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Syntheses of a series of fluorous amphiphiles and modulation of the relaxivity of gadolinium(III)-based contrast agents

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Syntheses of a series of fluorous amphiphiles and modulation of the relaxivity of gadolinium(III)-based contrast agents

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Syntheses of a Series of Fluorous Amphiphiles and Modulation of the Relaxivity of
Gadolinium(III)-Based Contrast Agents

By

Xinping Wu

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

Copyright 2014 Xinping Wu

ii
Dedication

To Mom and Dad.

iii
Acknowledgements

I joined the Williams group when the lab was still taking shape. I began to work on
establishing a system to modulate the relaxivity of contrast agents. I hope my work has
now pointed in the correct direction for future project participants. First of all, I would
like to thank my PhD advisor, Professor Travis Williams, for his mentorship on
chemistry and the merit of being a scholar. His brilliance in synthesis and his enthusiasm
in teaching were impressive to me. I received a tremendous amount of knowledge on
synthesis and NMR methods from him, which will continue to benefit me in the future.
Travis also set a solid standard for me as a chemist and pushed me to do my best. I would
also like to thank the rest of my lab, including Dr. Brian Conley, Dr. Emine Boz, Dr.
Anna Dawsey, Dr. Megan Pennington-Boggio, Dr. Vincent Li, Zhiyao Lu, Lily Zhang,
Jeff Celaje, Denver Guess, Christina Ratto, Blaine Bolibol, and Forrest Zhang. I am
thankful for Dr. Brian Conley for tirelessly educating me on chemistry and influencing
me with his positive attitude for the two years we worked in the same lab. I thank Jeff for
his enlightening discussion on chemistry and his selfless help whenever I needed it. I
thank Lily for the girly chats in the office and after work; my graduate school life would
not have been the same without you. All of your help is truly appreciated.
It has been eight years since I left my home country. I certainly would not have made it
without my family’s sacrifice and support. My father and my late mother had offered me
the largest financial sponsorship I could possibly obtain. They will always be my biggest
fans. My grandparents were my earliest educators on chemistry, which is a subject I
happened to be working on today. My boyfriend Allen acted as a soundboard during
iv
times of my success and failure, and I would like to thank him for his unique suggestions
and support. I feel blessed to have you guys as my family.
I’d also like to thank my committee members, Professor G. K. Surya Prakash and Dr.
Andy Chang, for reading this thesis and offering me with their valuable suggestions. I am
very thankful for Professor Prakash’s guidance on chemistry outside this thesis. I
appreciate your time and effort. Being a member of the LHI building and the chemistry
department, I thank the hard-working staff for keeping our institute and department
running, including Dr. Robert Anizfeld, Mr. David Hunter, Ms. Jessie May, Mrs. Carole
Phillips, Ms. Michele Dea, Ms. Katie McKissick and Ms. Susan Peterson. I also thank the
general chemistry and organic chemistry teaching staff, Dr. Elizabeth Erickson and Dr.
Jennifer Moore. I am grateful for the lessons learned for my teaching experience.
Thanks to my office buddies: Dr. Andrey Rudenko, Dr. Alejandra Beier and Besty
Melenbrink. You have made the office life truly enjoyable.
Many thanks to Dr. Anna Dawsey, Allen Chyr, Lily Zhang, and Dr. Andrey Rudenko,
for taking time to read this thesis. Last, I would like to thank the Robert and Mary Wright
Foundation for financial support.

v
Table of Contents
Dedication ii
Acknowledgements iii
List of Tables x
List of Figures xii
List of Schemes xvii
Preparative Procedures xviii
Abstract xxvi
Chapter 1. Fluorous Chemistry: Properties and Applications 1
1.1 Introduction 1
1.1.1 General Properties of Fluorine 1
1.1.2 Properties of the Fluorocarbons 2
1.2 Synthesis of Fluorous Molecules: Direct Fluorination and Incorporation of
Fluoroalkyl Building Blocks 4
1.2.1 Synthesis of Perfluorinated Molecules 4
1.2.2 Synthesis of Molecules Modified with Perfluoroalkyls 4
1.3 Applications of Fluorous Materials 7
1.3.1 Fluorous Surface 7
1.3.2 Fluorous Tags 9
1.4 Conclusion 11
1.5 Reference
12
Chapter 2. Synthesis of a Guanidine-Functionalized Fluorous Amphiphiles and Its
Binding to Phosphonates 16
vi
2.1 Introduction 16
2.1.1 End-Functionalized Fluoroalkanes with an Emphasis on Amine-
Functionalized Fluorocarbons 16
2.1.2 Goal and Hypothesis 17
2.2 Results and Discussion 18
2.2.1 Methodology Development: Synthesis of the Fluorous Amphiphile 18
2.2.2 Methodology Development: Fluoroalkyl Compounds 21
2.2.3 Binding Between the Fluorous Amphiphile and Phosphonates 24
2.2.4 pK
a
of the Amphiphile 26
2.3 Conclusion 29
2.4 References 30
Chapter 3. Synthesis of the Second-Generation Fluorous Amphiphile 32
3.1 Introduction 32
3.2 Results and Discussion 34
3.2.1 Synthesis of Fluorous Amphiphile with a Two-Methylene Spacer 34
3.2.2
19
F T
1
Measurements 37
3.3 Conclusion 49
3.4 References 49
Chapter 4. Introduction to Magnetic Resonance Imaging (MRI): Principles and
Contrast Agent Design 51
4.1 MRI Physics 51
4.1.1 T
1
and T
2
Relaxation 51
4.1.2 Principles of MRI 53
vii
4.1.3 Origin of T
1
- and T
2
-Weighted Images 54
4.1.4 T
1
Relaxation Mechanism 56
4.2 MRI Contrast Agents: Small Molecule Contrast Agents 58
4.2.1 Acyclic Small Molecule Contrast Agents 59
4.2.2 Cyclic Small Molecule Contrast Agnets 61
4.3 Relaxivity Optimization by q, τ
m
, τ
R
modulation 62
4.3.1 Relaxivity Enhancement via τ
R
Modulation 63
4.3.2 Relaxivity Enhancement via q Modulation 68
4.3.3 Relaxivity Enhancement via Outer-Sphere Relaxation 73
4.3.4 Relaxivity Enhancement via τ
m
Modulation 76
4.4 Conclusion 78
4.5 References 78
Chapter 5. A (Fluoroalkyl)Guanidine Modulates the Relaxivity of a Phosphonate-
Containing T
1
Contrast Agent 85
5.1 Introduction 85
5.2 Modulated Relaxivity in Gd-DOTP
5-
and Gd-DOTA
-
Systems 90
5.2.1 Relaxivity Measurements at 9.4 T 90
5.2.2 Relaxivity Measurements at 1.4 T 92
5.2.3 Luminescence Decay 95
5.2.4 Variable Temperature (VT)
17
O NMR 96
5.2.5 Electron Paramagnetic Resonance (EPR) 97
5.2.6
19
F NMR 100
5.2.7 Diffusion-Ordered NMR Spectroscopy (DOSY 2D) 106
viii
5.3 Conclusion 108
5.4 References 108
Chapter 6. Gold Nanoparticles in Drug Delivery 113
6.1 Introduction 113
6.1.1 Fabrication of Gold Nanoparticles and Surface Functionalization for
Drug Delivery 113
6.1.2 Gold Nanoparticles as Drug Carriers 114
6.2 Encapsulation of BODIPY in a Phosphonate-Coated Gold Nanoparticle and Its
Release in a Biphasic System 118
6.2.1 Characterization of the Loaded AuNPs 120
6.2.2 Non-Covalent Interaction between 2.9a and the Phosphonate Coated-
AuNPs 121
6.2.3 BODIPY Release from the Particles in a Toluene/H
2
O Biphasic
System 121
6.3 Conclusion 124
6.4 References 125
Chapter 7. Experimental Procedures and Spectral Data 127
7.1 General Procedures 127
7.1.1 Chemicals 127
7.1.2 Prepared Chemicals 128
7.1.3 Instrumentation 129
7.1.4 General Procedures for
1
H T
1
Measurements 130
7.15 General Procedures for
19
F T
1
Measurements 131
ix
7.1.6 pH Measurement 132
7.1.7 Dialysis 133
7.1.8 Other 133
7.2 Chapter 2 Experimental and Spectral Data 134
7.2.1 Preparation of Fluorous Amphiphiles 134
7.2.2 Preparation of Fluoroalkyl Compounds 195
7.3 Chapter 3 Experimental and Spectral Data 219
7.4 Chapter 5 Experimental and Spectral Data 237
7.4.1 Synthesis Ln-DOTP
5-
and Ln-DOTA
-
Compounds 237
7.4.2 Relaxivity (r
1
) Measurements at 9.4 T 249
7.4.3 Relaxivity (r
1
) Measurements at 1.4 T 258
7.4.4 Luminescence Decay Measurements 267
7.4.5 Variable-Temperature
17
O-NMR Data 269
7.4.6 EPR Spectroscopy 273
7.4.10 ICP-OES 276
7.5 Chapter 6 Experimental and Spectral Data 277
7.5.1 Synthesis of Phosphonate-Coated Gold Nanoparticles (1.5 nm Core in
Diameter) 277
7.5.2 BODIPY Encapsulation in the Surfactant-Coated Nanoparticles 280
7.5.3 BODIPY Loading Capacity 281
7.5.4 BODIPY Release in a Biphasic System 282
7.6 References 284

x
List of Tables

Table 1.1. List of γ
c
Values of Fluorinated Polyethylene. 8
Table 2.1. Synthesis of the Fluorous Amphiphile. 22
Table 2.2. Functionalized Ditriflate 2.10 and the Respective Yields. 25
Table 2.3. Linewidth Broadening of
31
P NMR as Evidence of Hydrogen Bond
Formation. 28
Table 3.1. Shortened
19
F T
1
in the Presence of Gold Nanoparticles. 43
Table 3.2. Shortened
19
F T
1
in the Presence of Gold Nanoparticles. 48
Table 4.1. Osmolalities, Viscosity, LogK
GdL
, q, τ
m
and τ
R
of Selected Contrast Agents. 61
Table 5.1. The r
1
of Gd-DOTP
5-
Titrated with 2.9a. (9.4 T). 91
Table 5.2. Relaxivity (r
1
) of Gd-DOTP
5-
through Sequential Addition of 2.9a, Urea,
and Sonication. (9.4 T) 91
Table 5.3. Relaxivity (r
1
) of Gd-DOTA
-
through Sequential Addition of 2.9a, Urea,
and Sonication (9.4T). 92
Table 5.4. Relaxivity (r
1
) of Gd-DOTP
5-
through Sequential Addition of 2.9a, Urea,
and Sonication (1.4 T). 93
Table 5.5. Relaxivity (r
1
) of Gd-DOTA
-
through Sequential Addition of 2.9a, Urea, and
Sonication (1.4 T). 94
Table 5.6. Summarized r
1
, τ
R
, τ
m
, and q for Gd-DOTP
5-
System. 103
Table 5.7. Summarized r
1
, τ
R
, τ
m
, and q for Gd-DOTA
-
System. 103
Table 5.8. Reductions in
19
F T
1
in the Presence of Gd-DOTP
5-
. 105
Table 5.9. Reductions in
19
F T
1
in the Presence of Gd-DOTA
-
. 105
xi
Table 5.10. Diffusion Coefficients (D) of 0.1%
t
BuOH and Y-DOTP
5-
measured by
DOSY 2D at 25
o
C. 107

xii
List of Figures

Figure 1.1. Fluorous amphiphiles, 1.10, are planted on a gold surface to form a non-
stick SAM. 8
Figure 1.2. The structure of (-)-dictyostatin. 10
Figure 2.1. a. Guanidiniums are capable of forming bidentate hydrogen bonds with
phosphonates. b. An example of the guanidine-rich molecular transporter.
The polycarbonate backbone is functionalized with guanidinium. 17
Figure 2.2. A general structure of the desired fluorous amphiphiles. 18
Figure 2.3.
31
P NMR evidence of linewidth broadening. 27
Figure 2.4. pH titration of 2.9a with 0.05 M NaOH. 29
Figure 3.1. First generation fluorous amphiphile with a one-methylene spacer (2.9a)
and second-generation fluorous amphiphile with a two-methylene spacer
(3.1). 33
Figure 3.2. Gold nanoparticles capped with a blend of phosphonic acids and Gd-DOTA-
type chelates. 33
Figure 3.3. Fluorous amphiphile 2.9a decorates the surface of the gold nanoparticles via
non-covalent interactions 34
Figure 3.4. pH titration of 3.1 in an aqueous solution. 37
Figure 3.5. The fluorous methylenes of 3.1 are labeled from a-d. 38
Figure 3.6. The four peaks displayed by 3.1 on
19
F NMR spectrum are labeled 1-4. 39
Figure 3.7.
1
H NMR spectrum of 3.1. (500 MHz, CDCl
3
)
19
F gCOSY spectrum of 3.1.
(500 MHz, CDCl
3
) 39
xiii
Figure 3.8.
13
C NMR spectrum of 3.1. (125 MHz, CDCl
3
) 40
Figure 3.9.
19
F gCOSY spectrum of 3.1. (125 MHz, CDCl
3
) 40
Figure 3.10.
19
F-
13
C gHMBC NMR spectrum of 3.1. (470 MHz in
19
F, CDCl
3
) 41
Figure 3.11.
1
H-
13
C gHSQCAD NMR spectrum of 3.1. (500 MHz in
1
H, CDCl
3
) 41
Figure 3.12.
1
H-
13
C gHMBCAD spectrum of 3.1. (500 MHz in
1
H, CDCl
3
) 42
Figure 3.13.
19
F assignment of 3.1. (470 MHz, CDCl
3
) 42
Figure 3.14. The four fluorous methylenes are labeled from a’-c’ in 2.9a. 42
Figure 3.15. The peaks on
19
F NMR spectrum of 2.9a are labeled as 1’-3’. (470 MHz
CD
3
CN) 44
Figure 3.16.
1
H NMR spectrum of 2.9a. (500 MHz CD
3
CN) 45
Figure 3.17.
13
C NMR spectrum of 2.9a. (125 MHz, CD
3
CN) 46
Figure 3.18.
19
F-
13
C gHMBC NMR spectrum of 2.9a. (470 MHz in
19
F, CD
3
CN) 46
Figure 3.19.
1
H-
13
C gHSQCAD NMR spectrum of 2.9a. (125 MHz CD
3
CN) 47
Figure 3.20.
1
H-
13
C gHMBCAD NMR assignment of 2.9a. (125MHz CD
3
CN) 47
Figure 3.21.
19
F NMR assignment of 2.9a. (470 MHz, CD
3
CN) 48
Figure 4.1. Measuring longitudinal relaxivity using inversion recovery. 52
Figure 4.2. A spin echo pulse sequence. 52
Figure 4.3. Gd-DTPA
2-
and some of its derivatives. 60
Figure 4.4. The isomerization equilibrium of the two diastereomers, M and m isomers,
in aqueous solution. The conformations of the cyclen are the same in the
two isomers. The arrangement of the acetate arms in m isomer are inverted
with regard to the M isomer. 62
xiv
Figure 4.5. To couple the Gd-OH
2
vector with the molecular motion, the Gd
3+
core was
assembled into the barycenter of the dendrimer. 64
Figure 4.6. The long chain alkyl conjugated Gd-DOTA forms micelle in solution
with 5.3 times of contrast enhancement. 65
Figure 4.7. The Biotin-tethered aptamer strand forms a 70 kDa hybrid with the Gd-
DOTA-tethered DNA strand, after streptavidin was added. 66
Figure 4.8. The structure of the doxorubicin-Gd chelate conjugate prodrug with an acid-
labile hydrazine linker (left), and the unconjugated Gd-DOTA-like chelate. 68
Figure 4.9. The first example of a smart MRI contrast agent, Egad. After removal of β-
galactopyranose, the number of bound water molecules in the inner-sphere
increased by 40%. 69
Figure 4.10. Ca
2+
-sensing smart MRI contrast agent based on the principle of
q-modulation. 71
Figure 4.11. A Cu
+
-sensing contrast agent consisting of a Cu-receptor site and a Gd-
core. 72
Figure 4.12. A q-modulated contrast agent switches between the spiropyran form and the
merocyanine form. 72
Figure 4.13. The CLADIO-NH-SP nanoparticles respond to visible light by shortening
their T
2
. 74
Figure 4.14. Structure of a contrast agent, DEVD-(Tm-DOTA) activated by caspase-3. 74
Figure 4.15. The structure of GdDOTA-4AmP
5-
. 75
Figure 4.16. A series of DOTA-based contrast agent with the ethylene bridge in cyclen
core gradually substituted by propylene bridge. 76
xv
Figure 4.17. A series of PEG-conjugated Gd-DOTA-like complexes show slowed down
water exchange rate at the metal center. 77
Figure 5.1. Structures of Gd-DOTA
-
, Gd-DOTP
5-
and the fluorous amphiphile (2.9a). 87
Figure 5.2. Expected outcome from formation of a non-covalent adduct between Gd-
DOTP
5-
and the fluorous amphiphile (2.9a). 88
Figure 5.3. Urea displaces 2.9a in the newly formed non-covalent adduct. r
1
, q and τ
m

are no longer modulated. 89
Figure 5.4. Relaxivity (r
1
) measurements on [Gd(DOTP)]
5-
treated with a. 0 equiv. of
2.9a, b. 4 equiv. of 2.9a, c. 4 equiv. of 2.9a + urea, d. 4 equiv. of 2.9a +
urea + sonication. (25
o
C, 9.4 T) 91
Figure 5.5. Relaxivity (r
1
) measurements on [Gd(DOTA)]
-
treated with a. 0 equiv. of
2.9a, b. 4 equiv. of 2.9a, c. 4 equiv. of 2.9a + urea, d. 4 equiv. of 2.9a + urea
+ sonication. (25
o
C, 9.4 T) 92
Figure 5.6. Relaxivity (r
1
) measurements on [Gd(DOTP)]
5-
with a. 0 equiv. 2.9a, b. 4
equiv. 2.9a, c.4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication.
(25
o
C, 1.4 T) 94
Figure 5.7. Relaxivity (r
1
) measurements on [Gd(DOTA)]
-
with a. 0 equiv. 2.9a, b. 4
equiv. 2.9a, c. 4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication.
(25
o
C, 1.4 T) 95
Figure 5.8. EPR spectrum of Gd-DOTP
5-
system treated with a. 0 equiv. 2.9a, b. 4 equiv.
2.9a, c. 4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication. 101
Figure 5.9. EPR study of [Gd(DOTP)]
5-
. a. [Gd(DOTP)]
5-
only. B. [Gd(DOTP)]
5-
+ 8
equiv. of 2.9a. c. [Gd(DOTP)]
5-
+ 25 equiv. of 2.9a. d. [Gd(DOTP)]
5-
+
xvi
50 equiv. of 2.9a. 102
Figure 5.10. Structures of meglumine and hexacyclen. 104
Figure 5.11.
19
F NMR of an aqueous solution of 2.9a before (top) and after (bottom)
the addition of Gd-DOTP
5-
. 105
Figure 5.12. DOSY 2D was acquired for samples containing Y-DOTP
5-
. 107
Figure 6.1. The hydrophobic phthalocyanine 4 is encapsulated in AuNPs capped with
HO-PEG-SH 5000. 115
Figure 6.2. A Zwitterion-stabilized AuNP loaded with BODIPY. 117
Figure 6.3. The surface charge of the gold nanoparticles is changed from positive to
negative after light irradiation. 118
Figure 6.4. a. BODIPY molecules are loaded into a phosphonic acid-capped Au NP.
b. Non-covalently decorating the particles blocks the diffusion of BODIPY
outside of the particle. 119
Figure 6.5. UV-Vis spectrum of the AuNPs loaded with BODIPY. 122
Figure 6.6. NaCN-induced decomposition of BODIPY-loaded AuNPs in THF/H
2
O
mixture. 123
Figure 6.7. BODIPY release from the AuNPs in a toluene/H
2
O biphasic system. The
release process is monitored by UV-Vis spectrometry. 123
Figure 6.8. The BODIPY-loaded AuNPs release BODIPY at the same speeds with or
without 2.9a (red and purple). When excess amount of 2.9a was added, the
rate of the release is accelerated (blue). 124
Figure 7.1. A sketch of a NMR tube ready for T
1
measurements. 131

xvii
List of Schemes

Scheme 1.1. Alkylation of 1.2 Affords Fluorous Amphiphile 1.3 in an Ether Synthesis. 5
Scheme 1.2. Synthesis of Fluoroalkyl p-nitrophenyl Ethers via Fluoroalkyl Sulfonate
Esters. 6
Scheme 1.3. FBS Catalysis: Hydroformylation of 1-Decene to Undecyl Aldehyde. 10
Scheme 2.1. Synthesis of Fluorous Amphiphiles. 19
Scheme 2.2. General Route to Functionalize the Fluorous Diol 2.1a. 24
Scheme 3.1. Retrosynthesis of 3.1. 35
Scheme 3.2. Synthesis of the Second-Generation Fluorous Amphiphile, 3.1. 36
Scheme 3.3. Overview of the Structural Assignment for 3.1. 38
Scheme 3.4. Overview of the Structural Assignment for 2.9a. 44

xviii
Preparative Procedures

PEG-Tosylate 2.3 126

PEG-Fluorinated Alcohol 2.4a 137

PEG-Fluorinated Alcohol 2.4b 141

PEG-Fluorinated Alcohol 2.4c 145

PEG-Fluorinated Triflate 2.5a 149

O
O S
O
O
4
O
O OH
FF
4 4
O
O OH
FF
3 4
O
O OH
FF
6 4
O
O O
FF
4 4
S CF
3
O
O
xix
PEG-Fluorinated Triflate 2.5b 152

PEG-Fluorinated Triflate 2.5c 156

PEG-Fluorinated Azide 2.6a 160

PEG-Fluorinated Azide 2.6b 163

PEG-Fluorinated Azide 2.6c 166

PEG-Fluorinated Amine 2.7a 169

O
O O
FF
3 4
S CF
3
O
O
O
O O
FF
6 4
S CF
3
O
O
O
O
FF
4 4
N
3
O
O
FF
3 4
N
3
O
O
FF
6 4
N
3
O
O
FF
4 4
NH
2
xx
PEG-Fluorinated Amines 2.7b 173

PEG-Fluorinated Amines 2.7c 177

PEG-Fluorinated Phthalimide 2.8 180

PEG-Fluorinated Guanidinium Mono TFA Salt 2.9b 183

PEG-Fluorinated Guanidinium Mono TFA Salts 2.9a 187

O
O
FF
3 4
NH
2
O
O
FF
6 4
NH
2
O
O
FF
4 4
N
O
O
O
O
FF
3 4
H
N
NH
NH
2
HO CF
3
O
O
O
FF
4 4
2.9a
H
N
NH
NH
2
HO CF
3
O
xxi
PEG-Fluorinated Guanidinium Mono TFA Salts 2.9c 191

Triflate 2.10 195

Dibromide 2.11 198

Malonocycloheptane 2.12 201

Azide 2.13 205

O
O
FF
4 4
H
N
NH
NH
2
HO CF
3
O
O O S S
O
O
CF
3
O
F
3
C
O FF
4
Br Br
FF
4
F
F
F
F F
F
F
F
O O
MeO OMe
2.12
N
3
N
3
FF
4
xxii
Phthalimide 2.14 208

Diamine 2.15 211

Triazole 2.16 215

PEG-Fluorinated Malonyl Ester 3.4 219

PEG-Fluorinated Dicarboxylic Acid 3.5 222

N N
FF
4
O
O
O
O
H
2
N NH
2
FF
4
N N
FF
4
N
N N
N
Ph Ph
O
O
FF
4 4
OMe O
O
OMe
O
O
FF
4 4
OH O
O
OH
xxiii
PEG-Fluorinated Monocarboxylic Acid 3.3 224

PEG-Fluorinated Benzyl Carbamate 3.6 227

PEG-Fluorinated Amine 3.2 230

PEG-Fluorinated Guanidine 3.1 (as TFA salt) 233

Na[Gd(DOTA)]·4H
2
O 5.1 237

O
O
FF
4 4
O
OH
O
O
FF
4 4
N
H
O
O
O
O
FF
4 4
NH
2
O
O
FF
4 4
N
H
NH
2
NH
HO CF
3
O
N
N
N
N
O
O O
O
O
Gd
O
O
O
Na H
2
O
xxiv
Na
5
[Gd(DOTP)]·9H
2
O 5.2 238

Na[Eu(DOTA)]·4H
2
O 5.3 240

Na
5
[Eu(DOTP)]·9H
2
O 5.4 241

N
N
N
N
P
P
P
P
Gd
O
O
O
O
O O
O
O
O
O
O
O
5
5Na 9H
2
O
N
N
N
N
O
O O
O
O
Eu
O
O
O
Na H
2
O
N
N
N
N
P
P
P
P
Eu
O
O
O
O
O O
O
O
O
O
O
O
5
5Na
9H
2
O
xxv
Na[Y(DOTA)]·4H
2
O 5.5 243

Na
5
[Y(DOTP)]·9H
2
O 5.6 244

Phosphonate-Coated Gold Nanoparticles 277

N
N
N
N
O
O O
O
O
Y
O
O
O
Na H
2
O
N
N
N
N
P
P
P
P
Y
O
O
O
O
O O
O
O
O
O
O
O
5
5Na 9H
2
O
xxvi
Abstract
The reversible activation of medical imaging agents continues to interest the MRI
contrast agent chemists. We have envisioned that by bringing self-associating
fluorocarbons close to a gadolinium(III)-based small molecule contrast agent, q and τ
m

(number and residence lifetime of the water molecules in the inner-sphere, respectively)
modulation can be achieved, thus enabling the design and optimization of a new class of
responsive contrast agents. The synthesis of a tri-segmented fluorous amphiphile
consisting of a guanidinium head, a fluorocarbon middle segment and a polyethylene
glycol tail was developed as a small molecule platform for this technology.
We have since developed the methodology to synthesize a novel class of fluorous
amphiphiles fulfilling the aforementioned requirements. The route described herein is
facile and applicable on a gram scale. The synthesis involves a versatile triflate
intermediate that enabled us to install a series of functionalities β- to the fluorocarbon.
The synthesis was concluded with guanylation of an extremely electron-deficient primary
amine with satisfying yield. Curious as to how varying length of methylene spacers
would affect the pK
a
value of the fluorous guanidinium, a new synthesis route placing a
two-methylene spacer between the guanidinium and fluorocarbon was devised. It was
concluded that an extra methylene spacer increases the pK
a
by 0.4 units.
Using Gd-DOTP
5-
as the study subject, it was demonstrated that the fluorous
amphiphile is capable of augmenting the r
1
of Gd-DOTP
5-
in an aqueous solution.
Mechanistic studies showed that although τ
m
was marginally attenuated, an enhancement
in τ
R
(rotational correlation time) was primarily accountable for the accentuated relaxivity.
xxvii
Additionally,
19
F T
1
evidence complies with a model in which a non-covalent conjugate is
formed between Gd-DOTP
5-
and the amphiphile.
The last portion of the project describes a drug delivery project in which the primary
objective is to decrease the diffusion rate of an encapsulated organic molecule from a
nanoparticle carrier by decorating the particles with the fluorous amphiphiles. Boron-
dipyrrolemethene (BODIPY) molecules were encapsulated in an alkyl-phosphonic acid
coated gold nanoparticle in order to probe these experimental parameters. No change in
the diffusion rate was observed upon addition of the amphiphile. Weak association
energy between the fluoroalkyls and high hydrophobicity of the fluorocarbon may
account for this observation.

1
Chapter 1. Fluorous Chemistry: Properties and Applications

1.1 Introduction
1.1.1 General Properties of Fluorine
Fluorine is an element of extreme properties. Fluorine occurs as the
19
F isotope in 100%
natural abundance. In natural compounds, it exists exclusively in the oxidation state of -1
and exhibits a strong tendency in capturing an additional electron to achieve the electron
configuration of neon.
19
F is the most electronegative element (electronegativity 3.98) on
Earth.
1
F
2
gas is highly reactive, since its homolytic dissociation energy is low (37.8 kcal
mol
-1
). Fluorine also has redox potentials of +3.06 and +2.87 eV, in acidic and basic
aqueous media.
2
In 1967, Olah et al. prepared the strongest known protic acid by mixing
anhydrous HF with strong Lewis acids such as AsF
5
, SbF
5
or SO
3
.
3
“Magic Acid”,
FSO
3
H-SbF
5
, is capable of extracting hydrides from weak bases such as n-butane.
Due to its high reactivity,
19
F can readily react with hot platinum or gold. It can be
prepared into noble gas compounds such as XeF
6
and KrF
2
. The first preparation of F
2

gas was reported in 1886.
4
Moissan prepared the gas by electrolyzing a HF-KF system,
and fluorine gas was collected at the positive electrode. This method was further
developed during the Manhattan Project, where pure fluorine was produced for uranium
enrichment. The method used for Manhattan Project is still being used for the industrial
production of F
2
gas today. The first purely chemical preparation of F
2
gas was reported
by Christe in 1986.
5

2
1.1.2 Properties of the Fluorocarbons
1.1.2.1 Physical Chemical Properties
From CH
3
F, CH
2
F
2
, CHCF
3
to CF
4
, the C-F bond length decreases, and the bond
strength increases progressively.
6
This property is unique to fluorinated methane and is
not to be found with other halogenated methanes. CHF
3
and CF
4
are less polar than CH
3
F
or CH
2
F
2
, due to the C-F cancellation of dipole moments. This leads to the
perfluoroalkane’s non-polarizability, while partially fluorinated alkanes are often more
polarized than the corresponding hydrocarbons. It is important to note that although
perfluoroalkanes are non-polar, perfluoroarenes do offer polar interactions.
In terms of bond rotation, perfluoroalkanes are considered to be more rigid than their
hydrocarbon counterparts. Unlike hydrocarbons, the C-C bond of perfluoroalkanes does
not show free rotation in the liquid state.
7

Due to the non-polarity, perfluorocarbon solvents are hardly miscible with hydrocarbon
solvents. The dipole-dipole interaction between the hydrocarbon solvent molecules is
much greater than that between fluorocarbon solvent molecules. Therefore, mixing
fluorocarbon with organic solvents induces significant loss in enthalpy and is not
energetically favored. Although the mixing process would lead to entropic gain, the loss
in enthalpy is too large to be compensated. The net result is the two solvents are not
miscible.
Since fluorocarbons are incapable of forming strong interactions, they interact mainly
by diffusion rather than attraction or repulsion.
8
In some cases, the phase behavior of
fluorous molecules are regarded as “fluorophilic”.
9
Although it is generally believed “like
dissolves like”, it is misleading to consider that fluorous solvents are capable of strong
3
intermolecular interactions. Immiscibility of fluorous compounds with organic solvents is
described as “molecular xenophobia”-separation of organic molecules capable of strong
interaction from fluorous molecules capable of only weak interactions.
10

The miscibility of fluorous solvents with hydrocarbon solvents improves as temperature
rises. When the temperature lowers, the two solvents are separated. The reversible
miscibility of fluorous solvents and hydrocarbon solvents can be used to chemists’
advantage, and it becomes the foundation of fluorous biphasic systems (FBS).

