University of Southern California Dissertations and Theses

The use of non-covalent interactions for the modification of nanoparticle surface and MRI contrast agent properties

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The use of non-covalent interactions for the modification of nanoparticle surface and MRI contrast agent properties

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Content Copyright 2014 Vincent Li
THE USE OF NON-COVALENT INTERACTIONS FOR THE MODIFICATION OF
NANOPARTICLE SURFACE AND MRI CONTRAST AGENT PROPERTIES

by

Vincent Li

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 2014

ii

Acknowledgements
I would like to express my great appreciation to my advisor, Professor Travis J. Williams
for providing me guidance throughout my journey through graduate school, and for teaching me
that success can often be found in the least likely of places.
I would like to thank my committee members, Professors G. K. Surya Prakash; Thieo
Hogen-Esch, Barry Thompson and Dr. Andy Y. Chang for their insightful discussions. I would
also like to thank Dr. Andy Y. Chang of Children's Hospital Los Angeles, and Professor Richard
J. Hooley of University of California, Riverside for their collaborative efforts.
Thanks to the Travis Williams group, past and present for insightful discussions: Brian
Conley, Ph.D., Emine Boz, Ph.D., Megan Pennington-Boggio, Ph.D., Xinping Wu, Zhiyao Lu,
Xingyue Zhang, Jeff Celaje, Kathryn Hathaway, Ana Victoria Salud Flores, Brock Malinoski,
Denver Guess, Lena Foellmer, and Nicky Terrille. I am particularly grateful for the assistance
provided by graduate students Anna Dawsey, Ph.D. and Xinping Wu, and to undergraduate
students Christina Ratto, Blaine Bolibol, and Forrest Zhang.
Assistance provided by 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, Katie McKissick, Magnolia Benitez, and Susan Peterson was greatly
appreciated.
I wish to thank Dr. Jennifer Moore, for providing an effective teaching environment and
allowing me numerous opportunities to teach beyond the lab. I would also like to thank all the
students whom I have had the pleasure to teach, for providing an entertaining environment in
which to teach.

iii

Table of Contents

Acknowledgements ii
List of Tables vii
List of Figures viii
List of Schemes xi
Preparative Procedures xii
Abstract xxi
Chapter 1. Introduction. Selective activation of imaging agents and nanotheranostics.
1.1. MRI contrast agents. 1
1.2. Biomolecule-responsive MRI contrast agents. 4
1.3. Metal ion-responsive MRI contrast agents. 6
1.4. pH-responsive contrast agents. 9
1.5.
19
F MRI. 10
1.6. Macromolecular drug delivery vehicles. 12
1.7. Nanotheranostic systems. 15
1.8. Conclusions. 16
1.9. References. 17
Chapter 2. A noncovalent, fluoroalkyl coating monomer for phosphonate-covered
nanoparticles.
2.1. Introduction. 26
2.2. Design and synthesis of the shell monomer 2.1. 27
2.3. NMR properties of the shell monomer 2.1. 29
iv

2.4. Design and synthesis of paramagnetic nanoparticles. 29
2.5. Particle-shell binding. 31
2.6. Particle sizes. 33
2.7. H
2
O
1
H T
1
behavior of particles. 34
2.8. Conclusion. 35
2.8. References. 35
Chapter 3. A responsive MRI contrast agent based on molecular self assembly.
3.1. Introduction. 39
3.2. MRI contrast agent masked by molecular self assembly. 39
3.3. Activation of masked MRI contrast agent 3.2a. 42
3.4. Control experiments. 43
3.5. Mechanism of observed behavior. 44
3.6. MRI phantoms. 49
3.7. In vivo behavior of cavitand masked MRI contrast agents. 49
3.8. Activation of contrast agent 3.2a using a trimethylammonium coated gold nanoparticles. 50
3.9. Conclusion. 52
3.10. References. 52
Chapter 4. An MRI pill for the detection of gastric motility disorders.
4.1. Introduction - gastroesophageal reflux disease and gastroparesis. 54
4.2. Current methods of diagnosis. 54
4.3. Design of MRI pill for detection of gastric motility disorders. 55
4.4. In vitro release of contrast agent from the MRI pill. 56 XX
4.5. In vivo behavior of the MRI pill. 57
v

4.6. Conclusion. 58
4.7. References. 59
Chapter 5. Introduction. Azoles as medicinal entities and methods of synthesis.
5.1. Azoles as pharmaceutical agents and issues with the current methods of synthesis. 61
5.2. Metal-mediated oxidation of azolines. 62
5.3. BrCCl
3
oxidation of azolines. 67
5.4. Aerobic oxidation of azolines. 68
5.5. Conclusion. 70
5.6. References. 71
Chapter 6. Copper-catalyzed oxidation of azolines to azoles.
6.1. Introduction. 73
6.2. Optimization of reaction conditions. 74
6.3. Oxidation of azolines to azoles. 75
6.4. Mechanistic observations of the reaction mechanism. 79
6.5. Scalability. 81
6.6. Conclusions. 82
6.7. References. 82
Chapter 7. Experimental and spectral data.
7.1. General Procedures. 83
7.2. Chapter 2 experimental and spectral data. 86
7.2.1. Synthetic procedures. 86
7.2.2. Measurement of
19
F T
1
of 2.1 coated particles
.
97

7.2.3. Representative
19
F T
1
inversion recovery spectra. 98
vi

7.2.4. Graphical DLS spectra. 99
7.3. Chapter 3 experimental and spectral data. 100
7.3.1. Synthetic detail. 100
7.3.2. Masking and unmasking gadolinium agent 3.2a. 114
7.3.3. Molar relaxivity curves. 117
7.3.4.
1
H NMR spectra of Y·DOTA titration experiments. 118
7.3.5. Graphical DLS data. 121
7.4. Chapter 4 experimental and spectral data. 123
7.4.2. Release of Gd·DOTA from a gelatin pill casing. 123
7.4.2. Experimental T
1
data. 124
7.4.4. Release of Gd·DOTA in vivo. 126
7.5. Chapter 6 experimental and spectral data. 127
7.6. References. 171

vii

List of Tables
Table 1.1. Summary of metal ion activated MRI contrast agents. 8
Table 2.1. Decrease of
19
F T
1
when particles are added to 2.1. 32
Table 3.1. Recovery of relaxivity using particle 3.12 as a triggering agent. 52
Table 5.1. Examples of nickel-mediated oxidation of azolines to azoles. 63
Table 5.2. Substrate scope for the MnO
2
mediated oxidation of thiazolines to thiazoles. 65
Table 5.3. Oxidation of azolines to azoles using BrCCl
3
. 68
Table 5.4. Aerobic oxidation of thiazolines to thiazoles. 69
Table 6.1. Cost analysis to synthesis 1 gram of 6.2a using catalyic conditions. 73
Table 6.2. Optimization of reaction conditions for the oxidation of 6.2 to 6.2a. 75
Table 6.3. Oxidation of thiazolines to thiazoles. 76
Table 6.4. Oxidation of azolines to azoles (continued). 78
Table 6.5. Scalability of the conversion of 6.2 to 6.2a. 81
Table 7.1. Titration of 3.1 into a 0.64 mM solution of 3.2a. 116
Table 7.2. Titration of choline into a 0.32 mM solution of 3.2a with 6 equiv. cavitand 3.1. 117
Table 7.3. Recovery of relaxivity using particle 3.12 as a triggering agent. 118
Table 7.4. Release of Gd·DOTA from a gelatin pill casing at pH=7. 126
Table 7.5. Release of Gd·DOTA from a gelatin pill casing at pH = 1. 127

viii

List of Figures
Figure 1.1. Examples of Gd(III) based MRI contrast agents and their commercial names. 2
Figure 1.2. The three important parameters governing the relaxivity of MRI contrast agents. 3
Figure 1.3. MS-325, a contrast agent activated in HSA. 5
Figure 1.4. HaloTag activated MRI contrast agent. 6
Figure 1.5. A Ca
2+
activated MRI contrast agent. 7
Figure 1.6. Metal-ion activated responsive MRI contrast agents. 8
Figure 1.7. Caspase-3 activated
19
F MRI Agent. 11
Figure 1.8. Examples of PEG modification on small molecule drugs to increase efficacy. 13
Figure 1.9. Cationic doxorubicin loaded gold nanoparticle 1.27 and anionic doxorubicin gold
nanoparticle 1.28. 14
Figure 1.10. Gadolinium-loaded gold nanoparticles as a bimodal MRI-CT contrast agent. 16
Figure 2.1. Structure of monomer 2.1. 27
Figure 2.2. Hypothetical interactions between phosphonate core and guanidine shell to yield
shell coated phosphonate particle 2.2. 28
Figure 2.3. NMR peak assignments of 2.1. 29
Figure 2.4. Composition of nanoparticles. 30
Figure 2.5. DLS of particles 2.8 and 2.1. 33
Figure 2.6. Small molecule contrast agents that have demonstrated r
1
modulation upon exposure
to 2.1. 34
Figure 2.7. H
2
O
1
H T
1
behavior of particles. 35
ix

Figure 3.1. (a) Water-soluble deep cavitand 3.1; (b) minimized structure of the cavitand 3.1:Gd
chelate 3.2a complex (SPARTAN, AM1 forcefield); (c) a representation of the responsive MRI
contrast process. 40
Figure 3.2. Cavitand masks contrast agent 3.2a. 42
Figure 3.3. Choline reveals contrast agent 3.2a. 43
Figure 3.4. Control experiments. 44
Figure 3.5. Upon introduction of Y agent 3.2b to cavitand 3.1, the
1
H NMR signals broaden. 45
Figure 3.6. Expelling guest 3.2b from host 3.1 with CD
3
CN. 46
Figure 3.7. DLS of cavitand 3.1 without (left) and with (right) trimethylammonium guest 3.2a. 48
Figure 3.8. Phantoms acquired in a Pharmascan 7 T MRI using Bruker’s FLASH sequence. 49
Figure 3.9. MRI of the bladder of a rat. 50
Figure 4.1. Gadodiamide, the gadolinium MRI contrast agent in Omniscan. 55
Figure 4.2. Na[Gd·DOTA], the MRI contrast agent used in this study. 56
Figure 4.3. Release of Gd·DOTA from gelatin pill casings. 57
Figure 4.4. MRI image planes of a rat highlighting the stomach (top) and intestines (bottom) at
various time points after introduction of the MRI pill. 58
Figure 5.1. Some examples of azole-containing natural products. 61
Figure 6.1. Crude reaction mixture of thiazoline 6.2 with K
2
CO
3
and catalyst 6.1. 80
Figure 7.1. Representative
19
F T
1
inversion recovery stacked spectra. 98
Figure 7.2. Representative T
1
curve. 98
Figure 7.3. DLS of particle 2.2. 99
Figure 7.4. Modulation of the T
1
relaxation rate of 3.2a by cavitand 3.1. 116
Figure 7.5. T
1
(H
2
O) variation upon addition of choline 3.6 to a solution of 3.1:3.2a. 117
x

Figure 7.6. Relaxivity of Gd agent 3.2a. 119
Figure 7.7. Relaxivity of Gd agent 3.2a with 6 equivalents of 3.1. 119
Figure 7.8. Relaxivity of Gd agent 3.2a with 6 equivalents 3.1 and excess choline. 119
Figure 7.9. Relaxivity of Gd·DOTA (3.5). 120
Figure 7.10. Relaxivity of 3.5 with 6 equivalents of 3.1. 120
Figure 7.11. Relaxivity of Gd·DOTA (3.5) with 6 equivalents of 3.1 and excess choline. 120
Figure 7.12.
1
H NMR spectra of the titration of Y·DOTA complex 3.2b into a solution of
cavitand 3.1. 121
Figure 7.13.
1
H NMR spectra of the titration of cavitand 3.1 into a solution of Y·DOTA
complex 3.2b. 121
Figure 7.14.
1
H NMR spectra of the titration of acetonitrile-d
3
into a 6:1 mixture of cavitand 3.1
and Y·DOTA complex 3.2b. 122
Figure 7.15. DLS histogram of cavitand 3.1. 123
Figure 7.16. DLS histogram of cavitand 3.1 with Gd agent 3.2a. 123
Figure 7.17. DLS histogram of cavitand 3.1 with Gd agent 2a upon exposure to choline. 124
Figure 7.18. Release of Gd·DOTA from a gelatin pill casing at pH = 7. 126
Figure 7.19. Release of Gd·DOTA from a gelatin pill casing at pH = 1. 127
xi

List of Schemes
Scheme 1.1. Activation of EGad using β-gal reveals a coordination site. 4
Scheme 1.2. A pH responsive MRI contrast agent. 9
Scheme 1.3. Masking Gd
3+
in a pH sensitive micelle. 10
Scheme 2.1. Synthesis of monomer 2.1. 28
Scheme 2.2. Synthesis of nanoparticles. 31
Scheme 3.1. Synthesis of agents 3.2a and 3.2b. 41
Scheme 3.2. Synthesis of trimethylammonium coated gold nanparticles. 51
Scheme 5.1. MnO
2
mediated oxidation of thiazolines to thiazoles. 64
Scheme 5.2. Copper (II) mediated oxidation of oxazolines to oxazoles. 66
Scheme 5.3. Proposed mechanism for the Cu(I)/Cu(II) oxidation of azolines to azoles. 67
Scheme 5.4. One pot cyclization of β-hydroxyamides and subsequent oxidation. 68
Scheme 5.5. Mechanism for the aerobic oxidation of thiazolines to thiazoles, as proposed by Yao
et al. 70
Scheme 6.1. Aerobic oxidation of thiazolines to thiazoles. 73
Scheme 6.2. Proposed mechanism for the oxidation of thiazolines to thiazoles. 79

xii

Preparative procedures
3-(Tritylthio)propan-1-amine 89

1,4,7-Tris(carboxymethyl)-10-[N-(3-trityl-thiopropyl)carbamoyl]1,4,7,10-tetraazacyclododecane 90

Gadolinium trityl-protected thiol 2.11 91

Gadolinium thiol 2.6 92

xiii

Particle 2.8 93

Particle 2.9 95

N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-
yl)acetamido)ethanaminium chloride ligand 3.4 100

xiv

Gadolinium(III) N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)acetamido)ethanaminium chloride guest 3.2a 103

Yttrium(III) N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-
yl)acetamido)ethanaminium chloride guest 3.2b 105

Na[Gd(DOTA)] 3.5 107

S-(10-bromodecyl) ethanethioate 3.8 108

xv

8-(Acetylthio)-N,N,N-trimethyloctan-1-aminium bromide 3.9 110

8-Mercaptotrimethylammonium bromide 3.10 112

Trimethylammonium coated gold nanoparticles 3.12 113

Fmoc-Cys(Trt)-OMe 129

Methyl 2-(4-fluorobenzamido)-3-(tritylthio)propanoate 130

xvi

Methyl 2-(4-cyanobenzamido)-3-(tritylthio)propanoate 132

Methyl 2-(indole-2-carboxamido)-3-(tritylthio)propanoate 134

Methyl 2-(1-methylindole-2-carboxamido)-3-(tritylthio)propanoate 136

Methyl 2-(2-naphthamido)-3-(tritylthio)propanoate 138

xvii

Methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate (6.2) 140

Methyl 2-(naphthalen-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.5) 142

Methyl 2-(4-fluorophenyl)thiazole-4-carboxylate (6.6) 144

Methyl 2-(4-cyanophenyl)thiazole-4-carboxylate (6.7) 146

Methyl 2-(indol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.12) 148

xviii

Methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.13) 150

Methyl 2-phenylthiazole-4-carboxylate (6.2a) 153

Methyl 2-(4-nitrophenyl)thiazole-4-carboxylate (6.3a) 154

Methyl 2-(4-methoxyphenyl)thiazole-4-carboxylate (6.4a) 156

Methyl 2-(naphthalen-2-yl)thiazole-4-carboxylate (6.5a) 158

xix

Methyl 2-(4-fluorophenyl)thiazole-4-carboxylate (6.6a) 160

Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate (6.7a) 162

Methyl 2-phenyloxazole-4-carboxylate (6.8a) 164

Methyl 2-(4-nitrophenyl)oxazole-4-carboxylate (6.9a) 165

Methyl 2-methylthiazole-4-carboxylate (6.10a) 166

xx

Methyl 2-phenethylthiazole-4-carboxylate (6.11a) 167

Methyl 2-(indol-2-yl)thiazole-4-carboxylate (6.12a) 169

Methyl 2-(1-methyl-indol-2-yl)thiazole-4-carboxylate (6.13a) 171

Methyl 4-hydroxy-2-phenyl-4,5-dihydrothiazole-4-carboxylate (6.15) 173

xxi

Abstract
The focus of this work is the non-covalent modulation of MRI based nanotheranostics.
This includes modification of nanoparticulate surface properties based on phosphonate-guanidine
hydrogen bonding, modulation of MRI contrast agent behavior based on guest-host interactions,
and masking of MRI contrast using a gelatin pill casing. Synthetic methodology for the
synthesis of potential pharmaceutical entities is also described.
Surface properties are key factors for controlling in vivo behavior of nanoparticulate drug
delivery vehicles. A general procedure for the surface modification of hybrid gadolinium-
phosphonate coated gold nanoparticles was achieved via hydrogen bonding interaction with an
amphiphilic fluoroalkyl monolayer. Along with an increase in hydrodynamic diameter, the
coating interaction was characterized by a significant decrease in
19
F T
1
of the coating monomer,
allowing for potential
19
F MRI based nanotheranostics.
Controllable, non-covalent interactions were also used to attenuate and reveal MRI
contrast agents by hiding them in molecular hosts. A container-shaped molecular host
compound, which had previously demonstrated selective and strong binding affinities towards
trimethylammonium functionality, was used. Modification of Gd·DOTA with the
trimethylammonium moiety resulted in a dramatic decrease of contrast upon exposure to the
molecular host. The masking event is accompanied by the assembly of contrast agent into a 7
nm micelle as characterized by DLS. The contrast agent guest was removed from the host via
competitive binding in the presence of a stronger binding agent, resulting in a 31% contrast
increase over the masked state.
The release of MRI contrast agent from a gelatin pill casing was characterized. The
masked MRI contrast agent was immediately revealed under acidic conditions, whereas it
xxii

remained masked under neutral conditions for over ten minutes. The release of the gadolinium-
based MRI contrast agent was characterized by the decrease of T
1
in vitro, and MRI contrast
observed in vivo. This MRI pill can potentially be used for imaging of gastric motility disorders
such as gastroesophageal reflux disease and gastroparesis.
Catalytic conditions for oxidation of azolines to azoles are also described. Azoles are
biologically active marine cyclopeptides in which its synthesis have historically involved
stoichiometric amounts of toxic reagents. By utilizing catalytic amounts of a copper catalyst and
base, inexpensive and environmentally benign conditions for this bond transformation were
achieved. Along with the catalytic copper conditions, copper-free conditions involving
stoichiometric amounts of base were also developed. These conditions demonstrated decreased
reaction times over the catalytic copper conditions.

1

Chapter 1. Introduction. Selective activation of imaging agents and
drug delivery vehicles.

1.1. MRI contrast agents.

Magnetic resonance imaging (MRI) is one of the most widely used techniques in
diagnostic medicine because of its ability to provide high resolution images utilizing non-
ionizing radiation.
1
Magnetization of bulk water protons in vivo are first aligned in a magnetic
field, which are then excited using a radiofrequency pulse. Signal is provided upon relaxation of
the excited protons. The strength of the MRI signal is dependent on both the longitudinal
relaxation rate (1/T
1
) and the transverse relaxation (1/T
2
). The signal is increased as 1/T
1

increases and 1/T
2
decreases.
2
MRI contrast is provided by the difference of 1/T
1
and 1/T
2
of
bulk water protons in various environments.
3

Contrast between healthy and diseased tissue is often times not high enough to
differentiate, and thus the need for contrast enhancement arises. Relaxation rates are accelerated
in the presence of paramagnetic species, which can be used to enhance contrast. Gadolinium(III)
based contrast agents are common because of their 7 unpaired electrons, and are used in over 10
million scans annually.
2
They enhance contrast by increasing 1/T
1
of bulk water protons.
4

Molar relaxivity (r
1
) is a measure of how much contrast enhancement an agent provides and is
defined as the increase of 1/T
1
per millimolar of contrast agent. This can be measured by simple
1
H NMR experiments with an inversion recovery pulse sequence to measure 1/T
1
at varying
concentrations. The resulting slope when 1/T
1
is plotted against concentration provides the
molar relaxivity value.
5

The size of Gd
3+
is similar to Ca
2+
and will rapidly transmetallate with Ca
2+
binding sites
in vivo. In order to prevent this toxicity, Gd
3+
are complexed to organic chelators prior to
2

administration.
6
Although a greater number of open water coordination sites is optimal for
contrast enhancement, the majority of coordination sites on Gd
3+
in clinically relevant contrast
agents are tightly ligated to nitrogen or oxygen ligands. Commonly used chelated Gd
3+
based
contrast agents are shown in Figure 1.1.
N N
N N
O
O
O
O
O
O
O
O
Gd
O
O
N
N
N
O
O
O
O
Gd
O
O
O
O
1.1 [Gd·(DOTA)]
-
1.3 [Gd·(DTPA)]
2-
2
N
N
N
O
O
Gd
O
O
O
O
O
HN
NH
O
1.2 Gd·(DTPA-BMA)
Dotarem
®
Omniscan
®
Magnevist
®

Figure 1.1. Examples of Gd(III) based MRI contrast agents and their commercial names.

