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Mechanism and synthesis of molecular building blocks in medicinal chemistry: aerobic azoline oxidation and ultrasound activated MRI contrast agents

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Mechanism and synthesis of molecular building blocks in medicinal chemistry: aerobic azoline oxidation and ultrasound activated MRI contrast agents

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MECHANISM AND SYNTHESIS OF MOLECULAR BUILDING BLOCKS IN
MEDICINAL CHEMISTRY: AEROBIC AZOLINE OXIDATION AND
ULTRASOUND ACTIVATED MRI CONTRAST AGENTS

by

Anna C. Dawsey

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

August 2013

Copyright 2013 Anna C. Dawsey

ii
Dedication

To Mom and Dad.

iii
Acknowledgements
I would like to thank my advisor, Professor Travis J. Williams for the knowledge
and guidance provided throughout my academic journey. He has taught me the
fundamental appreciation of the simplicity in the most complex of things and the
complexity in the most seemingly simple of things.
A special thank you to my qualifying exam and dissertation committee members,
Professors G. K. Surya Prakash; Kyung Jung; Barry Thompson and Dr. Andy Y. Chang
for their dedicated time and supportive discussions. Also, thanks to Prof. Hogen-Esch of
the University of Southern California, Dr. Andy Chang of Children’s Hospital Los
Angeles, Prof. Matthew Allen of Wayne State for collaboration and insightful
discussions. Indebted thanks to my undergraduate advisor and labmate, Prof. Charles
Beam and John D. Knight, Ph.D., without their guidance I would have followed a
different path. I can literally say I would not be where I am today without them.
Thank you to all past and present members of the Williams group for making the
lab an enjoyable and at times utterly entertaining environment for research: Brian Conley,
Ph.D., Megan Pennington-Boggio, Vincent Li, Xinping Wu, Zhiyao Lu, Jeff Celaje,
Xingyue Zhang, Kathryn Hathaway, Christina Ratto, Ana Victoria Flores, Brock
Malinoski, Denver Guess, Christine Epperson, and Emine Boz, Ph.D. Thanks to
Buddhima N. Siriwardena-Mahanama of Prof. Matt Allen’s lab for her diligent
collaborative effort.

iv
Thanks to the helpful staff of LHI and USC Department of Chemistry: Carole
Phillips, Jessy May, David Hunter, Dr. Robert Anizfeld, Michele Dea, Marie de la Torre,
Allan Kershaw, and Katie McKissick.
Thanks to my amazing friends: Abigail Joyce, Ciera Gerack, Alejandra Beier,
Jonathan Sommer, and Jessica Blankenship; without whom the experience would have
not been nearly as pleasant or sane.
Lastly, a sincere, heartfelt thank you to my loving and supportive family: mom,
dad, Richard, Tammy, Grandma Betty, Papa Reggie, Grandma Ona, Pa Cecil, Jason,
Heather, David, Elaina, Monnie and Al. You are the best family one could ever have, and
all of my accomplishments would not have been possible without your love and support.

v
Table of Contents
Dedication ii
Acknowledgements iii
List of Tables x
List of Figures xii
List of Schemes xiv
Preparative Procedures xvi
Abstract xxii
Chapter 1. Introduction. Aerobic oxidation of azolines to azoles and the applications
in medicinal chemistry. 1
1.1. Classical azoline oxidations and the challenges they present for medicinal scaffolds. 1
1.2. K
3
Fe(CN)
6
oxidation of azolines to azoles. 2
1.3. MnO
2
oxidation of azolines to azoles. 3
1.4. NiO
2
oxidation of azolines to azoles. 7
1.5. BrCCl
3
oxidation of azolines to azoles. 8
1.6. Cu(I)/Cu(II) oxidation of azolines to azoles and mechanistic details. 11
1.7. K
2
CO
3
oxidation of azolines to azoles. 13
1.8. Conclusions. 15
1.9. References. 15
Chapter 2. Synthesis and oxidation of azolines to azoles. 21
2.1. Introduction. Copper-catalyzed and copper-free, base-mediated aerobic oxidation of
azolines to azoles. 21

vi
2.2. Copper-catalyzed aerobic oxidation of azolines to azoles and optimization. 23
2.3. Copper-free, base-mediated aerobic oxidation of azolines to azoles and optimization.
26
2.4. Substrate scope. 27
2.5. Mechanistic evidence of catalytic and copper-free, base-mediated aerobic oxidation
of azoline to azole pathways. 31
2.6. Scalability of copper-catalyzed and copper-free, base-mediated aerobic oxidation of
azolines to azoles. 37
2.7. Conclusions. 37
2.8. References. 38
Chapter 3. Introduction. Magnetic resonance imaging contrast agents and
applications in medicinal chemistry. 39
3.1. MRI contrast agents and relaxation parameters. 39
3.2. “Smart” MRI contrast agents. 40
3.2.1. Activation of MRI contrast agents by small molecules and biomolecule
markers. 41
3.2.2. Activation of MRI contrast agents by metals. 47
3.2.3. Activation of MRI contrast agents by pH. 53
3.2.4. Activation of MRI contrast agents by light. 55
3.3. Externally activated MRI contrast agents. 57
3.4. References. 58

vii
Chapter 4. External activation of Magnetic Resonance Imaging contrast agents by
sonication. 62
4.1. Introduction. Magnetic Resonance Imaging and Nuclear Magnetic Resonance. 62
4.2. External activation and comparison of Gd–DOTA·Shell and Gd–DOTP systems. 65
4.3. Background and control experiments for Gd–DOTA·Shell system with urea and
sonication. 72
4.4. Phenolphthalein encapsulation by Shell-OH. 75
4.5. Conclusions. 76
4.6. References. 76
Chapter 5. A molar relaxivity experiment in a High School classroom. 78
5.1. Introduction. Magnetic Resonance Imaging: back to the basics. 78
5.2. MRI contrast agents. 79
5.3. Determination of relaxivity. 80
5.4. Drug efficacy vs. safety of MRI contrast agents. 85
5.5. Conclusions. 86
5.6. References. 86
Chapter 6. Polymer MRI contrast agents: structures, properties, and applications.
90
6.1. Introduction. Polymer MRI contrast agents. 90
6.2. Polymers and covalently bound MRI contrast agents. 91
6.3. Macromolecules and non-covalently bound MR contrast agents. 94

viii
6.4. Polyvinylphosphoric acid and Gd–DOTA as a potential non-covalently bound MRI
contrast agent. 97
6.4.1. Results: polyvinylphosphoric acid and Gd–DOTA. 97
6.5. References. 101
Chapter 7. Experimental and spectral data. 104
7.1. General procedures. 104
7.2. Chapter 2 experimental and spectral data. 105
7.2.1. Ligand screen. 105
7.2.2. Preparation of copper complexes. 105
7.2.3. Preparation of azoline precursors. 106
7.2.4. Preparation of azolines. 121
7.2.5. General procedure for catalytic oxidation. 133
7.2.6. General procedure for copper-free, base mediated oxidation. 134
7.2.7. Thaizoline to thiazole oxidation on 1000 mg scale. 156
7.2.8. Oxidation of thiazoline in the presence of H
2
18
O. 157
7.2.9. Oxidation of thiazoline with K
2
CO
3
. 158
7.3. Chapter 4 experimental data. 159
7.3.1. Preparation of small molecule contrast agents. 159
7.3.2. General procedure for assessment of the presence of free Gd
3+
. 163
7.3.3. Preparation of Gd-L·Shell and Gd-L·Shell with urea samples. 164
7.3.4. Construction of molar relaxivity curves. 165
7.3.5. Gd–DOTA and Gd–DOTP relaxivity response upon addition of shell. 167

ix
7.4. Chapter 5 experimental data. 168
7.4.1. Preparation of Gd
3+
complexes and standard solutions. 168
7.4.2. Molar relaxivity curves. 171
7.5. Chapter 6 experimental data. 172
7.6. References. 173
Appendix. 176

x
List of Tables
Table 1.1. Summary of selected K
3
Fe(CN)
6
thiazoline to thiazole oxidations. 3
Table 1.2. Summary of selected MnO
2
oxidations of thiazolines to thiazoles. 6
Table 1.3. Summary of selected NiO
2
oxidations of thiazolines to thiazoles. 8
Table 1.4. Summary of selected BrCCl
3
O
2
oxidations of thiazolines to thiazoles. 10
Table 2.1. Cost Analysis for the ynthesis of 1 gram of methyl 2-phenylthiazole-4-
carboxylate. 22
Table 2.2. Optimization of Cu(II)-catalyzed oxidation conditions. 24
Table 2.3. Ligand screen of copper catalyzed oxidation of thiazoline to thiazole. 25
Table 2.4. Aryl Thiazoline oxidation. 28
Table 2.5. Further examples of azoline oxidation. 30
Table 2.6. Scalability of the conversion of thiazoline to thiazole. 37
Table 3.1. T
1
(s) of cells at 9.4 T upon treatment of 50 mM of each compound and
incubated for 16 h. 43
Table 3.2. Longitudinal relaxivities of Free and Protein-Bound HaloTag Targeted
contrast agents 45
Table 3.3. Luminescence lifetime and water coordination number measurements of Nap-
DO3A. 52
Table 3.4. Relaxation time for aqueous CLADIO-NH-SP in the dark and with visible
light. 57
Table 4.1. Decrease in
19
F T
1
(ms) when Gd – DOTA is added to shell. 68

xi
Table 4.2. Observation of Gd–DOTA–Shell–Urea system with sonication in various
biological buffers. 74
Table 5.1. Measured T
1
values for samples prepared at Polytechnic School. 84
Table 6.1. Summary of properties of TTDA-BOM and TTDA-N’-BOM and interaction
with HSA. 95
Table 6.2. Summary of r
1
and k
ex
data for MS-325 isomers A and B at 37 ºC and 35 ºC.
96
Table 6.3. Summary of T
1
data for Gd–DOTA·PVPA·Shell system at 25 ºC. 98
Table 6.4. Summary of T
1
data for Gd–DOTA·PVPA·Shell system after heating at 70 ºC
overnight. 98
Table 6.5. Change in
19
F T
1
(ms) when PVPA is added to shell. 100
Table 7.1. Scalability of coversion of thiazoline to thiazole. 159

xii

List of Figures
Figure 1.1. Examples of thiazole containing bioactive compounds. 2
Figure 2.1. MALDI spectra of angular hydroxide thiazoline with reaction ran in H
2
16
O.
33
Figure 2.2. MALDI spectra of angular hydroxide thiazoline with reaction ran in H
2
18
O.
34
Figure 2.3.
1
H NMR spectra of intermediates in potassium carbonate-mediated oxidation.
36
Figure 3.1. Structure of select MRI contrast agents Gd–DTPA, Gd–DOTA, and Gd–
DOTP. 40
Firgure 3.2. T
2
-weighted MR image of samples demonstrating effect of increasing
adenosine. 44
Figure 3.3. Design of the modified ferritin (Fer-PPD) structure and illustration of the
assembly. 46
Firgue 3.4. Structure of Zn
2+
MRI contrast agents Gd–daa3 and Gd–apa2. 49
Figure 4.1. Varian’s “T1 Measure” Inversion Recovery Pulse Sequence. 62
Figure 4.2. Small molecule contrast agents Gd–DOTA and Gd–DOTP. 64
Figure 4.3. Guanidine-terminated fluorous amphiphilic shell molecule. 65
Figure 4.4. Plot of r
1
values for aqueous solution containing Gd–DOTA·Shellwith urea
and sonication. 66

xiii
Figure 4.5. Plot of r1 values for aqueous solution containing Gd–DOTP·Shell with urea
and sonication. 67
Figure 4.6.
19
F spectra of shell and Gd–DOTA·Shell (1:2). 69
Figure 4.7. MALDI mass spectra of [Gd(DOTA)Shell] and [Gd(DOTA)Shell
2
]. 70
Figure 4.8. Resulting r
1
values of aqueous solutions containing Gd–DOTA titration with
shell. 71
Figure 4.9. Resulting r
1
values of aqueous solutions containing Gd–DOTP titration with
shell. 71
Figure 4.10. T
1
of aqueous solutions containing Gd–DOTA upon addition of urea. 72
Figure 4.11. T
1
of aqueous solutions containing Gd–DOTA–Shell upon addition of urea.
73
Figure 4.12. Structure of Shell–OH. 75
Figure 5.1. Structures of the active agents in Dotarem and Magnevist. 81
Figure 5.2. Varian’s “T
1
Measure” inversion recovery pulse sequence. 82
Figure 5.3. Molar relaxivity curves of [Gd(DOTA)]
-
and [Gd(DTPA)]
2-
. 83
Figure 6.1. Structural representations of block, graft, dendritic, and micellar mCAs. 91
Figure 6.2. Stucture of PF-Gd. 92
Figure 7.1. Molar relaxivity curves for a) Gd–DOTA, b) Gd–DOTAShell, c) Gd–
DOTAShell with urea, and d) Gd–DOTAShell with urea and sonication. 168

xiv
List of Schemes
Scheme 1.1. Methanolysis of thiopeptide antibitotic sulfomycin to produce dimethyl
sulfomycinamate. 4
Scheme 1.2. Proposed ionic oxidation mechanism with Cu(I)/Cu(II). 11
Scheme 1.3. General Kharasch-Sosnovsky oxidation mechanism. 12
Scheme 1.4. Proposed radical oxidation mechanism with Cu(I) and Cu(II). 12
Scheme 1.5. Proposed radical oxidation mechanism with Cu(I), Cu(II), and Cu(III). 13
Scheme 1.6. Proposed mechanism with intermediate peroxy thiazoline. 14
Scheme 2.1. Oxidation of thiazoline to thiazole via catalytic-aerobic or base-mediated
oxidation. 21
Scheme 2.2.Synthesis and Molecular Structure of copper catalysts. 23
Scheme 2.3. Oxidation mechanism; comparison of DBU and K
2
CO
3
pathways. 31
Scheme 3.1. Structure of galactopyranose attached to Gd – DOTA and removal by b-
galactosidase. 42
Scheme 3.2. Conformational dependence of the DOPTA-Gd structure in the
presence/absence of Ca
2+
. 48
Scheme 3.3. Zn
2+
responsive bimodal MRI (GdL) and fluorescent probe ((GdL)2L). 52
Scheme 3.4. Doxorubicin-Gd complex designed to release doxorubicin when exposed to
low pH. 544
Scheme 3.5. Proposed isomerization of Gd-SPDO3A and Gd-MCDO3A. 566

xv
Scheme 4.1. T
1
inversion recovery measurements after addition of shell, urea, and
application of sonication to the Gd–DOTA–Shell–Urea system. 65
Scheme 6.1. Gd–DOTA–PVPA and Gd–PVPA xylenol orange complexometric indicator
results. 96

xvi
Preparative Procedures
Fmoc-Cys(trt)-OMe 106
N
H
STrt
OMe
O
Fmoc

7.1
Methyl 2-(4-Fluorobenzamido)-3-(tritylthio)propanoate 108
N
H
O
STrt
OMe
O
F

7.2
Methyl 2-(4-Cyanobenzamido)-3-(tritylthio)propanoate 110
N
H
O
STrt
OMe
O
NC

7.3
Methyl 2-(Indole-2-carboxamido)-3-(tritylthio)propanoate 112
O
OMe
H
N
STrt
O
NH

7.4
Methyl 2-(1-Methylindole-2-carboxamido)-3-(tritylthio)propanoate 115
O
OMe
H
N
STrt
O
N

7.5

xvii

Methyl 2-(2-Naphthamido)-3-(tritylthio)propanoate 118
N
H
O
STrt
OMe
O

7.6
2-Phenyl-4,5-dihydrothiazole-4-carboxylic acid 122
N
S
O
OH

7.7
Methyl 2-Phenyl-4,5-dihydrothiazole-4-carboxylate 122
N
S
OCH
3
O

2.2
Methyl 2-(Naphthalen-2-yl)-4,5-dihydrothiazole-4-carboxylate 123
N
S
OCH
3
O

2.5
Methyl 2-(4-Fluorophenyl)thiazole-4-carboxylate 125
N
S
OCH
3
O
F

2.6
Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate 127
N
S
OCH
3
O
C
N

2.7

xviii
Methyl 2-(Indol-2-yl)-4,5-dihydrothiazole-4-carboxylate 129
N
S
OCH
3
O
NH

2.12
Methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-carboxylate 131
N
S
OCH
3
O
N
CH
3

2.13
Methyl 2-Phenylthiazole-4-carboxylate 135
N
S OCH
3
O

Methyl 2-(4-Nitrophenyl)thiazole-4-carboxylate 136
N
S
OCH
3
O
O
2
N

2.3a
Methyl 2-(4-Methoxyphenyl)thiazole-4-carboxylate 138
N
S
O
OCH
3
H
3
CO

2.4a
Methyl 2-(Napthalen-2-yl)thiazole-4-carboxylate 140
N
S
OCH
3
O

2.5a
Methyl 2-(4-Fluorophenyl)thiazole-4-carboxylate 142
N
S
OCH
3
O
F

2.6a

xix

Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate 144
N
S
OCH
3
O
C
N

2.7a
Methyl 2-Phenyloxazole-4-carboxylate 146
N
O
OCH
3
O

2.8a
Methyl 2-(4-Nitrophenyl)oxazole-4-carboxylate 147
N
O
OCH
3
O
O
2
N

2.9a
Methyl 2-Methylthiazole-4-carboxylate 148
N
S
OCH
3
O

2.10a
Methyl 2-Phenethylthiazole-4-carboxylate 149
N
S
OCH
3
O

2.11a
Methyl 2-(Indol-2-yl)thiazole-4-carboxylate
151
N
S
OCH
3
O
NH

2.12a

xx

Methyl 2-(1-Methyl-indol-2-yl)thiazole-4-carboxylate 153
N
S
OCH
3
O
N
CH
3

2.13a
Methyl 4-hydroxy-2-phenyl-4,5-dihydrothiazole-4-carboxylate 155
N
S
OCH
3
O
OH

2.15
[(DOTA)Gd]
-
Na
+
159
N
N N
N
Gd
O
O
O
O
O
O
O
O
Gd-DOTA
Na

4.1
[Gd(DOTP)]
5-
5Na
+
160
N N
N N
Gd
P
O
P
O
O
O
P
O
O
P
O
O
O
O
O
O
5
5 Na

4.2

xxi

[CaCl]
+
[Gd(DOTA)]
-
· 2.5 H
2
O 167
N
N N
N
Gd
O
O
O
O
O
O
O
O
CaCl

5.1
2[H]
+
[Gd(DTPA)]
2-
· 2 H
2
O 169
N
N N Gd
O
O
O
O
O
O
O
O
O
O
2 2H

5.2

xxii
Abstract

Research laid out in this work describes the development of chemical mechanistic
insight and synthesis of molecular building blocks in medicinal and diagnostic fields. The
aerobic oxidation of azolines to azoles is of utmost significance in medicinal chemistry.
The azole core is a ubiquitous structural component in biologically active natural
products. Therefore, the necessity to efficiently and inexpensively synthesize these azole
targets is of interest to chemists and clinicians alike. This work describes a copper-
catalyzed aerobic oxidation of azolines to azoles that is high yielding and cost efficient.
Along with copper-catalyzed conditions, a second set of copper-free,
stoichiometric base-mediated conditions were developed for the entire substrate scope of
the azoline to azole transformation. Both catalytic and stoichiometric base-mediated
conditions demonstrate good yields with a substrate scope of thiazolines with aryl
substituents in the 2-position with a range of electron withdrawing and electron donating
groups. Catalytic conditions proved necessary for the transformation in the presence of
labile protons such as the N-H proton of indole. The oxidation of azolines to azoles with
both conditions were scaled to 1 g without a significant change in yield.
Additionally, this work describes the development and characterization of the first
ultrasound activated MRI contrast agent. The premise of an activatable MRI contrast
agent can be applied to many different therapeutic and diagnostics systems. A two-
component system has been developed in which contrast from a MRI contrast agent, Gd–
DOTA, is masked by a proprietary shell formulated to be water impenetrable yet water-

xxiii
soluble. The hydrogen bonding interaction holding the shell to the contrast agent prevents
water exchange with the paramagnetic gadolinium core, thus attenuating contrast.
At the desired time and location, the contrast agent-shell interaction can be
selectively disrupted externally with sonication to reveal the contrast.

1
Chapter 1. Introduction. Aerobic oxidation of azolines to azoles
and the applications in medicinal chemistry.

1.1. Classical azoline oxidations and the challenges they present for
medicinal scaffolds.
Azoles are ubiquitous structural components in biologically active natural
products with important medicinal properties that include anticancer
1
and antibiotic
activity
2
(Figure 1.1). Despite their importance, there were no previous catalytic
conditions for the synthesis of azoles by azoline oxidation.
3
Various conditions, which
involve either a toxic waste stream or a stoichiometric amount of reagent, affect
thiazoline oxidation. Such reagents include K
3
Fe(CN)
6
,
4
NiO
2
,
5
Cu(I)/Cu(II),
6
BrCCl
3
,
7

and MnO
2
.
8
In each of these cases, the stoichiometric metal or halogen waste stream
introduces disposal cost and environmental impact when these reactions are practiced at
production scale. Focusing primarily on thiazoline to thiazole oxidation, this chapter will
give a brief overview of classical and current azoline to azole transformations.
Recently, aerobic conditions for thiazoline oxidation based on DMF solutions of
stoichiometric potassium carbonate have recently been reported. These are efficient for
aerobic oxidation of many electron poor azolines.
9
Importantly, attempts to oxidize
electron rich azolines and azolines containing labile protons under base promoted
conditions are susceptible to production of undesired side products resulting in a
significant decrease in yield. Further, these reactions appear to proceed through the

2
intermediacy of a long-lived organic peroxide radical, which could be problematic if
these conditions were to be scaled up to production scale.
O
O OH O
HO
N
S
O
OH HO
HO
S
N
O
NH
2
Epothilone B Riboxamide (tiazofurin)
H
2
N O
O O
N
S
S
N
Myxothiazol
O
4
2
2
4
4
2
2
4

Figure 1.1. Examples of thiazole containing bioactive compounds.
1.2. K
3
Fe(CN)
6
oxidation of azolines to azoles.
Prior to Walker et al. in 1968, the only other reported examples of intentional
dehydrogenation of thiazolines to thiazoles we those of Schroder et al.
10
and White et
al.
11
Schroder effected high yielding dehydrogenation of alkyl substituted thiazolines by
heating with sulfur with reports of less effective dehydrogenations of thiazolines with
K
3
Fe(CN)
6
, K
2
Cr
2
O
7
, and H
2
O
2
.
10
White et al. reported qualitative evidence (chromato-
graphic identification) of dehydrogenation of the thiazoline moiety in firefly luciferin
with atmospheric oxygen and hot alkaline solution.
12
In 1968, Walker et al. used
stoichiometric amounts of K
3
Fe(CN)
6
to transform three 2-thiazoline-4carboxylic acids
to the corresponding thiazole-4-carboxylic acids. Reaction yields ranged
from 3-82% (Table 1.1).
13

3
Table 1.1. Summary of selected K
3
Fe(CN)
6
thiazoline to thiazole oxidations.
Entry Azoline (a) Oxidation
Conditions
Azole (b) Yield (%)
Walker (1968)
13

1
S
N
O
OH

K
3
Fe(CN)
6
NaOH
RT
7 h
S
N
O
OH

42
Impure
2
S
N
O
O
O
HN

K
3
Fe(CN)
6
NaOH
RT
1.5 h
S
N
O
O
O
HN

3
3
S
N
O
O

K
3
Fe(CN)
6
NaOH
reflux - RT
1 h
S
N
O
O

82
1.3. MnO
2
oxidation of azolines to azoles.
MnO
2
was used to oxidize isoxazolines to isoxazoles in 1960 by D'Alcontres et
al.
14
Both of these cases of oxidation involved phenyl groups at the 5 position of the
heterocycle. 4,5-dihydro-1,2-oxazoles were converted to the corresponding azoles in
> 97% yield with activated MnO
2
by Barco et al. in 1977.
15
Note that the 1,2 oxazole
formation is non-trivially different from the 1,3-oxazole formation. MnO
2
was employed
successfully to transform the thiazoline precursor to the thiazole moiety (65%) in the
Hecht synthesis of the antibiotic Bleomycin (Table 1.2, entry 1-2), which was under
investigation for anticancer activity.
16
Subsequently, NiO
2
was used to perform this
transformation with a significantly higher yield of 93% (Table 1.3 entry 1).
17

4
Not surprisingly in an effort to oxidize thiazolidines to thiazolines, MnO
2
was
used by Dunach et al. in 2001, which produced thiazoles as over oxidized side
products.
18,19
The oxazole-thiazole-pyridine product generated from the methanolysis of
sulfomycin (1.1) (thiopeptide antibiotic) by Bagley et al., dimethyl sulfomycinamate
(1.2) (Scheme 1.1), was produced with a key step being the oxidation of a thiazoline
precursor to a thiazole product via use of microwave assisted MnO
2
oxidation in CH
2
Cl
2

at 100 ºC (Table 1.2, entry 3).
20
Kelly et al. efficiently used MnO
2
in the key oxidation of
a thiazoline to thiazole (Table 1.2, entry 4) in 91% yield ultimately leading to the
synthesis of cytotoxic macrolide, didmolamide A.
21

NH
2
HN
NH
HN
O
O
O
O
N
N
S
O
N
O
N
H
HO
O
H
N
R
O N
O
HN
MeO
S
N
O
N
H
O
N NH
O
HN
O
R = CH(OH)Me
MeOH, H
+
O
N
N
S
O
N
O
CH
3
O
MeO
MeO
1.1
1.2

Scheme 1.1. Methanolysis of thiopeptide antibitotic sulfomycin (1.1) to produce
dimethyl sulfomycinamate (1.2).

5
Tiazofurin (Figure 1.1), a c-nucleoside active against various cancers, was
synthesized by Ramasamy et al. in 2000. The Δ
2
-thiazoline intermediate was oxidized
using MnO
2
to afford the key thiazole moiety intermediate in 88% yield (Table 1.2, entry
5).
22
However, in 2002 Dowden et al. developed a more concise route to Tiazofurin that
circumvented the need for protection with isopropylidene. Instead, benzoyl protected
esters allowed for clean conversion of thiazoline to thiazole with bromotrichloromethane
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Table 1.4, entry 4). Tiazofurin was
produced in unoptimized 61% yield in three steps from commercially available starting
materials.
23

In 2009, the Fu group expanded the substrate scope of MnO
2
oxidation of
thiazolines to thiazoles to include non-electron withdrawing and electron donating groups
in the 4 position of the thiazoline. This process required an excess of MnO
2
(10 equiv.).
Also, this reaction was extremely solvent and temperature dependent. Little to no yield
was achieved in DCM at room temperature over a period of 48 h. In DCE, CH
3
CN, and
benzene the reaction proceeded in up to > 95% yield within 12 h. The substrate scope
also included the versatile bis-thiazoline to bis-thiazole conversion in 70 – 80% yields
(Table 1.2, entry 6).
24
At this point, the oxidation of thiazolines to thiazoles was still
largely inconsistent and not universally applicable to a wide substrate scope.

6
Table 1.2. Summary of selected MnO
2
oxidations of thiazolines to thiazoles.
Entry Azoline (a)
Oxidation
Conditions
Azole (b) Yield (%)
Hecht (1977)
16

1
S
N
O
O
NH
O
H
3
C

MnO
2

CHCl
3

RT
4 d
S
N
O
O
NH
O
H
3
C

65
2
S
N
O
O
NH
O

MnO
2

CHCl
3

RT
4 d
S
N
O
O
NH
O

--
Bagley (2003)
20

3
N
S
N
N
O
O
O
CO
2
Me

MnO
2

µwave
100 ºC
CH
2
Cl
2

N
S
N
N
O
O
O
CO
2
Me

79
Kelly (2005)
21

4
N
S
FmocHN
OAllyl
O

activated
MnO
2
N
S
FmocHN
OAllyl
O

97
Ramasamy (2000)
22

5
S
N
O
O
O
BzO
O
O

MnO
2

Benzene
Reflux
24 h
S
N
O
O
O
BzO
O
O

88
Fu (2009)
24

6
S
N
S
N
N

MnO
2

DCE
Reflux
S
N
S
N
N

85

7
1.4. NiO
2
oxidation of azolines to azoles.
Although NiO
2
is a sufficient oxidant in many known organic reactions,

dehydrogenations of heterocyclic compounds using NiO
2
25

were only briefly reported.
26

in the late 1960s early 1970s. Hecht et al. investigated the oxidation of the heterocyclic
core of precursors for several potent antibiotics and anticancer compounds in 1978. NiO
2
was employed for the successful oxidation of several heterocycles, namely three
thazoline to thiazole conversions (60 – 93 % yield, 4h – 3 d) (Table 1.3, entry 1-3).
17

In 1979, Meyers et al. reported the oxidation of O-, S-, and N- containing
heterocycles by the use of NiO
2
.
27
The unprecedented oxidation of eight substituted 4,5-
dihydro-1,3-oxazolines to corresponding oxazoles was the highlight of this publication.
Thiazoline to thiazole oxidation was accomplished with NiO
2
with yields ranging from
43 – 93% and reaction times ranging from 4 h – 3 d depending on the temperature (25 ºC
– reflux) and solvent conditions (Table 1.3, entries 4-5).
28
Several thiazolines of
moderate complexity and functional diversity (Table 1.3, entries 1-5) were oxidized with
moderate yields rendering the NiO
2
method the most versatile and effective thiazoline
oxidation method to date at the time.
29

8
Table 1.3. Summary of selected NiO
2
oxidations of thiazolines to thiazoles.
Entry Azoline (a)
Oxidation
Conditions
Azole (b) Yield (%)
Hecht (1978)
17

1
S
N
O
O
NH
O
H
3
C

NiO
2

(2.4 equiv)
CHCl
3

3 d
RT
S
N
O
O
NH
O
H
3
C

93
a
2
S
N
O
O

NiO
2

(2.0 equiv)
CHCl
3
3 d
RT
S
N
O
O

81
3
N
S
S

NiO
2

(3.7 equiv)
C
6
H
6

4 h
Reflux
N
S
S

60
Meyers (1979)
28

4
N
S
HS

NiO
2
CHCl
3
12 h
25 ºC
N
S
HS

88
5
S
N
O
O
HN
O

NiO
2
CHCl
3
3 d
25 ºC
S
N
O
O
HN
O

93
b

a
65 % yield with MnO
2
.
b
69 % yield with MnO
2
.
1.5. BrCCl
3
oxidation of azolines to azoles.
Use of bromotrichloromethane (BrCCl
3
) for heterocycle oxidation was successful in
the Williams synthesis of funiculosin in 1997 when other oxidation methods such as
MnO
2
had failed to perform the transformation. BrCCl
3
(1.05 equiv.) with 1,5-

9
diazabicyclo[5.4.0]undecane (DBU) (2 equiv.) was used at 0 ºC in methylene chloride
for the mild dehydrogenations of the 5,6-dihydro pyridinone precursor to the 4-hydroxy-
2-pyridinone intermediate in a 78% yield. The substrate scope was expanded to include
several important oxazole and thiazole moieties with yields from 75 – 95% (Table 1.4,
entries 1-3).
30

Toward the syntheses of new thiazole analogues of pyochelin, siderophore of
Pseudomonas aeruginosa and Burkholderia cepacia, BrCCl
3
was employed by Mislin et
al. for the successful transformation of a Weinreb amide thiazoline precursor to the
thiazole intermediate in 87% yield (Table 1.4, entry 5). New oxidation conditions were
reported in this paper for the transformation in which NaH in dry MeOH at 20 ºC
produced 70% yield of not the Weinreb but the amide thiazole product. These NaH
conditions proved most useful in substrates with strongly withdrawing groups on the
adjacent phenyl system.
31

The Kelly group made progress toward oxidation of oxazolines to oxazoles in 2004
with BrCCl
3
and DBU in DCM with 34 – 70% yields over two, cyclization/oxidation,
steps. All but one of these substrates included a substituent other than hydrogen at the C5
position. Also, it is worth noting that these oxazoles were directly converted to the
carboxylic acid moieties at the C4 position. It was demonstrated that several compounds
in this series inhibit Transthyretin (TTR) amyloidogenesis in vitro and two bind TTR in
human plasma with some selectivity.
32

10
Table 1.4. Summary of selected BrCCl
3
oxidations of thiazolines to thiazoles.
Entry Azoline (a)
Oxidation
Conditions
Azole (b) Yield (%)
Williams (1997)
1
S
N
O
O

BrCCl
3

(1.05 equiv.)
DBU
(1.05-2 equiv.)
CH
2
Cl
2
S
N
O
O

95
2
S
N
O
O
Ph
O
N

BrCCl
3

(1.05 equiv.)
DBU
(1.05-2 equiv.)
CH
2
Cl
2

S
N
O
O
Ph
O
N

92
3
S
N
O
O

BrCCl
3

(1.05 equiv.)
DBU
(1.05-2 equiv.)
CH
2
Cl
2

S
N
O
O

95
Dowden (2002)
4
S
N
O
O
O
BzO
O
O

BrCCl
3
DBU
CH
2
Cl
2

0 ºC
S
N
O
O
O
BzO
O
O

63%
Over 3
Steps
Mislin (2004)
5
S
N
N
O
O
HO

BrCCl
3
DBU
CH
2
Cl
2

20 ºC
S
N
N
O
O
HO

87

11
1.6. Cu(I)/Cu(II) oxidation of azolines to azoles and mechanistic details.
The use of CuBr (2 equiv.) and DBU (2 equiv.) to oxidize oxazolines to oxazoles
was initiated by Barrish et al in 1993, who proposed an ionic mechanistic pathway
(Scheme 1.2).
33
Simultaneously, Meyer and Tavares were employing a radical oxidation
for the oxidation of 2-oxazolines to 1,3-oxazoles
34
and thiazoline to thiazoles.
35
N-
Bromosuccinimide (NBS)/AIBN and NBS/peroxide or light were initially used for the
oxidation of oxazolines.