1.1.2.2 Chemical Properties
Because the extreme reactivity of fluorine, the C-F bond is one of the most stable
covalent bonds, with bond strength measuring as high as 130 kcal/mol.
11
The stability of
C-F bond makes fluorocarbons suitable to work under harsh conditions. Fluorocarbon-
based oils are stable up to 400-500
o
C and are widely used in production of special
grease.
12

The high electronegativity of fluorine imposes strong electron withdrawing effect on
neighboring functional groups, resulting in the unique reactivity of fluorocarbons. One
can get a glimpse at the strong electron withdrawing effect by examining the pK
a
values
of fluorous acids and bases. The pK
a
values of CF
3
COOH, CF
3
CH
2
COOH,
CF
3
(CH
2
)
2
COOH are 0.23, 4.97, and 5.84 (25
o
C, aqueous solution).
13
The pK
a
values of
CF
3
CH
2
NH
3
+
and CF
3
(CH
2
)
2
NH
3
+
are 5.96 and 8.70 (25
o
C, aqueous solution).
14
These
values indicate the power of electron density attenuation imposed by fluorine. By looking
at these pK
a
values, we can deduce the approximate number of methylene functional
groups needed to insulate the electron withdrawing effect.
4
Another consequence of the strong electron withdrawing effect is dehydrofluorination.
When protons are vicinal to the fluorous segment, HF elimination is facile across the
CH
2
CF
2
junctions by strong bases. This is a phenomena commonly observed in fluorous
compound synthesis and should be avoided by judiciously choosing the type of base used
in the reaction.
15,16,17

1.2 Synthesis of Fluorous Molecules: Direct Fluorination and Incorporation of
Fluoroalkyl Building Blocks
1.2.1 Synthesis of Perfluorinated Molecules
Recently, low temperature gradient fluorination is used directly to fluorinate organic
molecules. This technique is referred to as LaMar (Lagow-Margrave) process. Organic
substrates are condensed at low temperature into a tube packed with copper turnings
through which highly diluted fluorine gas is passed.
18

The industrial synthesis of fluorinated compounds was developed during the Manhattan
Project, and issue of explosion was solved during this period. The large enthalpy gain in
fluorination is divided into two less exothermic processes. First, CoF
2
is converted to
CoF
3
on contact with F
2
gas at 350
o
C. Then, the organic compound is introduced and
fluorinated by CoF
3
at an appropriate temperature.

1.2.2 Synthesis of Molecules Modified with Perfluoroalkyls
Often unique properties, such as low surface tension enable long-chain fluorocarbons to
be incorporated into molecules to carry out specific functions. Simple coupling of the
5
linear fluorocarbon with molecules of interest will give the desired compounds.
19
The
main task is to find conditions compatible with perfluorocarbon.
Fluorous alcohols are considered to be cheap building blocks in fluorous synthesis. For
example, fluorous diol 1.1 is alkylated by polyethylene glycol 1.2 to afford amphiphile
1.3 via an ether synthesis (Scheme 1.1).
20
Compound 1.1 is a cheap byproduct from
Teflon synthesis, making it an economically efficient and cost effective starting material.
Radical conditions also tolerate the fluorocarbon, and radical-mediated electrophilic
alkylation with iodinated fluorocarbons is a common way to obtain fluorous materials.
21

Scheme 1.1. Alkylation of 1.2 Affords Fluorous Amphiphile 1.3 in an Ether Synthesis.

In 1965, Hansen reported that fluoroalkyl triflates are 10
4
times more reactive than the
corresponding tosylates in electrophilic fluoroalkylation reactions.
22
Břĭza et al. pointed
out that the rate and yield of electrophilic fluoroalkylation are usually determined by the
relative leaving group ability (nonaflate > triflate >> mesylate ≈ tosylate).
23
Prescher et
al. performed a systematic study on the potency of fluoroalkyl sulfonate esters, and their
results demonstrated the leaving group theory.
24
Fluoroalkyl sulfonate esters were used as
electrophile in ether synthesis, and sodium p-nitrophenyl oxide as the nucleophile
(Scheme 1.2). Fluoroalkyl sulfonates were synthesized from the corresponding fluorous
n = 3, 4, 6, 12, 13
HO O
F F
n 8
O O
OTs
1.1, KOH, dioxane
reflux
59-75%
1.2 1.3
HO OH
F F
8
= 1.1
n
6
alcohols (general structure CF
3
(CF
2
)
n
CH
2
OH, n = 0-6). When trifluoroethyl tosylate or
trifluoroethyl mesylate (1.4, n = 0, R = tosylate or mesylate) was used as the electrophile
in the ether synthesis, the mesylate analog afforded higher yield than the tosylate (70%
vs. 98%). The tosylate is more sterically hindered and more prone to hydrolysis than the
corresponding mesylate. Increasing the chain length of the fluoroalkyl renders the
mesylate 1.6 less reactive, and the yield of ether synthesis decreases drastically to 23%.
Adapting the triflate as the leaving group becomes necessary. Unlike the mesylates, the
reactivity of fluoroalkyl triflates 1.8 does not decrease as the fluoroalkyl chain lengthens:
For n = 2-6, the yield maintained between 80-89%.

Scheme 1.2. Synthesis of Fluoroalkyl p-nitrophenyl Ethers via Fluoroalkyl Sulfonate
Esters.

CF
3
(CF
2
)
n
CH
2
OR
HO NO
2
, NaH
1.4, n = 0, R = SO
2
PhCH
3
or R = SO
2
Me
CF
3
(CF
2
)
n
CH
2
O NO
2
a.
b.
1.5, n = 0
70% via tosylate
98% via mesylate
CF
3
(CF
2
)
n
CH
2
OR
1.6, n = 1, R = SO
2
Me
HO NO
2
, NaH
CF
3
(CF
2
)
n
CH
2
O NO
2
c.
CF
3
(CF
2
)
n
CH
2
OR
1.8, n = 2, R = SO
2
CF
3
n = 3, R = SO
2
CF
3
n = 4, R = SO
2
CF
3
n = 5, R = SO
2
CF
3
n = 6, R = SO
2
CF
3
HO NO
2
, NaH
CF
3
(CF
2
)
n
CH
2
O NO
2
1.7, n =1, 23%
1.9, n = 2, 85%
n = 3, 80%
n = 4, 98%
n = 5, 90%
n = 6, 95%
7
1.3 Applications of Fluorous Materials
Fluorous materials provide unique functionalities in material chemistry. The purpose of
using fluorous materials often fall into two categories: formation of a fluorous surface, or
labeling compounds with fluorous tags. Although fluorous materials are excellent
candidates for liquid crystalline formation, this application of fluoroalkyl will not be
discussed here.
Fluorous amphiphiles are an important class of materials. A fluorous amphiphile
consists of a fluoroalkyl segment and a hydrophobic or hydrophilic segment. Fluorous
amphiphiles behave similarly to their hydrocarbon counterparts but have lower critical
micelle concentration (CMC).
25
Because of the solvophobicity of fluoroalkyl, fluorous
amphiphiles such as C
n
F
2n+1
COOLi (n ≥ 6) can reduce the surface tension of water from
72 dyn cm
-1
to 15-20 dyn cm
-1
. The corresponding hydrocarbon amphiphile can only
reduce the surface tension to 25-35 dyn cm
-1
.
26

1.3.1 Fluorous Surface
Fluorinated surface has extremely low surface energy (γ
c
), which is associated with its
non-polarizable nature. Poly(tetrafluoroethylene) PTFE, Teflon) has a γ
c
value of 18.5
dyn cm
-1
, which accounts for its anti-stick and low-friction properties. This property is
often used to our advantage in fabrication of non-stick surfaces, such as Teflon coating
used in non-stick kitchenware. Because partially fluorinated alkyl groups are more
polarizable than perfluoroalkyls, the surface tension of fluoroalkyls is strictly correlated
with the degree of fluorination, as shown in Table 1.1.
26

8
Table 1.1. List of γ
c
Values of Fluorinated Polyethylene.
γ
c
(dyn cm
-1
)
poly(tetrafluoroethylene) 18.5
poly(difluoroethylene) 25
poly(fluoroethylene) 28
polyethylene 31

Kiessling et al. constructed a self-assembled monolayer (SAM) consisting of fluorous
amphiphile 1.10 planted on a gold surface. The resulting fluorous surface was used to
create a solvophobic and cytophobic surface that serves as the background for cell
adhesion assay (Figure 1.1).
27

Figure 1.1. Fluorous amphiphiles, 1.10, are planted on a gold surface to form a non-stick
SAM.

HS
CF
3
9
F F
7
F
3
C
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
F
3
C
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
F
3
C
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
F
3
C
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
1.10
Au
9
1.3.2 Fluorous Tags
The unique non-polarizable nature of fluorous tags allows incorporation into complex
molecule synthesis to accelerate the product purification. Conceptually, the fluoroalkyl-
tagged compounds should be purified on fluorous chromatography support. The
application of this concept has several variations, including FBS, fluorous mixture
synthesis (FMS), biological application of fluorous tags, etc, but all of them took
advantage of the solvophobic nature of fluoroalkyls.

1.3.2.1 Catalysis in Fluorous Biphasic Systems (FBS)
Modern organometallic catalysts typically contain expensive transition-metals and
precious ligands. Thus, it is desirable to recover the costly metal catalysts and reuse them.
FBS satisfies the need for catalyst recovery and easy separation of the product. The main
idea of fluorous biphasic system is to use a catalyst soluble in the fluorous phase in a
hydrocarbon/fluorous solvent system. Thus, a typical FBS catalysis reaction contains two
phases: a fluorous phase containing a fluorous catalyst, and a hydrocarbon phase
containing the substrate. Catalysis can occur in the fluorous phase or at the interface of
the two solvents. Under increased temperatures, the two phases are mixed into a single
phase, and catalysis can occur in the mixture as well. Lowering the temperature separates
the fluorocarbon catalyst and the product into two different phases. Recovering the
catalyst and harvesting the product are carried out by simply separating the two phases.
The first example of fluorous catalysis was reported by Horvăth et al. in
hydroformylation of 1-decene to undecyl aldehyde (Scheme 1.3).
28

10
Scheme 1.3. FBS Catalysis: Hydroformylation of 1-Decene to Undecyl Aldehyde.

1.3.2.2 Fluorous Mixture Synthesis (FMS)
Curran et al. widely used fluorous tags to accelerate product separation from a reaction
mixture. This process is referred to as FMS.
29
For synthesis of complex molecules, the
fluorous tag is often introduced at the early stage to label a specific stereoisomer. Each
isomer in a racemic mixture can be individually labeled with a different fluorous tag and
put through a racemic synthesis. During the last step, the racemic mixture is separated
based on their fluorous tag. For instance, in the synthesis of (-)-dictyostatin (1.11a,
Figure 1.2), the stereochemistry at C6 and C7 were varied to give four stereoisomers
(Scheme 1.4).
30
The isomers were labeled with four different fluorous tags at the early
stage of the synthesis. After the racemic synthesis, four stereoisomers were generated in
the end. (-)-Dictyostatin was obtained as one of the four isomers at 40% yield.

Figure 1.2. The structure of (-)-dictyostatin.
CH
3
5
Rh(CO)
2
(acac)
P[CH
2
CH
2
(CF
2
)
5
CF
3
]
3

C
6
F
11
CF
3
/toluene
10 bar CO/H
2
(1:1)
100
o
C
CH
3
5
OHC
O O
HO
OH OH
OH
6
7
(-)-Dictyostatin (1.11a)
11
1.3.2.3 Biological Application of Fluorous Tags
Fluoroalkyl tags have unique advantages over hydrocarbon tags. First because they can
be easily separated from the non-fluorous molecules using fluorous solid-phase support.
Also because synthesis of fluoroalkyl-tagged biomolecules is relatively easy and does not
require lengthy synthesis.
When fluoroalkyl-tagged biomolecules are used as the feedstock to organisms,
metabolites synthesized from the fluorous feedstock can be easily separated and
identified using fluorous separation. This concept has been demonstrated by the fluorous-
based carbohydrate microarrays.
31
In this example, cellular uptake of fluorous-tagged
sugar results in production of fluorous-tagged saccharides, which can be separated and
immobilized on a fluoroalkylsilyl derivatized glass slide. The immobilized saccharides
were probed with FITC-labeled lectins to reveal the binding target for lectin (FITC:
fluorescein isothiocyanate).

1.4 Conclusion
The extreme electronegativity endows fluorous compounds with their unique chemical
reactivity and physical properties. The non-polar nature of perfluoroalkyls provides the
theoretical basis for FMS and FBS. The most widely used practical applications are
Teflon materials and fluorous greases. As of yet, neither FMS or FBS has been adapted
into industrial-scale production, perhaps because of the cost of fluorous compounds.
Future direction could include expanding the application of fluorous chemistry in
material industry.

12
1.5 References

1
Jaccaud, M.; Faron, R.; Devilliers, D.; Romano, R. Fluorine. In Ullmann’s
Encyclopedia of Industrial Chemistry; Ullman, F., Ed.; Wiley-VCH: Weinheim, 2012;
Vol. 15, pp 381-395.
2
Kirsch, P. Modern Fluoroorganic Chemistry; Wiley-VCH: Weinheim, 2004; pp 7.
3
Olah, G. A. and Lukas, J. “Stable Carbonium Ions. XXXIX. Formation of
Alkylcarbonium Ions via Hydride Ion Abstraction from Alkanes in Fluorosulfonic Acid-
Antimony Pentafluoride Solution. Isolation of Some Crystalline Alkylcarbonium Ion
Salts” J. Am. Chem. Soc. 1967, 89, 2227-2228.
4
Moissan, H. “Action D’un Courant Électrique Sur L’acide Fluorhydrique Anhydre” C.
R. Acad. Sci. 1886, 102, 1543-1544.
5
Christe, K. O. “Chemical Synthesis of Elemental Fluorine” Inorg. Chem. 1986, 25,
3721-3722.
6
Liebman, J. F. and Greenberg, A. Studies of Organic Molecules. In Molecular Structure
and Energetics. VCH: Weinheim, 1986; Vol.3, pp 142.
7
Eaton, D. F. and Smart, B. E. “Are Fluorocarbon Chains Stiffer than Hydrocarbon
Chians? Dynamics of End-to-End Cyclization in a C8F16 Segment Monitored by
Fluorescence” J. Am. Chem. Soc. 1990, 112, 2821-2823.
8
Robb, I. D. Specialist Surfactants. Blackie Academic & Professional: London, UK,
1997, pp 112.
9
Shen, J. Perfluorocarbon Mediated Self-Assembly of Polymers. Ph.D. Thesis,
University of Southern California, August 2009.
13

10
Gladysz, J. A.; Curran, D. P.; Horváth, I. T. Handbook of Fluorous Chemistry. Wiley-
VCH: Weinheim, 2004; pp 19.
11
Lemal, D. M. “Perspective on Fluorocarbon Chemistry” J. Org. Chem. 2004, 69, 1-11.
12
Ishchuk, Y. L. Lubricating Grease Manufacturing Technology. New Age International
Publishers: New Delhi, 2010; pp 23.
13
Henne, A. L. and Fox, C. J. “Ionization Constant of Fluorinated Acids” J. Am. Chem.
Soc. 1951, 73, 2323-2325.
14
Henne, A. L. and Stewart, J. J. “Fluorinated Amines” J. Am. Chem. Soc. 1955, 77,
1901-1902.
15
Gladysz, J. A.; Curran, D. P.; Horváth, I. T. Handbook of Fluorous Chemistry. Wiley-
VCH: Weinheim, Germany, 2004; pp 43.
16
Rocaboy, C.; Rutherford, D.; Bennett, b.; Gladysz, J. A. “Strategy and Design in
Fluorous Phase Immobilizations: A Systematic Study of ‘Pony Tails’ (CH
2
)
3
(CF
2
)
n-1
CF
3
”
J. Phys. Org. Chem. 2000, 13, 596-603.
17
Knunyants, I. L.; Zeifman, Y. V.; Lushnikova, T. V.; Rokhlin, E. M.; Abduganiev, Y.
G.; Utebaev, U. “Dehydrofluorination with Triethylamine-Boron Trifluoride Adduct.
New Synthesis of Perfluoromethacrylic Acid Derivatives and Related Compounds” J.
Fluorine Chem. 1975, 6, 227-240.
18
Okazoe, T. “Overview on the History of Organofluorine Chemistry from the Viewpoint
of Material Industry” Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2009, 85, 276-289.
19
Holt, D. J.; Payne, R. J.; Abell, C. “Synthesis of Novel Fluorous Surfactants for
Microdroplet Stabilization in Fluorous Oil Streams” J. Fluorine Chem. 2010, 131, 398-
407.
14

20
Gentilini, C.; Boccalon, M.; Pasquato, L. “Straightforward Synthesis of Fluorinated
Amphiphilic Thiols” Eur. J. Org. Chem. 2008, 2008, 3308-3313.
21
Graupe, M.; Koini, T.; Wang, V. Y.; Nassif, G. M.; Colorado Jr., R.; Villazana, R. J.;
Dong, H.; Miura, Y.; Shmakova, O. E.; Lee, T. R. “Terminally Perfluorinated Long-
Chain Alkanethiols” J. Fluorine Chem. 1999, 93, 107-115.
22
Hansen, R. L. “Perfluoroalkanesulfonate Esters as Alkylating Agents” J. Org. Chem.
1965, 30, 4322-4324.
23
Bříza, T.; Král, V.; Martásek, P.; Kaplánek, R. “Electrophilic Polyfluoroalkylating
Agents based on Sulfonate Esters” J. Fluorine Chem. 2008, 19, 235-247.
24
Prescher, D.; Thiele, T.; Ruhmann, R. “Various Synthetic Approaches to Fluoroalkyl
p-nitrophenyl Ethers” J. Fluorine Chem. 1996, 79, 145-148.
25
Robb, I. D. Specialist Surfactants. Blackie Academic & Professional: London, UK,
1997, pp 115.
26
Kirsch, P. Modern Fluoroorganic Chemistry; Wiley-VCH: Weinheim, 2004; pp 11-12.
27
Orner, B. P.; Derda, R.; Lewis, R. L.; Thomson, J. A.; Kiessling, L. L. “Arrays for the
Combinatorial Exploration of Cell Adhesion” J. Am. Chem. Soc. 2004, 126, 10808-
10809.
28
Horváth, I. T. and Rábai, J. “Facile Catalyst: Separation Without Water: Fluorous
Biphase Hydroformylation of Olefins” Science 1994, 266, 72-75.
29
Curran, D. P. “Strategy-Level Separations in Organic Synthesis: From Planning to
Practice” Angew. Chem. Int. Ed. 1998, 37, 1174-1196.
15

30
Fukui, Y.; Brückner, A. M.; Shin, Y.; Balachandran, R.; Day, B. W.; Curran, D. P.
“Fluorous Mixture Synthesis of (-)-Dictyostatin and Three Stereoisomers” Org. Lett.
2006, 8, 301-304.
31
Ko, K.-S.; Jaipuri, F. A.; Pohl, N. L. “Fluorous-Based Carbohydrate Microarrays” J.
Am. Chem. Soc. 2005, 127, 13162-13163.
16
Chapter 2. Synthesis of a Guanidine-Functionalized Fluorous Amphiphile and Its
Binding to Phosphonates

With Dr. Emine Boz and Amy Sirkis. This work was published on a peer-reviewed
journal.
1

2.1 Introduction
2.1.1 End-Functionalized Fluoroalkanes with an Emphasis on Amine-
Functionalized Fluorocarbons
As discussed in Chapter 1, synthesis of fluorous materials often starts with fluorous
alcohols that are cheap byproducts from Teflon synthesis.
2,3
The alcohol is usually further
end-functionalized to impart desirable properties to the product. C-O bond cleavage is
considered difficult in these molecules due to the strong electron withdrawing effect of
fluorine. In order to end-functionalize fluoroalkanes, facile S
N
2 displacements are
enabled by converting the hydroxyl functional group into powerful leaving groups such
as sulfonates.
3,4
The triflate functionality is widely considered as the most popular
perfluoroalkylation reagent not only because -CF
3
is strongly electron withdrawing, but
also because its small size makes S
N
2 displacement less sterically hindered.
Historically, primary amines vicinal to fluorocarbons have been synthesized via
fluoroalkylation of ammonia with fluoroalkyl chloride, hydrogenation of azides,
utilization of the Gabriel synthesis, etc.
5
The hydrogenation of an azide is the most
desirable because of the azide’s low pK
a
value and high nucleophilicity. The product, a
primary amine, can be further transformed into a variety of functional groups such as
17
guanidine and amide. Guanidine is widely present in biological molecules and can be
synthesized via thiourea, isothiourea, carbodiimide starting materials.
6
It can also be
synthesized via guanyl triflate or guanyl pyrazole. Guanidine is known to form bidentate
hydrogen bonds with phosphonate (Figure 2.1a); this interaction is stable over a wide
range of pH values.
7
An excellent usage of this interaction is the guanidine-rich
molecular transporters (GRTs), in which polycarbonates are end-functionalized with
guanidine to assist crossing of the cell membrane (Figure 2.1b).
8

Figure 2.1. a. Guanidiniums are capable of forming bidentate hydrogen bonds with
phosphonates. b. An example of the guanidine-rich molecular transporter in which the
polycarbonate backbone is functionalized with guanidinium.

2.1.2 Goal and Hypothesis
Phosphonates can be found in many biological molecules including cell membranes,
DNA, RNA, and proteins. We were interested in constructing a fluorous amphiphile that
was capable of forming strong hydrogen bonds with phosphonates. It was envisioned that
such fluorous amphiphile could be constructed by attaching a water-soluble polyethylene
glycol segment to a Teflon segment. We then planned to install a guanidinium head
group next to the fluorocarbon so that the construct could bind to negatively charged
O
O
O
O
O
H
O
N
H
NH
NH
2
n
Probe/Drug
O CF
3
O
n

a.
b.

18
functional groups such as carboxylate and phosphonate. The envisioned fluorous
amphiphile has the structure depicted in Figure 2.2. As the amphiphile consists of the bio-
friendly polyethylene glycol and guanidinium, it could find useful applications in bio-
mimicking environment. To prove that the fluorous amphiphile can bind to phosphonates,
we hypothesize that the binding between the fluorous amphiphile and phosphonates will
cause an increase in the molecular weight of the phosphonates, which will be reflected by
the shortened T
1
of the phosphonates on
31
P NMR.

Figure 2.2. A general structure of the desired fluorous amphiphiles.

2.2 Results and Discussion
2.2.1 Methodology Development: Synthesis of the Fluorous Amphiphile
Fluorous diols are a cheap byproduct from the Teflon synthesis. We began begin with
the octafluoro analog (2.1a) and applied the experience to fluorocarbons of other lengths.
The first step in the synthesis was tosylation of tetraethylene glycol monomethyl ether.
Then fluorous diol 2.1a was monoalkylated with tosylated tetraethylene glycol
monomethyl ether (Scheme 2.1). By using three equivalents of the fluorous diol,
monoalkylation was attained in 51-60% yield. The fluorous alcohol 2.4a was smoothly
converted to triflate 2.5a, which was displaced by an azide to set up the nitrogen center
proximal to the fluorocarbon. Compound 2.6a was then hydrogenated to reveal amine
O
O
O
O
O N
H
FF
NH
2
NH
n
n ≥ 3
19
2.7a at 70% yield. The traditional Gabriel conditions afford the amine 2.7a in 93% yield.
Although the Gabriel synthesis was also readily available, the azide synthesis was
superior to phthalimide formation due to the lower pK
a
value of the azide is less prone to
induce dehydrofluorination. The amine was converted over an 11-days period into
guanidinium 2.9a at 92% yield. The long period suggested that proximal fluorocarbons
had greatly attenuated the nucleophilicity of the nitrogen. We chose 1H-guanyl-pyrazole
hydrochloride as our guanylation reagent, because the reaction is relatively clean and
facile.
9
For example, heavy metal is required to form the intermediate carbodiimide,
when guanidine is synthesized via a thiourea.

Scheme 2.1. Synthesis of Fluorous Amphiphiles.

a: n = 4, b: n = 3, c: n = 6

O OH
4
TsCl, Pyr
DCM
-20
o
C, 12 h
94%
O OTs
4
HO OH
n
F F
2.1a-c
, NaH, dioxane
90
o
C, 12 h
51-60%
O
O OH
F F
4 n
2.2 2.3
2.4a-c
TfCl, TEA
0
o
C - rt, 19 h
60-63%
O
O OTf
F F
4 n
2.5a-c
20

O
O OTf
F F
4 4
2.5a
84-99%
NaN
3
, DMF
rt, 12 h
O
O N
3
F F
4 n
2.6a, b
O
O NH
2
F F
4 n
2.7a-c
H
2
(balloon), Pd/C,
DMF or EtOH, 3 h
68-72%
1. NaN
3
, DMF, 6.5 h
2. H
2
(balloon), Pd/C, DMF
65-70%
Synthesis of the amine via azide intermediate:
O
O OTf
F F
4 4
2.5a
KN
O
O
, DMF, 65
o
C
84%
O
O
F F
4 4
2.8
N
O
O
H
2
NNH
2
H
2
O, EtOH, 65
o
C
93%
O
O NH
2
F F
4 4
2.7a
Amine synthesis via the phthalimide:
21

After completing the octafluoro analogs, the conditions were applied to hexafluoro and
dodecafluoro analogs. The results of the synthesis of the fluorous amphiphiles are
summarized in Table 2.1.

2.2.2 Methodology Development: Fluoroalkyl Compounds
Before synthesizing the target fluorous amphiphile, we studied the reactivities of
fluoroalkanes as the model reactions. We considered using the cheap fluorous diol 2.1a
as the source of fluorous segment and our model compound. Compound 2.1a can be
smoothly converted to ditriflate 2.10, which can be put through the route described in
Scheme 2.2. The results of model reactions are summarized in Table 2.2.
Initial alkylation reactions revealed that treating 2.10 with potassium enolate afforded a
cycloheptane 2.11 at moderate yield (Table 2.2, entry 1). Although fluorocarbons are
generally considered to be more rigid than their hydrocarbon counter part, this reaction
proved that octafluorododecanes are flexible to some degree.
O
O NH
2
F F
4 n
2.7a-c
N
N
HN
H
2
N
HCl
, DMF, DIEA, rt, 11 d
O
O
H
N
F F
4 n
2.9a-c
NH
2
NH
HO CF
3
O
56-92%
Guanylation of the amines:
22

Table 2.1. Synthesis of the Fluorous Amphiphile.

Entry
Starting
Material
Product Conditions Yield
1 2.2

2.3 TsCl, pyridine,
DCM, -20
o
C
94%
2 2.3

2.4a
NaH, dioxane, 90
o
C
62%
3 2.3

2.4b
NaH, dioxane, 90
o
C
51%
4 2.3

2.4c
NaH, dioxane, 90
o
C
60%
5 2.4a

2.5a
TfCl, THF, 0
o
C-rt
81%
6 2.4b

2.5b
TfCl, THF, 0
o
C-rt
60%
7 2.4c

2.5c
TfCl, THF, 0
o
C-rt
93%
8 2.5a

2.6a
NaN
3
, DMF, rt
84%
9 2.5b

2.6b
NaN
3
, DMF, rt
99%
10 2.5c

2.6c
NaN
3
, DMF, rt
87%

23
11 2.6a

2.7a
1. NaN
3
, DMF
2. H
2
(balloon),
Pd/C, rt
70%
12 2.6b

2.7b H
2
(balloon),
Pd/C, rt
72%
13 2.6c

2.7c
1. NaN
3
, DMF
2. H
2
(balloon),
Pd/C, rt
65%
14 2.5a

2.8 KN(phthal),
DMF, 85
o
C
84%
15 2.8

2.7a H
2
NNH
2
, EtOH,
65
o
C
93%
16 2.7a

2.9a
EtN(i-Pr)
2
, DMF,
rt
92%
17 2.7b

2.9b
EtN(i-Pr)
2
, DMF,
rt
57%
18 2.7c

2.9c
EtN(i-Pr)
2
, DMF,
rt
56%

Compound 2.10 can be easily displaced by Br
-
using NaBr and a catalytic amount of
18-crown-6 (Table 2.2,entry 2). Similarly, 2.10 was transformed cleanly into diazide 2.13
using NaN
3
(Table 2.2, entry 3). Displacement with phthalimide was also relatively facile
(Table 2.2, entry 4), and the resulting dithalimide 2.14 was transformed to the amine in
76% yield (Table 2.2, entry 5). Hydrogenation of 2.12 using Pd/C led to extensive
O
O NH
2
F F
4 4
O
O NH
2
F F
3 4
O
O NH
2
F F
6 4
O
O N
F F
4
O
O
4
O
O NH
2
F F
4 4
O
O
H
N
F F
4 4
NH
2
NH
TFA
N
N
NH
2
NH
HCl
O
O
H
N
F F
3 4
NH
2
NH
TFA
N
N
NH
2
NH
HCl
O
O
H
N
F F
6 4
NH
2
NH
TFA
N
N
NH
2
NH
HCl
24
degradation (Table 2.2, entry 6). Lindlar conditions with quinoline were employed to
attenuate the reactivity at Pd to obtain the diamine 2.15 (Table 2.2, entry 7). Additionally,
2.13 was converted into ditriazole 2.16 under classic Cu(I)-catalyzed click-chemistry
conditions (Table 2.2, entry 8).