The thermodynamic stability (K
St
) of gadolinium complexes can be described by the
equations 1 and 2. Acyclic chelators tend to be less stable than cyclic chelators. For example,
the measured logK
St
for cyclic Gd·DOTA 1.1 was 24.7, whereas acyclic Gd·DTPA-BMA 1.2
and GD·DTPA 1.3 were 16.9 and 22.5 respectively.
7
The FDA has issued warnings regarding the
dangers associated with the use of numerous acyclic gadolinium contrast agents such as
Omniscan and Magnevist.
8

Gd(H
2
O)
8
+ L GdL(H
2
O) + 7 H
2
O
(1)

(2)

The relaxivity of a contrast agent is controlled by three important parameters: the number
of open water coordination sites (q), water residency lifetime ( τ
M,
the reciprocal of water
exchange rate k
ex
), and rotational correlation time ( τ
R
) (Figure 1.2).
9
In order to achieve
3

maximum contrast, q and τ
R
should be maximized,
10
and τ
M
close to an optimal value of ~10
-8
s.
11

The mechanism of paramagnetic metal enhancement of T
1
relaxation can be attributed to both
inner-sphere and outer-sphere influences. Inner-sphere refers to a water molecule directly
coordinated to the metal whereas outer-sphere refers to water molecules in close proximity to the
paramagnetic center, but not directly coordinated.

Figure 1.2. The three important parameters governing the relaxivity of MRI contrast agents.

Some MRI contrast agents contain the added feature that contrast properties are
modulated upon exposure to specific environments including, but not limited to, the presence of
biomolecules,
12
metal ions,
13
pH change,
14
oxygen,
15
temperature change,
16
and light.
17
These
responsive contrast agents are designed such that one or more parameters (q, τ
M
, or τ
R
) are
affected upon exposure to stimulus, and thus the relaxivity behavior is altered. These systems
are advantageous in the selective imaging of certain disease states. This chapter will first discuss
some of the methods and mechanisms used to deactivate and activate contrast agents.
4

1.2. Biomolecule-responsive MRI contrast agents.

In 1997, the Meade research group demonstrated the first responsive MRI contrast agent
by utilizing a q blocking strategy (Scheme 1.1).
13a
The contrast agent, (4,7,10-tri(acetic acid)-l-
(2- β-galactopyranosylethoxy)-1,4,7,10-tetraazacyc1ododecane)gadolinium (EGad, 1.4), consists
of a modified cyclic DOTA-type chelator where the water coordination site is partially inhibited
by a galactopyranose residue. The partially inhibited water coordination site on masked agent
1.4 (q = 0.7) was revealed upon cleavage of the β-galactosyl linkage when incubated with β-
galactosidase (β-gal). The resulting contrast agent 1.5 (q = 1.2) displayed a 20% increase in
molar relaxivity.

Scheme 1.1. Activation of EGad using β-gal reveals a coordination site.

A different strategy for a biomolecule responsive MRI contrast agent utilizes the
receptor-induced magnetization enhancement effect (RIME). Upon small molecule binding to
biomacromolecules, τ
R
is increased and relaxivity is enhanced.
18
The clinical viability of this
method was demonstrated by Lauffer et al. who characterized the contrast enhancement upon
binding of MS-325 (1.6, Figure 1.3) to human serum albumin (HSA).
19
The Gd·DTPA derivative
displayed an increase of τ
R
from 115 ps when measure in phosphate buffered saline (PBS) to 10
5

ns when measured in 22.5% HSA.
20
As a result of the increased τ
R
, the molar relaxivity of agent
1.6 (r
1
= 6.6 mM
-1
s
-1
measured in PBS) was increased significantly when measured in human
plasma (r
1
= 53.5 mM
-1
s
-1
, an increase of 711%). MS-325 has been approved for clinical use in
MRI angiograms.
21

Figure 1.3. MS-325, a contrast agent activated in HSA.
The RIME strategy was also used by the Meade group in their development of an MRI
probe to track reporter protein expression.
22
Haloalkane modified gadolinium 1,4,7,10-
tetraazacyclododecane-1,4,7-trisacetic acid (1.7, Figure 1.4) noncovalently binds the HaloTag
reporter protein, dramatically increasing τ
R
. The relaxivity of the contrast agent (r
1
= 3.8 mM
-1
s
-1
)
was increased nearly six-fold upon binding to the HaloTag protein (r
1
= 22.0 mM
-1
s
-1
).

6

Figure 1.4. HaloTag activated MRI contrast agent.

1.3. Metal ion-responsive MRI contrast agents.

The first metal ion activated MRI contrast agent was also reported by the Meade group
and utilized a q blocking strategy.
9
The masked contrast agent (1.8, Figure 1.5) consists of a
known Ca
2+
chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),
linking 2 Gd·DO3A chelates.
13a
The design of this contrast agent is such that inner-sphere water
coordination site on gadolinium is inhibited by the BAPTA carboxylates until Ca
2+
is introduced.
The water coordination site to gadolinium is freed when Ca
2+
is chelated, activating the contrast
agent. The activated agent (1.9 r
1
= 5.76 mM
-1
s
-1
) displayed a relaxivity increase of 77% over
the unactivated agent (1.8 r
1
= 3.26 mM
-1
s
-1
) in the presence of 1 mM of Ca
2+
in solution.
Further studies showed the number of inner-sphere water molecules more than doubled upon
introduction of Ca
2+
and confirmed the mechanism of activation.
13b

7

Figure 1.5. A Ca
2+
activated MRI contrast agent.
A similar q blocking strategy has since been used in the development of numerous metal
ion activated contrast agents. Calcium(II),
23
zinc(II),
24
and copper(II),
25
activated MRI contrast
agents (Figure 1.6) have been reported, and the efficacy of these contrast agents are summarized
in Table 1.1.

8

Figure 1.6. Metal-ion activated responsive MRI contrast agents.

Compound Metal
ion
Amount of
metal
r
1
without
metal
r
1
with metal r
1

change
Ref.
1.10 Ca
2+
40 equiv. 3.3 mM
-1
s
-1
3.8 mM
-1
s
-1
15% 23a
1.11 Ca
2+
7.5 equiv. 5.4 mM
-1
s
-1
7.1 mM
-1
s
-1
31% 23b
1.12 Ca
2+
1 equiv. 3.5 mM
-1
s
-1
6.9 mM
-1
s
-1
97% 23c
1.13 Zn
2+
1 equiv. 2.3 mM
-1
s
-1
5.1 mM
-1
s
-1
122% 24
1.14 Cu
+
1 equiv. 1.5 mM
-1
s
-1
6.9 mM
-1
s
-1
360% 25a
1.15 Cu
+
1 equiv. 2.6 mM
-1
s
-1
11.4 mM
-1
s
-1
340% 25b
Table 1.1. Summary of metal ion activated MRI contrast agents.
9

1.4. pH-Responsive MRI Contrast Agents.
One strategy in the development of pH responsive MRI contrast agents is to block water
coordination sites on gadolinium using pH sensitive functional groups. This was demonstrated
by Sherry et al. in their development of the pH sensing agent gadolinium 1-methlyene-(p-
nitrophenol)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetate (Gd·NP-DO3A, 1.16).
14c
At pH =
9, the phenol functionality is unprotonated, blocking a water coordination site. The phenol
becomes protonated as the solution pH decreases from 9 to 5, opening the water coordination site
(1.17, Scheme 1.2). This results in a relaxivity increase from 4.1 mM
-1
s
-1
at pH = 9 to 7.0 mM
-
1
s
-1
at pH = 5.

Scheme 1.2. A pH responsive MRI contrast agent.

An alternate strategy is to modulate τ
R
with pH changes. One specific example is the
water soluble gadofullerene (Gd@C
60
(OH)
x
),
14d
which were stable at a high pH, but prone to
aggregation at lower pH conditions. The hydrodynamic diameter increased from 50 nm at pH =
9 to 1200 nm at pH = 4, as measured by dynamic light scattering (DLS). This was accompanied
by an increase in r
1
from 45.1 mM
-1
s
-1
to 74.9 mM
-1
s
-1
.
Recently, a strategy to mask gadolinium(III) within the interior of a micelle and its
release upon a change in pH was demonstrated.
14i
The pH sensitive micelle consists of contrast
agent methoxy poly(ethylene glycol)-b-poly(L-lactic acid)-diethylenetriaminopentaaceticacid
10

gadolinium (PEG-p(L-LA)-DTPA-Gd, 1.18) and pH sensitive polymer poly(ethylene-b-poly(L-
histidine) (PEG-p(L-His), 1.19). At physiological pH (7.4), the two polymers self assemble into
a 40 nm micelle (1.20). Upon decrease of pH from 7.4 to 6.5, 1.19 is protonated, effectively
disrupting the micelle (Scheme 1.3). This was accompanied by an increase of r
1
from 8.56 mM
-
1
s
-1
at pH = 7.4 to 12.01 mM
-1
s
-1
at pH = 6.5. The effectiveness of the micelle in inhibiting water
access to gadolinium was great enough to overcome the increased τ
R
of the micellar assembly.

Scheme 1.3. Masking Gd
3+
in a pH sensitive micelle.

1.5.
19
F MRI.

Although
1
H MRI is primarily used in the clinic, there are some limitations. One issue
with
1
H based MRI systems is the high concentration of the
1
H nucleus in vivo, which can yield
images with high background noise.
19
F MRI offers an alternate method of detection aimed to
alleviate the problem of background noise, as natural concentrations of
19
F in vivo are below
MRI detection limits.
26
With a high gyromagnetic ratio and 100% natural abundance,
19
F is the
2nd best nucleus for MRI detection, as its sensitivity is 0.83 of the
1
H nucleus.
27
Furthermore,
current MRI instruments used for
1
H detection can easily be tuned to detect
19
F.
In 1977, Holland et al. demonstrated the concept of
19
F MRI by acquiring phantoms of
sodium fluoride and perfluorotributylamine.
28
In 1985, the concept was further advanced by
11

McFarland et al., who acquired in vivo
19
F MRI images of rats after administration of 14%
perfluorodecalin and 6% perfluorotripropylamine (Fluosol). Numerous fluorinated compounds
such as sulfur hexafluoride,
29
perfluorooctyl bromide,
30
and fluorinated dendrimers
31
have since
been proposed for in vivo
19
F MRI based applications.
Responsive
19
F MRI based probes have also been demonstrated, utilizing a proximity
induced paramagnetic effect to detect protease cleavage of a peptide.
32
The contrast agent (1.21,
Gd-DOTA-DEVD-Tfb) consists of a Gd·DO3A modified with a caspase-3 substrate sequence
terminated with trifluromethoxy functionality for
19
F detection. In the absence of gadolinium
(DOTA-DEVD-Tfb), the measured
19
F T
1
and T
2
values were 1.910 and 0.326 s respectively.
Upon introduction of gadolinium, the T
1
and T
2
values were shortened to a point at which they
could not accurately be measured. The
19
F T
1
and T
2
values of the cleaved substrate were
measured to be 0.122 s and 0.032 s respectively, with the short relaxation times attributed to
intermolecular paramagnetic effects with gadolinium still in solution. Density-weighted
19
F
images of this process were also acquired, further demonstrated the viability of a
19
F MRI based
enzyme reporter.

Figure 1.7. Caspase-3 activated
19
F MRI Agent.

12

1.6 Macromolecular Drug Delivery Vehicles.
Common issues with the delivery of small molecule drug agents include nonspecific
delivery leading to undesirable side effects, low bioavailability due to short in vivo half life,
plasma stability, and water solubility.
33
The development of macromolecular drug delivery
vehicles aim to address these issues, resulting in lower drug dosage and lower systemic
toxicity.
34
Furthermore, surface modifications of delivery vehicles allow fine-tuning of
pharmacokinetics in both passive and active delivery systems.
Conjugation of low molecular weight drugs to non-toxic poly(ethylene glycol) (1.22,
PEG) has been shown to increase blood circulation lifetime of low molecular weight drugs.
35
As
PEG is a water-soluble macromolecule, this method can also be used to overcome the low water
solubility problem of organic drugs.
36
Some examples of PEGylated modifications of small
molecule drugs are demonstrated in Figure 1.8.
37
Numerous PEGylated formulations including
Doxil
®
(PEGylated doxorubicin),
38
PEG-Intron
®
(PEGylated interferon α-2b),
39
Pegasys
®

(PEGylated interferon α-2a)
40
and Adagen
®
(PEGylated adenosine deaminase)
41
have been
approved by the FDA and are used in the clinic.
13

Figure 1.8. Examples of PEG modification on small molecule drugs to increase efficacy.

Surface functionalized gold nanoparticles are another class of attractive macromolecular
drug delivery vehicles because of their low inherent toxicity, tunable surface, and controllable
size.
42
The synthesis of ligand stabilized colloidal Au
0
nanoparticles is well established, and
methods of synthesis ranging from 1.5 nm to 250 nm cores have been reported.
43
These gold
colloids can then be further functionalized via ligand exchange.
44
Drugs can either be covalently
conjugated to the particle,
45
or non-covalently loaded utilizing hydrophobic interactions.
46

Cancer drugs such as cis-platin,
47
doxorubicin,
48
and paclitaxel
49
have demonstrated increased
bioactivity upon loading in a gold nanoparticle.
14

Surface properties are important factors in drug delivery nanoparticles, as in vivo
behavior varies depending on surface coating functionality. This was demonstrated by Forbes et
al. who studied the effect of surface charge on in vivo distribution of drug loaded gold
nanoparticles.
50
Doxorubicin loaded cationic trimethylammonium coated particle 1.27 was
found to increase cellular uptake whereas analogous anionic carboxylate particle 1.28 was found
to have greater tissue penetration. This suggests that particle surfaces can be tuned to address
specific delivery needs.

Figure 1.9. Cationic doxorubicin loaded gold nanoparticle 1.27 and anionic doxorubicin gold nanoparticle 1.28.

Macromolecular vehicles also display advantages in tumor targeted delivery due to the
enhanced permeability and retention (EPR) effect.
51
While small molecule drugs suffer from
non-specific diffusion, the irregular lining of endothelial cells near a tumor allow for increased
accumulation and retention of macromolecules. This was first demonstrated by Maeda et al.
using the macromolecular anti-cancer agent poly(styrene-co-malelic acid half n-butylate)
neocarzinostatin (SMANCS).
52
72 hours after intravenous injection in tumor bearing mice, 4.7%
of the macromolecular drug was recovered in the tumor, compared to just 0.6% of the small
molecule neocarzinostatin. Although this phenomenon has demonstrated effectiveness for up to
particles 400 nm in diameter,
53
the greatest effectiveness is suggested to be less than 200 nm.
54

15

1.7 Nanotheranostic systems.

A class of nanoparticulate drug delivery vehicles are designed for the simultaneous
treatment and imaging of disease states, and are commonly known as nanotheranostic systems.
55

The inclusion of imaging agents in drug delivery vehicles offer further advantage from the added
ability to track biodistribution of the drug. Some examples of proposed theranostic systems
include paramagnetic nanoparticles for
1
H MRI imaging,
64
Cu loaded micelles for PET
imaging,
56
and polymer modified gadolinium-containing metal-organic frameworks for bimodal
MRI and fluorescent imaging.
57

One strategy used in the development of nanotheranostics is to utilize the inherent
imaging properties of the metal core. For example, the X-ray absorption properties of gold due
to its high atomic number enables gold nanoparticles used in drug delivery also be used for
simultaneous imaging via computed tomography (CT).
58
Another example are iron oxide based
nanoparticles.
59
The magnetic properties of iron oxide nanoparticles can be exploited as a T
2

weighted
1
H MRI imaging agent, while its macromolecular core is used as a drug delivery
vehicle.
60
Such particles have demonstrated effective tumor-targeted delivery of anti-cancer
agents such as doxorubicin with simultaneous imaging.
61

An alternate strategy is to load the drug delivery vehicle with an imaging agent. This
strategy can be used in drug delivery vehicles that contain no innate imaging properties, such as
liposomes.
56
Gadolinium-loaded liposomes have been used for real-time imaging of drug
delivery to the brain.
62
Imaging agents loaded onto macromolecular cores inherently containing
imaging properties can result in bimodal imaging platforms.
63
For example, thiol functionalized
GdDTPA derivative 1.29 was loaded onto gold nanoparticles, demonstrating an effective
bimodal MRI-CT contrast agent 1.30 (Figure 1.10).
64

16

Figure 1.10. Gadolinium-loaded gold nanoparticles as a bimodal MRI-CT contrast agent.

1.8 Conclusions.
This chapter highlighted some issues regarding drug delivery and imaging, and some
commonly employed techniques to overcome these issues. These strategies are mainly focused
around loading drug candidates into a nanoparticulate vehicle for delivery. Chapter 2 will focus
on surface modification of a potential drug delivery vehicle based on monolayer self assembly
around a phosphonate coated gold nanoparticle.
This chapter also highlighted the common strategies used in the design of responsive
MRI contrast agents, which mainly consist of q blocking and τ
R
modulation. Chapter 3 will
discuss a different approach in the design of responsive MRI contrast agents, where an MRI
contrast agent will be hidden in a host molecule via non-covalent, multicomponent assembly to
limit water access. These contrast agents are displaced in the presence of a stronger binder,
activating the contrast agent.

17

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26

Chapter 2. A noncovalent, fluoroalkyl coating monomer for
phosphonate-covered nanoparticles.

2.1. Introduction.

One key factor to controlling the in vivo behavior of nanoparticulate drug delivery
systems is the ability to control their surface properties.
1
Along these lines, monomeric organic
compounds that engage in predictable self-assembly around nanoparticulate molecular probes
and that have the ability to control the bioactivity of the former by modifying their size, surface,
and access to the external aqueous milieu can have potential applications as biomaterials.
2

A general strategy was developed for applying a (poly)ethyleneglycol (PEG)-terminated
fluoroalkyl coating to a particle with a phosphonate surface can utilize the same interaction(s) to
bind species 2.1, thus enabling the self-assembly of a monolayer coating around the particle
(Figure 2.1). Incorporating a paramagnetic species into such a particle can thus enable the use of
monomer 2.1 as a
19
F MRI contrast system for an appropriately filtered, T
1
-weighted image
plane. Although
19
F MRI-based nanotheranostic technology is not as developed as the analogous
1
H MRI based systems,
19
F MRI based systems are beginning to emerge,
3,4
and offer important
bioorthogonality unavailable to other imaging modalities.
5
The potential utility of monomer 2.1
as a flexible tool for the installation of
19
F groups on the surface of a phosphonate-covered
nanoparticle is shown here, which is illustrated by the corresponding modulation in
19
F T
1
upon
particle-2.1 binding. Also illustrated are 2.1’s selectivity for a phosphonate surface over a
phosphonate free particle and the coverage provided by 2.1 corresponds to a monolayer in
aqueous solution. This work was done in collaboration with Andy Y. Chang,
6
and Xinping Wu.
7

This work was published in 2013.
8

27

H
2
N N
H
NH
2
F F
O
O
O
O
O
F F
F F
F F
2.1
F
3
C O
-
O

Figure 2.1. Structure of monomer 2.1.

2.2. Design and synthesis of shell monomer 2.1.

Shell monomer 2.1 features a guanidinium head group, which is designed to participate in
hydrogen bonding with phosphonates on the periphery of a particle. This is attached to a fluorous
phase group
9
that prevents intercalation of the shell in to the interior of the particle and limits the
particle’s exposure to its surrounding. Appropriate fluorous starting materials for this design are
readily available side products from teflon synthesis.
10
A hydrophilic phosphonate-covered
nanoparticle agent and illustrate the use of bioorthogonal
19
F magnetic resonance relaxivity to
track the behavior of the coating monomer as it associates with the particle. The coating
monomer (Figure 2.1) exploits a guanidinium head group, which can interact with particle
surface phosphonate groups through a double hydrogen bonding system analogous to the one
used by the Wender molecular transporter system for its initial adhesion to cell surfaces.
11

Particles utilizing such core-shell interactions contain various clinical uses, such as the
facilitation of transfection in vivo for example.
12
Monomer 2.1 further features a fluoroalkane
region that enables the self-assembly of a fluorous, Teflon-like layer that can limit the particle’s
exposure to its aqueous surroundings.

28

Figure 2.2. Hypothetical interactions between phosphonate core and guanidine shell to yield shell coated
phosphonate particle 2.2.

The binding of monomer 2.1 with small-molecule phosphonates have been previously
characterized,
13
and hypothesized that a PEG chain is then appended as a solubilizing group.
Scheme 2.1 summarizes the synthesis of monomer 2.1, which is previously reported.
13
This route
relies on the intermediacy of a bench top-stable alkyl triflate (2.4), which is a versatile starting
material for fluoroalkyl amines.

Scheme 2.1. Synthesis of monomer 2.1.