However, NBS/AIBN required an additional step to halogen
metal exchange the 5-bromo-1,3-oxazoles, which detracted from the efficiency of the
method. Attempts to avoid the bromo- intermediate with use of a peroxide or light radical
initiator produced three oxazoles (62-83% yield) with three sets of reaction conditions.
N
O
R
Y
O
CuBr
-HBr
N
O
R
Y
O
Cu
L
Br
H
B:
Br Cu Br -HBr
N
O
R
Y
O
Y = NH(CH
2
)
4
CH
2

Scheme 1.2. Proposed ionic oxidation mechanism with Cu(I)/Cu(II) by Barrish et al.
33

Previously in 1959, Kharasch and Sosnovsky developed conditions for allylic
oxidation in which t-butyl peresters reacted smoothly with olefins in the presence of
copper or cobalt salt catalysts to produce allylic esters.
36
The Karasch-Sosnovsky
conditions typically involve the decomposition of a peroxyester with a copper catalyst
with subsequent reaction with a C-H substrate to generate a carbon-centered radical. The
radical is then oxidized and trapped by carboxylate anion (Scheme 1.3).

12
+ t-BuOOCOPh
Cu
+
Cu
2+
R
R
O
O
Ph
t-BuO
O
O
Ph
R
Cu
2+
R
Cu
+
+

Scheme 1.3. General Kharasch-Sosnovsky oxidation mechanism.
These Kharasch-Sosnovsky conditions
37
were utilized by Meyers and Tavares’ for
the oxidation of oxazolines to oxazoles with success using CuBr (1-1.1 equiv.) and t-
butyl perbenzoate (1.5 equiv.) in benzene at reflux (4.5 – 12 h, 55 – 83% yield). Meyers
and Tavares’ proposed a radical mechanism for the oxidation of oxazolines to oxazoles
via CuBr and t-butyl perbenzoate (Scheme 1.4).
34
Further studies by Meyers and Tavares
were done utilizing CuBr/peroxide toward the oxidation of oxazolines and thiazolines to
oxazole and thiazoles, which produced oxazoles and thiazoles containing amino groups
and stereocenters at the 2-(α) position without racemization during aromatization.
35
N
O
R
CO
2
Me
N
O
R
CO
2
Me
N
O
R
CH
2
O
2
Me
N
O
R
CO
2
Me
N
O
R
CO
2
Me
N
O
R
CO
2
Me t-BuOOCOPh
t-BuO
t-BuO
Cu
+
Cu
2+
Cu
2+
O
O
Ph
H
A
B

Scheme 1.4. Proposed radical oxidation mechanism with Cu(I) and Cu(II) by Meyer and
Tavares.
35

13
Later, Meyers and Tavares modified the previous Kharasch-Sosnovsky conditions
to include both Cu(I) and Cu(II) salts to promote the oxidation of thiazolines and
oxazolines to chiral and achiral oxazoles and thiazoles.
38
Cu(I) catalyzes decomposition
of the peroxyester resulting in the alkoxy radical. Cu(II) is proposed to enhance the rate
of oxidation and/or ligand transfer between the presumed radical (1.3) and the Cu(II)
salts. The proposed mechanism (Scheme 1.5) involves a Cu(III) species (1.4) formed via
oxidative addition, which is subsequently reductively eliminated to the acyloxy azoline
(1.5). Upon heating, syn elimination occurs to provide azole (1.6). This mechanism was
supported by the isolation of the acyloxy azoline intermediate (1.5).
N
X
R
CO
2
Me
N
X
R
CO
2
Me
N
X
R
CO
2
Me
N
X
R
CO
2
Me
Cu(OCCH
3
)
2
O
Cu
OCOR
OCOCH
3
-CuOAc
-PhCOOH
H
O
Ph
O
X = O or S
R = alkyl or aryl
1.3 1.4 1.5
1.6

Scheme 1.5. Proposed radical oxidation mechanism with Cu(I), Cu(II), and Cu(III) by
Meyer and Tavares.
38

1.7. K
2
CO
3
oxidation of azolines to azoles.
Simultaneously to our aerobic oxidation of azolines to azoles efforts
39
in 2010,
Yao et al. developed a facile, environmentally benign set of oxidation conditions with
K
2
CO
3
(3 equiv.), molecular sieves (4 Å, 200 weight %), and molecular oxygen

14
(atmosphere or O
2
) as the sole oxidant in dry DMF at 120 ºC to convert 4-carboxylate
thiazolines to 4-carboxylate thiazoles. A mechanism is proposed involving a peroxy
group at the C4 position of the thiazole (Scheme 1.6). These conditions are most effective
on thiazolines bearing electron-withdrawing groups at C2 and C4. These conditions will
be further discussed in Chapter 2.
9
Subsequently, in 2011 Yao et al. reported the
oxidation of 2-oxazoline-4-carboxamides and 2-oxazoline-4-carboxylates to the
corresponding 2-oxazole-4-carboxamides (6 – 8 h, 50 – 92% yield) and 2-oxazole-4-
carboxylates (5.5 – 8 h, 43 – 74% yield) by use of the same K
2
CO
3
mediated oxidation
conditions. These conditions also produced higher yields with more electron deficient
groups at the C2 position of the oxazoline ring. For the

2-oxazole-4-carboxylates,
temperatures had to be reduced to 100 ºC to prevent hydrolysis of the ester. Lastly, these
conditions were applied to the synthesis of the tri-substituted oxazole intermediate to
SC-ααδ9, a CDC25 phosphatase inhibitor, with success (14 h, 65%).
40

N
O
O
O
O
2
N
O
O
O
OOH
N
O
O
O
OH
N
O
O
O
K
2
CO
3
/DMF
Molecular Sieves
80
o
C, 91 %
1.7 1.8
1.9 1.10

Scheme 1.6. Proposed mechanism with intermediate peroxy thiazoline 1.8 by Yao et al.
9

15
1.8. Conclusions.
Various efficient azoline to azole oxidations have been developed using
K
3
Fe(CN)
6
, NiO
2
, Cu(I)/Cu(II),

BrCCl
3
,

and MnO
2
. However, in each of these cases,
halogen or metal disposal cost and environmental impact are introduced when these
reactions are practiced at production scale. Chapter 2 of this work details the first copper-
catalyzed aerobic oxidation of azolines to azoles; discussing in depth the optimization of
the catalyzed as well as base-mediated procedure for a vast substrate scope of thiazolines.
Additionally, thorough mechanistic investigation of the oxidation pathway and
intermediate will be detailed.
1.9. References.

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Additions of Cyclic α-carbonyl-azo-compounds. Bull. Chem. Soc. Jpn. 1970, 43,
3926-3927.
26) (a) Balachandran, K. S.; Bhatnagar, I.; George, M. V. Oxidation by Metal Oxides.
IV. Oxidation of Organic Compounds Using Nickel Peroxide. J. Org. Chem.
1968, 33, 3891-3895. (b) Takase, S.; Motoyama, T. Studies of Diels-Alder-type
Additions of Cyclic α-carbonyl-azo-compounds. Bull. Chem. Soc. Jpn. 1970, 43,
3926-3927.
27) (a) Evans, D. L.; Minster, D. K.; Jordis, U.; Hecht, S. M.; Mazzu, Jr. A. L.;
Meyers, A. I. Nickel Peroxide Dehydrogenation of Oxygen-, Sulfur-, and
Nitrogen – Containing Heterocycles. J. Org. Chem. 1979, 44, 497-501. (b)
Knight, D. W.; Pattenden, G.; Rippom, D. E. Synthesis of Tris-oxazole Ring
System of Ulapualides. Synlett. 1990, 1, 36-37. (c) Wipf, P.; Miller, C. P. A New
Synthesis of Highly Functionalized Oxazoles. J. Org. Chem. 1993, 58, 3604.

19

28) Knight, D. W.; Pattenden, G.; Rippom, D. E. Synthesis of Tris-oxazole Ring
System of Ulapualides. Synlett. 1990, 1, 36-37.

29) Wipf, P.; Miller, C. P. A New Synthesis of Highly Functionalized Oxazoles.
J. Org. Chem. 1993, 58, 3604.

30) (a) Williams, D. R.; Lowder, P. D.; Gu, Y. G.; Brooks, D. A. Studies of Mild
Dehydrogenations in Heterocyclic Systems. Tetrahedron Lett. 1997, 38, 331-334.

31) Mislin, G. L.; Burger, A.; Abdallah, M. A. Synthesis of New Thiazole Analogues
of Pyochelin, a Siderphore of Pseudomoas aeruginosa and Burkholderia cepacia.
A New Conversion of Thiazolines into Thiazoles. Tetrahedron 2004, 60, 12139-
12145.
32) Razavi, H.; Powers, E. T.; Purkey, H. E.; Adamski-Werner, S. L.; Chiang, K. P.;
Dendle, M. T. A.; Kelly, J. K. Design, Synthesis, and evaluation of Oxazole
Transthyretin Amyloidogenesis Inhibitors. Biorg. & Med. Chem. Lett. 2004, 15,
1075-1078.
33) Barrish, J. C.; Singh, J.; Spergel, W. –C. H.; Kissick, T. P.; Kronenthal, D. R.;
Mueller, R. H. Cupric Bromide Mediated Oxidation of 4-Carboxyoxazolines to
the Corresponding Oxazoles. J. Org. Chem. 1993, 58, 4494-4496.
34) Meyers, A. I.; Tavares, F. The Oxidation of 2-Oxazolines to 1,3-Oxazoles.
Tetrahedron Lett. 1994, 35, 2481.
35) Tavares, F.; Meyers, A. I. Further Studies on Oxazoline and Thiazoline
Oxidations. A Reliable Route to Chiral Oxazoles and Thiazoles. Tetrahedron Lett.
1994, 37, 6803-6806.

36) Khrasch, M. S.; Sosnovsky, G.; Yang, N. C. Reaction of t-Butyl Perestes. I. The
Reaction of Peresters with Olefins. J. Am. Chem. Soc. 1959, 81, 5819-5824.
37) Rawlinson, D. J.; Sosnovsky, G. One-step Substitutive Acyloxylation at Carbon.
I. Reactions Involving Peroxides. Synthesis 1973, 1, 1-28.
38) Tavares, F.; Meyers, A. I. Oxidation of Oxazolines and Thiazolines to Oxazoles
and Thiazoles. Application of the Kharasch-Sosnovsky Reaction. J. Org. Chem.
1996, 61, 8207-8215.
39) Dawsey, A. C.; Li, V.; Hamilton, K. C.; Wang, J.; Williams, T. J. Copper-
Catalyzed Aerobic Oxidation of Azolines to Azoles. Dalton Trans. 2012, 41,
7994-8002.

20

40) Huang, Y.; Ni, L.; Gan, H.; He, Y.; Xu, J.; Wu, X.; Yao, H. Environmental-
Benign Oxidation of 2-oxazolines to 2-oxazoles by Dioxygen as the Sole Oxidant.
Tetrahedron 2011, 67, 2066-2071.

21
Chapter
2.
Synthesis
and
oxidation
of
azolines
to
azoles.

2.1. Introduction. Copper-catalyzed and copper-free, base-mediated
aerobic oxidation of azolines to azoles.
We have developed conditions to transform azolines to azoles via two efficient
and economical aerobic oxidation routes. These reactions are applicable to a wide range
of substrates (from electron rich to electron poor), easy to use, involve little waste stream,
and are demonstrated on a reasonable laboratory scale. Stoichiometric base conditions
afford good yields in many cases, and copper-catalyzed conditions afford superior results
in most cases. This technology will be useful for building natural products and medicinal
entities containing one or more imbedded azole subunits, sensitive labile protons, and
electron rich species without the expense of stoichiometric metal oxidants.
10 mol% , 10 mol% DBU
DMF, 100 ºC
OR
1.1 equiv. DBU, DMF, 70 ºC
N
S OCH
3
O
N
S OCH
3
O
H
H
2.2 2.2a
Cu
N
Mes
Mes
N OH
2
2+
+ 2
-
OTf
OH
2
OH
2
2.1

Scheme 2.1. Oxidation of thiazoline 2.2 to thiazole 2.2a via catalytic-aerobic or
stoichiometric base-mediated oxidation.

22
We show here catalytic copper-based conditions for aerobic azoline oxidation that
improve the applicable scope of aerobic oxidation conditions to include electron donating
substituents and scalability of the reaction while minimizing metallic waste stream
(Scheme 2.1). Moreover, these conditions are low cost. For example compound 2.2a
(Scheme 2.1) is commercially available
1
for $22,500 g
-1
but can be prepared in our route
for < $28 g
-1
(Table 2.1)
Table 2.1. Cost Analysis for the Synthesis of 1 Gram of Methyl 2-Phenylthiazole-4-
Carboxylate.
Reagent List Price List Quantity Unit Price Amt. Needed Cost
Butanedione $90.60 500 g $0.18 g
-1
42 mg $0.01
Copper Triflate $190.00 25 g $7.60 g
-1
165 mg $1.25
2,4,6-dimethylaniline $530.00 2.5 kg $0.21 g
-1
134 mg $0.03
L-Cysteine $318.00 1 kg $0.32 g
-1
2.28 g $0.73
Benzonitrile $98.00 2.5 kg $0.04 g
-1
2.14 g $0.90
Iodomethane $455.16 500 g $0.87 g
-1
1.56 g $1.36
DBU $184.00 500 g $0.37 g
-1
69 mg $0.03
Phosphate Buffer 6.4 $74.38 4 L $18.60 L
-1
20.91 mL $0.39
MeOH $614.00 204 L $3.01 L
-1
20.91 mL $0.06
DMF $3,446.82 204 L $16.90 L
-1
232 mL $3.92
EtOAc $1,648.44 204 L $8.08 L
-1
1 L $8.08
Hexanes $916.12 204 L $4.49 L
-1
2.5 L $11.23
Total: $27.99
All prices are based on the VWR list price.

23
2.2. Copper-catalyzed aerobic oxidation of azolines to azoles and
optimization.
Copper complex 2.1 is prepared in two steps without need for purification via
chromatography from 2,3-butanedione and the corresponding trimethylaniline with the
intermediacy a known diazabutadiene ligand, [
Mes
DAB
Me
]
2
(Scheme 2.2). The structure of
2.1 is assigned by single-crystal X-ray diffraction (Scheme 2.2).
3a
In this case copper
adopts a distorted square pyramidal geometry
4
in which copper(II) appears to be a 19-
electron metal center. The analogous (4,7-diphenylphenanthroline)-ligated complex 2.1a
has a similar structure.
3b

NMes
NMes
[
Mes
DAB
Me
]
Cu(OTf)
2
CH
2
Cl
2
Cu
N
Mes
Mes
N
OH
2
OH
2
OH
2
2+
1
2
3
1
2

2.1
Scheme 2.2. Synthesis and molecular structure of complex 2.1. Ellipsoids are drawn at
the 50% probability level. Selected bond distances (Å): Cu-N1 = 1.99; Cu-N2 = 2.00;
Cu-O1 = 1.99; Cu-O2 = 1.97; Cu-O3 = 2.21.
2.1

24
Table 2.2 summarizes the optimization of catalytic aerobic oxidation conditions
for the transformation of thiazoline 2.2 to thiazole 2a. We found comparable results upon
screening other bidentate ligands for copper (vide infra); however, optimization and
scope studies were performed solely with catalyst 2.1. Entries 1-4 demonstrate that
although O
2
is essential for the reaction (entry 4), air is a more effective oxygen source
than 1 atmosphere of O
2
(entries 1 and 2). Repeating the O
2
experiment (entry 2) at 55
o
C
did not improve this reaction (entry 3).
Table 2.2. Optimization of Cu(II)-Catalyzed Oxidation Conditions.
S
N
S
N
Catalyst, base
[O]
H
H
O
OMe
O
OMe
2 2a

Entry
a
Cat. [O] Base Solvent Temp. (ºC) Time Yield (%)
b

1 2.1 Air

DBU DMF 100 8 h 88
2 2.1 O
2
DBU DMF 100 8 h 41
3 2.1 O
2
DBU DMF 55 7 d 47
4 2.1 N
2
DBU DMF 100 8 h 3
5 -- Air DBU DMF 100 8 h 36
6 2.1 Air DBU DCM reflux 8 h 41
7 2.1 Air DBU CH
3
CN reflux 8 h 4
8 2.1 Air DBU PhCH
3
100 8 h 14
9 2.1 Air Proton Sponge DMF 100 8 h 41
10 2.1 Air DIEA DMF 100 8 h 45
11 2.1 Air KOtBu DMF 100 8 h 22
12 2.1 Air K
2
CO
3
DMF 100 8 h 47
13 2.1 Air -- DMF 100 8 h 4
14 -- Air 1.1 eq. DBU DMF 70 30 min 66
a
Reaction conditions: 10 mol% [(
Mes
DAB
Me
)Cu
II
(OH
2
)
3
]
2+
[
-
OTf]
2
, 2.1, 10 mol% base
unless otherwise noted.
b

Isolated yields. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene.
2.2 2.2a

25
Table 2.3 summarizes that the ligand used on copper has little influence in the
outcome of the conversion of 2.2 to 2.2a. We found comparable results upon screening
several nitrogen-based ligands for copper (entries 1-7). Among these, the diimine system
found in 1 (entry 9) and ligand-free conditions (entry 8) afforded the best conversions,
with the former affording a superior isolated yield.
Table 2.3. Ligand Screen of Copper Catalyzed Oxidation of Thiazoline 2.2 to Thiazole
2.2a.
Entry
a
Ligand Conversion
b
(Isolated Yield)
1
N N

66%
2
N N

81%
3
NH
2
H
2
N

74%
4
N N
Ph Ph

81%
5
N
O O
N

86%
6
N
N N

64%
7
N N
tBu tBu

92% (68%)
8 none 100% (78%)
9
NMes MesN

90% (88%)
a
Conditions: Cu(OTf)
2
10 mol%, DBU (10 mol%), thiazoline (50 mM), DMF, 100 ºC,
8 h.
b
Based on starting material conversion by
1
H NMR.

26
2.3 Copper-free, base-mediated aerobic oxidation of azolines to azoles and
optimization.
The copper-free background reaction (Table 2.2, entry 5) occurs at an appreciable
rate and results in product formation in 36% yield. Along these lines, entry 14 illustrates
that in the presence of 1.1 molar equivalents DBU, oxidation reaches 66% yield (> 99%
conversion) in only 30 minutes. Solvents screening include DMF, DCM, CH
3
CN, and
PhCH
3
(entries 6-8); none was superior to the original DMF conditions. Neither Hünig’s
base (entry 10) nor t-butoxide (entry 11) is as effective as DBU in these conditions, but
both are superior to base-free conditions (entry 13). This result highlights the relative
utility of catalytic and base-promoted conditions with an electon-neutral substrate.
Importantly, we observe that in a direct comparison with thiazoline 2.2, DBU conditions
compare favorably to analogous K
2
CO
3
conditions (compare entries 1 and 12).
5

Many of these reactions produce reasonable yields in the presence of base alone
(Table 2.2, entry 14). Reactions run in the absence of copper with stoichiometric base
generally have lower, but comparable yields to their catalytic counterparts but with
advantageous, reduced reaction times. Yields for base promoted reactions are
summarized in tables 2.3 and 2.4 in conjunction with the results for catalytic oxidation.

27
2.4 Substrate scope.
Conditions were tested against a variety of thiazoline substrates (Table 2.4).
Substrates with aryl substituents in the 2-position demonstrated good yields with a range
of electron withdrawing and electron donating groups in the para-position. Electron-
withdrawing groups such as aryl fluoride and aryl nitrile (entries 5a, 6a) do not impede
oxidation; more importantly, an electron-rich thiazoline is tolerated (entry 3a). A
sensitive substrate and excellent synthetic handle such as the p-cyano thiazoline (2.7)
shows a significant advantage in yields 69% vs. 9% when using our catalytic method vs.
aerobic K
2
CO
3
.
5
Further, yields of 88% and 66% with DBU as base in the respective
presence and absence of copper are an interesting contrast to yields 47% and 30%
6
for
otherwise identical reactions run with K
2
CO
3
(1 equiv.) as base.
Oxazolines (Table 2.5, entries 1 and 2) were tested against the catalytic conditions
with less success. Yields of the corresponding oxazoles are lower than those of the
thiazole series. We are unsure of the reason for this difference, but we suspect that the
presence of a more polarizable sulfur center in an intermediate enolate (2.14, Scheme 2.2,
vide infra) facilitates oxygen transfer. We have not seen evidence of an S-oxidation
pathway, although such a mechanism cannot be eliminated.

28
Table 2.4. Aryl Thiazoline Oxidation.
Entry Substrate Conditions
a
Product Yield (%)
1a
N
S
OCH
3
O
2

Catalyzed
8 h
N
S
OCH
3
O
2a

88
b
1b
Base
30 min

66
b

2a
N
S
OCH
3
O
O
2
N
3

Catalyzed
3 h
N
S
OCH
3
O
O
2
N
3a

78
2b
Base
c

1 h

69
3a
N
S
OCH
3
O
H
3
CO
4

Catalyzed
8 h
N
S
OCH
3
O
H
3
CO
4a

68
3b
Base
4 h
58
4a
N
S
OCH
3
O
5

Catalyzed
8 h
N
S
OCH
3
O
5a

79
4b
Base
1 h

77
5a
N
S
OCH
3
O
F
6

Catalyzed
3 h
N
S
OCH
3
O
F
6a

58
5b
Base
45 min
44
6a
N
S
OCH
3
O
NC
7

Catalyzed
4 h
N
S
OCH
3
O
NC
7a

69
6b
Base
45 min
44
a
Catalyzed: 10 mol% 2.1, 10 mol% DBU, DMF, 100
o
C; Base: 1.1 equiv. DBU, DMF,
70
o
C.
b
Substituting K
2
CO
3
as base (e.g. ref 9) in these conditions affords yields of 47%
and 30%
6
respectively for the catalyzed and base-promoted reactions.
c
10 mol% DBU.
2.6
2.6a
2.7 2.7a
2.2
2.2a
2.3 2.3a
2.4 2.4a
2.5 2.5a

29
The base conditions demonstrate increased yields in both the 2-substituted alkyl
substrates (Table 2.5, entries 3b and 4b) and the oxazoline containing substrate
(entry 2b), while the catalytic conditions appear higher yielding in all other cases.
Particularly in situations of more electron rich thiazolines, catalytic conditions provide
increased yields. We suspect that the advantage in yield for the catalytic conditions is
related to the minimization of intermolecular side reactions.
When a thiazoline substrate with a 2-substituted heterocycle, e.g. indole
(Table 2.5, entry 5), is subjected to base conditions, no product formation is observed.
However, successful oxidation in 55% yield is achieved by application of catalytic
conditions. When Yao et al.’s conditions
5
were applied to the indole substrate
(2.12, Table 2.5, entry 5c) a yield of 36% was obtained. We suspect that this is because of
the labile acidic N-H proton. A N-methylindole-bearing substrate (2.13, entry 6) was
subsequently subjected to both catalytic and base conditions, which leads to good yields
in each case. These data show that in the presence of labile protons, as in indole, our
catalyst proves superior and necessary for thiazoline oxidation.

30
Table 2.5. Further examples of azoline oxidation.
Entry Substrate Conditions
a
Product Yield (%)
1a
N
O
OCH
3
O
8

Catalyzed
b

9 h
N
O
OCH
3
O
8a

18
1b
Base
6 h

16
2a
N
O
OCH
3
O
O
2
N
9

Catalyzed
b

12 h
N
O
OCH
3
O
O
2
N
9a

37
2b
Base
2 h

41
3a
N
S
OCH
3
O
2.10
Catalyzed
8 h
N
S
OCH
3
O
10a

24
3b
Base
5 h
39
4a
N
S
OCH
3
O
11

Catalyzed
12 h
N
S
OCH
3
O
11a

45
4b
Base
6 h

51
5a
N
S
OCH
3
O
NH
12

Catalyzed
6 h
N
S
OCH
3
O
NH
12a

55
5b
Base
1 h
0
6a
N
S
OCH
3
O
N CH
3
13

Catalyzed
b

14 h
N
S
OCH
3
O
N CH
3
13a

65
6b
Base
30 min
46
6c
K
2
CO
3
5
6 h
36
a
Catalyzed: 10 mol% 2.1, 10 mol% DBU, DMF, 100
o
C; Base: 1.1 equiv. DBU, DMF,
70
o
C.
b
30 mol% DBU.

2.8 2.8a
2.9 2.9a
2.10a
2.11a 2.11
2.12 2.12a

2.13a

2.13

31
2.5 Mechanistic evidence of catalytic and copper-free, base-mediated
aerobic oxidation of azoline to azole pathways.
We have made some observations that help us understand the reaction
intermediates (Scheme 2.3). We propose initial enolization of 2.2 followed by installation
of an angular hydroxide (2.15). Notably, isolation and characterization of 2.15 confirms
its presence in the reaction under copper-free conditions; independent conversion of 2.15
to 2.2a in the presence of DBU, with/without copper, provides evidence of its kinetic
competence. Thus, we propose 2.2 is enolized to form an intermediate 2.14, which is
oxidized either by a copper oxo species or O
2
itself to give angular hydroxide 2.15.
7

S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
S
N
Ph
O
O
OH
OOH
DBU
O
2
O
2
not observed
fast
slow
66% (DBU)
30% (K
2
CO
3
)
2.2 2.14
2.15 2.2a
2.16
observed via NMR and MALDI MS
OOH
K
2
CO
3

Scheme 2.3. Proposed oxidation mechanism; comparison of DBU and Yao et al. K
2
CO
3
5

pathways.

32
The angular hydroxide group of 2.15 comes from O
2
as opposed to H
2
16
O
(Figure 2.1) because we observe no incorporation of
18
O when the reaction is run in the
presence of H
2
18
O (Figure 2.2). Along these lines, the presence of a radical inhibitor
(BHT, butylated hydroxytoluene or tocopherol, vitamin E) does not affect the efficiency
of the copper-catalyzed or stoichiometric base-promoted oxidation of 2.2. With addition
of radical inhibitor vitamin E, the copper catalyzed reaction solution remains clear and
deep green in color. Which contrasts inhibitor-free copper catalyzed condition solutions,
which are clear and deep brown in color. Therefore, we do not suspect a long-lived
radical intermediate in either reaction. Addition of water (stoichiometric or excess) does
not provide increased yield or rate in either copper-catalyzed or stoichiometric base-
promoted oxidation of 2.2. Furthermore, the formation of 2.15 and 2.2a are not hindered
by the addition of molecular sieves to absorb any water in the reaction solution.
Oxidation reactions were performed with copper-catalyzed or stoichiometric base-
promoted conditions with the addition of 1 equivalent of H
2
O and were monitored by
1
H
NMR spectroscopy. Results were compared to
1
H NMR spectra from entries 1a and 1b of
Table 2.4. Further, we have found no evidence of a S-oxidation pathway or side reaction,
although such mechanisms cannot be eliminated.

33

Figure 2.2. MALDI spectra of oxidation of 2.15 in the presence of H
2
16
O.
S
N
OMe
O
16
OH
H
[MH]
+
: Theor: 238.05
g/mol, 100% relative intensity

[MH]
+
+ 1: Theor: 239.06
g/mol, 11.9% relative intensity

[MH]
+
+ 2: Theor: 240.05
g/mol, 4.5% relative intensity

34

Figure 2.3. MALDI spectra of oxidation of 2.15 in the presence of H
2
18
O with no
additional
18
O incorporation.
[MH]
+

35
Our conditions do not involve the intermediacy of a long-lived hydroperoxide
species. Yao et al. report that under potassium carbonate conditions,
5
a long-lived tertiary
peroxide intermediate (2.16) intervenes 2.14 and 2.15 in the oxidation mechanism as
characterized by TLC evidence (Scheme 2.2). By contrast, we observe no intermediate
species in the reaction mixture other than 2.15 and 2.2a when the oxidation is run with
either our copper catalyzed conditions or stoichiometric DBU. However, we do observe a
species consistent with 2.16 via NMR spectroscopy when we run the reaction under
potassium carbonate conditions (Figure 2.4).
Figure 2.4 illustrates intermediate 2.16 observed in potassium carbonate-mediated
oxidation. The thiazole product is evident from its methyl ester peak, highlighted in
green. Each of 2.15 (b) and the 2.16 (c) are represented both by their methyl esters at
3.9 ppm and by a pair of doublets corresponding to their C5 methylene protons. The low
concentration of a peroxide intermediate in our conditions is significant if this reaction is
to be practiced on scale. Nonetheless, it is essential to decompose any possible peroxide
in any aerobic oxidation before the product is isolated.

36

Figure 2.4.
1
H NMR spectra of intermediates in potassium carbonate-mediated oxidation
a) product 2.2 (-CO
2
Me); b) hydroxythiazoline 2.15; c) purportedly an
angular peroxide 2.16.
b a c c b c b

37
2.6 Scalability of copper-catalyzed and copper-free, base-mediated aerobic
oxidation of azolines to azoles.
The catalytic conditions are advantageous when the reaction is run on larger scale
(Table 2.5), which is important if this transformation is to be used for material
throughput. Thiazoline 2.2 is successfully oxidized on a 1 g scale to afford 80% yield of
the thiazole 2.2a when copper conditions are utilized (entry 2). The base-mediated
reaction is less efficient at this scale (entry 4). The 1000 mg scale base-mediated K
2
CO
3
oxidation of 2.2 to provide 2.2a with K
2
CO
3
was not performed by us or Yao et al.
9

Table 2.5. Scalability of the conversion of 2.2 to 2.2a.
10 mol% 2.1, 10 mol% DBU
DMF, 100 ºC
OR
1.1 equiv. DBU, DMF, 70 ºC
N
S OCH
3
O
N
S OCH
3
O
H
H
2.2 2.2a

Entry Scale (mg) Conditions

Time Isolated Yield (%)
1 20 Catalyzed

8 h 88
2 1000 Catalyzed 18 h 80
3 20 Base 1 h 66
4 1000 Base 2.5 h 55
2.7. Conclusions.
We have developed conditions to transform azolines to azoles via two efficient
and economical aerobic oxidation routes. These reactions are applicable to a wide range
of substrates (electron rich–electron poor), are easy to use, involve little waste, and are
demonstrated on a reasonable laboratory scale. Stoichiometric base conditions afforded

38
good yields in many cases, but copper catalyzed conditions afforded superior results in
most cases. This technology will be useful for building natural products and medicinal
entities containing one or more embedded azole subunits, sensitive labile protons, and
electron rich species without the expense of stoichiometric metal oxidants.
2.8. References.

