship’s lacunae (Figure 4.3A, arrows), whereas the higher-affinity
compound showed more uniform bone surface labeling (Figure 4.3A). This was most
pronounced in animals that had received the compounds with the largest difference in
affinity (ROX-RIS and AF647-3-PEHPC). Comparison of the distribution of AF647-RIS
and ROX-3-PEHPC, two compounds with a smaller difference in affinity for bone
mineral based on in vitro dentine binding (Figure 4.1), showed a higher degree of
colocalization, with preferential binding to distinct lacunar areas for both compounds
(Figure 4.3B, arrows). These findings were confirmed in animals treated with ROX-RIS
and AF647-RIS, in which AF647-RIS preferentially bound to distinct lacunar areas,
whereas the higher-affinity ROX-RIS again labeled the periosteal surface more uniformly
(Figure 4.3C). No clear differences in distribution between the highest- and lowest-
affinity compounds were observed at quiescent periosteal surfaces (Figure 4.3D). In
conclusion, at resorbing surfaces, the fluorescent BP anlogues showed preferential
deposition at resorption lacunae, as opposed to adjacent nonresorbed area; and this
preferential binding at resorption site is more evident with lower-affinity compounds.
This might be because that the high-affinity compounds became saturated at the
99
resorption sites, which may have diminished the difference in labeling between resorption
sites and the adjacent surfaces.
Figure 4.3 Distribution at resorbing and quiescent periosteal surfaces.
Optical sections, acquired by confocal microcopy, of resorbing (A-C) or quiescent (D) periosteal
surfaces from diaphysial cross-sections of tibiae of 9-week old rats 1 day after administration of
equimolar amounts of ROX-RIS (green), FAM-dRIS and AF647-3-PEHPC (red) (A, D), AF647-
RIS (green), FAM-dRIS and ROX-3-PEHPC (red) (B) or ROX-RIS (green), FAM-RIS and
AF647-RIS (red) (C). Only ROX and AF647-labeled compounds are shown for clarity. Left
panels show high affinity compounds and middle panels the low affinity compounds. Arrows
indicate resorbed areas with more intense labeling of lower affinity compounds. Scale bars = 50
merged ROX-RIS AF647-3-PEHPC
AF647-RIS ROX-3-PEHPC
AF647-RIS ROX-RIS
AF647-3-PEHPC ROX-RIS
A
B
C
D
merged
merged
merged
100
µm. Inset in merged image of (A) shows a higher magnification image of a different resorbing
site, demonstrating differential penetration (arrowheads) of the mineral by the two compounds
(scale bar = 20 mm). Adapted from ref. 4.
4.2.4 Mineral surface penetration of high- and low-affinity BP analogues
At bone-forming endocortical surfaces, penetration into the mineralizing osteoid
was found to inversely correlate with mineral affinity. In rats treated with ROX-RIS,
FAM-dRIS, and AF647-3-PEHPC, farthest penetration into the mineralizing surface was
observed with the lowest-affinity compound, AF647-3-PEHPC, whereas the high-affinity
ROX-RIS showed the lowest degree of penetration (Figure 4.4A, B). When the
fluorescent tags between the high-affinity RIS and low-affinity 3-PEHPC were switched
(ie, using compounds with a smaller difference in mineral affinity), the 3-PEHPC
analogue still showed the highest degree of mineral surface penetration, although the
differences between the three compounds were less pronounced (Figure 4.4C, D). FAM-
RIS, ROX-RIS, and AF647-RIS also showed differences in mineral surface penetration
when administered together, with the ROX conjugate showing decreased surface
penetration and the AF647 conjugate slightly increased penetration, relative to FAM-RIS
(Figure 4.4E, F). These findings are consistent with the differences in mineral affinity of
these compounds in vitro, showing ROX-labeled compounds to have a higher mineral
affinity than FAM- and AF647-labeled compounds (Figure 4.1). Similar differences in
surface penetration were also observed in trabecular bone, with the low-affinity AF647-3-
PEHPC again penetrating farthest into the bone matrix at sites of bone formation (Figure
4.4G). Furthermore, analysis of the tibia of a mouse treated with AF647-RIS, FAM-3-
PEHPC, and xylenol orange showed xylenol orange (even lower bone affinity than the 3-
PEHPC conjugates) to penetrate farthest, whereas AF647-RIS showed the most
101
superficial labeling (Figure 4.4H). Moreover, when dentine discs from elephant tusk, a
relatively poorly mineralized tissue, were labeled with fluorescent BP analogues in vitro,
a similar pattern of mineral penetration was observed, with FAM-3-PEHPC penetrating
further into the dentine surface compared to AF647-RIS, which in turn showed similar
surface penetration to FAM-RIS (Figure 4.4J, K). In contrast, no clear differences in
penetration between high- and low-affinity compounds were observed at quiescent bone
surfaces, and the seams of fluorescence at these areas were much thinner (Figure 4.4I).
Evidence of differential penetration was found at areas of resorption, although this was
less pronounced than at forming surfaces (Figure 4.3A).
These penetration differences are most likely a function of the degree of
mineralization, with high-affinity compounds less able to penetrate as mineral content
increases. One possible consequence of the deeper mineral penetration by lower-affinity
compounds might be that a greater amount of these compounds bind to mineralizing
surfaces than would be expected from their bone affinities. In addition, the low- and high-
affinity compounds colocalized in a narrow seam at the quiescent surfaces, indicating that
none of the compounds can detectably penetrate fully mineralized bone.
102
Figure 4.4 Penetration at mineral surfaces. Mineral surface penetration of fluorescent BP
analogues was compared in tibiae 1 day after administration.
(A-G, I) Nine-week old rats were treated with ROX-RIS (red), FAM-dRIS (green) and AF647-3-
A
*
B
0 10$12$14$16$18$
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intensity$
Distance$(µm)$
C
D
F E
0 5$ 6$ 7$ 8$ 9$
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intensity$
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*
Fluorescence$
intensity$
Distance$(µm)$
*
**(
*(
**(
0$ 10$ 12$ 14$
G
I(
*
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intensity$
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*
Fluorescence$
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10$ 12$ 14$
*
0 12$ 16$ 20$
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H
Average$bone$matrix$penetraDon$(µm)$$
relaDve$to$FAMFdRIS$
Average$bone$matrix$penetraDon$(µm)$$
relaDve$to$FAMFdRIS$
Average$bone$matrix$penetraDon$(µm)$$
relaDve$to$FAMFRIS$
FAM-3-PEHPC$
AF647-RIS $
0$ 5$ 10$
0$
25$
50$
75$
100$
125$
%(of(maxium(surface(
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Distance(from(denIne(surface(( µm)$
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J(
K(
103
PEHPC (blue) (A, B, G, I); AF647-RIS expressed as mean ± SD (n=2) (blue), FAM-dRIS (green)
and ROX-3-PEHPC (red) (C, D); or AF647-RIS (blue), FAM-RIS (green) and ROX-RIS (red) (E,
F). (H) A 3-month old mouse was treated with AF647-RIS (blue), FAM-3-PEHPC (green) and
xylenol orange (red). Optical sections showing labeling at forming endocortical (A, C, E, H),
trabecular (G) and quiescent endocortical bone surfaces (I). Asterisks indicate bone marrow.
Fluorescence intensity profiles shown below, each image was obtained along the white arrows.
Scale bars = 20 µm. (B, D, F) Average depth of penetration of compounds into the bone matrix,
relative to FAM-dRIS (B, D) or FAM-RIS (F). For each animal, ≥ 5 images and 6 fluorescence
profiles per image were analyzed. Data are shown as mean ± SD (n=3). * P < 0.05; ** P < 0.01.
(J, K) Penetration of fluorescent RIS and 3-PEHPC conjugates into the dentine surface in vitro.
Dentine discs were labeled with AF647-RIS and FAM-3-PEHPC (J) or AF647-RIS and FAM-
RIS (K) (100 µl; 10 µM each) for 1 h, and labeling of the dentine surface was analyzed by
confocal microscopy. Data shows mean fluorescence intensity for each compound plotted against
distance penetrated into the dentine surface. Adapted from ref. 4.
4.2.5 Penetration of the osteocyte canalicular network by high- and low-affinity BP
analogues
The binding of two fluorescent RIS analogues, FAM-RIS and AF647-RIS, to the
walls of osteocyte lacunae and within the canaliculi extending from the vascular channels
or bone surface to the osteocyte lacunae, was observed and previously reported.
2
Notably,
binding to bone mineral surrounding osteocytes may be a clinically relevant phenomenon
because very low concentrations of BPs have been reported to prevent apoptosis of
osteocytes in vitro and in vivo
14, 15
, which is in stark contrast to their pro-apoptotic effects
on osteoclasts and is mediated through distinct mechanisms.
16-19
These potential
interactions of BPs with osteocytes may contribute to their antifracture efficacy.
In addition, it has been hypothesized that high-affinity compounds show less
distribution to remote sites, including the vast osteocyte network, because these
compounds would be trapped by the first mineral they encounter.
6
The findings reported
here support the notion (Figure 4.5). The lower-affinity compounds (fluorescent
analogues of dRIS and 3-PEHPC, and xylenol orange) showed significantly increased
104
labeling of osteocyte lacunar walls, as well as canaliculi extending from the bone surface
or vascular channels, compared with the high-affinity fluorescent analogues of RIS
(Figure 4.5B, C; p < 0.05). In addition, coadministration of FAM-RIS, ROX-RIS, and
AF647-RIS to rats resulted in relatively low osteocyte lacunar wall labeling for all three
RIS conjugates (Figure 4.5D), confirming that the main determinant for osteocyte
network penetration is the affinity of the native compound.
It was previously showed that for fluorescent RIS, an inverse relationship exists
between degree of osteocyte lacunar labeling and distance of the osteocyte lacuna from
the nearest vascular channel or bone surface, ie, lacunae of osteocytes close to a vascular
channel showed a higher degree of labeling compared with lacunae from more distant
osteocytes. In the current study, similar inverse relationships between degree of labeling
and distance from the nearest vascular channel or bone surface was found for the lower-
affinity analogues FAM-dRIS and ROX-3-PEHPC (Figure 4.5E). This relationship was
found to be best described by a logarithmic function, with goodness-of-fit R
2
FAM-dRIS
0.24;R
2
ROX-3-PEHPC 0.23; and R
2
AF647-RIS 0.25. For AF647-3-PEHPC, such a
relationship was not apparent using the image analysis method employed (R
2
0.01),
possibly because its bone affinity is so low that it penetrates the osteocyte canalicular
network sufficiently freely so that osteocyte lacunar labeling no longer depends on
distance from the nearest blood supply.
105
Figure 4.5 Penetration of the osteocyte network.
(A) Xy maximum intensity projection image generated from a z series (25 mm depth) of images
of tibial cortical bone from a rat treated with FAM-RIS (green) and RhR-3-PEHPC (red),
acquired using confocal microscopy. Yellow indicates co-localization. Scale bar = 20 µm. (B-E)
Nine-week old rats were given single doses of ROX-RIS, FAM-dRIS and AF647-3-PEHPC (n=3;
B), AF647-RIS, FAM-dRIS and ROX-3-PEHPC (n=3; C, E), or AF647-RIS, FAM-RIS and
ROX-RIS (n=3, D) 1 day prior to sacrifice, and optical sections were acquired from cross-
sections through tibial cortical bone using confocal microscopy with detector gain settings
optimized to avoid saturation around osteocytes and vascular channels. For each rat, the mean
fluorescence intensity of >100 osteocyte lacunar walls, and nearby vascular channel walls, was
determined from 3 images and corrected for tissue autofluorescence. (B-D) Mean fluorescence
intensity of osteocyte lacunar walls relative to vascular channel walls, expressed as mean ± SD
(n=3). *p < 0.05. (E) Average fluorescence intensities of osteocyte lacunar walls, expressed as a
A
merged
E
*
FAM-RIS RhR-3-PEHPC
B
*
C
D
106
function of the distance to the nearest vascular channel or bone surface. Combined data from 3
animals is shown. Adapted from ref. 4.
4.2.6 Localization of high- and low-affinity BP analogues 7 days after
administration
The study of localization of differing affinity compounds 7 days after
administration showed similar results as discussed above. Analysis of tibial cortical bone
surfaces at day 8 showed that, at forming surfaces, both AF647-RIS and xylenol orange
were buried under a layer of newly formed bone, the surface of which was clearly
identified by labeling with calcein (Figure 4.6A). Differences in penetration at
mineralizing surfaces were also evident in the seams of AF647-RIS and xylenol orange
running through the cortical bone, with the very low-affinity xylenol orange clearly
buried deeper at the original bone surface compared with AF647-RIS (Figure 4.6A).
Similar results were seen at forming surfaces in tibias of rats, in which the difference in
penetration of the high-affinity (ROX-RIS) and low-affinity compound (AF647-3-
PEHPC) was still apparent 1 week after treatment (Figure 4.6B, C; arrows). No
differences in matrix penetration were found between high- and low-affinity compounds
at quiescent surfaces (Figure 4.6B, C; arrowheads), further confirming the observations
made at 1 day (Figure 4.4I). Finally, differential labeling of osteocyte lacunar walls was
still observed after 1 week, with lower-affinity 3-PEHPC analogues showing a higher
degree of labeling of the osteocyte network than RIS analogues (Figure 4.6F), similar to
at 1 day (Figure 4.5A).
The notion that BPs must recycle within bone is supported by published reports
that BPs are detectable in the body fluids long after they have been administered, which
107
has been proposed to explain the long duration of action of the drugs.
6
Further analysis
of these forming cortical bone surfaces using high detector gain settings revealed
evidence of very small amounts of the compounds being incorporated into bone that had
formed in the 7 days after administration of compounds, indicating that ‘‘recycling’’ had
occurred, albeit at very low levels. These data are in accordance with a recent study
looking at systemic recycling using a radiolabeled BP,
14
C-ibandronate.
20
This
‘‘recycling’’ was somewhat more evident for the 3-PEHPC analogues compared with the
RIS analogues (Figure 4.6D, E). Small amounts of fluorescent BP conjugates were also
found to be incorporated into newly formed bone matrix surrounding some vascular
channels, ie, between the deeper seam of the original label and the new bone surface
(Figure 4.6F). Again, this was more evident for the 3-PEHPC analogues. These
observations raise the question of whether recycling occurs to a sufficient degree to
contribute substantially to the prolonged actions of BPs in vivo, or whether other
mechanisms, such as prolonged suppression of osteoclast generation from progenitors
21-23
,
are responsible.
108
Figure 4.6 Distribution of fluorescent BP analogues one week after administration.
(A) Optical section showing endocortical surface labeling in the cortical bone of a 3-month old
mouse 7 days after administration of AF647-RIS (blue) and xylenol orange (red) and 1 day after
administration of calcein (green). Scale bar = 20 µm. (B, C) Optical sections of forming (arrows)
and quiescent (arrowheads) endocortical surfaces in the cortical bone of 9-week old rats 7 days
after administration of ROX-RIS (red), FAM-dRIS (green) and AF647-3-PEHPC (blue). Scale
bars = 20 µm. (D, E) Optical sections of forming endocortical surfaces in cortical bone of 9-week
old rats 7 days after administration of (D) AF647-RIS (green), ROX-3-PEHPC (red) and FAM-
dRIS or (E) ROX-RIS (green), AF647-3-PEHPC (red) and FAM-dRIS. Detector gain settings
were optimized to detect compounds in newly formed bone (resulting in saturation of
fluorescence at original site of binding). Only ROX and AF647-labeled compounds are shown for
clarity. Black arrows indicate bone surface. Fluorescence profiles shown on the right were
obtained along the white arrows in the merged images. Scale bars = 20 µm. (F) Optical section of
cortical bone of 9-week old rat 7 days after administration of FAM-RIS (green) and RhR-3-
PEHPC (red) showing labeling around osteocyte lacunar walls. Scale bars = 20 µm. Adapted
from ref. 4.
Fluorescence*
intensity*
Distance*(µm)*
0* 40* 60* 20*
D
*
*
*
*
Fluorescence*
intensity*
Distance*(µm)*
0* 40* 60* 20*
E
*
AF647;RIS* ROX;3;PEHPC*
ROX;RIS* AF647;3;PEHPC*
FAM;RIS* RhR;3;PEHPC*
Merged*
Merged*
Merged*
A
B
C
F
109
4.2.7 Conclusion
In summary, these studies support the concept that BPs that vary in their bone
mineral affinity behave differently in terms of distribution within bone in vivo. Three
parent compounds were utilized with relatively large differences in bone mineral affinity
to show differences in behavior in vivo in short-term experiments. Despite their subtle
differences in affinity, it is likely that clinically used BPs also exhibit differences in these
pharmacokinetic characteristics, which could become more apparent after repeated
administration, and may help to explain some of the clinical differences among BPs.
4.3 Mechanism study of osteonecrosis of the jaw by fluorescent bisphosphonate
**
Since 2003, phenomena collectively referred to as osteonecrosis of the jaw (ONJ,
Figure 1.3) have been reported as a rare but potentially severe adverse event in patients
receiving high dose of certain BPs, such as zoledronate, alendronate, and pamidronate.
24-
26
ONJ is clinically characterized as unresolved exposure of partially necrotic jawbone to
the oral cavity and is frequently associated with dentoalveolar procedures in cancer
patients receiving high doses of intravenously administered BPs.
25, 27-30
The etiology and
pathogenesis of ONJ remain poorly characterized, and there are still controversies on
whether ONJ is associated with BPs from drug research community and dental research
community, especially with the findings that ONJ was also reported in patients who
received the non-BP agent treatment, denosumab, an anti-RANKL antibody.
31
Numerous
hypotheses are proposed to explain the mechanism of ONJ, however, none is widely
**
The fluorescent bisphosphonates used in this study are 5-FAM-ZOL, 5(6)-FAM-RIS, and 6-ROX-RIS;
the 5-, 6-, 5(6)-prefix were omitted in the text of this part for simplicity.
110
accepted yet.