29

2.3. NMR properties of shell monomer 2.1.

Shell monomer 2.1 exhibits 3 different peaks in the
19
F NMR spectrum, which can be
rigorously assigned by a combination of
19
F-
13
C HMBC and
13
C-
1
H HSQC NMR spectra.
14
The
most downfield signal (-117.9 ppm, peak A) corresponds to the fluorine CF
2
group nearest the
guanidine of 2.1 and an upfield (-123.5 ppm, peak C) multiplet corresponds to the two central
CF
2
groups. The T
1
relaxation times were measured in 25 mM pH = 7.6 TRIS-HCl buffer and
were found to be 457(8) ms, 436(7) ms, and 497(7) ms for peaks A, B, and C respectively. These
data are summarized in Figure 2.3.

Figure 2.3. NMR peak assignments of 2.1.

2.4. Design and synthesis of paramagnetic nanoparticles.

Our design for a template nanoparticle is based on a gold-centered structure, which is
decorated with a periphery of thiol-terminated phosphonic acid surfactants (Figure 2.4). The
selection of this construction is based on its popularity in several scaffolds currently being
developed for drug delivery and clinical imaging applications.
15
It enables concise size control
and flexibility of surface functionalization.
16,17
Synthetic routes for paramagnetic particles
functionalized with both [Gd(DOTA)]
-
and phosphonate peripheries are presented in this section.
30

Figure 2.4. Composition of nanoparticles.

A 1.5 nm phosphine-stabilized gold core (2.10) was first synthesized based on previously
reported conditions.
18
Thiols 2.6 and 2.7 were assembled onto the gold core via interfacial ligand
exchange in a biphasic water/dichloromethane system (Scheme 2.2A).
19
The resulting particles
form visible aggregates at neutral pH, but are stable in a pH = 7.6 TRIS-HCl buffer. Monomer
2.1 spontaneously self assembles onto particles 2.8 immediately upon introduction to an aqueous
solution of 2.8 to yield hybrid gadolinium-phosphonate particles 2.2.
Phosphonate-free particles 2.9 are apparently unstable to aggregation when prepared
according to the procedure above, but in situ deprotection of trityl-protected thiol 2.11 followed
by treatment with HAuCl
4
can be used to generate these particles (Scheme 2.2B).
20
This method
yields particles that are of 7 nm in hydrodynamic diameter as measured by dynamic light
scattering (DLS).
19
F longitudinal relaxivity time constants (
19
F T
1
) provide evidence for the
31

binding of particle and monomer 2.1. Upon binding of 2.1 to the particle core, the fluorine nuclei
are brought into close proximity of the particle’s paramagnetic gadolinium centers, where the
paramagnetic gadolinium center causes a decrease in the T
1
relaxation times of the
19
F nuclei.
21

Scheme 2.2. Synthesis of nanoparticles.

2.5. Particle-shell binding.

Upon addition of an aqueous solution of particle 2.8 to 2.1, the
19
F T
1
relaxation times for
2.1 decreased to 332(14) ms, 348(4) ms and 385(4) ms respectively (Table 2.1). Significant
decrease of the observed T
1
values upon the addition of paramagnetic particles is consistent with
an intimate interaction between 2.1 and the particle core. To assess the role of the phosphonate
functionality on particle 2.8, phosphonate free particles 2.9 were also treated with 2.1. In this
experiment, the T
1
relaxation times were 434(6) ms, 431(7) ms, 481(5) ms. These data show that
2.9 exhibits lower changes in T
1
than those observed for particle 2.8. This suggests a more
32

intimate interaction between 2.1 and 2.8 versus 2.1 and 2.9. Furthermore, exposure of the shell
(2.1) to 1.0 mM GdCl
3
(0.25 molar equivalents relative to 2.1, 158 ppm wt/wt [Gd]) also resulted
in a less significant change in
19
F T
1
than our phosphonate coated particles (2.8, Table 2.1),
which shows that high [Gd] alone cannot account for the observed decrease in
19
F T
1
observed in
the presence of 2.8.

Peak A (-117.9 ppm) Peak C (-123.5 ppm) Peak B (-119.8 ppm)
2.1 457(8) ms 497(7) ms 436(7) ms
2.1 + 2.8 332(14) ms 385(4) ms 348(4) ms
2.1 + 2.9 434(6) ms 481(5) ms 431(7) ms
2.1 + GdCl
3
429(13) ms 466(6) ms 396(8) ms
Table 2.1. Decrease of
19
F T
1
when particles are added to 2.1.

T
1
time constants of H
2
O for aqueous solutions of particles 2.8 and 2.9 reveal that,
although 2.8 is a more effective
19
F T
1
contrast agent, 2.9 is a more efficient
1
H T
1
contrast agent.
H
2
O
1
H T
1
relaxation of the same particle solutions used for acquisition of the foregoing
19
F T
1

data were measured. Particle 2.8 showed a
1
H T
1
of 1.18(5) s, and particle 2.9 exhibited a T
1
of
0.495(3) s. The significantly lower H
2
O
1
H T
1
of 2.9 shows that it is a more relaxive
1
H contrast
agent, whereas particle 2.8 is a more relaxive
19
F T
1
contrast agent. This remarkable contrast
between
1
H and
19
F highlights the importance of a guanidinium-phosphate interaction in binding
1 to the particles: whereas phosphonate-rich 2.8 holds 2.1 in the proximity of the paramagnetic
gadolinium, phosphonate-free 2.9 does so less well, relative to a water standard. Moreover,
absolute gadolinium concentration measurements by ICP (ppm wt/wt) indicate [Gd] = 52 ppm in
the experimental solution of 2.8 and [Gd] = 37 ppm in the experimental solution of 2.9. Although
33

these values are similar, they are inconsistent with the relatively short
1
H T
1
of the 2.9 solution,
which can be attributed to slower tumbling of the larger particle.

2.6. Particle sizes.

The foregoing relaxivity-based shell binding data are supported by dynamic light
scattering (DLS) measurements. DLS data for particle 2.8 indicate monodisperse particles of
hydrodynamic diameter of 3.0 nm (Figure 2.5A). Addition of shell to the particle increased the
size of the particle to 4.0 nm (Figure 2.5B). The increase in diameter of 1 nm upon association of
2.1 is appropriate for the addition of two equivalents of 2.1, along with associated water, along
the diameter. Further, these data also show that the particles are not aggregating upon changing
their surface from phosphonate to PEG.

Figure 2.5. DLS of particles 2.8 and 2.1. (A) DLS of particle 2.8 (average hydrodynamic diameter = 3.0 nm) (B)
DLS of particle 2.2 (after the addition of monomer 2.1, hydrodynamic diameter = 4.0 nm).

34

2.7. H
2
O
1
H T
1
behavior of particles.

Shell monomer 2.1 had previously shown to modulate T
1
behavior of small molecule
contrast agents [Gd(DOTA)]
-
2.10 and [Gd(DOTP)]
3-
2.11. Shell monomer 2.1 was shown to
increase the r
1
of [Gd(DOTP)]
3-
from 3.04(16) to 3.67(30),
14
suggesting monomer 2.1 is binding
and increasing τ
R
. However, in the case of [Gd(DOTA)]
-
, the r
1
was decreased from 3.0(1) mM
-
1
s
-1
to 2.7(2) mM
-1
s
-1
,
22
suggesting the increased τ
R
upon binding of shell monomer is overcome
by the inhibition of water exchanged caused by the fluorous region of shell monomer 2.1.

Figure 2.6. Small molecule contrast agents that have demonstrated r
1
modulation upon exposure to 2.1.

The H
2
O
1
H T
1
responsiveness of gadolinium agents 2.8 and 2.9 when treated with shell
monomer 2.1 was determined via simple T
1
experiments. Neither particle exhibited strong
modulation of T
1
relaxation behavior. The T
1
relaxation of a freshly prepared sample of 2.8
increased from 1.18(3) s to 1.27(4) s upon introduction of 2.1, a minimal increase of 7.6%. The
T
1
relaxation of 2.9 increased from 0.480(4) s to 0.510(4) s, an increase of 6.3%.

35

Figure 2.7. H
2
O
1
H T
1
behavior of particles. (left)
1
H T
1
behavior of gadolinium agent 2.8 with shell monomer 2.1
(right)
1
H T
1
behavior of agadolinium agent 2.9 with shell monomer 2.1.

2.8. Conclusion.

In conclusion, the non-covalent coating of a gadolinium-containing gold nanoparticle
with a fluorous shell was characterized. The coating utilizes a phosphonate-guanidine interaction
and is characterized by both a decrease in
19
F T
1
and an appropriate increase in the particle’s
hydrodynamic radius.
1
H and
19
F NMR relaxivity data were further used to show that
phosphonate groups on the surface of a particle increase the particle’s affinity for
fluoroguanidine 2.1 relative to water. These observations describe a general procedure for the
selective self-assembly for fluoroaklyl groups around the surface of a phosphonate-covered
material, and they have potential applications in biomaterials and
19
F MRI-based nanotheranostic
systems.

2.9. References.

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Lucas, S.; Michiels, C.; Gallez, B.; Bonifazi, D. Antibody-Functionalized Polymer-
1.18
1.27
0
0.2
0.4
0.6
0.8
1
1.2
1.4
2.8 2.8+2.1
T
1
Relaxation Rate ( s )
Agent
0.48
0.51
0
0.2
0.4
0.6
0.8
1
1.2
1.4
2.9 2.9+2.1
T
1
Relaxation Rate ( s )
Agent
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41, 173-183.
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11) (a) Frankel, A. D.; Pabo, C. O. Cellular Uptake of the Tat Protein from Human
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Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider, E.; McGrane, P. L.; Wender, P. A.;
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Delivery and Inhibition of Inflammation. Nature Med. 2000, 6, 1253-1257. (d) Rothbard,
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Ph. D. Thesis, University of Southern California, Los Angeles, California, 2013.
39

Chapter 3. A responsive MRI contrast agent based on molecular self
assembly.

3.1. Introduction.
The strategies of the development of responsive MRI contrast agents discussed in chapter
1 typically involved q blocking or τ
R
modulation. An alternate strategy of T
1
attenuation would
be to “hide” the gadolinium center from water via non-covalent, multicomponent assembly.
Unfortunately, self-assembly in water is mainly controlled by concentration, a property not
amenable to external control. If the self-assembly could be triggered by a selective agent, simple
control of the visualization properties would be accessible. An example is reported in this
chapter: a new class of adaptive MRI contrast agents based on controlled, non-covalent
assembly. The system is based on a shape-selective dimerization of a functionalized gadolinium
chelate by a water-soluble host molecule. This reversible non-covalent binding of the relaxive
agent by the receptor allows displacement of the contrast agent by a stronger binder, and
activation of the contrast agent. This work was done in collaboration with Yoo-Jin Ghang
1
and
Richard J. Hooley,
2
and was published in 2014.
3

3.2. MRI contrast agent masked by molecular self assembly.
This process is illustrated in Figure 3.1. The relaxive agent (3.2a) is provided by
derivatization of Gd·DOTA to add an R-NMe
3
+
binding handle. This allows non-covalent
recognition by the water-soluble tetracarboxylate deep cavitand 3.1.
4
Cavitand 3.1 is capable of
selective recognition in water of guests displaying an R-NMe
3
+
handle,
5
lipid vesicles,
6

supported lipid bilayers
7
and living cells.
8
The binding generally occurs with high affinities (ca.
10
4
M
-1
in D
2
O), controlled by cation-π interactions, while the binding of larger organic cations
40

that cannot fit in the host (e.g. R-NEt
3
+
species) is minimal. Cavitand 3.1 displays properties of
lipids, including a hydrophobic body and a charged terminus, allowing self-assembly with
external lipids and incorporation into larger aggregates without loss of host behavior.

Figure 3.1. (a) Water-soluble deep cavitand 3.1; (b) minimized structure of the cavitand 3.1:Gd chelate 3.2a
complex (SPARTAN, AM1 forcefield); (c) a representation of the responsive MRI contrast process.

Addition of the R-NMe
3
+
binding handle to the contrast agent was performed using
known methods
9
from DOTA (3.3) and provides unmetalated scaffold 3.4 in 71% yield (Scheme
3.1). Treatment of 3.4 with GdCl
3
in water gives the MRI contrast agent 3.2a in 55% yield. The
same route furnished a diamagnetic surrogate of 3.2a for
1
H NMR measurements, yttrium
41

chelate 3.2b, in 36% yield. A minimized structure of the proposed 3.1:3.2a host:guest complex is
shown in Figure 3.1b.

Scheme 3.1. Synthesis of agents 3.2a and 3.2b.

Upon addition of host 3.1 to an aqueous solution of agent 3.2a, significant attenuation of
the relaxivity is observed. The T
1
of water of a 0.64 mM solution of agent 3.2a increased from
228(3) ms to 301(1) ms upon introduction of one molar equivalent of 3.1. As [3.1] increases, a
continuous increase in T
1
relaxation was observed through 6 molar equivalents, which increased
the T
1
of this solution to 344(1) ms. No further increase of T
1
was observed upon addition of
excess 3.1. These data are summarized in Figure 3.2 (left). Molar relaxivity decreased
accordingly (Figure 3.2, right): the relaxivity of 3.2a (5.1(4) mM
-1
s
-1
) is significantly lessened by
the addition of 6 molar equivalents of 3.1 (3.2(2) mM
-1
s
-1
), a decrease of 37%.

42

Figure 3.2. Cavitand masks contrast agent 3.2a. (Left): Titration of cavitand 3.1 to a 0.64 mM solution of contrast
agent 3.2a. (Right): Molar relaxivity curves of contrast agent 3.2a before ( •) and after ( ) the addition of 6 equiv.
cavitand 3.1.
3.3. Activation of contrast agent 3.2a.
The complexation of agent 3.2a and cavitand 3.1 with subsequent lessening of T
1
(the
“off” state) can be reversed by expelling agent 3.2a from the host, which can be achieved by
adding choline chloride (3.6).
10
It is fully encapsulated by the cavity of 3.1,
5
and so it displays a
greater K
a
than larger guests that protrude into the bulk water, exposing their hydrophobic
surface. When choline chloride (3.6) was titrated into a solution containing 3.2a (0.32 mM) and
6 equiv. of 3.1, a continuous decrease in T
1
was observed as [3.6] increased. The initial T
1

relaxation of 3.2a (576(4) ms) decreased to 420(5) ms after the addition of 100 molar equivalents
of choline (Figure 3.3, left). Molar relaxivities behave accordingly. The r
1
of the 3.1:3.2a
construct (3.2(2) mM
-1
s
-1
) is increased to 4.2(3) mM
-1
s
-1
upon introduction of choline, an
increase of 31% over the agent in its “off” state (Figure 3.3, right).
43

Figure 3.3. Choline reveals contrast agent 3.2a. (Left): Recovery of T
1
relaxation with the addition of choline
chloride 3.6 into a solution containing 0.32 mM agent 3.2a and 6 equiv. cavitand 3.1 (Right): Molar relaxivity
curves before( ) and after(o) the addition of choline chloride 3.6.

3.4. Control experiments.
The R-NMe
3
+
binding handle of 3.2a is essential to the behavior of the system. The
relaxivity-masking experiment above was repeated with unfunctionalized Na[Gd(DOTA)]
complex 3.5, which has little affinity for the cavitand due to the absence of the binding handle.
The molar relaxivity of 3.5 (2.8(2) mM
-1
s
-1
) was reduced upon addition of excess cavitand 3.1 by
only ca. 14% (r
1
= 2.4(1) mM
-1
s
-1
). In addition, there was no change in relaxivity upon exposure
of the 3.1:3.5 mixture to choline (r
1
= 2.5(1) mM
-1
s
-1
) within error (Figure 3.4, left). Without the
selective binding of the agent with its host, minimal reduction of molar relaxivity was observed,
and no reversal is possible upon treatment with choline.
44

Figure 3.4. Control experiments. (Left): Molar relaxivity curves of contrast agent 3.2a ( •), after the addition of 6
equiv. cavitand 3.1 ( ), and after the addition of 100 molar equivalents of choline 3.6 (o) (Right): Molar relaxivities
of agents 3.2a and 3.5 with sequential addition of 3.1 (6 equiv.) and 3.6 (100 equiv.).

3.5. Mechanism of observed behavior.
The exact mechanism of the relaxivity control is not immediately obvious.
Immobilization of gadolinium chelates on scaffolds of larger size (e.g. macromolecules) tends to
yield higher relaxivities due to reduced tumbling rate, but this system displays the opposite
behavior: relaxivity lessens upon binding of the cavitand to 3.2a. Simple
1
H NMR analysis could
show the binding, but these experiments are not practical with paramagnetic agent 3.2a. To
analyze the binding properties of 3.2a in the cavitand, the analogous diamagnetic yttrium
complex 3.2b was employed. Interestingly, upon addition of 3.2b to an aqueous solution of
cavitand 3.1, no characteristic peaks for the 3.1:R-NMe
3
+
complex were observed (Figure 3.5).
Instead, loss of signal was observed as the guest concentration increased. When cavitand 3.1 was
titrated into a solution of 3.2b, similar results were obtained. Only after addition of 6 equivalents
of 3.1 were sharp peaks observed, corresponding with those of free cavitand.
45

Figure 3.5. Upon introduction of Y agent 3.2b to cavitand 3.1, the
1
H NMR signals broaden.

The disappearance of signals for both host and guest and the lack of any observable
precipitate from the solution suggest that an aggregation of the lipid-like cavitand 3.1 is
occurring upon complexation with the amphiphilic guest 3.2b. This triggered self-assembly of
cavitand 3.1 is rare. Hydrocarbon guests and small R-NMe
3
+
species such as choline,
acetylcholine or NMe
4
Br form simple 1:1 complexes.
7
Longer alkyltrimethylammonium species
form micelles that incorporate the cavitand itself.
8
Guest 3.2b, however, is of intermediate size: it
is poorly capable of forming micelles itself, but a hydrophobic component of the guest protrudes
from the cavity into the bulk water upon binding, lowering the solubility of the complex. When
the host:guest binding event occurs, the 3.1:3.2b complex apparently initiates aggregation into a
larger assembly that displays slow tumbling, and so the
1
H signals are averaged. If the aggregate
is disrupted by addition of MeCN-d
3
to the solution, the individual peaks for both 3.1 and 3.2b
46

are observed again (Figure 3.6). The binding of 3.2b in 3.1 is relatively weak. While accurate
calculation of the K
a
(3.1:3.2b) is complicated by the aggregation phenomenon, it can be
estimated as ca. 250 M
-1
, two orders of magnitude less than that of choline. Hydrophobic guests
that are not completely “hidden” from the bulk water upon binding in 3.1 are known to display
lowered K
a
values,
10
so the relatively weak binding of 3.2b is not unexpected. This also explains
why the relaxivity of 3.2a ceases to decrease after the addition of 6 molar equiv. 3.1: that is the
amount of 3.1 required fully to bind all contrast agent molecules in the cavitand under the
conditions of the experiment. Lower cavitand concentration leads to free 3.2a molecules in the
“on” state.

Figure 3.6. Expelling guest 3.2b from host 3.1 with CD
3
CN.

47

The aggregation appears to be key to the lowered relaxivity of the contrast agent upon
cavitand binding, so the assembly events were further characterized by dynamic light scattering
(DLS, Figure 3.7). At the most effective 3.1:2.2a molar ratio of 6:1, discrete nanoparticle
aggregates with an average diameter of 7 nm were observed. There are some recent examples of
micelle formation by cavitands in aqueous solution,
11
but these involve the cavitand itself and are
not triggered by guest binding. The orientation of the 3.1:3.2a complex in this micellar aggregate
is not obvious: one would expect the tetracarboxylate rim of 3.1 to be oriented towards the bulk
solvent, but the Gd-DOTA agent 3.2a itself displays a lipophilic terminus. The relaxivity data
provides a possible explanation: even though the assembly is larger and presumably tumbles at a
slower rate than free 3.2a, the relaxivity of 3.2a is lowered upon binding to 3.1. In the aggregated
form, the gadolinium center of 3.2a is most likely hidden and has limited access to water.
12
This
indicates that the micellar aggregate displays the characteristics of the cartoon shown in Figure
3.1c, wherein the gadolinium center is oriented towards the center of the aggregate. The
nanoparticle aggregate is expected to have a slower tumbling rate, but the water-shielding
properties of the system ultimately dominates.
48

Figure 3.7. DLS of cavitand 3.1 without (left) and with (right) trimethylammonium guest 3.2a.

Upon addition of choline (3.6), guest 3.2a is liberated from the assembled aggregate,
leading to free 3.2a in solution that can resume its usual T
1
contrast properties. Unfortunately,
DLS analysis of the cavitand:choline complex was complicated by the appearance of larger
aggregates that occur upon addition of salt and/or varying the concentration of 1.9 At higher
concentrations of 3.1 and in the presence of 3.6, larger aggregates ranging from 20-100 nm and
900-1000 nm were observed. The aggregation phenomenon is evidently complex, but the nature
of the choline-containing aggregates is inconsequential to the T
1
modulation behavior: the more
strongly binding choline (3.6) occupies the cavitand aggregates and 3.2a is free to display strong
contrast again.