2.2.3 Binding Between the Fluorous Amphiphile and Phosphonates
Theoretically, binding of a large molecule to a small molecule would result in
significant increase in molecular weight and slower tumbling of the small molecule. The
increase in rotational correlation time (τ
R
) is conveniently reflected by line broadening on
a NMR spectrum. If fluorous guanidinium 2.9a is capable of forming strong hydrogen
bond with a phosphonate, the interaction would lead to broadening of the phosphonate
peak on a
31
P spectrum. Methyl phosphonic acid is the smallest phosphonate available
and is represented by a single peak at 21.5 ppm on
31
P spectrum. In its potassium salt

Scheme 2.2. General Route to Functionalize the Fluorous Ditriflate 2.10a.

HO OH
4
F F
TfCl, TEA
0
o
C to rt
94%
TfO OTf
4
F F
Nu:
Nu Nu
4
F F
2.1a 2.10
25
Table 2.2. Functionalized Ditriflate 2.10 and the Respective Yields.
Entry
Starting
Material
Nucleophile
or reagent
Conditions Product
Yiel
d
1 2.10 enolate
DMF, rt

2.11
57%
2 2.10 KBr DMF, 18-
Crown-6, rt

2.12
81%
3 2.10 NaN
3

DMF, rt

2.13
99%
4 2.10 Kphthal
DMF, 85
o
C

2.14
89%
5 2.14 H
2
NNH
2

EtOH, 65
o
C

2.15
76%
6 2.13 H
2
(balloon) EtOH,
Lindlar, rt
13%
7 2.13 H
2
(balloon)
EtOH,
Lindlar,
quinoline, rt
59%
8 2.13

DMF, CuI, 70
o
C

2.16
85%
F
F
F
F F
F
F
F
MeO
2
C CO
2
Me
Br
Br
F F
F F
F F
F F
N
3
N
3
F F
F F
F F
F F
F F
F F
F F
F F
phthal
phthal
F F
F F
F F
F F
NH
2
H
2
N
Ph H
F F
F F
F F
F F
triazole
triazole
26
form, methyl phosphonic acid was added to 1-3 equiv. of 2.9a. It was observed that the
peak broadened significantly, and little was changed in chemical shift (Figure 2.3a). For
comparison, MePO
3
K
2
was also added to HCl, NH
4
Cl and urea•HCl. Addition of HCl (1-
3 equiv.) induced a significant chemical shift change from 21.5 to 31 ppm with no peak
broadening (Figure 2.3b). Addition of NH
4
Cl caused a slight downfield chemical shift
(Figure 2.3c). Addition of urea•HCl induced a chemical shift of the peak downfield
without evident line broadening (Figure 2.3d). The observed broadening was not caused
by pH or formation of rapidly exchanging species, but rather attributed to formation of a
large molecular complex. The results of the
31
P linewidth study are listed in Table 2.3.

2.2.4 pK
a
of the Fluorous Amphiphile 2.9a
In order to determine the pK
a
of the fluorous amphiphile, a titration was performed
using 0.05M NaOH and 2.9a, which yielded a pK
a
of 10.56 (Figure 2.4). An ordinary
guanidinium’s pK
a
is 13.5. The smaller pK
a
value indicated a strong electron
withdrawing effect of the fluorocarbon region.

27
a. MePO
3
K
2
+ 2.9a

b. MePO
3
K
2
+ HCl

c. MePO
3
K
2
+ NH
4
Cl

d. MePO
3
K
2
+ Urea + HCl

Figure 2.3.
31
P NMR evidence of linewidth broadening.
28
Table 2.3. Linewidth Broadening of
31
P NMR as Evidence of Hydrogen Bond Formation.
Spectrum Content Chemical
shift (ppm)
Half-Height
Width (Hz)
A. 4 equiv MePO
3
K
2
+ 4 equiv. 2.9a 21.6 35.5
A. 2 equiv MePO
3
K
2
+ 2 equiv. 2.9a 21.6 20.0
A. 1 equiv MePO
3
K
2
+ 1 equiv. 2.9a 22.4 11.2
A. 0 equiv MePO
3
K
2
21.4 4.2
B. 3 equiv MePO
3
K
2
+ 3 equiv. HCl 30.4 3.4
B. 2 equiv MePO
3
K
2
+ 2 equiv. HCl 29.9 3.4
B. 1 equiv MePO
3
K
2
+ 1 equiv. HCl 27.8 3.8
B. 4 equiv MePO
3
K
2
21.4 4.2
C. 1 equiv MePO
3
K
2
+ 4 equiv. NH
4
Cl 22.1 4.2
C. 0 equiv MePO
3
K
2
+ 1 equiv. NH
4
Cl 21.8 3.6
C. 0 equiv MePO
3
K
2
21.4 4.2
D. 4 equiv MePO
3
K
2
+ 4 equiv. urea + 2 equiv. HCl 27.7 3.6
D. 1 equiv MePO
3
K
2
+ 1 equiv. urea + 2 equiv. HCl 27.6 3.3
D. 0 equiv MePO
3
K
2
21.4 4.2

29

Figure 2.4. pH titration of 2.9a with 0.05 M NaOH.

2.3 Conclusion
We have developed a relatively facile synthesis pathway to end-functionalize fluorous
alcohols into primary amines and guanidiniums. Through this endeavor, we successfully
avoided dehydrofluorination at vicinal protons by choosing a suitable alkylation reagent
(potassium malonate). The fluorous amphiphile showed promise in forming non-covalent
adduct with phosphonates. Following the initial report, we presented our finding on using
2.9a to modulate the relaxivity of some gadolinium(III)-chelate decorated gold
nanoparticles.
10
In the following chapters, we present interesting results on relaxivity
modulation involving a non-covalent adduct formed between contrast agents and 2.9a.
2.4 References
30

1
Wu, X.; Boz, E.; Sirkis, A. M.; Chang, A. Y.; Williams, T. J. “Synthesis and
Phosphonate Binding of Guanidine-Functionalized Fluorinated Amphiphiles” J. Fluorine
Chem. 2012, 135, 292-302.
2
Kasuya, M. C. Z.; Cusi, R.; Ishihara, O.; Miyagawa, A.; Hashimoto, K.; Sato, T.;
Hatanaka, K. “Fluorous-tagged Compound: a Viable Scaffold to Prime Oligoscharride
Synthesis by Cellular Enzymes” Biochem. Biophy. Res. Commun. 2004, 316, 599-604.
3
Prescher, D.; Thiele, T.; Ruhmann, R. “Various Synthetic Approaches to Fluoroalkyl p-
nitrophenyl Ethers” J. Fluorine Chem. 1996, 79, 145-148.
4
Bříza, T.; Král, V.; Martásek, P.; Kaplánek, R. “Electrophilic Polyfluoroalkylating
Agents based on Sulfonate Esters” J. Fluorine Chem. 2008, 129, 235-247.
5
a. For fluoroalkylation of ammonia with fluoroalkyl chloride: Henne, A. L.; Stewart, J.
J. “Fluorinated Amines” J. Am. Chem. Soc. 1955, 77, 1901-1902.
b. For example of hydrogenation of azide: Greenwald, R. B. “A Facile Preparation of
Highly Fluorinated Diamines” J. Org. Chem. 1976, 41, 1469-1470.
c. For example of Gabriel synthesis: Verez-Herrera, P.; Ishida, H. “Synthesis and
Characterization of Highly Fluorinated Diamines and Benzoxazines Derived therefrom”
J. Fluorine Chem. 2009, 130, 573-580.
6
For a comprehensive review, see: Katritzky, A. R. and Rogovoy, B. V. “Recent
Development in Guanylating Agents” ChemInform 2006, 37.
7
Dietrich, B.; Fyles, D. L.; Fyles, T. M.; Lehn, J.-M. “Anion Coordination Chemistry:
Polyguanidinium Salts as Anion Complexones” Helv. Chim. Acta 1979, 62, 2763-2787.
31

8
Rothbard, J. B.; Jessop, T. C.; Wender, P. A. “Adaptive Translocation: the Role of
Hydrogen Bonding and Membrane Potential in the Uptake of Guanidinium-Rich
Transporters into Cells” Adv. Drug Delivery Rev. 2005, 57, 495-504.
9
Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. “1H-Pyrazole-1-carboxamidine
Hydrochloride: An Attractive Reagent for Guanylation of Amines and Its Application to
Peptide Synthesis” J. Org. Chem. 1992, 57, 2497-2502.
10
Li, V.; Chang, A. Y.; Williams, T. J. “A Noncovalent, Fluoroalkyl Coating Monomer
for Phosphonate-Covered Nanoparticles” Tetrahedron 2013, 69, 7741-7745.
32
Chapter 3. Synthesis of the Second-Generation Fluorous Amphiphile

3.1 Introduction
Fluorine, the most electron negative element on earth, exerts strong electron
withdrawing effects on proximal functional groups.
1
As discussed in Chapter 1, proximal
perfluoroalkyl groups often cause a decrease in the pK
a
values of amines and acids.
2
Our
lab has previously synthesized a three-segmented fluorous amphiphile (2.9a, Figure 1)
which forms macromolecular assembly with phosphonates. The guanidinium and the
fluorocarbon being separated by a single methylene render the pK
a
of the fluorous
amphiphile at 10.56, which is much lower than the normal guanidinium (pK
a
= 13.5)
(Figure 3.1). Our lab has previously synthesized a class of gold nanoparticles that are
surface-coated with a blend of phosphonic acids and gadolinium(III)-chelates (Figure
3.2). Compound 2.9a was previously used to decorate the surface of gold nanoparticles
via non-covalent interaction. The result from 2.9a binding to the gold nanoparticles was
not satisfying, assumably due to the weak binding affinity between 2.9a and the
phosphonic acid. To improve the binding affinity, we planned to increase the pK
a
of the
guanidinium by placing a two-methylenes spacer between the guanidinium and the
fluorocarbon. The pK
a
of the new guanidinium will be higher than its predecessor. The
structure of the desired amphiphile is shown in Figure 3.1.

33

2.9a

3.1
Figure 3.1. First generation fluorous amphiphile with a one-methylene spacer (2.9a) and
second-generation fluorous amphiphile with a two-methylene spacer (3.1).

Figure 3.2. Gold nanoparticles capped with a blend of phosphonic acids and Gd-DOTA-
type chelates.

O
O
H
N NH
2
F F
4 4
NH
2
O CF
3
O
O
O
N
H
F F
4 4
NH
2
NH
2
O CF
3
O
Au
S P
P
OH
O
OH
P
OH
OOH
S
O
N
N
N
N
O
O
O
O
Gd
O
S
H
N
O
S
OH
OH
O
34

Figure 3.3. Fluorous amphiphile 2.9a decorates the surface of the gold nanoparticles via
non-covalent interactions.

3.2 Results and Discussion
3.2.1 Synthesis of Fluorous Amphiphile with a Two-Methylenes Spacer
We started with the cheap fluorous diol 2.1a as our starting material. As indicated in the
Chapter 2, we found that it is convenient to carry out transformations via a versatile
triflate intermediate 2.5a. The triflate can be displaced with potassium enolate to give a
malonyl ester intermediate 3.4 that can be converted to carboxylate 3.3. A Curtius
rearrangement can remove one carbon (Scheme 3.1) from carboxylate 3.3 and leave the
desired number of carbon atoms for the spacer.
The triflate intermediate 2.5a was synthesized as described in the previous chapter, and
it was put through the route described in Scheme 3.2. Malonyl ester intermediate 3.4 was
obtained through alkylation of 2.5a, and we attempted to convert 3.4 into a mono-ester
via Krapcho decarboxymethylation.
3
The harsh conditions led to rapid degradation of 3.4.
S P
P
O
OH
O
P
O
HOO
S
O
N
N
N
N
O
O
O
O
Gd
O
S
H
N
O
S
O
O
O
O
O
H
N
H
N
NH
F F
F F
F F
F F
H
H
O
O
O
O
O
H
N
H
N
NH
F F
F F
F F
F F
H
H
O
O
OH
O
O
O
O
O
H
N NH
NH
F F
F F
F F
F F
H
H
35
Scheme 3.1. Retrosynthesis of 3.1.

Alternatively, 3.4 could be saponified under strong basic conditions to afford 3.5,
which was heated in acidic medium to extrude CO
2
. The two-step synthesis produced 3.3
in a low yield of 27%, presumably because the conditions do not accommodate the
electron demands at the methine in the extrusion step and the basic conditions of
saponification were not compatible with fluoroalkyl functionalities. With the mono-
carboxylic acid 3.3 on hand, we proceeded to perform the Curtius rearrangement. This
transformation was performed previously on fluorocarbons using the highly hazardous
hydrazoic acid•pyridine complex.
4
Compound 3.3 is successfully transformed into 3.6
with 2-azido-1,3-dimethylimidazolinium (ADMC) as the azide source and proton sponge
as the base.
5,6
We observed that using old ADMC could lead to complete degradation of
the starting material. This is mainly caused by the short half-life of ADMC.
7
3.6 was
deprotected via hydrogenation to reveal primary amine 3.2, which was converted to 3.1
over two days.
O
O
N
H
F F
4 4
NH
2
NH
O
O
F F
4 4
NH
2
O
O
F F
4 4
OH
O
O
O
F F
4 4
OMe
O
O
OMe
O
O OTf
F F
4 4
3.1
3.2
3.3
3.4
2.5a
HO CF
3
O
36
Scheme 3.2. Synthesis of the Second-Generation Fluorous Amphiphile, 3.1.

O
O
F F
4 4
KH,
MeO
O O
OMe
80
o
C, THF, 12 h
77%
OTf
O
O
F F
4 4
OMe
O
O
OMe
aq. KOH:EtOH = 1:1
100
o
C, 4h
84%
O
O
F F
4 4
OH
O
O
OH
Dioxane:H
2
O = 7:3, 2% HCl
100
o
C, 2h
27%
O
O
F F
4 4
O
OH
1. ADMC, NaN
3
, CH
3
CN,
0
o
C, 30 min
2. THF, proton sponge
0
o
C, 1 h
3. Toluene, BnOH, 95
o
C
46 %
O
O
F F
4 4
NHCbz
Pd/C, quinoline,
EtOH, 1.5 h
73%
O
O
F F
4 4
NH
2
N
N NH
NH
HCl
, DIEA
DMF, 2 d
70%
O
O
F F
4 4
N
H
NH
2
NH
2
O CF
3
O
ADMC =
N N
Cl
Cl
2.9a 3.4
3.5
3.3 3.6
3.2
3.1
37
A titration experiment was performed using aqueous NaOH solution (0.05 M) and 3.1.
We found the pK
a
value of 3.1 was 10.96, a 0.4 unit enhancement over the predecessor
(Figure 3.4).

Figure 3.4. pH titration of 3.1 in an aqueous solution.

3.2.2
19
F T
1
Measurements
With the two-methylene spacer fluorous amphiphile in hand, we began to test its
binding affinity towards the gold nanoparticles decorated with Gd
3+
-chelates and
phosphonic acid. The four sets of fluorous methylenes in compound 3.1 displays four
peaks on
19
F NMR. To assign the fluorine on
19
F NMR, we used a combination of 1D and
2D NMR techniques (Scheme 3.3).
38
Scheme 3.3. Overview of the Structural Assignment for 3.1.

First, we labeled the four peaks of the
19
F NMR to be 1, 2, 3, 4, and the fluorine atoms
in 3.1 as -O-CH
2
-CF
a2
-CF
b2
-CF
c2
-CF
d2
-CH
2
-NH- (Figure 3.5, Figure 3.6). The
1
H and
13
C
NMR spectra of 3.1 are given in Figures 3.7 and 3.8. With the help of
19
F gCOSY, we
were able to clarify the
3
J
F,F
correlation of the fluorine atoms, and the order of fluorine
atoms along the alkane backbone is determined as 1 → 3 → 4 → 2 (Figure 3.9). On
19
F-
13
C gHMBC, peak 2 is correlated to a carbon at 68.89 ppm (Figure 3.10), which is shown
to have
1
J correlation with a proton at 3.98 on
1
H-
13
C gHSQCAD (Figure 3.11). The 3.98
proton is two bonds away from the polyethylene glycol carbons on
1
H-
13
C gHMBCAD
(Figure 3.12). Therefore, peak 2 represents the -CF
2
- closest to the polyethylene glycol
segment. In this way, we assigned peak 2 to F
a
. Peak 1 is assigned to F
d
, peak 3 to F
b
, and
peak 4 to F
c
(Figure 3.13).

Figure 3.5. The fluorous methylenes of 3.1 are labeled from a-d.
H
N NH
2
C
O
C O
F F
F F
F F
F F
NH
19
F-
13
C
gHMBC
H H H H
1
H-
13
C
gHMBCAD
1
H-
13
C
gHSQCAD
3.99 ppm
-120.07 ppm
68.89 ppm 72.09 ppm
O
O N
H
NH
2
4
F F
F F
F F
F F
NH
a c
b d
HO CF
3
O
39

Figure 3.6. The 4 peaks displayed by 3.1 on a
19
F NMR spectrum are labeled 1-4. (470
MHz, CDCl
3
)

Figure 3.7.
1
H NMR spectrum of 3.1. (500 MHz, CDCl
3
)

40

Figure 3.8.
13
C NMR spectrum of 3.1. (125 MHz, CDCl
3
)

Figure 3.9.
19
F gCOSY spectrum of 3.1. (470 MHz, CDCl
3
)

41

Figure 3.10.
19
F-
13
C gHMBC NMR spectrum of 3.1. (470 MHz in
19
F, CDCl
3
)

Figure 3.11.
1
H-
13
C gHSQCAD NMR spectrum of 3.1. (500 MHz in
1
H, CDCl
3
)

42

Figure 3.12.
1
H-
13
C gHMBCAD spectrum of 3.1. (500 MHz in
1
H, CDCl
3
)

Figure 3.13.
19
F assignment of 3.1. (470 MHz, CDCl
3
)

43
We then measured the reduction in
19
F T
1
in the presence of the gold nanoparticles. The
binding brings fluorine atoms close to the gadolinium center and thus shortens their T
1
.
We chose
19
F T
1
reduction over
1
H relaxivity measurements because the former gives a
direct estimate on the binding affinity between the guanidinium and the phosphonates.
An aliquot of gold nanoparticles is added to a solution of 3.1 (3.1 in ca. 6.3-fold excess),
and
19
F T
1
were recorded. We found when the gold nanoparticles are present, the
19
F T
1

were slightly shortened.
As shown in Table 3.1, the nanoparticles caused an 8.11% decrease to the
19
F T
1
of F
d
,
the fluorine atoms closest to the guanidinium. For F
a
to F
c
, the percentages of decrease
are 4.86%, 6.73%, and 7.37%, respectively.

Table 3.1. Shortened
19
F T
1
in the Presence of Gold Nanoparticles.
Fluorine 3.1 only 3.1 + gold nanoparticles percentage of reduction
F
d
0.4539 0.4171 8.11%
F
c
0.4711 0.4482 4.86%
F
b
0.5471 0.5103 6.72%
F
a
0.5575 0.5164 7.37%

We have previously used the gold nanoparticles to shorten the
19
F T
1
of 2.9a. The
four sets of fluorous methylenes are represented by three sets of peaks on
19
F NMR. We
were able to unambiguously assign the
19
F spectrum using 1D and 2D NMR methods
(Scheme 3.4). To do so, we first labeled the three peaks as 1’-3’ (Figure 3.14), and the
four methylenes were labeled as a’-c’ (Figure 3.15). The
1
H and
13
C NMR spectra of 2.9a
are given in Figures 3.16 and 3.17. The middle fluorous methylenes are considered to
44
have been represented by peak 3’. Using
19
F-
13
C gHMBC and
13
C NMR, we determined
that peak 2’ is correlated to a carbon atom at 68.55 ppm (Figure 3.18). This carbon atom
has
1
J correlation to a set of methylene protons at 4.07 ppm, as shown on
1
H-
13
C
gHSQCAD (Figure 3.19). The protons at 4.07 ppm are two bonds away from the
polyethylene carbons at 72.14 ppm, as shown on
1
H-
13
C gHMBCAD (Figure 3.20). The
19
F assignment is given in Figure 3.21.

Scheme 3.4. Overview of the Structural Assignment for 2.9a.

Figure 3.14. The four fluorous methylenes are labeled from a’-c’ in 2.9a.

N
H
NH
2
C
O
C O
F F
F F
F F
F F H H H H
-119.85 ppm
NH
68.5 ppm
4.07 ppm
1
H-
13
C
gHMBCAD
19
F-
13
C
gHMBC
1
H-
13
C
gHSQCAD
72.14 ppm
O
O
H
N
4
F F
F F
F F
F F
a' b'
b' c'
NH
NH
2
HO CF
3
O
45

Figure 3.15. The peaks on
19
F NMR spectrum of 2.9a are labeled as 1’-3’. (470HMz,
CD
3
CN)

Figure 3.16.
1
H NMR spectrum of 2.9a. (500 MHz, CD
3
CN)

46

Figure 3.17.
13
C NMR spectrum of 2.9a. (125 MHz, CD
3
CN)

Figure 3.18.
19
F-
13
C gHMBC NMR spectrum of 2.9a. (470 MHz in
19
F, CD
3
CN)

47

Figure 3.19.
1
H-
13
C gHSQCAD NMR spectrum of 2.9a. (500 MHz in
1
H, CD
3
CN)

Figure 3.20.
1
H-
13
C gHMBCAD NMR spectrum of 2.9a. (500 MHz in
1
H, CD
3
CN)

48

Figure 3.21.
19
F NMR assignment of 2.9a. (470 MHz, CD
3
CN)

Table 3.2. Shortened
19
F T
1
in the Presence of Gold Nanoparticles.
Fluorine 2.9a
2.9a + gold
nanoparticles
percentage of reduction
F
c’
0.4952 0.3317 27.4%
F
b’
0.4554 0.3477 23.6%
F
a’
0.5332 0.3851 22.5%

Based on the percentage of reduction in
19
F T
1
, it appears that 3.1 has lower binding
affinity towards the gold nanoparticles than 2.9a. The difference in the percentage of T
1

reduction for 2.9a and 3.1 is partly caused by the distance of fluorine atoms from
guanidinium. The fluorine atoms in 2.9a are closer to the guanidinium by one methylene
than the fluorine atoms in 3.1. Since the T
1
measurements did not show any improvement
in binding affinity, amphiphile 3.1 was not further employed in our project.
49
3.3 Conclusion
We have shown that perfluorocarbons can be alkylated using suitable carbon
nucleophiles. Using ADMC, a challenging Curtius rearrangement was successfully
carried out without using hazardous materials. Lastly, the incorporation of an additional
methylene between the fluorocarbon and the guanidinium, the pK
a
value of the
guanidinium increased by 0.4 units. However, this pK
a
value did not satisfy our need in
binding affinity and was not used to modulate the relaxivity of the gold nanoparticles
capped with phosphonic acid and gadolinium(III)-chelates. The well-below-average pK
a

value of the second-generation amphiphile demonstrated the powerful electron
withdrawing effect of the fluorocarbons.

3.4 References

1
Kirsch, P. Modern Fluoroorganic Chemistry: Synthesis, Reactivity, Applications.
Wiley-VCH: Weinheim, 2004; pp 5-7.
2
Henne, A. L.; Stewart, J. J. “Fluorinated Amines” J. Am. Chem. Soc. 1955, 77, 1901-
1902.
3
Krapcho, A. P.; Weimaster, J. F.; Eldridge, J. M.; Jahngen Jr, E. G. E.; Lovey, A. J.;
Stephens, W. P. “Synthetic Applications and Mechanism Studies of the
Decarbalkoxylations of Geminal Diesters and Related Systems Effected in Me
2
SO by
Water and/or by Water with Added Salts” J. Org. Chem. 1978, 43, 138-147.
4
Takakura, T.; Yamabe, M.; Kato, M. “Synthesis of Fluorinated Difunctional
Monomers” J. Fluor. Chem. 1988, 41, 173-183.
50

5
Gilman, J. W.; Otonari, Y. A. “Synthesis of Isocyanates from Carboxylic Acids Using
Diphenylphosphoryl Azide and 1,8-bis(Dimethylamino) naphthalene” Synthetic
Commun. 1993, 23, 335-341.
6
Kitamura, M.; Tashiro, N.; Takamoto, Y.; Okauchi, T. “Direct Synthesis of Acyl Azides
from Carboxylic Acids Using 2-Azido-1,3-dimethylimidazolinium Chloride” Chem. Lett.
2010, 39, 732-733.
7
Kitamura, M.; Norifumi, T.; Satoshi, M.; Tatuo, O. “2-azido-1,3-
dimethylimidazolinium Salts: Efficient Diazo-Transfer Reagents for 1,3-Dicarbonyl
Compounds” Synthesis 2011, 1037-1044.
51
Chapter 4 Introduction to Magnetic Resonance Imaging (MRI): Principles and
Contrast Agent Design

4.1 Introduction to MRI
MRI is a technology based on nuclear magnetic resonance (NMR). Like NMR, it
detects certain types of nuclei. In this case, it is the water protons ubiquitous in our body.
Sometimes, MRI is used to detect
19
F nuclei contrast agents, but
19
F MRI is far less
popular than
1
H MRI in clinical practice.
1
MRI has many advantages over other imaging
techniques. It provides superior soft tissue resolution over X-ray CT (computed
tomography). While CT utilizes harmful ionizing radiation, MRI uses innocent RF
pulses. MRI also penetrates deep into the tissues, creating high-resolution 3D image. In
this way, it is better at providing spatial resolution than optical methods such as
fluorescent imaging. The following sections will provide an introduction to MRI physics.

4.1.1 T
1
and T
2
Relaxation
Relaxation time plays an important role in NMR and in MRI. The process during
which the magnetization precesses around z-axis and revert to the beginning position is
called spin-lattice relaxation. And the time the spin takes for the process is called
longitudinal relaxation time (T
1
). To measure T
1
, a sequence called inversion recovery is
used. The magnetization is pulsed to xy plane, and the “recovered” magnetization is fitted
to an exponential growth curve as a function of time (Figure 4.1). The spin-spin
relaxation refers to a different process where the magnitude of the transverse
52

Figure 4.1. Measuring longitudinal relaxivity using inversion recovery. The nuclear spin
is pulsed on to the xy plane, and it slowly reverts back to z-axis following an exponential
rise growth.

Figure 4.2. A spin echo pulse sequence. It begins with an initial 90
o
pulse flipping the
magnetization to xy plane, followed by evolution time (where loss of phase coherence
occurs), and the 180
o
refocusing pulse. The refocused magnetization evolves for time
period τ and is finally pulsed to xy plane for detection.

z
y
x
B
0
Magnetization
T
1
90
o
180
o
90
o
τ
τ
53
magnetization (M
xy
) exponentially decays. This is due to the loss of phase coherence
among nuclei. The time dependence of this decay is measured by T
2
(spin-spin relaxation
time). T
2
is generally equal to or shorter than T
1
. Spin-spin relaxation often causes signal
to weaken during NMR acquisition. In order to minimize the loss of intensity, a spin-echo
pulse is usually applied before the detection pulse (Figure 4.2). As the spin rotates around
the z-axis, an 180
o
pulse is applied to refocus the spins in the opposite transverse plane.
The loss of phase is reverse in this process.

4.1.2 Principles of MRI
Although the fundamental physics of MRI is built upon that of NMR, there are still
great differences between a MRI scan and a routine NMR run. To leap from a 2D
spectrum to the 3D imaging, it is necessary to incorporate the theory of gradient field into
MRI.
2

Imagine a 3D coordinate system generated within the body, with z-axis being the axis
from head to toe. Generally speaking, the positions of protons along the z-axis are coded
by their precessing frequency. The position of a proton at a certain frequency (a certain
position on z-axis is determined by the so-called “phase-encoding”. To do so, two
gradient field scans that are phase-coded are performed individually along the y- and x-
axis, in order to pinpoint protons at each coordinate within a slice. This concludes how
the position of a proton is coded by its phase information as well as its precession
frequency.
Apparent, the intensity of signal at a certain position of the image is determined by the
number of spins (protons) present and their relaxation time of the proton. If we also take
54
chemical exchange into consideration, MRI can be further specialized to determine the
concentration of metabolites containing exchanging protons. This technique is referred to
as chemical exchange saturation transfer, CEST.
3
When a paramagnetic species is
involved to enhance the contrast of the exchanging proton, the technique is modified to
be paramagnetic chemical exchange saturation transfer, PARACEST.
4

4.1.3 Origin of T
1
- and T
2
- Weighted Images
Generally speaking, two types of MRI images are often acquired: T
1
-weighted and T
2
-
weighted images. Obviously, the T
1
-weighted images produces contrast based on the
differences in T
1
of the water protons, while the T
2
-weighted images are based on the
differences in T
2
. The mechanisms behind the acquisitions are inherently different.
In MRI, the time between the acquisition cycles is usually referred to as TR. In a simple
case, TR is adjusted to be close to a short T
1
value. After a 90
o
pulse, only protons with
this short T
1
value will appear on the image. The protons with long T
1
would not be able
to revert back to equilibrium and will look “dark” after the detection pulse.
Realistically speaking, the differences in T
1
between tissues are often not enough to be
distinguished by simply adjusting TR. Thus, a 180
o
pulse can be applied before the 90
o

pulse. After the 180
o
pulse, the magnetization will revert to back to the equilibrium.
During the relaxation process, a 90
o
pulse is applied. The evolution time between the
180
o
pulse and the 90
o
pulse is referred to at TI. Mathematically speaking, only protons
with T
1
close to TI will appear bright on the image. If TI = 0.7 T
1
, the protons are
considered to be “null” on the image. Protons with even longer T
1
will be dark on the
image. In this way, even protons without drastically different T
1
s can be distinguished.
55
If neither TR or TI adjustment is enough to achieve good resolution, paramagnetic
species should be employed. These are T
1
shortening agents that can be intravenously
injected into the patient. The paramagnetic species usually contains metal ions possessing
unpaired electrons, such as Gd
3+
, Mn
2+
, Co
2+
, and Cu
2+
can also be found in literature.
Since protons with short T
1
appear brighter on the image, applying T
1
contrast agents will
cause the area of interest to “light up”.
The acquisition mechanism for T
2
-weighted images is different from that of T
1
, but they
are conceptually similar. First of all, the differences in T
1
are eliminated by giving the
acquisition sequence a very long TR. In this way, the contrast is solely contributed by the
differences in T
2
. After the 90
o
excitation pulse, the protons will revert back to
equilibrium, and phase coherence is lost during this process. A spin-echo pulse is applied
to refocus the magnetization. The evolution time between the 90
o
excitation pulse and
spin-echo pulse is referred to as TE. If the T
2
of a proton is much longer than TE, then the
phase coherence is not completely lost as before the spin-echo pulse is applied. Thus,
protons with T
2
longer than TE will appear bright on the image. If the T
2
is much shorter
than TE, then the phase coherence is lost before the spin-echo pulse. These protons will
appear dark on the image. Sometimes, T
2
contrast agents are applied to accelerate the
spin-spin relaxation, and the area of interest will appear darker on the image.
The relationship among TR, TE, T
1
, and T
2
is described mathematically by Eq. 1,
given TE << TR:
5

[1]

S
SE
∝M
0
[1−e
(
−T
R
T
1
)
]e
(
−T
E
T
2
)
56
From this equation, we can infer a long TE and a long TR give a T
2
-weighted image. A
short TR and a short TE produce a T
1
-weighted image. A long TR and short TE produce
what is called a proton-weighted image.