27, 32, 33
Thus, more solid experimental evidence are highly desirable in the
mechanism study of ONJ and fluorescent BP probes are of great interest as a tool for the
mechanism study.
4.3.1 FAM-ZOL retained the pharmacological effect of ZOL in vivo
Previously, FAM-RIS has been shown to prevent prenylation of Rap1A of J774.2
macrophages in vitro (see Chapter 3 and ref.
1
). In the present study, to evaluate the effect
of FAM conjugation on the overall pharmacological efficacy of ZOL, a direct
comparison of FAM-ZOL vs. ZOL was carried out for relative anti-resorptive activity in
vivo. Nutritionally induced vitamin D deficiency (VitD(-)) increased osteoporosis-like
catabolic bone remodeling in rats.
34-36
Compared to the control VitD(-) group, the FAM-
ZOL group shows a significant (p < 0.05) increase in the trabecular bone volume and
improvements of trabecular structural indices, and the anti-resorptive effect of FAM-ZOL
in vivo was approximately 50% of that of ZOL (Figure 4.7B). This study provided direct
evidence for the first time that FAM-ZOL can retain anti-resorptive drug activity in vivo
albeit at reduced rates, which makes possible the mechanism study of ONJ.
111
Figure 4.7 FAM-ZOL anti-resorptive activity assay.
(A) Structure of 5-FAM-ZOL, 5(6)-FAM-RIS, and 6-ROX-RIS. (B) The pharmacological
function of FAM-ZOL was compared with ZOL in vitamin D deficient (VitD(-)) rats. ZOL,
FAM-ZOL or vehicle solution (0.9% NaCl) was injected via tail vein into VitD(-) rats (n=3 per
group). Six weeks after the injection, the trabecular bone morphology of femurs was evaluated by
micro-CT. FAM-ZOL significantly blocked catabolic bone remodeling resulting in increased
trabecular bone structure; however, the effectiveness of FAM-ZOL was reduced by
approximately 50% of ZOL *p < 0.05 by one-way ANOVA. Adapted from ref. 5.
4.3.2 BP adsorption kinetics to CaP disc in vitro
When increasing concentrations of FAM-ZOL in PBS were incubated with
commercially available synthetic calcium phosphate (CaP)-coated quartz substrate discs,
the amount of FAM-ZOL adsorbed also increased (Figure 4.8A) and there was no sign of
adsorption saturation observed up to 500 µM of applied drug. Since binding of FAM-
ZOL to hydroxyapatite crystals followed by the Langmuir adsorption isotherm has been
discussed in Chapter 3, CaP discs were used as an in vitro model for BP adsorption
studies.
112
One-time application of 50 µM FAM-ZOL consistently gave rise to
approximately 70 pixel-intensity fluorescence in CaP discs. Four repeated applications of
12.5 µM FAM-ZOL resulted in significantly less FAM fluorescence intensity in the CaP
discs than the single bolus application of 50 µM FAM-ZOL (Figure 4.8B), although
cumulative doses of FAM-ZOL were equivalent in these groups.
Because the Langmuir isotherm is influenced by the adsorption affinity, it was
postulated that the first application of BP to the CaP disc might condition the adsorption
affinity and thus interfere with the subsequent BP adsorption. To test this hypothesis, CaP
discs were alternately treated with A) ZOL followed by FAM-ZOL or B) FAM-ZOL
followed by ZOL. In the group of CaP discs pretreated via procedure A, the CaP disc
fluorescent intensity reached nearly at the level of FAM-ZOL application alone (Figure
4.8C). In the reverse addition order group (procedure B), the fluorescence intensity on the
CaP discs from pre-adsorbed FAM-ZOL decreased to an undetectable level after the
followed application of unlabeled ZOL (Figure 4.8C). By analyzing FAM fluorescence
intensity of these applied solutions, it was found that non-adsorbed FAM-ZOL appeared
to be effectively removed by sequential PBS washes to a negligible level (Figure 4.8D).
After unlabeled ZOL solution was applied, a small but distinct peak of FAM fluorescence
was detected in the ZOL solution (arrow in Figure 4.8D), suggesting that the loss of
FAM fluorescence intensity on CaP discs was likely to be due to displacement of
previously adsorbed FAM-ZOL from the discs.
It is shown in Chapter 3 that ROX-RIS has significantly higher affinity than
FAM-RIS in mouse tibiae. Taking advantage of the relative difference in affinities of
ROX-RIS (higher affinity) and FAM-RIS (lower affinity), it was further investigated
113
whether and how BP affinity influences the “displacement”. CaP-coated culture wells
were pre-treated by 50 µM ROX-RIS or FAM-RIS, giving rise to uniform adsorption as
detected by the ROX or FAM fluorescence. When the pre-adsorbed 50 µM ROX-RIS
was challenged by FAM-RIS, ROX-RIS was not completely replaced even at the same
molar concentration (Figure 4.9A). On the contrary, when the pre-adsorbed 50 µM
FAM-RIS was challenged by ROX-RIS, FAM-RIS was completely removed (Figure
4.9A). ROX-RIS showed initial increase in CaP adsorption and reached the plateau
between 10 µM and 50 µM (Figure 4.9B). It was noted that the adsorption concentration
of ROX-RIS did not seem to increase beyond the previously established adsorption
concentration between FAM-RIS and CaP.
Figure 4.8 Study of FAM-ZOL absorption on HAP.
(A) FAM-ZOL showed a dose-dependent linear absorption to CaP discs and did not saturate
below 500 µM in PBS. (B) Although the 4 repeated applications of 12.5µM FAM-ZOL should
deliver the equivalent cumulative dose of the single application of 50µM FAM-ZOL, the CaP
discs showed less fluorescent intensity level in the former protocol than those of single high-dose
application. *p < 0.05 by Fisher’s PLSD test. (C) Unlabeled ZOL (50µM) and FAM-ZOL (50µM)
114
were alternately applied to CaP discs. The fluorescence intensity of CaP discs was largely
influenced by the lastly applied compound. *p < 0.05 by Tukey-Kramer test. (D) The PBS wash
solutions collected from the group of FAM-ZOL followed by ZOL application in Fig. 4.8C
revealed a distinct peak of FAM fluorescence (arrow) during the second ZOL application,
suggesting the displacement of FAM-ZOL from CaP discs. Adapted from ref. 5.
Figure 4.9 Fluorescent BP “displacement” experiment.
CaP-coated wells were pre-treated with either ROX-RIS or FAM-RIS (50µM). After challenged
by serially diluted doses of FAM-RIS or ROX-RIS (from 0 to 50µM), respectively, ROX and
FAM fluorescence intensities of the CaP-coated wells were measured. (A) ROX-RIS in CaP (red
bars) remained near the pre-adsorbed 50 µM level until 5 µM or greater concentrations of FAM-
RIS challenged. On the contrary, FAM-RIS (green bars) was rapidly displaced by as low as 1 µM
ROX-RIS. (B) The pre-adsorbed FAM-RIS was completely replaced by the challenged ROX-RIS
between 10 µM to 50 µM range. Adapted from ref. 5.
115
4.3.3 Bio-distribution pattern of FAM-ZOL with different administration
protocols in vivo
In vivo bio-distribution of FAM-ZOL was examined in rats with nutritionally
induced vitamin D deficiency. A single injection of 180 µg/kg FAM-ZOL resulted in
localized intense fluorescence in the femur trabecular bone proximal to the poorly labeled
primary spongiosa located at the distal end of epiphysis (Figure 4.10A). The
fluorescence intensity varied significantly in the femur of this group. Contrarily, 4-
weekly injections of 45 µg/kg FAM-ZOL showed more diffused and uniform fluorescent
labeling in femurs (Figure 4.10A). The same results were also observed in the
mandibular ramus and alveolar bone (Figure 4.10B, C), with much stronger fluorescence
in the single injection group than in the repeated injection group, despite the fact that both
groups received the equivalent cumulative FAM-ZOL dose.
Figure 4.10 In vivo absorption pattern of FAM-ZOL in rat bone tissue.
Through the tail vein, a group of vitamin D-deficient rats (n=3) received 4 repeated-weekly
injections of 45µg/Kg FAM-ZOL, another group (n=3) received a single-injection of 180µg/Kg
FAM-ZOL and the control rats (n=3) received a single-injection of vehicle solution (0.9% NaCl).
Rat femurs and mandibles were harvested 8 weeks after the initial injection and subjected to the
fluorescent CCD detector imaging analysis. (A) Fluorescence distribution in femurs showed more
diffused and less intense adsorption of FAM-ZOL in the repeated injection group. In the single
116
application group, an intense fluorescence signal was found localized at the proximal (P) area of
femur primary spongiosa (arrowheads) contrasted to lesser labeling in the distal (D) primary
spongiosa of the more recent growth area. (B) Mandibular ramus specimens similarly indicated
bone growth-dependent discrepancy in FAM-ZOL bio-distributions at the anterior (A) and distal
(D) areas (arrowheads). (C) Mandibular molar alveolar bone (arrowheads; M3: the 3
rd
molar)
completed the growth by 6 to 8 weeks of age and showed the intense FAM-ZOL labeling in the
single-injection group. *p < 0.05 by Fisher’s PLSD test. Adapted from ref. 5.
4.3.4 Effect of different ZOL administration protocols on anti-catabolic bone
remodeling
The biological effect of different administration protocols with the equivalent
cumulative dose was examined in vitamin D deficient rats. All rats were treated by the
extraction of left maxillary molars. One group received 2 monthly-injections (70 µg/kg
ZOL per injection) and the other group received 8 weekly-injections (17.5 µg/kg ZOL per
injection). At the end of the experiment, whole blood, femur and maxillary bone samples
were harvested (Figure 4.11A).
The serum samples were examined for bone remodeling markers: alkaline
phosphatase
37
, carboxy-terminal collagen crosslinks (CTX)
37, 38
, and secreted tartrate-
resistant acid phosphatase 5b (TRACP-5b).
39
The weekly injection protocol significantly
decreased all serum bone remodeling markers compared to the monthly injection protocol
(Table 4.1). On the contrary, the micro CT bone morphometry data of the femur
trabecular bone clearly indicated the more robust increase in bone mass in the monthly
injection group (Figure 4.11B).
117
Figure 4.11 Different biological consequences to the modulated administration of ZOL.
(A) Experimental protocols involving vitamin D deficient treatment and ZOL injections in rats.
The equal cumulative ZOL dose of 140µg/Kg was given in 2 rat groups; however, one group
received 8 weekly-injections of 17.5µg/Kg ZOL (n=8), whereas the other group received 2
monthly-injections of 70µg/Kg ZOL (n=6). Control rats (n=4) received injections of vehicle
solution (0.9% NaCl). (B) Micro-CT bone morphometry of femur distal primary spongiosa
indicated significantly greater anti-catabolic effect by 70µg/Kg monthly-injections than by
17.5µg/Kg weekly-injections. *p < 0.05 by Fisher’s PLSD test. Adapted from ref. 5.
Table 4.1 Serum bone remodeling markers of vitamin D deficient rats with different
ZOL injection protocols. (Adapted from ref. 5.)
Parameters
ZOL Treatment
ALP
(U/L)
CTX
(ng/mL)
TRACP-5b
(U/L)
8 x 17.5µg/Kg 89.50±10.75
16.61±4.82
]*
2.95±0.69
]*
2 x 70.0µg/Kg 145.17±70.17 39.44±14.35 4.63±1.22
Reference (VD- rats) 270.20±46.82 63.85±13.26 14.37±3.81
*p < 10.05 Student t-test
118
4.3.5 Effect of different ZOL administration protocols on the development of ONJ-
like lesions in the rat maxilla
At the time of maxillary molar extraction and at the end of the experimental
period, the cumulative ZOL doses in both groups were equivalent (Figure 4.11A). Four
weeks after maxillary molar extraction, the maxillary tissues were harvested. The micro
CT scanning indicated that all of the 6 rats that received 2 injections of high dose ZOL
(70 µg/kg per injection) developed the abnormal bone sequestration, which appeared to
be separated from the palatal alveolar process at the maxillary molar extraction wound
(Figure 4.12A). The histological cross section of the maxilla revealed that the bone
sequestrum was indeed necrotic and associated with oral epithelial fistulation and
inflammatory cell infiltration (Figure 4.12B, C). There was 1 out of 8 rats (or 12.5%)
that received the 8 repeated weekly injections of low dose ZOL (17.5 µg/kg per injection)
developed the bone sequestration. Fisher's exact test showed the significant difference on
the presence of necrotic bone sequestration between 2 different ZOL administration
groups (p < 0.01) (Figure 4.12E). The buccal alveolar process of the tooth extraction side
was covered by the buccinators muscle and separated from the oral epithelium (Figure
4.12B). There were clusters of empty osteocyte lacunae in the buccal alveolar process
(Figure 4.12D). The percent of viable osteocytes was significantly lower in rats received
the single injection of high dose ZOL (Figure 4.12F). Thus, it is hypothesized that the
probability of developing ONJ, if associated with a BP, might be influenced by different
administration protocols. Furthermore, the fluorescent imaging studies above showed that
single injections of high dose ZOL resulted in the high concentration of BP at the
alveolar bone, which may contribute to the development of ONJ-like lesions.
119
Figure 4.12 Different dosing protocal results in different prevalence of ONJ-like lesions.
(A, C) Micro-CT evaluations of maxillary alveolar bone after molar extraction revealed the
partially necrotic bone sequestra (arrow) in rats received 70.0 µg/Kg monthly injections. (Scale
bar = 500 µm) (B) Histological cross section of maxilla of rats received 70.0 µg/Kg monthly
injections showed the necrotic bone sequestrum (arrow) that appeared to be derived from palatal
alveolar bone (P). The bony socket housed molar (M) was filled with new bone (Nu) between
palatal (P) and buccal (B) alveolar bone. (H & E staining; scale bar = 500 µm) (C) Necrotic bone
sequestrum was associated with abnormal hyperplasia of oral epithelium (arrows). (H & E
staining; scale bar = 100 µm) (D) Buccal alveolar process of rat maxilla received weekly
injections of 17.5 µg/Kg ZOL or monthly injections of 70.0 µg/Kg ZOL showed the different
distributions of empty osteocyte lacunae (arrowhead). (H & E staining; scale bar = 100 µm) (E)
The rate of rats developed necrotic bone sequestrum. **p < 0.01 by Fisher’s Exact test. (F) The
rate of viable osteocytes in the buccal alveolar process. **p < 0.01 by Student’s t test. Adapted
from ref. 5.
4.3.6 Conclusion
The results from this study collectively suggest that the establishment of a
120
dynamic equilibrium between BP and the CaP substrate may better explain the BP
adsorption pattern to bone tissue and its pharmacological effects than a simple dose
accumulation model. Repeated applications of low-dose FAM-ZOL did not produce the
adsorption level observed for a single high-dose FAM-ZOL application in vitro as well as
in vivo albeit at equivalent cumulative doses. Repeated vigorous washes in vitro or
creatinine clearance in rats
40
should not be the primary cause of the removal of the
adsorbed BP. Instead, the in vitro data suggest the possibility that the initially adsorbed
BP can be removed and replaced by subsequently applied BP depending on its affinity
and applied doses.
In conclusion, this study offers evidence for an equilibrium process of BP
adsorption to bone tissue. The postulated equilibrium model may better explain the
differences of BP bio-distribution, anti-catabolic bone remodeling and the prevalence of
ONJ between different dosing protocols. Furthermore, the possibility to remove adsorbed
BP molecules may suggest a novel opportunity for widening the therapeutic options.
4.4 Study of zoledronate distribution in cochlea by systemic and local delivery
Zoledronate is currently under consideration as a potential treatment for cochlear
otosclerosis, a disorder with a hereditary predisposition that is among the most common
causes of acquired hearing loss.
41, 42
The otic capsule is the densest bone in the body,
normally does not remodel, and its level of inclusion within the blood-cochlea barrier is
poorly understood.
43, 44
Thus zoledronate may not penetrate the bone of the cochlea after
systemic administration. A prerequisite for alternative local delivery of zoledronate will
be the ability to determine its distribution within the cochlea (Figure 4.13). Both
121
questions are addressed with the aid of the novel imaging probe, 6-FAM-ZOL (Figure
4.13), which is used to visualize zoledronate distribution in murine cochlea after systemic
delivery (i.p.), and also in Guinea pig cochlea after local delivery.
Figure 4.13 FAM-ZOL and otosclerosis.
(A) Low power H&E photomicrograph of human temporal bone showing otosclerosis around the
oval window, cochlea, and internal auditory canal. (B) 3D reconstruction of the guinea pig
cochlea showing location of cochleostomy (red dot) in the basal turn of the scala tympani for
intracochlear delivery method. (C) 6-FAM-ZOL structure (unpublished data of Woo Seok Kang,
David Jung, Alicia Quesnel, Shuting Sun, Adam Hacking, Charles McKenna, William Sewell,
and Michael McKenna).
Systemic delivery produces dose-related deposition of zoledronate in the bone of
the inner ear in concentrations comparable to that observed in the femur (Figure 4.14).
The concentration of zoledronate in the otic capsule increased with increasing systemic
dose between 1/3 to 10 times the equivalent weight-adjusted dose for humans (Figure
4.14A). The imaging results indicate that cochlear bone behaves similarly to other bones
N
N
P
P OH
OH
HO
O
OH
OH
O
OH
N
H
O
O
HO
COOH
O
ZOL
Linker
6-FAM
A B
C
122
with regard to accumulation of zoledronate, making systemic administration of the drug a
possible approach to therapy (Figure 4.14B-E). In addition, no ototoxicity was observed
from intraperitoneal 6-FAM-ZOL at doses equivalent to and 10 times the standard
clinical dose for humans (adjusted for weight in mouse, Figure 4.14F, G).
Figure 4.14 Systemic delivery and ototoxicity studies of 6-FAM-ZOL.