1) Quote from Aurora Fine Chemicals, San Diego, CA.
2) Zhong, H. A.; Labinger, J. A.; Bercaw. J. E. C-H Bond Activation by Cationic
Platinum(II) Complexes: Ligand Electronic and Steric Effects. J. Am. Chem. Soc.
2002, 124, 1378-1399.
3) CCDC (a) 761477 and (b) 761476 contain the supplementary crystallographic
data for these complexes. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
4) Other examples of cis (N—N)Cu
II
(OH
2
)
3
include (a) Xu, Z.; Thompson, L. K.;
Miller, D. O. Dicopper(II) Complexes Bridged by Single N-N Bonds. Magnetic
Exchange Dependence on the Rotation Angle between the Magnetic Planes.
Inorg. Chem. 1997, 36, 3985-3995; (b) Burkholder, E.; Wright, S.; Golub, V.;
O’Connor, C. J.; Zubieta, J. Solid State Coordination Chemistry of
Oxomolybdenum Organoarsonate Materials. Inorg. Chem. 2003, 42, 7460-7471.
5) (a) Huang, Y.; Gan, H.; Li, S.; Xu, J.; Wu, X.; Yao, H. Oxidation of 4-carboxylate
Thiazolines to 4-carboxylate Thiazoles by Molecular Oxygen. Tetrahedron Lett.
2010, 51, 1751-1753. (b) Huang, Y.; Ni, L.; Gan, H.; He, Y.; Xu, J.; Wu, X.; Yao,
H. Environmental-benign Oxidation of 2-oxazolines to Oxazoles by Dioxygen as
the Sole Oxidant. Tetrahedron 2011, 67, 2066-2071.
6) An additional portion of ca. 48% of azole-containing products were collected. See
Chapter 7 for detail.
7) Adam, W.; Ehrig, V. Cyclic peroxides. 42. Direct α-lithiation of 4,5-dihydro-
1,3-thiazole-4-carboxylic Acids and Electrophilic Substitution. Synthesis 1976,
12, 817-819.

39
Chapter
3.
Introduction.
Magnetic
resonance
imaging

contrast
agents
and
applications
in
medicinal
chemistry.

3.1. MRI contrast agents and relaxation parameters.
Magnetic resonance imaging (MRI) is a well-recognized method for non-
invasively imaging cells and human bodies (field strength = 0.2 – 3 T).
1
Offering an
alternative to light microscopy, three-dimensional MRI can be accomplished at the
cellular level (~ 10 µm). MRI contrast (brightness) is derived from variations in the
change in local water concentration, T
1
(spin-lattice), and T
2
relaxation (spin-spin) rates.
The interaction of the contrast agent and water molecules is described by parameters
including water coordination number (q), the exchange rate of the inner-sphere water
molecules (k
ex
), and the rotational correlation time (τ
r
) of the complex. The molar
relaxivity (r
1
) of a contrast agent is defined as a plot of 1/T
1
vs. concentration of the
contrast agent in solution. Therefore, r
1
and T
1
are inversely proportional. MRI contrast
agents enhance the T
1
and T
2
of water molecules, which in effect speed up the exchange
of the water molecules via interaction with a paramagnetic metal ion. Gd
3+
is the most
commonly used lanthanide metal for MRI contrast agents due to the fact that Gd
3+
has a
high magnetic moment (µ
2
= 63 µ
B
2
), seven unpaired electrons, and a symmetric ground
state making it ideal for magnetic resonance imaging.
2, 3

40
Small molecule Gd
3+
contrast agents (Figure 3.1) will be briefly discussed in this
chapter as well as Chapter 4 with more extensive focus on the activation of these contrast
agents. In the past two decades, the development of activatable or “smart” MRI contrast
agents that are responsive to physiological stimuli has naturally progressed to the
investigation of externally activated MRI contrast agents. The concept of external
activation allows for site selective MRI contrast in physiological systems at a desired
time and place chosen by the clinician.
N
N N Gd
O
O
O
O
O
O
O
O
O
O
N
N N
N
Gd
O
O
O
O
O
O
O
O
N N
N N
Gd
P
O
P
O
O
O
P
O
O
P
O
O
O
O
O
O
5
2
Gd-DTPA Gd-DOTA Gd-DOTP

Figure 3.1. Structures of select MRI contrast agents Gd–DTPA, Gd–DOTA, and Gd–
DOTP.
3.2. “Smart” MRI contrast agents.
Recent developments in the field of MRI contrast agents are focused on the
progress of smart contrast agents capable of producing contrast in response to their
physiological environment.
4
The ability of a MRI contrast agent to be activated stemmed
from the coordination number of the metal in the contrast agent and the inherent cause of
enhanced relaxivity generated by exchange of metal-bound water molecules with
surrounding bulk water molecules. Gd
3+
has nine coordination sites of which eight are

41
usually coordinated to a chelating ligand to prevent toxic Gd
3+
from escaping and
wrecking havoc on biological systems due to facile transmetallation with Ca
2+
. The one
remaining coordination site is available for inner-sphere coordination of a water
molecule. For contrast agents with a water coordination number (q) equal to 1, half of the
contrast agent’s relaxivity comes from the inner-sphere water coordination, and the other
half of the relaxivity comes from the second coordination sphere of water molecules.
Logically, the concept of inhibiting the exchange of water with the inner coordination
sphere of the metal (reducing the relaxivity) by a built-in ligand arm was conceived.
Subsequently, upon some chemical interaction, the blockage of the coordination site
would be removed; Thus, activating the contrast agent. These types of contrast agents are
referred to as activatable or “smart” MRI contrast agents.
5
A brief overview of
representative examples of each category of smart MRI contrast agent activation
(biomolecule markers, small molecules, metals, pH, light, etc.) will be discussed in this
work.
3.2.1. Activation of MRI contrast agents by small molecules and biomolecule
markers.
In 1997, Meade et al. pioneered the first responsive “smart” MRI contrast agent
to report on the metabolic state of cells and organs. The blocking agent described in this
system (galactopyranose) is designed for irreversible removal by the enzyme
β-galactosidase (β-gal) (Scheme 3.1). The water coordination number, q, was measured
for both blocked and cleaved complexes and determined to be 0.7 versus 1.2 respectively.
Consistent with q measurements, a 20% decrease in T
1
was observed upon addition of

42
β-gal to the galactopyranose blocked contrast agent demonstrating that the bond was
enzymatically cleaved revealing the coordination site to bulk water molecules. Magnetic
resonance images taken in 1.5 mm capillary tubes visually demonstrated the on-off states
of the galactopyranose system.
5
An application of the aforementioned system was
described in 2000 by the Meade group for the high-resolution in vivo imaging of opaque
animals to indicate reporter gene expression of β-galactosidase.
6

N
N N
N
CO
2
-
CO
2
-
-
O
2
C
O
OH
OH
OH
O
N
N N
N
CO
2
-
CO
2
-
-
O
2
C
H
2
O
β-gal HO
OH
H
2
O

Scheme 3.1. Structure of galactopyranose attached to Gd – DOTA and removal by β-
galactosidase (β-gal).

In 2005, the Meade group reported a steroid-conjugated contrast agent for
magnetic resonance imaging of cell signaling. This contrast agent was specifically
designed to monitor the activation of a mutated progesterone receptor (PR) pathway
important in the GAL4-UAS system. Synthetic progesterone antagonist RU-486, a steroid
hormone, was covalently attached to a Gd
3+
contrast agent. In vivo, the binding of the
Gd(III)-RU-486 substrate with the mutant receptor activates the endogenous gene
receptor pathway. A decrease in T
1
values from 3.18(11) s in PR cells with RU-486 to
2.06(11) s in PR(+) cells treated with the Gd(III)-RU-486 suggests that the binding of the
contrast agent to the receptor simultaneously reduces the T
1
of the contrast agent by

43
revealing the coordination site to water molecules (q) as well as increases the size of the
contrast agent. T1 results are summarized in Table 3.1. An increase in size of the contrast
agent increases the rotational correlation time, τ
r
, allowing more time for water molecules
to access the coordination site; Thus, increasing the relaxivity (r
1
) of the contrast agent.
7

The area of progesterone receptor pathway activation and monitoring by MRI contrast
agents was further developed and explored by Meade et al. in 2011 with the synthesis and
biological evaluation of water-soluble progesterone conjugated probes for MRI of
hormone related cancers. The lead promising results demonstrate PR(+) selective MRI
probes.
8

Table 3.1. T
1
(s) of cells at 9.4 T upon treatment of 50 µM of each compound and
incubated for 16 h.
7

Cells/Compounds T
1
(s)
PR(+) cells treated with RU-486 3.18(11)
PR(-) cells treated with RU-486 2.90(11)
PR(+) cells treated with Gd-RU-486 2.06(16)
PR(-) cells treated with Gd-RU-486 2.30(30)
media 3.03(16)

Lu et al. demonstrated “turn-on,” adenosine activated MRI contrast agents by
combining aptamer technology with superparamagnetic iron oxide nanoparticles
(SPIONs) in 2007. Aptamers have selective and sensitive binding properties that make
them ideal candidates for chemical and biological sensors. The aptamer of choice was the
adenosine DNA apatamer, which is functionalized onto SPIONs proposed to gain
relaxivity once exposed to adenosine. Indeed, the system showed enhancement of MRI

44
contrast in the presence of adenosine, which was confirmed with T
2
magnetic resonance
image plains in a 96-well micro plate. (Figure 3.2).
9

a)

b)

c)
two bases was used as control (Scheme 1, legend). This mutat-
ed linker has been shown not to bind adenosine.
[17,29]
As was
expected, no change in brightness was observed
with increasing adenosine concentration (Figure 2B).
In a separate control aimed at investigating the
ACHTUNGTRENNUNGselectivity of the system, the sensor was incubated
with 5 mm of cytidine, uridine, or guanosine. As
seen in Figure 2C, a significant change in contrast
was not observed in any of these three cases.
Quantitative analysis was performed by measuring
the T2 relaxation times of samples with different ad-
enosine concentrations. A clear increase in T2 values
from 36 to 63 ms was observed as the adenosine concentra-
tion was raised from 0 to 2.5 mm (Figure 3), and the initial T2
value of dispersed nanoparticles was reached.
[41]
Since the di-
ameter of the CLIO clusters can
be determined by using DLS,
the T2 value can be correlated
to the number of particles per
cluster.
[47]
In the lower concen-
tration range (0–1 mm) T2 was
linearly dependent on the con-
centration of adenosine, and
saturation was reached at
2.5 mm. To demonstrate utility
and stability of the current
system in vivo, the sensor was
prepared in the presence of
human serum (10%). The activi-
ty of the sensor was found to
be retained in the presence of
serum. Upon addition of adenosine the contrast within the
concentration gradient increased (Figure 4).
In conclusion, we have demonstrated a general method for
aptamer-based biosensing by using MRI. The adenosine apta-
mer-based design serves as a model system for similar smart
and functional CLIO contrast agents. Since aptamers
specific for a variety molecular markers of biological
functions and diseases can be obtained through
SELEX, this method can be applied for early molecu-
lar diagnosis for a number of targets.
Experimental Section
Materials: All DNA samples were purchased from Inte-
grated DNATechnologies Inc. (Coralville, IA, USA). Linker
DNA sequences were purified by HPLC whereas thiol-
modified DNA molecules were purified by the standard
desalting method. Adenosine, cytidine, uridine, and
guanosine were purchased from Aldrich (St. Louis, MO,
USA). Cross-linked dextran-coated superparamagnetic
iron oxide nanoparticles (CLIOs; 500mg Fe mL
!1
) were
synthesized and coupled to N-succinimidyl 3-(2-pyridyl-
dithio)-propionate (SPDP) according to literature proce-
dure and purified by using a PD-10 column.
[46]
Thiol-
modified oligonucleotides, 3’Adap (5’-TCACAGATGAGT-
A
12
-SH-3’) and 5’Adap (5’-SH-CCCAGGTTCTCT-3’) were
activated by being incubated with tris(2-carboxyethyl)
phosphine hydrochloride (TCEP; 8 equiv). Excess TCEP
was removed by desalting with a SepPak C-18 cartridge.
TCEP-activated thiol-modified DNA (50mm final concen-
tration) was mixed with CLIO–SPDP (400mg Fe mL
!1
) in
phosphate buffer (100 mm) at pH 8.0, overnight. Excess
DNA was removed by using a magnetic separation
Figure2.T2-weighted MR image of samples in a 96-well microplate. Effect of increasing adenosine concentration
in CLIO–DNA conjugates assembled either with A) linker–Adap, or B) mutated linker. C) Sensor with 5 mm adeno-
sine (A), cytidine (C), uridine (U), and guanosine (G).
Figure3.Disassembly of sensor clusters with increasing concentration of adenosine, as
detected by change in T2 relaxation times. The lower panel shows the T2-weighted MR
image of the samples.
Figure4.T2-weighted MR image of adenosine-induced disassembly of sensor in human
serum (10%).
ChemBioChem 2007, 8, 1675–1678 ! 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chembiochem.org 1677

Figure 3.2. T
2
-weighted MR image of samples in 96 well plate demonstrating the effect
of increasing adenosine on a) linker-adap, active adenosine aptamer; b) mutatated linker,
mutated adenosine aptamer; c) sensor + adenosine (A), cytodine (C), uridine (U), and
guanisine (G), active adenosine aptamer.
9

In 2011, Meade et al. synthesized four HaloTag reporter protein responsive Gd
3+

complexes aimed to increase specificity, versatility, and exploit the covalent interaction
between the substrate and the protein. A six-fold increase in r
1
(molar relaxivity) was
observed upon covalent binding of 2CHTGd (Table 3.2) and the target protein utilizing
the concept of receptor-induced magnetization enhancement (RIME). Once bound to the
protein, the τ
r
is increased, and in turn the relaxivity is increased.
10
The RIME concept
was also used in a similar system in which a small molecule contrast agent was used to
target human serum albumin (HSA).
11

45

Table 3.2. Longitudinal relaxivities of Free (F) and Protein-Bound (PB) HaloTag
Targeted contrast agents by Meade et al.
10

Gd
N N
N N
O
O
O
O
O
O
O
R
n = 1 : 1CHTGd
n = 2 : 2CHTGd
n = 3 : 3CHTGd
n = 4 : 4CHTGd
R =
HN
O
O
Cl n

Sample r
1
– F (mM
-1
s
-1
) r
1
– PB (mM
-1
s
-1
)
1CHTGd 3.6 ± 0.2 13.4 ± 1.3
2CHTGd 3.8 ± 0.1 22.0 ± 2.2
3CHTGd 5.1 ± 0.1 7.6 ± 0.7
4CHTGd 6.4± 0.5 8.6 ± 1.1

Nanocarriers and nanoparticles are an extension of small molecule contrast agents
and will only be briefly discussed in this chapter. However, the fields of nanoparticles,
imaging, therapeutics, and diagnostics are currently being combined into
“nanotheranostic” and “multimodality” scaffolds.
12
A nanocarrier T
2
MRI contrast agent
was developed by Matsumura et al. in 2011 that evolves its surface properties in the
presence of the tumor associated protease, matrix metalloproteinase-2 (MMP-2), to
initiate agglomeration. This modified ferritin (Fer-PPD) tumor-environment nanocarrier
contains a triad of modifiers including (i) a “sensing” segment (ii) a “hydrophobic”
segement and (iii) a “hydrophilic” segment (Figure 3.3). MMP-2 activity cleaves the
sensing segment exposing the hydrophobic segment initiating aggregation; thus,
enhancing the τ
r
and increasing the T
2
relaxivity. The enhancement of T
2
was measured

46
for Fer-PPD in the presence of MMP-2, and an 11% decrease in T
2
(ms) was observed
Zeta potential measurements as well as TEM images confirmed the manifestation of
aggregates of Fer-PPD in the presence of MMP-2. Ferritin was chosen as the nanocarrier,
which allowed for exploitation of the core inner space (8 nm) to load drug molecules.
13

Ferritin
C
H
N
O
HN
O
H
N
O
N
O
O
S
Cys-AhxAhxAhx-GlyAlaLeu-GlyLeuPro-Lys-FAM
NH
OH
O
O
n
PEG : 5 kDa
iii) Hydrophilic Segment
i) MMP-cleavage site
S
S
N
H
O
R
O
R = O
OH
O
OH
O
OH
H
2
N
OH O
O O OH
ii) Hydrophobic Segments
Figure 3.3. Design of the modified ferritin (Fer-PPD) structure and illustration of the
assembly: (i) a “sensing” segment (ii) a “hydrophobic” segment and (iii) a “hydrophilic”
segment.

47
3.2.2. Activation of MRI contrast agents by metals.
Monitoring of metals within physiological systems is a useful tool in
understanding pathways and diagnosing medical diseases associated with the presence of
an excess or deficiency of metal ions. Misregulation of metal ion stores is associated with
diseases such as cancer, heart disease, diabetes, and neurodegeneration. The merge of
MRI contrast agents and metal-sensing moieties in the late 1990s enabled a vast field of
metal activated MRI contrast agents to track metals within physiological systems and
report on biological metal ion processes and pathways.
14

Physiological flux in calcium concentration triggers changes in cellular
metabolism and is responsible for cell signaling and regulation. In 1999, Meade et al.
developed a calcium sensitive MRI contrast agent. In order to study cell signaling and
regulation, the macrocylic dimer Ca
2+
sensing MRI contrast agent, DOPTA-Gd
(Scheme 3.2), was created to provide information about physiological signals and
biochemical events. DOPTA is a chelating ligand synthesized by covalently joining
1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) and
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Upon chelation of
Ca
2+
, an 80% increase in relaxivity of the DOPTA-Gd is observed. The DOPTA-Gd
system demonstrates selectivity over biologically relevant Mg
2+
and H
+
ions.
15

48
a)
Gd
N N
N N
O
O
O
O
O
O
O
N
O
O
N
O
Gd
N N
N N
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
+ Ca
2+
- Ca
2+
Gd
N N
N N
O
O
O
O
O
O
O
N
O
O
N
O
Gd
N N
N N
O
O
O
O
O
O
C
O
O O
O
O
O
O
O Ca

Scheme 3.2. a) The proposed conformational dependence of the DOPTA-Gd structure in
the presence and absence of Ca
2+
.
15
Zn
2+
plays a vital role in biology with functions in gene transcription and
metalloenzyme function as well as modulating neuronal transmission in the brain. More
detrimentally, Zn
2+
has been shown to induce the formation the β-amyloid peptide.
β-amyloid plaque formation has been extensively studied as a potential cause for
Alzheimer’s disease. Zinc, copper, and iron metal ions within the brain have been shown
to contribute extensively to the formation of β-amyloid plaques. Additionally, zinc
monitoring is also useful in studying the etiology of diabetes due to the simultaneous

49
release of zinc with insulin by the pancreatic β-cells.
16
Furthermore, zinc deficiency has
been associated with prostate disease and diabetes.
In 2007, Meade and co-workers developed a Zn
2+
responsive MRI contrast agent,
Gd-daa3, that contained an iminodiacetate group for Zn
2+
binding (Figure 3.4). A drastic
122% increase in relaxivity was observed (r
1
= 2.3 to r
1
= 5.1 mM
-1
s
-1
) upon introduction
to Zn
2+
. The turn on effect was confirmed to be a water coordination number (q) effect by
luminescence lifetime experiment with Tb-daa3. Gd-daa3 (daa3, diaminoacetate
with 3 methylenes, gadolinium(III) carboxymethyl-{2-methyl-6-[3-[4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]phenylamino}acetic acid)
and modified Gd-apa3 (gadolinium(III) {{2-methyl-6-[3-[4,7,10-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-yl]propoxy]phenyl}pyridin-2-ylmethylamino}acetic acid)
(Figure 3.4) with one pyridine and one acetate arm both demonstrated selectivity for Zn
2+
in the presence of biologically relevant concentrations of Na
+
, K
+
, Mg
2+
, and Ca
2+

cations.
17

N
N
N
N
Gd
O
O
O
O
O
O
O
N
O
O
O
O
Gd-daa3 n = 3
Gd-daa2 n = 2
N
N
N
N
Gd
O
O
O
O
O
O
O
N
O
O
N
Gd-apa3 n = 3
n n

Figure 3.4. Structure of Zn
2+
MRI contrast agents Gd–daa3 and Gd–apa3.

50
In 2009, Leon-Rodriguez et al. developed a new gadolinium-based MRI Zn
2+

sensor based on a bis N,N-bis-(2-pyridyl-methyl) ethylene diamine (BPEN) diamide
ligand bound to Gd
3+
. Uniquely, the Gd-DOTA-diBPEN:Zinc (1:1) agent only binds
HSA weakly. However, the ternary complex Gd-DOTA-diBPEN:Zinc (1:2) complex
strongly binds HSA resulting in a 3-fold increase in the relaxivity of the complex;
detecting Zn
2+
concentrations as low as 30 µM in the presence of HSA. The affinity of
the Gd
3+
-Ligand (Gd-L) complex for Zn
2+
is similar to that of HSA indicating that in vivo
it is likely that the Gd-ligand complex will not disrupt normal physiological processes.
Selectivity of the Gd-Ligand complex was shown over Ca
2+
and Mg
2+
and a similar
selectivity for Cu
2+
, which would likely not be an issue in vivo due to the relatively low
concentrations of Cu
2+
in tissues.
16
A previous reported system demonstrated a reduction
in relaxivity (r
1
) with bound Zn
2+
from r
1
= 6.06 mM
-1
s
-1
to r
1
= 3.98 mM
-1
s
-1
. However,
the system returned to its original relaxivity once a second equivalent of zinc was added
rendering it useless in vivo.
18
This issue was rectified by the removal of two picolyl
substituents and replacement with carboxylic acid groups to encourage a stasis when
additional equivalents of Zn
2+
are added at the reduced relaxivity
(r
1
= 4.8 mM
-1
s
-1
to r
1
= 3.4 mM
-1
s
-1
).
19

“Dual-modality” or bimodal magnetic resonance imaging agents (combination of
two or more molecular imaging techniques) have emerged along with the development of
therapeutic diagnostics or theranostics. A Zn
2+
responsive bimodal MRI and fluorescent
imaging probe was synthesized and characterized by the Chen group in 2012. This Gd
3+

complex with bound tether 8-sulfonamidoquinoline chromophore (GdL) is fluorescent

51
(7-fold increase) in the absence of Zn
2+
and MRI active (55% enhancement) in the
presence of 0.5 equivalents Zn
2+
((GdL)
2
Zn) (Scheme 3.3). Excellent cell membrane
permeability and biocompatibility was demonstrated with in vitro studies. The relaxivity
increase was due to an increase in molecular weight as well as constraint of geometry;
both contributing to the increase in τ
R
. The GdL complex demonstrates satisfactory
selectivity over a wide range of biologically relevant cations and anions.
20

Gd N
N
N
N
O
O
O
O
O
O
H
N
O
S
NH
N
O
O
H
2
O
Zn
2+
- 2H
+
Gd N
N
N
N
O
O
O
O
O
O
HN
O
S
N
N
O
O
Gd N
N
N
N
O
O
O
O
O
O
H
N
O
S
N
N
O
O
Zn
GdL
(GdL)
2
Zn

Scheme 3.3. Zn
2+
responsive bimodal MRI (GdL) and fluorescent probe ((GdL)
2
L).
20

Recently, in 2012, Duan et al. developed a dual-functional gadolinium based Cu
2+

probe for selective MRI as well as fluorescent sensing. Similar to the Zn
2+
system
developed by Chen, this “dual modality” Nap-DO3-Gd complex has the capability to be
fluorescent in the absence of Cu
2+
and MRI active in the presence of Cu
2+
, which chelates

52
the naphthylamine (Nap) substituent removing the bulky hindrance from the
1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) chelated Gd
3+

center allowing water portioning and increasing MRI relaxivity. The luminescent
detection limit is 6 ppb; a relevant limit as demonstrated in in vitro studies.
The selectivity of the Nap-DO3A-Gd was tested over other biologically relevant metal
ions Zn
2+
, Mg
2+
, Ca
2+
, Cu
+
, K
+
, Na
+
, Fe
2+
, Fe
3+
, Mn
2+
and demonstrated impressive
selectivity for Cu
2+
.
Findings were corroborated with luminescence lifetime (τ
H2O
) and
water coordination number measurements (q) of Nap-DO3A in the presence and absence
of Cu
2+
(Table 3.3).
21

Table 3.3. Luminescence lifetime (τ
H2O
) and water coordination number measurements
(q) of Nap-DO3A in the presence and absence of Cu
2+
.
21

τ
H2O
/ms τ
D2O
/ms q
Nap-DO3A-Tb 0.73 0.77 0.28
Nap-DO3A-Tb +
Cu
2+

0.23 0.27 2.58

53
Iron, being the most abundant transition metal in the body, is of utmost concern to
track Fe
2+
and Fe
3+
ion pathways and destinations. Iron excess has been shown to
manifest in Alzheimer’s and Parkinson’s disease patients, and iron deficiencies are
detrimental to human health as well. Although several systems have been developed to
incorporate iron and gadolinium into a large contrast agent with a correspondingly large
rotational correlation time, no Fe
2+
or Fe
3+
specific responsive sensor has been
developed.
14

3.2.3. Activation of MRI contrast agents by pH.
Maintaining an acid-base homeostasis in a physiological system is critical as
certain pathologies are associated with a perturbed pH balance. The inherent nature of
extracellular pH in tumor tissues is acidic due to poor lymphatic drainage and increased
production of lactic acid. Physiological buffer bicarbonate has poor buffering capacity in
this pH range, which intensifies the situation. Another circumstance, in which high renal
and systemic pH levels are a complication, is due to pathologically altered renal
physiology. Renal tubular iron transport can be due to either inherited or acquired
deficiencies. Toward imaging the spatial distribution of pH and developing a noninvasive
assessment of disease extent, pH sensitive MRI contrast agents make a considerable
contribution to biomedical and clinical purposes.
In 2003, Sherry et al. developed a renal and systemic pH contrast enhanced MR
imaging agent. A simultaneous injection of pH sensitive Gd-DOTA-4AmP
5-
and the pH
insensitive tetraphosphonate analog Gd-DOTP
5-
allows monitoring of the

54
pharmacokinetics of Gd-DOTP
5-
prior and following injection of the Gd-DOTA-4AmP
5-
.
It was reasoned that the two contrast agents would have the same time dependent
distribution allowing for prediction of the concentration of the pH-sensitive agent. The
feasibility of imaging tissue pH in mice was successfully demonstrated allowing for
future advancement in the monitoring of renal tubular ion transport and pH distribution.
22

Meade et al. made a new approach toward prodrug-procontrast agents in 2006
with the synthesis and characterization of a Doxorubicin-Gd
3+
contrast agent conjugate.
Doxorubicin a sub-micromolar potent cancer therapeutic was attached to a Gd
3+
complex
using an acid labile hydrazine linker (Scheme 3.4); thus allowing for cleavage in the
acidic environment of tumor beds. Cleavage of the doxorubicin moiety caused a decrease
in the relaxivity due to the decrease in molecular weight and increase in the τ
R
. This
result was unexpected and less desirable than an increase in the water coordination
number or hydration state (q). Additionally, the MRI contrast agent would need to be
affective at a much lower concentration to parallel the doxorubicin dosage administered
clinically.
23

Gd
N N
N N
O
O
O
O
O
O
N
O
N
HO
O
OH
HO
O
O
O
OH
H
O
CH
3
OH
NH
2
O
HO
O
OH
HO
O
O
O
OH
O
CH
3
OH
NH
2
Gd
N N
N N
O
O
O
O
O
O
O
O
H
+

Scheme 3.4. Doxorubicin-Gd complex designed to release doxorubicin when exposed to
low pH and concomitantly undergo an increase in relaxivity.

55
Tan et al. achieved targeted chemotherapy and simultaneous magnetic resonance
imaging (T
2
) of cancer cells in vitro in 2011 via a smart multifunctional nanostructure
(SMN). The constituents of the SMN are a porous hollow magnetite nanoparticle
(PHMNP), a heterobifunctional polyethylene glycol (PEG) ligand, and an aptamer. The
PHMNP with acid-labile pores allowed for loading doxorubicin (DOX) for cancer
therapy, and the aptamers enabled targeting of receptors on the cell membrane and
endocytosis of the SMN into the cell where it is exposed to the lysosome pH enabling
DOX to be released. This advanced platform combines both passive targeting through the
EPR effect and active targeting through the aptamer functionalization promoting
decreased nonspecific killing of noncancerous cells in vitro as well as a means for
simultaneous magnetic resonance imaging.
24

3.2.4. Activation of MRI contrast agents by light.
The use of optical imaging to monitor bioluminescence is a helpful tool to
scientists and clinicians in the fields of cellular and molecular biology to monitor gene
expression and cell pathways. The major drawback to the optical imaging of
bioluminescence is the inherent limited tissue penetration depth of optical imaging. As a
natural parallel to the optical imaging of bioluminescence, light responsive MRI contrast
agents have been developed addressing the issue with noninvasive tissue depth
penetration. Applications of light responsive MRI include indicating gene expression
through exploitation of light emitting gene markers such as lucerase-luciferin. In 2006,
Louie et al. developed a novel photochromically-controlled, reversibly-activated light
responsive MRI contrast agent (Scheme 3.5).
25

56
Gd
N
N N
N
O
O
O
O
O
O
O
NO
2
N
Gd
N
N N
N
O
O
O
O
O
O
O
NO
2
N
Dark
Visible Light

Scheme 3.5. Proposed isomerization of Gd-SPDO3A (left) and Gd-MCDO3A (right).
25

The contrast agent Gd-MCDO3A (MC, merocyanine) (DO3A, 1,4,7-
tris(carboxymethylaza)cyclododecane-10-azaacetylamide) utilizes a light responsive
molecular switch derived from the spiropyran (SP) class of compounds. The electrostatic
interaction of the spiropyran moiety with the metal ion inhibits inner-sphere water
coordination; thus, the system responds to both ultra violet and visible light reversibly
with a 21% decrease in relaxivity of Gd-MCDO3A upon exposure to light to produce
isomer Gd-SPDO3A (Table 3.4).
25
In 2012, the Gd-SPDO3A (SP, spiropyran) system
was further developed, and LEDs were used to mimic firefly luciferase demonstrating
feasibility of in vivo systems.
26

The spiropyran (SP) system was extended to a T
2
MRI contrast agent by Louie et
al. when attached to dextran sulfate coated iron oxide nanoparticles (ADIO). In this
system, the SP is covalently bound to the surface of a cross-linked and aminated dextran
sulfate coated SPIONs. The emission wavelength of firefly luciferase and the absorbance
wavelength of the CLADIO-NH-SP magnetic nanoparticle were demonstrated to overlap

57
significantly. This system switches between hydrophilic and hydrophobic isomers upon
introduction to visible light producing an increase in relaxivity due to agglomeration of
the magnetic nanoparticles.
27

Table 3.4. Relaxation time for aqueous CLADIO-NH-SP in the dark and with visible
light.
27

CLADIO-NH-SP

T
1
(ms) T
2
(ms)
Dark 505 37.09 ± 3.09
Light 496 24.55 ± 1.86
3.3. Externally activated MRI contrast agents.
External activation of contrast agents has been achieved for ultrasound contrast
agents. Microbubbles (MB) and their various formulations are capable of being disrupted
via ultrasound; thus creating enhancement of the contrast.
28
Although ultrasound and
MRI have two very different mechanisms of contrast, microbubble application to MRI

58
contrast can be achieved through primarily a T
2
effect with relaxivity proportional to field
strength, microbubble volume fraction, and mircobubble size (diameter).
29

However, the field of external activation of gadolinium containing small molecule
MRI contrast agents is virtually uncharted scientific territory. External by our definition
applies to the “activation of the MRI contrast agent by a means physically outside of the
media in which the MRI contrast agent resides.” To the best of our knowledge, we have
developed the first switchable, sono-activated “On-Off-On” small molecule MRI contrast
agent. Chapter 4 of this work will outline: (1) the assembly of a Gd-DOTA-shell
compound in which the novel 3-layer fluorous shell serves to block water partitioning
from the gadolinium core, (2) the relaxivity measurements of the MRI contrast agent Gd-
DOTA, “on” state, Gd-DOTA-shell “off” state, Gd-DOTA-shell-urea (mimic for shell in
excess in solution), stasis, and Gd-DOTA-shell with applied sonication “on” state, and
(3) control experiments that validate the results.
3.4. References.