4.1.4 T
1
Relaxation Mechanism
As discussed in the previous section, the signal intensity of a T
1
-weighted image is
proportional to the number of protons present and inversely proportional to the T
1
of the
protons. The relaxation theory indicates that the observed relaxation time ((T
i
)
obs
) is a
sum of the paramagnetic relaxation time of the solvent and the diamagnetic contribution
(Eq. 2):
6

1
T
i
!
"
#
$
%
&
obs
=
1
T
i
!
"
#
$
%
&
p
+
1
T
i
!
"
#
$
%
&
d
,
[2]

The “p” and “d” subscripts refer to paramagnetic and diamagnetic, respectively. The
paramagnetic contribution is determined by the concentration of the paramagnetic
species. Relaxivity (r
i
) is defined as the slope of the concentration dependence.
Therefore, we can re-write the paramagnetic contribution in terms of r
i
, as shown in Eq.
3:
,
[3]

Relaxivity (mM
-1
s
-1
) is usually obtained as the slope by plotting (1/T
i
)
obs
vs. [Gd
3+
],
and the diamagnetic contribution is the intercept.
A T
1
contrast agent exerts its relaxivity in two ways: inner- and outer-sphere
relaxation. The inner-sphere relaxation defines how the paramagnetic species affects the
water molecules directly bound to the metal. The outer-sphere relaxation mainly deals
i=1,2
1
T
i
!
"
#
$
%
&
obs
=
1
T
i
!
"
#
$
%
&
d
+r
i
[Gd
3+
] i=1,2
57
with water molecules bound to the ligand via non-covalent interactions. Therefore,
(1/T
1
)
p
is re-written as the inner-sphere (IS) and outer-sphere (OS) contributions (Eq. 4):
1
T
1
!
"
#
$
%
&
p
=
1
T
1
IS
+
1
T
1
OS
[4]

The relationship of inner-sphere relaxation time can be represented by other inner-
sphere factors (Eq. 5):
[5]

Where q is the number of water molecules bound in the inner-sphere, τ
m
is the
residence life time of the water molecule, T
1m
is the relaxation enhancement experienced
by the inner-sphere water molecules, P
m
is the fraction of water molecules inside the
inner-sphere. When τ
m
<< T
1m
, the inner-sphere relaxivity is dominated by T
1m
. The
relaxation enhancement experienced by the inner-sphere water molecules is usually given
by two mechanisms, which are the dipole-dipole and scaler-coupling mechanisms (Eq. 6).
[6]
Because of the nature of the gadolinium(III)-H
2
O interaction is mainly dipolar, the
dipole-dipole mechanism is the main contributor to T
1m
. The dipole-dipole item is
determined by the local correlation time of the contrast agent (τ
1c
), which is expressed as
(Eqs. 7, 8):

, i = 1, 2 [7]
, i = 1, 2 [8]
1
T
1
IS
!
"
#
$
%
&
=
qP
m
T
1m
+τ
m
1
T
1m
=
1
T
1
DD
+
1
T
1
SC
1
τ
ci
=
1
T
ie
+
1
τ
m
+
1
τ
R
1
τ
ei
=
1
T
ie
+
1
τ
m
58
Where T
ie
is the electronic relaxation time of the unpaired electrons and τ
R
is the
rotational correlation lifetime. At field strength related to MRI (ca. 1.5 T), 1/T
ie
is large
enough so that contributions from other terms can be safely ignored. At higher field
strength, modulation from τ
m
and τ
R
must also be taken into consideration.
Thus, in order to maximize 1/ , τ
m
, τ
R
, q, r, T
1e
and T
2e
should be optimized
simultaneously. However, it is most convenient to reach maximum relaxivity by
controlling τ
m
, τ
R
, and q. While the relationship between q, τ
R
and relaxivity is
straightforward, the impact of τ
m
on relaxivity can be subtler. The predicted optimal value
of τ
m
at clinically relevant field strength is 10 ns for small molecule contrast agent, and
100 ps for macromolecular contrast agents. However, once τ
m
is optimized, other
parameters can become the limiting factor of reaching maximum relaxivity. So far, the
theoretically predicted maximum relaxivity (100 mM
-1
s
-1
at 20 MHz) has yet been
achieved.

4.2 MRI Contrast Agents: Small Molecule Contrast Agents
The goal of MRI is to distinguish different types of tissues, including healthy and
diseased. As mentioned in section 4.1.3, the contrast of an MR image arises from the
differences in proton density and proton relaxation rate between tissues. In cases where
the density and the relaxation rate of proton are not enough to distinguish between tissues,
it is necessary to apply contrast agents. The function of a contrast agent is to decrease the
longitudinal or transverse relaxation time of the water proton. In this way, a T
1
contrast
agent will “brighten up” the protons at its preferential location, and a T
2
contrast agent
will darken them. Nowadays, small molecule MRI contrast agents are indispensible to
T
1
IS
59
MRI for accurate diagnosis of diseases. FDA has approved several small molecule
contrast agents, including Dotarem (gadoterate meglumine, Guerbet), Omniscan (GE
Healthcare) and Magnevist (gadopentetate dimeglumine, Bayer). These contrast agents
are relatively stable inside the human body and are easy to use.
7

4.2.1 Acyclic Small Molecule Contrast Agents
Acyclic small molecule contrast agents, those based on diethylenetriamine backbone,
represent an important family of MRI contrast agents. At the time of discovery, Gd-
DTPA
-
had the highest stability constant (logK
GdL
= 22.5) among known Gd
3+
chelates.
6

The q, τ
m
, and τ
R
value of Gd-DTPA
2-
are listed in Table 1. In 1988, Gd-DTPA
2-
became
the first clinically approved MRI contrast agent. Gd-DTPA
2-
is often intravenously
injected into the patient before an MRI scan. Once inside our body, Gd-DTPA
2-
is carried
by blood stream and diffuses out of the veins. Because of the hydrophilic nature of Gd-
DTPA
2-
, it only resides in the extracellular space between cells and is quickly excreted
from human body via glomerular filtration. The circulation time of Gd-DTPA
2-
in blood
is 20 min.
8
Elongation of the circulation time of Gd-DTPA
2-
necessitates synthesis of
DTPA derivatives with decrease the charge and increased hydrophobicity. It is favorable
to modify DTPA with hydrophobic moieties because it allows binding to HSA (Figure
5).
9,10,11,12

The diamide analog of Gd-DTPA
2-
, Gd-DTPA-BMA, is commercially available as
Omniscan (Figure 4.3). Although replacing two of the chelating carboxylic oxygen atoms
with amide oxygen takes a significant toll in the stability constant, the osmolality and
viscosity of the contrast agent is also remarkably lowered (Table 4.1). Compared to Gd-
60
DTPA
2-
, Gd-DTPA-BMA it is more suitable for bolus injection through a small needle,
as the lower osmolality causes less pain in the patient.
13

Alternatively, the blood half-life is increased by covalently attaching Gd-DTPA
2-
to
macromolecules without loss in its stability constant. To modify protein with DTPA, the
free amino groups on the protein are often acylated with DTPA anhydride, followed by
ligation of Gd
3+
. Others have synthesized versatile built-in linker such as –NCS for
attaching Gd-DTPA
2-
to proteins macromolecules.
14,15

Gd-DTPA
2-
Gd-BOPTA
2-

Gd-DTPA-BMA Gd-DTPA-BMEA
Figure 4.3. Gd-DTPA
2-
and some of its derivatives.

N
N
N
O
O
O O
O O
O
O
O
O
Gd
2-
N
N
N
O
O
O O
O O
O
O
O
O
Gd
2-
O
N
N
N
O
O
O O
O O
NH
O
Gd
O
NH
N
N
N
O
O
O O
O O
NH
O
Gd
O
NH
O
O
61
Table 4.1. Osmolalities, Viscosity, LogK
GdL
, q, τ
m
and τ
R
of Selected Contrast Agents.
Gd-DTPA
2-
Gd-DTPA-BMA Gd-DOTA
-

Osmolality (kg
-1
)
a
1.96 0.65 1.35
Viscosity (cP)
a
2.9 1.4 2.0
logK
GdL
22.2 16.9 25.3
r
1
(mM
-1
s
-1
) 3.8 3.9 3.13
q
b
1.1 1.1 1.2
τ
m
(ns)
b
303 1000 108
τ
R
(ps)
b
103 25 56
reference 6,16, 17 6, 17, 18 6, 19
a
The osmolalities and viscosities were measured at 37
o
C in 0.5M contrast agent solution.
The osmolality of human plasma is 0.3 osmol kg
-1
.
b
q, τ
m
, τ
R
measured at 25
o
C.

4.2.2 Cyclic Small Molecule MRI Contrast Agents
Achieving higher stability constant requires synthesis of rigid backbone, such as those
used for macrocyclic contrast agents. These cyclic MRI contrast agents have much higher
stability constant over the acyclic ones. Gd-DOTA
-
is the representative member of this
family. The meglumine adduct of Gd-DOTA
-
is commercially available as Dotarem
(Guerbet), which was approved for clinical use in Europe in 1989. Dotarem was
approved by FDA in 2013. Gd-DOTA
-
has a short half-life due to its anionic charge.
The reported number of coordinated water on Gd-DOTA
-
ranges between 0.98 to 1.2,
indicating there is one water molecule bound in the inner-sphere of Gd-DOTA
-
. The
viscosity and osmolality are between Gd-DTPA
2-
and Gd-DTPA-BMA, indicating its
single negative charge nature (Table 4.1). Like all Ln-DOTA
-
complexes, Gd-DOTA
-

exists as m and M isomers in solution as a result of different torsion angles of the Ln-N-
C-COO
-
bonds.
20
The M isomer has an antiprismatic geometry, while the m isomer has a
twisted antiprismatic geometry. It is hard to consider the two isomers to be enantiomers.
62
In solution, the M and m isomers of all Ln-DOTA
-
complexes exist in equilibrium. Using
[Eu(DOTAM)(H
2
O)]
3+
as a model complex, it was found that the k
ex
of the m form is 50
times greater than that of the M form. The equilibrium constant between M and m forms
for [Eu(DOTAM)(H
2
O)]
3+
is K = [M]/[m] = 4.5, and m form accounts for 90% of the
water exchange rate (Figure 4.4).
21
Increasing the percentage of m isomer in the solution
structure of Gd-DOTA
-
should suffice the need for speeding up k
ex
. It was suggested that
DOTA-like ligands with α-substitution of the cyclen nitrogen favors the m form, which
can be helpful for designing future ligands.
22

Figure 4.4. The isomerization equilibrium of the two isomers, M and m, in aqueous
solution. The conformations of the cyclen are the same in the two isomers. The
arrangement of the acetate arms in m isomer are inverted with regard to the M isomer.

4.2 Relaxivity Optimization by q, τ
m
, τ
R
modulation
Safety and relaxivity enhancement are considered top priorities for contrast agent
design. It is equally important to increase the specificity and retention time of the contrast
agents. As indicated by Solomon-Bloembergen-Morgan equation, adjusting q, τ
R
, and τ
R

could enhance the innersphere relaxivity in predictable ways. Along these lines, a variety
N
N
N
N
O
R
O
R
O
R
O
R
Ln
N
N
N
N
Ln
R
O
R
O
R
O
R
O
M m
63
of novel contrast agents were designed, and they are superior in contrast enhancement or
specificity than their predecessors.
4.3.1 Relaxivity Enhancement via τ
R
Modulation
Attaching a small molecule contrast agent to a macromolecule often leads to slower
molecular motion of the contrast agent. If the metal-OH
2
vector successfully couples with
the molecular motion vector, the net result is an increased τ
R
in inner-sphere relaxation
and an enhancement in r
1
. This is a frequently employed strategy to achieve maximum
relaxivity of contrast agents. Macromolecule MRI contrast agents are also particularly
attractive for the purpose of MRI angiography, because the large size prevents early
excretion and elongates the retention time in the human body.
Early macromolecular MRI contrast agents were those consisting of a dendrimer
backbone with small molecule contrast agents covalently attached to the branches.
PAMAM dendrimer-DOTA-gadolinium chelates complexes were conveniently
synthesized by attaching Bz-NCS functionalized DO3A to the terminal amines of G3-G5
PAMAM dendrimers.
23
The resulting G5 dendrimer each carried up to 57 Gd
3+
ions; its
relaxivity reaches 18.8 mM
-1
s
-1
at 25 MHz, while the half-life in rat increases to 115(±8)
min. Using
17
O NMR, it was found that the water exchange rates for G3-G5 and Gd-
DO3A-Bz-NO
2
monomer were essentially the same, and relaxivity was only enhanced by
slower molecular tumbling.
Aime et al. designed a dendrimer-based contrast agent containing a Gd-DOTA like
barycenter (the center of mass).
24
In this construct, the motion of the Gd-DOTA like
barycenter coordinates with the molecular motion of the whole complex (Figure 4.5). The
relaxivity of PEO dendrimer-gadolinium chelates complex increases with the number of
64
dendrimer generation, reaching an r
1
as high as 19.6 mM
-1
s
-1
for G3; on the other hand,
τ
m
rose from 42 to 570 ns. Because the water exchange drops sharply with the number of
generation, outer sphere relaxation is considered the major contributor to r
1
.
Relaxivity can also be enhanced by the formation of micelles. The Merbach group
synthesized amphiphilic Gd-DO3A chelates that undergo self-assembly to give micelles
with greatly enhanced contrast (Figure 4.6).
25
The initial micelles containing purely
gadolinium(III) chelates were too mobile, and cholesterol was incorporated to render
rigidity. In the micelles, the amphiphilic gadolinium(III)-chelates have the same water

Figure 4.5. To couple the Gd-OH
2
vector with the molecular motion, the Gd
3+
core was
assembled into the barycenter of the dendrimer.

N
N
N
N
O O
O
O
O O
O
O
Gd
CONHR
RHNOC
CONHR
RHNOC
O
O
O
O
HO
HO
OH
OH
O
O
OH
OH
OH
OH
R=
R = Me, r
1
= 5.8 mM
-1
s
-1
, r
1
= 19.6 mM
-1
s
-1
65

Figure 4.6. The long chain alkyl conjugated Gd-DOTA forms micelle in solution with
5.3 times of contrast enhancement.

exchange rates as Gd-DOTA
-
. Due to its high τ
R
value (920 ± 40 ps), r
1
was enhanced to
18.01 mM
-1
s
-1
. The τ
R
and r
1
of ordinary Gd-DOTA
-
are 90 ± 15 ps and 3.4 mM
-1
s
-1
.
For MRI angiography purpose, it is desirable to design contrast agent that binds to
protein in blood serum. Small molecule MRI contrast agents can attach to protein via
covalent or noncovalent bond. For covalently bound gadolinium(III) chelates, it is
convenient to amidate DTPA anhydride with the amine groups of Lys or the terminal free
amine. BSA modified with DTPA anhydride and loaded with Gd
3+
carries an average of
5.4 Gd
3+
ions per protein molecule, with a r
1
of 19 mM
-1
s
-1
at 20 MHz.
26
Small molecule
contrast agents modified with hydrophobic moieties are capable of binding to protein via
non-covalent interactions. Sherry’s group designed and synthesized Gd-DOTP
5-

derivatives appended with octyl and undecyl side arms.
27
Although incapable of forming
micelles in solution, these Gd-DOTP
5-
derivatives can bind to five sites on HSA and the
relaxivity are immediately boosted with the addition of HSA; the relaxivity is boosted by
robust outer sphere relaxation, for the number of bound water in inner sphere is 0 (q = 0).
N
N
N
N
O O
O
O
O O
O
O
Gd
C
N
O
r
1
= 18.01 mM
-1
s
-1
66
Lu’s lab developed a T
1
-weighted contrast agent with attenuated contrast behavior in
the presence of adenosine (Figure 4.7).
28
They synthesized a 4 kDa DOTA-Gd-tethered
DNA strand and an adenosine-specific aptamer DNA strand. Aptamers are oligonucleic
acid that can specifically recognize and bind to a target molecule. The Gd-DOTA-
tethered strand is partially complementary to the aptamer strand. Thus, the two strands
and streptavidin can form a large 70 kDa hybrid molecule. The increase in molecular
weight leads to slower tumbling of the hybrid and an enhancement in relaxivity. The
DOTA-Gd-tethered strand is released from the hybrid when adenosine is added, thereby
restoring its molecular weight to 4 kDa. They proved this hypothesis by showing that the
r
1
of the hybrid decreases from 14.2 mM
-1
s
-1
to 9.3 mM
-1
s
-1
after a final concentration of
5 mM adenosine was added.
The Meade’s lab took a different approach. They combined a pH-sensing MRI contrast
agent and doxorubicin-prodrug into one modality.
29
Doxorubicin is an anti-cancer drug
widely used in clinic to treat non-Hodgkin’s Lymphoma and acute leukemia. The drug is
attached to a Gd-DOTA-like chelate via an acid-labile hydrazone linker (Figure 4.8). As
cancerous tissues often has lower pH than healthy tissues, this contrast agent can release
the drug at cancer tissue site. At 1 mM or higher, the r
1
of the doxorubicin-Gd chelate
conjugate is concentration-dependent, meaning that the conjugate aggregates at above
this concentration. The unconjugated Gd-DOTA-like chelate does not show concentration
dependence in r
1
. Because the unconjugated form has a smaller MW, its r
1
is also much
lower than the conjugated form. This difference in contrast behavior provided a
convenient handle to monitor the drug release process. In pH = 4.5 buffer, 90% of the
doxorubicin was released within 16 hours. This was indicated by a progressive drop in r
1
.
67

Figure 4.7. The Biotin-tethered aptamer strand forms a 70 kDa hybrid with the Gd-
DOTA-tethered DNA strand, after streptavidin was added. Adenosine binds to the
aptamer strand and separates the hybrid. The reduction in molecular weight causes the
relaxivity of the Gd-DOTA-tethered strand to decrease to 9.3 mM
-1
s
-1
.
strep
Gd
MW = 70 kDa
r
1
= 14.2 mM
-1
s
-1
A
strep
Gd
A
MW = 4 kDa
r
1
= 9.3 mM
-1
s
-1
= 3'-NH
2
-GTGACTGGACC
= 5'Biotin-CACTGACCTGGGGAGTATTGCGGAGGAAGGT
strep = strepavidin
Gd
=
N
N
N
N
O
O
O
O
Gd
O
O
O
A
=
N
N
N
N
NH
2
O
H OH
H H
H H
HO
68

Figure 4.8. The structure of the doxorubicin-Gd chelate conjugate prodrug with an acid-
labile hydrazine linker (left), and the unconjugated Gd-DOTA-like chelate.

Although it is not tested in vivo, this dual-functionality concept provided a new direction
for future contrast agent design.

4.3.2 Relaxivity Enhancement via q Modulation
q modulation is an often used strategy in designing and implementing enhanced
contrast for smart MRI contrast agents. Smart MRI contrast agents, those specifically
respond to certain stimuli by significantly altering their relaxivity, behave like probes to
detect changes in the environment. Stimuli include pH, enzyme, light, metal ion
concentration, temperature, etc. Some stimuli cause irreversible changes in the contrast
behavior, such as enzymes and metal ions. Other factors cause reversible changes, such
as light and pH. The goal of synthesizing smart MRI contrast agent is to detect certain
changes in the external environment.

N
N
N
N
O
O
O
O
Gd
O
O
O
N
H
N
HO
O
O O
HO O
OH
O
CH
3
NH
2
OH
N
N
N
N
O
O
O
O
Gd
O
O
O
N
H
NH
2
69
4.3.2.1 q-Modulation Involving Activation by Biomolecules
The first example of a MRI contrast agent activated by biomolecules (EGad), also the
first smart MRI contrast agent, was reported by Meade’s group in 1997 (Figure 4.9).
30

This can be regarded as the first example of q modulation. The DOTA-like contrast
agent’s inner-sphere coordination site is blocked by a galactopyranose, and the number of
bound water is 0.7. Removal of the galactopyranose by β-galactosidase recovers the H
2
O
binding site on the metal, and q increases from 0.7 to 1.2. The result is MRI contrast
enhancement as a result of enzymatic cleavage.

Figure 4.9. The first example of a smart MRI contrast agent, Egad. After removal of β-
galactopyranose, the number of bound water molecules in the inner-sphere increased by
40%.

4.3.2.2 q modulation Involving Activation by Metal Ions
Metal ions such as Ca
2+
play crucial roles in biological signaling process, and hence
metal ion-sensing MRI contrast agents were of considerable interest to contrast agent
chemists. The key to construct such contrast agent is to covalently modify the DO3A
backbone with a piece of metal ion binding moiety via a suitable linker. The binding
N N
N N
COO
-
COO
-
Gd
O -
OOC
O
CH
2
OH
O
O
O
H
H
β-galactosidase
N N
N N
COO
-
COO
-
Gd
-
OOC
HO
EGad
q = 0.7
Gad
q = 1.2
H
H
2
O H
2
O
70
module should be highly specific towards one type of ion, and the length of the linker
should allow the ion-binding module to mask the inner-sphere binding site on the metal.
After the initial report on enzyme-activated contrast agent, Meade et al. reported a
Ca
2+
-sensing MRI contrast agent in 1999 (Figure 4.10).
31
In their design, two equiv. of
DO3A were covalently attached to BAPTA via propyl linkers. When no Ca
2+
was present,
the four carboxylic arms of BAPTA bind to Gd
3+
and block the binding site of H
2
O.
When [Ca
2+
] increased from 0.1 to 10 µM, the relaxivity of DOPTA-Gd increased from
3.26 mM
-1
s
-1
to 5.76 mM
-1
s
-1
.
Chang et al. also constructed several types of metal ion-sensing MRI contrast agent
that are considered as model examples of ion-sensing contrast agents.
32
One of such
constructs, called Cu-Gad, comprises of a thiol-rich Cu-receptor site and a DOTA-like
Gd core (Figure 4.11). When Cu
+
is absent in the solution, the pyridine moiety in the Cu-
receptor prohibits water binding at the Gd core. After Cu
+
is added, the pyridine
transmetalates to Cu
+
and binds to the metal with other parts of the Cu-receptor (q = 0).
Therefore, water is able to access Gd
3+
(q = 2.2), and the relaxivity increases from 1.5
mM
-1
s
-1
to 6.9 mM
-1
s
-1
, mainly due to an increase in q.

4.3.2.3 q-Modulation Involving Activation by Light
An excellent example of light-switchable smart MRI contrast agent (Gd-SPDO3A)
was reported by Louie et al. in 1997.
33
Light reversibly changes the q value of this
contrast agent, therefore affecting its contrast behavior. The contrast agent consists of a
DOTA-like core attached with a spiropyran pendant arm (Figure 4.12). In Gd-SPDO3A,
a spiropyran moiety is attached to a DO3A backbone via a methylene linker; the oxygen
71
on the spiropyran blocks the binding site at Gd center. The blockage is removed by
visible light irradiation, and spiropyran is converted to the merocyanine isomer (Gd-
MCDO3A). As a result, light irradiation induced 21% loss in relaxivity for this complex.

Figure 4.10. Ca
2+
-sensing smart MRI contrast agent based on the principle of q-
modulation.

N
N
N
N
O
N
O
Gd
O
O
N
N
N
N
N
Gd
Ca
+ Ca
2+
- Ca
2+
DOPTA-Gd
O
O
O
O
O
O
O
O
O
O
O
O
O
-
O
O O
O
O
O O
N
N
N
N
O
N
O
Gd
O
O
N
N
N
N
N
Gd
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
2-
4-
72

Figure 4.11. A Cu
+
-sensing contrast agent consisting of a Cu-receptor site and a Gd-core.
The pyridine arm moves away from the Gd
3+
center after Cu
+
is added.

Figure 4.12. A q-modulated contrast agent switches between the spiropyran form and the
merocyanine form. The negatively charged oxygen atom in merocyanine form is more
coordinating than the neutral oxygen atom in the spiropyran form.

N N
N N
N
Gd
O
O
O
O
N
S
S
N N
N N
Gd
O
O
O
O
N
N
S
S
Cu
Cu
+
q = 0
r
1
= 1.6 mM
-1
s
-1
q = 2.00 ± 0.22
r
1
= 9.3 mM
-1
s
-1
O
O
O
O
H
2
O
O
2
H
N
N
N
N
O
O
O O
O
O
Gd
O
N
N
N
N
N
O
O
O O
O
O
Gd
O
NO
2
N
dark
visible light
Gd-MCDO3A
r
1
= 2.93 mM
-1
s
-1
Gd-SPDO3A
r
1
= 3.72 mM
-1
s
-1
NO
2
73
Recently, Louie group reported a class of light-switchable T
2
contrast agents based on
spiropyran functionalized dextran coated iron oxide nanoparticles (Figure 4.13).
34

Particles coated with hydrophobic spiropyran tend to aggregate in aqueous solution. The
high molecular weight slows down molecular motion and boosts T
2
. When these particles
are irradiated by light, the coating is isomerized into the hydrophilic merocyanine isomer.
The particles become soluble due to the surface charge and dissociates into monomeric
form. The decrease in molecular weight leads to an increase in T
2
. The size increase
induced a shortening of T
2
by 37%, and thus darker contrast was observed where no
visible light was applied. This particle design is novel among smart MRI contrast agent,
but the utility of this design can be limited by the shallow depth of penetration of visible
light.

4.3.3 Relaxivity Enhancement via Outer-Sphere Relaxation
Not all smart MRI contrast agents are designed based on the principle of q modulation.
Pagel et al reported a PARACEST MRI contrast agent activated by caspase-3, which is
an apoptosis-related cysteine peptidase and specifically cleaves the Asp-Glu-Val-Asp
(DEVD) sequence (Figure 4.14).
35
A segment of the DEVD peptide was engineered to
the arm of a Tm-DOTA
-
complex via an amide bond. The peptide caused a significant
PARACEST effect at -51 ppm. Removal of this peptide by caspase-3 reveals the amine
group α- to the cyclen nitrogen, which induces a minor PARACEST effect at 8 ppm.
When a solution of the uncleaved complex was selectively saturated at -51 ppm, a 14.5%
reduction in water MR signal was detected. No change was found in a solution of the
cleaved compound. Furthermore, the uncleaved complex showed increasingly greater
74

Figure 4.13. The CLADIO-NH-SP nanoparticles respond to visible light by shortening
their T
2
.

Figure 4.14. Structure of a contrast agent, DEVD-(Tm-DOTA) activated by caspase-3.
DEVD is a peptide fragment Asp-Glu-Val-Asp that can be specifically recognized by
caspase-3.
Dark or UV light
Visible light
O
N
NO
2
O
N
T
2
= 24.55 ms T
2
= 37.09 ms
NO
2
N
N
N
N
O
O O
O
O
Tm
O
O
O
N
H
DEVD
-
75
PARACEST effect with increasing pH, which can be only explained by proton chemical
exchange between an amide and water.
Because pH plays an important role in physiological processes, it is attractive to
synthesize pH-sensing smart contrast agent as a pH probe. Sherry’s group reported a case
of pH-sensing contrast agent, GdDOTA-4AmP
5-
, which was used in the first case of in
vivo renal and systemic pH imaging (Figure 4.15).
36,37
The pK
a
values of the four
phosphonates groups in the pendent arms range between 6.5 to 8. When pH is below 8,
the protonated phosphonates form hydrogen bond with the water molecule bound to Gd
3+

and catalytically exchange the T
1
-relaxed proton with protons from bulk water. The
relaxivity of GdDOTA-4AmP
5-
increases from 3.5 mM
-1
s
-1
at pH 9.5 to 5.3 mM
-1
s
-1
at
pH 6.3 due to outer-sphere relaxation. This contrast agent does not respond to the pH
change by adjusting its q value, and the contrast behavior is reversible.

Figure 4.15. The Structure of GdDOTA-4AmP
5-
. The pH-sensitive contrast agent
possesses three phosphonate groups in the pendant arms and responds to pH by changing
its r
1
via outer-sphere relaxation mechanism.
N
N
N
N HN
O
HN O
NH
O
NH O
P
P
P
P
GdDOTA-4AmP
5-
Gd
O
HO
O
O
OH
O
O
O
OH
O
O
HO
5-
76
4.3.4 Relaxivity Enhancement via τ
m
Modulation
τ
m
modulation is often seen as a “built-in” functionality of contrast agents. Allen et al.
has comprehensively reviewed the currently strategies of τ
m
modulation.
38
They pointed
out that tuning τ
m
at the metal center for DOTA-like type of ligands can be achieved by
modifying the following parameters: (1) the charge of the Lanthanide(III)-based complex;
(2) the steric crowding at the metal center; (3) the mechanism of water exchange; (3) the
ratio between the M and m isomers.

Figure 4.16. A series of DOTA-based contrast agent with the ethylene bridge in cyclen
core gradually substituted by propylene bridge. As the steric crowding increases at the
metal center, the water exchange rate is accelerated.