49

3.6. MRI phantoms.
The responsive MRI contrast agent system can be most effectively demonstrated by a
simple visualization of the contrast difference between the “on” and “off” states. To this end,
phantoms were acquired in a 7 T MRI imager using a FLASH sequence (Figure 3.8). Image 1
consists of distilled water in the absence of any gadolinium, and thus appears dark. Image 2
contains a 1 mM solution of gadolinium agent 3.2a and appears bright. Cavitand (image 3)
masks the contrast and the resulting image is darker. Upon treatment with choline, the contrast is
revealed (image 4). These observations are consistent with the molar relaxivity data obtained.

Figure 3.8. Phantoms acquired in a Pharmascan 7 T MRI using Bruker’s FLASH sequence. TR = 474 ms; TE = 6
ms. (1) H
2
O; (2) 0.64 mM solution of gadolinium agent 3.2a; (3) 0.64 mM solution of gadolinium agent 3.2a with 6
equivalents of cavitand 3.1; (4) 0.64 mM solution of gadolinium agent 3.2a with 6 equivalents of cavitand 3.1 upon
exposure to 100 equivalents of choline. A 30% difference in brightness between the masked agent (3) and the
revealed agent (4) was quantified using ImageJ.

3.7. In vivo behavior of masked gadolinium agent 3.2a.
In vivo images of a rat's bladder were acquired to gauge the in vivo responsiveness of the
masked MRI system (Figure 3.9). Image A illustrates the image of the rat prior to the injection
of any MRI contrast agent, and thus the bladder appears dark. Upon injection of ca. 1.0 mL of a
0.64 mM solution of 3.2a with 6 equivalents cavitand 3.1, the bladder becomes bright as contrast
agent is being introduced (Figure 3.9B). A minimal difference in brightness was detected once
choline was introduced into the system (Figure 3.9C). One possible explanation for this
observed behavior is the incompatibility of the masked contrast agent in vivo, such that contrast
is revealed prior to choline exposure. Since the injection of solid choline is not feasible, choline
was introduced as a concentrated solution. Another possible explanation for the observed lack of
contrast enhancement is the dilution of contrast agent.
50

Figure 3.9. MRI of the bladder of a rat A) without contrast agent B) upon injection of ~1.0 mL of 0.64 mM masked
agent 3.2a:3.1 and C) upon introducing ~0.1 mL of a 500 mM choline solution. A 2% difference in brightness
between the masked agent (B) and the revealed agent (C) was quantified using ImageJ.

3.8. Activation of contrast agent 3.2a using a trimethylammonium coated gold nanoparticle.
Although masked contrast agent 3.2a demonstrated responsiveness in the presence of
choline, an overwhelming excess of the competing guest is required for its activation.
Furthermore, trimethylammonium-containing small molecules may be biologically incompatible.
However, macromolecular trimethylammonium coated gold nanoparticles have demonstrated to
be biologically benign,
13
and can be used as a triggering agent in place of choline.
The synthesis of 8-mercaptotrimethylammonium bromide (8-MTAB) was performed
using a previously reported method.
14
Briefly, bromothioester 3.8 was obtained via refluxing
excess dibromooctane 3.7 with potassium thioacetate. Bromothioester 3.8 was then exposed to
trimethylamine and allowed to react for 13 days. Upon reaction completion, thiol 3.10 was
revealed upon ethanolysis with HBr. 3.10 was assembled onto 1.5 nm phosphine stabilized gold
nanoparticles via interfacial ligand exchange in a biphasic water/dichloromethane system
(Scheme 3.2).
15

51

Scheme 3.2. Synthesis of trimethylammonium coated gold nanparticles.

Surface coating of the trimethylammonium ligand on the gold nanoparticle is key. If the
ligand exchange is allowed to stir for 24 hours, no modulation of T
1
relaxation is observed upon
introduction into a solution of masked construct 3.2a:3.1. However, if the ligand exchange is
stopped after one hour, a change a recovery of T
1
relaxation is observed. The T
1
of 0.160 mL of
a 0.21 mM solution of 3.2a:3.1 (T
1
= 707.5 ms) was decreased by 31% when exposed to 0.25 mg
particles 3.12 (ca. 2 equiv. trimethylammonium with respect to cavitand 3.1 in solution, Table
3.1). The trimethylammonium concentration was estimated from thermogravimetric analysis,
which revealed ca. 50% w/w concentration of ligand 3.10 on particle 3.12. Thus it can be
52

estimated that 0.125 mg of ligand 3.10 was in solution to activate the masked agent 3.2a:3.1
([3.2a] = 0.21 mM, [3.1] = 1.26 mM, [trimethylammonium] = 2.75 mM, ca. 2 equiv. with respect
to 3.1). Comparatively, 100 equivalents of choline 3.6 is needed for the full activation of
3.2a:3.1. This demonstrates particle 3.12 as a potential biologically friendly triggering agent for
the masked 3.2a:3.1 system.
Contrast Agent T
1

3.1:3.2a (0.21 mM) 707.5 ms
3.1:3.2a + Choline 3.6 (100 equiv) 444.4 ms
3.1:3.2a + 3.12 (ca. 2 equiv. NMe
3
+
) 486.6 ms
Table 3.1. Recovery of relaxivity using particle 3.12 as a triggering agent. [2.1] = 0.21 mM, [3.1] = 1.26 mM
[trimethylammonium] = 2.75 mM.
3.9. Conclusion.
In conclusion, an MRI contrast system that is controlled solely by non-covalent
molecular recognition and self-assembly processes was developed. The self-assembly cascade
allows the contrast agent to be turned “off” by shielding the gadolinium center in a self-
assembled micellar aggregate with a suitable water-soluble cavitand host. The assembly is
triggered by the binding event, and can be disrupted by addition of a superior guest molecule,
freeing the contrast agent, regenerating its contrast abilities, and turning the agent “on” again.
This process occurs under mild aqueous conditions and shows great promise for applications in
biologically relevant environments.

3.10. References.

1 ) Richard J. Hooley research group. Department of Chemistry, University of California
Riverside. Riverside, CA 92521
2) Department of Chemistry, University of California Riverside. Riverside, CA 92521
3) Li, V.; Ghang, Y-J.; Hooley, R. J.; Williams, T. J. Non-Covalent Self Assembly Controls
the Relaxivity of Magnetically Active Guests. Chem. Commun. 2014, 50, 1375-1377
53

4) Hof, F.; Trembleau, L.; Ullrich, E. C.; Rebek Jr., J. Acetylcholine Recognition by a Deep,
Biomimetic Pocket. Angew. Chem. 2003, 115, 3258-3261; Angew. Chem. Int. Ed. 2003,
42, 3150-3153.
5) Biros, S. M.; Ullrich, E. C.; Hof, F.; Trembleau, L.; Rebek Jr., J. Kinetically Stable
Complexes in Water: The Role of Hydration and Hydrophobicity. J. Am. Chem. Soc.
2004, 126, 2870-2876.
6) Schramm, M. P.; Hooley, R. J.; Rebek Jr, J. Guest Recognition with Micelle-Bound
Cavitands. J. Am. Chem. Soc. 2007, 129, 9773-9779.
7) (a) Liu, Y.; Liao, P.; Cheng, Q.; Hooley, R. J. Protein and Small Molecule Recognition
Properties of Deep Cavitands in a Supported Lipid Membrane Determined by
Calcination-Enhanced SPR Spectroscopy. J. Am. Chem. Soc. 2010, 132, 10383-10390.
(b) Liu, Y.; Young, M. C.; Mosche, O.; Cheng, Q.; Hooley, R. J. A Membrane-Bound
Synthetic Receptor that Promotes Growth of a Polymeric Coating at a Bilayer-Water
Interface. Angew. Chem. 2012, 124, 7868-7871; Angew. Chem. Int. Ed. 2012, 51, 7748-
7751.
8) Ghang, Y-J.; Schramm, M. P.; Zhang, F.; Acey, R. A.; David, C. N.; Wilson, E. H.;
Wang, Y.; Cheng, Q.; Hooley, R. J. Selective Cavitand-Mediated Endocytosis of
Targeted Imaging Agents into Live Cells. J. Am. Chem. Soc. 2013, 135, 7090-7093.
9) Wängler, C.; Wängler, B.; Eisenhut, M.; Haberkorn, U.; Mier, W. Improved Syntheses
and Applicability of Different DOTA Building Blocks for Multiply Derivatized
Scaffolds. Bioorg. Med. Chem. 2008, 16, 2606-2616.
10) Hooley, R. J.; Anda, H. J. V.; Rebek Jr. J. Extraction of Hydrophobic Species into a
Water-Soluble Synthetic Receptor. J. Am. Chem. Soc. 2007, 129, 13464-13473.
11) Kubitschke, J.; Javor, S.; Rebek Jr., J. Deep Cavitand Vesicles - Multicompartmental
Hosts. Chem. Commun. 2012, 48, 9251-9253.
12) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Gadolinium(III) Chelates as
MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99,
2293-2352.
13) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Gold Nanoparticles
are Taken up by Human Cells but do not Cause Acute Toxicity. Small 2005, 1, 325-327.
14) Jong, L. I.; Abbott, N. L. Rate-Dependent Lowering of Surface Tension During
Transformations of Water-Soluble Surfactants from Bolaform to Monomeric Structures.
Langmuir 1998, 14, 2235-2237.
15) Warner, M. G.; Reed, S. M.; Hutchison, J. E. Small, Water-Soluble, Ligand-Stabilized
Gold Nanoparticles Synthesized by Interfacial Ligand Exchange Reactions. Chem. Mater.
2000, 12, 3316-3320.
54

Chapter 4. An MRI pill for the detection of gastric motility
disorders.

4.1. Introduction - gastroesophageal reflux disease and gastroparesis.

Gastroesophageal reflux disease (GERD) is a gastric motility disorder that is
characterized by the reflux of gastric contents from the stomach into the esophagus, which is a
frequent cause of heartburn.
1
The lower esophageal sphincter contains muscle fibers which
tighten to prevent reflux. However, these fibers are damaged in GERD patients, allowing such
contents to reflux back into the esophagus. GERD is a common condition, as 40% the
population experience heartburn, with 20% of the population experiencing at least weekly
occurrences of heartburn, the most common symptom of GERD.
2

Another gastric motility disorder, gastroparesis, is a condition characterized by the
delayed emptying of stomach contents and its symptoms include nausea, abdominal pain,
heartburn and bloating.
3
Although some cases are presented as an autoimmune or a neurological
condition, it is more commonly attributed to obesity and diabetes.
4
Because this condition is
linked to obesity and diabetes, the number of reported cases is increasing rapidly. Gastroparesis
related hospitalizations increased 158% between the years 1995-2004.
5
Although gastroparesis is
becoming a greater health concern, limited treatment options are available because the condition
is poorly characterized.
6
This work was done in collaboration with Andy Y. Chang.
7

4.2. Current methods of diagnosis.

A variety of treatment options exist for patients with GERD, so long as an accurate
diagnosis is made.
8
However, because current diagnostic tools are expensive or invasive,
9
the
main diagnostic tool for GERD is patient interview. If a patient displays symptoms consistent
with GERD, proton pump inhibitors (PPIs) are commonly prescribed to alleviate the condition.
10

55

If PPIs fail to relieve the symptoms, diagnostic tests such as 24-hour pH test or upper
gastrointestinal endoscopy can be performed.
11
These diagnostic tests are either invasive, utilize
ionizing radiation, or suffer from low accuracy.
10

A recent study by Kulinna-Cosentini et al. demonstrated the viability of using non-
invasive MRI as a method of GERD detection.
12
In the study, patients were asked to swallow a
cocktail containing a mixture of buttermilk and gadolinium-based MRI contrast agent Omniscan
(Figure 4.1). MRI images were then acquired with the patients lying in a supine position and the
results were corroborated with pH tests. This method of diagnosis resulted in 82% accuracy.
N
N
N
O
O
Gd
O
O
O
O
O
HN
NH
O

Figure 4.1. Gadodiamide, the gadolinium MRI contrast agent in Omniscan.

The most common method of gastroparesis diagnosis is gastric emptying scintigraphy.
13

In the typical procedure, patients are to ingest a meal containing
99
Tc labeled scrambled eggs. A
gamma camera is then used to quantify the amount of
99
Tc in the stomach, and images are
obtained at consistent time points over the course of hours to yield the rate at which
99
Tc exits
the stomach. Although able to quantify the rate of emptying, scintigraphy provides low
resolution images.

4.3. Design of MRI pill for detection of gastric motility disorders.

Although the study presented by Kulinna-Cosentini et al. demonstrated the ability to use
MRI for GERD detection,
12
the overall accuracy was only 82%, with 11% false positives and 7%
false negative. One possible explanation for the false positives is the use of an unmasked
56

contrast agent, and thus contrast seen on the MRI cannot reliably distinguish between ingestion
and reflux. In addition, since a high dose of contrast agent may result in a higher rate of false
positives, the maximum dosage of contrast agent is limited, thus providing a higher rate of false
negatives.
A masked MRI contrast agent which activates in the stomach can be used to minimize
both false positives and false negatives. This proposed agent will not be detected unless refluxed
from the stomach, thus eliminating false positives. Furthermore, higher dosing of contrast agent
can be used with the masked agent, which minimizes false negatives. The MRI contrast agent
used in this study was clinically relevant Na[Gd·DOTA] (Figure 4.2), which was then adsorbed
onto a biologically safe wheat chex breakfast cereal. The contrast was then loaded into a gelatin
pill casing, which physically blocks water from the gadolinium center and thus masking contrast.
The gelatin pill casing is delivered to the stomach via ingestion, where it will degrade in the
acidic gastric contents, revealing the contrast agent. MRI is then used to detect both reflux, and
gastic emptying.

Figure 4.2. Na[Gd·DOTA], the MRI contrast agent used in this study.

4.4. In vitro release of contrast agent from the MRI pill.

The effectiveness of the gelatin pill casing at masking and revealing contrast was
characterized via in vitro release of contrast agent. A 10:1 w/w mixture of breakfast cereal to
57

Gd·DOTA was first created and loaded into size #5 pill casings.
14
To assess pill behavior in both
the neutral esophageal and acidic gastric environments,
15
pills were then exposed to both neutral
and acidic (pH=1) aqueous environments. The release of contrast agent from the pills were
characterized by measuring T
1
relaxation of the solution over time.
The release of contrast agent is not immediate in a neutral environment. T
1
data suggests
that the agent is being released between 10 and 15 minutes after exposure to the environment as
demonstrated by the significant decrease in T
1
relaxation. However, this release appears to be
immediate in an acidic environment (Figure 4.3). These data suggest the pill casing is stable in
an neutral aqueous environment for a period of time long enough to be effective at masking
contrast in the esophagus until revealed in the acidic gastric environment.

Figure 4.3. Release of Gd-DOTA from gelatin pill casings.

4.5. In vivo behavior of the MRI pill.

To gauge in vivo behavior of the loaded gelatin pill, MRI images of a rat were acquired.
After a background scan (Figure 4.4A and Figure 4.4E), the rat was anesthetized, then fed a
0
0.5
1
1.5
2
2.5
3
0 10 20 30 40 50 60 70
T
1
(seconds)
Time (minutes)
pH=7
pH=1
58

loaded size #9 loaded pill capsule.
16
Prior to administration pill administration, the stomach
appeared dark, and undefined. Upon administration of the pill, it becomes brighter as the
contrast agent is introduced in the stomach (Figure 4.4B, Figure 4.4C and Figure 4.4D). In a
subsequent scan, the intestine was revealed (Figure 4.4H), which is an indication that gastric
emptying is being detected. More importantly, no contrast was observed in the esophagus of the
healthy rat, as GERD was not present. This suggests that the pill was masked as intended until
exposed to the acidic gastric environment.

Figure 4.4. MRI image planes of a rat highlighting the stomach (top) and intestines (bottom) at various time points
after introduction of the MRI pill.

4.6. Conclusion.

An MRI contrast agent was adsorbed onto wheat chex breakfast cereal and masked inside
a gelatin pill casing. The behavior of the pill in both neutral and acidic aqueous environments
was characterized in vitro. The release of contrast agent is delayed in neutral pH, but immediate
in acidic pH. MRI images of a rat demonstrated the contrast agent can be visualized
immediately in the stomach after ingestion, and eventually in the intestines after 60 minutes.
59

This suggests the MRI pill can be utilized for the detection of both the reflux of gastric contents
and gastric emptying.

4.7. References.

1) Kahrilas, P. J. GERD Pathogenesis, Pathophysiology, and Clinical Manifestations. Cleve.
Clin. J. Med. 2003, 70, 4-19.
2) Locke III, G. R.; Talley, N. J.; Fett, S. L.; Zinsmeister, A. R.; Melton III, L. J.
Prevalence and Clinical Spectrum of Gastroesophageal Reflux: A Population-Based
Study in Olmsted County, Minnesota. Gastroenterology 1997, 112, 1448-1456.
3) (a) Cherian, D.; Sachdeva, P.; Fisher, R. S.; Parkman, H. P. Abdominal Pain is a
Frequent Symptom of Gastroparesis. Clin. Gastroenterol. Hepatol. 2010, 8, 676 –681. (b)
Hasler, W. L.; Wilson, L. A.; Parkman, H. P.; Nguyen, L.; Abell, T. L.; Koch, K. L.;
Pasricha, P. J.; Snape, W. J.; Farrugia, G.; Lee, L.; Tonascia, J.; Unalp-Arida, A.;
Hamilton, F. Bloating in Gastroparesis: Severity, Impact, and Associated Factors. Am. J.
Gastroenterol. 2011, 106, 1492-1502.
4) Soykan, I.; Sivri, B.; Sarosiek, I.; Kiernan, B.; Mccallum, R.W. Demography, Clinical
Characteristics, Psychological and Abuse Profiles, Treatment, and Long-Term Follow-up
of Patients with Gastroparesis. Dig. Dis. Sci. 1998, 43, 2398-2404.
5) Wang, Y. R.; Fisher, R. S.; Parkman, H. P. Gastroparesis-Related Hospitalizations in the
United States: Trends, Characteristics, and the Outcomes. Am. J. Gastroenterol. 2008,
103, 313-322.
6) Hasler, W. L. Gastroparesis Pathogenesis, Diagnosis and Management. Nat. Rev.
Gastroenterol. Hepatol. 2011, 8, 438-453.
7) The Saban Research Institute of Children's Hospital Los Angeles. Los Angeles, CA
90027.
8) Tytgat, G. N. J. Review Article: Treatment of Mild and Severe Cases of GERD. Ailment
Pharmacol. Ther. 2002, 16, 73-78.
9) Storr, M.; Meining, A.; Allescher, H.-D. Pathophysiology and Pharmacological
Treatment of Gastroesophageal Reflux Disease. Dig. Dis. Sci. 2000, 18, 93-102.
10) DeVault, K. R.; Castell, D. O. Updated Guidelines for the Diagnosis and Treatment of
Gastroesophageal Reflux Disease. Am. J. Gastroenterol. 2005, 100, 190-200.
11) Patrick, L. Gastroesophageal Reflux Disease (GERD): A Review of Conventional and
Alternative Treatments. Altern. Med. Rev. 2011, 16, 116-133.
60

12) Kulinna-Cosentini, C.; Schima, W.; Lenglinger, J.; Riegler, M.; Kölblinger, C.; Ba-
Ssalamah, A.; Bischof, G.; Weber, M.; Kleinhansl, P.; Cosentini, E. P. Is There a Role for
Dynamic Swallowing MRI in the Assessment of Gastroesophageal Reflux Disease and
Oesophageal Motility Disorders? Eur. Radiol. 2012, 22, 367-370.
13) Seok, J. W. How to Interpret Gastric Emptying Scintigraphy. J. Neurograstroenterol.
Motil. 2011, 17, 189-191.
14 ) Size #5 pill casings measure 4.91 mm in outer diameter, 11.1 mm in length and 0.13 ml
volume.
15) Tutuian, R.; Castell, D. O. Gastroesophageal Reflux Monitoring: pH and Impedance. GI
Motility Online 2006, doi:10.1038/gimo31.
16) Size #9 pill casings measure 2.65 mm in outer diameter, 8.6 mm in length and 0.025 ml
volume.
61

Chapter 5. Introduction. Azoles as medicinal entities and methods of
synthesis

5.1. Azoles as pharmaceutical agents and issues with the current methods of synthesis.

Azoles are five-membered aromatic heterocycles containing one nitrogen atom and at
least one other heteroatom - either nitrogen, oxygen or sulfur. They are biologically active
cyclopeptides that are isolated from marine organisms and have been proposed as anticancer,
1

antifungal,
2
antiviral,
3
and antibacterial
4
agents. Some examples are shown in Figure 5.1.

Figure 5.1. Some examples of azole-containing natural products.
5

62

Despite their potential pharmaceutical applications, there are no catalytic approaches
toward their synthesis. Previous reaction conditions for the oxidation of azolines to azoles
consisted of either excess amounts of metals such as Ni,
6
Mn,
7
or Cu,
8
or the use of toxic
reagents such as BrCCl
3
.
9
The need for extreme quantities of toxic reagents introduces a
separatory issue for pharmaceutical studies along with high disposal costs. This chapter will
highlight the previous existing methods of azoline oxidation.