1) Brown, M. A.; Semelka, R. C.; MRI: Basic Principles and Applicaions, Wiley-
Liss, New York 2003.
2) Merbach, A. E.; Toth, E. The Chemistry of Contrast Agents in Medicinal
Magnetic Resonance Imaging; John Wiley & Sons Ltd.: Chichester, U.K., 2001,
471.
3) Leon-Rodriguez, L. M.; Lubag, A. J. M.; Malloy, C. R.; Martinez, G. V.;
Gilliwes, R. J.; Sherry, A. D. Responsive MRI Agents for Sensing Metabolism in
Vivo. Acc. Chem. Res. 2009, 42, 948-957.

59

4) (a) Tu, C.; Osborne, E. A.; Louie, A. Y. Activatable T
1
and T
2
Magentic
Resonance Imaging Contrast Agents. Annals Biomed. Eng. 2011, 39,1335-1348.
5) Moats, R. A.; Fraser, S. E.; Meade, T. J. A “Smart” Magnetic Resonance Imaging
Agent That Reports on Specific Enzymatic Activity. Angew. Chem., Int. Ed. 1997,
36, 726−728.
6) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats, R.; Jacobs, R.
E.; Fraser, S. E.; Meade, T. J. In vivo Visualization of Gene Expression Using
Magnetic Resonance Imaging. Nat. Biotechnol. 2000, 18, 321−325.
7) Lee, J.; Zylka, M. J.; Anderson, D. J.; Burdette, J. E.; Woodruff, T. K.; Meade, T.
J. A Steroid-Conjugated Contrast Agent for Magnetic Resonsance Imaging of
Cell Signaling. J. Am. Chem. Soc. 2005, 127, 13164-13166.
8) Sukerkar, P. A.; MacRenaris, K. W.; Townsend, T. R.; Ahmed, R. A.; Burdette, J.
E.; Meade, T. J. Synthesis and Biological Evaluation of Water-Soluble
Progesterone-Conjugated Probes for Magnetic Resonance Imaging of Hormone
Related Cancers. Bioconjugate Chem. 2011, 22, 2304-2316.
9) Yigit, M. V.; Mazumdar, D.; Kim, H. –K.; Lee, J. H.; Odintsov, B.; Lu, Y. Smart
“Turn-On” Magnetic Resonance Contrast Agents Based on Aptamer-
Functionalized Suparamagnetic Iron Oxide Nanopatricles. Chem. Bio. Chem.
2007, 8, 1675-1678.
10) Strauch, R. C.; Mastarone, D. J.; Sukerkar, P. A.; Song, Y.; Isaro, J. J.; Meade, T.
J. Reporter Protein-Trageted Probes for Magnetic Resonance Imaging. J. Am.
Chem. Soc. 2011, 133, 16346-16349.
11) Nivorozhkin, A. L.; Koloodziej, A. F.; Caravan, P.; Greenfield, M. T.; Lauffer, R.
B.; McMurry, T. J. Enzyme-Activated Gd
3+
Magnetic Resonance Imaging
Contrast Agents with a Prominent Receptor-Induced Magnetization Enhancement.
Angew. Chem. Int. Ed. 2001, 40, 2903-2906.
12) Louie, A. Multimodality imaging probes: design and challenges. Chem. Rev.
2010, 110, 3146–3195.
13) Matsumura, S.; Aoki, S.; Shiba, K. A Tumor-Environment-Responsive
Nanocarrier That Evolves Its Surface Properties upon Sensing Matrix
Metalloproteinase-2 and Initiates Agglomeration to Enhance T
2
Relaxivity for
Magnetic Resonance Imaging. Mol. Pharmaceutics 2011, 8, 1970-1974.

60

14) Que, E. L.; Chang, C. Responsive Magnetic Resonance Imaging Contrast Agents
as Chemical Sensors for Metals in Biology and Medicine. Chem. Soc. Rev. 2010,
39, 51-60.
15) Li, W. –H.; Fraser, S. E.; Meade, T. J. A Calcium-Sensitive Magnetic Resonance
Imaging Contrast Agent. J. Am. Chem. Soc. 1999, 121, 1413-1414.
16) Esqueda, A. C.; López, J. A.; Andreu-de-Riquer, G.; Alvarado-Monzón, J. C.;
Ratnakar, J.; Lubag, A. J. M.; Sherry, A. D.; De León-Rodriguez, L. M. A New
Gadolinium-Based MRI Sensor. J. Am. Chem. Soc. 2009, 131, 11387−11391.
17) (a) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. The Synthesis and in vitro
Testing of Zinc-Activated MRI Contrast Agent. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 13881-13886. (b) Major, J. L.; Boiteau, R. M.; Meade, T. J.
Mechanisms of ZnII-Activated Magnetic Resonance Imaging Agents. Inorg.
Chem. 2008, 47, 10788-10795.
18) Hanokam K.; Kikuchi, K.; Urano, Y.; Nagano, T. Selective Sensing with Zinc
Ions with a Novel Magentic Resonance Imaging Contrast agent. J. Chem. Soc.,
Perkin Trans. 2001, 2, 1840-1843.
19) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Narazaki, M.; Yokawa, T.; Sakamoto, S.;
Yamaguchi, K.; Nagano, T.; Design and Synthesis of a Novel Magnetic
Resonance Imaging Contrast Agent for Selective Sensing of Zinc Ion. Chem. Biol.
2002, 9, 1027-1032.
20) Luo, J. Li, W. –S.; Xu, P.; Zhnag, L. –I.; Chen, Z. –N. Zn
2+
Responsive Bimodal
Magnetic Resonance Imaging and Fluorescent Imaging Probe Based on a
Gadolinium(III) Complex. Inorg. Chem. 2012, 51, 9508-9516.
21) Zhang, X.; Jing, X.; Liu, T.; Han, G.; Li, H.; Duan, C. Dual-FunctionalFuctional
Gadolinium-Based Copper(II) Probe for Selective Magnetic Resonance Imaging
and Fluorescence Sensing. Inorg. Chem. 2012, 51, 2325-2331.
22) Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies, J. Renal and
Systemic pH Imaging by Contrast-Enhanced MRI. Magn. Reson. Med. 2003, 49,
249-257.
23) Frullano, L.; Tejerina, B.; Meade, T. J. Synthesis and Characterization of a
Doxorubicin-Gd(III) Contrast Agent Conjugate: A New Approach toward
Prodrug-Procontrast Complexes. Inorg. Chem. 2006, 45, 8489-8491.

61

24) Chen, T. Shukoor, M. I.; Wang, R.; Zhao, Z.; Yuan, Q.; Bamrungsap, S.; Xiong,
X.; Tan, W. Smart Multifunctional Nanostructure for Targeted Cancer
Chemotherapy and Magnetic Resonance Imaging. ACS Nano 2011, 5, 7866-7873.
25) Tu, C.; Louie, A. Y. Photochromically-Controlled, Reversibly-Activated MRI and
Optical Contrast Agent. Chem. Comm. 2007, 13, 1331-1333.
26) Kruttwig, K.; Yankelevich, D. R.; Brueggemann, C.; Tu, C.; L’Etoile, N.;
Knoesen, A.; Louie, A. Y. Reversible Low-Light Induced Photoswitching of
Crowned Spiropyran-DO3A Complexed with Gadolinium(III) Ions. Molecules
2012, 17, 6605-6624.
27) Osborne, E. A.; Jarrett, B. R.; Tu, C.; Louie, A. Y. Modulation of T
2
Relaxtion
Time by Light-Induced, Reversible Aggregation of Magnetic Nanoparticles. J.
Am. Chem. Soc. 2010, 132, 5934-5935.
28) (a) Grinstaff, M. W.; Suslick, K. S. Air-filled Proteinaceous Microbubbles –
Synthesis of an Echo – Contrast Agent. Proc. Natl. Acad. Sci. USA 1991, 88,
7708-7710. (b) Sirsi, S.; Borden, M. Microbubble Compositions, Properties and
Biomedical Applications. Bubble Sci. Eng. Technol. 2009, 1, 3-17.
29) Cai, X.; Yang, F.; Gu, N. Applications of Magnetic Microbubbles for
Theranostics. Theranostics 2012, 2, 103-112.

62
Chapter
4.
External
activation
of
Magnetic
Resonance

Imaging
contrast
agents
by
sonication.

4.1. Introduction. Magnetic Resonance Imaging and Nuclear Magnetic
Resonance.
The basic scientific principles of MRI are derived from nuclear magnetic
resonance (NMR). The development of clinical MRI from NMR by Paul Lauterbur and
Peter Mansfield pushed forward the forefront of medicinal chemistry.
1
The mechanism of
contrast enhancement utilized by gadolinium-based contrast agents involves accelerating
the T
1
, the spin-lattice relaxation time constant, of the bulk water surrounding the metal;
thus these are said to be “T
1
contrast agents.”
2
The degree of efficacy of the agent can be
quantified by the decrease in the T
1
of the water in which the contrast agent is dissolved.
T
1
values can be measured on any NMR spectrometer with a T
1
pulse sequence (Figure
4.1) making the assessment of the T
1
contrast agent relatively facile using basic NMR
tools.
3

Figure 4.1. Varian’s “T
1
Measure” Inversion Recovery Pulse Sequence. Axes are
transmit power versus time for the proton (T
x
) and carbon (Dec) channels for the three
portions of the pulse sequence, delay (A), pulse (B), and acquire (C). d1 = recycle delay
(s); p1 = broadband inversion pulse width (ms); d2 = interpulse delay (ms); pw =
detection pulse width, (ms); at = acquisition time (s).

63
Magnetic resonance imaging (MRI) is a well-recognized method for non-invasive
imaging of cells and human bodies.
4
Recent focus in the field of MRI contrast agents is
centered around the development of smart contrast agents that are capable of producing
contrast in response to small molecules, enzymes, pH, etc. within physiological
environments. These types of contrast agents are referred to as activatable or “smart MRI
contrast agents.”
5

Advancements in smart MRI contrast agents logically extend to
externally-activated MRI contrast agents. By our definition, an externally-activated MRI
contrast agent is an agent that is activated by means outside of the solution environment
in which the agent is residing. Thus, an external activation mechanism is different from
the ones triggered by environmental factors such as pH, biological markers, enzymes,
metal ions, etc. The ability to externally control MRI contrast and to site-selectively
“light up” an area of particular interest by non-invasive means is of great appeal to both
chemists and clinicians alike. Previously, a few UV/visible light activated MRI contrast
agents were developed by Louie et al.
6
However, these experiments are ultimately
intended for the purpose of activation by bioluminescence from luceferin-luciferase in
physiological systems to probe gene expression using non-invasive methods. This work
discusses the demonstration of the first small molecule Gd
3+
contrast agent·shell
interaction (Gd–DOTA·Shell) that attenuates the relaxivity of the small molecule contrast
agent, and upon external activation via sonication, the shell is removed and contrast of
Gd–DOTA is restored.

64
N N
N N
Gd
O
O
O
O
O
O
O
O
N N
N N
Gd
P
O
P
O
O
O
P
O
O
P
O
O
O
O
O
O
5
4.1 4.2

Figure 4.2. Small molecule contrast agents Gd–DOTA (4.1) and Gd–DOTP (4.2).
This work focuses on the interaction of two small molecules, Gd–DOTA (4.1)
and Gd–DOTP (4.2) (Figure 4.2), with a previously-reported tri-layered, guanidine-
terminated fluorous, amphiphilic shell molecule (4.3, Figure 4.3) with regards to T
1

values and respective r
1
curves.
7
The conceptual premise is that the shell (4.3) will hinder
water partitioning to the Gd
3+
center of the Gd–DOTA (4.1) or Gd–DOTP (4.2) via
interaction with the contrast agent through hydrogen bonding interactions of the guanyl
head hydrogen atoms and the oxygen atoms of the ligand (L) thus producing an “off-
state” of the MRI contrast agent. Shell 4.3 incorporates a guanidinium head group, which
is designed to participate in hydrogen bonding with phosphonates and carboxylates, a
fluorous phase group to inhibit water partitioning, and a hydrophilic PEG chain appended
as a solubilizing group.
7
Urea (isostere for guanyl group) is added to the Gd–DOTAShell
system. The excess of urea in the system is proposed to facilitate restoration of the
relaxivity of the Gd–DOTA contrast agent, “on-state”. Upon sonication through
interacting with the Gd–DOTA in the same fashion as the shell, the urea presence in
excess will prevent the reforming of the hydrogen bonds between the contrast agent and
4.3 by blocking the binding sites. Urea is less sterically bulky than the 4.3 and likely to
allow water partitioning to the Gd
3+
center more readily than 4.3.

65
H
2
N N
H
NH
2
F F
F F
F F
F F
O
O
O
O
O
O
O
F
3
C

Figure 4.3. Guanidine-terminated fluorous amphiphilic shell molecule (4.3).
7

4.2. External activation and comparison of Gd–DOTAShell and Gd–
DOTP systems.
Treatment of Gd–DOTA (4.1), with a functionalized guanidine, (4.3), reduces the
magnetic relaxivity of 4.1.
1
H inversion recovery was measured (Scheme 4.1) to generate
a relaxivity curve of 1/T
1
(s
-1
) vs. [Gd
3+
] (mM). The relaxivity of the Gd–DOTAShell
remains the same upon addition of urea. Upon treating the combination of 4.1, 4.3, and
urea (150 mM) with a bench top sonicator, a portion of the relaxivity is recovered.
N
N N
N
Gd
O
O
O
O
O
O
O
O
Shell (C
4
F
8
)
4 equiv.
Measure T
1
Sonication
Measure T
1
Urea
150 mM
Na
4.1
(4.3)
Measure T
1

Scheme 4.1. T
1
inversion recovery measurements taken after addition of shell (4.3), urea,
and application of sonication to the Gd–DOTAShell with Urea system.

66
The addition of 4.3 attenuates the contrast of 4.1 to produce an “off-state” as
observed by the decrease in r
1
in aqueous solution at 400 MHz at room temperature,
plotted from 0–1.0 mM, from 3.0(1) mM
-1
s
-1
to 2.7(2) mM
-1
s
-1
indicating a blocking of
H
2
O exchange at the Gd
3+
center. Upon addition of urea the r
1
value remains constant at
2.7(2) mM
-1
s
-1
. Upon sonication of the Gd–DOTAShell system with urea, the
r
1
increases to 3.0(1) mM
-1
s
-1
indicating the return of the contrast by removal of shell 4.3.
Results are summarized in Figure 4.4.

Figure 4.4. Plot of r
1
values for aqueous solutions containing Gd–DOTA (4.1), Gd–
DOTAShell, Gd–DOTAShell with urea, and Gd–DOTAShell with urea and
sonication obtained from water-proton relaxation data obtained for 0–1 mM [Gd
3+
] with 4
equiv. shell (4.3) relative to [Gd
3+
] and 150 mM [urea].
Xinping Wu demonstrated using
31
P NMR that the shell binds to methyl
phosphonic acid to form a macromolecular assembly, conceivably through a bidentate
salt bridge.
7
Findings presented in this work establish that unlike Gd–DOTA (4.1), the
relaxivity of Gd–DOTP (4.2) is not diminished but enhanced in the presence of the shell.
Slower tumbling of the macromolecular Gd–DOTPShell assembly most likely results in

67
an increase rotational correlation time (τ
R
) contributing to the enhancement of r
1
. The
relaxivity of Gd–DOTP (4.2) is 3.04(16) mM
-1
s
-1
in aqueous solution at 400 MHz at
room temperature, which is consistent with literature values.
8
Upon addition of shell
(4.3), the relaxivity of Gd–DOTP (4.2) (3.67(30) mM
-1
s
-1
) was enhanced. Addition of
urea to Gd–DOTP–Shell causes a slight decrease in r
1
to 3.56(22) mM
-1
s
-1
. However, the
decrease was within error and considered insignificant. The subsequent sonication of the
Gd–DOTPShell system with urea does not significantly affect r
1
(3.62(24) mM
-1
s
-1
)
(Figure 4.5.).

Figure 4.5. Plot of r1 values for aqueous solutions containing Gd–DOTP (4.2),
Gd–DOTPShell, Gd–DOTPShell with urea, and Gd–DOTPShell with urea and
sonication obtained from water-proton relaxation data obtained for 0 – 1 mM [Gd
3+
] with
4 equiv. shell relative to [Gd
3+
] and 150 mM [urea].
The binding of 4.3 to small molecule phosphonates is characterized and
demonstrates an intimate interaction between 4.2 and 4.3.
7
The interaction of
phosphate-coated gold nanoparticles containing a Gd–DOTA functionalized thiol and 4.3
was characterized via
19
F T
1
and DLS data.
9

19
F T
1
data of a 1 mM solution of 4.1 and

68
Gd–DOTAShell corroborates binding of 4.3 to 4.1. The T
1
(s) values of the three
19
F
signals (Peaks A, B, and C) of shell (4.3) (1 mM) are 501(16), 474(15), and 527(8) ms,
which are reduced to 150(3), 221(6), and 213(4) ms respectively. The decrease in
19
F T
1

values of the shell fluorine atoms indicates a close proximity to the Gd
3+
metal center.
Additionally, Vincent Li demonstrated that exposure of the shell (4.3) to 1.0 mM GdCl
3

(0.25 molar equivalents relative to 4.3) resulted in a less significant change in
19
F T
1
than
Gd–DOTAShell (Table 4.1), which shows that high [Gd
3+
] alone can not account for the
observed decrease in
19
F T
1
in the Gd–DOTAShell system.
9

Table 4.1. Decrease in
19
F T
1
(ms) when Gd – DOTA is added to (4.3).
Compilation of
Matter
Peak A
-117.9 ppm
Peak B
-119.8 ppm
Peak C
-123.5 ppm
4.3 501(16) ms 474(15) ms 527(8) ms
4.3 + 4.1 150(3) ms 221(6) ms 213(4) ms
4.3 + GdCl
3
429(13) ms 396(8) ms 466(6) ms
Further, upon titration of 4.1 into an aqueous solution of 4.3 to a final 1:2
equivalents, 4.1:4.3, a shift in the
19
F NMR spectrum was observed. The
19
F NMR
spectrum of 4.3 without Gd–DOTA contains peaks at -118.5, -120.4, -124.1, and 124.2
ppm, which shifted to -118.3, -120.4, and -123.9 ppm respectively upon addition of 4.1
with each spectrum referenced to an external CFCl
3
standard (Figure 4.6). This indicates
that the fluorous region of 4.3 is in close proximity to the Gd
3+
metal ion causing a shift
downfield and broadening of Peak C (Figure 4.6).

69
H
2
N N
H
NH
2
F F
F F
F F
F F
O
O
O
O
F
3
C
N
N N
N
Gd
O
O
O
O
O
O
O
O
Na 4 H
2
O
ACQUIRE
19
F
4

Peak A Peak B Peak C
Figure 4.6.
19
F spectra of Shell (bottom) and Gd–DOTAShell (1:2) (top).
Additionally, evidence of shell proximity to the Gd
3+
metal center is corroborated
by MALDI MS data where Gd–DOTA and Gd–DOTAShell mass peaks can be
distinguished (Figure 4.7). Gd–DOTAShell mass peaks can be distinguished clearly at
[M+H]
+
= 1052.96 g/mol (theor. 1052.27 g/mol).

70

Figure 4.7. MALDI mass spectra of [HGd(DOTA)Shell]
+
.
In order to determine the optimal amount of shell (4.3) required to achieve the
maximum contrast between the Gd–DOTA and Gd–DOTAShell aqueous solutions, a
titration experiment was performed in which T
1
(s) values were obtained for Gd–DOTA
(0.5 mM) with addition of 0, 1, 2, 3, and 4 equivalents of shell. The initial increase in the
T
1
(s) value of the aqueous solution containing Gd–DOTA (T
1
= 0.299(3) mM
-1
s
-1
) upon
addition of 1 equivalent of 4.3 (T
1
= 0.452(12) mM
-1
s
-1
) from was maintained upon
additional equivalents of shell (2, 3, 4 equivalents, T
1
= 0.455(13), 0.412(10), and
484(5) mM
-1
s
-1
respectively). T
1
values represented as the inverse, 1/T
1
s
-1
, for Gd–
DOTA and Gd–DOTA + Shell (0, 1, 2, 3, 4 equivalents) are summarized in Figure 4.8.
[M+H]
+

1052.96
Na
+
[M+H]
+

1072.90

71

Figure 4.8. Resulting T
1
values (mM
-1
s
-1
) Gd–DOTA
a
titration with 0, 1, 2, 3, and 4
equivalents of shell.
a
[CaCl]
+
[Gd(DOTA)]
-
· 2.5 H
2
O (see Ch. 7.4.1. for synthesis).
The shell titration experiment was also performed for the Gd–DOTP (4.2) analog;
the r
1
value of the aqueous solution containing Gd–DOTP gradually increases to
3.86(22) mM
-1
s
-1
from 3.04(16) mM
-1
s
-1
upon addition of 1, 2, 4, and 8 equivalents of
shell (Figure 4.9).

Figure 4.9. Resulting r
1
values (mM
-1
s
-1
) (0-1.0 mM [Gd
3+
]) of 4.2 titration with 1, 2, 4,
and 8 equivalents of shell.

72
4.3. Background and control experiments for Gd–DOTAShell–Urea–
Sonication system.
Determination of the background T
1
is important to generate an accurate
representation of the r
1
curve. In order to achieve an understanding of the background
contrast, a series of experiments was performed. Urea (50, 150, and 250 mM) was added
to Gd–DOTA (4.1) (0.5 mM) to determine the affect of urea on the T
1
(s) of an aqueous
solution containing 4.1. Addition of urea to 4.1 increases the T
1
of an aqueous solution
containing Gd–DOTA from 371(6) ms to 448(9) ms, 456 (8) ms, and 434(10) ms for 50,
150, and 250 mM of urea respectively (Figure 4.10). The contrast of 4.1 in the presence
of urea exhibits an increase in T
1
. However, this increase is not concentration dependent
in [urea]. This result is consistent with the slight increase in the T
1
value observed upon
addition of urea to the Gd–DOTAShell solution (Figure 4.7).

Figure 4.10. T
1
(s) value of an aqueous solution containing Gd–DOTA (4.1) (0.5 mM)
upon addition of urea (50, 150, and 250 mM).

73
The affect of urea on the Gd–DOTAShell system was determined by addition of
urea across a biologically relevant concentration range. Urea does not have a significant
effect on the Gd–DOTAShell system as demonstrated by the T
1
values of an aqueoues
solution of Gd–DOTA (1 mM)–Shell (4 equiv.) with [Urea] = 50, 150, and 250 mM
(Figure 4.11).

Figure 4.11. T
1
(s) of an aqueous solution containing Gd–DOTAShell (0.5 mM) upon
addition of urea (50, 150, and 250 mM).
The ideal system would be effective at physiological pH. Previous data in this
chapter are reported for non-buffered aqueous systems. However, early studies were
conducted to probe the effectiveness of the on-off-on Gd–DOTAShell system with
sonication in the presence of several biological buffers (HEPES and Tris) as well as
NaHCO
3
. Results in Table 4.2 are given as an increase or decrease in T
1
with respect to
the previous acquisition.

74
Table 4.2. Preliminary observations of Gd–DOTAShell system with urea and sonication
in various biological buffer systems.
Buffer
Results of Shell
Addition (T
1
increase)
Results of Urea
Addition (T
1

increase)
Results of Sonication
(T
1

decrease)
NaHCO
3
34-77% 0% 30%
HEPES
7%
10% 0%
Tris 0% - - - -
Tris
a
6% - - - -
None
a
7-47% 0-1% 2-3%
NaHCO
3
a
55% 5% 4%
a
Gd–DOTA synthesized from Gd
2
O
3
; therefore, no HCl byproduct in solution.
The buffering of the Gd–DOTAShell system with urea is important because
guanidine-carboxylate and guanidine-phosphate binding should only work effectively in
a fixed pH range: the guanidine should be protonated, and the phosphate or carboxylate
should not be fully protonated. Although NaHCO
3
was useful for the acquisition of the
results described above, it is not representative of physiological conditions.
Table 4.2 shows a summary of our observations of T
1
values of the Gd–
DOTAShell system with urea and sonication in HEPES and Tris buffers. Our system is
sufficiently effective in neither. In a HEPES solution, it appears that the shell binds to
Gd–DOTA as indicated by an increase in T
1
, but the relaxivity does not change when the
complex is sonicated in the presence of urea. In Tris buffer, the shell does not appear to
bind the Gd–DOTA. In the absence of HCl byproduct, no buffer seems to be required for
the Gd–DOTAShell system with urea to respond in the desired on-off-on fashion. It is
important to note that these experiments were performed prior to the synthesis of

75
Gd–DOTA as described in Chapter 7.3.; thus these preliminary assessments of buffer
systems may need revisiting to best conclude the most appropriate buffer system.
4.4. Phenolphthalein encapsulation by Shell-OH.
Phenolphthalein (phth) is a chemical indicator that reacts with NaOH to produce a
characteristic fuchsia colored solution. With the objective of encapsulating the dye, phth,
in a structure of sorts formed with a fluorous alcohol molecule (Shell–OH, 4.4,
Figure 4.9), two identical samples were created with the exception that the experimental
sample contains the Shell–OH molecule and the control does not contain Shell–OH. Both
samples contain 4 mL of H
2
O and 2 µL of phenolphthalein. It was apparent that the shell
alcohol in solution visually caused a significantly decreased appearance of the fuchsia
color indicated by the reaction of phth with NaOH, which produces a bright fuchsia color
in the control sample. Visually, appears that the phenolphthalein is caged in some
fashion due to loss of color in solution. Next, the experimental and control vials were
sonicated. At which point, the experimental solution seemed to appear more colored upon
initial sonication (2 x 15 sec) bursts. After continued sonication ~ 2 min the experimental
solution seemed to get less colored than the original experimental solution prior to
sonication (nearly colorless). The control solution remained the same color throughout.
F F
F F
F F
O
O
O
O
O
F F
F F
F F
HO

4.4
Figure 4.2. Structure of Shell–OH.

76
Lastly, a control experiment was performed to ensure that the difference in
reaction of NaOH with indicator (phth) was not purely a pH argument in which the shell-
OH was acting as an acid and reacting with NaOH (base) causing no appearance of the
fuchsia indicator. In this experiment, and equal amount of trifluoroethanol (pKa = 12.5)
was added to the control sample. This experiment proceeded identically to the previous
experiment until the sonication step, where no return of fuchsia color was visualized in
the experimental sample.
4.5. Conclusions.
We have developed a two-component system in which contrast from a MRI contrast
agent, Gd–DOTA, is masked by a proprietary shell formulated to be water impenetrable
yet water soluble. The hydrogen bonding interaction holding the shell to the contrast
agent prevents water exchange with the paramagnetic gadolinium core, thus attenuating
contrast. At the desire time and location, the contrast agent-shell interaction can be
selectively disrupted externally with sonication to restore the contrast (11% restored).
These properties have been studied extensively by
1
H NMR T
1
inversion recovery
experiments.
4.6. References.

1) (a) Mewis, R. E.; Archibald, S. J. Biomedical Applications of Macrocyclic Ligand
Complexes. Coordination Chem. Rev. 2010, 254, 1686-1712. (b) Bonnet, C. S.;
Toth, E. Smart MRI Imaging Agents Relevant to Potential Neurological
Applications. Am. J. Neurodiol. 2010, 31, 401-409.
2) (a) de Haen, C. Conception of the First Magnetic Resonance Imaging Contrast
Agents: A Brief History. Topics Magn. Reson, Imaging 2001, 12, 221-230. (b)
Geraldes, C. F.; Laurent, S. Classification and Basic Properties of Contrast Agents
for Magnetic Resonance Imaging. Contrast Media Mol. Imaging, 2009, 4, 1-23.

77

(c) Mewis, R. E.; Archibald, S. J. Biomedical Applications of Macrocyclic Ligand
Complexes. Coordination Chem. Rev. 2010, 254, 1686-1712. (e) Caravan, P.
Strategies for Increasing the Sensitivity of Gadolinium Based MRI Contrast
Agents. Chem. Soc. Rev. 2006, 35, 512-523.
3) (a) Manus, L. M.; Strauch, R. C.; Hung, A. H.; Eckermann, A. L.; Meade, T. J.
Analytical Methods for Characterizing Magnetic Resonance Probes. Analytical
Chem. 2012, 84, 6278-6287. (b) Fry, C. G. The Nobel Prize in Medicine for
Magnetic Resonance Imaging. J. Chem. Educ. 2004, 81, 922-932.
4) (a) Brown, M. A.; Semelka, R. C.; MRI: Basic Principles and Applications,
Wiley-Liss, New York 2003.
5) (a) Tu, C.; Osborne, E. A.; Louie, A. Y. Activatable T
1
and T
2
Magnetic
Resonance Imaging Contrast Agents. Ann. Biomed. Eng. 2011, 39, 1335-1348. (b)
Sosnovik, D. E.; Weissleder, R. Emerging Concepts in Molecular MRI. Curr.
Opin. Biotechnol. 2007, 18, 4-10. (c) Lowe, M. P. Activated MR Contrast Agents.
Current Pharmaceutical Biotech. 2004, 5, 519-528.
6) Tu, C.; Louie, A. Y. Photochromically-Controlled, Reversibly-Activated MRI and
Optical Contrast Agent. Chem. Comm. 2007, 13, 1331-1333. (b) Kruttwig, K.;
Yankelevich, D. R.; Brueggemann, C.; Tu, C.; L’Etoile, N.; Knoesen, A.; Louie,
A. Y. Reversible Low-Light Induced Photoswitching of Crowned Spiropyran-
DO3A Complexed with Gadolinium(III) Ions. Molecules 2012, 17, 6605-6624.
7) Wu, X.; Boz, E.; Sirkis, A. M.; Chang, A. Y.; Williams, T. J. Synthesis and
Phosphonate Binding of Guanidine-Functionalized Fluorinated Amphiphiles. J.
Fluor. Chem. 2012, 135, 292-302.
8) Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies, R. J. Renal and
Systemic pH Imaging by Contrast-Enhanced MRI. Magn. Reson. Med. 2003, 49,
249-257.
9) Li, V.; Chang, A. Y.; Williams, T. J. A Noncovalent, Fluorylakyl Coating
Monomer for Phosphonate-Covered Nanoparticles. Tetrahedron, submitted.

78
Chapter
5.
A
molar
relaxivity
experiment
in
a
High
School

classroom.

5.1. Introduction. Magnetic Resonance Imaging: back to the basics.
This experiment requires access to a nuclear magnetic resonance (NMR)
spectrometer or relaximeter, which are tools that are not generally available to high
school teachers. However, the U. S. National Science Foundation has prioritized both
cyber-enabling of chemical instrumentation
1
and K-12 outreach
2
to promote interaction
between K-12 students and educators and NSF-sponsored investigators and facilities. It is
in this spirit that we offer this as a high school lab experiment.
3
The work described
within this chapter was published essentially as is with minor changes in the
Journal of Chemical Education.
4
The experiments described were performed in
collaboration with Kathryn Hathaway, High School teacher, Susie Kim, and a class of
students at Polytechnic School in Pasadena, California. Kathryn helped to synthesize and
characterize the MRI contrast agents as well as ran the remote operation of the NMR
console. The experiment may be easily adopted for any introductory chemistry class or
undergraduate analytic chemistry lab.
Magnetic resonance imaging is a common imaging modality in modern
medicine,
5
and thus is a technique with which most high school students have at least
passing familiarity. Many MRI images are acquired with the aid of a contrast agent drug.