(A) Zoledronate concentration measured in bone 3 days after IP injection of mice with saline
(n=7), 1/3 of standard human clinical dose (0.06 mg/kg) (n=7), equivalent clinical dose (0.18
mg/kg, 1X systemic dose) (n=7), and 10 times clinical dose (1.8 mg/kg)(n=7). Average and
standard deviation are shown. (B-E) Fluorescent images of mouse hemicochlea, tibia, and
calvarium from control (B) and mice injected with 0.06 mg/kg (C), 0.18 mg/kg (D) and 1.8mg/kg
(E) of 6-FAM-ZOL. (F-G) Distortion product otoacoustic emission (DP) and auditory brainstem
response (ABR) thresholds for mice injected IP with saline (n=5), 6-FAM-ZOL dose equivalent
to human clinical dose (0.185 mg/kg), and 10 times clinical dose (1.85 mg/kg) measured 3 days
after injection. Average ± standard error shown (unpublished data of Woo Seok Kang, David
Jung, Alicia Quesnel, Shuting Sun, Adam Hacking, Charles McKenna, William Sewell, and
Michael McKenna).
Potential side effects may occur with systemic administration of zoledronate in
humans
45
, thus the distribution study of local delivery approach is of great importance.
A B
C
D
E
F
G
123
The alginate hydrogel was selected as a delivery system, which has been studied in other
biological contexts, including drug delivery to the inner ear. The round window was used
as a portal of entry into the inner ear, given the well-described permeability of the round
window in both animal and human studies.
Two locally delivered doses of 6-FAM-ZOL, 10% of the 1X systemic dose and
30% of the 1X systemic dose were compared. It was found that placement of 6-FAM-
ZOL hydrogel on the round window membrane resulted in delivery of 6-FAM-ZOL
throughout the otic capsule to levels comparable to the 1X systemic dose (Figure 4.15,
4.16C). Statistical comparison of the amount of fluorescence seen at each half-turn of the
cochlea found that there was no difference in fluorescence as a function of distance from
the round window to the cochlear apex. Critically, no change in ABR or DPOAE
following 6-FAM-ZOL delivery was observed, suggesting no ototoxicity was found at
the dose used for local delivery (Figure 4.16A, B). Thus it is concluded that uniform
delivery of 6-FAM-ZOL throughout the cochlea could be achieved with local delivery of
30% of the systemic dose, and that local delivery of 6-FAM-ZOL to the cochlea was not
ototoxic.
Figure 4.15 Local delivery studies of 6-FAM-ZOL.
Fluorescent photomicrographs taken at mid-modiolar sections of the cochlea are shown for
A B C
D E F
124
untreated cochlea (A), treatment with 10% of the 1X systemic dose (B), and treatment with 30%
of the 1X systemic dose (C). D-F show close-up fluorescent photomicrographs taken of the oval
window region from sections A-C. As these are mid-modiolar sections, the round window is not
well seen in these sections (unpublished data of Woo Seok Kang, David Jung, Alicia Quesnel,
Shuting Sun, Adam Hacking, Charles McKenna, William Sewell, and Michael McKenna).
Figure 4.16 Ototoxicity and quantification of fluorescence by local 6-FAM-ZOL treatment.
(A, B) DPOAE and ABR measurements are shown local delivery experiments. The y-axis reports
the level of threshold shift seen upon hearing re-evaluation immediately prior to animal sacrifice
and analysis, relative to the initial hearing evaluation immediately prior to treatment. (C)
Fluorescence values on photomicrographs were measured using ImageJ software. Measurements
were taken at each of six cochlear half-turns from base to apex. The data summarize eight
independent experiments. Statistical significance was measured using ANOVA (unpublished
data of Woo Seok Kang, David Jung, Alicia Quesnel, Shuting Sun, Adam Hacking, Charles
McKenna, William Sewell, and Michael McKenna).
In conclusion, the fluorescent probe 6-FAM-ZOL makes the drug distribution and
delivery study of zoledronate possible in cochlea; systemic administration of 6-FAM-
ZOL demonstrated that zoledronate binding in the cochlea is similar to other bones.
Cochlear deposition increased with increasing delivery concentration. In addition, a
concentration gradient from base to apex was not identified in the cochlea following
A B
C D
Systemi
c
Systemi
c
Local Local
A B
C D
Systemi
c
Systemi
c
Local Local
A
B
C
125
placement of 6-FAM-ZOL to the round window. The absence of a concentration gradient
in the findings most likely reflects the fact that this system detects the accrued amount of
6-FAM-ZOL present at any given location within the cochlea. It may be instructive in
additional experiments to see if 6-FAM-ZOL is present in a concentration gradient within
the cochlea at shorter time scales.
4.5 Conclusion
The above biological applications demonstrate the versatility of the fluorescent
bisphosphonate imaging probe “toolkit”. The different mineral binding affinity,
pharmacological activity as well as various spectroscopic properties of these probes
provide numerous options for diverse biological studies. The applications of these probes
in tumor imaging, stem cell therapy and other bone related studies are currently in
progress.
4.6 Experimental
4.6.1 Study of bisphosphonate distribution pattern in bone skeleton by simultaneous
imaging approach
The biological experiments were done by Dr. Anke Roelofs, et al.; for detailed
experimental methods, please refer to ref. 4.
4.6.2 Mechanism study of osteonecrosis of the jaw by fluorescent bisphosphonate
The biological experiments were done by Dr. Ichiro Nishimura’s laboratory; for
detailed experimental methods, please refer to ref. 5.
126
4.6.3 Study of zoledronate distribution in cochlear by systemic and local delivery
The biological experiments were done by Dr. Michael McKenna’s laboratory; the
results have not been published yet, thus the experimental details provided by Dr. Woo
Seok Kang, Dr. David Jung, Dr. Alicia Quesnel, Dr. Adam Hacking, Dr. William Sewell,
Dr. Michael McKenna, are included here:
Systemic injection experiments: Mice were injected intraperitoneally with saline
(controls) (n=7) or 6-FAM-ZOL at one of three concentrations (n=7 for each of three
concentrations). Three days later, DPOAE and ABR were measured, and then the animals
were euthanized. The tibia, calvarium, and bilateral cochleae were harvested, and the
cochleae were prepared as a mid-modiolar specimen. Fluorescent images were captured
and measured as below.
Local delivery experiments: Zoledronate is infused with a single 5 mg dose for
human patients with osteoporosis. Based on this human systemic dose, 0.185 mg/kg of 6-
FAM-ZOL was diluted into 100 mL of PBS and intraperitoneally injected through 0.5 cm
incision in the abdomen of guinea pigs, which we call “1X 6-FAM-ZOL”. For “3X 6-
FAMZOL”, we used 0.555 mg/kg of 6-FAM-ZOL diluted into 1 mL of PBS. 1 mL of
PBS was injected in the control animals. DPOAEs and ABRs were measured
immediately prior to surgery and 3 weeks later immediately before sacrificing the guinea
pigs.
Either artificial perilymph (AP, 130 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl
2
, 5.5
mM glucose, 20 mM HEPES, pH 7.4) or PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na
2
HPO
4
, 1.8 mM KH
2
PO
4
, pH 7.4) was used as a solvent. For local delivery, 2% (w/v)
127
solution of sodium alginate was prepared in AP or PBS. 2 µL of 2% sodium alginate
solution was placed on a microscope slide. For 1X bead (10% of 1X 6-FAM-ZOL), 1.3
µL of 4 µg/µL 6-FAM-ZOL was mixed with 1.3 µL of 2% sodium alginate solution. 3X
beads (30% of 1X 6-FAM-ZOL), were made by mixing 1 µL of 15 µg/µL 6-FAM-ZOL
with 1.3 µL of 2% sodium alginate solution. A few drops of 0.2 M CaCl
2
were placed
over this mixture using a pipette, which then formed a hydrogel with a diameter of 1-
2 mm. Each hydrogel was made right before the surgery.
The round window was visualized via a bullectomy approach and the beads were
placed on the round window membrane (RWM). Animals were sacrificed 3 weeks after
hydrogel placement to allow for middle ear inflammation to subside.
Fluorescent imaging and measurements: Fluorescent images were collected with
an inverted fluorescence microscope (Zeiss Axiovert), UV lamp, FITC (fluorescein)
filters, and a 16 bit camera. Images were analyzed with image J by measuring average
pixel intensity over regions of the bone. Calibration curves were constructed from
measurements of 10 fold fluorescein dilutions, and used to convert arbitrary fluorescence
units measured on image J to 6-FAM-ZOL concentrations.
4.7 References
1. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.
G.; Coxon, F. P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled
risedronate and related analogues: "magic linker" synthesis. Bioconjug Chem 2008, 19
(12), 2308-10.
2. Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.;
Nancollas, G. H.; Sun, S.; Blazewska, K. M.; Bala, J. L.; Kashemirov, B. A.; Khalid, A.
B.; McKenna, C. E.; Rogers, M. J., Fluorescent risedronate analogues reveal
bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in
128
vivo. J Bone Miner Res 2010, 25 (3), 606-16.
3. Turek, J.; Ebetino, F. H.; Lundy, M. W.; Sun, S. T.; Kashemirov, B. A.; McKenna,
C. E.; Gallant, M. A.; Plotkin, L. I.; Bellido, T.; Duan, X. C.; Triffitt, J. T.; Russell, R. G.
G.; Burr, D. B.; Allen, M. R., Bisphosphonate Binding Affinity Affects Drug Distribution
in Both Intracortical and Trabecular Bone of Rabbits. Calcif Tissue Int 2012, 90 (3), 202-
210.
4. Roelofs, A. J.; Stewart, C. A.; Sun, S. T.; Blazewska, K. M.; Kashemirov, B. A.;
McKenna, C. E.; Russell, R. G. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon,
F. P., Influence of Bone Affinity on the Skeletal Distribution of Fluorescently Labeled
Bisphosphonates In Vivo. J Bone Miner Res 2012, 27 (4), 835-847.
5. Hokugo, A.; Sun, S. T.; Park, S.; McKenna, C. E.; Nishimura, I., Equilibrium-
dependent bisphosphonate interaction with crystalline bone mineral explains anti-
resorptive pharmacokinetics and prevalence of osteonecrosis of the jaw in rats. Bone
2013, 53 (1), 59-68.
6. Russell, R. G. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J., Mechanisms of
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7. Ebetino, F. H.; Hogan, A. M. L.; Sun, S. T.; Tsoumpra, M. K.; Duan, X. C.;
Triffitt, J. T.; Kwaasi, A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M.
W.; Boyde, A.; Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. G., The relationship
between the chemistry and biological activity of the bisphosphonates. Bone 2011, 49 (1),
20-33.
8. Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.;
Golub, E.; Rodan, G. A., Bisphosphonate action. Alendronate localization in rat bone and
effects on osteoclast ultrastructure. J Clin Invest 1991, 88 (6), 2095-105.
9. Azuma, Y.; Sato, H.; Oue, Y.; Okabe, K.; Ohta, T.; Tsuchimoto, M.; Kiyoki, M.,
Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and
in experimental hypercalcemia models. Bone 1995, 16 (2), 235-45.
10. Masarachia, P.; Weinreb, M.; Balena, R.; Rodan, G. A., Comparison of the
distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19
(3), 281-90.
11. Christiansen, C.; Phipps, R.; Burgio, D.; Sun, L.; Russell, D.; Keck, B.; Kuzmak,
B.; Lindsay, R., Comparison of risedronate and alendronate pharmacokinetics at clinical
doses. Osteoporos Int 2003, 14, S38-S38.
12. Lin, J. H., Bisphosphonates: a review of their pharmacokinetic properties. Bone
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129
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bone mineral. J Bone Miner Res 2007, 22 (8), 1208-1216.
14. Plotkin, L. I.; Weinstein, R. S.; Parfitt, A. M.; Roberson, P. K.; Manolagas, S. C.;
Bellido, T., Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and
calcitonin. J Clin Invest 1999, 104 (10), 1363-1374.
15. Follet, H.; Li, J. L.; Phipps, R. J.; Hui, S.; Condon, K.; Burr, D. B., Risedronate
and alendronate suppress osteocyte apoptosis following cyclic fatigue loading. Bone 2007,
40 (4), 1172-1177.
16. Plotkin, L. I.; Manolagas, S. C.; Bellido, T., Dissociation of the pro-apoptotic
effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on
osteoblasts/osteocytes with novel analogs. Bone 2006, 39 (3), 443-452.
17. Plotkin, L. I.; Bivi, N.; Bellido, T., A bisphosphonate that does not affect
osteoclasts prevents osteoblast and osteocyte apoptosis and the loss of bone strength
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18. Plotkin, L. I.; Manolagas, S. C.; Bellido, T., Transduction of cell survival signals
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19. Plotkin, L. I.; Lezcano, V.; Thostenson, J.; Weinstein, R. T. S.; Manolagas, S. C.;
Bellido, T., Connexin 43 Is Required for the Anti-Apoptotic Effect of Bisphosphonates
on Osteocytes and Osteoblasts In Vivo. J Bone Miner Res 2008, 23 (11), 1712-1721.
20. Aya-Ay, J.; Athavale, S.; Morgan-Bagley, S.; Bian, H.; Bauss, F.; Kim, H. K.,
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Vantreslongdegroot, H. B.; Bijvoet, O. L. M., Migration and Phenotypic Transformation
of Osteoclast Precursors into Mature Osteoclasts - the Effect of a Bisphosphonate. J Bone
Miner Res 1988, 3 (2), 185-192.
22. Hughes, D. E.; Macdonald, B. R.; Russell, R. G. G.; Gowen, M., Inhibition of
Osteoclast-Like Cell-Formation by Bisphosphonates in Long-Term Cultures of Human-
Bone Marrow. J Clin Invest 1989, 83 (6), 1930-1935.
23. Van Beek, E. R.; Lowik, C. W. G. M.; Papapoulos, S. E., Bisphosphonates
suppress bone resorption by a direct effect on early osteoclast precursors without
affecting the osteoclastogenic capacity of osteogenic cells: The role of protein
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precursors. Bone 2002, 30 (1), 64-70.
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24. Marx, R. E.; Cillo, J. E., Jr.; Ulloa, J. J., Oral bisphosphonate-induced
osteonecrosis: risk factors, prediction of risk using serum CTX testing, prevention, and
treatment. J Oral Maxillofac Surg 2007, 65 (12), 2397-410.
25. Khosla, S.; Burr, D.; Cauley, J.; Dempster, D. W.; Ebeling, P. R.; Felsenberg, D.;
Gagel, R. F.; Gilsanz, V.; Guise, T.; Koka, S.; McCauley, L. K.; McGowan, J.; McKee,
M. D.; Mohla, S.; Pendrys, D. G.; Raisz, L. G.; Ruggiero, S. L.; Shafer, D. M.; Shum, L.;
Silverman, S. L.; Van Poznak, C. H.; Watts, N.; Woo, S. B.; Shane, E., Bisphosphonate-
associated osteonecrosis of the jaw: report of a task force of the American Society for
Bone and Mineral Research. J Bone Miner Res 2007, 22 (10), 1479-91.
26. Ruggiero, S. L., Bisphosphonate-related osteonecrosis of the jaw (BRONJ): initial
discovery and subsequent development. J Oral Maxillofac Surg 2009, 67 (5 Suppl), 13-8.
27. Reid, I. R.; Cornish, J., Epidemiology and pathogenesis of osteonecrosis of the
jaw. Nat Rev Rheumatol 2012, 8 (2), 90-6.
28. Hoff, A. O.; Toth, B. B.; Altundag, K.; Johnson, M. M.; Warneke, C. L.; Hu, M.;
Nooka, A.; Sayegh, G.; Guarneri, V.; Desrouleaux, K.; Cui, J.; Adamus, A.; Gagel, R. F.;
Hortobagyi, G. N., Frequency and risk factors associated with osteonecrosis of the jaw in
cancer patients treated with intravenous bisphosphonates. J Bone Miner Res 2008, 23 (6),
826-36.
29. Urade, M.; Tanaka, N.; Furusawa, K.; Shimada, J.; Shibata, T.; Kirita, T.;
Yamamoto, T.; Ikebe, T.; Kitagawa, Y.; Fukuta, J., Nationwide survey for
bisphosphonate-related osteonecrosis of the jaws in Japan. J Oral Maxillofac Surg 2011,
69 (11), e364-71.
30. Barasch, A.; Cunha-Cruz, J.; Curro, F. A.; Hujoel, P.; Sung, A. H.; Vena, D.;
Voinea-Griffin, A. E.; Beadnell, S.; Craig, R. G.; DeRouen, T.; Desaranayake, A.; Gilbert,
A.; Gilbert, G. H.; Goldberg, K.; Hauley, R.; Hashimoto, M.; Holmes, J.; Latzke, B.;
Leroux, B.; Lindblad, A.; Richman, J.; Safford, M.; Ship, J.; Thompson, V. P.; Williams,
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T.; Diel, I. J.; Takahashi, S.; Shore, N.; Henry, D. H.; Barrios, C. H.; Facon, T.; Senecal,
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32. Liberman, U. A., Long-term safety of bisphosphonate therapy for osteoporosis: a
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33. Silverman, S. L.; Landesberg, R., Osteonecrosis of the jaw and the role of
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Greenspan, S. L., Serum CTX: A new marker of bone resorption that shows treatment
effect more often than other markers because of low coefficient of variability and large
changes with bisphosphonate therapy. Calcif Tissue Int 2000, 66 (2), 100-103.
39. Rissanen, J. P.; Suominen, M. I.; Peng, Z. Q.; Halleen, J. M., Secreted tartrate-
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133
Chapter 5
Design and Synthesis of Dual Functional Clickable
Bisphosphonate for Preparation of Fluorescent Imaging
Probes
5.1 Background
The concept of “click chemistry” has gained lots of attention after it was first
proposed by Sharpless, et al.,
1
who also identified a number of reactions that meet the
criteria for click chemistry, namely, reactions that ‘‘are modular, wide in scope, high
yielding, create only inoffensive by-products (that can be removed without
chromatography), are stereospecific, simple to perform and that require benign or easily
removed solvent’’. Of all these click reactions, the Huisgen 1,3-dipolar cycloaddition
reaction of alkynes and azides to yield 1,2,3-triazoles is arguably “the cream of the crop”
and stands at the “central stage”
2, 3
, especially when Cu(I) catalysis was found to
dramatically accelerate the reaction as well as offer high regioselectivity.