5.2. Metal mediated oxidation of azolines.

Nickel mediated oxidation of thiazolines to the corresponding thiazoles was reported by
Hecht et al.
6
The reaction required 2-4 molar equivalents of NiO
2
with temperatures ranging
from room temperature to refluxing benzene. This reaction was further explored by Evans et al.,
who expanded the substrate scope to include a broad array of thiazolines, as well as oxazolines.
10

Although a variety of substrates were compatible with the reaction conditions, they were
noticeably harsh, as some reactions were refluxed for days in the presence of up to 6 molar
equivalents of NiO
2
(Table 5.1).

63

Starting Material Product Equiv.
NiO
2

Conditions Yield Ref.

5.5

5.5a
2.0
CHCl
3
, room
temp. 3 days
81% 6

5.6

5.6a
3.7
C
6
H
6
, reflux,
4h
60% 6

5.7

5.7a
2.4
CHCl
3
, room
temp. 3 days
93% 6

5.8

5.8a
1.0 CHCl
3
, room
temp. 12h
88% 10

5.9

5.9a
1.5 Hexanes,
16h, reflux
53% 10

5.10

5.10a
5.0 C
6
H
6
, 7h,
reflux
28% 10

5.11

5.11a
6.0 Cyclohexane,
5 days, reflux
19% 10
Table 5.1. Examples of nickel-mediated oxidation of azolines to azoles.

64

As an alternative to NiO
2
, MnO
2
can also be used as an oxidant. MnO
2
promoted
oxidation of azolines to azoles was also demonstrated in 1977 by Hecht et al., who converted 5.7
to 5.7a by exposing 5.7 to an excess of MnO
2
at room temperature for 4 days.
7
Although the
desired product 5.7a was obtained, the yield observed was only 65%, and NiO
2
was ultimately
used for the desired bond transformation. MnO
2
was later utilized for the oxidation of
thiazolines bearing a carboxylate at the 4-position and demonstrated to be compatible with
various carbamate protecting groups (Table 5.2).
11
Fu et al. later expanded the use of MnO
2
to
oxidize a wide scope of 2,4-disubstituted thiazolines to thiazoles (Scheme 5.1).
12
However, the
reaction conditions were once again harsh, as 10 molar equivalents of MnO
2
with respect to
thiazoline in refluxing dichloroethane solvent for 12 hours were required to effect this oxidation.
Yields ranging from 76-99% were obtained (Table 5.2).

Scheme 5.1. MnO
2
mediated oxidation of thiazolines to thiazoles.

65

Pattenden et al.
a

Entry Starting Material Product Yield
1
N
S
O
O
NH Cbz

5.12
N
S
O
O
NH Cbz

5.12a
91%
2
N
S
O
O
NH Alloc

5.13
N
S
O
O
NH Alloc

5.13a
50%
3
N
S
O
O
NH Boc

5.14
N
S
O
O
NH Boc

5.14a
88%
Fu et al.
b

4

5.15

5.15a
95%
6

5.16

5.16a
99%
7

5.17

5.17a
95%
8

5.18

5.18a
76%
9

5.19

5.19a
85%
Table 5.2. Substrate scope for the MnO
2
mediated oxidation of thiazolines to thiazoles.
a
Reactions demonstrated by
Pattenden et al.
11
Reaction was stirred with 10 molar equivalents of MnO
2
in DCM solvent for up to 18 hours.
b
Reactions demonstrated by Fu et al.
12
Reactions were refluxed in dichloroethane and 10 molar equivalents of MnO
2

for 12 hours.
66

Copper was also demonstrated to be an effective oxidant in the conversion of azolines to
azoles as demonstrated by Barrish et al. with the oxidation of oxazoline 5.20 to oxazole 5.21
using 4 equivalents each of CuBr
2
, 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) and
hexamethyltetramine (HMTA).
8
This reaction was particularly effective for the oxidation of 4-
carboxyoxazolines, providing yields up to 82%.

Scheme 5. 2. Copper(II) mediated oxidation of oxazolines to oxazoles.

Meyers later developed a Cu(I)/Cu(II) radical-pathway oxidation of azolines to azoles
based on the Kharasch-Sosnovsky allylic oxidation system.
13
This reaction required 1.1 molar
equivalents of both copper(I) and copper(II) under benzene reflux temperatures. A proposed
mechanism is shown in Scheme 5.3. The mechanism initiates with the formation of the α radical
with tert-butyl peroxybenzoate and copper(I), followed by ligand transfer from copper(II)
benzoate. Elimination of benzoate yields the thiazole product.
67

Scheme 5.3. Proposed mechanism for the Cu(I)/Cu(II) oxidation of azolines to azoles.

5.3. BrCCl
3
oxidation of azolines.

Oxidation of azolines to their corresponding azoles using DBU and BrCCl
3
were
developed by Williams et al.
9
The reactions were done in dichloromethane solvent at 0 °C,
using 1.05 equivalents of BrCCl
3
and up to 2 equivalents of DBU. The reaction demonstrated
compatibility with both oxazoline and thiazoline substrates, as isolated yields were as high as
95% (Table 5.3). A modified one-pot cyclization of β-hydroxyamides to oxazolines using either
diethylaminosulfur trifluoride (DAST) or bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-
Fluor) and its subsequent oxidation using BrCCl
3
was later developed (Scheme 5.4).
14

68

Starting Material Product Yield
N
S
O
O
5.22
N
S
O
O
5.22a
95%
N
O
O
O
5.23
N
O
O
O
5.23a
75%
N
S
O
O
Ph
O
N
5.24
N
S
O
O
Ph
O
N
5.24a
92%
N
O
O
O
O
N

5.25
N
O
O
O
O
N

5.25a
87%
Table 5.3. Oxidation of azolines to azoles using BrCCl
3
.
9
Reactions were done in dichloromethane solvent at 0 °C
using 1.05 equivalents BrCCl
3
and 1.05-2 equivalents DBU.
R
1
O
N
H
R
2
R
3
HO Deoxo-Fluor, -20
o
C
or DAST, -78
o
C
N
O
R
1
R
2
R
3
DBU, BrCCl
3
0
o
C - 22
o
C
N
O
R
1
R
2
R
3

Scheme 5.4. One pot cyclization of β-hydroxyamides and subsequent oxidation.
14

5.4. Aerobic oxidation of azolines.

Aerobic oxidations of thiazolines to thiazoles were recently reported by Yao et al.
15
The
reaction conditions required 3 molar equivalents of K
2
CO
3
, 4Å molecular sieves (200 weight %)
and was heated to 80 °C in the presence of air to afford the corresponding thiazoles. A limited
substrate scope was demonstrated, as mainly electron deficient thiazolines were used (Table 5.4).
The electron rich alkyl substrates that were demonstrated required an O
2
balloon to provide
comparable yields of the corresponding thiazoles.

69

Starting Material Product Yield
N
S
CO
2
Et

5.26
N
S
CO
2
Et

5.26a
77%
a

N
S
CO
2
Et
NO
2

5.27
N
S
CO
2
Et
NO
2

5.27a
83%
a

N
S
CO
2
Et
O
2
N

5.28
N
S
CO
2
Et
O
2
N

5.28a
82%
a

N
S
CO
2
Et
O
2
N

5.29
N
S
CO
2
Et
O
2
N

5.29a
77%
a

N
S
CO
2
Et
F

5.30
N
S
CO
2
Et
F

5.30a
80%
a

N
S
CO
2
Et
F
3
C

5.31
N
S
CO
2
Et
F
3
C

5.31a
91%
a

N
S
CO
2
Et

5.32
N
S
CO
2
Et

5.32a
67%
b

N
S
CO
2
Et

5.33
N
S
CO
2
Et

5.33a
67%
b

Table 5.4. Aerobic oxidation of thiazolines to thiazoles. Reactions were run in DMF solvent, with 4Å molecular
sieves (200 weight %), 3 equivalents K
2
CO
3
and heated to 80 °C.
a
Reactions were run in air.
b
Reactions were run
under an O
2
balloon.

70

In their mechanistic report, hydroperoxide intermediate 5.34 (Scheme 5.5) was proposed
upon exposure to air. This intermediate was reportedly characterized via thin-layer
chromatography (TLC). Peroxide intermediate 5.34 then collapses to hydroxide intermediate
5.35, which can be isolated under reduced reaction temperatures. Dehydration of 5.35 yields the
thiazole final product 5.26a. This mechanism will be discussed in greater detail in chapter 6.

Scheme 5.5. Mechanism for the aerobic oxidation of thiazolines to thiazoles, as proposed by Yao et al.

5.5. Conclusion.

Although azoles are an attractive class of bioactive natural products, previously known
methods of synthesis involve excess amounts of toxic reagents, such as metals or halogenated
reagents. Chapter 6 will discuss two methods of aerobic azoline oxidation - one catalytic in both
copper and base, and one base promoted. The reactions conditions were environmentally benign,
compatible with a wide substrate scope, and demonstrated scalability.

71

5.6. References.

1) (a) Kanoh, K.; Matsuo,Y.; Adachi, K.; Imagawa, H.; Nishizawa, M.; Shizuri, Y. J.
Mechercharmycins A and B, Cytotoxic Substances from Marine-Derived
Thermoactinomyces sp. YM3-251. Antibiot. 2005, 58, 289-292. (b) Höfle, G.; Bedorf,
N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H. Epothilone A and B-
Novel 16-Membered Macrolides with Cytotoxic Activity: Isolation, Crystal Structure and
Conformation in Soltuion. Angew. Chem. Int. Ed. 1996, 35, 1567-1569. (c) Robins, R.
K.; Srivastava, P. C.; Narayanan, V. L.; Plowman, J.; Paull, K. D. 2-β-D-
ribofuranosylthiazole-4-carboxaminde, a Novel Potential Antitumor Agent for Lung
Tumors and Mestases. J. Med. Chem. 1982, 25, 107-108. (d) Lu, Y.; Li, C.; Wang, Z.;
Ross, C. R. 2nd; Chen, J.; Dalton, J. T.; Li, W.; Miller, D. D. Discovery of 4-Substituted
Methoxybenzoyl-aryl-thiazole as a Novel Anticancer Agents: Synthesis, Biological
Evaluation, and Structure – Activity Relationships. J. Med. Chem. 2009, 52, 1701-1711.
2) (a) Heeres, J.; Backx, J. J.; Mostmans, J. H.; Cutsem, J. V. Antimycotic Imidazoles. Part
4. Synthesis and Antifungal Activity of Ketoconazole, a New Potent Orally Active
Broad-Spectrum Antifungal Agent. J. Med. Chem. 1979, 22, 1003-1005. (b) Pfaller, M.
A.; Messer, S. A.; Hollis, R. J.; Jones, R. N. Antifungal Activities of Posaconazole,
Ravuconazole, and Voriconazole Compared to Those of Itraconazole and Amphotericin
B against 239 Clinical Isolates of Asperillus spp. and Other Filamentous Fungi: Report
from SENTRY Antimicrobial Surveillance Program, 2000. Antimicrob. Agents
Chemother. 2002, 46, 1032-1037. (c) Herbrecht, R.; Denning, D. W.; Patterson, T. F.;
Bennett, J. E.; Greene, R. E.; Oestmann, J-W.; Kern, W. V.; Marr, K. A.; Ribaud, P.;
Lortholary, O.; Sylvester, R.; Rubin, R. H.; Wingard, J. R.; Stark, P.; Durand, C.; Caillot,
D.; Thiel, E.; Chandrasekar, P. H.; Hodges, M. R.; Schlamm, H. T.; Troke, P. F.; De
Pauw, B. Vorinconazole Versus Amphotericin B for Primary Therapy of Invasive
Aspergillosis. N. Engl. J. Med. 2002, 347, 408-415.
3) (a) Srivastava, P. C.; Pickering, M. V.; Allen, L. B.; Streeter, D. G.; Campbell, M. T.;
Witkowski, J. T.; Sidwell, R. W.; Robins, R. K. Synthesis and Antiviral Activity of
Certain Thiazole C-nucleosides. J. Med. Chem. 1977, 20, 256-262. (b) Sebastián
Barradas, J.; Errea, M. I.; D'Accorso, N. B.; Sepúlveda, C. S.; Talarico, L. B.; Damonte,
E. B. Synthesis and Antiviral Activity of Azoles Obtained from Carbohydrates.
Carbohydr. Res. 2008, 22, 2468-2474
4) (a) de Souza, M. Synthesis and Biological Activity of Natural Thizaoles: An Important
Class of Heterocyclic Compounds. J. Sulfur Chem. 2005, 26, 429-449. (b) Gerth, K.;
Irschik, H.; Reichenbach, H.; Trowitzsch, W. Myoxthiazol, an Antibiotic from
Myxococcus fulvus (Myxobacterales). J. Antibiot. 1980, 33, 1474-1479. (c) Hughes, R.
A.; Moody, C. J. From Amino Acids to Heteroaromatics—Thiopeptide Antibiotics,
Nature’s Heterocyclic Peptides. Angew. Chem. Int. Ed. 2007, 46, 7930-7930. (d) Bagley,
M. C.; Dale, J. W.; Merrittm E. A.; Xiong, X. Thiopeptide Antibiotics. Chem. Rev. 2005,
105, 685-714.
5) (a) Wipf, P. Synthetic Studies of Biologically Active Marine Cyclopeptides. Chem. Rev.
1995, 95, 2115-2134. (b) Tanaka, J.; Yan, Y.; Choi, J.; Bai, J.; Klenchin, V. A.; Rayment,
72

I.; Marriott, G. Biomolecular Mimicry in the Actin Cytoskeleton: Mechanisms
Underlying the Cytotoxicity of Kabiramide C and Related Macrolides. Proc. Natl. Acad.
Sci. U. S. A. 2003, 100, 13851-13856. (c) Popsavin, M; Torovic, L.; Svircev, M.; Kojic,
V.; Bogdanovic, G.; Popsavin, V. Synthesis and Antiproliferative Activity of Two New
Tiazofurin Analogues with 2'-amido Functionalities. Bioorg. Med. Chem. Lett. 2006, 16,
2773-2776.
6) Minster, D. K.; Jordis, U.; Evans, D. E.; Hecht, S. M. Thiazoles from Cysteinyl Peptides.
J. Org. Chem. 1977, 43, 1624-1626.
7) McGowan, D. A.; Jordis, U.; Minster, D. K.; Hecht, S. A. A Biomimetic Synthesis of the
Bithiazole Moiety of Bleomycin. J. Am. Chem. Soc. 1977, 99, 8078-8079.
8) Barrish, J. C.; Singh, J.; Spergel, S. H.; Han, W-C.; 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.
9) 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.
10) 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, 4, 497-501.
11) North, M.; Pattenden, G. Synthetic Studies Towards Cyclic Peptides. Concise Synthesis
of Thiazoline and Thiazole Containing Amino Acids. Tetrahedron 1990, 46, 8267-8290.
12) Yu, Y-B.; Chen, H-L.; Wang, L-Y.; Chen, X-Z.; Fu, B.; A Facile Synthesis of 2,4-
Disubstituted Thiazoles Using MnO
2
. Molecules 2009, 14, 4858-4865.
13) Kharasch, M. S.; Sosnovsky, G.; Yang, N. C. Reactions of t-Butyl Peresters. I. The
Reaction of Peresters with Olefins J. Am. Chem. Soc. 1959, 81, 5819-5824.
14) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Synthesis of
Functionalized Oxazolines and Oxazoles with DAST and Deoxo-Fluor. Org. Lett. 2000,
8, 1165-1168.
15) 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.
73

Chapter 6. Copper-catalyzed oxidation of azolines to azoles.

6.1. Introduction.
Environmentally benign conditions for the oxidation of azolines to azoles were developed
to address the toxicity issues associated with azoline oxidation. These aerobic oxidation reaction
conditions feature either catalytic amounts of copper catalyst 6.1
1
and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) , or stoichiometric amounts of base at reduced
temperatures without the need for metal catalyst (Scheme 6.1). Additionally, these conditions
are low cost (Table 6.1), as cost analysis revealed commercially available 6.2a ($22,500 g
-1
)
2
can
be prepared for <$28 g
-1
. This work was done in collaboration with Anna C. Dawsey.
3

Scheme 6.1. Aerobic oxidation of thiazolines to thiazoles.
Reagent List Price List Quantity Unit Price Amt. Needed Cost
Butanedione $90.60 500 g $0.18 g
-1
42.7 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
69.4 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

Table 6.1. Cost analysis to synthesis 1 gram of 6.2a using catalyic conditions.
74

6.2. Optimization of reaction conditions.

Table 6.2 shows the optimization of reaction conditions for the conversion of 6.2 to 6.2a.
The initial reaction conditions (entry 1) produced desired product in 88% isolated yield. Despite
O
2
being necessary for the reaction to proceed (entry 4), the yield was decreased when an O
2

balloon was used (entries 2 and 3). One possible explanation is that large concentration of O
2

can lead to the facile formation of undesirable insoluble byproducts, which accounts for the
observed loss in yield. DMF was demonstrated to be the optimal solvent, as switching to
dichloromethane (DCM), acetonitrile or toluene all resulted in drastically lower yields (entries 6-
8). Switching the base from DBU to proton sponge, diisopropylethylamine (DIEA), potassium
tert-butoxide or potassium carbonate all produced significantly reduced yields (entries 9-12).
Removal of base from the reaction conditions resulted in almost no product formed (entry 13).
Interestingly, a 36% yield was obtained when the reaction was run in the absence of catalyst 6.1.
Further investigation revealed 1.1 equivalents of DBU as a base in the absence of copper at a
reduced reaction temperature can provide a 66% yield of 6.2a at a much reduced reaction time of
30 minutes. Because of the efficiency observed when the reaction was run in the absence of
copper, substrate scope studies were carried out using both copper catalyzed and stoichiometric
base promoted conditions.

75

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

1 1 Air DBU DMF 100 8h 88
2 1 O
2
DBU DMF 100 8h 41
3 1 O
2
DBU DMF 55 7d 47
4 1 N
2
DBU DMF 100 8h 3
5 - Air DBU DMF 100 8h 36
6 1 Air DBU DCM Reflux 8h 41
7 1 Air DBU CH
3
CN Reflux 8h 4
8 1 Air DBU PhCH
3
100 8h 14
9 1 Air Proton Sponge DMF 100 8h 41
10 1 Air DIEA DMF 100 8h 45
11 1 Air KOtBu DMF 100 8h 22
12 1 Air K
2
CO
3
DMF 100 8h 47
13 1 Air - DMF 100 8h 4
14 - Air 1.1 equiv. DBU DMF 70 30 min 66
a
Reaction conditions: 10 mol% 6.1, 10 mol% base unless otherwise noted.
b
Isolated yields.
Table 6.2. Optimization of reaction conditions for the oxidation of 6.2 to 6.2a.

6.3. Oxidation of azolines to azoles
Thiazoline substrates were subjected to both the catalytic and base promoted oxidation
conditions (Table 6.3). Electron withdrawing (entries 2, 5, and 6) and electron donating (entry 3)
substrates both tolerated the reaction conditions well. The reaction conditions also demonstrated
compatibility with sensitive functional groups such as fluoro (entry 5) and nitrile (entry 6). The
catalytic conditions typically provided higher yields, albeit with slower reaction rates.

76

Entry Substrate Conditions
a
Product Yield (%)
1a

1b

1c

Catalyzed
8h
Base
30 min
K
2
CO
3

2h

88

66

37
2a

2b

Catalyzed
3h
Base
b

1h

78

69
3a

3b

Catalyzed
8h
Base
4h

68

58
4a

4b

Catalyzed
8h
Base
1h

79

77
5a

5b

Catalyzed
3h
Base
45 min

58

44
6a

6b

6c

Catalyzed
4h
Base
45 min
K
2
CO
3

2h

69

44

9
a
Catalyzed: 10 mol% 6.1, 10 mol% DBU, DMF, 100 °C; Base: 1.1 equiv. DBU, DMF, 70 °C.
b
10 mol% DBU.

Table 6.3. Oxidation of thiazolines to thiazoles.

77

Oxazolines (Table 6.4, entries 1 and 2) and alkyl substrates (entries 3 and 4) both proved
more difficult to oxidize than the aryl thiazoline series. Interestingly, when the base promoted
oxidation conditions were tested against indole substituted thiazoline 6.12 (entry 5), no product
was observed, whereas the catalytic conditions provided a 55% yield. One possible explanation
is the incompatibility of the base promoted conditions towards substrates containing labile
protons. To test this hypothesis, the reaction conditions were tested against N-methyl substituted
indole thiazoline 6.13 (entry 6), which reaction was complete in just 30 minutes under base
promoted conditions. This demonstrates the advantage of the copper catalyzed conditions over
the base promoted conditions for substrates containing labile protons.

78

Entry Substrate Conditions
a
Product Yield (%)
1a

1b

Catalyzed
b

9h
Base
6h

18

16
2a

2b

Catalyzed
b

12h
Base
6h

37

41
3a

3b

Catalyzed
8h
Base
5h

24

39
4a

4b

Catalyzed
12h
Base
6h

45

51
5a

5b

5c

Catalyzed
6h
Base
1h
K
2
CO
3

6h

55

0

36
6a

6b

Catalyzed
14h
Base
30 min

65

46
a
Catalyzed: 10 mol% 1, 10 mol% DBU, DMF, 100 °C; Base: 1.1 equiv. DBU, DMF, 70 °C.
b
30 mol% DBU.
Table 6.4. Oxidation of azolines to azoles (continued).