79
In this experiment we introduce the concepts of MRI and MRI contrast agents in a high
school classroom by measuring the molar relaxivity curves for two clinically available
MRI contrast agents. In the process of this experiment, students gain familiarity with
magnetic relaxivity, exposure to simple data treatment, and basic analytical chemistry
skills. At the conclusion of the lab, they learn that the more relaxive of the two clinical
agents is also the more toxic. This enables a practical discussion of drug efficacy versus
drug safety and science in society.
5.2. MRI Contrast Agents.
In many clinical applications, contrast of MRI images is enhanced by the use of
one of several MRI contrast agents.
6
A highly paramagnetic metal ion, Gd
3+
in particular,
which is chelated with an appropriate ligand, provides the basis of most of the current
clinically available contrast agents in the United States.
7
Because it is paramagnetic, Gd
3+

promotes the rapid relaxation of nuclear spins that are excited in the MRI experiment.
With an appropriate pulse sequence, this results in the amplification of MRI signals from
aqueous regions that are in the proximity of the gadolinium agent.
8

The basic principles of relaxation involved in MRI are those of NMR The
mechanism of contrast enhancement utilized by gadolinium-based agents involves
accelerating the T
1
, the spin-lattice relaxation time constant,
9
of the water surrounding the
metal; thus these are said to be “T
1
contrast agents.”
10
The degree of efficacy of the
contrast agent at any given concentration can thus be quantified by how short it can make
the T
1
of the water in which it is dissolved. T
1
values can be easily measured on any

80
NMR spectrometer. Therefore it is simple to assay an agent’s efficacy using basic NMR
tools.
11

Clinical use of gadolinium-based MRI contrast agents is limited by the toxicity of
the Gd
3+
ion.
12
Because Gd
3+
is an isostere for Ca
2+
,
13
gadolinium-based MRI contrast
agents are associated with acute renal toxicity side effects, including nephrogenic
systemic fibrosis (NSF).
14
This toxicity can be mitigated by the design of the supporting
chelating ligand in which the gadolinium ion is caged.
7a
This is the basis for ongoing
research in medicinal chemistry.
15

5.3. Determination of relaxivity.
In this experiment we introduce the dual issues of MRI contrast and drug efficacy
versus drug safety. In the first part of the experiment, students prepare samples on which
T
1
values can be measured for active ingredients in two MRI contrast agents, Dotarem,
[Gd(DOTA)]
-
(5.1), and Magnevist, [Gd(DTPA)]
2-
(5.2) (Figure 5.1). In the second part
of the experiment, students process their T
1
data and find that [Gd(DTPA)]
2-
(5.2), the
active ingredient in Magnevist, is a more potent T
1
contrast agent. Upon making this
finding, they are shown the FDA warning
16
on Magnevist and taught why Dotarem is a
safer clinical agent. This enables a discussion of safety versus efficacy of these agents.

81
N
N
N
N
O
O
O
O
O
O
O
O
Gd

N
N
N
O
O
O
O
Gd
O
O
O
O
O
O

[Gd(DOTA)]
-
Anion (5.1) [Gd(DTPA)]
2-
Dianion (5.2)
Figure 5.1. Structures of the active agents in Dotarem (5.1) and Magnevist (5.2).
For this experiment we used basic laboratory glassware, standard liquid 5 mm
NMR tubes, coaxial insert tubes for 5 mm NMR tubes, disposable plastic syringes (or
equivalent), disposable needles (or equivalent) with tips removed as desired, 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid, GdCl
3
, CaCO
3
, diethylenetri-
aminepentaacetic acid, Gd
2
O
3
, D
2
O, and deionized H
2
O. Note that the water must be
deionized; bottled drinking water is effective in our experience.
T
1
data were collected on a Varian 400-MR NMR spectrometer equipped with a
7600AS autosampler. T
1
data were processed with the VNMRJ 2.3 native T
1
processing
regime and reported as measured (VNMRJ = Varian Nuclear Magnetic Resonance Java).
Remote operation of the 400-MR spectrometer was enabled using VNC (Virtual Network
Computing). The VNC server application native to Red Hat Linux was adequate to serve
a VNC session from the instrument; the session is conveniently viewed, in our
experience, with the freeware programs Chicken of the VNC for Mac (by Jason Harris)
or RealVNC or TightVNC for Windows.

82
NMR T
1
values are readily measured by an inversion-recovery pulse sequence,
which is available in any spectrometer’s sequence library (Figure 5.2).
9
Conceptually, the
method in which this experiment works is that a 180º broadband pulse (p1) is delivered to
the sample, and then a variable-length relaxation delay (d2) follows. During the delay,
some of the protons lose their 180º excitation, which is measured by a follow-up 90º
pulse (pw). Tracking the integration of the resulting NMR signal as a function of the
interpulse delay (d2) gives an exponential recovery curve with time constant T
1
.

Figure 5.2. Varian’s “T
1
Measure” inversion recovery pulse sequence. Axes are
transmit power versus time for the proton (T
x
) and carbon (Dec) channels for the three
portions of the pulse sequence, delay (A), pulse (B), and acquire (C). d1 = recycle delay
(s); p1 = broadband inversion pulse width (ms); d2 = interpulse delay (ms); pw =
detection pulse width, (ms); at = acquisition time (s).
Stock solutions of gadolinium chelates can be prepared either by the instructors
ahead of time or by the students as part of the experiment. Stock solutions of each active
agent (1 mM) were provided to students for dilution to 0.25, 0.50, and 0.75 mM. Detailed
synthetic procedures for the preparation of these solutions are presented in Supporting
Information. Students were supplied with standard 1 mM solutions and prepared dilutions
of them to the respective final concentration and final volume of 1 mL with disposable
1 mL syringes with blunted needle tips. A portion (100 mL) of each of the samples was

83
then transferred to an NMR coaxial insert tube, which was inserted into a standard 5 mm
NMR tube containing 700 mL of D
2
O.
The samples were remotely queued on a Varian 400 NMR spectrometer, and data
were acquired in automation. An initial
1
H NMR spectrum followed by a T
1
inversion
recovery experiment was acquired for each sample. FIDs were obtained and processed
using the instrument’s native VNMRJ software. T
1
data were presented to the students
with fit error, as reported by VNMRJ. Students then plotted molar relaxivity curves, T
1
-1

vs. concentration, in the classroom using Microsoft Excel (see Chapter 7.4.2). The T
1

data from groups 1–4 were combined in a comparative molar relaxivity plot to visualize
the difference in the relaxivity between the two contrast agents (Figure 5.3). This
experiment requires approximately two hours of class time.

Figure 5.3. Molar relaxivity curves of [Gd(DOTA)]
-
(5.1) (lower curve, groups 1 and 2)
and [Gd(DTPA)]
2-
(5.2) (upper curve, groups 3 and 4). Error bars describe fit errors in the
T
1
data, not measurement errors that may have been introduced by the students.

84
T
1
values were acquired for [Gd(DOTA)]
-
(5.1) vs. [(Gd(DTPA)]
2-
(5.2) (Table
5.1). We found that the average student obtained relaxivities are r
1
= 2.6(2) mM-1s-1 and
r
1
= 4.3(4) mM
-1
s
-1
for 5.1 and 5.2 respectively (Figure 5.3). With a higher r
1
(molar
relaxivity) [Gd(DTPA)]2- (5.2), the active ingredient in Magnevist, was found to be the
more relaxive contrast agent.
Table 5.1. Actual measured T
1
values for samples prepared at Polytechnic School.
Active Agent [Gd] / mM T
1
(s
-1
)
a

[Gd(DOTA)]
-
(5.1) 0.25 0.679(26)
0.50 0.488(16)
0.75 0.344(6)
1.00 0.284(3)
0.25 0.699(30)
0.50 0.484(16)
0.75 0.394(79)
1.00 0.304(34)
[Gd(DTPA)]
2-
(5.2) 0.25 0.364(9)
0.50 0.288(3)
0.75 - -
b
1.00 0.157(1)
0.25 0.339(3)
0.50 0.291(3)
0.75 0.205(3)
1.00 0.181(1)
a
T
1
values and their standard fit errors generated by Varian’s VNMRJ 2.3c software.
Standard errors are represented in parentheses in msec.
b
This sample did not conform to a
T
1
fit due to an acquisition error.

85
5.4. Drug efficacy vs. safety of MRI contrast agents.
The primary objective of high school science courses is to provide basic
knowledge of the fundamental principles of science, mainly through the study of physics,
biology, and chemistry. This often translates into a cursory investigation of each
discipline. Students gravitate towards the acquisition and memorization of concepts and
are bereft of the experience to appreciate the practical application of these ideas in real
life. Thus the opportunity to engage in a research experiment enables students to exercise
their newfound knowledge and helps foster retention of the information. In this particular
experiment, students acquired empirical data to determine drug efficacy, which they then
had to compare with actual results in the form of drug safety: in 2007 the FDA issued a
boxed warning about renal toxicity associated with Magnevist and other gadolinium-
based contrast agents.
16
Dotarem, by contrast, is designed to avoid this toxicity
problem.
17

When faced with the notion that other factors need to be accounted for in data
analysis, students then are forced to realize that science is not simply about learning
formulae and plugging in numbers, but that its utility comes in assimilating knowledge
with reality. Thus, we use this type of exercise to encourage students to recognize that
beyond the basics taught in the classroom, there is real practical application of these
concepts in our everyday lives.
Kathryn Hathaway a High School student at Polytechnic High School working in
our lab, assessed the student experience in this exercise with student interviews. She

86
found that students were engaged and prompted to consider the broader implications of
the subject they study all year in school. Although not all students grasped every detail,
whether or not students walk away with an advanced understanding of MRI seemed a
secondary issue. The primary purpose of this activity was to demonstrate an aspect of
chemistry that is not immediately obvious to students and to get them thinking about
considerations of drug efficacy vs. safety inherent in medicine and pharmacology.
Without a doubt, the students enjoyed the lab and walked away with a new appreciation
for the numerous applications of chemistry.
5.5. Conclusions.
The procedures and data in this manuscript should enable instructors to use
standard T
1
measurement techniques to demonstrate molar relaxivity measurements in an
educational laboratory. Furthermore, use of the provided T
1
data could be the basis of an
analytical problem set that demonstrates the process of calculating molar relaxivities.
5.6. References.

1) Atkins, D. E.; Droegemeier, K. K.; Feldman, S. I.; Garcia-Molina, H.; Klein, M.
L.; Messerschmitt, D. G.; Messina, P.; Ostriker, J. P.; Wright, M. H.
Revolutionizing Science and Engineering Through Cyberinfrastructure: Report of
the National Science Foundation Blue-Ribbon Advisory Panel on
Cyberinfrastructure; National Science Foundation: Arlington, VA, 2003.
2) Empowering the Nation Through Discovery and Innovation: NSF Strategic Plan
for Fiscal Years 2011-2016; National Science Foundation: Arlington, VA, 2011,
pp 14.
3) (a) For a prior report of a molar relaxivity experiment in an undergraduate
physical chemistry lab, see Nestle, N.; Dakkouri, M.; Rauscher, H.

87

Superoxygenated Water as an Experimental Sample for NMR Relaxometry.
J. Chem. Educ. 2004, 81, 1040-1041. (b) For a classroom experiment involving
computational modeling of NMR contrast agents, see Ramos, M. J.; Fernandes, P.
A. Computer Modeling and Research in the Classroom. J. Chem. Educ. 2005, 82,
1021-1025.
4) Dawsey, A. C.; Hathaway, K. L.; Kim, S.; Williams, T. J. Introductory
Chemistry: A Molar Relaxivity Experiment in a High School Classroom.
J. Chem. Ed. 2012, ASAP.
5) (a) Squire, L. F., Novelline R. A. Squire's Fundamentals of Radiology, 5th ed.;
Hrvard UP: Cambridge, 1997. (b) Hendee, W. R., Morgan, C. J. Magnetic
Resonance Imaging. Part I–Physical Principles. West J. Med. 1984, 141, 491–500.
6) (a) Raymond, K. N.; Pierre, V. C. Bioconjugate Chem. Next Generation High
Relaxivity Gadolinium MRI Agents. 2005, 16, 3-8. (b) Caravan, P.; Ellison, J. J.;
McMurry, T. J.; Lauffer, R. B. Chem. Rev. Gadolinium(III) Chelates as MRI
Contrast Agents: Structure, Dynamics, and Applications 1999, 99, 2293-2352.
7) (a) For a current list, see FDA Drug Safety Communication: New Warnings for
Using Gadolinium-Based Contrast Agents in Patients with Kidney Dysfunction;
U.S. Food and Drug Administration: Silver Spring, MD, 2010. Note that Dotarem
is not yet approved in the US market, although it is currently used in other
countries. (b) For a history, see Haën, C. Top. Magn. Reson. Imaging. 2001, 12,
221-230.
8) Anelli, P. L., Lattuada, L., Visigalli, M. Biomedical Imaging: The Chemistry of
Labels, Probes and Contrast Agents; Royal Society of Chemistry: London, 2012,
pp 173-174.
9) Keeler, J. Understanding NMR Spectroscopy; Wiley: West Sussex, UK, 2005.
10) Geraldes, C. F.; Laurent, S. Classification and Basic Properties of Contrast Agents
for Magentic Resonance Imaging. Contrast Media Mol Imaging 2009, 4, 1-23.
11 Fry, C. G. The Nobel Prize for Medicine for Magnetic Resonance Imaging.
J. Chem. Educ. 2004, 81, 922-932.
12) Colletti, P. M. Nephrogenic Systemic Fibrosis and Gadolinium: A Perfect Storm.
Am. J. Roentgenology 2008, 191, 1150-1153.
13) Bellin, M. F. MR Contrast Agents, the Old and the New. Eur. J. Radiol. 2006, 60,
314-323. (b) Lurusso, V.; Pascolo, L.; Fernetti, C.; Anelli, P. L.; Uggeri, F.;

88

Tiribelli, C. Magnetic Resonance Contrast Agents: From the Bench to the
Patient. Curr. Pharm. Des. 2005, 11, 4079-4098. (c) Perazella, M. A. Current
Status of Gadolinium Toxicity in Patients with Kidney Disease. Clin. J. Am. Soc.
Nephrol. 2009, 4, 461-469. (d) Broome, D. R.; Girguis, M. S.; Baron, P. W.;
Cottrell, A. C.; Kjellin, I.; Kirk, G.A. Gadodiamide-Associated Nephrogenic
Systemic Fibrosis: Why Radiologist Should Be Concerened. Am. J. Roentol.
2007, 586-592. (e) Herborn, C. U.; Honold, E.; Wolf, M.; Kemper, J.; Kinner, S.;
Adam, G.; Barkhausen, J. Clinical Safety and Diagnostic Value of Gadolinium
Chelate Gadoterate Meglumine (Gd-DOTA). Invest. Radiol. 2007, 42, 58-62. (f)
Haylor, J.; Dencausse, A.; Vickers, M.; Nutter, F. Jestin, G.; Slater, D.; Idee, J.;
Morcos, S. Nephrogenic Gadolinium Biodistribution and Skin Cellularity
Following a Single Injection of Omniscan in the Rat. Invest. Radiol. 2010, 45,
507-512.
14) (a) Grobner, T. Gadolinium-a Specific Trigger for the Development of
Nephrogenic Fibrosing Dermopathy and Nephrogenic Systemic Fibrosis?
Nephrol. Dial. Transplant. 2006, 21, 1104–1108. (b) Marckmann, P; Skov, L;
Rossen, K, Dupont, A.; Damholt, M. B.; Heaf, J. G.; Thomsen, H. S. Nephrogenic
systemic fibrosis: suspected causative role of gadodiamide used for contrast-
enhanced magnetic resonance imaging J. Am. Soc. Nephrol. 2006, 17, 2359–2362.
15) (a) Caravan, P.; Zhang, Z. Structure-Relaxivity Relationships Among Targeted
MR Contrast Agents. Eur. J. Inorg. Chem. 2012, 1916-1923. (b) Kueny-Stotz, M.;
Garofalo, A.; Felder-Flesch, D. Manganese-Enhanced MRI Contrast Agents:
From Small Chelates to Nanosized Hybrids. Eur. J. Inorg. Chem. 2012, 1987-
2005. (c) Peters, J. A.; Djanashvili, K. Lanthanide Loaded Zeolites, Clays, and
Mesoporous Silica Materials as MRI Probes. Eur. J. Inorg. Chem. 2012, 1961-
1974. (d) Laurent, S.; Henoumont, C.; Vander Elst, L.; Muller, R. N. Synthesis
and Physiochemical Characterisation of Gd-DTPA Derivatives as Contrast Agents
for MRI. Eur. J. Inorg. Chem. 2012, 1889-1915. (e) Caravan, P.; Zhang, Z.
Structure-Activity Relationships between Targeted MR Contrast Agents. Eur. J.
Inorg. Chem. 2012, 1916-1923.
16) (a) FDA Requests Boxed Warning for Contrast Agents Used to Improve MRI
Images; U.S. Food and Drug Administration: Silver Spring, MD, 2007. (b)
Information for Healthcare Professionals: Gadolinium-Based Contrast Agents for
Magnetic Resonance Imaging (marketed as Magnevist, MultiHance, Omniscan,
OptiMARK, ProHance); U.S. Food and Drug Administration: Silver Spring, MD,
2007.
17) Morcos, S. K. Nephrogenic Systemic Fibrosis Following the Administration of
Extracellular Gadolinium Based Contrast Agents: Is the Stability of the Contrast

89

Agent Molecule an Important Factor in the Pathogenesis of this Condition?
British J. Radiology 2007, 80, 73–76.

90
Chapter 6. Polymer MRI contrast agents: structures,
properties, and applications.

6.1. Introduction. Polymer MRI contrast agents.
Gadolinium chelates conjugated to macromolecules (polymers, dendrimers,
liposomes, etc.) are referred to as macromolecular contrast agents (mCAs). As an
emerging field in MRI contrast agents, mCAs are aimed to improve contrast efficiency,
enable targeting of tissues and tumors, and confer new applications for MRI. Covalent
attachment of an MRI contrast agent onto a macromolecule inhibits the rotational motion
of the contrast agent; thus, effectively increasing the τ
R
and in turn the relaxivity of the
contrast agent. Langereis et al. demonstrated that the rigidity of the bound polymer
dictates the significance of the enhancement of the τ
R
of the contrast agent. More rigid
polymers have demonstrated a greater increase in τ
R
than flexible polymers such as
polyethylene glycol (PEG). Additionally, conjugation of contrast agents to polymers can
serve to decrease the water coordination number (q) through inhibiting water access to
the Gd
3+
core.
1
This chapter will briefly discuss current mCAs, which can be categorized
into four main groups: 1) block mCAs 2) graft mCAs 3) dendritic mCAs and 4) micellar
mCAs (Figure 6.1) as well as non-covalent polymer–Gd–DOTA interactions being
investigated in our laboratory.
2

91
a) b)
c) d)
= Gd-Chelate

Figure 6.1. Structural representations of a) block b) graft c) dendritic and d) micellar
mCAs.
6.2. Polymers and covalently bound MRI contrast agents.
Graft mCAs are synthesized via conjugation of chelates onto side arms of polymer
chains (Figure 6.1b). Linear polymers such as polylysine, polyornithine, poly(glutamic
acic), poly[N-(2-hydroxypropyl)methacrylamide], poly(methacrylic acid),
polysuccinimide, etc. are used to generate graft mCAs. Recently, a conjugate polymer,
PF–Gd, with a delocalized structure was developed by Wang et al. in which single and
multiple bonds alternate along the backbone creating rigidity in the polymer (Figure 6.2).
Increased rigidity resulted in increased τ
R
and increased r
1
.
3

92
O
O
O
O
O
OH
O
O
O
HO
N N
N
Gd
O
O
O
O
O
O
N
O
O
O
O

Figure 6.2. Stucture of PF-Gd (r
1
= 12.57 mM
-1
s
-1
).
3

Block mCAs are formed by inserting chelators into the backbone of a polymer to
chelate Gd
3+
(Figure 6.1a). The DTPA di-ester or DTPA bisamide copolymers are
synthesized by condensation polymerization with DTPA dianhydride with diol or
diamine monomers. This type of block polymerization imparts rigidity to the polymer
once GdCl
3
is incorporated.
4
The restricted rotation of the polymer serves to increase the
τ
R
and enhance r
1
. Molecular weight of the polymer has little effect on the enhancement
of relaxivity. Additionally, incorporation of other polymers (PEG) to the existing
polymer via grafting results in similar r
1
values.
5

Unique tree-like, nanosized, three-dimensional polymers called dendrimers have been
modified to incorporate MRI contrast agents as well (Figure 6.1c). The inherent
branched, spherical, compact structure of dendrimers gives them advantages over other

93
polymers. Surface modification to introduce functional groups is also a distinct advantage
with dendrimers. Like branched and draft polymers, DTPA and DOTA chelates are
mainly attached through the amino groups. The branch structure provides even more
rigidity further enhancing the properties discussed for the linear polymers. Therefore, the
contrast efficiencies are even higher in dendritic polymers than linear polymers.
Synthesis of micellar mCAs is another extension of mCAs. Advantages of micellar
mCAs include high Gd loading capacity as well as tunable sizes. Both of these
characteristics make micellar mCAs the ideal candidates for drug loading and delivery.
The hydrophobic core of the micelle is capable of loading drugs. Additionally, tunable
sizing of micellar mCAs lends to enhancing the EPR effect of the micelles. The
techniques employed to synthesize micellar mCAs incvolcve emulsion polymerization
and assembly of mCAs with the Gd chelates either in the core or shell of the micellar
structures (Figure 6.1d). Depending on the assemblage, the micellar mCAs can either
have enhanced τ
R
and r
1
values due to restricted rotation or attenuated q and r
1
values due
to restricted access of water to the Gd
3+
core. Physiologically responsive micellar CAs
with changeable r1 values can be achieved via designing environment-sensitive micellar
structures. Changes in shape via aggregation, pH induced cleavage, etc. can alter r1
values. Importantly, the micellar mCAs also have different pharmacokinetics and
biodistribution compared to linear and dendritic polymer mCAs due to their larger size
lending longer blood retention times.
6

94
6.3. Macromolecules and non-covalently bound MR contrast agents.
Most relevant to our scientific endeavors with the field of macromolecular MRI
contrast agents, a few examples of polymer or macromolecules and non-covalently bound
MRI contrast agent interactions have been described. In 2006, Wang et al. described the
synthesis and physiochemical characterization of two gadolinium(III) complexes as well
as their interaction with human albumin serum (HSA). Two derivatives of 3,6,10-
tri(carboxymethyl)-3,6,10-triazzdodecanedioic acid (TTDA), TTDA–BOM and
TTDA–N’–BOM were complexed with Gd
3+
. The water exchange rates (k
ex
), rotational
correlation times (τ R), and mean bound relaxivity (r 1
b
) when bound to HSA of these
complexes were compared to those of Gd–DTPA and Gd–BOPTA (Table 6.1).
The kinetic stability of Gd-TTDA-BOM and Gd-TTDA-N’-BOM toward transmetallation
with Zn
2+
was assessed and determined to be significantly higher than that of
[Gd(DTPA–BMA)–(H
2
O)], which demonstrates potential for use as MRI contrast
agents.
7

95
Table 6.1. Summary of properties of TTDA–BOM and TTDA–N’–BOM as well as
interaction with HSA measured by Wang et al.
O
N
COOH
HOOC
N N COOH
COOH
COOH
N HOOC
HOOC
N N COOH
COOH
COOH
O
TTDA-BOM TTDA-N'-BOM
HOOC N
N
N
HOOC
COOH
COOH
COOH
HOOC N
N
N
HOOC
COOH
COOH
COOH
O
DTPA BOPTA

Complex k
ex
τ R r 1 (mM
-1
s
-1
) r 1
b
(mM
-1
s
-1
)
Gd–TTDA–BOM 117 x 10
6
s
-1
119 ps 4.42±0.02 65.8±2.7

Gd–TTDA–N’–BOM 131 x 10
6
s
-1
125 ps 4.44±0.03 61.5±1.8
Gd–DTPA 4.1 x 10
6
s
-1
103 ps 3.89±0.03 44
Gd–BOPTA 3.45 x 10
6
s
-1
104 ps 3.85±0.03 33
Water exchange rates (k
ex
), rotational correlation times (τ R), and mean bound relaxivity
(r 1
b
) when bound to HSA.
Similarly, in 2007, Caravan et al. described the albumin binding, relaxivity, and water
exchange kinetics of the diastereomers of a Gd
3+
-based magnetic resonance angiography
(MRA) contrast agent. The isolable diastereomers, A and B, of MS-325 ((trisodium-{(2-
(R)-[4,4-diphenylcyclohexyl) phosphonooxylmethyl] diethylenetriaminepentaacetato)

96
(aquo) gadolinium(III)}] slowly interconvert. At physiological conditions, the two
isomers do not exhibit a significant difference relaxivity or HSA affinity. However, at
lower temperatures the relaxivities of the two isomers were determined to statistically
significantly different (Table 6.2). The differences in the relaxivities of the complexes
while interacting with HSA are attributed to the differing water exchange rates by the
author.
Table 6.2. Summary of r
1
and k
ex
data presented by Caravan et al. for MS-325 isomers A
and B at 37 ºC and 35 ºC.
Gd
N N
N
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
*
*
Gd
N N
N
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
*
*
2R,4R
MS-325B
2R,4S
MS-325A

Complex k
ex
25 ºC (s
-1
) r 1 at 37 ºC (mM
-1
s
-1
)
MS–325A 5.9±2.8 x 10
-6
6.91±0.49
MS–325B 3.2±2.8 x 10
-6
6.86±0.49
MS–325A-HSA 42±1
MS–325B-HSA 38±1
Water exchange rates (k
ex
) relaxivity (r 1) at 20 MHz in phosphate buffered saline (PBS).

97
6.4. Polyvinylphosphoric acid and Gd–DOTA as a potential
non-covalently bound MRI contrast agent.
Parallel to our small molecule MRI contrast agent work discussed in Chapter 4, we
have some preliminary results on a polyvinylphosphoric acid (PVPA) and non-covalently
bound Gd–DOTA MRI contrast agent in collaboration with the Hogen-Esch group at the
University of Southern California, T
1
data has been obtained for Gd–DOTA–PVPA as
well as Gd–DOTA–PVPA–shell. Further investigation into this system is ongoing to
determine the interaction of the Gd–DOTA with the PVPA.
6.4.1. Results: polyvinylphosphoric acid and Gd–DOTA.
In order to understand and determine the relaxivities of the Gd–DOTA–PVPA–
Shell system a series of T
1
inversion recovery experiments were performed. Initially, a
series of T
1
inversion recovery experiments, similar to those ran for the small molecule
Gd–DOTA–Shell–Urea system, were executed at 25 ºC. A very slight increase in
relaxivity is observed upon addition of PVPA to Gd–DOTA (T
1
= 237(2) ms) to form
Gd–DOTA–PVPA (T
1
= 230(2) ms). However, no significant change in T
1
was observed
upon addition on shell to form Gd–DOTA–PVPA–Shell (T
1
= 229(3) ms) (Table 6.3).

98
Table 6.3. Summary of T
1
data for Gd–DOTA–PVPA–Shell system at 25 ºC.
P HO OH
O
n
N N
N N
Gd
O
O
O
O
O
O
O
O
Measure T
1
Shell
4 equiv.
Measure T
1

Complex T
1
(ms) (error)
Gd–DOTA 237(2)
PVPA 1048(32)
Gd–DOTA–PVPA 230(2)
Gd–DOTA–PVPA–Shell 229(3)

Additionally, a set of T
1
inversion recovery experiments was performed after
heating the Gd–DOTA–PVPA–Shell system at 70 ºC overnight. A significant increase in
the T
1
of the Gd–DOTA–PVPA at 25 ºC versus Gd–DOTA–PVPA after heating at 70 ºC
overnight was observed from 230(2) ms to 69(9) ms respectively (Table 6.).
Table 6.4. Summary of T
1
data for Gd–DOTA–PVPA–Shell system after heating at
70 ºC overnight.
Complex T
1
(ms) (error)
Gd–DOTA 242(2)
PVPA 1196(39)
Gd–DOTA–PVPA 69(9)
Gd–DOTA–PVPA–Shell 74(9)

99
Determination of the fate of the Gd
3+
metal ion was of interest due to the
possibility that the Gd
3+
could either be free in solution or potentially ligand exchanged
the DOTA for the PVPA. In order to elucidate some information toward this end,
Gd–DOTA and PVPA were heated overnight to provide Gd–DOTA–PVPA, which was
then exposed to a xylenol orange indicator test to determine whether or not free Gd
3+
was
present in solution. Similarly, the experiment was ran with nonchelated GdCl
3
and
PVPA. The xylenol orange complexometric indicator test was negative for presence of
free Gd
3+
in either the Gd–DOTA–PVPA or the Gd–PVPA (Scheme 6.1). This
experiment did not elucidate the destiny of the Gd
3+
metal ion conclusively; however, it
was shown that both DOTA and PVPA are effective chelators of Gd
3+
. Whether PVPA is
capable of extracting the bound gadolinium from Gd–DOTA is yet to be determined.
N N
N N
Gd
O
O
O
O
O
O
O
O
Xylenol Orange
Negative
P HO OH
O
n
Δ
a)

P HO OH
2
O
n
b)
GdCl
3 Xylenol Orange
Negative
Δ

Scheme 6.1. Gd–DOTA–PVPA and Gd–PVPA xylenol orange complexometric indicator
results.

100
Additionally, interaction of shell with PVPA was monitored by
19
F NMR
spectroscopy. The results suggest that the shell interacts with PVPA with peak A (closest
to the guanyl head) of the shell
19
F spectrum being most affected (Table 6.5); a good
indication that shell is binding to PVPA in the end on hydrogen bonding fashion that we
propose.
Table 6.5. Change in
19
F T
1
(ms) when PVPA is added to shell.
Compilation of Matter
Peak A
-117.9 ppm
Peak B
-119.8 ppm
Peak C
-123.5 ppm
Shell 501(16) ms 474(15) ms 527(8) ms
PVPA–Shell 324(47) ms 460(10) ms 409(20) ms
Gd–DOTA–PVPA–Δ–Shell 186(10) ms 212(7) ms 211(44) ms

101
6.5. References.