Merbach et al. demonstrated that faster water exchange can be achieved by increasing
the steric crowding at the metal center for nine-coordinated Gd
3+
-poly(amino
carboxylates).
39
It is known that these Gd
3+
complexes undergo dissociated mechanism in
water exchange. They successfully replaced the ethylene bridge in the cyclen backbone
N
N
N
N
O
O O
O
O
Gd
O
O
O
-
N
N
N
N
O
O O
O
O
Gd
O
O
O
-
N
N
N
N
O
O O
O
O
Gd
O
O
O
-
Gd-DOTA
-
k
ex
= 4.1 x 10
6
s
-1
Gd-TRITA
-
k
ex
= 270 x 10
6
s
-1
Gd-TETA
-
no inner-sphere H
2
O
77
of DOTA with a propylene bridge (Figure 4.16). After two of such replacements, the
water exchange rate ( ) gradually decreases from 4.1 × 10
6
s
-1
, 270 × 10
6
s
-1
to no
inner-sphere H
2
O. The steric crowding of at the metal center assisted the dissociation of
H
2
O from the metal center.

Figure 4.17. A series of PEG-conjugated Gd-DOTA-like complexes show slowed down
water exchange rate at the metal center.

To fine-tune the water exchange rate at the metal center, Allen et al. synthesized a
series of PEG-conjugated DOTA-like ligands (Figure 4.17). They showed with variable
temperature
17
O NMR experiments that by increasing the length (molecular weight) of
the PEG chain, it is possible to slow down the water exchange rate.
40
In other words,
bringing hydrophilic moieties in the proximity to the metal center causes the water
exchange rate to slow down. Although their approaching provided an interesting direction
for future research, slowing down the water exchange rate is not desirable for synthesis of
contrast agents. The k
ex
of existing contrast agents is already two orders of magnitude
k
ex
298
N
N
N
N
O
O
O
O
Gd
O
O
O
N
H
NHR
R =
R =
R =
R =
O
O
OCH
3
O
3
H
N
O
CH
3
17
O
O
O
O
CH
3
O
O
110
, k
ex
= 2.7 x 10
6
s
, k
ex
= 1.5 x 10
6
s
, k
ex
= 0.83 x 10
6
s
, k
ex
= 0.67 x 10
6
s
78
slower than the theoretically predicted optimal value (10
6
s
-1
compared with the optimal
10
8
s
-1
). However, no hydrophobic fragments have been brought close to the metal center
before. It will be interesting to study if these fragments could slow down or accelerate k
ex

for DOTA-like ligands.

4.4 Conclusion
MRI is a unique type of modern diagnostic technology. It is safe, minimally invasive
and offers high spatial resolution. MRI contrast agents improve the resolution of the
image by speeding up the relaxation rate of water protons. Current strategies to improve
the efficiency of contrast agents fall in two categories: q modulation and τ
R
modulation.
τ
m
modulation is more complicated than the other two and is less appreciated in contrast
agent design. However, to achieve maximum relaxivity, all three parameters must be
optimized simultaneously.
As τ
m
modulation is a less exploited area, we have sought to understand the effect of τ
m

modulation by placing fluorocarbon segment in proximity to the metal center. In Chapter
4, we show the τ
m
and τ
R
of Gd-DOTP
5-
are modulated via formation of a non-covalent
adduct with a fluorous amphiphile. To our knowledge, this is the first case where τ
m
is
modulated by non-covalent interaction and by bringing fluorocarbon moieties close to the
metal center.

4.5 References

1
Cabello, J. R.; Barnett, B. P.; Bottomley, P. A.; Bulte, J. W. M. “Fluorine (
19
F) MRS
and MRI in Biomedicine” NMR Biomed. 2011, 24, 114-129.
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Freeman, R. Magnetic Resonance in Chemistry and Medicine, 1
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Van Zijl, P.C.M and Yadav, N. N. “Chemical Exchange Saturation Transfer (CEST):
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Zhang, S.; Merritt, M; Woessner, D. E.; Lenkinski, R. E.; Sherry, A. D. “PARACEST
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Baert, A. L.; Buckley, D. L.; Parker, G. J. M. Dynamic Contrast-Enhanced Magnetic
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Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. “Gadolinium(III) Chealtes as
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Clarkson, R. B. Contrast Agents I. In Topics in Current Chemistry. Krause, W., Ed.;
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8
Weinmann, H.-J.; Brasch, R. C.; Press, W.-R.; Wesbey, G. E. “Characteristics of
Gadolinium-DTPA Complex: A Potential NMR Contrast Agent” Am. J. Roentgenol.
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9
Weber, R. W. Paramagnetic DTPA and EDTA Alkoxyalkylamide Complexes as MRI
Agents. U.S. Patent 5,130,120, July 14, 1992.
10
Elst, L. V.; Maton, F.; Laurent, S.; Seghi, F.; Chapelle, F.; Muller, R. N. “A
Multinuclear MR Study of Gd-EOB-DTPA: Comprehensive Preclinical Characterization
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11
Uggeri, F.; Aime, S.; Anelli, P. L.; Botta, M.; Brocchetta, M.; de Haën, C.; Ermondi,
G.; Grandi, M.; Paoli, P. “Novel Contrast Agent for Magnetic Resonance Imaging.
Synthesis and Characterization of the Ligand BOPTA and Its Ln(III) Complexes (Ln =
Gd, La, Lu). X-ray Structure of Disodium (TPS-9-145337286-C-S)-[4-Carboxy-5,8,11-
tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oato(5−)]gadolinite(2−) in a
Mixture with Its Enantiomer” Inorg. Chem. 1995, 34, 633-642.
12
Aime, S.; Botta, M.; Dastrú, W.; Fasano, M; Panero, M.; Arnelli, A. “Synthesis and
Characterization of a Novel DTPA-like Gadolinium(III) Complex: A potential Reagent
for the Determination of Glycated Proteins by Water Proton NMR Relaxation
Measurements” Inorg. Chem. 1993, 32, 2068-2071.
13
Runge, V. M. Contrast-Enhanced Clinical Magnetic Resonance Imaging. In Eighteenth
Century Novels by Women; Runge, V. M.; Scott, S.; Rizzo, B, Eds.; The University
Press of Kentucky: Kentucky, 1996; pp 15.
14
Brechbiel, M. W.; Gansow, O. A.; Atcher, R. W.; Scholm, J.; Esteban, J.; Simpson, D.
E.; Colcher, D. “Synthesis of 1-(p-Isothiocyanatobenzyl) Derivatives of DTPA and
EDTA. Antibody Labeling and Tumor-Imaging Studies” Inorg. Chem. 1986, 25, 2772-
2781.
15
Keana, J. F. W. and Mann, J. S. “Chelating Ligands Functionalized for Facile
Attachment to Biomolecules. A Convenient Route to 4-Isothiocyanatobenzyl Derivatives
of Diethylenetriaminepentaacetic Acid and Dethylenediaminetetraacetic Acid” J. Org.
Chem. 1990, 55, 2868-2871.
16
Ou, M.-H.; Cheng, T.-H.; Liu, G.-C.; Wang, Y.-M. “Physicochemical Characterization
of Four Gadolinium(III) DTPA-like Complexes” J. Chin. Chem. Soc. 2005, 52, 895-906.
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17
Tweedle, M. F.; Hagan, J. J.; Kumar, K.; Mantha, S.; Chang, C. A. “Reaction of
Gadolinium Chelates with Endogenously Available Ions” Magn. Reson. Imaging 1991, 9,
409-415.
18
Vogler, H.; Platzek, J.; Schuhmann-Giampieri, G.; Frenzel, T.; Weinmann, H.-J.;
Radüchel, B.; Press, W.-R. “Pre-clinical Evaluation of Gadobutrol: a New, Neutral,
Extracellular Contrast Agent for Magnetic Resonance Imaging” Euro. J. Radiol. 1995,
21, 1-10.
19
Tweedle, M. F. “Physicochemical Properties of Gadoteridol and Other Magnetic
Resonance Contrast Agents” Invest. Radiol. 1992, 27, Suppl 1:S2-6.
20
Aime, S.; Botta, M.; Ermondi, G. “NMR Study of Solution Structures and Dynamics of
Lanthanide(III) Complexes of DOTA” Inorg. Chem. 1992, 31, 4291-4299.
21
Dunand, F. A.; Aime, S.; Merbach, A. E. “First
17
O NMR Observation of Coordinated
Water on Both Isomers of [Eu(DOTAM)(H
2
O)]
3+
: A Direct Access to Water Exchange
and its Role in the Isomerization” J. Am. Chem. Soc. 2000, 122, 1506-1512.
22
Dunand, F. A.; Dickins, R. S.; Parker, D.; Merbach, A. E. “Towards Rational Design of
Fast Water-Exchanging Gd(dota-Like) Contrast Agents? Importance of the M/m ratio”
Chem. Eur. J. 2001, 7, 5160-5167.
23
Margerum, L. D.; Campion, B. K.; Koo, M.; Shargill, N.; Lai, J.-J.; Marumoto, A.;
Sontum. P. C. “Gadolinium(III) DO3A Macrocycles and Polyethylene Glycol Coupled to
Dendrimers: Effect of Molecular Weight on Physical and Biological Properties of
Macromolecular Magnetic Resonance Imaging Contrast Agents” J. Alloy Comp. 1997,
249, 185-190.
82

24
Fulton, D. A.; O’Halloran, M.; Parker, D.; Senanayake, K.; Botta, M.; Aime, S.
“Efficient Relaxivity Enhancement in Dendritic Gadolinium Complexes: Effective
Motional Coupling in Medium Molecular Weight Conjugates” Chem. Commun. 2005,
474-476.
25
André, J. P.; Tóth, É.; Fischer, H.; Seelig, A.; Mäcke, H. R.; Merbach, A. E. “High
Relaxivity for Monomeric Gd(DOTA)-Based MRI Contrast Agents, Thanks to Micellar
Self-Organization” Chem. Eur. J. 1999, 5, 2977-2983.
26
Niemi, P.; Reisto, T.; Hemmila, I.; Kormano, M. “Magnetic Field Dependence of
Longitudinal Relaxation rates of solutions of Various Protein-Gaodlinium
3+
Chelate
Conjugates” Invest. Radiol. 1991, 26, 820-824.
27
Caravan, P.; Greenfield, M. T.; Li, X.; Sherry, A. D. “The Gd
3+
Complex of a Fatty
Acid Analogue of DOTP Binds to Multiple Albumin Sites with Variable Water
Relaxivities” Inorg. Chem. 2001, 40, 6580-6587.
28
Xu, W. and Lu, Y. “A Smart Magnetic Resonance Imaging Contrast Agent Responsive
to Adenosine Based on a DNA Aptamer-Conjugated Gadolinium Complex” Chem.
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29
Frullano, L.; Tejerina, B.; Meade, T. J. “Synthesis and Characterization of a
Doxorubicin-Gd(III) Contrast Agent Conjugate: A New Approach toward Prodrug-
Procontrast Complexes” Inorg. Chem. 2006, 45, 8489-8491.
30
Moats, R. A.; Fraser, S. E.; Meade, T. J. “A ‘Smart’ Magnetic Resonance Imaging
Agent That Reports on Specific Enzymatic Activity” Angew. Chem. Int. Ed. 1997, 36,
726-727.
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31
Li, W.-H.; Fraser, S. E.; Meade, T. J. “A Calcium-Sensitive Magnetic Resonance
Imaging Contrast Agent” J. Am. Chem. Soc. 1999, 121, 1413-1414.
32
Que, E. L.; Gianolio, E.; Baker, S. L.; Wong, A. P.; Aime, S.; Chang, C. J. “Copper-
Responsive Magnetic Resonance Imaging Contrast Agents” J. Am. Chem. Soc. 2009,
131, 8527-8536.
33
Tu, C. and Louie, A. Y. “Photochromically-controlled, Reversibly-activated MRI and
Optical Contrast Agent” Chem. Commun. 2007, 1331-1333.
34
Osborne, E. A.; Jarrett, B. R.; Tu, C.; Louie, A. Y. “Modulation of T
2
Relaxation Time
by Light-Induced, Reversible Aggregation of Magnetic Nanoparticles” J. Am. Chem. Soc.
2010, 132, 5934-5935.
35
Yoo, B. and Pagel, M. D. “A PARACEST MRI Contrast Agent to Detect Enzyme
Activity” J. Am. Chem. Soc. 2006, 128, 14032-14033.
36
Zhang, S.; Wu, K.; Sherry, A. D. “A Novel pH-Sensitive MRI Contrast Agent” Angew.
Chem. Int. Ed. 1999, 38, 3192-3194.
37
Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies, R. J. “Renal and
Systemic pH Imaging by Contrast-Enhanced MRI” Magn. Reson. Med. 2003, 49, 249-
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38
Siriwardena-Mahanama, B. N. and Allen, M. J. “Strategies for Optimizing Water-
Exchange Rates of Lanthanide-Based Contrast Agents for Magnetic Resonance Imaging”
Molecules 2013, 18, 9352-9381.
39
Ruloff, R.; Tóth, É.; Scopelliti, R.; Tripier, R.; Handel, H.; Merbach, A. E.
“Accelerating Water Exchange for GdIII Chelates by Steric Compression around the
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84

40
Siriwardena-Mahanama, B. N. and Allen, M. J. “Modulating Water-exchange Rates of
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85
Chapter 5. A (Fluoroalkyl)Guanidine Modulates the Relaxivity of a Phosphonate-
Containing T
1
Contrast Agent

This work is in collaboration with Dr. Buddhima Siriwardena-Mahanama and Dr. Matt
Allen at Wayne State University. Dr. Anna Dawsey performed synthesis of Ln-DOTA
-

complexes and T
1
measurements on Gd-DOTA
-
at 9.4T .

5.1 Introduction
MRI belongs to the family of high-end diagnostic technologies and is considered
superior to X-ray CT in providing soft tissue resolution. However, contrast agents are
often employed in the diagnostic process and are indispensible to MRI. This is because
the inherent T
1
differences between tissues are often not enough to provide satisfying
contrast. Contrast agents in clinical use are almost exclusively Gd
3+
species complexed
with linear or macrocyclic polyamino(polycarboxylate)-type ligands, such as Gd-DOTA
(Figure 5.1).
1

Responsive MRI contrast agents, also referred to as smart MRI contrast agents, are
those that can respond to certain stimuli by significantly changing their contrast
enhancement. They were developed to increase the specificity and efficiency of contrast
agents, and they can be used as probes to detect certain changes in the environment.
Some stimuli cause irreversible changes in the contrast enhancement, those include
enzymes, metal ions, and small biomolecules; others, such as light and pH, cause
reversible changes in the contrast enhancement.
2

86
The Solomon-Bloembergen-Morgan equation points out that by adjusting inner-sphere
factors such as q, τ
m
and τ
R
, relaxivity of the contrast agents can be modulated in
predictable ways. Allen et al. has comprehensively reviewed τ
m
modulation. They
concluded the four most frequently used methods leading to τ
m
modulation: (1)
modifying the charge of the contrast agent, (2) changing the accessibility of the metal
center to water, (3) modifying the mechanism of water exchange, and (4) changing the
ratio between M and m isomers for DOTA-like chelates.
3
In a separate publication, Allen
et al. demonstrated that τ
m
modulation could be achieved by placing hydrophilic moieties
such as PEG in the proximity of the metal center.
4

Our lab is interested in the development of responsive MRI contrast agents using non-
covalent interactions. We chose non-covalent interactions over covalent types, because
the contrast enhancement caused by the former is reversible. We envision that τ
m

modulation can be achieved by placing a hydrophobic fluorocarbon piece in the
proximity of the metal center. Along these lines, we have designed and synthesized a
three-segmented fluorocarbon amphiphile (2.9a) consisting of a PEGlated tail, a
fluorocarbon segment (-C
4
F
8
-), and a guanidinium head group (Figure 5.1).
5

Guanidinium and phosphonate are known to form robust, bidentate hydrogen bonds that
are stable over a wide pH range.
6
Thus, our fluorous amphiphile is expected to form non-
covalent adduct with phosphonate-containing contrast agents such as Gd-DOTP
5-
(Figure
5.1). By placing the fluorocarbon proximal to the metal center, we hoped to observe a
decrease in r
1
caused by q and τ
m
modulation (Figure 5.2). Urea structurally resembles
guanidine and forms similar bidente hydrogen bond with phosphonates. Therefore, we
envision 2.9a in the non-covalent adduct can be easily displaced by urea, especially when
87
urea is present in excess amount, and external stimulation such as sonication is applied
(Figure 5.3). Because urea does not contain the fluoroalkyl segment like 2.9a, it does not
exert q and τ
m
modulation in the way 2.9a does. Thus, r
1
, q and τ
m
values in the newly
formed non-covalent adduct will be restored. We present our results showing that the
fluorous amphiphile caused a significant contrast enhancement for Gd-DOTP
5-
. The
carboxylate analog, Gd-DOTA
-
, is not similarly affected under the same conditions. Dr.
Buddhima Siriwardena-Mahanama and Dr. Matt Allen at Wayne State University
performed experiments including r
1
measurements at 1.4 T, luminescence decay
experiments, variable temperature
17
O NMR and electronic paramagnetic resonance on
Gd-DOTA
-
. Dr. Emine Boz and Amy Sirkis contributed greatly in developing the
synthesis route for 2.9a.
5
Dr. Emine Boz was the earliest participant in our lab to carry
out projects on sono-activated MRI contrast agents. Our design of the sono-activated
MRI contrast agents was subsequently patented.
7

Gd-DOTA
-
(5.1) Gd-DOTP
5-
(5.2)

2.9a
Figure 5.1. Structures of Gd-DOTA
-
, Gd-DOTP
5-
and the fluorous amphiphile (2.9a).
N
N
N
N
O O
O
O
O O
O
O
Gd
-
OH
2
N
N
N
N
P
P
P
P
O
O
-
O
O
O
-
O O O
O
-
O
-
O
O
Gd
5-
OH
2
O
O
O
O
O
F F
F F
F F
F F
H
N
NH
2
NH
2
O
O
CF
3
88

Figure 5.2. Expected outcome from formation of a non-covalent adduct between Gd-
DOTP
5-
and the fluorous amphiphile (2.9a).
N
N
N
N
P
P
P
P
O
O
-
O
O
O
-
O O O
O
-
O
-
O
O
Gd
5-
OH
2
+
N N
N N
Gd
H
H
HN
NH
NH
F
F
H
H
NH
NH
HN
P
P
P
P
O
O
O
O
O
O
O
O
O
O
O
O
H
H
HN
NH
NH
F
F
F
F
H
H
NH
NH
HN
F
F
↓ r
1
, ↓ q ,↓ τ
m
r
1
, q, τ
m
O
O
O
O
O
F F
F F
F F
F F
H
N
NH
2
NH
2
-
O
O
CF
3
89

Figure 5.3. Urea displaces 2.9a in the newly formed non-covalent adduct. r
1
, q and τ
m
are
no longer modulated.

N N
N N
Gd
H
H
HN
NH
NH
F
F
H
H
NH
NH
HN
P
P
P
P
O
O
O
O
O
O
O
O
O
O
O
O
H
H
HN
NH
NH
F
F
F
F
H
H
NH
NH
HN
F
F
O
H
2
N NH
2
,
sonication
N N
N N
Gd
H
H
HN
NH
H
H
NH
NH
P
P
P
P
O
O
O
O
O
O
O
O
O
O
O
O
H
H
HN
NH
H
H
HN
NH
O
O
O
O
r
1
↑, q ↑, τ
m
↑
90
5.2 Modulated Relaxivity in Gd-DOTP
5-
and Gd-DOTA
-
Systems
5.2.1 Relaxivity Measurements at 9.4 T
Assuming 2.9a can cause relaxivity modulation in Gd-DOTP
5-
, we started by titrating
an aqueous solution of Gd-DOTP
5-
with 2.9a and took T
1
measurement at 9.4 T.
Relaxivity gradually increased as more 2.9a was added. r
1
rose from 3.04(14) to 3.86(22)
mM
-1
s
-1
as 0-8 equiv. of the fluorous amphiphile was added (Table 5.1). 4 eq. of 2.9a
was determined to be the optimal amount, because it ensured a significant enhancement
in relaxivity (20%), and a relatively small amount of the material was used. After
observing the contrast enhancement, we used urea to reverse the contrast enhancement.
Addition of an excess amount of urea is likely to substitute the fluorous amphiphile,
which should restore the relaxivity to 3.04 mM
-1
s
-1
. An excess amount of urea (150 mM
final concentration, 36.5-fold excess than 2.9a) was added to the solution containing 1
equiv. of Gd-DOTP
5-
and 4 equiv. of 2.9a, but no change in r
1
was observed. Sonication
often assists in breaking non-covalent interaction, and it was applied to this sample.
However, the relaxivity was still unaffected after sonication (Table 5.2, Figure 5.4).
We then repeated our procedure on Gd-DOTA
-
with 4 equiv. of 2.9a. We found r
1

decreased from 2.96(6) to 2.69(21) mM
-1
s
-1
at 9.4 T after 4 equiv. of 2.9a was added,
and the addition of urea and sonication restored r
1
to 2.95(7) mM
-1
s
-1
(Table 5.3, Figure
5). Although the data implies that a non-covalent interaction occurred between 2.9a and
Gd-DOTA
-
that was disrupted by sonication, the observed error bars are too large be
conclusive.

91
Table 5.1. Relaxivity (r
1
) of Gd-DOTP
5-
Titrated with 2.9a. (9.4 T)
r
1
(mM
-1
s
-1
) error
0 equiv. 3.04 0.16
1 equiv. 3.22 0.23
2 equiv. 3.52 0.19
4 equiv. 3.67 0.3
8 equiv. 3.86 0.22

Table 5.2. Relaxivity (r
1
) of Gd-DOTP
5-
through Sequential Addition of 2.9a, Urea, and
Sonication (9.4 T).
r
1
(mM
-1
s
-1
) error
0 equiv. 3.04 0.16
4 equiv. 3.67 0.30
4 equiv. + urea 3.56 0.22
4 equiv. + urea + sonication
3.62 0.24

Figure 5.4. Relaxivity (r
1
) measurements on [Gd(DOTP)]
5-
treated with a. 0 equiv. of
2.9a, b. 4 equiv. of 2.9a, c. 4 equiv. of 2.9a + urea, d. 4 equiv. of 2.9a + urea +
sonication. (25
o
C, 9.4 T)
2
2.5
3
3.5
4
4.5
a b c d
r
1
(mM
-1
s
-1
)
92
Table 5.3. Relaxivity (r
1
) of Gd-DOTA
-
through Sequential Addition of 2.9a, Urea, and
Sonication. (9.4T)
r
1
(mM
-1
s
-1
) error
0 equiv. 2.97 0.06
4 equiv. 2.69 0.21
4 equiv. + urea 2.67 0.18
4 equiv. + urea + sonication 2.95 0.07

Figure 5.5. Relaxivity (r
1
) measurements on [Gd(DOTA)]
-
treated with a. 0 equiv. of
2.9a, b. 4 equiv. of 2.9a, c. 4 equiv. of 2.9a + urea, d. 4 equiv. of 2.9a + urea +
sonication. (25
o
C, 9.4 T)

5.2.2 Relaxivity Measurements at 1.4 T
Because the value of r
1
is dependent on field strength, we determined r
1
of both Gd-
DOTP
5-
and Gd-DOTA
-
systems at low field strength. Dr. Buddhima Siriwardena-
Mahanama and Dr. Matt Allen measured relaxivity using similar procedure at 1.4 T.
2
2.2
2.4
2.6
2.8
3
3.2
a b c d
r
1
(mM
-1
s
-1
)
93
Molar relaxivity r
1
for the Gd-DOTP
5-
system increased from 3.17(2) to 3.65(0) mM
-1
s
-1

after 4 equiv. of 2.9a was added. Additionally, 150 mM urea and sonication could not
restore r
1
to 3.17 mM
-1
s
-1
, and r
1
slightly increased to 3.75(1) mM
-1
s
-1
. Thus, results on
Gd-DOTP
5-
at low field strength were similar to those obtained at high field strength
(Table 5.4, Figure 5.6).
At 1.4 T, the r
1
of Gd-DOTA
-
dropped slightly after the addition of 2.9a, from 3.11(0)
to 3.05(0) mM
-1
s
-1
. Addition of urea and sonication did not affect r
1
, which stayed at
3.05(0) mM
-1
s
-1
(Table 5.5, Figure 5.7). Therefore, 2.9a has little or no effect on the r
1
of
Gd-DOTA
-
at low field strength (1.4 T). The results suggest that 2.9a does not strongly
interact with Gd-DOTA
-
.

Table 5.4. Relaxivity (r
1
) of Gd-DOTP
5-
through Sequential Addition of 2.9a, Urea, and
Sonication (1.4 T).
r
1
(mM
-1
s
-1
) error
0 equiv. 3.17 0.02
4 equiv. 3.65 0.00
4 equiv. + urea 3.75 0.01
4 equiv. + urea + sonication 3.75 0.01

94

Figure 5.6. Relaxivity (r
1
) measurements on [Gd(DOTP)]
5-
with a. 0 equiv. 2.9a, b. 4
equiv. 2.9a, c. 4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication. (25
o
C, 1.4 T)

Table 5.5. Relaxivity (r
1
) of Gd-DOTA
-
through Sequential Addition of 2.9a, Urea, and
Sonication (1.4 T).
r
1
(mM
-1
s
-1
) error
0 equiv. 3.11 0.00
4 equiv. 3.05 0.00
4 equiv. + urea 3.05 0.00
4 equiv. + urea + sonication 3.05 0.00

2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
a b c d
r
1
(mM
-1
s
-1
)
95

Figure 5.7. Relaxivity (r
1
) measurements on [Gd(DOTA)]
-
with a. 0 equiv. 2.9a, b. 4
equiv. 2.9a, c. 4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication. (25
o
C, 1.4 T)

5.2.3 Luminescence Decay
The luminescence decay experiments were kindly performed by Dr. Buddhima
Siriwardena-Mahanama and Dr. Matt Allen at Wayne State University with
europium(III) analogs (Eu-DOTA
-
5.3, Eu-DOTP
5-
5.4) synthesized at University of
Southern California. Luminescence decay measurements enable determination of the
number of inner sphere coordinated water molecules. The water coordination number (q)
for Gd
3+
complex is calculated using Horrock’s equation and luminescence-decay
measurements obtained from corresponding europium(III) or thulium(III) complexes.
Europium(III) is considered a better substitute because it is only 0.9% different from
gadolinium(III) in atomic radius.
8,9,10

2
2.2
2.4
2.6
2.8
3
3.2
a b c d
r
1
(mM
-1
s
-1
)
96
Our results show that despite sequential addition of 2.9a, addition of urea, and
sonication, q remained between 0.10 and 0.12 for the Gd-DOTP
5-
system (Table 6).
Similarly, q remained between 0.85-0.87 for the Gd-DOTA
-
system (Table 7). Gd-
DOTP
5-
is known to have a low q value and is largely an outer-sphere relaxation contrast
agent.
11,12,13
Overall, our luminescence-decay studies indicate that proximal fluorocarbon
does not induce significant change to the number of coordinated water molecules in the
inner sphere for Gd-DOTP
5-
or Gd-DOTA
-
.

5.2.4 Variable Temperature (VT)
17
O NMR
The VT
17
O NMR experiments were performed by Dr. Buddhima Siriwardena-
Mahanama and Dr. Matt Allen at Wayne State University using yttrium(III) and
gadolinium(III) analogs synthesized at University of Southern California. Water
exchange rate (k
ex
, which is τ
m
-1
) is usually calculated from VT
17
O NMR data.
14
Merbach
and coworkers extended this method to extract k
ex
for aqueous solutions of Gd
3+

chelates.
15
To obtain k
ex
, NMR linewidth at half height of the
17
O-labelled bulk water
peak obtained from 1%
17
O-enriched aqueous solutions of Y-DOTA
-
(5.5), Gd-DOTA
-
,
Y-DOTP
5-
(5.6), and Gd-DOTP
5-
were fitted to the Swift and Connick equation.
4,16

For Gd-DOTP
5-
system, adding 4 equiv. of 2.9a to a solution of Gd-DOTP
5-
caused
water exchange to slow down, and τ
m
rose from 8.00 to 10.6 ns. This increase is likely
caused by non-covalent interactions between 2.9a and Gd-DOTP
5-
. Urea and sonication
further increased τ
m
to 19.4 ns for reasons we don’t fully understand.
For Gd-DOTA
-
system, τ
m
increased with the addition of 2.9a, from 236 to 268 ns.
2.9a caused the water exchange to slow down at the metal center for Gd-DOTA
-
. After
97
addition of urea and sonication, τ
m
dropped by more than 37% to 131 ns, a value even
shorter than before 2.9a was added. The results indicate that urea has likely displaced
2.9a from Gd-DOTA
-
, restoring the water exchange at the metal center.
Longer τ
m
negatively affects r
1
and decreases the contrast, and because 2.9a elongates
τ
m
for the Gd-DOTP
5-
system, τ
R
is likely to be the main reason for enhanced relaxivity.
The calculated τ
m
for both systems are listed in Tables 5.6 and 5.7.