79

6.4. Mechanistic observations of the reaction mechanism.

A proposed mechanism is shown in Scheme 6.2. Consistent with the observation of
slower reaction rates when electron donating groups are present, it's plausible that a key step in
the mechanism is the base-promoted enolization of 6.2 to 6.14. This intermediate is then
converted to intermediate 6.16 when exposed to air. Intermediate 6.16 can be isolated and
characterized by
1
H NMR. The hydroxyl oxygen in intermediate 6.16 is believed to come from
O
2
as opposed to residual H
2
O in the solvent, as reaction run in the presence of H
2
18
O resulted in
no additional incorporation of
18
O in intermediate 6.16. Dehydration of 6.16 yields desired
product 6.2a.

Scheme 6.2. Proposed mechanism for the oxidation of thiazolines to thiazoles.

80

Interestingly, the reaction was not inhibited when run in the presence of radical inhibitors
such as butylated hydroxytoluene (BHT), tocopherol or vitamin E. Thus, a long lived radical is
not believed to be present in the reaction mechanism.
In the aerobic oxidiation of thiazolines to thiazoles using K
2
CO
3
reported by Yao et al.,
4

hydroperoxide intermediate 6.15 was reportedly observed via TLC, whereas this intermediate
was not observed when DBU was used as a base. This hydroperoxide intermediate is then
collapsed to provide hydroxide intermediate 6.16, which was observed by NMR. In order to
provide a better understanding of these reaction intermediates, the copper catalyzed oxidation
conditions were repeated using K
2
CO
3
as the base instead of DBU and the contents were then
analyzed by
1
H NMR and MALDI.

Figure 6.1. Crude reaction mixture of thiazoline 6.2 with K
2
CO
3
and catalyst 6.1. (A) Hydroxythiazoline 6.14; (B)
hydroperoxythiazoline 6.15; (C) product 6.2a.

Thiazole product 6.2a, hydroxide intermediate 6.16 and peroxide 6.15 were all observed
in the
1
H NMR spectrum of the crude reaction mixture (Figure 6.1). The green arrow
81

corresponds to thiazole product 6.2a, the red arrows correspond to hydroxide intermediate 6.16
and the blue arrows correspond to hydroperoxide intermediate 6.15. This demonstrates the
existence of a long-lived hydroperoxide intermediate if K
2
CO
3
is used as a base, whereas if this
intermediate is formed in the presence of DBU it is quickly decomposed to hydroxide
intermediate 6.16.
As organic peroxides are hazardous and can potentially decompose starting material or
product, rapid decomposition of any peroxide intermediates formed is essential for wide
applicability of reaction conditions. Attempted replication of oxidation of 6.2 to 6.2a using the
reaction conditions reported by Yao et al. produced only a 37% yield of thiazole 6.2a (Table 6.3,
entry 1c). Furthermore, sensitive substrates 6.7 (Table 6.3, entry 6c) and 6.12 (Table 6.4, entry
5c) also displayed significant decreases in yield when exposed to the K
2
CO
3
conditions
compared with the catalytic conditions. This demonstrates the limitations of the reaction scope if
long lived peroxides are present.

6.5. Scalability.
Demonstration of reaction scalability is essential if the reaction were to be used on
industrial scale. Oxidation of 6.2 to on a 1 gram scale produced 80% of thiazole 6.2a under
catalytic conditions, which is comparable to the 88% obtained on a small scale. The base
promoted conditions scaled up less efficiently, only providing a 55% yield (Table 6.5).
Entry Scale(mg) Conditions
a
Time Isolated Yield(%)
1 20 Catalyzed 8 hr 88
2 1000 Catalyzed 18 hr 80
3 20 Base 1 hr 66
4 1000 Base 2.5 hr 55
a
Catalyzed: 10 mol% Cu
II
, 10 mol% DBU, DMF, 100 °C; Base: 1.1 equiv. DBU, DMF, 70 °C.
Table 6.5. Scalability of the conversion of 6.2 to 6.2a.
82

6.6. Conclusion.
Two sets of environmentally benign aerobic oxidation conditions for the oxidation of
azolines to azoles were developed. One set of conditions are catalytic in base and copper,
whereas the other conditions require stoichiometric amounts of base, but is metal-free. These
conditions are compatible with a wide range of substrates, although catalytic conditions were
demonstrated to be more effective with substrates bearing labile protons. Mechanistic studies
revealed a hydroxide intermediate, which was consistent with previously reported observations,
although no hydroperoxide intermediate was observed. These low cost reaction conditions will
be useful in the synthesis of azole-containing medicinal building blocks.

6.7. References.

1) For the synthesis and characterization of copper catalyst 6.1, see: (a) Dawsey, A. C.; Li,
V.; Hamilton, K. C.; Wang, J.; Williams, T. J. Copper-Catalyzed Oxidation of Azolines
to Azoles. Dalton Trans. 2012, 41, 7994-8002. (b) Dawsey, A. C. Mechanism and
Synthesis of Molecular Building Blocks in medicinal Chemistry: Aerobic Azoline
Oxidation and ultrasound Activated MRI Contrast Agents. Ph. D. Thesis, University of
Southern California, Los Angeles, California, 2013.
2) Price quote from Aurora Fine Chemicals, San Diego, CA. January 2011.
3 ) Travis J. Williams research group. University of Southern California, Department of
Chemistry. Los Angeles, CA 90089.
4) 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.
83

Chapter 7. Experimental and spectral data.

7.1. General procedures.

Chemicals.

Deuterated NMR solvents were purchased from Cambridge Isotopes Labs and used as
received. Distilled water was purchased from Arrowhead water. Dichloromethane, chloroform
and methanol were purchased from Macron Chemicals. (12-mercaptododecyl)phosphonic acid
was purchased from SiKÉMIA. Chloroauric acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) were purchased from STREM Chemicals. TRIS buffer was purchased
from Angus Chemicals. Triethylsilane, gadolinium(III) chloride hexahydrate, yttrium chloride n-
hydrate, N-(3-bromopropyl)phthalamide, trifluoroacetic acid (TFA), 1M trimethylamine in THF
solution, 47% HBr in ethanol solution, choline chloride, diisopropylethylamine, indole-2-
carboxylic acid, hydrazine monohydrate, triphenylmethyl mercaptan, calcium carbonate, 2,5-
dihydroxybenzoic acid, L-cysteine, L-Cys(Trt)-OH , TiCl
4
and iodomethane, were purchased
from Alfa Aesar. Ethanol was purchased from Koptec. Dicyclohexylcarbodiimide (DCC) and
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from Lancaster. Acetonitrile,
magnesium sulfate, hexanes, dimethylformamide and tetrahydrofuran (THF) were purchased
from EMD. 2-aminoethyltrimethylammonium chloride hydrochloride, benzonitrile, 4-
fluorobenzoyl chloride, 2-naphthoyl chloride and 4-cyanobenzyol chloride were purchased from
Sigma Aldrich. 1,8-dibromooctane and potassium thioacetate were purchased from Acros. Size
#5 pill casings were purchased from thrivingpets.com Size #9 pill casings were purchased from
Torpac. Wheat Chex breakfast cereal was purchased from Ralph's grocery store. Diethylamine
and pyridine were purchased from J.T. Baker. N-methylmorpholine and xylenol orange were
purchased from TCI America. 1-hydroxybenzotriazole (HOBt) was purchased from Chem-
Impex International. Acetic acid, ammonium hydroxide, and pH=6.4 phosphate buffer was
purchased from EM Science. Potassium carbonate, sodium hydroxide, sodium chloride, sodium
bicarbonate and ethyl acetate were purchased from Mallinckcrodt. 0.45 μm polyethersulfone
syringe filters were purchased from VWR international. Fmoc-Cys(trt)-OH was purchased from
BA Chem. All chemicals were used as received.

Prepared reagents.

1-methyl-indole-2-carboxylic acid was prepared according to literature procedure.
1

Phosphine stabilized gold nanoparticles 2.10 and 3.11 were synthesized according to literature
procedure
2
and graciously provided by Anna C. Dawsey.
3
Cavitand 3.1 was synthesized
according to literature procedures
4
and graciously provided by Yoo-Jin Ghang.
5
Copper catalyst
6.1 was prepared according to reported conditions.
6
Methyl 2-(4-nitrophenyl)-4,5-
dihydrothiazole-4-carboxylate 6.3, methyl 2-(4-methoxyphenyl)-4,5-dihydrothiazole-4-
carboxylate 6.4 and 2-phenethyl-4,5-dihydrothiazole-4-carboxylate 6.11 were prepared
according to reported conditions.
16
Methyl 2-methyl-4,5-dihydrothiazole-4-carboxylate 6.10 was
prepared according to reported conditions.
7

84

Instrumentation.

Flash chromatography was performed on a Teledyne Combiflash Rf. Normal phase
(SiO
2
) and reverse-phase (C18) columns were purchased as pre-packed columns from Teledyne.
NMR spectra were recorded on a Varian Mercury 400, 400MR, VNMRS 500, or VNMRS 600
spectrometer at 25 °C. 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.
All
19
F T
1
data was recored on a Varian NMRS 500 spectrometer. All H
2
O
1
H T
1
data were
recorded on a Varian 400MR spectrometer. 5 mm NMR tubes and coaxial inserts were
purchased from Norell at nmrtubes.com (NI5CCI-V). 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. ICP-MS data were collected at the University
of Illinois at Urbana Champaign at the School of Chemical Sciences Microanalysis Laboratory.
DLS were acquired on a Wyatt Dynapro Titan instrument. Optical absorption spectra were
acquired on a Shimadzu UV-1800 spectrometer with the help of Jannise Buckley.
8
Infrared
spectroscopy data were acquired on a Bruker Vertex 80v. MRI images were acquired on a
Bruker Pharmascan 7 T MRI. Thermogravimetric analysis (TGA) was performed on TA
instruments TGA-Q50 with the help of Jannise Buckley.

General procedures for the measurement of
19
F T
1
relaxation rates.

To measure the
19
F T
1
, a solution was prepared and placed inside a 3 mm diameter
coaxial NMR tube insert, with D
2
O in the 5 mm outer tube. A
19
F NMR direct detect spectrum
was acquired at 376 MHz with a 3763 Hz window.
19
F T
1
s were then acquired using a
broadband inversion recovery pulse sequence over the same spectral window. In a representative
experiment, the interpulse delay was varied logarithmically among 12 points ranging from 2.5
ms to 5.1 s. Peak heights for the corresponding spectra were tabulated and fitted to a 3-parameter
exponential growth model by Varian Nuclear Magnetic Resonance Java (VnmrJ v. 3.2.) to give
experimental values and errors for T
1
for each line in the spectrum.

General procedures for the measurement of
1
H T
1
relaxation rates.

1
H T
1
data were obtained on a Varian 400MR spectrometer using a standard T
1
inversion
recovery pulse sequence with 4 scans and a 10 sec interpulse delay. Peak heights for the
corresponding spectra were tabulated and fitted to a 3-parameter exponential growth model, by
either VnmrJ v. 3.2. or Mestrenova v. 8.1. as indicated, to give experimental values and errors
for T
1
for each line in the spectrum.

85

General procedures for the determination of free gadolinium(III) in solution via the xylenol
orange test.
9

An acetate buffer was first prepared by dissolving acetic acid (1.4 mL) in distilled water
(400 mL). The pH was adjusted to 5.8 using 1M NH
4
OH, and distilled water was added to
produce a total volume of 500 mL. Gadolinium complex (0.3 mg) was dissolved in acetate
buffer (0.1 mL), to which xylenol orange (3 mL of a 16 μM solution in acetate buffer) was
added. The presence of free metal is indicated by a color change from orange to violet. No
detectable color change indicates no free Gd
3+
present in solution.

Lyopholizing samples.

Aqueous samples were first prepared in glass vials. The samples were then frozen using
liquid nitrogen, with the vials tilted to prevent cracking of the glass. The frozen samples were
then placed in a Millrock Technology BT85 lyopholizer for at least 8 hours, until dry.

Other.

Molecular modeling (semi-empirical calculations) was performed using the AM1 force
field using SPARTAN by Richard J. Hooley.
10
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.
86

7.2. Chapter 2 experimental and spectral data.

7.2.1. Synthetic procedures.

Synthesis of shell monomer 2.1.

Shell monomer 2.1 was synthesized according to literature procedure
11
and graciously
provided by Xinping Wu.
3

87

1
H NMR (500 MHz, CDCl
3
)

19
F NMR (470 MHz, CD
3
CN)

88

COSY (500 MHz, CDCl
3
)

1
H-
13
C HSQC (CDCl
3
)

19
F-
13
C HMBC

89

Synthesis of 3-(tritylthio)propan-1-amine.
12

Triphenylmethyl mercaptan (8.7g, 31.4 mmol) was dissolved in DMF (40 mL). Stirring
at room temperature, sodium hydride (1.5g of a 60% w/w dispersion in mineral oil, 38 mmol)
was slowly added. This solution was added dropwise to a solution of N-(3-
bromopropyl)phthalamide (10.0g, 35.4 mmol) in DMF (30 mL). The reaction was stirred at
room temperature for 15 hours. Diethyl ether (150 mL) was added to the reaction to initiate
precipitation of NaBr. The reaction was filtered, and the filtrate was concentrated under reduced
pressure. The crude product was used for the next step without further purification.
Crude N-(3-tritylthio)phthalamide prepared from previous step (2.4 g) was suspended in
ethanol (30 mL). Stirring at 40 °C, hydrazine monohydrate (1.8 mL, 39 mmol) was added. The
reaction was stirred for 2 hours until it turned into a thick paste. The reaction mixture was
diluted with ethanol (90 mL) and filtered. The filtrate was collected and concentrated under
reduced pressure. Chloroform (30 mL) was added to the reaction mixture and stirred for 30
minutes at room temperature. The reaction was filtered and the filtrate was concentrated to
provide 3-(tritylthio)propan-1-amine.
1
H NMR Data are consistent with previously characterized
compound.
12

1
H NMR (400 MHz, CDCl
3
) δ: 7.40-7.43 (m, 6H), 7.28-7.30 (m, 6H), 7.18-7.23 (m, 3H), 2.63 (t,
2H, J = 8 Hz), 2.20 (t, 2H, J = 8 Hz) 1.53 (p, 2H, J = 8 Hz) 1.45 (sb, 2H)

90

Synthesis of 1,4,7-tris(carboxymethyl)-10-[N-(3-trityl-thiopropyl)carbamoyl] 1,4,7,10-
tetraazacyclododecane.

3-(tritylthio)propan-1-amine (248 mg, 0.74 mmol) and DOTA (300 mg, 0.74 mmol) were
dissolved in aqueous acetonitrile (50%, 20 mL). A solution of DCC (152 mg, 0.74 mmol) in
pyridine (4.5 ml) was added dropwise to the reaction. The reaction was stirred for 2 days at
room temperature. The reaction was concentrated under reduced pressure and purified via
reversed phase column chromatography (C18 silica, 0-50% CH
3
CN:H
2
O) to provide 1,4,7-
tris(carboxymethyl)-10-[N-(3-trityl-thiopropyl)carbamoyl] 1,4,7,10-tetraazacyclododecane as a
white solid (100 mg, 0.18 mmol, 24%). Mass data is consistent with a known sample.
12

MS (MALDI) m/z calc'd for C
38
H
49
N
5
O
7
S:719.34 g/mol, found 719.96 g/mol.

91

Synthesis of gadolinium trityl-protected thiol 2.11.

1,4,7-tris(carboxymethyl)-10-[N-(3-trityl-thiopropyl)carbamoyl]1,4,7,10-
tetraazacyclododecane (50 mg, 0.07 mmol) and GdCl
3
·6H
2
O (27 mg, 0.07 mmol) were dissolved
in distilled water (10.0 mL). Stirring at reflux, CaCO
3
(11 mg, 0.11 mmol) was added and
allowed to stir for 2 hours. The sample was lyopholized and purified via reversed-phase
chromatography (C18 silica, 10-70% MeOH:H
2
O) to provide 2.11 as a white solid (31.4 mg,
52%) No free Gd
3+
was detected in solution by xylenol orange test. Mass data for 2.11 is
consistent with a known sample.
12

MS MALDI m/z calc'd for C
38
H
46
GdN
5
O
7
S: 874.24 g/mol, found 874.85 g/mol. Measured
isotopic distribution matches calculated prediction.

92

Synthesis of gadolinium thiol 2.6.

N N
N N
C
O
N
H
Gd
O
O O
O
O
O
SH
2.6
N N
N N
C
O
N
H
Gd
O
O O
O
O
O
STrt
2.11
TFA, Et
3
SiH
DCM

Gadolinium thiol 2.11 (100 mg, 0.11 mmol) was dissolved in DCM (3 mL). Stirring at
room temperature, TFA (0.214 mL, 2.28 mmol) and Et
3
SiH (0.035 mL, 0.23 mmol) were added.
The reaction was stirred for 1 hour at room temperature. The reaction was concentrated under
reduced pressure and redissolved in ethyl acetate (5 mL). The organic layer was extracted with
distilled water (3 mL 3 times) and discarded. The aqueous layer was lyopholized and the
product was purified via reversed phase column chromatography (C18 silica, 0-15%
MeOH:H
2
O) to provide 2.6 as a white solid (61 mg, 84%). Mass data for 2.6 is consistent with a
known sample.
12

MALDI for C
19
H
32
GdN
5
O
7
S: Calculated 632.13 g/mol; found 631.94 g/mol. Measured isotopic
distribution matches calculated prediction.

93

Synthesis of Particle 2.8.

Phosphine stabilized gold nanoparticles 2.10 (1.0 mg) were dissolved in dichloromethane
(1 mL), and TRIS-HCl buffer (1 mL of 25 mM pH = 7.6) was added to form a biphasic mixture.
While stirring at room temperature, gadolinium thiol 2.6 (40 L of a 10 mM aqueous solution)
was added, and the solution was stirred for 4 hours. (12-mercaptododecyl)phosphonic acid was
then added and the reaction was stirred until the brown color partitioned completely into the
aqueous layer. The aqueous layer was diluted with TRIS-HCl buffer (1.0 mL of a 25 mM
solution at pH = 7.6) and extracted with dichloromethane (5 mL, 5 times), then filtered through a
0.45 m polyethersulfone syringe filter to yield an aqueous solution of particle 2.8.

ICP-MS data for aqueous solution of particle 2.8: [Gd] = 52.3 ppm

94

UV-Vis Spectrum:

DLS Histogram:

95

Synthesis of particle 2.9.

Trityl-protected gadolinium complex 2.11 (33.4 mg, 38 mol) was suspended in
tetrahydrofuran (0.4 mL). Chloroauric acid (HAuCl
4
, 13.0 mg, 38 mol) was added to the
solution at room temperature and the reaction was stirred for 1 hour. Triethylsilane (Et
3
SiH, 4.2
mg, 38 mol) was then added, and the solution stirred for 18 hours at room temperature. The
solution was filtered and ethanol (100%, 10 mL) was added to initiate precipitatation. The
precipitate was washed with ethanol (5 times, 10 mL), and the filter paper was sonicated in H
2
O
(10 mL) to dissolve precipitate. The resulting aqueous solution was then filtered through a 0.45
mm polyethersulfone syringe filter to yield particles 9 as an aqueous solution. DLS
measurements on the resulting solution showed a monodisperse nanoparticulate material with
hydrodynamic diameter of 7 nm.

ICP-MS data for aqueous solution of particle 2.9: [Gd] = 37.4 ppm

96

UV-Vis Spectrum:

DLS Histogram:

0
0.05
0.1
0.15
0.2
0.25
400 450 500 550 600 650 700
Abs
λ (nm)
97

7.2.2. Measurement of
19
F T
1
of 2.1 coated particles.

A portion of 2.1 (3 L of a 100 mM solution) was lyophilized to an oil in a 1/2 dram
glass vial. A 125 L aliquot of aqueous particle solution was delivered to the vial containing
lypholized 2.1, stirred, then placed in a 3 mm diameter coaxial NMR tube insert and
19
F T
1
time
constants were acquired using the general procedure on p. 84.

98

7.2.3. Representative
19
F T
1
inversion recovery spectra.

Figure 7.1. Representative
19
F T
1
inversion recovery stacked spectra.

Figure 7.2. Representative T
1
curve.

99

7.2.4 Graphical DLS Spectra.

Figure 7.3. DLS of particle 2.2.

100

7.3. Chapter 3 experimental and spectral data.

7.3.1. Synthetic detail.

Synthesis of N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-
yl)acetamido)ethanaminium chloride ligand 3.4.