4, 5
The Cu(I)
catalyzed alkyne-azide coupling (CuAAC) reaction has become undoubtedly the most
powerful click chemistry reaction and has been applied widely in drug discovery,
polymer and materials science, as well as bioconjugation.
6-11
It was known that the nitrogen atom in the R
2
side chain of clinically used
bisphosphonates plays a pivotal role in their anti-resorptive pharmacological activity as
discussed previously. CuAAC click reaction is a very efficient way to introduce 1,2,3-
triazole heterocycle into a molecule, suggesting its potential for development of novel
134
bisphosphonates. In addition, BPs can be used as “magic bullets” in drug delivery and
imaging probe studies, and CuAAC reactions of alkynyl bisphosphonates or azido
bisphosphonates should have applications in these studies, because the CuAAC reaction
conditions are usually compatible with biological systems.
11
However, interest in preparing alkynyl/azido-containing bisphosphonates (Figure
5.1) only started in the last few years. It was not until 2007 that Osipov and
Röschenthaler
12
reported the first application of CuAAC click chemistry in
bisphosphonate synthesis using tetraethyl but-3-yne-1,1-diyldiphosphonate (Figure 5.1, 1)
or tetraethyl hepta-1,6-diyne-4,4-diyldiphosphonate (Figure 5.1, 2), which is five years
after the introduction of CuAAC reaction; although a series of azidoalkylphosphonates, -
phosphinates and -phosphine oxides were synthesized and their 1,3-dipolar cycloaddition
reactions were investigated earlier.
13
Wiemer et al. recently reported the use of the
CuAAC reaction for syntheses of triazole-based inhibitors of geranylgeranyltransferase II
from the same alkynyl bisphosphonate starting material 1 (Figure 5.1).
14
Guénin et al.
synthesized HMBPyne (Figure 5.1, 3) and applied the compound in the coating of an
iron oxide nanoparticle γ-Fe
2
O
3
to act as an anchored scaffold ready for further “click”
modification.
15
McKenna et al. reported the first examples of α-azido bisphosphonate
esters and acids (Figure 5.1, 4a-d) and applied the α-azido bisphosphonic acid in the
preparation of novel nucleotide analogues containing a CHN
3
or C(CH
3
)N
3
at either the
α,β or β,γ bridging position; but their CuAAC reactions have not been reported.
16
Herczegh et al. utilized the O-Silylated 3-azidopropyl-tetraethyl bisphosphonate (Figure
5.1, 6) and synthesized a series of 1,2,3-triazolelinked hydrobisphosphonate derivatives
of ciprofloxacin as antibacterial agents.
17
Chen et al. recently reported the synthesis of β-
135
azido bisphosphonate (5) in EtOH-water (1:1) (Figure 5.1, 5), and investigated one-pot
synthesis of triazole bisphosphonates which were proposed via the intermediate
compound 5.
18
It should be noted that Szajnman and Rodriguez et al. reported that
compound 5 was not afforded in solvents such as methanol, methanol-water (1:1) or
acetonitrile, from the same starting materials as Chen et al. used.
19
Figure 5.1 Alkynyl bisphosphonates and azido bisphosphonates.
Another way to introduce an alkynyl or azido group into some bisphosphonates,
which could be further conjugated with target molecules of interest by the CuAAC
reaction, is by direct coupling of the alkynyl/azido containing reagents (Figure 5.2) with
the terminal amino group of bisphosphonates such as alendronate and pamidronate.
20-23
However, to the best of our knowledge, there is no report on alkynyl/azido-containing N-
heterocyclic bisphosphonates yet in literature.
P P
OEt
O
EtO
O
EtO OEt
P P
OEt
O
EtO
O
EtO OEt
1
2
P P
OR
O
RO
O
RO OR
N
3
X
4a: X = CH
3
, R = i-Pr
4b: X = H, R = i-Pr
4c: X = CH
3
, R = H
4d: X = H, R = H
P P
OEt
O
EtO
O
EtO OEt
N
3
5
P P
OEt
O
EtO
O
EtO OEt
N
3
TBDMSO
6
P P
OH
O
HO
O
HO OH
3
HO
136
Figure 5.2 Examples of some alkynyl/azido containing reagents.
In this chapter, the synthesis of a first example of bifunctional amino/azido-
containing N-heterocyclic bisphosphonate (amino-azido-para-dRIS, Figure 5.3, 7) will
be reported with its clickable reactivity for the preparation of fluorescent probes
discussed. The N-heterocyclic BP used here is an analogue of deoxy-RIS (para-dRIS),
and two functionalities, amino and azido groups, were introduced sequentially, which
makes it possible for dual-conjugation, e.g., reacting one group with an imaging tag and
the other one with a drug, peptide, protein, oligonucleotide, matrix, et al. In addition, the
introduced alkyl chain only has three carbons, in order to minimize the potential effect to
HAP binding affinity caused by the intrinsic hydrophobic property of alkyl chains.
Figure 5.3 Bifunctional azido-containing N-heterocyclic bisphosphonate (amino-azido-para-
dRIS).
OR
O
N
H
N
3
N
3
O
OH
O
OR
O
N
3
OR
O
OR
O
N
3
O
O
O
O O
O
N
O
O
R = H, NHS
Fluorescent tag
Drug molecule of interest
Peptide
Protein
Oligonucletide
Matrix
(nanoparticles, polymer,
et al.)
... ...
N
P
P
OH HO
O
OH
OH
O
N
3
NH
2
7
N
P
P
OH HO
O
OH
OH
O
N
N
H
N
N
137
5.2 Results and discussion
5.2.1 Synthesis of bifunctional azido-containing N-heterocyclic bisphosphonate
(amino-azido-para-dRIS, 1-(3-amino-2-azidopropyl)-4-(2,2-
diphosphonoethyl)pyridin-1-ium)
The synthesis of amino-azido-para-dRIS is outlined in Scheme 1. The readily
available tetraisopropyl methylene bisphosphonate (8) was treated with NaH to generate
carbanion and was then reacted with 4-(Chloromethyl)pyridine (9), yielding para-dRIS
(10). The reaction is rather slow at room temperature (r.t.) probably due to steric
hindrance, thus temperature is increased to 70 ̊C to accelerate the reaction and improve
yield. In addition, besides mono-substituted compound 10, a little di-substituted
compound was also formed. Compound 10 was first purified by column chromatography
and partially dealkylated product was observed, suggesting the phosphonate ester is
hydrolyzed on silica gel column. Since the ester protection is necessary for the following
reactions, an extraction/wash procedure was developed to avoid the column
chromatography.
Three linkers (Figure 5.4) were then investigated for the conjugation with para-
dRIS (10). Different solvents were screened for each linker reaction. As for the linker 9,
it was found that there was no reaction in CHCl
3
; reaction profile is complicated in
ethanol and methanol, namely, more side products were observed even after a short
reaction time; and isopropanol proved to be the best solvent in this reaction. When
temperature increased to 100 ̊C, 50-80% of product 12 yielded. As for epichlorohydrin
linker 15, reaction was very fast at r.t. in D
2
O with 5 eq. of linker and 0.5 eq. of DIEA
used, however, both products 17 and 18 were observed that were afforded by
138
displacement of chloride and epoxide ring-opening, and it is apparently different from the
case of epichlorohydrin reacting with zoledronate where we observed only epoxide ring-
opening product (see Chapter 2). The reaction of linker 16 was comparable with linker 9
regarding reaction rate and side product percentage, but one more step needs to be done
to obtain the N-protected para-dRIS-linker intermediate 20 for the following reactions,
thus linker 9 was finally selected in the synthetic route.
Scheme 5.1 Synthesis of bifunctional azido-containing N-heterocyclic
bisphosphonate.
Figure 5.4 Linkers tested for para-dRIS-linker synthesis.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
P P
O-iPr
O-iPr
O
iPr-O
O-iPr
O
N
Cl
NaH, DMF/THF
NHBoc
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OH
isopropanol, 100 °C
O
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
N
3
MsCl, TEA, CH
2
Cl
2
1) BTMS, CH
3
CN, rt, 24 hrs
2) MeOH
N
P
P
O OH
OH
O
OH
OH
NH
2
N
3
NaN
3
, DMF, 50 °C
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OMs
8 10 12
11
14 7
9
13
45-55% 50-75%
Quant.
75-85%
70-80%
O
NHBoc
O
Cl
Br Br
OH
9
15
16
N
P
O
iPr-O
O-iPr
12
P
O
iPr-O
iPr-O
N
P
O
iPr-O
O-iPr
17
P
O
iPr-O
iPr-O
O
HO NHBoc
N
P
O
iPr-O
O-iPr
P
O
iPr-O
iPr-O
HO Cl
+
18
19
N
P
O
iPr-O
O-iPr
P
O
iPr-O
iPr-O
HO Br
Phthalimide, NaHCO
3
100 °C
N
P
O
iPr-O
O-iPr
P
O
iPr-O
iPr-O
HO N
O
O
20
139
Compound 12 obtained after extraction purification was used for preparation of
NHBoc-azido-para-dRIS (14) via the intermediate compound 13. It was found that at
temperature > 60 ̊C, ~21% of compound para-dRIS (10) was observed in the reaction
mixture, suggesting the C-N bond cleavage (Figure 5.5). Notably, compound 12 is fairly
stable and no C-N bond cleavage was seen, implying the azido group in proximity has a
potentially catalytic role in the C-N bond cleavage. When the temperature decreased to
50 ̊C, C-N bond cleavage was minimized, indicating the stability of compound 14 is
temperature dependent.
Figure 5.5
31
P NMR trace of reaction 13→14.
Finally, the isopropyl groups were deprotected by BTMS method after
optimization, and the products were further purified by HPLC, giving the target molecule
amino-azido-para-dRIS (7). It should be noted that this synthetic route is also applicable
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
N
3
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OMs
140
to other N-heterocyclic deoxy-bisphosphonates and related analogues, such as dRIS,
dRISPC, etc.
5.2.2 Clickable reactivity studies of amino-azido-para-dRIS (7)
The clickability of amino-azido-para-dRIS (7) was investigated by reacting with
an alkyne-containing fluorescent dye (5(6)-FAM-alkyne, synthesized according to
Scheme 5.2). Reactions in H
2
O at different temperatures (r.t. and 55 ̊C) were tried first
and < 10% of triazole product (24) was found after 48 hrs. Obvious precipitates could be
observed before and after the reaction, which were proposed as complexes with Cu(II)
from CuSO
4
that has not been converted to Cu(I) completely yet or oxidized later by tiny
amount of O
2
; thus I later tried the reaction by adding Cu(I) catalyst that was already
prepared ahead of time and running the reaction under vacuum line to avoid the
introduced O
2
, however, precipitates still existed after all reactants were mixed. It was
then found out that the precipitates were due to poor solubility of 5(6)-FAM-alkyne (23)
and triazole product (24) in aqueous solutions if pH was lower than 8. Thus 0.2 M
triethylammonium bicarbonate buffer (pH 8.0) was used as reaction medium and no
precipitates were observed along the reaction went on. TLC analysis (100% MeOH as
eluent) of the reaction mixture suggested that almost all 5(6)-FAM-alkyne were
consumed and converted to triazole product (24) after overnight incubation at either r.t.
or 45 ̊C.
141
Scheme 5.2 Synthesis of 5(6)-FAM-alkyne (23).
Scheme 5.3 Click reaction of amino-azido-para-dRIS (7) and 5(6)-FAM-alkyne (23).
Since pH also influences the solubility of triazole product (24), a precipitation
procedure by adjusting pH is used to purify the compound. The pH of reaction mixture
was adjusted to 3.0 by 0.5 M HCl until no more precipitate formed. Precipitates were
then collected by centrifuging and then washed sequentially by acetone (0.5 mL × 2) and
diluted HCl (pH = 3.0, 0.25 mL × 2). 5- and 6-isomers of product 24 were further
separated by semi-preparative reverse phase HPLC (Figure 5.6).
O O HO
COOH
O
O
N
O
O
DMF
r.t.
O O HO
COOH
NH
O
NH
2
21
22
23
O O HO
COOH
NH
O
N
P
P
O OH
OH
O
OH
OH
NH
2
N
3
+
O O HO
COOH
NH
N
N
N
H
2
N
N
P
P
OH
O HO
O
OH
OH
O
CuSO
4
,
sodium ascorbate
0.2 M triethylammonium bicarbonate buffer
pH 8.0
7 23 24
142
Figure 5.6 HPLC separation of (A) 5- and 6-isomers of triazole product (24) and (B) FAM-
alkyne (23).
Conditions: Beckman Ultrasphere ODS C18 column (250 x 10 mm, 5 µm, 80 Å pore size), flow
rate 4.0 mL/min, UV detection at 256 nm, mobile phase: buffer A (0.1 M TEAB in 10% methanol,
pH 8.0) and buffer B (0.1M TEAB in 75% methanol, pH 8.0). Gradient as follows: linearly
increase from 0% of buffer B to 100% of buffer B in 20 min.
5.3 Conclusion
In conclusion, the first example of bifunctional amino/azido-containing N-
heterocyclic bisphosphonate (amino-azido-para-dRIS, 7) has been synthesized; and it has
been successfully applied for the preparation of fluorescent probes via CuAAC click
reaction. The synthetic strategy developed based on para-dRIS, should be adaptable to
other N-heterocyclic deoxy-bisphosphonates and related analogues, such as dRIS,
dRISPC, et al. In addition, two functionalities, azido and amino group, were introduced
together via the strategy, which makes it possible for dual-conjugation, and the
introduced alkyl chain only has three carbons, which could minimize the potential effect
on HAP binding affinity caused by the intrinsic hydrophobic property of alkyl chains.
B
A
6-isomer triazole
6-FAM-alkyne
5-FAM-alkyne
5-isomer triazole
143
5.4 Experimental
5.4.1 General
Reagents and Spectral Measurements: 5(6)-carboxyfluorescein, succinimidyl
ester (5(6)-FAM, SE) were purchased from Invitrogen, US. 4-(Chloromethyl)pyridine-
HCl, NaH (60% in oil) and methanesulfonyl chloride (MsCl) were purchased from
Aldrich. Tetraisopropyl methylenebisphosphonate was kind gift from Rhodia, Inc. Linker
11 was synthesized according to the procedure reported in Chapter 2. Triethylamine
(TEA) was distilled from KOH; and CH
2
Cl
2
was distilled from P
2
O
5
. DriSolv® DMF and
THF were purchased from VWR International LLC. All other compounds were used as
supplied by the manufacturer. Thin layer chromatography was performed on Merck Silica
Gel 60 F
254
plates, and the developed plates were visualized under a UV lamp at 354 nm.
HPLC separations were performed on a Shimadzu LC-8A Preparative HPLC with
Shimadzu SPD-20A Prominence UV-Vis detector, Shimadzu CBM-20A Prominence
communications bus module. NMR spectra were recorded on either 400 MHz Varian,
500 MHz Varian, 600 MHz Varian or 500 MHz Bruker spectrometers. UV spectra were
recorded on a DU 800 spectrometer, and fluorescence emission spectra were taken on
Jobin Yvon Nanolog fluorimeter (Jobin Yvon Inc). Mass spectra were taken on ESI
Thermo-Finnigan LCQ DECA XPmax Ion Trap LC/MS/MS spectrometer.
5.4.2 Synthesis of bifunctional azido-containing N-heterocyclic bisphosphonate
(amino-azido-para-dRIS, 7, 1-(3-amino-2-azidopropyl)-4-(2,2-
diphosphonoethyl)pyridin-1-ium)
Synthesis of para-dRIS (10, tetraisopropyl (2-(pyridin-4-yl)ethane-1,1-
diyl)bis(phosphonate)):
144
To NaH (420 mg, 60% in oil, 9.63 mmol, 1.1 eq.) in 10 mL of dry DMF was
added 4-(chloromethyl)pyridine-HCl (1.43 g, 8.71 mmol, 1 eq.) in 15 mL of dry DMF at
0 °C with stirring under N
2
. In another flask, to 1.03 g of NaH (60% in oil, 25.78 mmol, 3
eq.) dispersed in 15 mL of dry THF was added tetraisopropyl methylenebisphosphonate
(6.0 g, 17.42 mmol, 2 eq.) drop-wise at 0 °C under N
2
, and stirring was continued at 0 °C
for 30 - 45 min then for 1 h at room temperature. The 4-(chloromethyl)pyridine solution
was added to the tetraisopropyl methylenebisphosphonate carbanion solution at 0 °C and
stirred for 8 h at 70 °C. The reaction was quenched by the addition of 100 - 200 µL of
EtOH, cooled in freezer for 0.5 h, then dispersed in 100 mL of chilled H
2
O, and extracted
with chilled CH
2
Cl
2 (100 mL × 2). The organic CH
2
Cl
2
phase was then dispersed in 150
mL of chilled 0.25 M HCl solution; shake well and the aqueous phase was collected, and
further extracted by CHCl
3
. The CHCl
3
phase was collected and dried over MgSO
4
, and
then concentrated to obtain 1.9 g of 10, 50% yield.
1
H NMR (D
2
O): δ 8.56 (d, J = 6.8 Hz,
2H), 7.88 (d, J = 6.8 Hz, 2H), 4.62 – 4.52 (m, 4H), 3.33 (td, J = 15.9, 7.1 Hz, 2H), 2.98
(tt, J = 23.9, 7.1 Hz, 1H), 1.14 (ddd, J = 25.2, 6.2, 1.4 Hz, 24H).
31
P NMR (D
2
O): δ 19.83
(s). MS (positive ion MALDI): calcd 435.1 m/z, found [M+Na]
+
= 458.1 m/z.