5.2.5 Electron Paramagnetic Resonance (EPR)
Electron paramagnetic resonance (EPR) spectra were recorded at X-band. The goal is
to find out the peak-to-peak line width (ΔH
pp
) and the Landé g-factor (g) of the EPR
spectrum, which can be used to extract information on the rotational correlation time (τ
R
)
of the inner-sphere.
Dr. Buddhima Siriwardena-Mahanama and Dr. Matt Allen at Wayne State University
recorded the EPR spectra for the Gd-DOTA
-
system, using Gd-DOTA
-
synthesized by
Dr. Anna Dawsey at USC. Gd-DOTA
-
displayed well-defined line shape on EPR
spectrum, and ΔH
pp
and g were easily extracted. Dr. Siriwardena-Mahanama was able to
calculate τ
R
using these data. The results showed that τ
R
was almost unaffected by the
sequential addition of 2.9a, addition of urea, or sonication (Table 5.7), and the non-
covalent interaction between 2.9a and Gd-DOTA
-
was too weak to affect the molecular
motion of Gd-DOTA
-
.
The EPR spectra of Gd-DOTP
5-
system were acquired at USC. We found that EPR
spectrum with well-defined line shape could not be obtained by simply reproducing
literature procedures; most of the EPR spectrum obtained using literature procedures
98
resulted in distorted spectrum, showing large zero-field splitting effects (ZFS).
17,18
ZFS
is caused by aggregation of the metal complexes, and EPR spectra acquisition
necessitates a preparation that does not lead to formation of aggregates. We found
refluxing Gd-DOTP
5-
with EDTA
4-
appeared to have disintegrated the aggregates, giving
well-resolved EPR spectra. The stability constant (LogK
GdL
) of Gd-EDTA
-
is only 17.7,
which is far less than that of Gd-DOTP
5-
(28.8).
1
Therefore, Gd-EDTA
-
cannot form in
the presence of Gd-DOTP
5-
, and any already formed Gd-EDTA
-
will be quickly
converted to Gd-DOTP
5-
.
We proceeded to take EPR spectra of Gd-DOTP
5-
in the EDTA
4-
buffer, with 0 or 4
equiv. of 2.9a. We found that the two samples produced exactly the same EPR spectrum
(Figure 5.8). The negatively charged EDTA
4-
contains four carboxylate pendant arms,
and EDTA
4-
is highly likely to bind to the positively charged 2.9a. When both EDTA
4-

and Gd-DOTP
5-
are present, it is possible that they will compete for the 2.9a. However, if
the Gd-DOTP
5-
solution buffered with EDTA
4-
is titrated with more equivalents of the
fluorous amphiphile, the peak-to-peak line width of the EPR spectrum is likely to change.
The Gd-DOTP
5-
solution buffered with EDTA
4-
was titrated with 8, 25, and 50 equiv. of
the fluorous amphiphile (Figure 5.8). On the EPR spectrum, we observed no change in
the line width, which stayed between 225-227 G. We deduced that 2.9a does not cause
changes to ΔH
pp
, and we proceeded to record EPR on samples treated with urea and
sonication (Figure 5.9). Finally, our collaborator was able to extract τ
m
and τ
R
values with
ΔH
pp
and g obtained from the EPR spectrum (Tables 5.6 and 5.7).
After both τ
m
and τ
R
were solved, it became clear that τ
R
is the main contributor to the
enhanced relaxivity. For Gd-DOTP
5-
system, τ
m
was elongated from 0.8 × 10
-9
to 1.06 ×
99
10
-9
sec after 4 equiv. of 2.9a was added, implying that bringing fluorocarbon to the
proximity of Gd
3+
center induces slower water exchange. Addition of urea and sonication
further slowed down the exchange rate due to reasons we do not fully understand. τ
R
was
also elongated in the presence of 2.9a, indicating a non-covalent adduct has formed
between 2.9a and Gd-DOTP
5-
. 2.9a is known to slow down the molecular motion of
methylphosphonic acid by forming a macromolecular adduct, which was shown to be a
property unique to 2.9a and not to urea or ammonium ion.
5
In this study, 2.9a formed a
non-covalent adduct with Gd-DOTP
5-
and caused slower molecular motion similar to the
methylphosphonic acid case. In the presence of 36.5 fold excess of urea and after
sonication, τ
R
increased only slightly, indicating the non-covalent interaction between
2.9a and Gd-DOTP
5-
is very robust. Overall, our results indicated that the impact of τ
R

overwhelmed τ
m
, and the net result is enhanced relaxivity for Gd-DOTP
5-
system.
For Gd-DOTA
-
system, τ
m
increased from 2.36 × 10
-7
to 2.68 × 10
-7
s in the presence
of 2.9a, meaning that proximal fluorocarbon also caused a reduction in the water
exchange rate at the Gd
3+
center. Addition of urea and sonication caused τ
m
to decrease
significantly for unspecified reasons. τ
R
remained almost unchanged through sequential
addition of 2.9a and urea, and sonication. The molecular motion of Gd-DOTA
-
was
unaffected by 2.9a, indicating that no non-covalent adduct was formed, and the
interaction between 2.9a and the carboxylates of Gd-DOTA
-
is relatively weak.
Gd-DOTP
5-
is known as an outer-sphere relaxation contrast agent. The Gd
3+
ion-
proton distance is longer in Gd-DOTP
5-
than in Gd-DOTA
-
(3.26 Å vs. 3.00 to 3.16 Å).
Aime et al. demonstrated that the relaxivity of Gd-DOTP
5-
is accentuated in the presence
of meglumine, and the relaxivity becomes responsive to pH in the presence of 40-fold
100
excess of meglumine. Similar contrast enhancement was found when hexacyclen was
added to Gd-DOTP
5-
.
19
Molecules like hexacyclen or meglumine (Figure 5.10) forms an
ion pair with Gd-DOTP
5-
. A large number of water molecules are sandwiched between
the ion pair via an extensive hydrogen bond network. These water molecules serve as
second-sphere and outer-sphere water and assist Gd-DOTP
5-
to exert its relaxivity. We
did not observe similar contrast enhancement behavior when 4 equiv. of 2.9a and 36.5
fold excess urea were present in the Gd-DOTP
5-
solution. Moreover, Gd-DOTP
5-
was
previously used by Raghunand et al. as a non-pH sensitive probe in renal and systemic
imaging.
2f
Although copious amount of urea and other metabolites exist in the renal
imaging environment, no relaxivity enhancement was reported for Gd-DOTP
5-
by
Raghunand et al. We suspect this is because unlikely meglumine or exacyclen, 2.9a or
urea does not have functional groups capable of forming hydrogen bonds evenly
distributed along the backbone. This structural difference renders 2.9a or urea unable to
form the extensive hydrogen bond network that harbors the large number of second-
sphere and outer-sphere water molecules.

5.2.6
19
F NMR
Binding between 2.9a and Gd
3+
complexes should cause
19
F T
1
to decrease. Three
different peaks on
19
F NMR represent the four sets of fluorous methylenes in 2.9a. Their
assignment is discussed in Chapter 3 and shown in Figure 5.11. We measured
19
F T
1
for a
sample containing 4:1 ratio of 2.9a to Gd-DOTP
5-
. The fluorous methylene peaks
broadened significantly, and the chemical shifts moved slightly downfield. Gd-DOTP
5-

caused remarkable reduction in
19
F T
1
, with the fluorous methylene close to the
101

Figure 5.8. EPR spectrum of Gd-DOTP
5-
system treated with: a. 0 equiv. 2.9a, b. 4
equiv. 2.9a, c. 4 equiv. 2.9a + urea, d. 4 equiv. 2.9a + urea + sonication.

102

Figure 5.9. EPR study of [Gd(DOTP)]
5-
. a: [Gd(DOTP)]
5-
only. b: [Gd(DOTP)]
5-
+ 8
equiv. of 2.9a. c. [Gd(DOTP)]
5-
+ 25 equiv. of 2.9a. d. [Gd(DOTP)]
5-
+ 50 equiv. of 2.9a.

-10000
8000
2600 4600
Intensity
Field Strength [G]
0 equiv.
8 equiv
25 equiv
50 equiv
103
Table 5.6. Summarized r
1
, τ
R
, τ
m
, and q for Gd-DOTP
5-
System.
Gd-DOTP
5-

Gd-DOTP
5-

+ 2.9a
Gd-DOTP
5-

+ 2.9a +
urea
Gd-DOTP
5-

+ 2.9a +
urea, after
sonication
r
1
(mM
–1
s
–1
)
a
3.04(16) 3.67(30) 3.56(22) 3.62(24)
r
1
(mM
–1
s
–1
)
b
3.17(2) 3.65(0) 3.75(1) 3.75(1)
q 0.12(1) 0.10(1) 0.12(1) 0.12(1)
τ
m
× 10
-9
(s)
0.800 1.06 1.82 1.94
k
ex
× 10
7
(s
–1
) 12.5 9.45 5.50 5.16
T
1e
× 10
–8
(s) 0.345 1.26 0.711 0.710
T
2e
× 10
–10
(s) 2.94 2.97 2.96 2.96
g 1.97 1.96 1.96 1.96
ΔH
pp
(G) 227 225 225 225
τ
R
× 10
–12
(s)
c
119 136 140 140
a
Acquired at 9.4 T.
b
Acquired at 1.4 T.
c
Assuming 56% second sphere and 44% outer
sphere contribution.
19

Table 5.7. Summarized r
1
, τ
R
, τ
m
, and q for Gd-DOTA
-
System.
Gd-DOTA
-

Gd-DOTA
-

+ 2.9a
Gd-DOTA
-

+ 2.9a +
urea
Gd-DOTA
-

+ 2.9a +
urea, after
sonication
r
1
(mM
–1
s
–1
)
a
2.97(6) 2.69(21) 2.67(18) 2.95(7)
r
1
(mM
–1
s
–1
)
b
3.11(0) 3.05(0) 3.05(0) 3.05(0)
q 0.87(1) 0.86(1) 0.85(1) 0.85(1)
τ
m
× 10
-7
(s) 2.36 2.68 1.69 1.31
k
ex
× 10
6
(s
–1
) 4.24 3.73 5.91 7.65
T
1e
× 10
–7
(s) 1.83 7.44 9.14 3.88
T
2e
× 10
–10

(s)
1.44 1.49 2.68 1.09
g 1.98 1.99 1.98 1.98
ΔH
pp
(G) 460.4 443.8 247.3 60.61
τ
R
× 10
–12
(s) 58.7 57.7 58.5 58.6
a
Acquired at 9.4 T.
b
Acquired at 1.4 T.

104

Figure 5.10. Structures of meglumine and hexacyclen.

guanidinium displaying the greatest reduction. T
1
of F
c’
was reduced to 5% of the original
value.
19
F T
1
of F
b’
decreased by 90.3%, and F
a’
was reduced by the least extent (Table
5.8). Based on the percentage of reduction in
19
F T
1
, the relative position of these fluorous
methylenes in solution can be arranged as (from close to far away from the guanidinium):
F
c’
, F
b’
, F
a’
. Our observation complies with a model where binding has occurred between
the phosphonates and the guanidinium.
For Gd-DOTA
-
,
19
F T
1
was measured using the same set of conditions. Broadening of
the fluorine peaks and slight downfield shifts were also observed.
19
F T
1
was significantly
shortened. For T
1
of F
a’
, F
b’
, and F
c’
the percentage of reduction are 53.4%, 59.6% and
70.1%, respectively (Table 5.9). Again, the distance from the guanidinium appeared to
have determined the degree of reduction. This is similar to what was observed for Gd-
DOTP
5-
, and the order indicates binding occurred between 2.9a and the phosphonates.
Because the percentage of reduction in
19
F T
1
is greater for the Gd-DOTP
5-
system than
for Gd-DOTA
-
, we suspected that the binding affinity between 2.9a and Gd-DOTP
5-
is
greater than that between 2.9a and Gd-DOTA
-
.

H
N
OH
OH
OH
OH
OH
NH
N
H
HN
HN NH
H
N
meglumine hexacyclen
105

Figure 5.11.
19
F NMR of an aqueous solution of 2.9a before (top) and after (bottom) the
addition of Gd-DOTP
5-
.

Table 5.8. Reductions in
19
F T
1
in the Presence of Gd-DOTP
5-
.
T
1
(ms) F
a’
F
b’
F
c’

2.9a only 417 507 443
2.9a + Gd-DOTP
5-
70.8 49.2 24.1
percentage of
reduction
83.0% 90.3% 95.2%

Table 5.9. Reductions in
19
F T
1
in the Presence of Gd-DOTA
-
.
T
1
(ms) F
a’
F
b’
F
c’

2.9a only 474 527 501
2.9a + Gd-DOTA
-
221 213 150
percentage of
reduction
53.4% 59.6% 70.1%
106
5.2.7 Diffusion-Ordered NMR Spectroscopy (DOSY 2D)
Aside from EPR, there are other ways to obtain information on the size and motion of
a molecule. DOSY 2D NMR is a convenient method to resolve the diffusion coefficient
(D) of individual components in a mixture.
20
It can be used to obtain diffusion coefficient
of an analyte.
21,22
Formation of a non-covalent adduct between Gd-DOTP
5-
and the
fluorous amphiphile can cause an increase the molecular weight, and this change will be
reflected in the diffusion coefficient.
To support our hypothesis, Y-DOTP
5-
was synthesized. The diffusion coefficient of
the end methyl group of 2.9a was recorded using 0.1% t-BuOH as a reference. Then the
diffusion coefficient of the methyl group was recorded for a sample containing Y-DOTP
5-

and 2.9a in 1:4 ratio. The results shows while D of the reference was the same for both
samples, D of the methyl group decreased slightly after 2.9a was added (Figure 5.12,
Table 5.10). The DOSY 2D results showed that the amphiphile formed a non-covalent
adduct with Y-DOTP
5-
.

-2
-1
0
0 1 2 3 4
(G)
2
ln(I/I
0
)
t
BuOH

Y-DOTP
5-

a.
107

Figure 5.12. DOSY 2D was acquired for samples containing Y-DOTP
5-
. 0.1%
t
BuOH
was used as an external reference. a. Y-DOTP
5-
only. b. Y-DOTP
5-
:2.9a = 1:4. Results
from the DOSY 2D NMR are plotted to obtain D value. G: Gradient Field Strength. I:
Integral of the peak at a given G. I
0
: Integral of the peak at G = 0.

Table 5.10. Diffusion Coefficients (D) of 0.1%
t
BuOH and Y-DOTP
5-
measured by
DOSY 2D at 25
o
C.
D (× 10
-12
m
2
s
-1
)

t
BuOH Y-DOTP
5-

0 equiv. of 2.9a 4.960 ± 0.01738 2.221 ± 0.02059
4 equiv. of 2.9a 4.962 ± 0.009305 2.088 ± 0.01625

5.3 Conclusion
In this study, we observed how Gd-DOTP
5-
responds to 2.9a by accentuating its
contrast enhancement. The increase in relaxivity was caused by formation of a non-
covalent adduct between Gd-DOTP
5-
and amphiphile 2.9a. Placing the fluorocarbon
proximal to the Gd
3+
center caused an increase in τ
m
and decelerated water exchange in
-2
-1.5
-1
-0.5
0
0 1 2 3 4
(G)
2
ln(I/I
0
)
t
BuOH

Y-DOTP
5-
+ 4 equiv. 2.9a
b.
108
the inner-sphere. However, formation of the non-covalent adduct also increased the
molecular weight of Gd-DOTP
5-
and slowed down the molecular motion. The increase in
τ
R
overwhelmed the increase in τ
m
, and the net result is contrast enhancement for Gd-
DOTP
5-
.
Unlike Gd-DOTP
5-
, r
1
of Gd-DOTA
-
was unaffected by 2.9a. The proximal
fluorocarbon also causes a decrease in k
ex,
but r
1
was unaffected in this case. In the
presence of 2.9a, the molecular motion of Gd-DOTA
-
was also unaffected, presumably
because the binding between 2.9a and Gd-DOTA
-
is weak and no long-lived non-
covalent adduct was formed. The polyphosphonate and polycarbonate-based contrast
agents respond to the fluorous amphiphile in distinctive ways because their binding
affinities towards guanidinium are very different.

5.4 References

1
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.
2
For examples of MRI contrast agent responsive to enzyme: a. Moats, R. A.; Fraser, S.
E.; Meade, T. J. “A ‘Smart’ Magnetic Resonance Imaging Agent That Reports on
Specific Enzymatic Activity” Angew. Chem. Int. Ed. 1997, 36, 726-728. b. Stavila, V.;
Allali, M.; Canaple, L.; Stortz, Y.; Franc, C.; Marin, P.; Beuf, O.; Dufay, O.; Samarut, J.;
Janier, M.; Hasserodt, J. “Significant Relaxivity Gap Between a Low-Spin and a High-
Spin Iron(II) Complex of Structural Similarity: an Attractive Off-On system for the
Potential Design of Responsive MRI Probes” New J. Chem. 2008, 32, 428-435.
109

For examples of MRI contrast agent responsive to ion concentration: c. Que, E. L.;
Gianolio, E.; Baker, S. L.; Wong, A. P.; Aime, S.; Chang, C. J. “Copper-Responsive
Magnetic Resonance Imaging Contrast Agents” J. Am. Chem. Soc. 2009, 131, 8527-8536.
d. 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.
For examples of MRI contrast agent responsive to pH: e. Zhang, S.; Wu, K.; Sherry, A.
D. “A Novel pH-Sensitive MRI Contrast Agent” Angew. Chem. Int. Ed. 1999, 38, 3192-
3194. f. Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies, R. J. “Renal and
Systemic pH Imaging by Contrast-Enhanced MRI” Magn. Reson. Med. 2003, 49, 249-
257.
For an example of MRI contrast agent responsive to temperature: g. Fossheim, S. L.; Il’-
yasov, K. A.; Hennig, J; Bjornerud A. “Thermosensitive Paramagentic Liposomes for
Temperature Control During MR Imaging-Guided Hyperthermia: In Vitro Feasibility
Studies” Acad. Radiol. 2000, 7, 1107-15.
For examples of MRI contrast agent responsive to light: h. Tu, T.; Louie, A. Y.
“Photochromically-Controlled, Reversibly-Activated MRI and Optical Contrast Agent”
Chem. Commun. 2007, 1131-1133. i. Osborne, E.; Jarett, B. R.; Tu, C.; Louie, A. Y.
“Modulation of T
2
Relaxation Time by Light-Induced, Reversible Aggregation of
Magnetic Nanoparticles” J. Am. Chem. Soc. 2010, 137, 5934-5935.
3
Siriwardena-Mahanama, B. N. and Allen M. J. “Strategies for Optimizing Water-
Exchange Rates of Lanthanide-Based Contrast Agents for Magnetic Resonance Imaging”
Molecules 2013, 18, 9352-9381.
110

4
Siriwardena-Mahanama, B. N.; Allen, M. J. “Modulating Water-Exchange Rates of
Lanthanide(III)-Containing Polyaminopolycarboxylate-type Complexes using
Polyethylene Glycol” Dalton Trans. 2013, 42, 6724-6727.
5
Wu, X.; Boz, E.; Sirkis, A.; Chang, A.; Williams, T. J. “Synthesis and Phosphonate
Binidng of Guanidine-Functionalized Fluorinated Amphiphiles” J. Fluorine Chem. 2012,
135, 665-669.
6
Dietrich, B.; Fyles, D. L.; Fyles, T. M.; Lehn, J.-M. “Anion Coordination Chemistry:
Polyguanidinium Salts as Anion Complexones” Helv. Chim. Acta 1979, 62, 2763-2878.
7
Chang, A. Y.; Williams, T. J.; Boz, E. “Ultrasound-Activated Nanoparticles as Imaging
Agents and Drug Delivery Vehicles” PCT Int. Appl. WO 2011079317, Jun 30, 2011.
8
Supkowski, R. M.; Horrocks Jr., W. D. “On the Determination of the Number of Water
Molecules, q, Coordinated to Europium (III) Ions in Solution from Luminescence Decay
Lifetimes” Inorg. Chim. Acta 2002, 340, 44-48.
9
Dissanyayake, P.; Mei, Y.; Allen, M. J. “Luminescence-Decay as an Easy-to-Use Tool
for the Study of Lanthanide-Containing Catalysts in Aqueous Solutions” ACS Catal.
2011, 1, 1203-1212.
10
Ulrich, B. D. Peptide-based Magnetic Resonance Imaging Probes for Detection of
Enzyme Activity. Ph.D. Thesis, Northwestern University, December 2008.
11
Literature reports on this value are scattered and have high error. See (a) Geraldes, C.
F. G. C.; Brown III, R. D.; Cacheris, W. P.; Koenig, S. H.; Sherry, A. D.; Spiller, M.
“Evaluation of Polyaza Macrocylic Methylene Phosphonate Chelates of Gd
3+
Ions as
MRI Contrast Agents” Magn. Reson. Med. 1989, 9, 94-104. (b) Anelli, P. L.; Balzani, V.;
111

Prodi, L.; Uggeri, F. “Luminescence Properties of Eu
3+
Complexes of Highly Polydentate
Ligands” Gazz. Chim. Ital. 1991, 121, 359-364.
12
Aime, S.; Botta, M; Terreno, E.; Anelli, P. L.; Uggeri, F. “Gd(DOTP)
5-
Outer-Sphere
Relaxation Enhancement Promoted by Nitrogen Bases” Magn. Reson. Med. 1993, 30,
583-591.
13
Avecilla, F.; Peter, J. A.; Geraldes, C. F. G. C. “X-ray Crystal Structure of a Sodium
Salt of [Gd(DOTP)]
5-
: Implications for Its Second-Sphere Relaxivity and the
23
Na NMR
Hyperfine Shift Effects of [Tm(DOTP)]
5-
” Eur. J. Inorg. Chem. 2003, 4179-4186.
14
Swift, T. J. and Connick, R. E. “NMR (Nuclear Magnetic Resonance)-Relaxation
Mechanism of O
17
in Aqueous Solutions of Paramagnetic Cations and the Lifetime of
Water Molecules in the First Coordination Sphere” J. Chem. Phys. 1962, 37, 307-320.
15
Micskei, K.; Helm, L.; Brücher, E.; Merbach, A. E. “
17
O NMR Study of Water
Exchange on [Gd(DTPA)(H
2
O)]
2-
and [Gd(DOTA)(H
2
O)]
-
Related to NMR Imaging”
Inorg. Chem. 1993, 32, 3844-3850.
16
a. Urbanczyk-Pearson, L. M.; Femia, F. J.; Simith, J.; Parigi, G.; Duimstra, J. A.;
Eckerman, A. L.; Lucinat, C.; Meade, T. J. “Mechanistic Investigation of β-
Galactosidase-Activated MR Contrast Agents” Inorg. Chem. 2008, 47, 56-68. b. Garcia,
J.; Neelavalli, J.; Haacke, E. M.; Allen, M. J. “Eu
II
-Containing Cryptates as Contrast
Agents for Ultra-High Field Strength Magnetic Resonance Imaging” Chem. Commun.
2011, 47, 12858-12860.
17
Pereira, G. A.; Ball, L.; Sherry, A. D.; Peters, J. A.; Geraldes, C. F. G. C. “NMR
Characterization of Lanthanide(3+) Complexes of Tetraazatetrakisphosphinato and
Tetraazatetrakisphophonato Ligands” Helv. Chim. Acta 2009, 92, 2532-2551.
112

18
Geraldes, C. F. G. C.; Brown III, R. D.; Cacheris, W. P.; Koenig, S. H.; Sherry, A. D.;
Spiller, M. “Evaluation of Polyaza Macrocyclic Methylene Phosphonate Chelates of Gd
3+

Ions as MRI Contrast Agents” Magn. Reson. Med. 1989, 9, 94-104.
19
Botta, M. “Second Coordination Sphere Water Molecules and Relaxivity of
Gadolinium(III) Complexes: Implications for MRI Contrast Agents” Eur. J. Inorg. Chem.
2000, 2000, 399-407.
20
Freeman, R. Magnetic Resonance in Chemistry and Medicine; Oxford University
Press: New York, 2003; pp 176-178.
21
McArthur, D.; Butts, C. P.; Lindsay, D. M. “A Dialkylborenium Ion via Reaction of N-
heterocyclic Carbene-Organoborane with Brønsted Acids-Synthesis and DOSY NMR
Studies” Chem. Commun. 2011, 47, 6650-6652.
22
Li, W.; Kagan, G.; Hopson, R.; Williard, P. G. “Measurement of Solution Viscosity via
Diffusion-Order NMR Spectroscopy (DOSY)” J. Chem. Ed. 2011, 88, 1331-1335.
113
Chapter 6. Gold Nanoparticles in Drug Delivery

6.1 Introduction
As the science of drug discovery is reaching maturity in the past century, the medicinal
chemistry community has shifted their focus to the development of drug delivery systems
(DDS). The goal is to develop more efficient and specific systems to minimize toxic side
effects and lower the required drug dose. Nanoparticles are attractive drug carriers
because they are capable of carrying large drug load, and can be easily manipulated to
increase their selectivity.
1
Liposomes, dendrimers, mesoporous silica, and gold
nanoparticles (AuNPs) have been thoroughly explored as drug delivery systems. AuNPs
are particularly attractive as potential drug carriers, because of their ease to synthesize
and their stability in physiological environments.
2
This introduction will focus on the
developments in AuNPs as drug delivery systems.

6.1.1 Fabrication of Gold Nanoparticles and Surface Functionalization for Drug
Delivery
Various approaches have been developed for AuNP synthesis. The two major methods
are the Turkevich citrate-reduction method, and the two-phase synthesis established by
Brust and Schiffrin et al.
3,4
The latter is more advantageous for the construction of drug
delivery systems because it allows the generation of relatively small and monodispersed
AuNPs (diameter 1.5 - 5.2 nm) and the surface functionalization can be attained in one-
pot with the synthesis of the core. The size of the gold core is tuned by changing the ratio
114
of HAuCl
4
to NaBH
4
. Both thiol and phosphine ligands are commonly used to stabilize
the AuNPs. Substitution of the thiol ligands can be achieved by the place exchange
reaction, in which a proportion of the original thiol ligands are displaced by newly added
thiols in the system. This process has revolutionary impact on the functionalization of
AuNPs.
5

6.1.2 Gold Nanoparticles as Drug Carriers
6.1.2.1 Drug Release by Passive Diffusion
As the naked gold core is unable to carry drug molecules, it is the surface coating that
determines the type and amount of drug loading. Polyethylene glycol is the most popular
surface coating for AuNP due to its high aqueous solubility and bio-friendly nature. Since
polyethylene glycol (PEG) does not interact with proteins, it is not absorbed by
reticuloendothelial system. Burda et al. prepared a type of AuNP capped with HO-PEG-
SH 5000 (Figure 6.1).
6
The particles were loaded with a hydrophobic anti-caner drug,
phthalocyanine 4 (Pc 4), which is a photosensitizer targeting cancer cells. By forming the
Pc 4/AuNP conjugate, the hydrophobic drug is rendered “soluble” in aqueous
environment. It was estimated that each PEGylated AuNP carries up to 30 Pc 4
molecules. Tumor-bearing mouse were injected with the Pc 4-loaded gold nanoparticles.
These large AuNPs (core diameter: 5.0 ± 2 nm) were able to diffusion into the tumor site
after the injection due to EPR (enhanced permeability and retention effect). In an in vitro
study, they demonstrated using a biphasic system that the drug gradually diffused from
the gold nanoparticles over a 6-hour period.
115

Figure 6.1. The hydrophobic phthalocyanine 4 is encapsulated in AuNPs capped with
HO-PEG-SH 5000. The phthalocyanine 4 is absorbed onto the gold surface via the Au-N
bond.
= HO-PEG-SH 5000
=
N
N N
N
N
N
N
N Si
OSi
HO
N
Pc 4
116
Rotello et al. prepared a series of neutral gold nanoparticles functionalized with
zwitterionic ligands.
7
To prepare the drug-carrier AuNP, they synthesized a tetra-
segmented surfaced coating consisting of a thiol tail, an undecyl alkyl segment, a
tetraethylene glycol segment, and a zwitterionic head (Figure 6.2).
8
The alkyl segment
provides the hydrophobic environment needed for encapsulating the hydrophobic drugs.
They chose the highly hydrophobic small molecules including BODIPY (boron-
dipyrromethene) (LogP = 4.0) as the guest to be encapsulated. By monitoring the
fluorescence from BODIPY, they were able to track the release of the molecule in a
biphasic system over 24 hours. No release of the fluorescent molecules was detected for
24 hours, when the loaded AuNPs were placed in PBS buffer. This result demonstrated
that “tight” drug loading can be realized by using suitable alkyls to functionalize AuNP.
The drug loading capacity is 4 molecules of BODIPY per particle. Anti-cancer drugs
TAF (tamoxifen, LogP = 3.6) and LAP (β-lapachone, LogP =2.7) were loaded at 10 and
1.5 molecules/particle capacity, indicating that the amount of drug loading is not merely
determined by the hydrophobicity.

6.1.2.2 Drug Release Trigger by External Factors
Light-triggered release of DNA from AuNP carriers was first reported by Rotello et al.
They synthesized a cell-permeable, positively charged nanoparticle that forms a complex
with negatively charged dsDNA (double-stranded) via Columbic interactions (Figure
6.3).
9
These positively-charged particles bear light-sensitive o-nitrobenzene ester
linkages. The charge of the nanoparticle is reversed by light irradiation, which in turn
releases the DNA duplex. In this way, the DNA can be carried into the nucleus and
117

Figure 6.2. A Zwitterion-stabilized AuNP loaded with BODIPY. The molecules are
encapsulated in the hydrophobic pockets formed by the alkyl coating.

selectively released. They validated their hypothesis by proving that in vitro DNA
transcription occurred after light irradiation. They also showed that DNA is translocated
inside the cell, when no irradiation was applied.
It is known that the intracellular environment is more reducing that the extracellular
environment, because glutathione (GSH) is present inside the cell. Rotello et al.
synthesized AuNPs that are triggered by GSH to release their surface coating. The
mechanism behind the release is the ligand place exchange reaction. In this case, it is
GSH that replaces the original ligands.

S
O
N
+
S
O
-
O
O
S
O
N
+
S
O
-
O
O
N
B
N
F F
S
O
N
+
S
O
-
O
O
S
O
N
+
S
O
- O
O
4
S
O
N
+
S
O
-
O
O
Au
118

Figure 6.3. The surface charge of the gold nanoparticles is changed from positive to
negative after light irradiation.

6.2 Encapsulation of BODIPY in a Phosphonate-Coated Gold Nanoparticle and Its
Release in a Biphasic System
Alkylphosphonic acid-coated gold nanoparticles are suitable carriers for hydrophobic
small molecules such as BODIPY. We chose BODIPY because of its large molar
extinction coefficient, relatively small size, and strongly hydrophobic nature (LogP = 2).
We planned to load BODIPY into a (12-mercaptododecyl)phosphonic acid-coated AuNP
(Figure 6.4a). When these particles are added to a biphasic system, the loaded particles
can freely release BODIPY into the organic phase. To stall this free diffusion, the
BODIPY-loaded particles would be decorated with a fluorous amphiphile (2.9a) via non-
S O
O
O
9
4
O NO
2
O
N
Au
Light
S O
O
9
4
O
NO
2
O
N
Au
O
H
O
+
Br
Br
119

Figure 6.4. a. BODIPY molecules are loaded into a phosphonic acid-capped Au NP. b.
Non-covalently decorating the particles blocks the diffusion of BODIPY outside of the
particle.
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
load
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
Diffusion is blocked
Loaded particle
S = -S-C
12
H
24
PO
3
H
2
= O
O
H
N NH
2
FF
4 4
NH
2
-
O CF
3
O
a
b
Loaded particle
=
N
B
N
F F
120
covalent interactions. The nature of the interaction is the stable bidentate hydrogen bond
between guanidinium and phosphonic acid. Because perfluoroalkyl groups tend to diffuse
together, it is possible that surrounding the particle with 2.9a will lead to formation of a
Teflon-like layer. Due to its high lipophobicity, the fluorous amphiphile might stop
BODIPY from diffusing out of the particle (Figure 6.4b). Thus, the BODIPY release
from the particle is controlled. Our findings on this system are presented in the following
sections.