Aqueous acetonitrile (50%, 10 mL) was added to 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid. (DOTA, 100 mg, 0.25 mmol), and 2-aminoethyltrimethylammonium
chloride hydrochloride (43.5 mg, 0.25 mmol) and stirred at room temperature until fully
dissolved (15 minutes). DCC (dicyclohexylcarbodiimide, 51 mg, 0.31 mmol) was dissolved in
pyridine (2 mL) and added dropwise to the reaction mixture. The reaction was stirred for 2 days
in a sealed flask at room temperature. The resulting precipitate was filtered, and the filtrate was
purified via reversed phase column chromatography (C18

Silica, H
2
O eluent) to provide 3.4 as a
white powder (93 mg, 71%).

1
H NMR (500 MHz, D
2
O) δ: 3.90-3.08 (m, broad 26H), 3.20 (s, 9H).

13
C NMR (125 MHz, D
2
O) δ: 174.6, 172.3, 170.3, 64.1, 56.2, 55.5, 53.6, 51.6, 50.9, 49.0, 48.5,
33.7.

FT-IR (cm
−1
): ν = 3384 (b), 3095, 2917, 2849, 1653, 1559, 1457.

MS (MALDI) m/z calcd for C
21
H
41
N
6
O
7
+
: 489.30 g/mol, found 489.09 g/mol.

101

Chromatography Trace

1
H NMR (500 MHz, D
2
O)

102

13
C NMR

103

Synthesis of gadolinium(III) N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)acetamido)ethanaminium chloride guest 3.2a.

To a solution of 3.4 (6.8 mg, 0.01 mmol) in H
2
O (1 mL) was added GdCl
3
·6H
2
O (4.8 mg,
0.01 mmol). The resulting solution was then stirred at 50 ºC for 20 hours. The pH of the solution
was neutralized to pH ~ 7 by the addition of NaOH (0.1 M aq) every hour over the first ten
hours. The reaction mixture was then lyopholized to powder and purified via reversed phase
column chromatography (C18 silica, H
2
O eluent) to provide 3.2a as a white powder (4.6 mg,
55%). A single chromatography trace was observed. The product was dissolved in distilled
water and the final Gd
3+
concentration was determined by ICP-MS to be 0.64 mM. No free Gd
3+

was detected in solution by xylenol orange test. NMR for this compound cannot be recorded
because this compound is paramagnetic.

MS (MALDI) m/z calcd for C
21
H
38
GdN
6
O
7
: 644.20 g/mol; found 643.94 g/mol. Measured
isotopic distribution matches calculated prediction.

FT-IR (cm
−1
): ν = 3384 (b), 2917, 2850, 1617, 1541, 1396.

104

Chromatography trace

105

Synthesis of yttrium(III) N,N,N-trimethyl-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)acetamido)ethanaminium chloride guest 3.2b.

Ligand 3.4 (148 mg, 0.282 mmol) and YCl
3
·xH
2
O (64 mg) were dissolved in H
2
O (5
mL). The reaction was stirred at room temperature for 12 hours. The solution was neutralized
over the course of the reaction to pH ~ 7 by the addition of NaOH (0.1 M aq). The product was
purified via reversed phase column chromatography (C18 silica, 0-5% MeOH:H
2
O) to yield
product as a white solid (63 mg, 36%).

1
H NMR (500 MHz, D
2
O, 25 ºC): δ 3.96-3.25 (m, broad, 16H), 3.21 (s, 9H), 2.81, (s, broad, 7H),
2.61-2.50 (m, broad, 6H).

13
C NMR was not obtained as significant broadening of the peaks rendered the signal:noise ratio
too low for detection.

FT-IR (cm
−1
): ν = 3393, 2987, 2878, 1604, 1437, 1806.

MS (MALDI) m/z calc'd for C
21
H
38
N
6
O
7
Y: 575.19 g/mol; found 574.94 g/mol.
106

1
H NMR (500 MHz, D
2
O, 25 ºC).

107

Synthesis of Na[Gd(DOTA)] 3.5.

DOTA (24.3 mg, 0.06 mmol) and GdCl
3
·6H
2
O were dissolved in 3 mL distilled H
2
O.
The solution was neutralized over the course of the reaction to pH ~ 7 by the addition of NaOH
(0.1 M aq). The reaction was stirred until the pH was constant for 1 hour (4 hour total reaction
time). The solution was then adjusted to pH ~ 11 by the addition of NaOH (0.1 M aq) and the
reaction was stirred for 20 minutes more, then filtered through a 0.45 m syringe filter. The
product was lyopholized to powder, then purified via reversed phase column chromatography
(C18 silica, 0-5% MeOH:H
2
O) to yield Gd·DOTA as a white solid (10.2 mg, 31%). NMR for
this compound cannot be recorded because this compound is paramagnetic. Mass data for 2.6 is
consistent with a known sample.
13

MS (MALDI) m/z calc'd for C
16
H
25
GdN
4
NaO
8
+
: 582.1 g/mol, found 582.0 g/mol. Measured
isotopic distribution matches calculated prediction.

108

Synthesis of S-(10-bromodecyl) ethanethioate 3.8.

1,8-Dibromooctane (2g, 7.3 mmol) and potassium thioacetate (168 mg, 1.47 mmol) were
dissolved in acetonitrile (3.0 mL) and the reaction was refluxed for 15 hours. The crude product
was purified via flash chromatography (SiO
2
, 0-40% hexanes:ethyl acetate) to yield 3.8 as a
colorless oil (283 mg, 1.06 mmol, 72%).

GC-MS for C
10
H
19
BrOS: calculated [MH]
+
267.03 g mol
-1
, found 267.06 g mol
-1
.

1
H NMR (500 MHz, CDCl
3
): δ = 3.39 (t, 2H, J = 10 Hz), 2.85 (t, 2H, J = 10 Hz), 2.31 (s, 3H),
1.84 (p, 2H, J = 10 Hz), 1.56 (p, 2H, J = 10 Hz), 1.43-1.28 (m, 8H).

13
C NMR (125 MHz, CDCl
3
): δ = 196.1, 34.1, 32.1, 30.8, 29.6, 29.2, 29.0, 28.8, 28.7, 28.2.

FT-IR (cm
−1
): ν = 2930, 2855, 1693, 1463, 1353, 1134.

109

1
H NMR (500 MHz, CDCl
3
)

13
C NMR (125 MHz, CDCl
3
)

110

Synthesis of 8-(acetylthio)-N,N,N-trimethyloctan-1-aminium bromide 3.9.

To a glass vial containing S-(10-bromodecyl) ethanethioate 3.8 (190 mg, 0.70 mmol), 1M
trimethylamine in THF solution was added (7.0 mL). The reaction was stirred at room
temperature for 13 days. The white precipitate was filtered and washed with THF (20.0 mL) to
yield 3.9 as a white solid. (128 mg, 0.39 mmol, 56%).

MS (MALDI) m/z calcd for C
13
H
28
NOS
+
: 246.19 g/mol, found 246.60 g/mol.

1
H NMR: δ = 3.60 (m, 2H), 3.47 (s, 9H), 2.83 (t, 2H, J = 10 Hz), 2.31 (s, 3H), 1.75 (m, 2H), 1.54
(m, 2H), 1.31-1.36 (mb, 8H).

13
C NMR: δ = 196.2, 67.1, 53.5, 30.8, 29.5, 29.04, 29.01, 28.8, 28.5, 26.1, 23.2.

FT-IR (cm
−1
): ν = 3016, 2923, 2854, 1691, 1485, 1354.

111

1
H NMR (CDCl
3
, 500 MHz)

13
C NMR (125 MHz, CDCl
3
)

112

Synthesis of 8-Mercaptotrimethylammonium bromide 3.10.

To a glass vial containing 8-(acetylthio)-N,N,N-trimethyloctan-1-aminium bromide 3.9
(60 mg, 0.18 mmol), a solution of 47% HBr in EtOH solution was added (1.8 ml) and stirred for
3 days at room temperature. The crude reaction mixture was concentrated under reduced
pressure. Desired product was obtained upon trituration with hexanes (45 mg, 0.16 mmol, 87%).
Data are consistent with a previously characterized compound.
14

1
H NMR (500 MHz, CDCl
3
): δ = 3.59 (m, 2H) 3.45 (s, 9H), 2.51 (sb, 2H), 1.76 (sb, 2H), 1.59 (p,
2H, J = 8.0 Hz), 1.24-1.43 (mb, 9H).

113

Synthesis of trimethylammonium coated particles 3.12.

Phosphine stabilized gold nanoparticles 3.11 (1.0 mg) were dissolved in dichloromethane
(1 mL), and distilled water (1.0 mL) was added to form a biphasic mixture. While stirring at
room temperature, 8-mercaptotrimethylammonium bromide 3.10 was added and the reaction was
stirred for 1 hour. The aqueous layer was diluted with distilled water (1.0 mL) and extracted
with dichloromethane (5 mL, 5 times), then filtered through a 0.45 m syringe filter. The
particles were lyopholized to a solid, and then dissolved in water to make a 1.0 mg/1.0 mL
solution.

114

DLS histogram

Thermogravimetric analysis (TGA)

115

UV-Vis

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
250 350 450 550 650 750
Abs
λ (nm)
116

7.3.2. Masking and unmasking gadolinium agent 3.2a.

3.2a (125 µl of a 0.64 mM solution) was added to six 0.5 dram glass vials, which were
lyophilized to dryness. Cavitand 3.1 (5 mg, 3.6 µmol) was dissolved in 368 µL H
2
O. The
cavitand solution (12.5 µL per equivalent of Gd) was added to each vial to make vials containing
gadolinium agent 3.2a with 1.6, 3.1, 4.7, 6.3, 7.8, and 15.6 equivalents cavitand, respectively. An
appropriate volume of water was added to each vial to bring the total volume of water to 125 µL.
The solutions were then transferred to a 3 mm diameter coaxial NMR tube insert for T
1

measurements. T
1
data were processed using VnmrJ.

Equiv. Cavitand 3.1 T
1
(ms)
0 228(3)
1.6 301(1)
3.1 304(4)
4.7 317(1)
6.3 344(1)
7.8 349(1)
15.6 349(1)
Table 7.1. Titration of 3.1 into a 0.64 mM solution of 3.2a

Figure 7.4. Modulation of the T
1
relaxation rate of 3.2a by cavitand 3.1. a) T
1
(H
2
O) variation upon increasing [3.1]
([3.2a] = 0.64 mM).

200
220
240
260
280
300
320
340
360
0 2 4 6 8 10 12 14 16
T
1
(ms)
Equiv. Cavitand 3.1
117

Effect of choline on masked contrast agent 3.2a.

To 10 glass vials was distributed choline chloride (10 mM aq). These were lyopholized to
dryness. A solution of agent 3.2a with 6 equivalents of cavitand 3.1 (125 µL, 0.32 mM [Gd],
1.92 mM [3.1]) was added to the vials, which were then transferred to a 3 mm diameter coaxial
NMR tube insert for
1
H T
1
measurements. T
1
data were processed using VnmrJ.
Equiv. Choline T
1
(ms)
0 558(10)
1 553(10)
2 548(3)
3 527(3)
4 523(4)
10 503(3)
20 486(12)
30 479(6)
40 460(4)
100 397(4)
200 386(1)
Table 7.2. Titration of choline into a 0.32 mM solution of 3.2a with 6 equiv. cavitand 3.1.

Figure 7.5. T
1
(H
2
O) variation upon addition of choline 3.6 to a solution of 3.1:3.2a (H
2
O, [3.2a] = 0.32 mM, [3.1] =
1.8 mM).

300
350
400
450
500
550
600
0 50 100 150 200
T
1
(ms)
Equiv. Choline
118

Effect of particles 3.11 on masked contrast agent 3.2a.

250 µL of a 1.0 mg/1.0 mL solution of particle 3.11 was lyopholized to dryness. A
solution of agent 3.2a (0.21 mM) along with 6 equiv. of cavitand 3.1 (160 µL, 0.21 mM [Gd])
was added to the vials, which were then transferred to a 3 mm diameter coaxial NMR tube insert
for T
1
measurements. T
1
data were acquired using the procedures above, and processed using
VnmrJ.

Contrast agent T
1

3.1:3.2a (0.21 mM) 707.5 ms
3.1:3.2a + choline 3.6 (100 equiv.) 444.4 ms
3.1:3.2a + 3.12 (ca. 2 equiv. NMe
3
+
) 486.6 ms
Table 7.3. Recovery of relaxivity using particle 3.12 as a triggering agent.

119

7.3.3. Molar relaxivity curves.

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
0.64 0.228(3) 4.388(57)
0.32 0.352(8) 2.840(61)
0.21 0.473(10) 2.115(45)
0.16 0.500(20) 1.999(80)

Figure 7.6. Relaxivity of Gd agent 3.2a.

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
0.64 0.355(1) 2.817(7)
0.32 0.596(2) 1.678(5)
0.21 0.702(5) 1.425(10)
0.16 0.758(6) 1.319(11)

Figure 7.7. Relaxivity of Gd agent 3.2a with 6 equiv. 3.1.

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
0.64 0.272(2) 3.682(25)
0.32 0.458(6) 2.183(30)
0.21 0.510(7) 1.962(26)
0.16 0.610(12) 1.640(32)

Figure 7. 8. Relaxivity of Gd agent 3.2a with 6 equiv. 3.1 and
excess choline.

y = 5.102x + 1.1348
R² = 0.9947
0
1
2
3
4
5
0 0.2 0.4 0.6 0.8
1/T
1
( s
-1
)
[Gd] (mM)
y = 3.1867x + 0.7473
R² = 0.9911
0
0.5
1
1.5
2
2.5
3
0 0.2 0.4 0.6 0.8
1/T
1
( s
-1
)
[Gd] (mM)
y = 4.1754x + 0.9771
R² = 0.9883
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8
1/T
1
( s
-1
)
[Gd] (mM)
120

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
1 0.277(6) 3.617(74)
0.5 0.439(16) 2.279(84)
0.33 0.522(23) 1.917(83)
0.25 0.730(28) 1.370(52)

Figure 7.9. Relaxivity of Gd·DOTA (3.5).

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
1 0.286(4) 3.494(51)
0.5 0.446(12) 2.242(60)
0.33 0.509(22) 1.963(84)
0.25 0.588(43) 1.701(125)

Figure 7.10. Relaxivity of 3.5 with 6 equiv. 3.1.

[Gd] (mM) T
1
(s) 1/T
1
(s
-1
)
1 0.298(3) 3.357(29)
0.5 0.476(7) 2.103(31)
0.33 0.621(14) 1.611(36)
0.25 0.657(26) 1.522(60)

Figure 7.11. Relaxivity of Gd·DOTA (3.5) with 6 equiv. 3.1 and
excess choline.

y = 2.8205x + 0.8269
R² = 0.9811
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
1/T
1
(s
-1
)
[Gd] (mM)
y = 2.358x + 1.1224
R² = 0.9963
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
1/T
1
(s
-1
)
[Gd] (mM)
y = 2.5118x + 0.84
R² = 0.9966
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
1/T
1
(s
-1
)
[Gd] (mM)
121

7.3.4.
1
H NMR spectra of Y-DOTA titration experiments.

Figure 7.12.
1
H NMR spectra of the titration of Y·DOTA complex 3.2b into a solution of cavitand 3.1 ([3.1] = 2
mM, D
2
O, 400 MHz, 298 K).

Figure 7.13.
1
H NMR spectra of the titration of cavitand 3.1 into a solution of Y·DOTA complex 3.2b ([3.2b] = 2
mM, D
2
O, 400 MHz, 298 K).

122

Figure 7.14.
1
H NMR spectra of the titration of acetonitrile-d
3
into a 6:1 mixture of cavitand 3.1 and Y·DOTA
complex 3.2b ([3.2b] = 2 mM, D
2
O, 400 MHz, 298 K).

123

7.3.5. Graphical DLS data.

Figure 7.15. DLS histogram of cavitand 3.1.

Figure 7.16. DLS histogram of cavitand 3.1 with Gd agent 3.2a.

124

Figure 7.17. DLS histogram of cavitand 3.1 with Gd agent 3.2a upon exposure to choline.

125

7.4. Chapter 4 experimental and spectral data.

7.4.1. Release of Gd·DOTA from a gelatin pill casing.

A 10:1 w/w mixture of Wheat chex/Gd·DOTA was loaded inside two size #5 gelatin pill
casings. The loaded pills were simultaneously placed in 2 vials of water - one containing 5.0 mL
distilled water and one containing 5.0 mL 0.1 M HCl. 200 µl aliquots of the solution were
removed at various time points, and the release of Gd·DOTA was determined by measuring the
H
2
O
1
H T
1
of the aliquots. T
1
data were processed using Mestrenova.

126

7.4.2. Experimental T
1
data.

Release of Gd·DOTA from a gelatin pill casing at pH = 7.

Time(min) T
1
(s) T
1
Error (s)
0 2.309469 0.066671
5 2.270663 0.079917
10 2.005214 0.021311
15 1.204274 0.004971
20 0.88574 0.00739
30 0.424809 0.001387
40 0.26137 0.00043
50 0.186776 0.000178
60 0.12639 0.000369
Table 7.4. Release of Gd·DOTA from a gelatin pill casing at pH = 7.

Figure 7.18. Release of Gd·DOTA from a gelatin pill casing at pH = 7.

0
0.5
1
1.5
2
2.5
0 10 20 30 40 50 60 70
T
1
(s)
Time (Minutes)
127

Release of Gd·DOTA from a gelatin pill casing at pH = 1.

Time(min) T
1
T
1
Error
0 2.70106 0.012695
5 1.490313 0.071999
10 0.727802 0.002219
15 0.312793 0.000264
20 0.175009 0.000212
30 0.083794 0.000256
40 0.048736 1.73E-05
50 0.029861 0.00234
Table 7.5. Release of Gd·DOTA from a gelatin pill casing at pH = 1.

Figure 7.19. Release of Gd·DOTA from a gelatin pill casing at pH = 1.

0
0.5
1
1.5
2
2.5
3
0 10 20 30 40 50 60
T
1
(s)
Time (Minutes)
128

7.4.3. Release of Gd·DOTA in vivo.

A Sprague-Dawley rat was anesthetized with isoflurane and a background scan was
acquired with the rat lying in a prone position. FLASH sequence, TR= 912 ms; TE = 10 ms;
slice thickness = 2 mm. A size #9 pill loaded with a 10:1 w/w mixture of Gd·DOTA:Wheat
Chex was placed in the back of the rat's mouth and the rat was allowed to wake up. Once the pill
was swallowed, the rat was anesthetized again and images were acquired consecutively using the
same parameters.

129

7.5. Chapter 6 experimental and spectral data.

Synthetic procedures.

Fmoc-Cys(Trt)-OMe

Fmoc-Cys(trt)-OH (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 5 times
with 1:1 brine: H
2
O (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.
15

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

General Procedure for Preparation of Triphenylmethyl-Protected Cysteine Amides.
16

Fmoc-Cys(trt)-OMe was dissolved in acetonitrile (0.16 M solution), to which
diethylamine (65 equiv.) 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.) 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.

130

Methyl 2-(4-Fluorobenzamido)-3-(tritylthio)propanoate.

Prepared from Fmoc-Cys(trt)-OMe (4.6 g, 7.67 mmol) and 4-fluorobenzoyl chloride
(1.34 g, 8.43 mmol) according to the general procedure to give product as white crystals (1.66 g,
43%).

1
H NMR (400 MHz, CDCl
3
): δ = 7.77 (m, 2H), 7.38 (m, 6H), 7.23 (m, 9H), 7.13 (m, 2H), 6.59
(d, 1H, J = 7.6 Hz), 4.81 (m, 1H), 3.75 (s, 3H), 2.80 (dd, 1H, J
1
= 12.4 Hz, J
2
= 5.6 Hz), 2.74 (dd,
1H, 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
):  = 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

131

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

132

Methyl 2-(4-Cyanobenzamido)-3-(tritylthio)propanoate.

Prepared from Fmoc-Cys(trt)-OMe (5 g, 8.33 mmol) and 4-cyanobenzoyl chloride (1.52
g, 9.17 mmol) to give product as white crystals (2.58 g, 56%).

1
H NMR (400 MHz, CDCl
3
):  = 7.84 (m, 2H, J
1
= 8 Hz), 7.75 (m, 2H, J
1
= 8 Hz), 7.35-7.40 (m,
6H), 7.17-7.26 (m, 9H), 6.65 (d, 1H, J = 8 Hz), 4.79 (dt, 1H, J
1
= 8 Hz, J
2
= 5 Hz), 3.77 (s, 3H),
2.83 (dd, 1H, J
1
= 12 Hz, J
2
= 4 Hz), 2.75 (dd, 1H, J
1
= 12 Hz, J
2
= 4 Hz).

13
C NMR (100 MHz, CDCl
3
):  = 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
):  = 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

133

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

134

Methyl 2-(Indole-2-carboxamido)-3-(tritylthio)propanoate.