Synthesis of para-dRIS-linker-OH (12, 4-(2,2-bis(diisopropoxyphosphoryl)ethyl)-
1-(3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)pyridin-1-ium):
500 mg of para-dRIS (10, 1.15 mmol, 1 eq.) was dissolved in 2 mL of isopropanol
in a pressure-tight glass vial. Then 100 µL of DIEA (0.57 mmol, 0.5 eq.) was added to
the solution by syringe followed by adding linker 11 (790 mg, 4.56 mmol, 4 eq.) in 1 mL
of isopropanol. The reaction mixture was stirred at 100 °C for 16 h. Solvent was removed
under vaccuo and 12 mL of CHCl
3
was added in the reaction mixture, which was then
dispersed in 3-4 mL of H
2
O; shake well and collect the aqueous phase (repeat for 4-5
145
times). The aqueous phase was further washed by ether to remove excess linker 11, and
re-extracted with CHCl
3
. The final CHCl
3
phase was dried over MgSO
4
, and then
concentrated to obtain 520 mg of 12, 75% yield.
1
H NMR (CDCl
3
): δ 9.38 – 9.27 (m, 2H),
7.83 (d, J = 6.4 Hz, 2H), 6.14 (m, 2H), 5.09 – 4.99 (m, 1H), 4.80 – 4.64 (m, 5H), 4.21 –
4.04 (m, 1H), 3.39 – 3.19 (m, 4H), 2.50 (tt, J = 23.6, 6.4 Hz, 1H), 1.36 (s, 9H), 1.28 –
1.22 (m, 24H).
31
P NMR (CDCl
3
): δ 18.73 (s). MS (positive ion MALDI): calcd 609.3
m/z, found M
+
= 609.1 m/z.
Synthesis of para-dRIS-linker-N3-ester (14, 1-(2-azido-3-((tert-
butoxycarbonyl)amino)propyl)-4-(2,2-bis(diisopropoxyphosphoryl)ethyl)pyridin-1-ium):
450 mg of para-dRisBP-linker-OH (12, 0.74 mmol, 1 eq.) was dissolved in 4 mL
of anhydrous CH
2
Cl
2
in a pressure-tight glass vial. Then 225 µL of TEA (1.6 mmol, 2.2
eq.) was added to the solution by syringe. Add 100 µL of MsCl (1.29 mmol, 1.7 eq.)
drop-wise into the mixture at ice/water bath and stir the reaction mixture for 1.5 h until
compound 12 was converted to intermediate 13 completely (monitored by MS and
31
P
NMR). Reaction mixture was briefly filtered and the filtrate was pumped to dryness
under vacuo, quantitatively yielding brown oily intermediate 13.
350 mg of intermediate 13 (0.51 mmol, 1 eq.) was dissolved in 5 mL of
anhydrous DMF, to which added in 330 mg NaN
3
(5.1 mmol, 10 eq.) and stir the reaction
mixture vigorously at 50 °C oil bath for 30 hrs. The reaction was monitored by MS and
31
P NMR (Figure B8).
The above mixture was filtered and the filtrate was concentrated under vacuo to
give brown oil. 3 mL of CHCl
3
was used to dissolve the oil and filter off the insoluble
solid. Remove the solvent of CHCl
3
and the residues were purified by silica column
146
chromatography (R
f
= 0.3, CHCl
3
/MeOH, 5:1). 220 mg of compound 14 was obtained,
70% yield.
1
H NMR (400 MHz, CDCl
3
) δ 9.31 (s, 2H), 7.91 (s, 2H), 6.35 (s, 1H), 5.37 (s,
1H), 4.72 (dh, J = 24.3, 6.3 Hz, 4H), 4.55 – 4.32 (m, 2H), 3.66 – 3.45 (m, 2H), 3.45 –
3.24 (m, 2H), 2.61 (t, J = 25.3 Hz, 1H), 1.38 (s, 9H), 1.29 – 1.21 (m, 24H).
31
P NMR
(CDCl
3
): δ 18.76 (s). MS (positive ion MALDI): calcd 634.3 m/z, found M
+
= 634.1 m/z.
Synthesis of amino-azido-para-dRIS (7, 1-(3-amino-2-azidopropyl)-4-(2,2-
diphosphonoethyl)pyridin-1-ium):
50 mg of para-dRIS-linker-N3-ester (14, 0.08 mmol) was dissolved in 1 mL of
CH
3
CN followed by adding 0.3 mL of BTMS in a pressure-tight glass vial. Stir the
mixture at r.t. for 24 hrs. Then the mixture was pumped under vacuo to dryness, which
was added with 0.5 mL of methanol and stirred for 0.5 h. Remove the methanol to give
the crude product for further HPLC purification. Preparative C18 column (Phenomenex
Luna 5µ C18 column, 100 Å, 21.2 mm x 250 mm, 5 µ), flow rate: 8 mL/min, UV-VIS
detection at 260 nm. Sample was eluted with A: 0.1 M triethylammonium bicarbonate
(TEAB), pH 7.8, B: CH
3
CN, using a gradient that was increased from 0-3% of eluent B
over 20 min, and then increased to 100% of eluent B from 20 - 24 min followed by
decreasing to 0% of eluent B from 24 – 25 min. The peak eluting at 17.6 min was
collected (the retention time has ±1.0 min error between different runs), and solvents
were evaporated, obtained 23 mg of compound 7, 80% yield.
1
H NMR (500 MHz, D
2
O):
δ 8.55 (d, J = 6.6 Hz, 2H), 7.95 (d, J = 6.6 Hz, 2H), 4.63 (d, J = 3.2 Hz, 1H), 4.30 (dd, J =
13.9, 9.7 Hz, 1H), 4.06 – 3.96 (m, 1H), 3.34 – 3.19 (m, 2H), 2.90 (d, J = 4.5 Hz, 1H),
2.73 (dd, J = 13.7, 7.4 Hz, 1H), 2.23 – 2.07 (tt, J = 20.8, 7.3 Hz, 1H).
31
P NMR (D
2
O): δ
14.8. MS (negative/positive ion MALDI): calcd 366.1 m/z, found [M-2H]
-
= 364.4 m/z,
[M+Na]
+
= 388.0, [M+2Na]
+
= 410.0 m/z.
147
5.4.3 Clickable reactivity studies of amino-azido-para-dRIS (7)
Synthesis of 5(6)-FAM-alkyne (23, 5-FAM-alkyne: 2-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)-5-(prop-2-yn-1-ylcarbamoyl)benzoic acid; 6-FAM-alkyne: 2-(6-hydroxy-
3-oxo-3H-xanthen-9-yl)-4-(prop-2-yn-1-ylcarbamoyl)benzoic acid):
4.2 µL (0.065 mmol, 3 eq.) of propargylamine was added to a solution of 10.4 mg
(0.022 mmol, 1 eq.) of 5(6)-carboxyfluorescein, succinimidyl ester in DMF (0.5 mL).
After 5 h of stirring at r.t., the solvent was removed under vacuo and the crude mixture
was purified by a silica gel TLC plate (R
f
= 0.7, acetone/CH
2
Cl
2
, 1:1) to give 7.7 mg
(85%) of 5(6)-FAM-alkyne (23).
1
H NMR (500 MHz, Methanol-d
4
): δ 8.98 (dt, J = 1.5,
0.6 Hz, 1H), 8.73 (dd, J = 8.0, 1.7 Hz, 1H), 8.70 – 8.62 (m, 2H), 8.18 (dt, J = 1.3, 0.5 Hz,
1H), 7.86 (dt, J = 8.0, 0.6 Hz, 1H), 7.28 – 7.20 (m, 7H), 7.11 (ddd, J = 9.0, 6.9, 2.4 Hz,
4H), 4.76 (d, J = 2.6 Hz, 2H), 4.64 (d, J = 2.6 Hz, 2H), 3.19 (td, J = 2.6, 0.4 Hz, 1H),
3.11 (td, J = 2.5, 0.5 Hz, 1H). MS (negative/positive ion MALDI): calcd 413.1 m/z, found
[M-H]
-
= 412.3 m/z, [M+H]
+
= 414.4 m/z.
Synthesis of 5(6)-FAM-triazole-para-dRIS (24, 5-isomer: 1-(3-amino-2-(4-((3-
carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)methyl)-1H-1,2,3-triazol-1-
yl)propyl)-4-(2,2-diphosphonoethyl)pyridin-1-ium; 6-isomer: 1-(3-amino-2-(4-((4-
carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)methyl)-1H-1,2,3-triazol-1-
yl)propyl)-4-(2,2-diphosphonoethyl)pyridin-1-ium):
Dissolve 4.6 mg of amino-azido-para-dRIS (7, 12.6 µmol, 1.8 eq.) and 3 mg of
5(6)-FAM-alkyne (23, 7 µmol, 1 eq.) in water in a pressure-tight vial. Add 0.5 M
triethylammonium bicarbonate (TEAB) buffer, pH 8.0, to final concentration as 0.2 M
and volume of 1 mL. Add 28 µL of CuSO
4
/sodium ascorbate solution (50 mM/75 mM in
D
2
O, 20% eq. Cu catalyst). Degas the solution by pumping and then flushing with argon,
repeat three times. Split the solution into two halves. Incubate them at room temperature
and 45 °C water bath overnight.
148
The reaction was quenched by adding Chelex resin followed by 2 h of
ultrasonication. Then the mixture was monitored by TLC (eluent 100% methanol) which
shows almost absence of reactant 5(6)-FAM-alkyne (23). Reaction mixture was then
adjusted to pH 3.0 by 0.5 M HCl until no more precipitates formed. Precipitates were
collected by centrifuging and then washed sequentially by acetone (0.5 mL × 2) and
diluted HCl (pH 3.0, 0.25 mL × 2). Obtained 3 mg, 57% yield.
31
P NMR spectrum
indicates the purity of 5(6)-FAM-triazole-para-dRIS product (24) is >98%. MS
(negative/positive ion MALDI): calcd 779.2 m/z, found [M-2H]
-
= 777.5 m/z, [M+Na]
+
=
801.3 m/z.
The 5- and 6-isomers were further separated by reverse phase HPLC. The
precipitated product was re-dissolved in 0.1 M TEAB buffer and separated by semi-
preparative reverse phase HPLC using the following conditions: Beckman Ultrasphere
ODS C18 (250 x 10 mm, 5 µm, 80 Å pore size), flow rate 4.0 mL/min, UV detection at
256 nm and 370 nm, mobile phase: buffer A (0.1 M TEAB in 10% methanol, pH 8.0) and
buffer B (0.1M TEAB in 75% methanol, pH 8.0). Gradient as follows: linearly increase
from 0% of buffer B to 100% of buffer B in 20 min. 5- and 6-isomers were eluted at very
different retention time, 7.2 min and 9.6 min. Collect each peak and remove solvent
under vacuo. 5-isomer:
1
H NMR (400 MHz, D
2
O): δ 8.26 (d, J = 5.7 Hz, 2H), 7.99 –
7.75 (m, 5H), 7.58 (s, 1H), 7.06 (d, J = 9.0 Hz, 2H), 6.64 – 6.47 (m, 4H), 5.25 (s, 1H),
5.13 – 4.89 (m, 3H), 3.47 (d, J = 20.7 Hz, 2H), 3.26 – 3.17 (m, 1H), 3.01 – 2.87 (m, 2H),
2.19 (s, 1H).
31
P NMR (400 MHz, D
2
O): δ 17.05. 6-isomer:
1
H NMR (400 MHz, D
2
O): δ
8.30 (d, J = 6.6 Hz, 2H), 8.10 (d, J = 1.7 Hz, 1H), 7.93 (s, 1H), 7.88 (t, J = 6.3 Hz, 3H),
7.34 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 9.2 Hz, 2H), 6.64 – 6.55 (m, 4H), 5.36 (s, 1H), 5.14
149
(d, J = 10.0 Hz, 1H), 5.08 – 4.91 (m, 1H), 4.58 (d, J = 2.4 Hz, 1H), 3.72 – 3.46 (m, 2H),
3.22 (s, 2H), 2.94 (d, J = 7.6 Hz, 1H), 2.23 (s, 1H).
31
P NMR (400 MHz, D
2
O): δ 17.03.
UV-VIS absorption and fluorescence emission spectra measurement of compound
24:
The 6- and 5-isomer of compound 24 were dissolved in water and diluted with 0.1
M phosphate buffer (pH 7.2). Assuming that two isomers of compound 24 have the same
extinction coefficient (ε) as the carboxylic acid of the free fluorescent label, the final
concentrations for labeled products are calculated from UV-VIS absorption spectra at λ =
493 nm (ε = 73000 M
-1
cm
-1
at pH 7.2).
Emission spectra were recorded using an excitation wavelength of 493 nm. The
excitation slit and emission slit were set as 1 nm; and integration time and increment
were determined to get optimal spectra for each compound respectively, depending on
the sample concentration and spectrometer used.
5.5 References
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3. Meldal, M.; Tornoe, C. W., Cu-catalyzed azide-alkyne cycloaddition. Chem Rev
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4. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B., A stepwise
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and terminal alkynes. Angew Chem Int Ed 2002, 41 (14), 2596-2599.
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6. Kolb, H. C.; Sharpless, K. B., The growing impact of click chemistry on drug
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bisphosphonate as clickable and solid anchor to elaborate multifunctional iron oxide
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17. McPherson, J. C.; Runner, R.; Buxton, T. B.; Hartmann, J. F.; Farcasiu, D.;
Bereczki, I.; Roth, E.; Tollas, S.; Ostorhazi, E.; Rozgonyi, F.; Herczegh, P., Synthesis of
osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials. Eur J
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18. Chen, X. L.; Li, X.; Yuan, J. W.; Qu, L. B.; Wang, S. H.; Shi, H. Y.; Tang, Y. C.;
Duan, L. K., Simple, efficient one-pot method for synthesis of novel N-attached 1,2,3-
triazole containing bisphosphonates. Tetrahedron 2013, 69 (20), 4047-4052.
151
19. Ferrer-Casal, A.; Barboza, A. P.; Szajnman, S. H.; Rodriguez, J. B., 1, 3-Dipolar
Cycloadditions of the Versatile Intermediate Tetraethyl Vinylidenebisphosphonate.
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20. Liu, X. M.; Thakur, A.; Wang, D., Efficient synthesis of linear multifunctional
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21. Liu, X. M.; Lee, H. T.; Reinhardt, R. A.; Marky, L. A.; Wang, D., Novel
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152
Chapter 6
Conclusions and Perspectives
6.1 Summary
Bisphosphonates (BPs) are widely used in the clinic as therapeutic agents for
treatment of bone diseases characterized by excessive bone resorption. In addition, due to
their specific bone binding affinity, BPs are also used as a targeting moiety in drug
delivery and imaging probe design. The lack of knowledge on BPs’ skeletal, tissue and
cellular distribution as well as the unclear mechanisms of some side effects observed in
patients receiving BP treatment have stimulated the design and synthesis of fluorescent
bisphosphonate imaging probes. In this dissertation, a fluorescent imaging “toolkit” with
more than 20 probes derived from all three heterocyclic N-BP drugs (risedronate,
zoledronate and minodronate) and their related analogues have been successfully
synthesized. A linking strategy with two routes was applied under exceedingly mild
conditions and introduced a terminal amino group in drug-linker intermediates able to
conjugate with the commercially available activated ester of fluorescent dyes. Route A
was previously demonstrated for the synthesis of carboxyfluorescein-risedronate and
related conjugates (FAM-RIS), and has been further developed in this dissertation for
preparation of new near-infrared risedronate conjugates, fluorescent zoledronate,
minodronate and related analogues. Route B adopted a commercially available epoxide
linker, epichlorohydrin, which enhanced the reaction rate significantly. All the
fluorescent probes were prepared in good yield (50-77%) and high purity (> 95%), and
are fully characterized by HPLC, UV-VIS and fluorescence emission spectroscopy,
1
H
153
and
31
P NMR and high-resolution MS.
Both the mineral binding affinity and pharmacological cellular activity were
tested for the fluorescent imaging probe “toolkit”, which provide the first full
characterization of the probes. The imaging probes generally retain substantial affinity for
bone mineral, reflecting the varying affinities of their parent drug components. The
conjugated fluorophores also have important influences (generally slight reduction but in
some cases enhancement) on mineral affinity of the probes. In addition, we have obtained
evidence that certain probes (e.g. the FAM- and ROX- conjugates) have anti-prenylation
and anti-resorptive effects in vitro and in vivo, albeit weaker than their parent drug
compounds, which demonstrated that the probes could retain some biological activities of
the parent BP drugs. However, the near infrared conjugates (AF647-RIS and 800CW-
ZOL) do not show clear activity, which may be due to the interference to their
interactions with the enzymatic target caused by the larger sized near infrared
fluorophores. Therefore, a series of fluorescent BP conjugates with a different length of
linker
1
may be synthesized to provide a systematic structure-activity relationship study,
which may help to explain the various pharmacological activities of different fluorescent
BP probes. In addition, since the anti-prenylation and anti-resorptive activity is an overall
effect, the enzyme target of fluorescent bisphosphonate probes is not necessarily the same
as their parent BP drugs. Thus it will be of great interest to perform the inhibition assays
of the “toolkit” towards specific enzyme targets, e.g. FPPS, in order to confirm the exact
mechanism of their cellular activity, and these results could also provide useful
information to “tune” the desired activity of the fluorescent BP probes by structural
modifications.
154
Due to their diverse spectroscopic and pharmacological properties, the fluorescent
imaging probe “toolkit” has been applied in various biological studies including
osteoclast imaging, drug distribution at bone skeleton and cellular level, mechanism of
osteonecrosis of the jaw (ONJ) studies, drug delivery studies in cochlea, et al., and
several papers have been published based on the obtained results; in addition, the
applications of the imaging probes in cancer imaging, stem cell therapy and other dental
related studies are also in progress. Notably, because of the strong binding affinity of
fluorescent BP imaging probes to bone mineral, specifically hydroxyapatite (HAP), it is
highly conceivable that fluorescent BP imaging probes could be utilized in all bone-
related imaging as well as soft tissue calcification studies
2-4
, including metastatic
calcification and dystrophic calcification
3, 5-8
; and the applications of fluorescent
pamidronate probes have already been demonstrated in breast cancer microcalcification
9
,
atherosclerosis
10, 11
, and calcium urolithiasis.