6.2.1 Characterization of the Loaded AuNP
The gold core of the nanoparticles were synthesized using a slightly modified Brust and
Shriffin’s method.
10
These triphenylphosphine-stabilized AuNPs have a very small core
size (diameter = 1.5 nm). The AuNPs were functionalized with (12-
mercaptododecyl)phosphonic acid via a ligand exchange reaction. Because of the small
core size, the AuNPs gave a broad, weakly-absorbing plasmon resonance band on the
UV-Vis spectrum. Thermogravimetric analysis (TGA) showed that the metal accounts for
ca. 65% of the mass of the particles. After the particles were loaded with BODIPY, the
loaded particles showed a similar weakly-absorbing plasmon resonance band on UV-Vis
spectrometry (Figure 6.5).
The mass of a single particle can therefore be calculated based on the density of an fcc
packing cell consisting of gold atoms.
11

Knowing the mass of a single alkyl phosphonic
acid-coated particle allowed us to calculate the moles of gold nanoparticles in any given
sample. Then, we estimate the number of BODIPY molecules in a given particle sample
by performing a cyanide decomposition. Decomposition of the gold nanoparticles by
121
NaCN appeared to be complete at 5 hours (Figure 6.6) and afforded a homogenous
solution. By measuring the absorbance of the decomposition solution, the molarity of
BODIPY in the cuvette was calculated. BODIPY has a molar extinction coefficient of
119.4 mM
-1
in THF : H
2
O = 1 : 1 mixture (λ
max
= 506 nm). Now that both the quantity
of the gold nanoparticles and BODIPY were known, the number of BODIPY per gold
nanoparticle was calculated. It was estimated that each particle contains 27 molecules of
BODIPY.

6.2.2 Non-Covalent Interaction between 2.9a and the Phosphonate Coated- AuNPs
Binding of the guanidinium-ended 2.9a to the nanoparticle showed increased the
hydrodynamic radius of the particles.
12
The change in radius was detected by dynamic
light scattering (DLS). The gold nanoparticles capped with alkyl phosphonic acid has an
average diameter of 3.0 nm. After 2.9a is added, an average diameter of 4.0 nm was
observed on DLS (see section 7.5.1 for DLS histograms), indicating the interaction
between 2.9a and the particles has occurred.

6.2.3 BODIPY Release from the Particles in a Toluene/H
2
O Biphasic System
BODIPY encapsulated in the nanoparticles was released in a toluene/H
2
O biphasic
system (Scheme 2).
6

The UV-Vis cuvette depicted in Scheme 2 is fitted with a magnetic
stir bar. The encapsulated BODIPY is released at the interface and into the toluene layer.
The concentration of the BODIPY was monitored by UV-Vis absorbance at 510 nm. The
absorbance at 510 nm vs. time was plotted (Figure 6.5). The concentration of BODIPY
reached maximum after about 2 hours of stirring, and gradually decreased afterwards.
122
The decrease in absorption was presumably due to oxidation at the 5- position of
BODIPY.
After the addition of 2.9a, the particles aggregated and precipitated to the bottom of the
cuvette. The rate of BODIPY release was not slowed down by the addition of 2.9a, as
shown by the similar shape of the curve with or without 2.9a (Figure 6.8, red and purple).
We attempted to use an excess amount of 2.9a to stall BODIPY diffusion. When 20 times
excess amount of 2.9a was applied, the speed of BODIPY release appeared to have been
accelerated (Figure 6.8, blue). We suspect this is because the fluoroalkanes have higher
hydrophobicity than hydrocarbons, and fluoroalkyl monolayers are more prone to
solubilize hydrophobic materials. Lucarini et al. showed that para-substituted benzyl
hydroxyalkyl nitroxides probes tend to diffuse more readily into the fluoroalkyl-coated
gold nanoparticles than the aliphatic alkyl-coated gold nanoparticles.
13

Figure 6.5. UV-Vis spectrum of the AuNPs loaded with BODIPY.

0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
400 450 500 550 600 650 700
Absobance @510 nm
Wavelength (nm)
123

Figure 6.6. NaCN-induced decomposition of BODIPY-loaded AuNPs in THF/H
2
O
mixture.

Figure 6.7. BODIPY release from the AuNPs in a toluene/H
2
O biphasic system. The
release process is monitored by UV-Vis spectrometry.

0.09
0.095
0.1
0.105
0.11
0.115
0.12
0.125
0.13
0 5 10 15 20 25 30
Absorbance @510 nm
Time (hour)
= Loaded gold nanoparticles in H
2
O
toluene phase
UV/Vis absorbance
Toluene
H
2
O
124

Figure 6.8. The BODIPY-loaded AuNPs release BODIPY at the same speeds with or
without 2.9a (red and purple). When excess amount of 2.9a was added, the rate of the
release is accelerated (blue).

6.3 Conclusion
We have successfully prepared and loaded fluorescent small molecule BODIPY into a
class of alkyl phosphonate coated gold nanoparticles. The loading capacity of these
particles appears to be larger than gold nanoparticles of similar diameter and similar
length of alkyl capping group.
8

BODIPY molecules diffuse out of the particles into
toluene phase in a toluene/H
2
O biphasic system. When 2.9a is applied, the partition rate
did not change significantly. Our results showed that 2.9a did not stop BODIPY diffusion
out of the particles. We suspect this is due to the high hydrophobicity of fluoroalkyls.

0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0.00 2.00 4.00 6.00 8.00
Normalized UV-Vis Absorption
Time (h)
Excess 2.9a
2.9a
No 2.9a
125
6.4 References

1
Wilczewska, A. Z.; Niemirowicz, K.; Markiewicz, K.H.; Car, H. “Nanoparticles as
Drug Delivery Systems” Pharmacol. Rep. 2012, 64, 1020-1037.
2
Duncan, B.; Kim, C.; Rotello, V. M. “Gold Nanoparticle Platforms as Drug and
Biomacromolecule Delivery Systems” J. Control. Release 2010, 148, 122-127.
3
Turkevich, J.; Stevenson, P. C.; Hillier, J. “A Study of the Nucleation and Growth
Processes in the Synthesis of Colloidal Gold” Discuss. Faraday. Soc. 1951, 11, 55-75.
4
Brust, M. W. M.; Bethell, D.; Schriffrin, D. J.; Whyman, R. “Synthesis of Thio-
Derivatised Gold Nanoparticles in a Two-Phase Liquid-Liquid Systems” J. Chem. Soc.-
Chem. Commun. 1994, 801-802.
5
Hostetler, M. J.; Templeton, A. C.; Murray, R. W. “Dynamics of Place-Exchange
Reactions on the Monolayer-Protected Gold Cluster Molecules” Langmuir 1999, 15,
3782-3789.
6
Cheng, Y.; Samia, A. C.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. “Highly
Efficient Drug Delivery with Gold Nanoparticle Vectors for in Vivo Photodynamic
Therapy of Cancer” J. Am. Chem. Soc. 2008, 130, 10643-10647.
7
Zhu, Z. -J.; Ghosh, P. S.; Miranda, O. R.; Vachet, R. W.; Rotello, V. M. “Multiplexed
Screening of Cellular Uptake of Gold Nanoparticles Using Laser Desorption/Ionization
Mass Spectrometry” J. Am. Chem. Soc. 2008, 130, 14139-14143.
8
Kim, C. K.; Ghosh, P.; Pagliuca, C.; Zhu, Z.-J.; Menichetti, S.; Rotello, V. M.
“Entrapment of Hydrophobic Drugs in Nanoparticle Monolayers with Efficient Release
into Cancer Cells” J. Am. Chem. Soc. 2009, 131, 1360-1361.
126

9
Han, G.; You, C.-C.; Kim, B.-J.; Turingan, R. S.; Forbes, N. S.; Martin, C. T.; Rotello,
V. M. “Light-Regulated Release of DNA and Its Delivery to Nuclie by Mean sof
PHotolabile Gold Nanoparticles” Angew. Chem. Int. Ed. 2006, 45, 3165-3169.
10
Wang, R.; Zheng, Z.; Koknat, F. W.; Marko, D. J.; Müller, A.; Das, S. K.;
Krickemeyer, E.; Kuhlmann, C.; Therrien, B.; Plasseraud, L.; Süss-Fink, G.; Pasquale, A.
D.; Lei, X.; Fehlner, T. P.; Diz, E. L.; Haak, S.; Cariati, E.; Dragonetti, C.; Lucenti, E.;
Roberto, D.; Lee, C. Y.; Song, H.; Lee, K.; Park, B. K.; Park, J. T.; Hutchison, J. E.;
Foster, E. W.; Warner, M. G.; Reed, S. M.; Weare, W. W. Cluster and Polynuclear
Compounds. In Inorganic Syntheses; Shapley, J. R., Ed.; John Wiley & Sons, Inc.:
Hoboken, NJ, USA, 2004; Vol. 34, pp 228-234.
11
Liu, X.; Atwater, M.; Wang, J.; Huo, Q. “Extinction Coefficient of Gold Nanoparticles
with Different Sizes and Different Capping Ligands” Colloid. Surface. B 2007, 58, 3-7.
12
Li, V.; Chang, A. Y.; Williams, T. J. “A Noncovalent, Fluoroalkyl Coating Monomer
for Phosphonate-Covered Nanoparticles” Tetrahedron, 2013, 69, 7741-7745.
13
Lucarini, M.; Franchi, P.; Pedulli, G. F.; Pengo, P.; Scrimin, P.; Pasquato, L. “EPR
Study of Diakyl Nitroxides as Probes to Investigate the Exchange of Solutes between the
Ligand Shell of Monolayers of Protected Gold Nanoparticles and Aqueous Solutions” J.
Am. Chem. Soc. 2004, 126, 9326-9329.

127
Chapter 7. Experimental Procedures and Spectral Data

7.1 General Procedures
7.1.1 Chemicals
Deuterated NMR solvents were purchased from Cambridge Isotope Laboratories and
used as received. CDCl
3
was stored over 4Å molecular sieves. Tetraethyleneglycol
monomethyl ether, triethylamine, trifluoromethanesulfonyl chloride, 2,2,3,3,4,4-
hexafluoropentn-1,5-diol, 2,2,3,3,4,4,5,5-octafluorohexan-1,6-diol,
2,2,3,3,4,4,5,5,6,6,7,7-decafluorooctane-1,8-diol, triphenyl phosphine, urea, p-
toluenesulfonyl chloride, Hunig’s base, Amberlite IRN-78 ion exchange resin, dimethyl
malonate, phenylacetylene, Pd/C, sodium borohydride, calcium hydride, potassium
hydride (30-35%, wt%, suspension in mineral oil), oxalyl chloride, formaldehyde (37%
aqueous solution, stabilized with MeOH), phosphorus acid, phosphoric acid, and
potassium phthalimide were purchase from Alfa Aesar. Trifluoroacetic acid was
purchased from Oakwood Products. Ethyl acetate, hexanes, acetonitrile, pentane,
benzene, isopropyl alcohol, dimethylformamide (DMF, DriSolv), ammonium chloride,
tetrahydrofuran (THF), 4 Å molecular sieves, concentrated HCl solution, magnesium
sulfate (anhydrous), alumina, silica gel, and celite were purchased from EMD.
Chloroform, dichloromethane (DCM), toluene, sodium hydroxide, potassium hydroxide,
sodium bicarbonate, sodium chloride and HPLC grade H
2
O were purchased from
Macron. MeOH, pyridine, and dioxane were purchased from Mallinckrodt. Acetone and
diethyl ether were purchased from BDH. 1H-pyrazole-1-carboxamidine hydrochloride,
quinoline, and 1,3-dimethyl-2-imidazolidinone were purchased from Arcos Organics.
128
Sodium hydride, proton sponge, copper(I) iodide, hydrazine monohydrate, and sodium
cyanide were purchased from Sigma-Aldrich. Sodium azide was purchased from MP
Biomedicals. Absolute ethanol (200 proof pure ethanol) was purchased from KOPTEC.
Lindlar catalyst (Pd, 5wt% on calcium carbonate, poisoned by lead) was purchased from
TCI. Hexahydrates of GdCl
3
, YCl
3
, and EuCl
3
were obtained from REacton. (12-
mercaptododecyl)phosphonic acid was purchased from SiKÉMIA. Chloroauric acid
(unknown number of hydrate), cyclen and DOTA were purchased from Strem Chemicals.
Distilled water was purchased from Arrowhead. H
4
EDTA (EMD) was a generous gift
from Dr. Peter Qin’s lab. Dionized water was generated from a PURELAB Ultra Mk2
water purifier (Elga) at Wayne State University.

7.1.2 Prepared Reagents
DCM and pentane were dried over calcium hydride and distilled using standard
Schlenk distillation techniques. THF and dioxane were first passed through a plug of
alumina to remove the radicals before drying. THF, dioxane, benzene, pyridine and
toluene were dried over sodium/benzophenone and distilled using standard Schlenk
distillation techniques. Acetonitrile was dried over 4 Å molecular sieves and distilled
using standard Schlenk distillation techniques. DMF was purchased from EMD
(DriSolv®) and used as received. Brine solutions were prepared as saturated NaCl
solutions.
H
8
DOTP was prepared according to literature procedures.
1
2-Azido-1,3-
dimethylimidazolinium chloride (ADMC, 75.8 mg, 0.438 mmol) was prepared according
to literature procedures.
2
The phosphine-stabilized gold nanoparticles (diameter = 1.5
129
nm) were synthesized according to literature procedures and stored in a scintillation vial
at -20
o
C.
3,4
These particles were graciously provided by Dr. Anna C. Dawsey. Boron-
dipyrrolemethene (BODIPY) was a generous gift from Dr. Cong Tran.

7.1.3 Instrumentation
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 referred to
the
13
C in the solvent and delimited by carbon peaks, not carbon count.
19
F spectra are
referenced to an external standard of CFCl
3
solution (10% in CDCl
3
). 5 mm NMR tubes
(535-pp-7) were purchased from Wilmad.
1
H T
1
measurements were taken on a Varian
400MR magnetic resonance spectrometer at 9.4 T, or on a Bruker mq 60 NMR Analyzer
at 1.4 T.
19
F T
1
measurements were taken on a Varian VNMRS 500 magnetic resonance
spectrometer at 11.7 T. DOSY 2D were acquired on a Varian VNMRS 500 magnetic
resonance spectrometer at 11.7 T using the Doneshot sequence. Melting points were
taken on a Mel Temp apparatus and are uncorrected. Lyophilization was carried out on a
Millirock BT85A lyophilizer. Normal phase flash chromatographies were carried out
using RediSep® normal-phase silica flash columns on a Combiflash RF system
(Teledyne Isco), unless otherwise indicated. Reverse-phase flash chromatographies were
performed using RediSep Rf Gold® high performance C18 columns on the Combiflash
RF system (Teledyne Isco). Bulk solvent removal was carried out on a Laborota 4000
Efficient rotary evaporator (Heidolph) attached to a membrane pump (Welch 2022B-01).
130
A belt-drive pump was used when high vacuum was need. Centrifugation was performed
on an Eppendorf 5415D centrifuge using 1.5 mL Eppendorf tubes (Eppendorf
International). DLS measurements were performed on a DynaPro Titan instrument
(Wyatt Technology). TGA was performed on Q50 instrument (TA Instrument) using
ceramic crucibles. UV-Vis measurements were carried out either on a Shimadzu UV
spectrophotometer (UV-1800) or Cary 14 UV-Vis-NIR spectrophotometer using a quartz
cuvette (10 mm, 3.5 mL, Science Outlet). EPR spectra were recorded on a Bruker EMX
instrument at 25
o
C, using borosilicate glass tubes (0.6 I.D. × 0.84 O.D.). GC-MS was
performed on a DSQ
TM
II series single quadrupole GC-MS instrument (Thermo
Scientific). MALDI-TOF 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. The ratio between the matrix and the analyte is 20:1 (w:w).
Luminescence-decay measurements were acquired using a HORIBA Jobin Yvon
Fluoromax-4 spectrofluorometer in decay by delay scan mode using the phosphorescence
lifetime setting at Wayne State University. CHN analyses were carried out at the School
of Chemical Sciences Microanalysis Laboratory, University of Illinois at Urbana-
Champaign.

7.1.4 General Procedures for
1
H T
1
Measurements
To measure relaxivity at 9.4 T, an apparatus is set up as described in Figure 7.1. 150
µL solution of sample was pipetted into a coaxial insert (4 mm tube with 60 mm stem,
Norell), which was gently spun to allow all liquid to settle on the bottom of the tube;
another NMR tube (5 mm diam., Wilmad) was filled with D
2
O, and the coaxial insert was
131
placed inside this 5 mm NMR tube. The finished assembly enables the thinner end of
coaxial insert to be positioned at the detection window of the NMR probe. This assembly
is ready for T
1
measurement.

Figure 7.1. A sketch of a NMR tube ready for T
1
measurements.

7.1.5 General Procedures for
19
F T
1
Measurements

19
F T
1
measurements were performed using a previously described procedure.
5
To
measure the
19
F T
1
, a solution was prepared and placed inside a 5 mm diameter coaxial
NMR tube insert. A
19
F NMR direct detect spectrum was acquired on a VNMRS 500 nmr
D
2
O
Sample
132
spectrometer at 470 MHz. The window was adjusted to view the peaks of interest, and
the experiment was converted to T
1
measurement experiment using the top drop-down
window.
19
F T
1
s were then acquired using a broadband inversion recovery pulse
sequence. 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.

7.1.6 General Procedures for pH Measurement
For pH titrations, the pH of the solution was recorded using an Orion 210A+ (Thermo)
pH meter. For the synthesis of Gd-DOTP
5-
, Eu-DOTP
5-
, and Y-DOTP
5-
, the pH of the
solutions were checked by Panpeha pH paper (Whatman, purchased from Sigma-
Aldrich). All other solutions’ pH were checked by universal pH indicator paper (Sargent-
Welch).

7.1.7 General Procedures for Dialysis
Dialysis bags (Thermo Scientific SnakeSkin Pleated Dialysis tubing, 3500 MWCO, 22
mm dry diameter (ID)) were generous gifts from Professor Matthew Pratt’s lab. The bags
were soaked in HPLC grade H
2
O for 24 hours and rinsed in HPLC grade H
2
O before use.
To set up a dialysis, one end of the dialysis was folded and clamped. The sample solution
to be dialyzed was added to the bag, and the other end of the bag was also folded and
clamped. One clamp was attached to a Styrofoam block to ensure the dialysis bag is
133
afloat in the dialysis solution. The dialysis solution (in which the sample will be dialyzed)
is dispensed into a 600 mL beaker, and the dialysis bag is placed in this solution. The
solution was stirred constantly, and the dialysis solution was changed every 8 hours. The
dialysis was considered to have reached completion once a total of three dialysis solution
changes were finished.

7.1.7 General Procedures for Lyophilization
To prepare samples for lyophilization, 8 dr. vials containing aqueous solutions were
placed in liquid nitrogen. The container of the aqueous solution was tilted to avoid
creaking of the glass. The frozen samples were then placed in a thick-wall flask and
attached to a Millrock Technology BT85 lyophilizer. The time of lyophilization was
dependent on the volume of the sample. The end of the lyophilization is determined by
visual appraisal.

7.1.8 Other General Procedures
All air and water sensitive procedures were carried out using standard Schlenk
techniques.

134
7.2 Chapter 2 Experimental and Spectral Data
7.2.1 Preparation of Fluorous Amphiphiles
PEG-Tosylate 2.3

Tetraethyleneglycol monomethyl ether (2.2) (10.0 g, 48.0 mmol) was added via a
syringe to a flamed-dried three-neck round bottom flask fitted with a stir bar. To this
flask were added sequentially dry CH
2
Cl
2
(170 mL) and pyridine (84.0 mL, 1.04 mmol)
under nitrogen. Solid p-toluenesulfonyl chloride (22.0 g, 115.4 mmol) was added portion-
wise at -20 ºC under nitrogen. The resulting reaction mixture was stirred for 2 days at -20
ºC. Then, the reaction mixture was allowed to warm to room temperature, and water (200
mL) was added. The aqueous layer was extracted with CH
2
Cl
2
(150 mL × 3). The
combined organic fractions were dried over MgSO
4
and the solvent was removed under
reduced pressure. The crude product was purified by chromatography on silica (1 : 1
EtOAc : hexanes; R
f
= 0.3) to yield 2.3 as a colorless oil, 16.4 g, 94 %.
1
H NMR (500 MHz, CDCl
3
): δ = 7.80 (d, Ar, 2H), 7.34 (d, Ar, 2H), 4.16 (t, 2H), 3.66
(t, 2H), 3.62-3.65 (m, 6H), 3.58 (s, 4H), 3.532 - 3.56 (m, 2H), 3.34 (s, 3H), 2.43 (s, 3H).
13
C NMR (125 MHz, CDCl
3
): δ = 144.71 (s, CSO
2
O), 132.94 (s, CH
3
CCH), 129.74 (s,
CHCHCSO
2
), 127.89 (s, CCHCH), 71.79, 70.57, 70.46, 70.44, 70.38, 70.36, 69.20,
68.52, 58.94 (CH
3
OCH
2
), 21.56 (CH
3
CHCH).
Data are consistent with a previously reported compound.
6

O
O S
O
O
4
2.3
135
1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

136
FT-IR (cm
-1
, neat)

137
PEG-Fluorinated Alcohol 2.4a

Diol 2.1a (10.85 g, 41.4 mmol) was added to a 50 mL flame-dried three-neck round
bottom flask under nitrogen. To the solid in the flask was added sequentially dry dioxane
(236 mL) and NaH powder (0.563 g, 23.46 mmol) under nitrogen. The reaction was
stirred for 30 min at room temperature, after which the flask was placed in a 90 °C oil
bath for two hours. At the end of the two hours, a solution of 2.3 (5 g, 13.8 mmol) in dry
dioxane (20 ml) was added drop-wise from an oven-dried addition funnel. The reaction
was stirred overnight. The reaction was cooled to room temperature thereafter. The
solution was treated with hydrochloric acid (2 M in diethyl ether, 4.33 mL), and the
solvent was removed under reduced pressure. The residual was collected and dissolved in
dichloromethane (200 mL). A resulting white precipitate was removed via filtration. The
filtrate was collected and the solvent was removed under reduced pressure to give the
crude product, which was purified by flash chromatography (2:1 ethyl acetate: hexanes,
R
f
= 0.33) to yield the monosubstituted product as a clear oil, 3.86 g, 62%.
1
H NMR (400 MHz, CDCl
3
): δ = 4.09-3.96 (m, 4H, OCH
2
(CF
2
)
4
CH
2
OH), 3.78-3.74
(m, 2H), 3.67-3.61 (m, 12H), 3.55-3.52 (m, 2H), 3.36 (s, 3H, OCH
3
), 3.12 (t,
3
J
H,H
= 7.2
Hz, 1H, CH
2
OH).
13
C NMR (125 MHz, CDCl
3
): δ = 118.08-109.06 (m, CF
2
), 72.37, 72.00, 70.80, 70.77,
70.67, 70.63, 70.53, 68.3 (t,
2
J
C,F
= 24.9 Hz, CF
2
CH
2
OCH
2
), 60.59 (t,
2
J
C,F
= 25.4 Hz,
CF
2
CH
2
OH), 59.07 (s, CH
3
O).
O
O OH
FF
4 4
2.4a
138
19
F NMR (376 MHz, CDCl
3
): δ = (-124.1)-(-124.1) (m, 4F), -123.0 (p,
3
J
F,F
= 214.8 Hz,
2F), -120.4 (p,
3
J
F,F
= 13.3 Hz, 2F).
FT-IR (cm
-1
, neat): ν = 3415, 2882, 1457, 1351, 1177-1119, 946, 865, 762.
MALDI-TOF for C
15
H
24
F
8
O
6
[MNa]
+
: calculated 475.13 g/mol, found 475.10 g/mol.

1
H (400 MHz, CDCl
3
)

PEG-Fluorinated Alcohol 4a
!
O
O
4
OH
FF
4

1
H NMR

13
C NMR

139
13
C (125 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

19
F NMR

140
FT-IR (cm
-1
, neat)

141
PEG-Fluorinated Alcohol 2.4b

Diol 2.1b (5.27 g, 24.8 mmol) was added to a flame-dried 100 mL three-neck round-
bottom flask under nitrogen. To this solid in the flask were sequentially added dry
dioxane (66.2 mL) and NaH powder (0.298 g, 12.4 mmol) under nitrogen. The reaction
was stirred for 30 min at room temperature. Then the flask was placed in a 90 °C oil bath
for 2 hours. A solution of 2.3 (2.96 g, 8.28 mmol) in dry dioxane (4.1 ml) was added
drop-wise from an oven-dried addition funnel thereafter. The solution was stirred
overnight. The reaction was cooled to room temperature, and the solution was treated
with hydrochloric acid (2 M in diethyl ether, 2.07 mL). The solvent was removed under
reduced pressure. The residual was collected and dissolved in dichloromethane (200 mL),
and a resulting white precipitate was removed via filtration. The filtrate was collected and
the solvent was removed under reduced pressure to give the crude product, which was
purified by flash chromatography (1:1 ethyl acetate: hexanes, R
f
= 0.48) to yield the
monosubstituted product as a clear oil, 5.39 g, 51%.
1
H NMR (400 MHz, CDCl
3
): δ = 4.07 (td,
3
J
H,F
= 15.32 Hz,
3
J
H,H
= 7.3, 4H,
HOCH
2
(CF
2
)
3
), 4.00 (t,
3
J
H,F
= 14.3 Hz, 4H, HOCH
2
(CF
2
)
3
CH
2
), 3.77-3.76 (m, 2H),
3.68-3.63 (m, 12H), 3.56-3.54 (m, 2H), 3.38 (s, 3H, OCH
3
), 2.91 (t,
3
J
H,H
= 7.3 Hz, 1H,
CH
2
OH).
O
O OH
FF
3 4
2.4b
142
13
C NMR (125 MHz, CDCl
3
): δ = 118.19 - 109. 41 (m, (CF
2
)
3
), 72.06, 71.94, 70.66-
70.48 (OCH
2
CH
2
O), 68.20 (t,
2
J
C,F
= 25.4 Hz, CF
2
CH
2
OCH
2
), 60.31 (t,
2
J
C,F
= 25.4 Hz,
CF
2
CH
2
OH), 59.01 (s, CH
3
O).
19
F NMR (376 MHz, CDCl
3
): δ = -120.46 (m, 2F), -123.05 (m, 2F), -127.47 (m, 2F).
FT-IR (cm
-1
, neat): ν = 3421, 2881, 1460, 1350, 1285-1307, 937, 886, 850, 771, 668.
MALDI-TOF for C
14
H
24
F
6
O
6
[MNa]
+
: 425.14 g/mol, found 425.03 g/mol.

1
H (400 MHz, CDCl
3
)

PEG-Fluorinated Alcohol 4b
O
O
4
OH
FF
3

1
H NMR

13
C NMR

143
13
C (125 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

PEG-Fluorinated Alcohol 4b
O
O
4
OH
FF
3

1
H NMR

13
C NMR

19
F NMR

144
FT-IR (cm
-1
, neat)

145
PEG-Fluorinated Alcohol 2.4c

Diol 2.1c (2.997 g, 8.28 mmol) was added to a flame-dried 50 mL 3-neck round bottom
flask under nitrogen. To this solid in the flask was added sequentially dry dioxane (22.1
mL) and NaH powder (0.340 g, 4.69 mmol) under nitrogen. The solution was stirred for
30 min at room temperature. Then the flask was placed in a 90 °C oil bath for 2 hours. A
solution of 2.3 (0.991 g, 2.76 mmol) in dry dioxane (4.5 ml) was added drop-wise from
an oven-dried addition funnel thereafter. The solution was stirred overnight. The reaction
was cooled to room temperature, and the solution was treated with hydrochloric acid (2
M in diethyl ether, 4.03 mL). The solvent was removed under reduced pressure. The
residual was collected and dissolved in dichloromethane (200 mL), and a resulting white
precipitate was removed via filtration. The filtrate was collected and the solvent was
removed under reduced pressure to give the crude product, which was purified by flash
chromatography (1:2 ethyl acetate: hexanes, R
f
= 0.31) to yield the monosubstituted
product as a clear oil, 0.837g, 60%.
1
H NMR (400 MHz, CDCl
3
): δ = 4.09 (td,
3
J
H,F
= 14.3 Hz,
3
J
H,H
= 7.6, 4H,
HOCH
2
(CF
2
)
6
), 4.04 (t,
3
J
H,F
= 14.2 Hz, 4H, HOCH
2
(CF
2
)
6
CH
2
),

3.79-3.77 (m, 2H), 3.69-
3.63 (m, 12H), 3.56-3.54 (m, 2H), 3.38 (s, 3H, OCH
3
), 2.19 (t,
3
J
H,H
= 7.6 Hz, 1H,
CH
2
OH).
O
O OH
FF
6 4
2.4c
146
13
C NMR (125 MHz, CDCl
3
): δ = 117.82-108. 76 (m, (CF
2
)
3
), 72.41, 72.00, 70.82-
70.56 (OCH
2
CH
2
O), 68.44 (t,
2
J
C,F
= 24.4 Hz, CF
2
CH
2
OCH
2
), 60.60 (t,
2
J
C,F
= 25.4 Hz,
CF
2
CH
2
OH), 59.08 (s, CH
3
O).
19
F NMR (470 MHz, CDCl
3
): δ = -120.25 (m, 2F), -122.60 (m, 4F), -122.83 (m, 2F), -
124.08 (m, 4F).
FT-IR (cm
-1
, neat): ν = 3408, 2884, 1645, 1457, 1197-1106, 944, 846, 758-726.
MALDI-TOF for C
17
H
24
F
12
O
6
[MNa]
+
: 575.13 g/mol, found 575.08 g/mol.