1) a) Kobayashi, H.; Brechbiel, M. W. Dendrimer-based Macromolecular MRI
Contrast Agents: Characteristics and Application. Molecular Imaging 2003, 2, 1–
10. b) Kobayashi H.; Brechbiel M. W. Nano-sized MRI Contrast Agents with
Dendrimer Cores. Advanced Drug Delivery Reviews 2005, 57, 2271–86. c)
Langereis, S.; de Lussanet, Q. G.; van Genderen, M. H. P.; Backes, W. H.;
Meijer, E. W. Multivalent Contrast Agents Based on
Gadoliniumdiethylenetriaminepentaacetic Acid-terminated Poly(propylene imine)
Dendrimers for Magnetic Resonance Imaging. Macromolecules 2004, 37, 3084–
91. d) Toth, E.; van Uffelen, I.; Helm, L.; Merbach, A. E.; Ladd, D.; Briley Saebo,
K.; Kellar, K. E. Gadolinium-based Linear Polymer with Temperature
Independent Proton Relaxivities: a Unique Interplay between the Water Exchange
and Rotational Contributions. Magn. Reson. Chem. 1998, 36, S125–34. e) Doble,
D. M. J.; Botta, M., Wang, J.; Aime, S.; Barge, A.; Raymond, K. N. Optimization
of the relaxivity of MRI Contrast Agents: Effect of Poly(ethylene glycol) Chains
on the Water-exchange Rates of Gd-III Complexes. J. Am. Chem. Soc. 2001, 123,
10758–9.
2) Tang, J.; Sheng, Y.; Hu, H.; Shen, Y. Macromolecular MRI Contrast Agents:
Structures, Properties, and Applications. Prog. Polym. Sci. 2013, 38, 462-502.
3) Xu, Q. L.; Zhu, L. T.; Yu, M. H.; Feng, F. D.; An, L. L.; Xing, C. F.; Wang, S.
Gadolinium(III) Chelated Conjugated Polymer as a Potential MRI Contrast
Agent. Polymer 2010, 51, 1336–1340.
4) a) Ouyang, M.; Zhuo, R. X.; Fu, G. C. Study on Synthesis and Relaxivity of
Paramagnetic Polyester Metal Complexes for MRI. Chinese J. Reaction Polym.
1995, 4, 99–106. b) Duarte, M. G. Gil, M. H.; Peters, J. A.; Colet, J. M. Vander
Elst, L.; Muller, R. N.; Geraldes, C. F. G. C. Synthesis, Characterization, and
Relaxivity of Two Linear Gd(DTPA)-polymer Conjugates. Bioconjugate Chem.
2001, 12, 170–177. c) Lu, Z. R.; Parker D. L.; Goodrich, K. C.; Wang, X. H.;
Dalle, J. G.; Buswell, H. R. Extracellular Biodegradable Macromolecular
Gadolinium(III) Complexes for MRI. Magn. Reson. Medicine, 2004, 51, 27–34.
d) Lucas, R. L.; Benjamin, M.; Reineke, T. M. Comparison of a Tartaric Acid
Derived Polymeric MRI Contrast Agent to a Small Molecule Model Chelate.
Bioconjugate Chem. 2008, 19, 24–7. e) Ladd, D. L.; Hollister, R.; Peng, X.; Wei,
D.; Wu, G.; Delecki, D.; Snow, R. A.: Toner, J. L.; Kellar, K.; Eck, J.; Desai, V.
C.; Raymond, G.; Kinter, L. B.; Desser, T. S.; Rubin, D. L. Polymeric
Gadolinium Chelate Magnetic Resonance Imaging Contrast Agents: Design,
Synthesis, and Properties. Bioconjugate Chem. 1999, 10, 361–70. f) Mohs, A. M.;

102

Wang, X. H.; Goodrich, K. C.; Zong, Y. D.; Parker, D. L.; Lu, Z. R. PEG-g-
poly(GdDTPA-co-l-cystine): a Biodegradable Macromolecular Blood Pool
Contrast Agent for MR Imaging. Bioconjugate Chem. 2004, 15, 1424–1430.
5) a) Zong, Y. Guo, J.; Ke, T.; Mohs, A. M.; Parker, D. L. Lu, Z. R. Effect of Size
and Charge on Pharmacokinetics Enhancement of Biodegradable and in vivo MRI
Contrast Polydisulfide Gd(III) Complexes. J. Control. Release 2006, 112, 350–
356. b) Mohs, A. M.; Zong, Y. D.; Guo, J. Y.; Parker, D. L.; Lu, Z. R. PEG-g-
poly(GdDTPAco- l-cystine): Effect of PEG Chain Length on in vivo Contrast
Enhancement in MRI. Biomacromolecules, 2005, 6, 2305–2311.
6) Specific examples of micellar mCAs: a) Zhang, G.; Zhang, R.; Wen, X.; Li, L.;
Li, C. Micelles Based on Biodegradable poly(L-glutamic acid)-b-polylactide with
Paramagnetic Gd Ions Chelated to the Shell Layer as a Potential Nanoscale MRI–
visible Delivery System. Biomacromolecules 2007, 9, 36–42. b) Reynolds, C. H.;
Annan, N.; Beshah, K.; Huber, J. H.; Shaber, S. H.; Lenkinski, R. E.; Wortman, J.
A. Gadolinium-loaded Nanoparticles: New Contrast Agents for Magnetic
Resonance Imaging. J. Am. Chem. Soc. 2000, 122, 8940–8945. c) Turner, J. L.;
Pan, D. P. J.; Plummer, R.; Chen, Z. Y.; Whittaker, A. K.; Wooley, K. L.
Synthesis of Gadolinium-labeled Shell-crosslinked Nanoparticles for Magnetic
Resonance Imaging Applications. Adv. Funct. Mater. 2005, 15, 1248–1254. d)
Gong, P. Chen, Z. Y.; Chen, Y. Y.; Wang, W.; Wang, X. S.; Hu, A. G. High
Relaxivity MRI Contrast Agents Prepared from Miniemulsion Polymerization
Using Gadolinium(III)-based Metallosurfactants. Chem. Commun. 2011, 47,
4240–4242. e) Cheng, Z. L.; Thorek, D. L. J.; Tsourkas, A. Gadolinium-
conjugated Dendrimer Nanoclusters as a Tumor-targeted T
1
Magnetic Resonance
Imaging Contrast Agent. Angew. Chem. Int. Ed. 2010, 49, 346–350. f) Kaida, S.;
Cabral, H.; Kumagai, M.; Kishimura, A.; Terada, Y.; Sekino, M.; Aoki, L.;
Nishiyama, N.; Tani, T.; Kataoka, K. Visible Drug Delivery by Supramolecular
Nanocarriers Directing to Single-platformed Diagnosis and Therapy of Pancreatic
Tumor Model. Cancer Res. 2010, 70, 7031–7041. g) Nakamura, E.; Makino, K.;
Okano, T.; Yamamoto, T.; Yokoyama, M. A Polymeric Micelle MRI Contrast
Agent with Changeable Relaxivity. J. Control. Release 2006, 114, 325–333. h)
Shiraishi, K.; Kawano, K.; Maitani, Y.; Yokoyama, M. Polyion Complex Micelle
MRI Contrast Agents from poly(ethylene glycol)-β-poly(L-lysine) Block
Copolymers having Gd-DOTA; Preparations and Their Control of T(1)-
relaxivities and Blood Circulation Characteristics. J. Control. Release 2010, 148,
160–167. i) Shiraishi, K.; Kawano, K.; Minowa, T.; Maitani, Y.; Yokoyama, M.
Preparation and in vivo Imaging of PEG-poly(L-lysine)-based Polymeric Micelle
MRI Contrast Agents. J. Control. Release 2009, 136, 14–20.

103

7) Ou, M. H.; Tu, C. H.; Tsai, S. C.; Lee, W. T.; Liu, G. C.; Wang, Y. M. Synthesis
and Physicochemical Characterization of Two Gadolinium(III) TTDA-like
Complexes and Their Interaction with Human Serum Albumin. Inorg. Chem.
2006, 45, 244–254.

104
Chapter
7.
Experimental
and
spectral
data.

7.1. General procedures.
All air and water sensitive procedures were carried out either in a Vacuum
Atmospheres glove box under nitrogen (2-10 ppm O
2
for all manipulations) or using
standard Schlenk techniques under nitrogen. Deuterated NMR solvents were purchased
from Cambridge Isotopes Labs and used as received. Other organic solvents and bulk
inorganic reagents were purchased from EM Science and used as received, except where
indicated. Distilled water used was distilled Arrowhead water. Iodomethane was
purchased from Alfa Aesar and stored, as received, over copper shot. Copper(II) triflate
was purchased from Alfa Aesar and used as received. Silica gel (230-400 mesh) was
purchased as pre-packed columns from Teledyne.
NMR spectra were recorded on a Varian Mercury 400, 400MR, VNMRS 500, or
VNMRS 600 spectrometer. All chemical shifts are reported in units of ppm and
referenced to the residual
1
H in the solvent and line-listed according to (s) singlet, (sb)
broad singlet, (d) doublet, (t) triplet, (dd) double doublet, etc.
13
C spectra are delimited by
carbon peaks, not carbon count. Chapter 4-6
1
H and spectra and T
1
data were obtained on
a Varian 400 MR spectrometer outfitted with as AS7600 autosampler. 5 mm NMR tubes
and coaxial inserts were purchased from Norell at nmrtubes.com (NI5CCI-V). Disposable
syringes, needles, syringe filters, GdCl
3
, DOTA, and DOTP were acquired from VWR
scientific. For experiments in Chapter 5, needles were blunted by cutting off the tips with
a Dremel rotary tool. Melting points were obtained on a mel-temp apparatus and are
uncorrected. MALDI mass spectra were obtained on an Applied Biosystems Voyager
spectrometer using the evaporated drop method on a coated 96 well plate. The matrix was
2,5-dihydroxybenzoic acid. In a standard preparation, ca. 1 mg analyte and ca. 20 mg
matrix were dissolved in a suitable solvent and spotted on the plate with a micro-pipetter.
Electrospray ionization (ESI) high-resolution mass spectra were collected at the
University of California, Riverside Mass Spectrometry Facility. CHN elemental analyses
were collected at the University of Illinois at Urbana Champaign at the School of
Chemical Sciences Microanalysis Laboratory.
Catalytic azoline oxidation: see section 7.2.5.
Copper-free, base mediated azoline oxidation: see section 7.2.6.

105
7.2. Chapter 2 experimental and spectral data.
7.2.1. Ligand screen.
Various ligands were screened for the oxidation of thiazoline 2.2 to thiazole 2.2a.
In a representative procedure, the ligand (10 mol%) and Cu(OTf)
2
(10 mol%) were
dissolved in N,N-dimethylformamide (DMF) and stirred at room temperature for
30 minutes. Thiazoline 2.2 (50 mM) and DBU (10 mol%) were added at room
temperature. The reaction was stirred at 100 °C in air for 8 hours. Results, as determined
by NMR spectroscopy, are summarized in Table 2.3.
7.2.2. Preparation of copper complexes.
[(
Mes
DAB
Me
)Cu
II
(OH
2
)
3
]
2+
2TfO
-
(2.1). [
Mes
DAB
Me
] ligand (321 mg, 1.0 mmol)
and Cu(OTf)
2
(289 mg, 0.8 mmol) were dissolved in dry dichloromethane (3.0 mL) under
a dry N
2
atmosphere and was allowed to stir at room temperature overnight. The product
was precipitated upon addition of hexanes and the crystals were washed with hexanes
multiple times to yield product as a dark green crystalline solid (577 mg, 98%).
1
H NMR (400 MHz, CDCl
3
): δ = 0.88 (sb, 6 H), 2.28 (sb, 12 H), 2.35 (sb, 6 H), 6.90
(sb, 4 H).
13
C NMR cannot be recorded because this compound is paramagnetic.
19
F NMR (376 MHz, CDCl
3
): δ = -78.8.
X-ray crystal structure ORTEP report in Appendix.

106
7.2.3. Preparation of azoline precursors.
Fmoc-Cys(trt)-OMe
N
H
STrt
OMe
O
Fmoc

7.1
Fmoc-Cys(trt)-OH
1
(10.0 g, 17.1 mmol) was dissolved in DMF (30 mL) at 0 °C,
to which K
2
CO
3
(2.6 g, 18.8 mmol) was added. The reaction was allowed to stir for 30
minutes before iodomethane (2.13 mL, 34.1 mmol) was added to the reaction, which was
then allowed to warm to room temperature and stirred until complete by TLC (80
minutes, TLC eluted with 1:1 hexanes: ethyl acetate). The solution was diluted with ethyl
acetate (50 mL) then washed 1:1 brine: H
2
O (5·25 mL). Solvent was removed from the
remaining organic fraction under reduced pressure to leave fluffy white crystals
(9.69 g, 95%). Data are consistent with a previously characterized compound.
2

1
H NMR (400 MHz, CDCl
3
): δ = 7.77 (m, 2 H), 7.61 (m, 2 H), 7.41-7.19 (m, 19 H), 5.23
(d, 1 H, J = 8 Hz), 4.36 (m, 3 H), 4.23 (t, 1 H, J = 8 Hz), 3.72 (s, 3 H), 2.67 (d, 2 H, J =
5.2 Hz).

107
General Procedure for Preparation of Triphenylmethyl-Protected Cysteine Amides
3

Fmoc-Cys(trt)-OMe (7.1) was dissolved in acetonitrile (0.16 M solution), to
which diethylamine (65 equiv.)
4
was added and the reaction was stirred at room
temperature for 30 minutes. Completion of this reaction was verified by TLC, eluting
with 3:1 hexanes: ethyl acetate. The solution was then concentrated under reduced
pressure, and the residue was suspended in 1:1 dichloromethane: acetonitrile (20 mL).
Diisopropylethylamine (2.1 equiv.)
9
and the appropriate acyl chloride (1.1 equiv.) were
then added while stirring at room temperature, and the reaction was stirred overnight. The
reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate,
washed twice with saturated aqueous NaHCO
3
, and dried over MgSO
4
. The product was
purified via flash chromatography on silica gel, eluting with an ethyl acetate/ hexanes
mixture.

108
Methyl 2-(4-Fluorobenzamido)-3-(tritylthio)propanoate
N
H
O
STrt
OMe
O
F

7.2
Prepared from Fmoc-Cys(trt)-OMe (7.1) (4.6 g, 7.67 mmol) and 4-fluorobenzoyl
chloride (1.34 g, 8.43 mmol)
11
according to the general procedure for preparation of
triphenylmethyl-protected cysteine amides to give product as white crystals (1.66 g,
43%).
1
H NMR (400 MHz, CDCl
3
): δ = 7.77 (m, 2 H), 7.38 (m, 6 H), 7.23 (m, 9 H), 7.13 (m, 2
H), 6.59 (d, 1 H, J = 7.6 Hz), 4.81 (m, 1 H), 3.75 (s, 3 H), 2.80 (dd, 1 H, J
1
= 12.4 Hz, J
2

= 5.6 Hz), 2.74 (dd, 1 H, J
1
= 12.6 Hz, J
2
= 4.8 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.1, 166.4, 165.9, 163.9, 144.4, 129.7 (d, J
C-F
= 37.2
Hz), 129.6, 128.2, 127.1, 115.8 (d, J
C-F
= 86.8 Hz), 67.1, 53.0, 51.6, 34.2.
FT-IR (cm
-1
): ν = 3031, 2933, 1746, 1812.
ESI-HRMS for C
30
H
26
FNO
3
S: calculated [MNa]
+
522.1617 g/mol, found 522.1510
g/mol.
M.P. 135-137 ºC
1
H NMR, 400 MHz, CDCl
3
, 25 °C

109
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

110
Methyl 2-(4-Cyanobenzamido)-3-(tritylthio)propanoate
N
H
O
STrt
OMe
O
NC

7.3
Prepared from Fmoc-Cys(trt)-OMe (7.1) (5 g, 8.33 mmol) and 4-cyanobenzoyl chloride
(1.52 g, 9.17 mmol)
11
according to the general procedure for preparation of
triphenylmethyl-protected cysteine amides to give product as white crystals
(2.58 g, 56%).
1
H NMR (400 MHz, CDCl
3
): δ = 7.84 (m, 2 H, J
1
= 8 Hz), 7.75 (m, 2 H, J
1
= 8 Hz), 7.35-
7.40 (m, 6 H), 7.17-7.26 (m, 9 H), 6.65 (d, 1 H, J = 8 Hz), 4.79 (dt, 1 H, J
1
= 8 Hz, J
2
= 5
Hz), 3.77 (s, 3 H), 2.83 (dd, 1 H, J
1
= 12 Hz, J
2
= 4 Hz), 2.75 (dd, 1 H, J
1
= 12 Hz, J
2
= 4
Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 170.8, 165.2, 144.3, 137.7, 132.6, 129.6, 128.2, 128.1,
127.1, 118.1, 115.6, 67.2, 53.1, 51.8, 33.9.
FT-IR (cm
-1
): ν = 3058, 2953, 2232, 1746, 1654.
ESI-HRMS for C
31
H
26
N
2
O
3
S: calculated [MNa]
+
529.1664 g/mol, found 529.1556 g/mol.
M.P. 164-167 ºC

111
1
H NMR, 400 MHz, CDCl
3
, 25 °C

13
C NMR, 100 MHz, CDCl
3
, 25 °C

112
Methyl 2-(Indole-2-carboxamido)-3-(tritylthio)propanoate
O
OMe
H
N
STrt
O
NH

7.4
Fmoc-Cys(Trt)-OMe (7.1) (3.0 g, 5.0 mmol) was dissolved in 36 mL acetonitrile.
Diethylamine (20.2 mL, 195 mmol) was added while stirring at room temperature. The
reaction was stirred until complete by TLC (30 minutes, 3:1 hexanes: ethyl acetate) and
the solvents were removed under reduced pressured. The residue was suspended in 8:1
dichloromethane: DMF (45 mL), to which indole-2-carboxylic acid
9
(886 mg, 5.5 mmol)
and N-methylmorpholine (1.65 mL, 15 mmol)
5
were added. While stirring at 0 ºC, a
solution of DCC (N,N’-dicyclohexylcarbodiimide, 1.24 g, 6.0 mmol)
6
and HOBt
7
(1-
hydroxybenzotriazole, 946 mg, 7 mmol)
7
in 8:1 dichloromethane: DMF solution (15 mL)
was added. The resulting solution was brought to room temperature and allowed to stir
for 15 hours. It was then filtered, and the filtrate was washed with saturated aqueous
NaHCO
3
(30 mL x 3) and H
2
O (30 mL) then dried over MgSO
4
. The reaction mixture
was then concentrated and purified via flash chromatography (gradient 5-30% ethyl
acetate in hexanes) to give product as white crystals (1.39 g, 2.64 mmol, 53%).
1
H NMR (400 MHz, CDCl
3
): δ = 9.33 (s, 1 H), 7.68 (dd, 1 H, J
1
= 8 Hz, J
2
= 0.8 Hz),
7.43 (dd, 1 H, J
1
= 8 Hz, J
2
= 0.8 Hz), 7.39 (m, 6 H), 7.30 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz,
J
3
= 0.8 Hz), 7.24-7.15 (m, 10 H), 6.93 (dd, 1 H, J
1
= 1.2 Hz, J
2
= 0.8 Hz), 6.78 (d, 1 H,
J
1
= 8 Hz), 4.84 (dt, 1 H, J
1
= 8 Hz, J
2
= 4.8 Hz), 3.77 (s, 3 H), 2.84 (dd, 1 H, J
1
= 12 Hz,
J
2
= 5.4 Hz), 2.74 (dd, 1 H, J
1
= 12 Hz, J
2
= 4.8 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 170.9, 161.1, 144.4, 136.6, 129.9, 129.6, 128.2, 127.8,
127.1, 124.9, 122.3, 120.9, 112.1, 103.4, 67.2, 53.6, 51.3, 34.3.
FT-IR (cm
-1
): ν = 3057, 2949, 1741, 1645, 1541.
ESI-HRMS for C
32
H
28
N
2
O
3
S: calculated [MNa]
+
543.1821 g/mol, found 543.1708 g/mol.
M.P. 145-148 ºC (Decomposition temperature)

113
1
H NMR , 400 MHz, CDCl
3
, 25 °C

13
C NMR, 100 MHz, CDCl
3
, 25 °C

114
IR

115
Methyl 2-(1-Methylindole-2-carboxamido)-3-(tritylthio)propanoate
O
OMe
H
N
STrt
O
N

7.5
Fmoc-Cys(Trt)-OMe (7.1) (3.0 g, 5.0 mmol) was dissolved in 36 mL acetonitrile.
Diethylamine (20.2 mL, 195 mmol) was added while stirring at room temperature. The
resulting solution was stirred until complete by TLC (30 minutes, 3:1 hexanes: ethyl
acetate) and the solvents were removed under reduced pressured. The residue was
suspended in 8:1 dichloromethane: DMF (45 mL), to which 1-methyl-indole-2-carboxylic
acid (963 mg, 5.5 mmol)
8
and N-methylmorpholine (1.65 mL, 15 mmol) was added.
While stirring at 0 ºC, a solution of DCC (1.24 g, 6.0 mmol) and HOBt (946 mg, 7 mmol)
in 8:1 dichloromethane: DMF (15 mL) was added. The resulting solution was brought to
room temperature and allowed to stir for 15 hours. The product mixture was then filtered,
and the filtrate was washed with saturated aqueous NaHCO
3
(30 mL x 3) and H
2
O (30
mL) then dried over MgSO
4
. This was then concentrated and purified via flash
chomatography (gradient 5-30% ethyl acetate in hexanes) to give product as a dark
yellow solid (1.23 g, 2.3 mmol, 46%).
1
H NMR (400 MHz, CDCl
3
): δ = 7.67 (d, 1 H, J = 8 Hz), 7.41-7.15 (m, 18 H), 6.93 (s, 1
H), 6.69 (d, 1 H, J = 8 Hz), 4.79 (dt, 1 H, J
1
= 8 Hz, J
2
= 4.8 Hz), 4.02 (s, 3 H), 3.77 (s, 3
H), 2.80 (dd, 1 H, J
1
= 12 Hz, J
2
= 6 Hz), 2.74 (dd, 1 H, J
1
= 12 Hz, J
2
= 6 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 170.9, 161.9, 144.2, 139.1, 131.0, 129.5, 128.0, 126.9,
126.0, 124.3, 122.0, 120.5, 110.1, 104.8, 67.0, 52.8, 52.0, 34.1, 31.5.
FT-IR (cm
-1
): ν = 3060, 2948, 1742, 1660, 1526.
MALDI for C
33
H
30
N
2
O
3
S: calculated [MNa]
+
557.20 g/mol, found 557.02 g/mol.
M.P. 65-68 ºC

116
1
H NMR, 400 MHz, CDCl
3
, 25 °C

13
C NMR, 100 MHz, CDCl
3
, 25 °C

117
IR

118
Methyl 2-(2-Naphthamido)-3-(tritylthio)propanoate
N
H
O
STrt
OMe
O

7.6
L-Cys(Trt)-OH (727 mg, 2.0 mmol)
9
was dissolved in acetonitrile (10.0 mL) and
dichloromethane (5.0 mL). Diisopropylethylamine
9
(732 mL, 4.2 mmol) was added and
the solution was stirred at room temperature for 14 hours until complete by TLC (eluting
with methanol). The reaction was washed three times with saturated aqueous NaHCO
3

(10 mL) and dried over MgSO
4
. Solvent was removed from the resulting organic fraction
under reduced pressure to yield product as white crystals (727 mg), which were used for
the next step without further purification.
Product prepared from previous step (400 mg, 0.77 mmol) was dissolved in DMF
(1.36 mL). K
2
CO
3
(117.1 g, 0.85 mmol, 1.1 equiv.) was added while stirring at 0 ºC.
After stirring for 30 minutes, iodomethane (0.101 mL, 1.62 mmol, 1.9 equiv.) was added
and the solution was brought to room temperature and stirred until complete by TLC
(eluting with 1:1 hexanes: ethyl acetate). The reaction was diluted with ethyl acetate (10
mL) and washed with deionized water: brine (1:1) (3·10 mL), and the combined organic
fractions were dried over MgSO
4
. This was then concentrated and purified via flash
chromatography (gradient 5-25% ethyl acetate in hexanes) to yield product as white
crystals (203 mg, 35% over 2 steps).
1
H NMR (400 MHz, CDCl
3
): δ = 8.29 (s, 1 H), 7.93 (m, 3 H), 7.82 (dd, 1 H, J
1
= 6.8 Hz,
J
2
= 2 Hz), 7.58 (m, 2 H), 7.39 (d, 6 H, J = 7.2 Hz), 7.23 (d, 6 H, J = 8.0 Hz), 7.20 (t, 3 H,
J = 8.0 Hz), 6.82 (d, 1 H, J = 8.0 Hz), 4.90 (dt, 1 H, J
1
= 8.0 Hz, J
2
= 4.8 Hz), 3.78 (s, 3
H), 2.84 (dd, 1 H, J
1
= 12 Hz, J
2
= 4 Hz), 2.78 (dd, 1 H, J
1
= 12 Hz, J
2
= 4 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.2, 167.0, 144.4, 135.1, 132.7, 131.0, 129.7, 129.6,
129.2, 128.6, 128.2, 127.9, 127.1, 127.0, 126.7, 123.8, 67.1, 52.9, 51.7, 34.3.
FT-IR (cm
-1
): ν = 3057, 2951, 1813, 1744.
ESI-HRMS for C
34
H
29
NO
3
S: calculated [MNa]
+
554.1868 g/mol, found 554.1760 g/mol.
M.P. 75-80 ºC

119
1
H NMR, 400 MHz, CDCl
3
, 25 °C

13
C NMR, 100 MHz, CDCl
3
, 25 °C

120
IR

121
7.2.4. Preparation of azolines

General Procedure for Thiazoline Preparation.
3

Trityl-protected amide was dissolved in dry dichloromethane (0.05 M solution).
Stirring under N
2
, a solution of TiCl
4
(1 M in dichloromethane, 3 equiv.)
9
was added and
stirred at room temperature overnight until completion. The reaction mixture was then
washed with sat. aq. NaHCO
3
twice and dried over MgSO
4
. The product was purified via
flash chromatography on silica, eluting with ethyl acetate and hexanes.

122
2-Phenyl-4,5-dihydrothiazole-4-carboxylic acid
10

N
S
O
OH

7.7
Benzonitrile (8.24 mL, 80 mmol)
11
and L-cysteine (10.9 g, 90 mmol, 1.1 equiv.)
9

were dissolved in 1:1 methanol: pH 6.4 phosphate buffer (200 mL),
12
and the reaction
was stirred at 40 °C for 3 days. A white precipitate was filtered out and the resulting clear
yellow solution was acidified to ca. pH 4 with 1 M HCl and extracted three times with
dichloromethane (25 mL). The solution was concentrated under reduced pressure, and
crude product was obtained as a yellow solid upon its sonication in ethyl acetate. This
solid was purified by recrystallization in ethyl acetate to yield product as white solid
(4.97 g). Crude product was used in the next step without further purification.
Methyl 2-Phenyl-4,5-dihydrothiazole-4-carboxylate (2.2) via (7.7)
N
S
OCH
3
O

(2.2) is prepared from crude 2-phenyl-4,5-dihydrothiazole-4-carboxylic acid (7.7)
(3.5 g, 16.9 mmol) dissolved in 28 mL DMF at 0 °C, to which potassium carbonate (2.57
g, 18.6 mol)
13
was added. After stirring for 30 minutes, iodomethane (2.21 mL, 35.5
mmol) was added and the solution was brought to room temperature and stirred for 1.5
hours until completion by TLC (eluting with 3:1 hexanes: ethyl acetate). The reaction
mixture was then diluted in ethyl acetate (40 mL), washed with brine 5 times, and dried
over MgSO
4
. The crude product mixture was then concentrated under reduced pressure
and purified via flash chromatography (5-25% ethyl acetate in hexanes) to yield product
as white solid (2.92 g, 13.2 mmol, 23%, 2 steps). Data are consistent with a previously
characterized compound.
3

1
H NMR (400 MHz, CDCl
3
): δ = 7.87 (m, 2 H), 7.47 (m, 1 H), 7.41 (m, 2 H), 5.29 (t, 1
H, J = 8.8 Hz), 3.84 (s, 3 H), 3.73 (dd, 1 H, J
1
= 11.2 Hz, J
2
= 8.8 Hz), 3.62 (dd, 1 H, J
1
=
11.2 Hz, J
2
= 8.8 Hz).
All other thiazolines were prepared via a route reported by Kelly et al.
3

123
Methyl 2-(Naphthalen-2-yl)-4,5-dihydrothiazole-4-carboxylate (2.5)
N
S
OCH
3
O

2.5 is prepared from N-(2-napthoyl)-Cys(Trt)-OMe (7.6, 177 mg, 0.33 mmol)
according to the general procedure for thiazoline (Ch. 7.2.4) preparation to give product
as oil (23 mg, 26%).
1
H NMR (400 MHz, CDCl
3
): δ = 8.31 (s, 1 H), 8.02 (dd, 1 H, J
1
= 8.0 Hz, J
2
= 2.0 Hz),
7.91 (dd, 1 H, J
1
= 8.0 Hz, J
2
= 1.6 Hz), 7.86 (d, 2 H, J = 8.0 Hz), 7.54 (m, 2 H), 5.35 (t, 1
H, J = 8.0 Hz), 3.86 (s, 3 H), 3.78 (dd, 1 H, J
1
= 12 Hz, J
2
= 8.0 Hz), 3.69 (dd, 1 H, J
1
=
12 Hz, J
2
= 8.0 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.5, 171.1, 135.0, 132.8, 130.2, 129.8, 129.1, 128.4,
127.9, 127.8, 126.8, 125.0, 78.7, 53.0, 35.6.
FT-IR (cm
-1
): ν = 2954, 2929, 1742, 1604.
ESI-HRMS for C
15
H
13
NO
2
S: calculated [MH]+ 272.0667 g/mol, found 272.0740 g/mol.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

124
13
C NMR, 100 MHz, CDCl
3
, 25 °C

125
Methyl 2-(4-Fluorophenyl)thiazole-4-carboxylate (2.6)
N
S
OCH
3
O
F

2.6 is prepared from N-(4-fluorobenzoyl)-Cys(trt)-OMe (7.2, 750 mg, 1.5 mmol)
according to general procedure for thiazoline preparation to give product as white solid
(220 mg, 61%).
1
H NMR (500 MHz, CDCl
3
): δ = 7.87 (ddd, 2 H, J
1
= 8.5 Hz, J
2
= 5.5 Hz, J
3
= 2.0 Hz),
7.1 (ddd, 2 H, J
1
= 8.5 Hz, J
2
= 8 Hz, J
3
= 2 Hz), 5.28 (t, 1 H, J = 8.5 Hz), 3.84 (s, 3 H),
3.73 (dd, 1 H, J
1
= 11 Hz, J
2
= 9 Hz), 3.65 (dd, 1 H, J
1
= 11 Hz, J
2
= 9 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.4, 166.3, 163.7, 131.0, 129.1 (d, J
C-F
= 37.2 Hz),
115.8 (d, J
C-F
= 86.8 Hz), 78.6, 53.0, 35.8.
FT-IR (cm
-1
): ν = 2953, 1742, 1666, 1603, 1505.
ESI-HRMS for C
11
H
10
FNO
2
S: calculated [MH]
+
240.0416 g/mol, found 240.0487.
M.P. 103-105 ºC.

1
H NMR, 500 MHz, CDCl
3
, 25 °C

126
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

127
Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate (2.7)
N
S
OCH
3
O
C
N

2.7 is prepared from N-(2-cyanophenyl)-Cys(trt)-OMe (7.3, 2.03 g, 4 mmol)
according to the general procedure for thiazoline preparation to give product as white
solid (151 mg, 15%).
1
H NMR (500 MHz, CDCl
3
): δ = 7.96 (dt, 2 H, J
1
= 8.5 Hz, J
2
= 2.0 Hz), 7.71 (dt, 2 H, J
1

= 9.0 Hz, J
2
= 2.0 Hz), 5.32 (t, 1 H, J = 9 Hz), 3.85 (s, 3 H), 3.79 (dd, 1 H, J
1
= 11.5 Hz,
J
2
= 9.0 Hz), 3.70 (dd, 1 H, J
1
= 11.3 Hz, J
2
= 9.0 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 170.9, 147.6, 136.6, 132.4, 129.3, 118.2, 115.2, 78.7,
53.1, 35.9.
IR (cm
-1
): ν = 2953, 2920, 2230, 1743.
ESI-HRMS for C
12
H
10
N
2
O
2
S: calculated 247.0463 g/mol, found 247.0536 g/mol.
M.P. 107-108 ºC.

1
H NMR, 500 MHz, CDCl
3
, 25 °C

128
13
C NMR, 100 MHz, CDCl
3
, 25 °C

129
Methyl 2-(Indol-2-yl)-4,5-dihydrothiazole-4-carboxylate (2.12)
N
S
OCH
3
O
NH

2.12 is prepared from methyl 2-(indole-2-carboxamido)-3-(tritylthio)propanoate
(7.4, 200 mg, 0.38 mmol) according to the general procedure for thiazoline preparation to
give product as white solid (40 mg, 0.15 mmol, 40%).
1
H NMR (400 MHz, CDCl
3
): δ = 9.20 (s, 1 H), 7.65 (dd, 1 H, J
1
= 8 Hz, J
2
= 1.2 Hz),
7.35 (dd, 1 H, J
1
= 8 Hz, J
2
= 1.2 Hz), 7.29 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz),
7.13 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 6.98 (d, 1 H, J = 1 Hz), 5.26 (t, 1 H, J =
8 Hz), 3.84 (s, 3 H), 3.76 (dd, 1 H, J
1
= 12 Hz, J
2
= 8 Hz), 3.68 (dd, 1 H, J
1
= 12, Hz, J
2
=
8 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.3, 163.0, 137.1, 130.2, 127.9, 152.2, 122.1, 120.2,
111.7, 108.7, 77.7, 53.0, 35.6.
FT-IR (cm
-1
): ν = 3061, 2951, 1739, 1603, 1518.
ESI-HRMS for C
13
H
12
N
2
O
2
S: calculated [MH]
+
261.0619 g/mol, found 261.0691 g/mol.
M.P. 141-142 ºC.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

130
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

131
Methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-carboxylate (2.13)
N
S
OCH
3
O
N
CH
3

2.13 is prepared from methyl 2-(1-methylindole-2-carboxamido)-3-
(tritylthio)propanoate (7.5, 1.1 g, 2.1 mmol) according to the general procedure for
thiazoline preparation to give product as white solid (120 mg, 0.44 mmol, 21%).
1
H NMR (400 MHz, CDCl
3
): δ = 7.64 (dt, 1 H, J
1
= 8 Hz, J
2
= 1.2 Hz), 7.37 (d, 1 H, J =
8 Hz), 7.33 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 7.14 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8
Hz, J
3
= 1.2 Hz), 7.16 (s, 1 H), 5.37 (dd, 1 H, J
1
= 8 Hz, J2 = 8 Hz), 4.12 (s, 3 H), 3.84 (s,
3 H), 3.65 (dd, 1 H, J
1
= 8 Hz, J
2
= 12 Hz), 3.59 (dd, 1 H, J
1
= 8 Hz, J
2
= 12 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.6, 163.3, 139.9, 131.0, 126.7, 124.7, 122.0, 120.5,
110.3, 110.2, 79.1, 52.9, 34.8, 32.3.
FT-IR (cm
-1
): ν = 3060, 2951, 1741, 1660, 1603, 1510.
ESI-HRMS for C
14
H
14
N
2
O
2
S: calculated [MH]
+
275.0776 g/mol, found 275.0850 g/mol.
M.P. 78-80 ºC.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

132
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

133
7.2.5. General procedure for catalytic oxidation.
Azoline was dissolved in DMF at room temperature (50 mM). After the addition
of (DAB)Cu
II
complex 1 (10 mol%) and DBU (10 mol%, or other if specified), the
reaction was allowed to stir at 100 °C in air until complete by TLC (eluting with 3:1
hexanes: ethyl acetate). The solution was then diluted with ethyl acetate, washed with
distilled water, and dried over MgSO
4
. The crude product was purified via flash
chromatography on silica, eluting with 0-20% ethyl acetate in hexanes, to give the
corresponding azole.