12
Furthermore, fluorescent imaging is non-
invasive, non-toxic and highly sensitive, which is an ideal tool for disease diagnosis; thus
the potential use of the versatile fluorescent BP imaging probes in disease diagnosis as
well as the image-guided surgery
13
which received keen interest recently will
significantly widen the application scope of fluorescent BP imaging probes.
The first example of a bifunctional amino/azido-containing N-heterocyclic
bisphosphonate (amino-azido-para-dRIS, compound 7 in Chapter 5) has been
synthesized; and it has been successfully applied for the preparation of new fluorescent
probes via Cu(I) catalyzed alkyne-azide coupling (CuAAC) click reaction. The synthetic
strategy developed based on para-dRIS, should be adaptable to other N-heterocyclic
deoxy-bisphosphonates and related analogues, such as dRIS, dRISPC, et al. In addition,
155
two functionalities, azido and amino group, were introduced sequentially through the
strategy, thus dual-conjugation of the molecule is possible, providing the opportunity for
novel cleavable imaging agents and drug delivery system design.
In summary, studies in this dissertation provided a fluorescent bisphosphonate
imaging probe “toolkit” with various options, and also demonstrated the versatile
applications of these probes in bone and dental related imaging investigations. In addition,
a bifunctional amino/azido-containing N-heterocyclic bisphosphonate was first
introduced; this molecule scaffold was not only applied successfully in the synthesis of
new fluorescent BP imaging probe, but also opened a door to next generation of imaging
probe design as well as drug delivery system development.
6.2 References
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APPENDIX A. Chapter 2 Supporting Data
Figure A1.
1
H NMR (CDCl
3
) spectrum of tert-butyl-allylcarbamate.
Figure A2.
1
H NMR (CDCl
3
) spectrum of epoxide linker 5.
H
N
O
O
H
N
O
O
O
174
Figure A3.
1
H NMR (D
2
O) spectrum of RIS-linker 4a.
Figure A4.
31
P NMR (D
2
O) spectrum of RIS-linker 4a.
N
P
P
HO
OH
HO
O
OH HO
O
OH
NH
2
4a
N
P
P
HO
OH
HO
O
OH HO
O
OH
NH
2
4a
175
Figure A5.
1
H NMR (D
2
O) spectrum of RISPC-linker 4b.
Figure A6.
31
P NMR (D
2
O) spectrum of RISPC-linker 4b.
N
P
C
HO
OH
HO
O
OH
O
OH
NH
2
4b
N
P
C
HO
OH
HO
O
OH
O
OH
NH
2
4b
176
Figure A7.
1
H NMR (D
2
O) spectrum of dRIS-linker 4c.
Figure A8.
31
P NMR (D
2
O) spectrum of dRIS-linker 4c.
N
P
P
OH
HO
O
OH HO
O
OH
NH
2
4c
N
P
P
OH
HO
O
OH HO
O
OH
NH
2
4c
177
Figure A9.
1
H NMR (D
2
O) spectrum of ZOL-linker 4d.
Figure A10.
31
P NMR (D
2
O) spectrum of ZOL-linker 4d.
!
N
N
P
P
HO
O
OH
OH
HO
OH
O
OH
NH
2
4d
!
N
N
P
P
HO
O
OH
OH
HO
OH
O
OH
NH
2
4d
178
Figure A11.
1
H NMR (D
2
O) spectrum of ZOL-N,O-dilinker P1.
Figure A12.
31
P NMR (D
2
O) spectrum of ZOL-N,O-dilinker P1.
!
N
N
P
P
HO
O
O
OH
O
OH
OH
OH
NH
2
HO
NH
2
P1
!
N
N
P
P
HO
O
O
OH
O
OH
OH
OH
NH
2
HO
NH
2
P1
179
Figure A13. Mass spectrum (+) of ZOL-N,O-dilinker P1.
N
N
P
P
HO
O
O
OH
O
OH
OH
OH
NH
2
HO
NH
2
P1
Chemical Formula: C 11H25N4O9P2
+
Exact Mass: 419.11
Molecular Weight: 419.28
180
Figure A14.
1
H NMR (D
2
O) spectrum of P3 of HPLC trace (ZOL).
Figure A15.
31
P NMR (D
2
O) spectrum of P3 of HPLC trace (ZOL).
!
P3
N
N
P
P
HO
O
OH
OH
O
OH
OH
!
P3
N
N
P
P
HO
O
OH
OH
O
OH
OH
181
Figure A16.
1
H NMR (D
2
O) spectrum of MIN-linker 4e.
Figure A17.
31
P NMR (D
2
O) spectrum of MIN-linker 4e.
N
N
P
P
HO
OH
O
OH
HO
OH
O
OH
NH
2
4e
N
N
P
P
HO
OH
O
OH
HO
OH
O
OH
NH
2
4e
182
Figure A18.
1
H NMR (D
2
O) spectrum of MINPC-linker 4f.
Figure A19.
31
P NMR (D
2
O) spectrum of MINPC-linker 4f.
N
N
P
C
HO
OH
O
OH
OH
O
OH
NH
2
4f
N
N
P
C
HO
OH
O
OH
OH
O
OH
NH
2
4f
183
Figure A20. N, C(P)-O-dilinker-MINPC side products.
Figure A21.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-RIS, 7a1.
31
P NMR
1
H NMR
N
N
P
C
HO
O
O
OH
O
OH
OH
NHBoc
OH
NHBoc
N
N
P
C
HO
O
OH
OH
O
O
OH
NHBoc
NHBoc
OH
O O HO
CO
2
H
O
HN
HO
N
P
OH
O HO
HO
P
O
HO
HO
7a1
184
Figure A22.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-RIS, 7a1.
Figure A23.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-RISPC, 7b1.
O O HO
CO
2
H
O
HN
HO
N
P
OH
O HO
HO
P
O
HO
HO
7a1
!
O O HO
CO
2
H
O
HN
HO
N
P
OH
O HO
HO
C
O
HO
7b1
185
Figure A24.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-RISPC, 7b1.
Figure A25. HPLC trace of 5(6)-FAM-RISPC, 7b1.
!
O O HO
CO
2
H
O
HN
HO
N
P
OH
O HO
HO
C
O
HO
7b1
!
Excess RIS/
RISLinker
5(6)-FAM-RISPC
186
Figure A26.
1
H NMR (D
2
O) spectrum of 5(6)-FAM-dRIS, 7c1.
Figure A27.
31
P NMR (D
2
O) spectrum of 5(6)-FAM-dRIS, 7c1.
O O HO
CO
2
H
O
HN
HO
N
P
H
O HO
HO
P
O
HO
HO
7c1
O O HO
CO
2
H
O
HN
HO
N
P
H
O HO
HO
P
O
HO
HO
7c1
187
Figure A28.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-RIS, 7a4.
Figure A29.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-RIS, 7a4.
O N N
SO
3
H
NH
OH
N
P
O
OH
OH P
O
OH
OH
SO
2
NH
O
HO
7a4
O N N
SO
3
H
NH
OH
N
P
O
OH
OH P
O
OH
OH
SO
2
NH
O
HO
7a4
188
Figure A30. Mass spectrum of 5(6)-RhR-RIS, 7a4.
KBIV-223
Measured Mass 1010.2866
Element Low Limit High Limit
C 38 48
H 50 70
N4 6
O 14 16
P0 2
S0 3
Formula Calculated Mass mDaError ppmError RDB
C44 H59 N4 O15 P S3 1010.2871 -0.5 -0.5 18
C40 H62 N5 O15 P2 S3 1010.2874 -0.8 -0.8 13.5
C46 H54 N6 O14 S3 1010.2855 1.1 1.1 23
C43 H58 N5 O15 P2 S2 1010.2841 2.5 2.5 18.5
C47 H55 N4 O15 P S2 1010.2838 2.8 2.8 23
845.0 964.2 1083.4 1202.6 1321.8 1441.0
Mass ( m/z)
0
2.0E+4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 M C = > B C = > N F 0 . 7 = > B C = > N F 0 . 7 [ B P = 1 0 1 0 . 3 , 1 9 6 4 3 ]
McKenna (USC) KBIV-223 H2O/CHCA/MIX1A
1010.2866
904.4682
1296.6854
799.0 919.2 1039.4 1159.6 1279.8 1400.0
Mass ( m/z)
0
4.4E+4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 [ BP = 1 0 1 0 . 2 , 4 4 4 4 1 ]
McKenna (USC) KBIV-223 H2O/CHCA
1010
1011
1012
928
982
1048
929
Page 1
189
Figure A31.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-RISPC, 7b2.
Figure A32.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-RISPC, 7b2.
O N N
SO
3
H
NH
OH
N
C
O
OH
P
O
OH
OH
SO
2
NH
O
HO
7b2
O N N
SO
3
H
NH
OH
N
C
O
OH
P
O
OH
OH
SO
2
NH
O
HO
7b2
190
Figure A33.
1
H NMR (D
2
O) spectrum of 5(6)-RhR-dRIS, 7c2.
Figure A34.
31
P NMR (D
2
O) spectrum of 5(6)-RhR-dRIS, 7c2.
O N N
SO
3
H
NH
OH
N
P
O
OH
OH P
O
OH
OH
SO
2
NH
O
H
7c2
O N N
SO
3
H
NH
OH
N
P
O
OH
OH P
O
OH
OH
SO
2
NH
O
H
7c2
191
Figure A35. Mass spectrum of 5(6)-RhR-dRIS, 7c2.
KBIV-109
Measured Mass 994.2872
Element Low Limit High Limit
C 38 48
H 50 70
N3 7
O 12 16
P0 2
S0 2
Formula Calculated Mass mDaError ppmError RDB
C45 H53 N7 O13 P S2 994.2875 -0.3 -0.3 23.5
C46 H54 N5 O14 P2 S 994.2858 1.4 1.4 23.5
C47 H55 N4 O14 P S2 994.2888 -1.6 -1.6 23
C43 H58 N5 O14 P2 S2 994.2891 -1.9 -2.0 18.5
C38 H58 N7 O16 P2 S2 994.2851 2.1 2.1 14.5
C42 H55 N6 O16 P S2 994.2848 2.4 2.4 19
C46 H52 N5 O16 S2 994.2845 2.7 2.7 23.5
C45 H52 N6 O16 P2 994.2910 -3.8 -3.8 24
765.0 894.2 1023.4 1152.6 1281.8 1411.0
Mass (m/z)
0
6.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C [ B P = 1 2 9 6 . 7 , 5 9 8 6 4 ]
McKenna (USC) KBIV-109 H2O/CHCA/MIX1A
1296.6853
904.4681
994.2872
499.0 659.2 819.4 979.6 1139.8 1300.0
Mass (m/z)
0
1.4E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 [ BP = 9 9 4 . 2 , 1 4 0 8 0 ]
McKenna (USC) KBIV-109 H2O/CHCA
994
995
996
977
966
1016
1056
997
978 1017
1054 998
Page 1
192
Figure A36.
1
H NMR (D
2
O) spectrum of 5-ROX-RIS, 7a5.
Figure A37.
31
P NMR (D
2
O) spectrum of 5-ROX-RIS, 7a5.
7a5
N O N
C
O
OH
OH
N
P
P
HO
O
OH
OH
O
OH
OH
O
N
H
7a5
N O N
C
O
OH
OH
N
P
P
HO
O
OH
OH
O
OH
OH
O
N
H
193
Figure A38. Mass spectrum of 5-ROX-RIS, 7a5.
SUN-II-22
Measured Mass 873.2647
Element Low Limit High Limit
C 38 48
H 35 55
N3 5
O 10 14
P0 2
Formula Calculated Mass mDaError ppmError RDB
C47 H44 N3 O12 P 873.2657 -1.0 -1.2 28
C43 H47 N4 O12 P2 873.2660 -1.3 -1.5 23.5
C42 H44 N5 O14 P 873.2617 3.0 3.4 24
C46 H41 N4 O14 873.2614 3.3 3.8 28.5
C47 H45 N3 O10 P2 873.2575 7.2 8.3 28
602.0 675.6 749.2 822.8 896.4 970.0
Mass (m/z)
0
3.4E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 [ BP = 1 2 9 6 . 7 , 3 9 3 2 1 ]
McKenna (USC) SUN-II-22 H20/MeOH/CHCA/MIX1A
904.4681
660.3469
873.2647
599.0 719.2 839.4 959.6 1079.8 1200.0
Mass (m/z)
0
2.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 [ BP = 8 7 3 . 0 , 1 9 5 1 3 ]
McKenna (USC) SUN-II-22 H20/MeOH/CHCA
873
874
871
872
791
869
895
792 656
Page 1
194
Figure A39. HPLC trace of 5-ROX-RIS, 7a5.
Figure A40.
1
H NMR (D
2
O) spectrum of 5-ROX-RISPC, 7b3.
Chrom. 1 0.0 mins. 60.0 mins.
1
Excess RIS/
RISLinker
ROX-RIS
ROX residues
O N
CO
2
H
N
N
H
O
OH
N
P
C
HO
O
OH
OH
O
OH
7b3
195
Figure A41.
31
P NMR (D
2
O) spectrum of 5-ROX-RISPC, 7b3.
O N
CO
2
H
N
N
H
O
OH
N
P
C
HO
O
OH
OH
O
OH
7b3
196
Figure A42. Mass spectrum of 5-ROX-RISPC, 7b3.
KBV-229
Measured Mass 835.2733
Element Low Limit High Limit
C 39 49
H 35 55
N0 5
O 9 13
P0 2
Formula Calculated Mass mDaError ppmError RDB
C48 H41 N3 O11 835.2747 -1.4 -1.6 30
C44 H44 N4 O11 P 835.2750 -1.7 -2.0 25.5
C40 H47 N5 O11 P2 835.2753 -2.0 -2.4 21
C43 H41 N5 O13 835.2706 2.7 3.2 26
C46 H46 N O12 P 835.2763 -3.0 -3.6 25
C42 H49 N2 O12 P2 835.2766 -3.3 -4.0 20.5
2
x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
- Scan (0.438-0.603 m in, 15 scans) 12080930t.d Subtract (1)
835.2733
903.2591
871.2489
893.2288
Counts vs. Mass-to-Charge (m / z)
780 790 800 810 820 830 840 850 860 870 880 890 900 910 920 930 940 950 960
2
x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
- Scan (0.438-0.603 m in, 15 scans) 12080930t.d Subtract (1)
835.2733
1680.5406
260.0349
Counts vs. Mass-to-Charge (m / z)
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
Page 1
197
Figure A43. HPLC trace of 5-ROX-RISPC, 7b3.
Figure A44.
1
H NMR (D
2
O) spectrum of AF647-RIS, 7a6.
Chrom. 1 0.0 mins. 44.1 mins.
1
Excess RISPC/
RISPCLinker
ROX-RISPC
ROX residues
!
N
O
NH
OH
N
P
HO
O
OH
OH
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
HO
3
S SO
3
H
P
O
OH
OH
7a6
198
Figure A45.
31
P NMR (D
2
O) spectrum of AF647-RIS, 7a6.
!
N
O
NH
OH
N
P
HO
O
OH
OH
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
HO
3
S SO
3
H
P
O
OH
OH
7a6
199
Figure A46. Mass spectrum of AF647-RIS, 7a6.
AF647RIS
Measured Mass 1197.2358
Element Low Limit High Limit
C 41 51
H 55 75
N3 5
O 19 23
P0 2
S3 5
Formula Calculated Mass mDaError ppmError RDB
C49 H59 N5 O20 S5 1197.2352 0.6 0.5 23
C47 H64 N3 O21 P S5 1197.2368 -1.0 -0.8 18
C43 H67 N4 O21 P2 S5 1197.2371 -1.3 -1.1 13.5
C46 H63 N4 O21 P2 S4 1197.2337 2.1 1.7 18.5
C50 H60 N3 O21 P S4 1197.2334 2.4 2.0 23
C49 H58 N4 O23 P S3 1197.2386 -2.8 -2.4 23.5
C42 H64 N5 O23 P S5 1197.2328 3.0 2.5 14
C45 H61 N5 O23 P2 S3 1197.2389 -3.1 -2.6 19
C46 H61 N4 O23 S5 1197.2325 3.3 2.8 18.5
C49 H59 N4 O21 P2 S3 1197.2304 5.4 4.5 23.5
C50 H59 N3 O23 S4 1197.2417 -5.9 -4.9 23
850.0 957.8 1065.6 1173.4 1281.2 1389.0
Mass (m/z)
0
2.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 [ BP = 9 0 4 . 5 , 2 0 1 2 7 ]
McKenna (USC) AF647RIS H2O/CHCA/MIX1A
904.4681
1197.2358
1296.6853
900 1000 1100 1200 1300 1400
Mass (m/z)
0
1.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 = > BC = > N F 0 . 7 [ BP = 1 1 9 7 . 2 , 1 0 0 5 1 ]
McKenna (USC) AF647RIS H2O/CHCA
1197
1198
1221
1235
1199
1222
1119
1223
1115
1237
1200
1037 1143
1259
1121 1219
1144
Page 1
200
Figure A47. HPLC trace of AF647-RIS, 7a6.
Chrom. 1 0.0 mins. 52.4 mins.
4
3
2
1
Chrom. 1 0.0 mins. 52.4 mins.
4
3
2
1
A
B
A: Channel A, UV-VIS detector set at 260nm
B: Channel B, UV-VIS detector set at 598nm
Ris/Rislinker
AF647-RIS
free
dye
201
Figure A48.
1
H NMR (D
2
O) spectrum of AF647-RISPC, 7b4.
Figure A49.
31
P NMR (D
2
O) spectrum of AF647-RISPC, 7b4.
!
N
O
NH
OH
N
P
HO
O
OH
OH
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
HO
3
S SO
3
H
C
O
OH
7b4
!
N
O
NH
OH
N
P
HO
O
OH
OH
C
H
C
H
C
H
C
H
C
H N
SO
3
H HO
3
S
HO
3
S SO
3
H
C
O
OH
7b4
202
Figure A50. Mass spectrum of AF647-RISPC, 7b4.