Fmoc-Cys(Trt)-OMe (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 (886 mg, 5.5 mmol) and N-methylmorpholine (1.65 mL,
15 mmol) were added. While stirring at 0 ºC, a solution of DCC (N,N’-
dicyclohexylcarbodiimide, 1.24 g, 6.0 mmol) and HOBt (1-hydroxybenzotriazole, 946 mg, 7
mmol) 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
(SiO
2
, 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
):   s, 1H), 7.68 (dd, 1H, J
1
= 8 Hz, J
2
= 0.8 Hz), 7.43 (dd,
1H, J
1
= 8 Hz, J
2
= 0.8 Hz), 7.39 (m, 6H), 7.30 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 0.8 Hz), 7.24-
7.15 (m, 10H), 6.93 (dd, 1H, J
1
= 1.2 Hz, J
2
= 0.8 Hz), 6.78 (d, 1H, J
1
= 8 Hz), 4.84 (dt, 1H, J
1
=
8 Hz, J
2
= 4.8 Hz), 3.77 (s, 3H), 2.84 (dd, 1H, J
1
= 12 Hz, J
2
= 5.4 Hz), 2.74 (dd, 1H, J
1
= 12 Hz,
J
2
= 4.8 Hz).

13
C NMR (100 MHz, CDCl
3
):  = 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
):  = 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)

135

13
C NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

136

Methyl 2-(1-Methylindole-2-carboxamido)-3-(tritylthio)propanoate.

Fmoc-Cys(Trt)-OMe (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) 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 (SiO
2
, 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
):  = 7.67 (d, 1H, J = 8 Hz), 7.41-7.15 (m, 18H), 6.93 (s, 1H), 6.69
(d, 1H, J = 8 Hz), 4.79 (dt, 1H, J
1
= 8 Hz, J
2
= 4.8 Hz), 4.02 (s, 3H), 3.77 (s, 3H), 2.80 (dd, 1H,
J
1
= 12 Hz, J
2
= 6 Hz), 2.74 (dd, 1H, J
1
= 12 Hz, J
2
= 6 Hz).

13
C NMR (100 MHz, CDCl
3
): = 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
):  = 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

137

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

138

Methyl 2-(2-Naphthamido)-3-(tritylthio)propanoate.

L-Cys(Trt)-OH (727 mg, 2.0 mmol)

and 2-naphthoyl chloride (419 mg, 2.2 mmol) were
dissolved in acetonitrile (10.0 mL) and dichloromethane (5.0 mL). Diisopropylethylamine (732
L, 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 times (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, 1H), 7.93 (m, 3H), 7.82 (dd, 1H, J
1
= 6.8 Hz, J
2
= 2
Hz), 7.58 (m, 2H), 7.39 (d, 6 H, J = 7.2 Hz), 7.23 (d, 6H, J = 8.0 Hz), 7.20 (t, 3H, J = 8.0 Hz),
6.82 (d, 1H, J = 8.0 Hz), 4.90 (dt, 1H, J
1
= 8.0 Hz, J
2
= 4.8 Hz), 3.78 (s, 3H), 2.84 (dd, 1H, J
1
=
12 Hz, J
2
= 4 Hz), 2.78 (dd, 1H, 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
):  = 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

139

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

140

Methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate (6.2).

Benzonitrile (8.24 mL, 80 mmol) and L-cysteine (10.9 g, 90 mmol, 1.1 equiv.) were
dissolved in 1:1 methanol: pH 6.4 phosphate buffer (200 mL), 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 2-phenyl-4,5-dihydrothiazole-4-carboxylic acid product as white solid (4.97 g).
Crude product was used in the next step without further purification.
Crude 2-phenyl-4,5-dihydrothiazole-4-carboxylic acid (3.5 g, 16.9 mmol) was dissolved
in 28 mL DMF at 0 °C, to which potassium carbonate (2.57 g, 18.6 mol) was added. After
stirring for 30 min, iodomethane (2.21 mL, 35.5 mmol) was added and the solution was brought
to room temperature and stirred for 1.5 h 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 the
product as a white solid (2.92 g, 13.2 mmol, 23%, 2 steps).

1
H NMR (400 MHz, CDCl
3
): δ = 7.87 (m, 2H), 7.47 (m, 1H), 7.41 (m, 2H), 5.29 (t, 1H, J = 8.8
Hz), 3.84 (s, 3H), 3.73 (dd, 1H, J
1
= 11.2 Hz, J
2
= 8.8 Hz), 3.62 (dd, 1H, J
1
= 11.2 Hz, J
2
= 8.8
Hz).

Data are consistent with a previously characterized compound.
16

All other thiazolines were prepared via a route reported by Kelly et al.
16

141

General procedure for thiazoline preparation.
16

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.) was added and stirred at room
temperature overnight until completion. The reaction mixture was then washed with saturated
aqueous NaHCO
3
twice and dried over MgSO
4
. The product was purified via flash
chromatography on silica, eluting with ethyl acetate and hexanes.

142

Methyl 2-(naphthalen-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.5).

6.5 was prepared from N-(2-naphthoyl)-Cys(Trt)-OMe (177 mg, 0.33 mmol) according to
the general procedure for thiazoline preparation to give the product as an oil (23 mg, 26%).

1
H NMR (400 MHz, CDCl
3
): δ = 8.31 (s, 1H), 8.02 (dd, 1H, J
1
= 8.0 Hz, J
2
= 2.0 Hz), 7.91 (dd,
1H, J
1
= 8.0 Hz, J
2
= 1.6 Hz), 7.86 (d, 2H, J = 8.0 Hz), 7.54 (m, 2H), 5.35 (t, 1H, J = 8.0 Hz),
3.86 (s, 3H), 3.78 (dd, 1H, J
1
= 12 Hz, J
2
= 8.0 Hz), 3.69 (dd, 1H, 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
-1
, found 272.0740 g mol
-1
.

143

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

144

Methyl 2-(4-fluorophenyl)thiazole-4-carboxylate (6.6).

6.6 was prepared from N-(4-fluorobenzoyl)-Cys(trt)-OMe (750 mg, 1.5 mmol) according
to the general procedure for thiazoline preparation to give the product as a white solid (220 mg,
61%).

1
H NMR (500 MHz, CDCl
3
): δ = 7.87 (ddd, 2H, J
1
= 8.5 Hz, J
2
= 5.5 Hz, J3 = 2.0 Hz), 7.1 (ddd,
2H, J
1
= 8.5 Hz, J
2
= 8 Hz, J
3
= 2 Hz), 5.28 (t, 1H, J = 8.5 Hz), 3.84 (s, 3H), 3.73 (dd, 1H, J
1
=
11 Hz, J
2
= 9 Hz), 3.65 (dd, 1H, 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.

M.P. 103–105 °C

ESI-HRMS for C
11
H
10
FNO
2
S: calculated [MH]
+
240.0416 g mol
-1
, found 240.0487 g mol
-1
.

145

1
H NMR (500 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

146

Methyl 2-(4-cyanophenyl)thiazole-4-carboxylate (6.7).

6.7 was prepared from N-(2-cyanophenyl)-Cys(trt)-OMe (2.03 g, 4 mmol) according to
the general procedure for thiazoline preparation to give the product as a white solid (151 mg,
15%).

M.P. 107–108 °C

1
H NMR (500 MHz, CDCl
3
): δ = 7.96 (dt, 2H, J
1
= 8.5 Hz, J
2
= 2.0 Hz), 7.71 (dt, 2H, J
1
= 9.0
Hz, J
2
= 2.0 Hz), 5.32 (t, 1H, J = 9 Hz), 3.85 (s, 3H), 3.79 (dd, 1H, J
1
= 11.5 Hz, J
2
= 9.0 Hz),
3.70 (dd, 1H, 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
-1
, found 247.0536 g mol
−1
.

147

1
H NMR (500 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

148

Methyl 2-(indol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.12).

6.12 was prepared from methyl 2-(indole-2-carboxamido)-3-(tritylthio) propanoate (200
mg, 0.38 mmol) according to the general procedure for thiazoline preparation to give the product
as a white solid (40 mg, 0.15 mmol, 40%).

Melting point: 141–142 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 9.20 (s, 1H), 7.65 (dd, 1H, J
1
= 8 Hz, J
2
= 1.2 Hz), 7.35 (dd,
1H, J
1
= 8 Hz, J
2
= 1.2 Hz), 7.29 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 7.13 (ddd, 1H, J
1
=
8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 6.98 (d, 1H, J = 1 Hz), 5.26 (t, 1H, J = 8 Hz), 3.84 (s, 3H), 3.76
(dd, 1H, J
1
= 12 Hz,
J2
= 8 Hz), 3.68 (dd, 1H, 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
-1
, found 261.0691 g mol
-1
.

149

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

150

Methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.13).

6.13 was prepared from methyl 2-(1-methylindole-2-carboxamido)- 3-
(tritylthio)propanoate (1.1 g, 2.1 mmol) according to the general procedure for thiazoline
preparation to give the product as a white solid (120 mg, 0.44 mmol, 21%).

M.P. 78–80 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 7.64 (dt, 1H, J
1
= 8 Hz, J
2
= 1.2 Hz), 7.37 (d, 1H, J = 8 Hz),
7.33 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 7.14 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2
Hz), 7.16 (s, 1H), 5.37 (dd, 1H, J
1
= 8 Hz, J
2
= 8 Hz), 4.12 (s, 3H), 3.84 (s, 3H), 3.65 (dd, 1H, J
1

= 8 Hz, J
2
= 12 Hz), 3.59 (dd, 1H, 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
-1
, found 275.0850 g mol
-1
.

151

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

152

General procedure for catalytic oxidation.

Azoline was dissolved in DMF at room temperature (50 mM). After the addition of
(DAB)Cu
II
complex 6.1 (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 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.

General procedure for base-promoted oxidation of azolines.

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 stir 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.

153

Methyl 2-phenylthiazole-4-carboxylate (6.2a).

6.2a was prepared from methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate (6.2, 22 mg,
0.1 mmol) according to the catalytic procedure (8 h, 19 mg, 87%) or base-promoted procedure
(0.5 h, 15 mg, 66%) to give 6.2a as white solid.

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

Data are consistent with a previously characterized compound.
17

154

Methyl 2-(4-nitrophenyl)thiazole-4-carboxylate (6.3a).

6.3a was prepared from methyl 2-(4-nitrophenyl)-4,5-dihydrothiazole-4-carboxylate (6.3,
53 mg, 0.2 mmol) according to the general catalytic procedure (3 h, 41 mg, 78%) or a variant of
the base-promoted procedure wherein only 10 mol% of DBU is incorporated (1 h, 36 mg, 69%).

Melting point: 224–227 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 8.31 (d, 2H, J = 8 Hz), 8.29 (s, 1H), 8.18 (d, 2H, J = 8 Hz),
3.98 (s, 3H).

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
-1
, found: 265.0278 g mol
-1
.

155

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

156

Methyl 2-(4-methoxyphenyl)thiazole-4-carboxylate (6.4a).

6.4a was prepared from methyl 2-(4-methoxyphenyl)-4,5-dihydrothiazole-4-carboxylate
(6.4, 25 mg, 0.1 mmol) according to the general catalytic procedure (8 h, 17 mg, 68%) or base-
promoted procedure (4 h, 14 mg, 58%).

Melting point: 67–79 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 8.10 (s, 1H), 7.96 (d, 2H, J = 8 Hz), 6.97 (d, 2H, J = 8 Hz),
3.97 (s, 3H), 3.87 (s, 3H).

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
−1
, found 250.0532 g mol
−1
.

157

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

158

Methyl 2-(naphthalen-2-yl)thiazole-4-carboxylate (6.5a).

6.5a was prepared from methyl 2-(naphthalen-2-yl)-4,5-dihydrothiazole-4-carboxylate
(6.5, 20 mg, 0.74 mmol) according to the catalytic procedure (8.5 h, 16 mg, 79%) or base-
promoted procedure (1 h, 15 mg, 77%) to give 6.5a.

1
H NMR (400 MHz, CDCl
3
): δ = 8.52 (s, 1H), 8.22 (s, 1H), 8.09 (d, 1H, J = 8 Hz), 7.93 (m, 2
H), 7.85 (m, 1H), 7.54 (m, 2H), 4.01 (s, 3H).

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
−1
, found 270.0583 g mol
−1
.

159

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

160

Methyl 2-(4-fluorophenyl)thiazole-4-carboxylate (6.6a).

6.6a was prepared from methyl 2-(4-fluorophenyl)-4,5-dihydrothiazole-4-carboxylate
(6.6, 20 mg, 0.084 mmol) according to the catalytic procedure (2 h, 12 mg, 58%) or base-
promoted oxidation (45 min, 9 mg, 44%) to give 6.6a.

1
H NMR (400 MHz, CDCl
3
): δ = 8.16 (s, 1H), 8.00 (m, 2H), 7.15 (t, 2H, J = 8.4 Hz), 3.98 (s,
3H).

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
−1
, found 238.0333 g mol
−1
.

161

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz CDCl
3
)

162

Methyl 2-(4-Cyanophenyl)thiazole-4-carboxylate (6.7a).

6.7a was prepared from methyl 2-(4-cyanophenyl)-4,5-dihydrothiazole-4-carboxylate (6.7, 20
mg, 0.81 mmol) according to the catalytic procedure (4 hours, 14 mg, 69%) or base-promoted
procedure 7.2.6 (45 minutes, 9 mg, 44%) to give 6.7a as a white crystalline solid.

Melting point: 199–201 °C.

1
H NMR (500 MHz, CDCl
3
): δ = 8.27 (s, 1H), 8.13 (dd, 2H, J
1
= 8.5 Hz, J
2
= 2.5 Hz), 7.76 (dd,
2H, J
1
= 8.5 Hz, J
2
= 2.5 Hz), 4.0 (s, 3H).

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
−1
, found 245.0379 g mol
−1
.

163

1
H NMR (500 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

164

Methyl 2-phenyloxazole-4-carboxylate (6.8a).

6.8a was prepared from methyl 2-phenyl-4,5-dihydrooxazole-4-carboxylate (6.8, 20 mg, 0.1
mmol)
18
according to a variant of the catalytic procedure wherein 30 mol% of base is added (9
hours, 4 mg, 18%) or base-promoted procedure (6 h, 3 mg, 16%).

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

Data are consistent with a previously characterized compound.
18

165

Methyl 2-(4-nitrophenyl)oxazole-4-carboxylate (6.9a).

6.9a was prepared from methyl 2-(4-nitrophenyl)-4,5-dihydrooxazole-4-carboxylate
19
(6.9, 20
mg 0.08 mmol) according to a variant of the catalytic procedure wherein 30 mol% of base is
added (12 h, 7 mg, 37%) or base-promoted procedure (2 h, 8 mg,41%).

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

Data are consistent with a previously characterized compound.
20

166

Methyl 2-methylthiazole-4-carboxylate (6.10a).

6.10a was prepared from methyl 2-methyl-4,5-dihydrothiazole-4-carboxylate (6.10, 20 mg, 0.12
mmol) according to the catalytic procedure (8 h, 5 mg, 24%) or base-promoted procedure (5 h, 8
mg, 39%).

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

Data are consistent with a previously characterized compound.
21

167

Methyl 2-phenethylthiazole-4-carboxylate (6.11a).

6.11a was prepared from methyl 2-phenethyl-4,5-dihydrothiazole-4-carboxylate (6.11, 20 mg,
0.08 mmol) according to the catalytic procedure (12 h, 9 mg, 45%) or base-promoted procedure
(6 h, 10 mg, 51%).

1
H NMR (400 MHz, CDCl
3
): δ = 8.05 (s 1H), 7.3 (m, 2H), 7.21 (m, 3H), 3.96 (s, 3H), 3.38 (t,
2H, J = 8 Hz), 3.18 (t, 2H, 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
−1
, found 248.0740 g mol
−1
.

168

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

169

Methyl 2-(indol-2-yl)thiazole-4-carboxylate (6.12a).

6.12a was prepared from methyl 2-(indol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.12, 20 mg,
0.077 mmol) according to the catalytic procedure (6 h, 11 mg, 55%).

Melting point: 69–71 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 9.33 (s, 1 H), 8.13 (s 1H), 7.65 (dd, 1H J
1
= 8 Hz, J
2
= 0.8 Hz),
7.40 (dd, 1H J
1
= 8 Hz, J
2
= 0.8 Hz), 7.28 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 0.8 Hz), 7.15 (ddd,
1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 0.8 Hz), 7.05 (dd, 1H, J
1
= 2 Hz, J
2
= 0.8 Hz), 3.99 (s, 3H).

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
−1
, found 259.0536 g mol
−1
.

170

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

171

Methyl 2-(1-methyl-indol-2-yl)thiazole-4-carboxylate (6.13a).

6.13a was prepared from methyl 2-(1-methylindol-2-yl)-4,5-dihydrothiazole-4-carboxylate (6.13,
20 mg, 0.073 mmol) according to the catalytic procedure (14 h, 13 mg, 65%) or base-promoted
procedure (30 min, 9 mg, 45%).

Melting point: 124–127 °C.

1
H NMR (400 MHz, CDCl
3
): δ = 8.15 (s, 1H), 7.64 (d, 1H, J = 8 Hz), 7.40 (d, 1H, J = 8.8 Hz),
7.32 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2 Hz), 7.16 (ddd, 1H, J
1
= 8 Hz, J
2
= 8 Hz, J
3
= 1.2
Hz), 7.04 (s, 1H), 4.21 (s, 3H), 3.98 (s, 3H).

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
−1
, found 273.0697 g mol
−1
.

172

1
H NMR (400 MHz, CDCl
3
)

13
C NMR (100 MHz, CDCl
3
)

173

Methyl 4-hydroxy-2-phenyl-4,5-dihydrothiazole-4-carboxylate (6.15).

6.15 was prepared from methyl 2-phenyl-4,5-dihydrothiazole-4-carboxylate via the general
procedure for base-promoted oxidation in which the reaction was stopped after 15 min.

1
H NMR (400 MHz, CDCl
3
): 7.89 (dd, 2H, J = 8 Hz, J = 1.2 Hz), 7.51 (tt, 1H, J = 8 Hz, J = 8
Hz), 7.42 (tt, 2H, J = 8 Hz, J = 1.2 Hz), 4.18 (s, 1H), 4.02 (dd, 2H, J = 12 Hz, J = 1.2 Hz), 3.89
(s, 3H), 3.55 (d, 1H, J = 12 Hz).

MALDI for C
11
H
11
NO
3
S: calculated [MH]
+
238.04 g mol
−1
, found 238.00 g mol
−1
.

1
H NMR (400 MHz, CDCl
3
)

7.6. References.

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175

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Abstract (if available)

Abstract The focus of this work is the non‐covalent modulation of MRI based nanotheranostics. This includes modification of nanoparticulate surface properties based on phosphonate‐guanidine hydrogen bonding, modulation of MRI contrast agent behavior based on guest‐host interactions, and masking of MRI contrast using a gelatin pill casing. Synthetic methodology for the synthesis of potential pharmaceutical entities is also described. ❧ Surface properties are key factors for controlling in vivo behavior of nanoparticulate drug delivery vehicles. A general procedure for the surface modification of hybrid gadolinium‐phosphonate coated gold nanoparticles was achieved via hydrogen bonding interaction with an amphiphilic fluoroalkyl monolayer. Along with an increase in hydrodynamic diameter, the coating interaction was characterized by a significant decrease in ¹⁹F T₁ of the coating monomer, allowing for potential ¹⁹F MRI based nanotheranostics. ❧ Controllable, non‐covalent interactions were also used to attenuate and reveal MRI contrast agents by hiding them in molecular hosts. A container‐shaped molecular host compound, which had previously demonstrated selective and strong binding affinities towards trimethylammonium functionality, was used. Modification of Gd•DOTA with the trimethylammonium moiety resulted in a dramatic decrease of contrast upon exposure to the molecular host. The masking event is accompanied by the assembly of contrast agent into a 7 nm micelle as characterized by DLS. The contrast agent guest was removed from the host via competitive binding in the presence of a stronger binding agent, resulting in a 31% contrast increase over the masked state. ❧ The release of MRI contrast agent from a gelatin pill casing was characterized. The masked MRI contrast agent was immediately revealed under acidic conditions, whereas it remained masked under neutral conditions for over ten minutes. The release of the gadolinium‐based MRI contrast agent was characterized by the decrease of T₁ in vitro, and MRI contrast observed in vivo. This MRI pill can potentially be used for imaging of gastric motility disorders such as gastroesophageal reflux disease and gastroparesis. ❧ Catalytic conditions for oxidation of azolines to azoles are also described. Azoles are biologically active marine cyclopeptides in which its synthesis have historically involved stoichiometric amounts of toxic reagents. By utilizing catalytic amounts of a copper catalyst and base, inexpensive and environmentally benign conditions for this bond transformation were achieved. Along with the catalytic copper conditions, copper‐free conditions involving stoichiometric amounts of base were also developed. These conditions demonstrated decreased reaction times over the catalytic copper conditions.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Li, Vincent (author)

Core Title The use of non-covalent interactions for the modification of nanoparticle surface and MRI contrast agent properties

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 06/30/2014

Defense Date 06/03/2014

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

Tag cavitand,guest‐host,MRI contrast agent,nanoparticle MRI,OAI-PMH Harvest,responsive MRI

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 vinli11@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-426051

Unique identifier UC11286532

Identifier etd-LiVincent-2587.pdf (filename),usctheses-c3-426051 (legacy record id)

Legacy Identifier etd-LiVincent-2587.pdf

Dmrecord 426051

Document Type Dissertation

Format application/pdf (imt)

Rights Li, Vincent

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