134
7.2.6. General procedure for copper-free, base mediated oxidation.
Azoline was dissolved in DMF at room temperature (50 mM). After the addition
of DBU (1.1 equiv., or other if specified), the reaction was allowed to stirring at 70 °C in
air until complete by TLC (eluting with 3:1 hexanes:ethyl acetate). The solution was then
diluted with ethyl acetate, washed with deionized water and dried over MgSO
4
. The
crude product was purified via flash chromatography on silica, eluting with 0-20% ethyl
acetate in hexanes, to give the corresponding azole.

135
Methyl 2-Phenylthiazole-4-carboxylate (2.2a)
N
S OCH
3
O

2.2a is prepared from methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate
(2.2, 22 mg, 0.1 mmol) according to the catalytic procedure (8 hours, 19 mg, 87%) or
base-promoted procedure (0.5 hours, 15 mg, 66%) to give 2.2a as white solid. Data are
consistent with a previously characterized compound.
14

1
H NMR (400 MHz, CDCl
3
): δ = 8.37 (s, 1 H), 7.98 (m, 2 H), 7.47 (m, 3 H), 3.91
(s, 3 H).

136
Methyl 2-(4-Nitrophenyl)thiazole-4-carboxylate (2.3a)
N
S
OCH
3
O
O
2
N

2.3a is prepared from methyl 2-(4-nitrophenyl)-4,5-dihydrothiazole-4-carboxylate
(2.3, 53 mg, 0.2 mmol)
3
according to the general catalytic procedure 7.2.5
(3 hours, 41 mg, 78%) or a variant of the base-promoted procedure 7.2.6 wherein only
10 mol% of DBU is incorporated (1 hour, 36 mg of yellow, orange crystalline solid,
69%).
1
H NMR (400 MHz, CDCl
3
): δ = 8.31 (d, 2 H, J = 8 Hz), 8.29 (s, 1 H), 8.18 (d, 2 H, J = 8
Hz), 3.98 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 161.8, 148.8, 138.3, 129.7, 129.0, 127.9, 124.6, 52.9,
29.9.
IR (cm
-1
): ν = 3125, 3092, 1721.
ESI-HRMS for C
11
H
8
N
2
O
4
S: calculated [MH]
+
265.0205 g/mol, found: 265.0278 g/mol.
Melting Point: 224-227 ºC.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

137
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

138
Methyl 2-(4-Methoxyphenyl)thiazole-4-carboxylate (2.4a)
N
S
O
OCH
3
H
3
CO

2.4a is prepared from methyl 2-(4-methoxyphenyl)-4,5-dihydrothiazole-4-
carboxylate (2.4, 25 mg, 0.1 mmol)
3
according to the general catalytic procedure 7.2.5 (8
hours, 17 mg, 68%) or base-promoted procedure 7.2.6 (4 hours, 14 mg, 58%) to give a
white crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 8.10 (s, 1 H), 7.96 (d, 2 H, J = 8 Hz), 6.97 (d, 2 H, J = 8
Hz), 3.97 (s, 3 H), 3.87 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 169.0, 162.2, 161.8, 147.6, 128.7, 126.7, 125.8, 114.4,
55.6, 52.6.
FT-IR (cm
-1
): ν = 3119, 3025, 1740, 1710.
ESI-HRMS for C
12
H
11
NO
3
S: calculated 250.0460 g/mol, found 250.0532 g/mol.
M.P. 67-79 °C.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

139
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

140
Methyl 2-(Napthalen-2-yl)thiazole-4-carboxylate (2.5a)
N
S
OCH
3
O

2.5a is prepared from methyl 2-(naphthalen-2-yl)-4,5-dihydrothiazole-4-
carboxylate (2.5, 20 mg, 0.74 mmol) according to the catalytic procedure 7.2.5 (8.5
hours, 16 mg, 79%) or base-promoted procedure 7.2.6 (1 hour, 15 mg, 77%) to give 2.5a
as a white crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 8.52 (s, 1 H), 8.22 (s, 1 H), 8.09 (d, 1 H, J = 8 Hz), 7.93
(m, 2 H), 7.85 (m, 1 H), 7.54 (m, 2 H), 4.01 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 169.3, 162.2, 148.1, 134.6, 133.3, 130.3, 129.1, 129.0,
128.1, 127.6, 127.2, 126.9, 124.3, 52.8, 29.9.
FT-IR (cm
-1
): ν = 3138, 3048, 1733.
ESI-HRMS for C
15
H
11
NO
2
S: calculated [MH]
+
270.0510 g/mol, found 270.0583 g/mol.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

141
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

142
Methyl 2-(4-Fluorophenyl)thiazole-4-carboxylate (2.6a)
N
S
OCH
3
O
F

2.6a is prepared from methyl 2-(4-fluorophenyl)-4,5-dihydrothiazole-4-
carboxylate (2.6, 20 mg, 0.084 mmol) according to the catalytic procedure 7.2.5 (2 hours,
12 mg, 58%) or base-promoted oxidation 7.2.6 (45 minutes, 9 mg, 44%) to give 2.6a as a
white crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 8.16 (s, 1 H), 8.00 (m, 2 H), 7.15 (t, 2 H, J = 8.4 Hz),
3.98 (s, 3 H).
13
C NMR (100 MHz CDCl
3
) δ: 165.7, 163.2, 162.1, 147.9, 129.30, 129.1 (d, J
C-F
= 33.6
Hz), 127.5, 116.3 (d, J
C-F
= 88.4 Hz), 52.7.
FT-IR (cm
-1
): ν = 3133, 3108, 1750.
ESI-HRMS for C
11
H
8
FNO
2
S: calculated [MH]
+
238.0260 g/mol, found 238.0333 g/mol.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

143

13
C NMR, 100 MHz, CDCl
3
, 25 °C

144
Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate (2.7a).
N
S
OCH
3
O
C
N

2.7a is prepared from methyl 2-(4-cyanophenyl)-4,5-dihydrothiazole-4-
carboxylate (2.7, 20 mg, 0.81 mmol) according to the catalytic procedure 7.2.5 (4 hours,
14 mg, 69%) or base-promoted procedure 7.2.6 (45 minutes, 9 mg, 44%) to give 2.7a as a
white crystalline solid.
1
H NMR (500 MHz, CDCl
3
): δ = 8.27 (s, 1 H), 8.13 (dd, 2 H, J
1
= 8.5 Hz, J
2
= 2.5 Hz),
7.76 (dd, 2 H, J
1
= 8.5 Hz, J
2
= 2.5 Hz), 4.0 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 166.6, 161.8, 148.6, 136.7, 133.0, 128.7, 127.6, 118.4,
114.3, 52.9.
FT-IR (cm
-1
): ν = 3133, 2233, 1747.
ESI-HRMS for C
12
H
8
N
2
O
2
S: calculated [MH]
+
245.0306 g/mol, found 245.0379 g/mol.
M.P. 199-201 ºC.

1
H NMR, 500 MHz, CDCl
3
, 25 °C

145
13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

146
Methyl 2-Phenyloxazole-4-carboxylate (2.8a)
N
O
OCH
3
O

2.8a is prepared from methyl 2-phenyl-4,5-dihydrooxazole-4-carboxylate (2.8,
20 mg, 0.1 mmol)15

according to a variant of the catalytic procedure 7.2.5 wherein
30 mol% of base is added (9 hours, 4 mg, 18%) or base-promoted procedure 7.2.6
(6 hours, 3 mg, 16%) to give 2.8a as a white crystalline solid. Data are consistent with a
previously characterized compound.
15

1
H NMR (400 MHz, CDCl
3
): δ = 8.31 (s, 1 H), 8.13 (d, 2 H, J = 8 Hz), 7.49 (m, 3 H),
3.97 (s, 3 H).

147
Methyl 2-(4-Nitrophenyl)oxazole-4-carboxylate (2.9a)
N
O
OCH
3
O
O
2
N

2.9a is prepared from methyl 2-(4-nitrophenyl)-4,5-dihydrooxazole-4-carboxylate
(2.9, 20 mg 0.08 mmol)
16
according to a variant of the catalytic procedure 7.2.5 wherein
30 mol% of base is added (12 hours, 7 mg, 37%) or base-promoted procedure 7.2.6
(2 hours, 8 mg, 41%) to give 2.9a as a white crystalline solid. Data are consistent with a
previously characterized compound.
17

1
H NMR (400 MHz, CDCl
3
): δ = 8.38 (s, 1 H), 8.36 (dt, 2 H, J
1
= 8 Hz, J
2
= 2.4 Hz), 8.31
(dt, 2 H, J
1
= 8 Hz, J
2
= 2.4 Hz), 3.99 (s, 3 H).

148
Methyl 2-Methylthiazole-4-carboxylate (2.10a)
N
S
OCH
3
O

2.10a is prepared from methyl 2-methyl-4,5-dihydrothiazole-4-carboxylate (2.10,
20 mg, 0.12 mmol)
18
according to the catalytic procedure 7.2.5 (8 hours, 5 mg, 24%) or
base-promoted procedure 7.2.6 (5 hours, 8 mg, 39%) to give 2.10a as a white crystalline
solid. Data are consistent with a previously characterized compound.
19

1
H NMR (400 MHz, CDCl
3
): δ = 8.05 (s, 1 H), 3.95 (s, 3 H), 2.77 (s, 3 H).

149
Methyl 2-Phenethylthiazole-4-carboxylate (2.11a).
N
S
OCH
3
O

2.11a is prepared from methyl 2-phenethyl-4,5-dihydrothiazole-4-carboxylate
(2.11, 20 mg, 0.08 mmol)
3
according to the catalytic procedure 7.2.5 (12 hours, 9 mg,
45%) or base-promoted procedure 7.2.6 (6 hours, 10 mg, 51%) to give 2.11a as a white
crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 8.05 (s 1 H), 7.3 (m, 2 H), 7.21 (m, 3 H), 3.96 (s, 3 H),
3.38 (t, 2 H, J = 8 Hz), 3.18 (t, 2 H, J = 8 Hz).
13
C NMR (100 MHz, CDCl
3
): δ = 171.1, 162.1, 146.6, 140.0, 128.8, 128.6, 127.4, 126.7,
52.6, 36.1, 35.4.
FT-IR (cm
-1
): ν = 3119, 2954, 1721.
ESI-HRMS for C
13
H
13
NO
2
S: calculated [MH]
+
248.0667 g/mol, found 248.0740 g/mol.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

150

13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

151
Methyl 2-(Indol-2-yl)thiazole-4-carboxylate (2.12a)
N
S
OCH
3
O
NH

2.12a is prepared from methyl 2-(indol-2-yl)-4,5-dihydrothiazole-4-carboxylate
(2.12, 20 mg, 0.077 mmol) according to the catalytic procedure 7.2.5 (6 hours, 11 mg,
55%) to give 2.12a as a white crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 9.33 (s, 1 H), 8.13 (s 1 H), 7.65 (dd, 1 H J
1
= 8 Hz, J
2
=
0.8 Hz), 7.40 (dd, 1 H J
1
= 8 Hz, J
2
= 0.8 Hz), 7.28 (ddd, 1 H, J
1
= 8 Hz, J
1
= 8 Hz, J
3
=
0.8 Hz), 7.15 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 0.8 Hz), 7.05 (dd, 1 H, J
1
= 2 Hz, J
2
=
0.8 Hz), 3.99 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 161.8, 161.1, 147.2, 136.8, 130.6, 128.4, 126.8, 124.7,
121.6, 121.0, 111.6, 104.3, 52.7.
FT-IR (cm
-1
): ν = 2921, 2852, 1732, 1717.
ESI-HRMS for C
13
H
10
N
2
O
2
S: calculated [MH]
+
259.0463 g/mol, found 259.0536 g/mol.
M.P. 69-71 ºC.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

152

13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

153
Methyl 2-(1-Methyl-indol-2-yl)thiazole-4-carboxylate (2.13a)
N
S
OCH
3
O
N
CH
3

2.13a is prepared from methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-
carboxylate (2.13, 20 mg, 0.073 mmol) according to the catalytic procedure 7.2.5 (14
hours, 13 mg, 65%) or base-promoted procedure 7.2.6 (30 minutes, 9 mg, 45%) to give
2.13a as a white crystalline solid.
1
H NMR (400 MHz, CDCl
3
): δ = 8.15 (s, 1 H), 7.64 (d, 1 H, J = 8 Hz), 7.40 (d, 1 H, J =
8.8 Hz), 7.32 (ddd, 1 H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 7.16 (ddd, 1 H, J
1
= 8 Hz, J
2
=
8 Hz, J
3
= 1.2 Hz), 7.04 (s, 1 H), 4.21 (s, 3 H), 3.98 (s, 3 H).
13
C NMR (100 MHz, CDCl
3
): δ = 162.0, 161.6, 147.6, 139.5, 131.6, 127.2, 127.1, 124.1,
121.5, 120.7, 110.3, 106.1, 52.6, 32.1.
FT-IR (cm
-1
): ν = 2953, 2925, 1732, 1552.
ESI-HRMS for C
14
H
12
N
2
O
2
S: calculated [MH]
+
273.0619 g/mol, found 273.0697 g/mol.
M.P 124-127 ºC.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

154

13
C NMR, 100 MHz, CDCl
3
, 25 °C

IR

155
Methyl 4-hydroxy-2-phenyl-4,5-dihydrothiazole-4-carboxylate (2.15)
N
S
OCH
3
O
OH

2.15 was isolated and characterized via the general procedure for base-promoted
oxidation 7.2.6 of methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate 2.2 in which the
reaction was stopped after 15 minutes and cooled to room temperature. Product was
purified via automated column chromatography (hexanes:ethyl acetate) to give 2.15 as a
white solid. Results of further characterization and mechanistic details are summarized in
Chapter 2.5.
1
H NMR: 7.89 (dd, 2 H, J = 8 Hz, J = 1.2 Hz), 7.51 (tt, 1 H, J = 8 Hz, J = 8 Hz), 7.42
(tt, 2 H, J = 8 hz, J =1.2 Hz), 4.18 (s, 1 H), 4.02 (dd, 2 H, J = 12 Hz, J = 1.2 Hz), 3.89
(s, 3 H), 3.55 (d, 1 H, J = 12 Hz).
MALDI for C
11
H
11
NO
3
S: Calculated [MH]
+
238.04 g/mol, found 238.00 g/mol.

1
H NMR, 400 MHz, CDCl
3
, 25 °C

156
7.2.7. Thiazoline to thiazole oxidation on 1000 mg scale.
In a 3-neck round bottom flask, N,N'-(butane-2,3-diylidene)bis(2,4,6-
trimethylaniline) (145 mg, 0.45 mmol) and copper(II) triflate (164 mg, 0.45 mmol),
forming [(
Mes
DAB
Me
)Cu
II
(OH
2
)
3
]
2+
[
-
OTf]
2
in situ, were stirred in DMF at room
temperature for 30 minutes. DBU (0.068 mL, 0.45 mmol) and methyl 2-phenyl-4,5,-
dihydrothiazole-4-carboxylate 2.2 (1.0 g, 4.5 mmol) were added sequentially. A
condenser was then attached to the flask, which was then placed in a 100 ºC oil bath. A
gentle stream of compressed air was bubbled into the reaction, which was stirred for
18 hours. The reaction mixture was diluted with ethyl acetate and washed with deionized
water three times then dried over MgSO
4
. The crude reaction mixture was then
concentrated under reduced pressure and purified via column chromatography
(5-25% hexanes in ethyl acetate) to yield desired product 2.2a as a white crystalline solid
(791 mg, 3.6 mmol, 80%). Base-promoted oxidation of 2.2 to 2.2a was performed on a
1000 mg scale according to general base-promoted procedure 7.2.6 to give 2.2a as a
white crystalline solid (545 mg, 2.5 mol, 55 %).
Table 7.1. Scalabilty of the conversion of thiazoline to thiazole.
10 mol% 2.1, 10 mol% DBU
DMF, 100 ºC
OR
1.1 equiv. DBU, DMF, 70 ºC
N
S OCH
3
O
N
S OCH
3
O
H
H
2.2 2.2a

Entry Scale (mg) Conditions

Time Isolated Yield (%)
1 20 Catalyzed

8 h 88
2 1000 Catalyzed 18 h 80
3 20 Base 1 h 66
4 1000 Base 2.5 h 55

157
7.2.8. Oxidation of thiazoline in the presence of H
2
18
O.
Thiazoline was dissolved in DMF (dried over calcium hydride)
9
at room
temperature (50 mM). H
2
18
O (1.2 equiv.) and DBU (1.1 equiv.) were added and the
reaction was stirred at 70 °C in air for 30 minutes. An aliquot of the reaction mixture was
analyzed by MALDI and compared to an isolated sample of angular hydroxide thiazoline
2.15 made as a reaction intermediate by the general procedure for base-promoted
oxidation 7.2.6. Vanishingly little additional incorporation of
18
O was observed. Spectra
available in Chapter 2.5.

158
7.2.9. Oxidation of thiazoline with K
2
CO
3
.
Reaction of thiazoline 2.2 with K
2
CO
3
(1 equiv.) and catalyst 2.1 in DMF (2 mL)
produced thiazole 2.2a in 30% yield. The remaining mixture contained angular hydroxide
2.15 and an unknown intermediate, which is purportedly an angular peroxide,
20
in a ratio
of ca. 1:1.3 ratio, 22% and ca. 26% isolated yields respectively. We were unable to detect
this third compound via MALDI spectrometery, possibly because of photolytic
decomposition of the purported peroxide.

159
7.3. Chapter 4 experimental data.
7.3.1. Preparation of small molecule contrast agents.

[(DOTA)Gd]
-
Na
+

N
N N
N
Gd
O
O
O
O
O
O
O
O
Gd-DOTA
Na

4.1
[(DOTA)Gd]
-
Na
+
(4.1) was prepared according to modified literature
procedures.
21
Ligand (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
(24.3 mg, 0.06 mmol) was dissolved in distilled Arrowhead® water (3 mL).
GdCl
3
• 6 H
2
O (22.3 mg, 0.06 mmol) was added to the solution of ligand while stirring.
After the addition of GdCl
3
, pH = 2.12, adjust the pH of the resulting reaction mixture to
between 5.5 and 7.0 by adding 0.1 M NaOH. The reaction is complete when the pH
remains constant for > 1 h. The reaction mixture pH was adjusted with 1 M NaOH pH to
a final pH ≥ 11 to precipitate any uncomplexed metal as the insoluble hydroxide. The
solution was filtered through a 0.45 µm filter and lyophilized to dryness.The dried solid
purified via reverse phase column chromatography (H
2
O:MeOH). [(DOTA)Gd]
-
Na
+
•
4 H
2
O (4.1) was produced as a white solid (21.3 mg) in a 54 % yield. Xylenol orange test
(Chapter 7.3.2) indicated no presence of free Gd
3+
.
Elemental analysis: calc’d C, 29.44; H, 4.94; Gd, 24.09; N, 8.58; Na, 3.52; O, 29.42;
found C, 29.73; H, 4.46; N, 8.41.
MALDI MS for [Gd(DOTA)]
-
m/z: [H
2
Gd(DOTA)]
+
calc’d 560.1 g/mol, found 560.0
g/mol; [HNaGd(DOTA)]
+
calc’d 582.1 g/mol, found 582.0 g/mol; [Na
2
Gd(DOTA)]
+

calc’d 604.1 g/mol, found 603.9 g/mol; [Na
2
Gd(DOTA)(H
2
O)]
+
calc’d 622.1g/ mol,
found 621.9 g/mol.

160
[Gd(DOTP)]
5-
5 Na
+

N N
N N
Gd
P
O
P
O
O
O
P
O
O
P
O
O
O
O
O
O
5
Gd-DOTP
5 Na

4.2
[Gd(DOTP)]
5-
5 Na
+
(4.2) were prepared and crystallized according to literature
procedures.
22, 23, 24
[Gd(DOTP)]
5-
5 Na
+
(107.3 mg) prepared following the literature
procedure and was subjected to slow-diffusion crystallization (H
2
O:isopropanol). After
slow-diffusion crystallization, the crystals were placed under high vacuum at 35
o
C
overnight to give white solid product (82.7 mg, 77.1%). Elemental analysis is consistent
with the formula [Gd(DOTP)]
5-
5 Na
+
• 9 H
2
O.
Elemental analysis: calc’d C, 14.79; H, 4.34; N, 5.75; Na, 11.79; found C, 14.57;
H, 4.02; N, 5.36; Na, 11.88.
MALDI MS for [(DOTP)Gd]
5-
(uncalibrated) m/z: [(DOTP)H
6
Gd]
+
calc’d 704.00 g/mol,
found 703.71 g/mol; [(DOTP)H
5
NaGd]
+
calc’d 725.99 g/mol, found 725.67 g/mol;
[(DOTP)H
4
Na
2
Gd]
+
calc’d 747.96 g/mol, found 747.64 g/mol; [(DOTP)H
3
Na
3
Gd]
+

calc’d 769.95 g/mol, found 767.54 g/mol.

161
Dilution of [(DOTA)Gd]
-
Na
+
• 4 H2O (4.1) and Na
5
[Gd(DOTP)] • 9 H
2
O (4.2):
Solutions of [(DOTA)Gd]
-
Na
+
• 4 H2O (4.1) and [Gd(DOTP)]
5-
5 Na
+
• 9 H
2
O (4.2)
were separately prepared. The Gd–L is dissolved in distilled water to a final
concentration of 1 mM. The 1 mM stock solution is then diluted to 0.75, 0.5, and 0.25
mM solutions using a microsyringe to measure [(DOTA)Gd]
-
Na
+
• 4 H2O (4.1) or
[Gd(DOTP)]
5-
5Na
+
• 9 H
2
O (4.2) 1 mM solution and water.

162
Shell Synthesis:
H
2
N N
H
NH
2
F F
F F
F F
F F
O
O
O
O
O
O
O
F
3
C

4.3
Shell was synthesized according to literature procedure.
25

163
7.3.2. General procedure for assessment of the presence of free Gd
3+
.
The metal complex (see below for structures and results) (0.3 mg) was dissolved
in acetate buffer (Buffer preparation: dissolve 1.4 mL of acetic acid
12
in 400 mL water,
adjust the pH to 5.8 with 1 M NH
4
OH,
12
and add water to produce a total volume of 500
mL) and xylenol orange
5
(3 mL, 16 µM xylenol orange in pH 5.8 acetate buffer) was
added. The presence of free metal is detected via observation of a color change of the
indicator from yellow/orange to violet. No detectable orange to violet color change
indicates minimal to no free Gd
3+
present.
[(DOTA)Gd]
-
Na
+

N
N N
N
Gd
O
O
O
O
O
O
O
O
Gd-DOTA
Na

4.1
Result of procedure 7.3.2 for 4.1 was negative for presence of free Gd
3+
with minimal
color change from orange to yellow/orange.
[Gd(DOTP)]
5-
5 Na
+

N N
N N
Gd
P
O
P
O
O
O
P
O
O
P
O
O
O
O
O
O
5
Gd-DOTP
5 Na

4.2
Result of procedure 7.3.2 for 4.2 was negative for presence of free Gd
3+
with minimal
color change from orange to yellow/orange.

164
7.3.3. Preparation of Gd–LShell and Gd-L·Shell with Urea samples
(L = DOTA or DOTP).

Gd–DOTAShell (4.1–4.3)
A stock solution of shell (4.3) in distilled water was prepared by dispensing 4.3 as
an oil into a tared vial and diluting to 100 mM in distilled H
2
O (15 mg in 250 mL
distilled H
2
O). An aliquot of shell (4.3) solution corresponding to 4 equivalents of 4.3
relative to 4.1 (example calculation: 1 µL of 100 mM shell solution for 100 µL of 0.25
mM Gd–DOTA) was added to a 0.5 dram vial. Separate vials of 4.3 solution were made
for each concentration of Gd–DOTA (4.1) solution (0.25, 0.5, 0.75, and 1.0 mM 4.1 in
distilled H
2
O, see 7.3.1 for preparation). The H
2
O was removed under vacuum leaving a
film of 4.3 in the vial. Subsequently, 100 µL of Gd–DOTA of corresponding
concentration (0.25, 0.5, 0.75, or 1.0 mM 4.1 in distilled H
2
O) was used to rinse the
respective vials of shell in order to combine 4.1 and 4.3 to form 4.1–4.3.
Gd–DOTPShell (4.2–4.3)
A stock solution of shell (4.3) in distilled water was prepared by dispensing 4.3 as
an oil into a tared vial and diluting to 100 mM in distilled H
2
O (15 mg in 250 mL
distilled H
2
O). An aliquot of shell (4.3) solution corresponding to 4 equivalents of 4.3
relative to 4.2 (example calculation: 1 µL of 100 mM shell solution for 100 µL of 0.25
mM Gd–DOTP) was added to a 0.5 dram vial. Separate vials of 4.3 solution were made
for each concentration of Gd–DOTP (4.2) solution (0.25, 0.5, 0.75, and 1.0 mM 4.2 in
distilled H
2
O, see 7.3.1 for preparation). The H
2
O was removed under vacuum leaving a
film of 4.3 in the vial. Subsequently, 100 µL of Gd–DOTP of corresponding
concentration (0.25, 0.5, 0.75, or 1.0 mM 4.1 in distilled H
2
O) was used to rinse the
respective vials of shell in order to combine 4.2 and 4.3 to form 4.2–4.3.
Gd–DOTAShell (4.1–4.3) with Urea and Gd–DOTPShell (4.2–4.3) with Urea
Gd–DOTAShell (4.1–4.3) with Urea and Gd–DOTPShell (4.2–4.3) with Urea were
prepared as describe above for Gd–DOTAShell (4.1–4.3) and Gd–DOTPShell (4.2–4.3)
with the addition of urea (0.9 mg in 100 µL, 150 mM) to (4.1–4.3) or (4.2–4.3).

165
7.3.4. Construction of r
1
molar relaxivity curves.
Gd–L, Gd–Lshell, and Gd–Lshell with urea solutions (L = DOTA or DOTP)
were prepared via 7.3.3 (0.25, 0.50, 0.75, and 1.0 mM in H
2
O) and 100 µL of each were
dispensed into 1 mm coaxial inserts inside 5 mm standard NMR tubes with D
2
O (700 µL)
in the outer tube.
1
H and T
1
data were obtained on a Varian 400MR spectrometer using
standard T
1
inversion recovery pulse sequences with 4 scans and a 5 sec interpulse delay
and processed using Varian Nuclear Magnetic Resonance Java (VNMRJ). Peak heights
for the corresponding spectra were tabulated and fitted to a 3-parameter exponential
growth model to give experimental values and errors for T1 for each line in the spectrum.
The Gd–Lshell with urea solution tubes (0.25, 0.50, 0.75, and 1.0 mM) were removed
after the first T
1
measurement (urea) acquisition, sonicated with a benchtop sonicator (3 x
3 s followed by 1 x 1 s insertions in the sonicator water bath), and an additional T
1
was
acquired. A background T
1
measurement was taken with Distilled water in the inner
coaxial insert and D
2
O in the outer 5mm NMR tube. Resulting T
1
values are summarized
in Figure 7.1.

166
1
1.5
2
2.5
3
3.5
4
4.5
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
y = m1 + m2 * M0
Error Value
0.039042 1.1391 m1
0.063756 2.9651 m2
NA 0.0076216 Chisq
NA 0.99931 R
1
1.5
2
2.5
3
3.5
4
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
y = m1 + m2 * M0
Error Value
0.13045 1.0201 m1
0.21303 2.6861 m2
NA 0.085089 Chisq
NA 0.9907 R
1
1.5
2
2.5
3
3.5
4
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
y = m1 + m2 * M0
Error Value
0.11192 1.0348 m1
0.18277 2.6627 m2
NA 0.062632 Chisq
NA 0.99301 R
1
1.5
2
2.5
3
3.5
4
4.5
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
y = m1 + m2 * M0
Error Value
0.045251 1.0956 m1
0.073894 2.9524 m2
NA 0.010238 Chisq
NA 0.99906 R

Figure 7.1. Molar relaxivity curves for a) Gd–DOTA, b) Gd–DOTAShell,
c) Gd–DOTAShell with urea, and d) Gd–DOTAShell with urea and sonication. Plotted
using Kaleidagraph (Synergy Software 2006, Version 4.03) with m2 = r
1
and error in m2
representing the standard error in the molar relaxivity.
a) b)
c) d)

167
7.3.5. Gd–DOTA (4.1) and Gd–DOTP (4.2) relaxivity response upon addition
of Shell (4.3).
Separate vials of shell 4.3 (1, 2, 4, and 6 equivalents relative to 1.0, 0.75, 0.5, and
0.25 mM Gd–DOTA 4.1) dispensed as 100 mM solution in H
2
O were added via
mircosyringe to 0.5 dram vials and evaporated to dryness under vacuum. The vials
containing dried 4.3 were rinsed with 100 µL of the corresponding concentration of 4.1
solution and pipetted into a 1 mm NMR coaxial insert, which was inserted into a 5 mm
NMR tube with 700 µL of D
2
O.
1
H and T
1
data were obtained on a Varian 400MR
spectrometer using standard T
1
inversion recovery pulse sequences with 4 scans and a
5 sec interpulse delay for each sample, processed using VNMRJ; T
1
values for 4.1 r
1

values for 4.2 and were generated and compared using Microsoft Excel. Peak heights for
the corresponding spectra were tabulated and fitted to a 3-parameter exponential growth
model to give experimental values and errors for T
1
for each line in the spectrum. The
experiment was repeated with 1, 2, 4, and 8 equivalents relative to 1.0, 0.75, 0.5, and 0.25
mM Gd–DOTP 4.3. A summary of these plots is given Chapter 4.2.

168
7.4. Chapter 5 experimental and spectral data.
7.4.1. Preparation of Gd
3+
complexes and standard solutions.