AF647RISPC
Measured Mass 1161.2596
Element Low Limit High Limit
C 42 52
H 55 75
N3 5
O 19 21
P0 1
S3 5
Formula Calculated Mass mDaError ppmError RDB
C48 H63 N3 O20 S5 1161.2603 -0.7 -0.6 19
C44 H66 N4 O20 P S5 1161.2606 -1.0 -0.9 14.5
C47 H62 N4 O20 P S4 1161.2572 2.4 2.0 19.5
C51 H59 N3 O20 S4 1161.2569 2.7 2.3 24
C50 H58 N4 O20 P S3 1161.2539 5.7 4.9 24.5
850.0 963.6 1077.2 1190.8 1304.4 1418.0
Mass (m/z)
0
5.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C [ BP = 9 0 4 . 5 , 5 0 0 0 1 ]
McKenna (USC) AF647RISPC H2O/CHCA/MIX1A
904.4677
1296.6853
1161.2596
799.0 919.2 1039.4 1159.6 1279.8 1400.0
Mass ( m/z)
0
1.7E+4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 = > BC = > N F 0 . 7 [ BP = 1 1 6 1 . 2 , 1 6 7 6 6 ]
McKenna (USC) AF647RISPC H2O/CHCA
1161
1162
1163
1079
1083
1164
1185
1080
1186 1085
802
Page 1
203
Figure A51. HPLC trace of AF647-RISPC, 7b4.
Figure A52.
1
H NMR (D
2
O) spectrum of 5-FAM-ZOL, 7d1.
HPLC trace of AF647-RISPC
1-3: channel A 260nm
4-6: channel B 598nm
Chrom. 1 0.0 mins. 58.0 mins.
6
5
4
3
2
1
RisPC/RisPClinker
AF647-RISPC
free dye
O O HO
CO
2
H
N
HO
P
OH
P
O
OH
OH
O
OH
OH
N
NH O
7d1
204
Figure A53.
31
P NMR (D
2
O) spectrum of 5-FAM-ZOL, 7d1.
O O HO
CO
2
H
N
HO
P
OH
P
O
OH
OH
O
OH
OH
N
NH O
7d1
205
Figure A54. Mass spectrum of 5-FAM-ZOL, 7d1.
Sun-I-260-FZP2
Measured Mass 704.1013
Element Low Limit High Limit
C 24 34
H 20 40
N0 5
O 12 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C31 H23 N5 O13 P 704.1025 -1.2 -1.6 23.5
C24 H28 N5 O16 P2 704.1001 1.2 1.7 14.5
C28 H25 N4 O16 P 704.0998 1.5 2.2 19
C32 H22 N3 O16 704.0995 1.8 2.6 23.5
C33 H25 N2 O14 P 704.1038 -2.5 -3.5 23
C29 H28 N3 O14 P2 704.1041 -2.8 -4.0 18.5
625.0 692.4 759.8 827.2 894.6 962.0
Mass (m/z)
0
2.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C [ BP = 7 0 4 . 1 , 2 0 0 2 2 ]
McKenna (USC) Sun-I-260-FZP2 H2O/CHCA/MIX1A
704.1013
660.3468
904.4676
499.0 619.2 739.4 859.6 979.8 1100.0
Mass (m/z)
0
4.1E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 [ BP = 7 0 4 . 2 , 4 1 2 7 6 ]
McKenna (USC) Sun-I-260-FZP2 H2O/CHCA
704
705
706
726
622
500
748 627
Page 1
206
Figure A55.
1
H NMR (D
2
O) spectrum of 6-FAM-ZOL, 7d2.
Figure A56.
31
P NMR (D
2
O) spectrum of 6-FAM-ZOL, 7d2.
O O HO
CO
2
H
N
HO
P
OH
P
O
OH
OH
O
OH
OH
N
NH
O
7d2
O O HO
CO
2
H
N
HO
P
OH
P
O
OH
OH
O
OH
OH
N
NH
O
7d2
207
Figure A57. Mass spectrum of 6-FAM-ZOL, 7d2.
Sun-I-260-FZP2
Measured Mass 704.1027
Element Low Limit High Limit
C 24 34
H 20 40
N0 5
O 12 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C31 H23 N5 O13 P 704.1025 0.2 0.4 23.5
C33 H25 N2 O14 P 704.1038 -1.1 -1.6 23
C29 H28 N3 O14 P2 704.1041 -1.4 -2.0 18.5
C24 H28 N5 O16 P2 704.1001 2.6 3.7 14.5
C31 H30 O15 P2 704.1054 -2.7 -3.9 18
C28 H25 N4 O16 P 704.0998 2.9 4.2 19
C32 H22 N3 O16 704.0995 3.2 4.6 23.5
610.0 683.6 757.2 830.8 904.4 978.0
Mass (m/z)
0
3.4E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C [ BP = 6 6 0 . 3 , 3 3 8 0 5 ]
McKenna (USC) Sun-I-260-FZP1 H2O/CHCA/MIX1A
660.3467
704.1027
904.4680
499.0 619.2 739.4 859.6 979.8 1100.0
Mass (m/z)
0
1.5E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 [ BP = 7 0 4 . 2 , 1 5 4 6 1 ]
McKenna (USC) Sun-I-260-FZP1 H2O/CHCA
704
705
726
706
727 538 505
622
656
501
748
728
Page 1
208
Figure A58.
1
H NMR (D
2
O) spectrum of AF647-ZOL, 7d3.
Figure A59.
31
P NMR (D
2
O) spectrum of AF647-ZOL, 7d3.
!
N
O
NH
OH
N
C
H
C
H
C
H
C
H
C
H
N
SO
3
H HO
3
S
HO
3
S SO
3
H
N
P
P HO
O OH
OH
O
OH
HO
7d3
!
N
O
NH
OH
N
C
H
C
H
C
H
C
H
C
H
N
SO
3
H HO
3
S
HO
3
S SO
3
H
N
P
P HO
O OH
OH
O
OH
HO
7d3
209
Figure A60. Mass spectrum of AF647-ZOL, 7d3.
SUN-II-88-AF647ZOL
Measured Mass 1186.2337
Element Low Limit High Limit
C 39 49
H 50 70
N4 6
O 20 22
P1 2
S3 5
Formula Calculated Mass mDaError ppmError RDB
C41 H66 N5 O21 P2 S5 1186.2324 1.3 1.1 12.5
C45 H63 N4 O21 P S5 1186.2321 1.6 1.4 17
C48 H60 N4 O21 P2 S3 1186.2382 -4.5 -3.8 22
C44 H62 N5 O21 P2 S4 1186.2290 4.7 4.0 17.5
C48 H59 N4 O21 P S4 1186.2287 5.0 4.2 22
877 973 1069 1165 1261 1357
Mass (m/z)
0
5279.9
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > B C = > N F 0 . 7 = > BC = > N F 0 . 7 = > M C [ B P = 9 0 4 . 5 , 5 2 8 0 ]
McKenna (USC) SUN-II-88-AF647ZOL MeOH/CHCA/Mix1A
904.4681
1210.1991
1296.6853
905.4703
1297.6891
1211.1995
1212.1965
1298.6958
1186.2337
1240.1589
699.0 829.2 959.4 1089.6 1219.8 1350.0
Mass (m/z)
0
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 = > B C = > N F 0 . 7 = > BC = > N F 0 . 7 [ B P = 1 2 1 0 . 3 , 1 1 1 8 4 ]
McKenna (USC) SUN-II-88-AF647ZOL MeOH/CHCA
1210
1211
1212
1186
1213
1240
Page 1
210
Figure A61. HPLC trace of AF647-ZOL, 7d3.
Figure A62.
1
H NMR (D
2
O) spectrum of 800CW-ZOL, 7d4.
!
ZOL-Linker
AF647-ZOL
AF647 dye residues
0 - 10 min: 230 nm
10 - 50 min: 598 nm
!
N
-
O
3
S
N
SO
3
H
O
SO
3
H
SO
3
Na
O
H
2
C
HO
N
N
P
P
OH
O
OH
HO
O
HO
HO
HN
7d4
211
Figure A63.
31
P NMR (D
2
O) spectrum of 800CW-ZOL, 7d4.
!
N
-
O
3
S
N
SO
3
H
O
SO
3
H
SO
3
Na
O
H
2
C
HO
N
N
P
P
OH
O
OH
HO
O
HO
HO
HN
7d4
212
Figure A64. Mass spectrum of 800CW-ZOL, 7d4.
SUN-II-110-CW800ZOL
Measured Mass 1330.2885
Element Low Limit High Limit
C 49 59
H 60 80
N4 6
O 21 23
S3 5
P1 2
Formula Calculated Mass mDaError ppmError RDB
C55 H71 N4 O22 P S5 1330.2896 -1.1 -0.8 23
C51 H74 N5 O22 P2 S5 1330.2899 -1.4 -1.0 18.5
C54 H70 N5 O22 P2 S4 1330.2865 2.0 1.5 23.5
C58 H67 N4 O22 P S4 1330.2862 2.3 1.7 28
C57 H66 N5 O22 P2 S3 1330.2831 5.4 4.0 28.5
855.0 970.6 1086.2 1201.8 1317.4 1433.0
Mass (m/z)
0
717.4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 M C = > B C = > N F 0 . 7 = > BC = > N F 0 . 7 [ B P = 1 2 9 7 . 7 , 7 1 7 ]
McKenna (USC) SUN-II-110-CW800ZOL MeOH/CHCA (+ve)/MIX1A
1296.6853
1330.2885
1354.2621
904.4681
799.0 936.8 1074.6 1212.4 1350.2 1488.0
Mass (m/z)
0
8030.9
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 [ BP = 1 3 5 4 . 4 , 8 0 3 1 ]
McKenna (USC) SUN-II-110-CW800ZOL MeOH/CHCA (+ve) 1354
1330
1355
1331
1356
1332
1384
1126 1003
1357 1252
1385 1333
1127
1004
Page 1
213
Figure A65. HPLC trace of 800CW-ZOL, 7d4.
Figure A66.
1
H NMR (D
2
O) spectrum of Sulfo-Cy5-ZOL, 7d5.
ZOL-Linker
800CW-ZOL
IRDye 800CW dye residues
0 - 8 min: 230 nm
10 - 50 min: 598 nm
N N
NaO
3
S
SO
3
-
NH
O
OH N N P
P
OH
HO
OH
O
HO
HO
O
7d5
214
Figure A67.
31
P NMR (D
2
O) spectrum of Sulfo-Cy5-ZOL, 7d5.
N N
NaO
3
S
SO
3
-
NH
O
OH N N P
P
OH
HO
OH
O
HO
HO
O
7d5
215
Figure A68. Mass spectrum (+) of Sulfo-Cy5-ZOL, 7d5.
216
Figure A69. HPLC trace of Sulfo-Cy5-ZOL, 7d5.
Figure A70.
1
H NMR (D
2
O) spectrum of 5-FAM-MIN, 7e1.
!
ZOL-Linker
sulfo-Cy5-ZOL
sulfo-Cy5 dye residues
0 - 12 min: 230 nm (UV-VIS detector)
12 -50 min: 598 nm (UV-VIS detector)
O O HO
CO
2
H
H
N
HO
N
N
P
HO
P OH
O OH
OH
O
OH
O
7e1
217
Figure A71.
31
P NMR (D
2
O) spectrum of 5-FAM-MIN, 7e1.
O O HO
CO
2
H
H
N
HO
N
N
P
HO
P OH
O OH
OH
O
OH
O
7e1
218
Figure A72. Mass spectrum of 5-FAM-MIN, 7e1.
5-Fammin
Measured Mass 754.1178
Element Low Limit High Limit
C 28 38
H 20 40
N0 5
O 11 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C35 H25 N5 O13 P 754.1181 -0.3 -0.4 26.5
C37 H27 N2 O14 P 754.1194 -1.6 -2.2 26
C33 H30 N3 O14 P2 754.1198 -2.0 -2.6 21.5
C28 H30 N5 O16 P2 754.1157 2.1 2.7 17.5
C32 H27 N4 O16 P 754.1154 2.4 3.2 22
C36 H24 N3 O16 754.1151 2.7 3.6 26.5
C35 H32 O15 P2 754.1211 -3.3 -4.4 21
591.0 668.2 745.4 822.6 899.8 977.0
Mas s (m/z)
0
5708.0
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 = > BC = > N F 0 . 7 [ BP = 1 2 9 6 . 7 , 7 3 3 5 ]
McKenna (USC) 5-Fammin MeOH/CHCA/Mix1A
904.4681
754.1178
660.3469
499.0 619.2 739.4 859.6 979.8 1100.0
Ma ss (m/z)
0
6060.5
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 = > BC = > N F 0 . 7 [ BP = 7 5 3 . 8 , 6 0 6 1 ]
McKenna (USC) 5-Fammin MeOH/CHCA
754
755
756
777
776
Page 1
219
Figure A73.
1
H NMR (D
2
O) spectrum of 6-FAM-MIN, 7e2.
Figure A74.
31
P NMR (D
2
O) spectrum of 6-FAM-MIN, 7e2.
O O HO
CO
2
H
HN
HO
N
N
P
HO
P OH
O OH
OH
O
OH
O
7e2
O O HO
CO
2
H
HN
HO
N
N
P
HO
P OH
O OH
OH
O
OH
O
7e2
220
Figure A75. Mass spectrum of 6-FAM-MIN, 7e2.
6-Fammin
Measured Mass 754.1187
Element Low Limit High Limit
C 28 38
H 20 40
N0 5
O 11 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C35 H25 N5 O13 P 754.1181 0.6 0.8 26.5
C37 H27 N2 O14 P 754.1194 -0.7 -1.0 26
C33 H30 N3 O14 P2 754.1198 -1.1 -1.4 21.5
C35 H32 O15 P2 754.1211 -2.4 -3.2 21
C28 H30 N5 O16 P2 754.1157 3.0 3.9 17.5
C32 H27 N4 O16 P 754.1154 3.3 4.3 22
C36 H24 N3 O16 754.1151 3.6 4.8 26.5
C36 H28 N4 O11 P2 754.1224 -3.7 -5.0 26
606.0 682.2 758.4 834.6 910.8 987.0
Ma ss (m/z)
0
1.7E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 [ BP = 7 5 4 . 1 , 1 7 0 7 5 ]
McKenna (USC) 6-Fammin MeOH/CHCA/Mix1A
754.1187
904.4680
660.3469
499.0 619.2 739.4 859.6 979.8 1100.0
Ma ss (m/z)
0
1049.8
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 = > BC = > N F 0 . 7 = > B C = > N F 0 . 7 [ BP = 7 5 3 . 8 , 1 0 5 0 ]
McKenna (USC) 6-Fammin MeOH/CHCA
754
755
673
672
776
756
538
Page 1
221
Figure A76. HPLC separation of 5-FAM-MIN (7e1) and 6-FAM-MIN (7e2).
Figure A77.
1
H NMR (D
2
O) spectrum of 5-FAM-MINPC, 7f1.
!
MIN-Linker
6-FAM-MIN
5-FAM-MIN
FAM dye residues
0 - 8 min: 280 nm (UV-VIS detector)
8 -50 min: 598 nm (UV-VIS detector)
O O HO
CO
2
H
H
N
HO
N
N
C
HO
P OH
O OH
O
OH
O
7f1
222
Figure A78.
31
P NMR (D
2
O) spectrum of 5-FAM-MINPC, 7f1.
O O HO
CO
2
H
H
N
HO
N
N
C
HO
P OH
O OH
O
OH
O
7f1
223
Figure A79. Mass spectrum of 5-FAM-MINPC, 7f1.
5-FAMMinPC
Measured Mass 718.1399
Element Low Limit High Limit
C 28 38
H 20 40
N0 5
O 11 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C35 H28 N O16 718.1403 -0.4 -0.5 22.5
C29 H29 N5 O15 P 718.1392 0.7 0.9 18.5
C31 H31 N2 O16 P 718.1406 -0.7 -0.9 18
C33 H26 N4 O15 718.1389 1.0 1.4 23
C36 H24 N5 O12 718.1416 -1.7 -2.4 27.5
C38 H26 N2 O13 718.1429 -3.0 -4.2 27
C34 H29 N3 O13 P 718.1433 -3.4 -4.7 22.5
C36 H32 O12 P2 718.1364 3.5 4.9 22
644.0 704.2 764.4 824.6 884.8 945.0
Ma ss (m/z)
0
2.1E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 [ BP = 7 1 8 . 1 , 2 1 1 5 5 ]
McKenna (USC) 5-FAMMinPC MeOH/CHCA/Mix1A
718.1399
904.4681
660.3469
499.0 619.2 739.4 859.6 979.8 1100.0
Ma ss (m/z)
0
1055.7
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 = > BC = > N F 0 . 7 = > B C = > N F 0 . 7 [ BP = 7 1 7 . 8 , 1 0 5 6 ]
McKenna (USC) 5-Fammin in PC MeOH/CHCA
718
740
550
741
542
638
742
Page 1
224
Figure A80.
1
H NMR (D
2
O) spectrum of 6-FAM-MINPC, 7f2.
Figure A81.
31
P NMR (D
2
O) spectrum of 6-FAM-MINPC, 7f2.
O O HO
CO
2
H
HN
HO
N
N
C
HO
P OH
O OH
O
OH
O
7f2
O O HO
CO
2
H
HN
HO
N
N
C
HO
P OH
O OH
O
OH
O
7f2
225
Figure A82. Mass spectrum of 6-FAM-MINPC, 7f2.