[CaCl]
+
[Gd(DOTA)]
-
· 2.5 H
2
O (CaCl[5.1])
N
N N
N
Gd
O
O
O
O
O
O
O
O
CaCl

5.1
To an 8-dram vial equipped with magnetic stir bar was added DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 24.3 mg, 0.06 mmol), GdCl
3

(22.3 mg, 0.06 mmol), and H
2
O (6 mL). The reaction mixture was stirred at reflux for 4 h
by partial immersion in a 100
o
C oil bath. Once at room temperature, CaCO
3
(90 mg,
0.9 mmol, 15 equiv.) was added. Insoluble precipitate was filtered using a disposable
45 µm PTFE syringe filter. The resulting aqueous solution was lyophilized to dryness to
yield 40 mg (99%) as a colorless solid, mp = 315-320
o
C (decomp.). Absent the ability to
lyophilize 6 mL of H
2
O, liquid water can be removed under reduced pressure or by
leaving it to evaporate in evaporating dish under a stream of dry air.
Elemental analysis for C
19
H
31
CaClGdN
4
O
8
· 2.5 H
2
O calc’d: C 27.96%, H 4.40%,
N 8.15%, Ca 5.83%; found C 27.85(26)%, H 4.34(7)%, N 7.87(9)%, Ca 6.88%. Error
bars represent standard deviation over 4 measurements.
MALDI MS for [Gd(DOTA)]
-
m/z: [H
2
Gd(DOTA)]
+
calc’d 560.1, found 560.0;
[HNaGd(DOTA)]
+
calc’d 582.1, found 582.0; [Na
2
Gd(DOTA)]
+
calc’d 604.1, found
603.9; [Na
2
Gd(DOTA)(H
2
O)]
+
calc’d 622.1, found 621.9.
The resulting solid was dissolved in deionized water to give a 1 mM solution for
use in the experiment.

169
Experimental Design Note:
We use calcium carbonate as a base here because we observe that synthesis of
[Gd(DOTA)]
-
salts from Gd
2
O
3
or GdCl
3
plus a sodium base give solutions of poorly
reproducible relaxivities on this scale. We observe the relaxivity of [Gd(DOTA)]
-
to have
significant pH sensitivity, but this calcium prep is more reliable and reproducible in our
hands. Relaxivity (r
1
) values of [H]
+
[Gd(DOTA)]
-
(2.9(1) mM
-1
s
-1
) and
[CaCl]
+
[Gd(DOTA)]
-
· 2.5 H
2
O (2.8(1) mM
-1
s
-1
) are in agreement. Thus, we believe that
[CaCl]
+
[Gd(DOTA)]
-
is a suitable, more convenient substitute for the free acid in this
experiment and its comparison to 2 [H]
+
[Gd(DTPA)]
2-
· 2 H
2
O is fair.

170
2[H]
+
[Gd(DTPA)]
2-
· 2 H
2
O (H
2
[5.2])
26

N
N N Gd
O
O
O
O
O
O
O
O
O
O
2 2H

5.2
To an 8-dram vial equipped with magnetic stir bar, diethylenetriaminepentacetic
acid (DTPA, 19.7 mg, 0.05 mmol), Gd
2
O
3
(54.3 mg, 0.15 mmol), and H
2
O (5 mL). The
reaction mixture was stirred at reflux 16 h by partial immersion in a 100
o
C oil bath, then
cooled to room temperature. Once at room temperature, excess Gd
2
O
3
was filtered using a
45 µm PTFE syringe filter.
27
The resulting solution was lyophilized to dryness to give
29 mg (%), mp > 300
o
C (decomp.).
MALDI MS for [Gd(DTPA)]
-
m/z: [H
3
Gd(DTPA)]
+
calc’d 549.0, found 548.9;
[H
2
NaGd(DTPA)]
+
calc’d 571.0, found 570.9; HNa
2
Gd(DTPA)]
+
calc’d 593.0, found
592.9; [Na
3
Gd(DTPA)]
+
calc’d 615.0, found 615.0. T
1
data of this material are consistent
with literature values for H
2
[Gd(DTPA)].
28

The resulting solid was dissolved in deionized water to give a 1 mM solution for
use in the experiment.

171
7.4.2. Molar relaxivity curves

Molar relaxivity curves of [Gd(DOTA)]
-
(5.1) with r
1
= 2.8(1) mM
-1
s
-1
and r
1
= 2.4(1)
mM
-1
s
-1
for Groups 1 and 2 respectively and [Gd(DTPA)]
2-
(5.2) with r
1
= 5.0(7) mM
-1
s
-1
and r
1
= 3.7(5) mM
-1
s
-1
for Groups 3 and 4 respectively. Error bars describe fit errors in
the T
1
data, not measurement errors that might have been introduced by the students.
Groups 1, 2, 3, and 4 consisted of 3-4 High School students at Polytechnic School.

172
7.5. Chapter 6 experimental data.

Gd–DOTA (4.1):
Gd-DOTA (100 µL, 1 mM) solution prepared as described in section 7.3.1 was
pipetted into a 1 mm coaxial NMR tube insert inside of a 5 mm NMR tube with
700 µL of D
2
O.
1
H and T
1
data were obtained on a Varian 400MR spectrometer using
standard T
1
inversion recovery pulse sequences with 4 scans and a 5 sec interpulse delay
for each sample and processed using VNMRJ. Results are summarized in
Chapter 6.4.1.
Polyvinylphosphoric acid (PVPA, 7.8):
2

Polyvinylphosphonic acid (~ 20 kDa, 200 mg PVPA in 10 mL H
2
O) solution was
pipetted into a 1 mm coaxial NMR tube insert inside of a 5 mm NMR tube with 700 µL
of D
2
O.
1
H and T
1
data were obtained on a Varian 400MR spectrometer using standard T
1

inversion recovery pulse sequences with 4 scans and a 5 sec interpulse delay for each
sample and processed using VNMRJ. Results are summarized in Chapter 6.4.1.
Gd–DOTAPVPA:
200 µL of PVPA (7.8, 200 mg PVPA in 10 mL H
2
O) was lyophilized to dryness
overnight. To the dried PVPA, 200 µL of Gd–DOTA (1 mM in H
2
O) was added. The
Gd–DOTAPVPA was pipetted into a 1 mm coaxial NMR tube insert inside of a 5 mm
NMR tube with 700 µL of D
2
O.
1
H and T
1
data were obtained on a Varian 400MR
spectrometer using standard T
1
inversion recovery pulse sequences with 4 scans and a 5
sec interpulse delay for each sample and processed using VNMRJ. Results are
summarized in Chapter 6.4.1.
Gd–DOTAPVPAShell:
200 µL of PVPA (7.8) (1 mM in H
2
O) was lyophilized to dryness overnight. To
the dried PVPA, 200 µL of Gd–DOTA (4.1) (1 mM in H
2
O) was added. To the
Gd–DOTAPVPA solution, shell (4.3) (4 equiv., 8 mL of 100 mM solution). The
Gd–DOTAPVPAShell solution was pipetted into a 1 mm coaxial NMR tube insert
inside of a 5 mm NMR tube with 700 µL of D
2
O.
1
H and T
1
data were obtained on a
Varian 400MR spectrometer using standard T
1
inversion recovery pulse sequences with 4
scans and a 5 sec interpulse delay for each sample and processed using VNMRJ. Results
are summarized in Chapter 6.4.1.

173
Heated Gd–DOTA (4.1), PVPA (7.8), Gd–DOTAPVPA, Gd–DOTAPVPAShell:
Separate solutions of Gd–DOTA (4.1), PVPA (7.8), Gd–DOTAPVPA,
Gd–DOTAPVPAShell solutions (200 µL, 1 mM) were prepared as described above in
0.5 dram vials and heated in an oil bath at 70 ºC for 15 h overnight. The solutions were
cooled to room temperature and pipetted into a 1 mm coaxial NMR tube insert and placed
inside of a 5 mm NMR tube with 700 µL of D
2
O.
1
H and T
1
data were obtained on a
Varian 400MR spectrometer using standard T
1
inversion recovery pulse sequences with 4
scans and a 5 sec interpulse delay for each sample and processed using VNMRJ. Results
are summarized in Chapter 6.4.1.
Xylenol orange determination of free Gd
3+
:
Presence of Gd
3+
in solution was determine via xylenol orange indicator test 7.3.2.
7.6. References.

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.
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176
Appendix

X-Ray Crystal Structure Solutions of 2.1 and 2.1a

Figure A1. ORTEP Diagram of Complex 1. Ellipsoids are Drawn at the 50% Probability
Level.

177
Table A1. Crystal Data and Structure Refinement 2.1.
Empirical formula C
24
H
28
Cu F
6
N
2
O
9
S
2

Formula weight 730.14
Temperature 250(2) K
Wavelength 0.71073 Å
Crystal system Monoclinic
Space group P2(1)
Unit cell dimensions a = 10.4067(13) Å α= 90°
b = 12.5305(15) Å β= 91.443(2)°
c = 12.5733(16) Å γ = 90°
Volume 1639.1(4) Å
3

Z 2
Density (calculated) 1.479 Mg/m
3

Absorption coefficient 0.876 mm
-1

F(000) 746
Theta range for data collection 1.62 to 27.49°
Index ranges -13 ≤ h ≤ 13, -12 ≤ k ≤ 5, -15 ≤ l ≤ 6
Reflections collected 10158
Independent reflections 5429 [R(int) = 0.0389]
Completeness to theta = 27.49° 97.5%
Absorption correction Empirical
Transmission factors min/max: 0.697
Refinement method Full-matrix least-squares on F
2

Data / restraints / parameters 5429 / 1 / 405
Goodness-of-fit on F
2
1.021
Final R indices [I>2sigma(I)] R1 = 0.0485, wR2 = 0.1149
R indices (all data) R1 = 0.0640, wR2 = 0.1231
Absolute structure parameter 0.001(16)
Largest diff. peak and hole 0.574 and -0.298 e Å
-3

Table A2. Atomic Coordinates (x 10
4
) and Equivalent Isotropic Displacement Parameters
(Å
2
x10
3
) for 2.1. U(eq) is Defined as one Third of the Trace of the Orthogonalized U
ij

Tensor.
x y z U(eq)
C(1) 7484(5) 5854(4) 304(4) 35(1)
C(2) 7787(5) 5459(5) -693(4) 36(1)
C(3) 8536(6) 6038(5) -1386(4) 43(1)
C(4) 9002(5) 7014(5) -1059(4) 42(2)
C(5) 8738(5) 7452(4) -63(4) 34(1)
C(6) 7956(4) 6863(5) 598(3) 28(1)
C(7) 6670(5) 5208(5) 1055(4) 41(1)
C(8) 8854(7) 5588(5) -2476(4) 57(2)
C(9) 9269(6) 8538(5) 260(4) 47(2)

178
N(1) 7625(3) 7243(3) 1632(3) 27(1)
C(10) 6744(5) 7927(4) 1736(3) 29(1)
C(11) 6402(5) 8204(4) 2868(3) 30(1)
N(2) 7088(4) 7715(3) 3569(3) 26(1)
C(14) 6847(4) 7856(4) 4691(3) 30(1)
C(15) 6235(4) 7015(5) 5211(3) 34(1)
C(16) 6029(5) 7146(5) 6302(4) 45(2)
C(17) 6394(5) 8061(6) 6839(4) 51(2)
C(18) 7003(6) 8866(6) 6298(4) 51(2)
C(19) 7259(5) 8792(5) 5196(4) 37(1)
C(20) 7881(6) 9699(5) 4638(5) 49(2)
C(21) 6105(7) 8202(7) 8006(4) 72(2)
C(22) 5757(6) 6065(5) 4630(5) 49(2)
C(13) 5341(5) 8949(5) 3074(4) 41(1)
C(12) 5993(5) 8455(5) 835(4) 40(1)
C(23) 7330(6) 2860(6) 3887(5) 57(2)
C(24) 7188(6) 1660(6) 9685(6) 65(2)
Cu(1) 8476(1) 6787(1) 2999(1) 28(1)
F(1) 7099(4) 2129(3) 3154(3) 76(1)
F(2) 6963(4) 2463(4) 4809(4) 83(1)
F(3) 6604(4) 3704(3) 3652(4) 82(1)
F(4) 8364(4) 1515(4) 10056(4) 105(2)
F(5) 6730(5) 2506(4) 10164(3) 89(2)
F(6) 6505(6) 858(5) 9973(5) 126(3)
O(1) 8889(3) 6145(3) 4416(2) 35(1)
O(2) 9561(3) 5723(3) 2291(2) 34(1)
O(3) 10040(3) 7979(3) 3144(3) 37(1)
O(4) 7901(5) 930(5) 7889(6) 95(2)
O(5) 5827(3) 1791(5) 7952(3) 55(1)
O(6) 7772(5) 2847(4) 8079(3) 56(1)
O(7) 9650(4) 2233(3) 4171(3) 55(1)
O(8) 9080(4) 3990(3) 4833(3) 50(1)
O(9) 9238(4) 3685(3) 2946(3) 52(1)
S(1) 9014(1) 3233(1) 3965(1) 39(1)
S(2) 7161(1) 1830(2) 8238(1) 54(1)

179
Table A3. Bond lengths [Å] for 2.1.
C(1)-C(2) 1.391(7)
C(1)-C(6) 1.402(8)
C(1)-C(7) 1.518(7)
C(2)-C(3) 1.388(8)
C(3)-C(4) 1.374(8)
C(3)-C(8) 1.526(7)
C(4)-C(5) 1.401(7)
C(5)-C(6) 1.390(7)
C(5)-C(9) 1.520(8)
C(6)-N(1) 1.434(5)
N(1)-C(10) 1.264(6)
N(1)-Cu(1) 1.997(3)
C(10)-C(12) 1.512(6)
C(10)-C(11) 1.516(6)
C(11)-N(2) 1.277(6)
C(11)-C(13) 1.474(7)
N(2)-C(14) 1.450(6)
N(2)-Cu(1) 2.001(4)
C(14)-C(19) 1.397(7)
C(14)-C(15) 1.401(7)
C(15)-C(16) 1.404(6)
C(15)-C(22) 1.476(8)
C(16)-C(17) 1.379(9)
C(17)-C(18) 1.380(9)
C(17)-C(21) 1.515(7)
C(18)-C(19) 1.421(7)
C(19)-C(20) 1.492(8)
C(23)-F(1) 1.317(7)
C(23)-F(2) 1.326(8)
C(23)-F(3) 1.328(8)
C(23)-S(1) 1.814(7)
C(24)-F(6) 1.288(8)
C(24)-F(4) 1.311(7)
C(24)-F(5) 1.314(9)
C(24)-S(2) 1.831(7)
Cu(1)-O(2) 1.974(3)
Cu(1)-O(1) 1.992(3)
Cu(1)-O(3) 2.214(3)
O(4)-S(2) 1.440(5)
O(5)-S(2) 1.427(4)
O(6)-S(2) 1.441(5)
O(7)-S(1) 1.437(4)
O(8)-S(1) 1.447(4)
O(9)-S(1) 1.425(4)
Symmetry transformations used to generate equivalent atoms.

180
Table A4. Bond angles [°] for 2.1.
C(2)-C(1)-C(6) 118.2(5)
C(2)-C(1)-C(7) 120.9(5)
C(6)-C(1)-C(7) 120.9(4)
C(3)-C(2)-C(1) 121.6(5)
C(4)-C(3)-C(2) 118.5(5)
C(4)-C(3)-C(8) 121.0(5)
C(2)-C(3)-C(8) 120.5(6)
C(3)-C(4)-C(5) 122.7(5)
C(6)-C(5)-C(4) 117.2(5)
C(6)-C(5)-C(9) 122.0(5)
C(4)-C(5)-C(9) 120.8(5)
C(5)-C(6)-C(1) 121.8(4)
C(5)-C(6)-N(1) 121.5(5)
C(1)-C(6)-N(1) 116.6(4)
C(10)-N(1)-C(6) 120.7(4)
C(10)-N(1)-Cu(1) 114.3(3)
C(6)-N(1)-Cu(1) 125.0(3)
N(1)-C(10)-C(12) 125.6(4)
N(1)-C(10)-C(11) 116.1(4)
C(12)-C(10)-C(11) 118.3(4)
N(2)-C(11)-C(13) 126.2(4)
N(2)-C(11)-C(10) 113.5(4)
C(13)-C(11)-C(10) 120.3(4)
C(11)-N(2)-C(14) 120.4(4)
C(11)-N(2)-Cu(1) 115.3(3)
C(14)-N(2)-Cu(1) 124.3(3)
C(19)-C(14)-C(15) 123.9(4)
C(19)-C(14)-N(2) 119.1(5)
C(15)-C(14)-N(2) 117.0(4)
C(14)-C(15)-C(16) 116.8(5)
C(14)-C(15)-C(22) 121.8(4)
C(16)-C(15)-C(22) 121.3(5)
C(17)-C(16)-C(15) 121.9(6)
C(16)-C(17)-C(18) 119.4(5)
C(16)-C(17)-C(21) 120.8(7)
C(18)-C(17)-C(21) 119.7(7)
C(17)-C(18)-C(19) 122.2(5)
C(14)-C(19)-C(18) 115.8(5)
C(14)-C(19)-C(20) 123.9(5)
C(18)-C(19)-C(20) 120.3(5)
F(1)-C(23)-F(2) 107.4(6)
F(1)-C(23)-F(3) 107.7(5)
F(2)-C(23)-F(3) 108.6(6)
F(1)-C(23)-S(1) 112.1(5)
F(2)-C(23)-S(1) 110.4(4)
F(3)-C(23)-S(1) 110.5(5)
F(6)-C(24)-F(4) 108.0(6)
F(6)-C(24)-F(5) 106.9(7)
F(4)-C(24)-F(5) 107.1(6)
F(6)-C(24)-S(2) 112.0(6)
F(4)-C(24)-S(2) 111.1(6)
F(5)-C(24)-S(2) 111.4(4)
O(2)-Cu(1)-O(1) 90.97(14)
O(2)-Cu(1)-N(1) 92.97(14)
O(1)-Cu(1)-N(1) 165.13(15)
O(2)-Cu(1)-N(2) 168.64(15)
O(1)-Cu(1)-N(2) 93.20(14)
N(1)-Cu(1)-N(2) 80.43(15)
O(2)-Cu(1)-O(3) 93.72(15)
O(1)-Cu(1)-O(3) 93.27(14)
N(1)-Cu(1)-O(3) 100.77(14)
N(2)-Cu(1)-O(3) 96.58(15)
O(9)-S(1)-O(7) 115.1(3)
O(9)-S(1)-O(8) 114.2(3)
O(7)-S(1)-O(8) 115.0(2)
O(9)-S(1)-C(23) 103.5(3)
O(7)-S(1)-C(23) 103.0(3)
O(8)-S(1)-C(23) 103.7(3)
O(5)-S(2)-O(4) 114.9(3)
O(5)-S(2)-O(6) 115.2(3)
O(4)-S(2)-O(6) 114.2(3)
O(5)-S(2)-C(24) 103.7(3)
O(4)-S(2)-C(24) 102.6(4)
O(6)-S(2)-C(24) 104.2(3)
Symmetry transformations used to generate equivalent atoms.

181
Figure A2. ORTEP Diagram of Complex 2.1a. Ellipsoids are Drawn at the 50%
Probability Level.

182
Table A5. Crystal Data and Structure Refinement for 2.1a.
Empirical formula C
26
H
16
Cu F
6
N
2
O
9
S
2

Formula weight 742.07
Temperature 148(2) K
Wavelength 0.71073 Å
Crystal system Orthorhombic
Space group Pbca
Unit cell dimensions a = 8.5687(12) Å α = 90°
b = 26.708(4) Å β = 90°
c = 26.793(4) Å γ = 90°
Volume 6131.6(15) Å
3

Z 8
Density (calculated) 1.608 Mg/m
3

Absorption coefficient 0.939 mm
-1

F(000) 2984
Crystal size 0.21 x 0.11 x 0.03 mm
3

Theta range for data collection 1.52 to 27.58°.
Index ranges -10 ≤ h ≤ 11, -34 ≤ k ≤ 33, -31 ≤ l ≤ 34
Reflections collected 35920
Independent reflections 7016 [R(int) = 0.1111]
Completeness to theta = 27.58° 98.7%
Absorption correction None
Refinement method Full-matrix least-squares on F
2

Data / restraints / parameters 7016 / 0 / 415
Goodness-of-fit on F
2
1.007
Final R indices [I>2sigma(I)] R1 = 0.0620, wR2 = 0.1399
R indices (all data) R1 = 0.1290, wR2 = 0.1662
Largest diff. peak and hole 0.895 and -0.721 e Å
-3

Table A6. Atomic Coordinates (x 10
4
) and Equivalent Isotropic Displacement
Parameters (Å
2
x10
3
) for 2.1a. U(eq) is Defined as One Third of the Trace of the
Orthogonalized U
ij
Tensor.
x y z U(eq)
Cu(1) -1795(1) 3431(1) 1570(1) 26(1)
O(1) 428(3) 3570(1) 1996(1) 37(1)
O(2) -2592(4) 4096(1) 1783(1) 35(1)
O(3) -2658(3) 3090(1) 2166(1) 29(1)
N(1) -948(4) 3710(1) 936(1) 25(1)
N(2) -1288(4) 2785(1) 1234(1) 23(1)
C(1) -630(5) 4183(2) 823(2) 30(1)
C(2) -18(6) 4319(2) 368(2) 33(1)
C(3) 290(5) 3975(2) -5(2) 29(1)
C(4) 50(5) 3462(2) 123(2) 25(1)
C(5) 490(5) 3049(2) -187(2) 29(1)
C(6) 312(5) 2567(2) -33(2) 30(1)

183
C(7) -340(5) 2443(2) 446(2) 25(1)
C(8) -526(5) 1950(2) 638(2) 26(1)
C(9) -1097(5) 1900(2) 1121(2) 27(1)
C(10) -1462(5) 2319(2) 1403(2) 28(1)
C(11) -761(5) 2848(2) 760(2) 24(1)
C(12) -553(5) 3352(2) 599(2) 23(1)
C(13) 832(5) 4129(2) -505(2) 28(1)
C(14) 76(5) 3960(2) -934(2) 36(1)
C(15) 550(6) 4118(2) -1402(2) 40(1)
C(16) 1794(6) 4441(2) -1451(2) 38(1)
C(17) 2565(6) 4609(2) -1027(2) 35(1)
C(18) 2082(6) 4457(2) -559(2) 33(1)
C(19) -132(5) 1497(2) 339(2) 29(1)
C(20) -718(5) 1435(2) -141(2) 32(1)
C(21) -354(6) 1006(2) -410(2) 41(1)
C(22) 597(6) 648(2) -203(2) 47(1)
C(23) 1185(6) 710(2) 270(2) 45(1)
C(24) 812(5) 1131(2) 547(2) 36(1)
S(1) 3413(2) 4603(1) 1887(1) 41(1)
O(4) 4683(4) 4521(1) 1546(1) 44(1)
O(5) 3779(5) 4443(2) 2393(1) 72(1)
O(6) 1900(5) 4482(2) 1718(2) 83(2)
C(25) 3387(6) 5282(2) 1950(2) 43(1)
F(1) 4717(5) 5455(2) 2104(2) 102(2)
F(2) 3105(4) 5489(1) 1510(1) 73(1)
F(3) 2279(4) 5436(1) 2251(1) 69(1)
S(2) 3920(2) 3023(1) 1492(1) 46(1)
O(7) 4147(4) 3178(1) 2002(1) 42(1)
O(8) 5325(4) 3033(2) 1198(1) 65(1)
O(9) 2534(4) 3226(2) 1260(1) 63(1)
C(26) 3452(7) 2383(3) 1555(3) 78(2)
F(4) 4628(5) 2112(2) 1747(2) 108(2)
F(5) 2225(5) 2303(2) 1854(2) 87(1)
F(6) 3084(5) 2169(2) 1116(2) 115(2)

184
Table A7. Bond Lengths [Å] for 2.1a.
Cu(1)-O(3) 1.982(3)
Cu(1)-O(2) 1.985(3)
Cu(1)-N(1) 1.992(3)
Cu(1)-N(2) 1.995(3)
Cu(1)-O(1) 2.251(3)
N(1)-C(1) 1.327(5)
N(1)-C(12) 1.359(5)
N(2)-C(10) 1.334(5)
N(2)-C(11) 1.357(5)
C(1)-C(2) 1.376(6)
C(2)-C(3) 1.382(6)
C(3)-C(4) 1.427(6)
C(3)-C(13) 1.478(6)
C(4)-C(12) 1.406(6)
C(4)-C(5) 1.433(6)
C(5)-C(6) 1.361(6)
C(6)-C(7) 1.438(6)
C(7)-C(11) 1.417(6)
C(7)-C(8) 1.422(6)
C(8)-C(9) 1.390(6)
C(8)-C(19) 1.490(6)
C(9)-C(10) 1.386(6)
C(11)-C(12) 1.427(6)
C(13)-C(18) 1.391(6)
C(13)-C(14) 1.394(6)
C(14)-C(15) 1.385(6)
C(15)-C(16) 1.378(7)
C(16)-C(17) 1.389(7)
C(17)-C(18) 1.382(6)
C(19)-C(24) 1.386(6)
C(19)-C(20) 1.390(6)
C(20)-C(21) 1.389(6)
C(21)-C(22) 1.374(7)
C(22)-C(23) 1.374(7)
C(23)-C(24) 1.384(7)
S(1)-O(6) 1.411(4)
S(1)-O(4) 1.439(3)
S(1)-O(5) 1.456(4)
S(1)-C(25) 1.821(6)
C(25)-F(1) 1.297(6)
C(25)-F(3) 1.312(6)
C(25)-F(2) 1.323(6)
S(2)-O(8) 1.439(4)
S(2)-O(7) 1.442(3)
S(2)-O(9) 1.445(4)
S(2)-C(26) 1.765(9)
C(26)-F(5) 1.339(7)
C(26)-F(4) 1.343(7)
C(26)-F(6) 1.346(7)

Symmetry transformations used to generate equivalent atoms

185
Table A8. Bond Angles [°] for 2.1a.
O(3)-Cu(1)-O(2) 92.95(12)
O(3)-Cu(1)-N(1) 174.36(13)
O(2)-Cu(1)-N(1) 92.06(13)
O(3)-Cu(1)-N(2) 92.68(13)
O(2)-Cu(1)-N(2) 168.00(13)
N(1)-Cu(1)-N(2) 81.92(13)
O(3)-Cu(1)-O(1) 89.04(11)
O(2)-Cu(1)-O(1) 89.91(13)
N(1)-Cu(1)-O(1) 93.57(13)
N(2)-Cu(1)-O(1) 100.77(13)
C(1)-N(1)-C(12) 117.8(4)
C(1)-N(1)-Cu(1) 128.8(3)
C(12)-N(1)-Cu(1) 113.2(3)
C(10)-N(2)-C(11) 118.1(4)
C(10)-N(2)-Cu(1) 129.0(3)
C(11)-N(2)-Cu(1) 112.8(3)
N(1)-C(1)-C(2) 122.2(4)
C(1)-C(2)-C(3) 122.4(4)
C(2)-C(3)-C(4) 116.0(4)
C(2)-C(3)-C(13) 122.0(4)
C(4)-C(3)-C(13) 122.0(4)
C(12)-C(4)-C(3) 118.1(4)
C(12)-C(4)-C(5) 117.5(4)
C(3)-C(4)-C(5) 124.2(4)
C(6)-C(5)-C(4) 121.6(4)
C(5)-C(6)-C(7) 122.1(4)
C(11)-C(7)-C(8) 117.5(4)
C(11)-C(7)-C(6) 117.0(4)
C(8)-C(7)-C(6) 125.4(4)
C(9)-C(8)-C(7) 117.7(4)
C(9)-C(8)-C(19) 120.2(4)
C(7)-C(8)-C(19) 122.1(4)
C(10)-C(9)-C(8) 120.7(4)
N(2)-C(10)-C(9) 122.8(4)
N(2)-C(11)-C(7) 123.2(4)
N(2)-C(11)-C(12) 116.2(4)
C(7)-C(11)-C(12) 120.6(4)
N(1)-C(12)-C(4) 123.2(4)
N(1)-C(12)-C(11) 115.6(4)
C(4)-C(12)-C(11) 121.2(4)
Symmetry transformations used to
generate equivalent atoms

C(18)-C(13)-C(14) 118.5(4)
C(18)-C(13)-C(3) 120.7(4)
C(14)-C(13)-C(3) 120.8(4)
C(15)-C(14)-C(13) 120.8(5)
C(16)-C(15)-C(14) 120.2(5)
C(15)-C(16)-C(17) 119.6(4)
C(18)-C(17)-C(16) 120.4(5)
C(17)-C(18)-C(13) 120.6(4)
C(24)-C(19)-C(20) 120.0(4)
C(24)-C(19)-C(8) 119.3(4)
C(20)-C(19)-C(8) 120.8(4)
C(21)-C(20)-C(19) 119.8(5)
C(22)-C(21)-C(20) 119.8(5)
C(21)-C(22)-C(23) 120.5(5)
C(22)-C(23)-C(24) 120.5(5)
C(23)-C(24)-C(19) 119.5(5)
O(6)-S(1)-O(4) 117.1(3)
O(6)-S(1)-O(5) 115.4(3)
O(4)-S(1)-O(5) 112.6(2)
O(6)-S(1)-C(25) 104.3(3)
O(4)-S(1)-C(25) 102.7(2)
O(5)-S(1)-C(25) 102.1(2)
F(1)-C(25)-F(3) 109.2(5)
F(1)-C(25)-F(2) 107.1(5)
F(3)-C(25)-F(2) 106.5(4)
F(1)-C(25)-S(1) 111.9(4)
F(3)-C(25)-S(1) 112.2(4)
F(2)-C(25)-S(1) 109.7(4)
O(8)-S(2)-O(7) 113.6(2)
O(8)-S(2)-O(9) 116.5(2)
O(7)-S(2)-O(9) 114.2(2)
O(8)-S(2)-C(26) 105.1(3)
O(7)-S(2)-C(26) 102.5(3)
O(9)-S(2)-C(26) 102.7(3)
F(5)-C(26)-F(4) 105.9(7)
F(5)-C(26)-F(6) 105.8(6)
F(4)-C(26)-F(6) 106.4(6)
F(5)-C(26)-S(2) 113.0(5)
F(4)-C(26)-S(2) 112.9(5)
F(6)-C(26)-S(2) 112.3(6)

Abstract (if available)

Abstract Research laid out in this work describes the development of chemical mechanistic insight and synthesis of molecular building blocks in medicinal and diagnostic fields. The aerobic oxidation of azolines to azoles is of utmost significance in medicinal chemistry. The azole core is a ubiquitous structural component in biologically active natural products. Therefore, the necessity to efficiently and inexpensively synthesize these azole targets is of interest to chemists and clinicians alike. This work describes a copper-catalyzed aerobic oxidation of azolines to azoles that is high yielding and cost efficient. ❧ Along with copper-catalyzed conditions, a second set of copper-free, stoichiometric base-mediated conditions were developed for the entire substrate scope of the azoline to azole transformation. Both catalytic and stoichiometric base-mediated conditions demonstrate good yields with a substrate scope of thiazolines with aryl substituents in the 2-position with a range of electron withdrawing and electron donating groups. Catalytic conditions proved necessary for the transformation in the presence of labile protons such as the N-H proton of indole. The oxidation of azolines to azoles with both conditions were scaled to 1 g without a significant change in yield. ❧ Additionally, this work describes the development and characterization of the first ultrasound activated MRI contrast agent. The premise of an activatable MRI contrast agent can be applied to many different therapeutic and diagnostics systems. A two-component system has been developed in which contrast from a MRI contrast agent, Gd–DOTA, is masked by a proprietary shell formulated to be water impenetrable yet water-soluble. The hydrogen bonding interaction holding the shell to the contrast agent prevents water exchange with the paramagnetic gadolinium core, thus attenuating contrast. At the desired time and location, the contrast agent-shell interaction can be selectively disrupted externally with sonication to reveal the contrast.

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Asset Metadata

Creator Dawsey, Anna Christine (author)

Core Title Mechanism and synthesis of molecular building blocks in medicinal chemistry: aerobic azoline oxidation and ultrasound activated MRI contrast agents

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/23/2013

Defense Date 05/13/2013

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

Tag aerobic,catalytic,magnetic resonance imaging,OAI-PMH Harvest,oxidation,sonication,thiazole

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Williams, Travis J. (committee chair), Chang, Andy Y. (committee member), Prakash, G. K. Surya (committee member)

Creator Email acdawsey@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-295066

Unique identifier UC11293843

Identifier etd-DawseyAnna-1814.pdf (filename),usctheses-c3-295066 (legacy record id)

Legacy Identifier etd-DawseyAnna-1814.pdf

Dmrecord 295066

Document Type Dissertation

Format application/pdf (imt)

Rights Dawsey, Anna Christine

Type texts

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

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

Repository Name University of Southern California Digital Library

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