6-FAMMinPC
Measured Mass 718.1416
Element Low Limit High Limit
C 28 38
H 20 40
N0 5
O 11 16
P0 2
Formula Calculated Mass mDaError ppmError RDB
C36 H24 N5 O12 718.1416 0.0 0.0 27.5
C31 H31 N2 O16 P 718.1406 1.0 1.4 18
C35 H28 N O16 718.1403 1.3 1.9 22.5
C38 H26 N2 O13 718.1429 -1.3 -1.9 27
C34 H29 N3 O13 P 718.1433 -1.7 -2.3 22.5
C30 H32 N4 O13 P2 718.1436 -2.0 -2.7 18
C29 H29 N5 O15 P 718.1392 2.4 3.3 18.5
C33 H26 N4 O15 718.1389 2.7 3.7 23
C36 H31 O14 P 718.1446 -3.0 -4.2 22
C32 H34 N O14 P2 718.1449 -3.3 -4.6 17.5
499.0 619.2 739.4 859.6 979.8 1100.0
Ma ss (m/z)
0
1249.9
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r S p e c # 1 = > BC = > N F 0 . 7 = > B C = > N F 0 . 7 [ BP = 7 1 8 . 9 , 1 2 5 0 ]
McKenna (USC) 6-FAMMinPC MeOH/CHCA 719
718
565
638 566
636
756 564
720
639.0 705.6 772.2 838.8 905.4 972.0
Ma ss (m/z)
0
1.0E+ 4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Vo y a g e r Sp e c # 1 M C = > BC = > N F 0 . 7 [ BP = 7 1 9 . 1 , 1 0 1 7 3 ]
McKenna (USC) 6-Fammin in PC MeOH/CHCA/Mix1A
718.1416
904.4681
660.3469
Page 1
226
Figure A83. HPLC separation of 5-FAM-MINPC (7f1) and 6-FAM-MINPC (7f2).
Figure A84. UV-VIS, fluorescence emission spectra of compounds 7a1 – 7f2
UV Absorption of 5(6)-FAM-RIS (7a1)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 17.6 µM (λmax = 493 nm).
Recorded on DU 800 spectrometer. Extinction coefficient: 73,000M
-1
cm
-1
!
MINPC-Linker
6-FAM-MINPC
5-FAM-MINPC
FAM dye residues
0 - 8 min: 280 nm (UV-VIS detector)
8 -50 min: 598 nm (UV-VIS detector)
227
Emission Spectrum of 5(6)-FAM-RIS (7a1)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 1 µM. Recorded on Jobin Yvon
Horiba Fluoromax-3 fluorometer (λmax = 519 nm).
UV Absorption of 5(6)-RhR-RIS (7a4)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 8.1 µM. Recorded on DU 800
spectrometer. λmax = 567.5 nm. Extinction coefficient: 114,850M
-1
cm
-1
.
228
Emission Spectrum of 5(6)-RhR-RIS (7a4)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 1.05 µM. λmax = 589
nm.Recorded on Jobin Yvon Horiba Fluoromax-3 Fluorometer.
UV Absorption of ROX-RIS (7a5)
Sample in 0.1 M phosphate buffer, pH 8, with concentration at 5.48 µM. Recorded on DU 800
spectrometer. Extinction coefficient: 72,000M
-1
cm
-1
229
Emission Spectrum of ROX-RIS (7a5)
Sample in 0.1 M phosphate buffer, pH 8, with concentration at 1.37 µM. Recorded on photon
technology international quanta master model C-60SE spectrofluorimeter.
UV Absorption of AF647-RIS (7a6)
Sample in 0.1 M phosphate buffer, pH 7.0, with concentration at 3.6 µM. Recorded on DU 800
spectrometer. Extinction coefficient: 240,000M
-1
cm
-1
230
Emission Spectrum of AF647-RIS (7a6)
Sample in 0.1 M phosphate buffer, pH 7.0, with concentration at 0.9 µM. Recorded on Jobin
Yvon Horiba Fluoromax-3 Fluorometer.
UV Absorption of 5(6)-FAM-RISPC (7b1)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 16.7 µM. Recorded on DU 800
spectrometer. Extinction coefficient: 73,000M
-1
cm
-1
231
Emission Spectrum of 5(6)-FAMRISPC (7b1)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 8.39 µM. Recorded on Jobin
Yvon Horiba Fluoromax-3 Fluorometer.
UV Absorption of 5(6)-RhR-RISPC (7b2)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 9.16 µM. Recorded on DU 800
spectrometer. λmax = 568 nm. Extinction coefficient: 114,850M
-1
cm
-1
.
232
Emission Spectrum of 5(6)-RhR-RISPC (7b2)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 1.4 µM. λmax = 589
nm.Recorded on Jobin Yvon Horiba Fluoromax-3 Fluorometer.
UV Absorption of ROX-RISPC (7b3)
Sample in 0.1 M phosphate buffer, pH 8, with concentration at 10.4 µM. Recorded on DU 800
spectrometer. Extinction coefficient: 73,000M
-1
cm
-1
233
Emission Spectrum of ROX-RISPC (7b3)
Sample in 0.1 M phosphate buffer, pH 8, with concentration at 2.0 µM. Recorded on Jobin Yvon
Horiba Fluoromax-3 Fluorometer.
UV Absorption of AF647-RISPC (7b4)
Sample in 0.1 M phosphate buffer, pH 7.0, with concentration at 4.8 µM. Recorded on DU 800
spectrometer. Extinction coefficient: 240,000M
-1
cm
-1
234
Emission Spectrum of AF647-RISPC (7b4)
Sample in 0.1 M phosphate buffer, pH 7.0, with concentration at 1.2 µM. Recorded on Jobin
Yvon Horiba Fluoromax-3 Fluorometer.
UV Absorption of 5(6)-RhR-dRIS (7c2)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 12.5 µM. Recorded on DU 800
spectrometer. λmax = 567.5 nm. Extinction coefficient: 114,850M
-1
cm
-1
.
235
Emission Spectrum of 5(6)-RhR-dRIS (7c2)
Sample in 0.1 M phosphate buffer, pH 7.5, with concentration at 3.1 µM. λmax = 589
nm.Recorded on Jobin Yvon Horiba Fluoromax-3 Fluorometer.
UV-VIS Spectrum of 5-FAM-ZOL (7d1)
In 0.1 M Phosphate buffer (pH 7.2), 22.6 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer
236
Fluorescent Emission Spectrum of 5-FAM-ZOL (7d1)
In 0.1 M Phosphate buffer (pH 7.2), 5.66 µM
Recorded on Jobin Yvon Fluorolog Spectrometer
UV-VIS Spectrum of 6-FAM-ZOL (7d2)
In 0.1 M Phosphate buffer (pH 7.2), 21.8 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer
237
Fluorescent Emission Spectrum of 6-FAM-ZOL (7d2)
In 0.1 M Phosphate buffer (pH 7.2), 5.45 µM
Recorded on Jobin Yvon Fluorolog Spectrometer
UV-VIS Spectrum of AF647-ZOL (7d3)
In 0.1 M Phosphate buffer (pH 7.0), 3.8 µM, ε= 240,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer
238
Fluorescent Emission Spectrum of AF647-ZOL (7d3)
In 0.1 M Phosphate buffer (pH 7.0), 0.95 µM
Recorded on Jobin Yvon Fluorolog Spectrometer
UV-VIS Spectrum of 800CW-ZOL (7d4)
In 1 х PBS (pH 7.4), 6.79 µM, ε= 240,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer
239
Fluorescent Emission Spectrum of 800CW-ZOL (7d4)
In 0.1 M Phosphate buffer (pH 7.0), 1.70 µM
Recorded on SHIMADZU spectrofluorophotometer RF-5301PC (corrected based on IRDye
800CW)
UV Absorption of Sulfo-Cy5-ZOL (7d5)
Sample in 1 х PBS, pH 7.4, with concentration at 3.39 µM (λmax = 644 nm). Recorded on DU
800 spectrometer. Extinction coefficient: 240000M
-1
cm
-1
0.00E+00
1.00E+02
2.00E+02
3.00E+02
4.00E+02
5.00E+02
6.00E+02
7.00E+02
732 782 832 882
Intensity
Wavelength (nm)
240
Emission Spectrum of Sulfo-Cy5-ZOL (7d5)
Sample in 0.1M phosphate buffer, pH 7.0, with concentration at 0.85 µM. Recorded on Jobin
Yvon Nanolog spectrofluorimeter (λmax = 663 nm).
UV-VIS Spectrum of 5-FAM-MIN (7e1)
In 0.1 M Phosphate buffer (pH 7.2), 13.8 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer (λ = 493 nm)
-0.1
1999999.9
3999999.9
5999999.9
7999999.9
9999999.9
12000000
14000000
16000000
18000000
600 650 700 750 800
Intensity
Wavelength (nm)
241
Fluorescent Emission Spectrum of 5-FAM-MIN (7e1)
In 0.1 M Phosphate buffer (pH 7.2), 3.45 µM
Recorded on PTI QuantaMaster model C-60SE Spectrometer equipped with a 928 PMT detector
(λem = 522 nm)
UV-VIS Spectrum of 6-FAM-MIN (7e2)
In 0.1 M Phosphate buffer (pH 7.2), 14.0 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer (λ = 493 nm)
242
Fluorescent Emission Spectrum of 6-FAM-MIN (7e2)
In 0.1 M Phosphate buffer (pH 7.2), 3.5 µM. Recorded on PTI QuantaMaster model C-60SE
Spectrometer equipped with a 928 PMT detector (λem = 518 nm)
UV-VIS Spectrum of 5-FAM-MINPC (7f1)
In 0.1 M Phosphate buffer (pH 7.2), 10.1 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer (λ = 493 nm)
243
Fluorescent Emission Spectrum of 5-FAM-MINPC (7f1)
In 0.1 M Phosphate buffer (pH 7.2), 2.53 µM. Recorded on PTI QuantaMaster model C-60SE
Spectrometer equipped with a 928 PMT detector (λem = 522 nm)
UV-VIS Spectrum of 6-FAM-MINPC (7f2)
In 0.1 M Phosphate buffer (pH 7.2), 15.1 µM, ε= 73,000 M
-1
cm
-1
Recorded on DU 800 Spectrophotometer (λ = 493 nm)
244
Fluorescent Emission Spectrum of 6-FAM-MINPC (7f2)
In 0.1 M Phosphate buffer (pH 7.2), 3.78 µM. Recorded on PTI QuantaMaster model C-60SE
Spectrometer equipped with a 928 PMT detector (λem = 517 nm)
245
APPENDIX B. Chapter 5 Supporting Data
Figure B1.
1
H NMR (400 MHz, D
2
O) spectrum of para-dRIS, 10.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
10
246
Figure B2.
31
P NMR (162 MHz, D
2
O) spectrum of para-dRIS, 10.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
10
247
Figure B3. MS(+) spectrum of para-dRIS, 10.
248
Figure B4.
1
H NMR (400 MHz, CDCl
3
) spectrum of para-dRIS-linker-OH, 12.
Figure B5.
31
P NMR (162 MHz, CDCl
3
) spectrum of para-dRIS-linker-OH, 12.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OH
12
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OH
12
249
Figure B6. MS(+) spectrum of para-dRIS-linker-OH, 12.
250
Figure B7a.
31
P NMR (162 MHz, CDCl
3
) spectrum of intermediate 13.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
OMs
13
251
Figure B7b.
MS(+) spectrum of intermediate 13.
252
Figure B8.
31
P NMR (162 MHz, CDCl
3
) monitoring of reaction intermediate 13→14.
Figure B9.
1
H NMR (400 MHz, CDCl
3
) spectrum of para-dRIS-linker-N3-ester, 14.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
N
3
14
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
10
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
N
3
14
253
Figure B10.
31
P NMR (162 MHz, CDCl
3
) spectrum of para-dRIS-linker-N3-ester, 14.
N
P
P
O O-iPr
O-iPr
O
O-iPr
O-iPr
NHBoc
N
3
14
254
Figure B11. MS spectrum of para-dRIS-linker-N3-ester, 14.
255
Figure B12a.
1
H NMR (500 MHz, D
2
O) spectrum of amino-azido-para-dRIS, 7.
N
P
P
O OH
OH
O
OH
OH
NH
2
N
3
7
256
Figure B12b. gCOSY spectrum of amino-azido-para-dRIS, 7.
257
Figure B13.
31
P NMR (203 MHz, D
2
O) spectrum of amino-azido-para-dRIS, 7.
N
P
P
O OH
OH
O
OH
OH
NH
2
N
3
7
258
Figure B14a. MS(+) spectrum of amino-azido-para-dRIS, 7.
259
Figure B14b. MS(-) spectrum of amino-azido-para-dRIS, 7.
260
Figure B15.
1
H NMR (500 MHz, Methanol-d
4
) spectrum of 5(6)-FAM-alkyne, 23.
O O HO
COOH
NH
O
23
261
Figure B16a. MS(+)spectrum of 5(6)-FAM-alkyne, 23.
262
Figure B16b. MS(-)spectrum of 5(6)-FAM-alkyne, 23.
263
Figure B17.
1
H NMR (400 MHz, D
2
O) spectrum of 6-FAM-triazole-para-dRIS, 24a.
Figure B18.
31
P NMR (162 MHz, D
2
O) spectrum of 6-FAM-triazole-para-dRIS, 24a.
O O HO
COOH
NH
N
N
N
H
2
N
N
P
P
OH
O HO
O
OH
OH
6-isomer
O
O O HO
COOH
NH
N
N
N
H
2
N
N
P
P
OH
O HO
O
OH
OH
6-isomer
O
264
Figure B19.
1
H NMR (400 MHz, D
2
O) spectrum of 5-FAM-triazole-para-dRIS, 24b.
Figure B20.
31
P NMR (162 MHz, D
2
O) spectrum of 5-FAM-triazole-para-dRIS, 24b.
O O HO
COOH
NH
N
N
N
H
2
N
N
P
P
OH
O HO
O
OH
OH
5-isomer
O
O O HO
COOH
NH
N
N
N
H
2
N
N
P
P
OH
O HO
O
OH
OH
5-isomer
O
265
Figure B21a. MS(+) spectrum of 5(6)-FAM-triazole-para-dRIS, 24.
266
Figure B21b. MS(-) spectrum of 5(6)-FAM-triazole-para-dRIS, 24.
267
Figure B22. UV-VIS, fluorescence emission spectra of compound 24
UV absorption of 6-FAM-triazole-para-dRIS (24a, 6-isomer)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 2.68 µM (λmax = 493
nm). Recorded on DU 800 spectrometer. Extinction coefficient: 73,000M
-1
cm
-1
Emission Spectrum of 6-FAM-triazole-para-dRIS (24a, 6-isomer)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 2.68 µM. Recorded on
Jobin Yvon Nanolog spectrofluorimeter (λmax = 518 nm).
268
UV Absorption of 5-FAM-triazole-para-dRIS (24b, 5-isomer)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 2.81 µM (λmax = 493
nm). Recorded on DU 800 spectrometer. Extinction coefficient: 73,000M
-1
cm
-1
Emission Spectrum of 5-FAM-triazole-para-dRIS (24b, 5-isomer)
Sample in 0.1 M phosphate buffer, pH 7.2, with concentration at 2.81 µM. Recorded on
Jobin Yvon Nanolog spectrofluorimeter (λmax = 522 nm).
Abstract (if available)
Abstract
Bisphosphonates (BPs) are therapeutic agents for treatment of bone disorders such as osteoporosis and Paget’s disease. Several nitrogen-containing bisphosphonates (N-BPs) and phosphonocarboxylate (PC) analogues also have potential as anti-cancer agents. However, details of the skeletal distribution, cellular uptake and mechanisms of these drugs remain to be elucidated, stimulating the creation of imaging probes which mimic some or all of their pharmacological properties. ❧ A fluorescent imaging "toolkit" with more than 20 probes derived from all three heterocyclic N-BP drugs (risedronate, zoledronate and minodronate) and related analogues have been successfully synthesized. A linking strategy with two routes was applied under exceedingly mild conditions and introduced a terminal amino group in drug-linker intermediates capable to conjugate with the commercially available activated ester of fluorescent dyes. All the fluorescent probes were prepared in good yield (50-77%) and high purity (> 95%), and are fully characterized by HPLC, UV-VIS and fluorescence emission spectroscopy, ¹H and ³¹P NMR and high-resolution MS. ❧ The "toolkit" contains a series of fluorescent probes ranging from visible to near infrared optical window and the probes generally retain substantial affinity for bone mineral reflecting the varying affinities of their parent drug components, depending on the structure of the conjugating fluorescent dye. In addition, we have obtained evidence that certain probes (e.g. the FAM- and ROX- conjugates) have anti-prenylation and anti-resorptive effects in vitro and in vivo, demonstrating that the probes could retain biological activities of the parent BP drugs. ❧ Due to their diverse spectroscopic and pharmacological properties, the fluorescent imaging probe "toolkit" has been successfully utilized in various biological researches including osteoclast imaging, drug distribution at bone skeleton and cellular level, mechanism studies of osteonecrosis of the jaw (ONJ), cancer imaging, et al., and several papers have been published based on the obtained results, demonstrating the versatility of the imaging probe "toolkit". ❧ The first example of a bifunctional amino/azido-containing N-heterocyclic bisphosphonate (amino-azido-para-dRIS, compound 7 in Chapter 5) has been synthesized
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Creator
Sun, Shuting
(author)
Core Title
Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Chemistry
Publication Date
10/13/2015
Defense Date
08/30/2013
Publisher
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(original),
University of Southern California. Libraries
(digital)
Tag
azido bisphosphonate,bisphosphonates,BPs,cancer imaging,click chemistry,cochlear imaging,drug distribution,fluorescent bisphosphonates,fluorescent imaging,imaging probes,minodronate,N-BPs,nitrogen-containing bisphosphonates,OAI-PMH Harvest,ONJ,osteonecrosis of the jaw,osteoporosis,otosclerosis,phosphonocarboxylate,risedronate,zoledronate
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McKenna, Charles E. (
committee chair
), Qin, Peter Z. (
committee member
), Shen, Wei-Chiang (
committee member
)
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shutings@usc.edu,shutingssun@gmail.com
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Tags
azido bisphosphonate
bisphosphonates
BPs
cancer imaging
click chemistry
cochlear imaging
drug distribution
fluorescent bisphosphonates
fluorescent imaging
imaging probes
minodronate
N-BPs
nitrogen-containing bisphosphonates
ONJ
osteonecrosis of the jaw
osteoporosis
otosclerosis
phosphonocarboxylate
risedronate
zoledronate