1
H (400 MHz, CDCl
3
)

PEG-Fluorinated Alcohol 4c

O
O
4
OH
FF
6

1
H NMR

13
C NMR

147
13
C (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

PEG-Fluorinated Alcohol 4c

O
O
4
OH
FF
6

1
H NMR

13
C NMR

19
F NMR

148
FT-IR (cm
-1
, neat)

149
PEG-Fluorinated Triflate 2.5a

Compound 2.4a (1 g, 2.21 mmol) was added to a septum-sealed, oven-dried 8 dr. vial
via a syringe under nitrogen. To this vial were sequentially added dry THF (3.3 ml) and
triethylamine (0.68 ml, 4.862 mmol) under nitrogen. The mixture was stirred for 10 min,
after which the vial was cooled to 0
o
C. Trifluoromethanesulfonyl chloride (0.47 ml, 4.42
mmol) was added via a syringe, and the reaction was stirred for 14 hours. The solvent
was removed under reduced pressure. The residual was collected and dissolved in ether.
A resulting white precipitate was removed by filtration. The filtrate was collected and the
solvent was removed under reduced pressure. The crude product was purified by flash
chromatography (2 : 1 ethyl acetate : hexanes, R
f
= 0.42) to give 2.5a as a clear, oily
liquid (1.05 g). Yield: 81%, conversion: 95%.
1
H NMR (400 MHz, CDCl
3
): δ = 4.79 (t,
3
J
H,F
= 12.6 Hz, 2H, CF
3
SO
2
OCH
2
CF
2
), 4.01
(t,
3
J
H,F
= 13.9 Hz, 2H, CH
2
OCH
2
CF
2
), 3.77-3.73 (m, 2H), 3.66-3.59 (m, 12H), 3.53-3.50
(m, 2H), 3.35 (s, 3H, CH
3
O).
13
C NMR (100 MHz, CDCl
3
): δ = 118.4 (q,
1
J
C,F
= 318.9 Hz, SO
2
CF
3
), 117.89-110.77
(m, CF
2
), 72.2, 71.8, 70.62, 70.59, 70.51, 70.49, 70.42, 68.45 (t,
2
J
C,F
= 27.3 Hz,
CH
2
OTf), 68.16 (t,
2
J
C,F
= 24.9 Hz, CH
2
OCH
2
CF
2
), 58.9 (s, CH
3
O).

O
O O
FF
4 4
2.5a
S CF
3
O
O
150
19
F NMR (376 MHz, CDCl
3
): δ = -124.0 (m, 4F), -120.5 (p, 2F,
2
J
C,F
= 12.4 Hz,
OCH
2
CF
2
), -120.3 (m, 2F, CF
2
CH
2
OTf), -74.6 (s, 3F, CF
3
SO
2
).
FT-IR (cm
-1
, neat): ν = 2885, 1428, 1215-1134, 1017, 958, 838, 611.
MALDI-TOF for C
16
H
23
F
11
O
8
S [MNa]
+
: calculated 607.08 g/mol, found 606.85 g/mol.

1
H (400 MHz, CDCl
3
)

19
F NMR

151
13
C (100 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

PEG-Fluorinated Triflate 5a

O
O
4
OTf
FF
4

1
H NMR

13
C NMR

19
F NMR

152
PEG-Fluorinated Triflate 2.5b

Compound 2.4b (0.100 g, 0.249 mmol) was added to a septum-sealed, oven-dried 8 dr.
vial via a syringe under nitrogen. To this vial were sequentially added dry THF (0.62 ml)
and triethylamine (52.0 µL, 0.374 mmol) under nitrogen. The mixture was stirred for 10
min, after which the vial was cooled to 0
o
C. Trifluoromethanesulfonyl chloride (34.5 µL,
0.324 mmol) was added via a syringe, and the reaction was stirred for 25 min. The
solvent was removed under reduced pressure. The residual was collected and dissolved in
ether. A resulting white precipitate was removed by filtration. The filtrate was collected
and the solvent was removed under reduced pressure. The crude product was purified by
flash chromatography (2 : 1 ethyl acetate : hexanes, R
f
= 0.36) to give 2.5b as a yellow,
oily liquid (78.3 mg). Yield: 60%. Conversion: 100%.
1
H NMR (500 MHz, CDCl
3
): δ = 4.82 (t,
3
J
H,F
= 13.2 Hz, 2H, CF
3
SO
2
OCH
2
CF
2
), 4.03
(t,
3
J
H,F
= 14.2 Hz, 2H, CH
2
OCH
2
CF
2
), 3.77-3.76 (m, 2H), 3.67-3.63 (m, 12H), 3.55-3.53
(m, 2H), 3.37 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 118.57 (q,
1
J
C,F
= 318.6 Hz, SO
2
CF
3
), 117.4-108.8
(m, CF
2
), 72.35, 72.07, 70.83, 70.76, 70.74, 70.71, 70.65, 69.03 (t,
2
J
C,F
= 26.4 Hz,
CH
2
OTf), 68.14 (t,
2
J
C,F
= 26.4 Hz, CH
2
OCH
2
CF
2
), 58.9 (s, CH
3
O).

O
O O
FF
3 4
2.5b
S CF
3
O
O
153
19
F NMR (470 MHz, CDCl
3
): δ = (-123.9)-(-124.1) (m, 4F, CF
2
CF
2
CH
2
), -120.5 (2F,
OCH
2
CF
2
), (-120.2)-(-120.35) (m, 2F, CF
2
CH
2
OTf), -74.6 (s, 3F, CF
3
SO
2
).
FT-IR (cm
-1
, neat): ν = 2878, 1427, 1216, 1144 1008, 969, 613.
MALDI-TOF for C
15
H
23
F
9
O
8
S [MNa]
+
: calculated 557.09 g/mol, found 556.90 g/mol.

1
H (500 MHz, CDCl
3
)

PEG-Fluorinated Triflate 5b

O
O
4
OTf
FF
3

1
H NMR

13
C NMR

154
13
C (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

PEG-Fluorinated Triflate 5b

O
O
4
OTf
FF
3

1
H NMR

13
C NMR

19
F NMR

155
FT-IR (cm
-1
, neat)

156
PEG-Fluorinated Triflate 2.5c

Compound 2.4c (1.00 g, 1.81 mmol) was added to a flame-dried round bottom flask via
a syringe. To this flask were sequentially added dry THF (2.26 ml) and triethylamine
(43.0 mL, 3.077 mmol) under nitrogen. The mixture was stirred for 10 min, after which
the flask was cooled to 0
o
C. Trifluoromethanesulfonyl chloride (34.5 µL, 0.324 mmol)
was added via a syringe, and the reaction was stirred for 12 hours. The solvent was
removed under reduced pressure. The crude product was collected and dissolved in ether.
A resulting white precipitate was removed by filtration. The filtrate was collected and the
solvent was removed under reduced pressure. The crude product was purified by flash
chromatography (ethyl acetate : hexanes = 3 : 1, R
f
= 0.48) to give 2.5c as a clear, oily
liquid (1.13 g). Yield: 93%. Conversion: 95% conversion.
1
H NMR (500 MHz, CDCl
3
): δ = 4.80 (t,
3
J
H,F
= 12.2 Hz, 2H, CF
3
SO
2
OCH
2
CF
2
), 4.02
(t,
3
J
H,F
= 13.4 Hz, 2H, CH
2
OCH
2
CF
2
), 3.77-3.75 (m, 2H), 3.66-3.60 (m, 12H), 3.53-3.51
(m, 2H), 3.35 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 118.52 (q,
1
J
C,F
= 320.7 Hz, SO
2
CF
3
), 118.03-108.41
(m, CF
2
), 72.41, 72.02, 70.82-70.60, 68.37 (t,
3
J
C,F
= 28.9 Hz, CH
2
OTf), 68.13 (t,
3
J
C,F
=
28.9 Hz, CH
2
OCH
2
CF
2
), 59.0 (s, CH
3
O).

O
O O
FF
6 4
2.5c
S CF
3
O
O
157
19
F NMR (470 MHz, CDCl
3
): δ = -120.16 (m, 4F), -122.5 (m, 2F), -122.48 (m, 2F), -
123.43 (m, 2F), -74.4 (s, 3F, CF
3
SO
2
).
FT-IR (cm
-1
, neat): ν = 2884, 1427, 1203, 1142, 1012, 956, 821, 612.
MALDI-TOF for C
18
H
23
F
15
O
8
S [MNa]
+
: calculated 707.08 g/mol, found 706.73 g/mol.

1
H (500 MHz, CDCl
3
)

PEG-Fluorinated Triflate 5c

O
O
4
OTf
FF
6

1
H NMR

13
C NMR

158
13
C (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

PEG-Fluorinated Triflate 5c

O
O
4
OTf
FF
6

1
H NMR

13
C NMR

19
F NMR

159
FT-IR (cm
-1
, neat)

160
PEG-Fluorinated Azide 2.6a

Compound 2.5a (194 mg, 0.34 mmol) was added to a septum-sealed, oven-dried 8 dr.
vial via a syringe under nitrogen. To this vial were added sequentially dry DMF (1.5 mL)
and sodium azide (26.7 mg, 0.41 mmol) under nitrogen. The solution was stirred for 6.5
hours. The reaction was poured over H
2
O (2 mL) and extracted with Et
2
O (2 mL × 3).
The combined ether fractions were washed with H
2
O (6 mL × 3) and dried over MgSO
4
.
The solvent was removed under reduced pressure to obtain 2.6a as a light-yellow liquid
(136 mg, 84%).
1
H NMR (500 MHz, CDCl
3
): δ 4.02 (t,
3
J
H,F
= 14.3 Hz, 2H, CH
2
OCH
2
CF
2
), 3.79-3.77
(m, 2H), 3.75 (t,
3
J
H,F
= 14.6 Hz, 2H, N
3
CH
2
CF
2
), 3.68-3.63 (m, 12H), 3.55-3.53 (m, 2H),
3.38 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 117.97-108.79 (m, CF
2
), 72.38, 72.00, 70.79, 70.75,
70.68, 70.66, 70.58, 68.38 (t,
3
J
C,F
= 24.6 Hz, CF
2
CH
2
OCH
2
), 59.04 (s, CH
3
O), 50.18 (t,
3
J
C,F
= 23.9 Hz, CH
2
N
3
).
19
F NMR (470 MHz, CDCl
3
): δ = -118.08 (m, 2F), -120.32 (m, 2F), -123.95 (m, 2F), -
124.11 (m, 2F).
FT-IR (cm
-1
, in CDCl
3
): ν = 2881, 2113, 1456, 1123, 960, 857.
MALDI-TOF for C
15
H
24
F
8
N
3
O
5
[MH]
+
, calculated 478.16 g/mol, found 478.02 g/mol.

O
O
FF
4 4
2.6a
N
3
161
1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

PEG-Fluorinated Azide 8a

O
O
4
N
3
FF
4

1
H NMR

13
C NMR

PEG-Fluorinated Azide 8a

O
O
4
N
3
FF
4

1
H NMR

13
C NMR

162
19
F (470 MHz, CDCl
3
)

19
F NMR

163
PEG-Fluorinated Azide 2.6b

Compound 2.5b (512.9 mg, 0.96 mmol) was cannula transferred to a flame-dried 25
mL three-neck round bottom flask under nitrogen. To this flask were added dry DMF
(2.11 mL) and sodium azide (74.9 mg, 1.15 mmol) under nitrogen. The solution was
stirred for 11.5 hours. The reaction was poured over H
2
O (4 mL) and extracted with Et
2
O
(4 mL × 3). The combined ether fractions were washed with H
2
O (12 mL × 3) and dried
over MgSO
4
. The solvent was removed under reduced pressure to obtain 2.6b as a light-
yellow liquid (409.4 mg, > 99%).
1
H NMR (500 MHz, CDCl
3
): δ 4.01 (t,
3
J
H,F
= 14.4 Hz, 2H, CH
2
OCH
2
CF
2
), 3.78-3.75
(m, 2H), 3.77 (t,
3
J
H,F
= 14.6 Hz, 2H, N
3
CH
2
CF
2
), 3.68-3.63 (m, 12H), 3.55-3.53 (m, 2H),
3.37 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 117.97-108.58 (m, CF
2
), 72.13, 71.84, 70.68, 70.60,
70.54, 70.51, 70.49, 70.41, 68.04 (t,
3
J
C,F
= 24.9 Hz, CF
2
CH
2
OCH
2
), 58.83 (s, CH
3
O),
50.06 (t,
3
J
C,F
= 23.5 Hz, CH
2
N
3
).
19
F NMR (470 MHz, CDCl
3
): δ = -118.30 (m, 2F), -120.21 (m, 2F), -126.43 (m, 2F).
FT-IR (cm
-1
, in CDCl
3
): ν = 2881, 2114, 1653, 1457, 1306, 1148, 956.
MALDI-TOF for C
14
H
24
F
6
N
3
O
5
[MH]
+
, calculated 428.1620 g/mol, found 428.1664
g/mol.

O
O
FF
3 4
2.6b
N
3
164
1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

PEG-Fluorinated Azide 8b

O
O
4
N
3
FF
3

1
H NMR

13
C NMR

PEG-Fluorinated Azide 8b

O
O
4
N
3
FF
3

1
H NMR

13
C NMR

165
19
F (470 MHz, CDCl
3
)

19
F NMR

166
PEG-Fluorinated Azide 2.6c

Compound 2.5c (95.5 mg, 0.140 mmol) was added to a septum-sealed, oven-dried 8 dr.
vial via a syringe under nitrogen. To this vial were sequentially added DMF (0.61 mL)
and sodium azide (11.2 mg, 0.167 mmol) under nitrogen. The solution was stirred for 6.5
hours. The reaction was poured over H
2
O (2 mL) and extracted with Et
2
O (2 mL × 3).
The combined ether fractions were washed with H
2
O (6 mL × 3) and dried over MgSO
4
.
The solvent was removed under reduced pressure to obtain 2.6c as a light-yellow liquid
(80.6 mg, 87%).
1
H NMR (500 MHz, CDCl
3
): δ = 4.04 (t,
3
J
H,F
= 14.0 Hz, 2H, CH
2
OCH
2
CF
2
), 3.80-
3.77 (m, 2H), 3.77 (t,
3
J
H,F
= 15.6 Hz, 2H, N
3
CH
2
CF
2
), 3.68-3.63 (m, 12H), 3.56-3.54 (m,
2H), 3.38 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 117.97-108.58 (m, CF
2
), 72.13, 71.84, 70.68, 70.60,
70.54, 70.51, 70.49, 70.41, 68.04 (t,
3
J
C,F
= 24.9 Hz, CF
2
CH
2
OCH
2
), 58.83 (s, CH
3
O),
50.06 (t,
3
J
C,F
= 23.5 Hz, CH
2
N
3
).
19
F NMR (470 MHz, CDCl
3
): δ = -117.88 (m, 2F), -120.19 (m, 2F), -122.30 (m, 2F), -
122.50 (m, 2F), -123.66 (m, 2F), -123.88 (m, 2F).
FT-IR (cm
-1
, in CDCl
3
): ν = 2881, 2114, 1456, 1142, 958.
MALDI-TOF for C
17
H
24
F
12
N
3
O
5
[MH]
+
, calculated 578.1524 g/mol, found 577.9730
g/mol.

O
O
FF
6 4
2.6c
N
3
167
1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

PEG-Fluorinated Azide 8c

O
O
4
N
3
FF
6

1
H NMR

13
C NMR

PEG-Fluorinated Azide 8c

O
O
4
N
3
FF
6

1
H NMR

13
C NMR

168
19
F (470 MHz, CDCl
3
)

19
F NMR

169
PEG-Fluorinated Amine 2.7a

Compound 2.8 (3.8 g, 6.5 mmol) was cannula transferred to a 250 mL three-neck round
bottom flask under nitrogen. To this flask was cannula transferred hydrazine
monohydrate (2.05 mL, 65.2 mmol) in absolute ethanol (150 mL). The flask was place in
a 65 ºC oil bath overnight. The reaction was cooled to room temperature. A resulting
white precipitate was removed by filtration. The filtrate was collected and the solvent was
removed under reduced pressure. Chloroform (200 mL) was added to the residual, and
the solution was stirred for 30 minutes. Additional white precipitate was removed by
filtration. The filtrate was collected and the solvent was removed under reduced pressure
to yield the product as light yellow oil (2.74 g, 93%).
Alternatively, 2.7a can be obtained by one-pot hydrogenation from 2.5a:
Compound 2.5a (190 mg, 0.33 mmol) in dry DMF (1.5 mL) was cannula transferred to
a septum-sealed 8 dr. vial under nitrogen. To this vial was added sodium azide (26.6 mg,
0.41 mmol). The reaction was stirred for 6.5 hours, and Pd/C (2.4 mg, 10% w/w) was
added. A balloon filled with hydrogen gas was attached to the flask. The solution was
stirred for 3 hours. The suspension was filtered over a pad of celite. The filtrate was
collected and poured over 1M aqueous HCl (2 mL). The aqueous phase was washed with
ether (1.5 mL × 2), and the pH was adjusted to basic with saturated NaOH solution. The
product was extracted from the basic aqueous phase with ether (2 mL × 3). The ether
fractions were combined and washed with brine (2 mL × 1) anf dried over MgSO
4
. The
O
O
FF
4 4
2.7a
NH
2
170
solvent was removed under reduced pressure. 2.7a was obtained as a yellow, oily liquid
(102.8 mg). Yield: 70%.
1
H NMR (400 MHz, CDCl
3
): δ = 4.02 (t,
3
J
H,F
= 14.3 Hz, 2H, CH
2
OCH
2
CF
2
), 3.80-
3.75 (m, 2H), 3.69-3.61 (m, 12H), 3.56-3.51 (m, 2H), 3.37 (s, 3H, CH
3
O), 3.24 (t,
3
J
H,F
=
15.8 Hz, 2H, NCH
2
CF
2
), 1.36-1.21 (br, 2H, CF
2
CH
2
NH
2
).
13
C NMR (100 MHz, CDCl
3
): δ = 118.4-109.6 (m, CF
2
), 72.3, 71.9, 70.71, 70.68,
70.65, 70.59, 70.57, 70.50, 68.3 (t,
3
J
C,F
= 24.6 Hz, CF
2
CH
2
OCH
2
), 58.99 (s, CH
3
O),
42.92 (t,
3
J
C,F
= 24.1 Hz, CF
2
CH
2
NH
2
).
19
F NMR (376 MHz, CDCl
3
): δ = -124.5 (m, 2F), -124.21 (m, 2F), -122.17 (m, 2F), -
120.50 (m, 2F).
FT-IR (cm
-1
, in CDCl
3
): ν = 3409, 3340, 2878, 1632, 1460, 1352, 1232−1115, 956,
858.
MALDI-TOF for C
15
H
25
F
8
NO
5
[MH]
+
, calculated 452.17 g/mol, found 451.96 g/mol.

1
H (400 MHz, CDCl
3
)

PEG-Fluorinated Amine 7a

O
O
4
NH
2
FF
4

1
H NMR

13
C NMR

171
13
C (100 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

PEG-Fluorinated Amine 7a

O
O
4
NH
2
FF
4

1
H NMR

13
C NMR

19
F NMR

172
FT-IR (cm
-1
, in CDCl
3
)

173
PEG-Fluorinated Amines 2.7b

Compound 2.7b was prepared via hydrogenation of azide 2.6b. Two procedures were
performed using either EtOH or DMF as the solvent.
Method using EtOH as the solvent: 2.6b (105.4 mg) was transferred to a 3 mL Schlenk
flask via a syringe. To this flask were sequentially added absolute EtOH (0.5 mL) and
Pd/C (1.3 mg, 10% w/w). A H
2
-filled balloon was attached to the Schlenk flask, and the
solution was stirred for 3 hours. The reaction was filtered over a pad of celite. The
solvent was reduced under reduced pressure to afford 2.7b as a clear liquid (67 mg, 68%)
Method using DMF as the solvent: 2.6b (92.9 mg) was transferred to a 3 mL Schlenk
flask via a syringe. To this flask were sequentially added dry DMF (0.5 mL) and Pd/C
(1.2 mg, 10% w/w). A H
2
-filled balloon was attached to the Schlenk flask, and the
solution was stirred overnight. The solution was filtered over a pad of celite thereafter.
The filtrate was collected and poured over 1M HCl (1 mL). The aqueous phase was
washed with ether (1 mL × 2), and the pH was adjusted to basic using saturated NaOH.
The product was extracted from the basic aqueous solution using ether (1 mL × 2). The
combined ether fractions were washed with brine (1 mL × 1) and dried over MgSO
4
. The
solvent was removed under reduced pressure to afford 2.7b as a clear liquid (63.2 mg,
72%)
O
O
FF
3 4
2.7b
NH
2
174
1
H NMR (500 MHz, CDCl
3
): δ = 3.99 (t,
3
J
H,F
= 14.3 Hz, 2H, CH
2
OCH
2
CF
2
), 3.78-
3.76 (m, 2H), 3.70-3.63 (m, 12H), 3.56-3.54 (m, 2H), 3.39 (s, 3H, CH
3
O), 3.31 (t,
3
J
H,F
=
15.3 Hz, 2H, NCH
2
CF
2
), 1.56 (br, 2H, CF
2
CH
2
NH
2
).
13
C NMR (125 MHz, CDCl
3
): δ = 120.26-109.56 (m, CF
2
), 72.34, 72.02, 70.79-70.60,
68.36 (t,
2
J
C,F
= 24.5 Hz, CF
2
CH
2
OCH
2
), 59.08 (s, CH
3
O), 43.05 (t,
3
J
C,F
= 24.6 Hz,
CF
2
CH
2
NH
2
).
19
F NMR (470 MHz, CDCl
3
): δ = -119.84 (m, 2F), -121.62 (m, 2F,), -122.04 -122.01
(m, 2F), -122.15 (m, 2F), -123.56 (m, 2F), -123.70 (m, 2F).
FT-IR (cm
-1
, in CHCl
3
): ν = 3408, 3340, 2875, 1632, 1456, 1350, 1284, 1234, 1141,
956, 881.
MALDI-TOF for C
14
H
25
F
6
NO
5
[MH]
+
: calculated 402.17 g/mol, found 402.04 g/mol.

1
H (500 MHz, CDCl
3
)

PEG-Fluorinated Amine 7b

O
O
4
NH
2
FF
3

1
H NMR

13
C NMR

175
13
C (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

PEG-Fluorinated Amine 7b

O
O
4
NH
2
FF
3

1
H NMR

13
C NMR

19
F NMR

176
FT-IR (cm
-1
, in CHCl
3
)

177
PEG-Fluorinated Amines 2.7c

Compound 2.7c was prepared via one-pot hydrogenation from 2.5c
.
Compound 2.5c
(0.298 g, 0.438 mmol) was added to a 25 mL Schlenk flask via a syringe under nitrogen.
To this flask were sequentially added dry DMF (0.96 mL) and NaN
3
(35.3 mg, 0.526
mmol). The reaction was stirred for 6.5 hours. Pd/C (37.9 mg, 10% w/w) was added and
a balloon charged with H
2
was attached to the flask thereafter. Upon completion, the
reaction was filtered over a pad of celite. The filtrate was collected and poured over 1M
HCl (1mL). The aqueous phase was washed with ether (2 mL × 2), and the pH was
adjusted to basic using saturated NaOH. The product was extracted with ether (3 mL × 2)
from the basic aqueous phase. The combined ether fractions were washed with brine (2
mL × 1) and dried over MgSO
4.
The solvent was removed under reduced pressure to
afford 2.7c as a yellow, oily liquid (156 mg, 65.0% yield).
1
H NMR (500 MHz, CDCl
3
): δ 4.02 (t,
3
J
H,F
= 14.0 Hz, 2H, CH
2
OCH
2
CF
2
), 3.77-3.75
(m, 2H), 3.66-3.61 (m, 12H), 3.53-3.52 (m, 2H), 3.36 (s, 3H, CH
3
O), 3.24 (t,
3
J
H,F
= 15.8
Hz, 2H, NCH
2
CF
2
), 1.28 (br, 2H, CF
2
CH
2
NH
2
).
13
C NMR (125 MHz, CDCl
3
): δ = 118.78-108.75 (m, CF
2
), 72.43, 72.03, 70.83-70.58,
68.43 (t,
2
J
C,F
= 25.43 Hz, CF
2
CH
2
OCH
2
), 59.08 (s, CH
3
O), 42.96 (t,
3
J
C,F
= 23.5 Hz,
CF
2
CH
2
NH
2
).
19
F NMR (470 MHz, CDCl
3
): δ = -119.84 (m, 2F), -121.62 (m, 2F,), -122.04 -122.01
(m, 2F), -122.15 (m, 2F), -123.56 (m, 2F), -123.70 (m, 2F).
O
O
FF
6 4
2.7c
NH
2
178
FT-IR (cm
-1
, in CHCl
3
): ν = 3411, 3348, 2881, 1632, 1458, 1351, 1200−1141, 960,
840.
MALDI-TOF for C
17
H
25
F
12
NO
5
[MNa]
+
: calculated 574.14 g/mol, found 574.12 g/mol.

1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

179
19
F (470 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

180
PEG-Fluorinated Phthalimide 2.8

Compound 2.5a (6.5 g, 11.1 mmol) was cannula transferred to a flame-dried 500 mL
three-neck round bottom flask under nitrogen. To this flask were sequentially added dry
DMF (223 mL) and potassium phthalimide salt (4.12 g, 22.3 mmol). The resulting
suspension was stirred at 65 ºC overnight under nitrogen, then it was cooled to room
temperature. The solvent was removed using a rotary evaporator (elevated temperature,
membrane pump). Chloroform (200 mL) was added to the residue, and a resulting white
precipitate was filtered. The filtrate was collected, and the solvent was removed under
reduced pressure to give the crude compound, which was purified on a silica gel column
with a solvent gradient (4 : 1 ethyl acetate: hexanes R
f
= 0.4) to yield a clear oil 5.4 g,
84%.
1
H NMR (400 MHz, CDCl
3
): δ = 7.92 (q,
3
J
H,H
= 2.8 Hz, Ar, 4H), 7.78 (q,
3
J
H,H
= 2.8
Hz, Ar, 4H), 4.35 (t,
3
J
H,F
= 15.8 Hz, 2H, NCH
2
CF
2
), 4.03 (t,
3
J
H,F
= 14.4 Hz, 2H,
CH
2
OCH
2
CF
2
), 3.80-3.75 (m, 2H), 3.69-3.61 (m, 12H), 3.56-3.51 (m, 2H), 3.36 (s, 3H,
CH
3
O).
13
C NMR (100 MHz, CDCl
3
): δ = 166.9 (s, Ar), 134.5 (s, Ar), 131.6 (s, Ar), 123.9 (s,
Ar), 118.4-109.2 (m, CF
2
), 72.3, 71.9, 70.71, 70.67, 70.64, 70.58, 70.56, 70.52, 70.48,
70.44, 68.3 (t,
2
J
C,F
= 24.9 Hz, CF
2
CH
2
OCH
2
), 58.97 (s, CH
3
O), 37.45 (t,
2
J
C,F
= 23.5 Hz,
CF
2
CH
2
N).
O
O
FF
4 4
2.8
N
O
O
181
19
F NMR (376 MHz, CDCl
3
): δ = -124.13 (m, 2F), -123.73 (m, 2F,), -120.35 (m, 2F), -
116.6 (m, 2F).
FT-IR (cm
-1
, in CDCl
3
): ν = 3154, 2985, 2903, 2254, 1793, 1472, 1378, 1382, 1099,
910, 733.
MALDI-TOF for C
23
H
27
F
8
NO
7
[MNa]
+
: calculated 604.1557 g/mol, found 603.8150
g/mol.

1
H (400 MHz, CDCl
3
)

PEG-Fluorinated Phthalimide 6

O
O
4
N
FF
4
O
O

1
H NMR

13
C NMR

182
13
C (100 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

PEG-Fluorinated Phthalimide 6

O
O
4
N
FF
4
O
O

1
H NMR

13
C NMR

19
F NMR

183
PEG-Fluorinated Guanyldinium Mono TFA Salt 2.9b

Compound 2.7b (161.6 mg, 0.4 mmol) was added to a flame-dried 15 mL three-neck
round bottom flask via a syringe under nitrogen. To this flask were sequentially added
dry DMF (0.4 mL), Hünig’s base (0.14 mL, 0.80 mmol), and 1H-pyrazole-1-
carboxamidine hydrochloride (117.3 mg, 0.80 mmol) under nitrogen. The reaction was
stirred vigorously. Additions of Hünig’s base (0.14 mL, 0.80 mmol) and 1H-pyrazole-1-
carboxamidine hydrochloride (117.3 mg, 0.80 mmol) were made at 72 hours, 144 hours,
and every 24 hours thereafter to drive the reaction to completion. After 11 days, the
solvent was removed under reduced pressure (35
o
C overnight, high vacuum). The
residual was obtained as a yellow gum, which was suspended in H
2
O and passed through
a column packed with Amberlite IRN-78 ion exchange resin. The aqueous fractions were
collected, and the solvent was removed under a stream of nitrogen to yield a reddish
viscous liquid. The resulting crude product was purified using reverse phase
chromatography (MeOH/H
2
O, 0.1% TFA) to yield a light yellow, highly viscous liquid
as the product in mono-TFA salt form (126.8 mg, 57%).
1
H NMR (500 MHz, 55
o
C, CDCl
3
): δ 8.62 (br, 1H, H
2
NCNHNH
2
), 7.37 (br, 4H,
H
2
NCNHNH
2
), 3.96 (t,
3
J
H,F
= 13.4 Hz, 2H, CH
2
OCH
2
CF
2
), 3.92 (NHCH
2
CF
2
), 3.77 –
3.75 (m, 2H), 3.65-3.62 (m, 12H), 3.55-3.53 (m, 2H), 3.34 (s, 3H, CH
3
O).
O
O
FF
3 4
2.9b
H
N
NH
NH
2
HO CF
3
O
184
13
C NMR (125 MHz, CDCl
3
): δ = 158.9 (s, C=N), 117.9-109.2 (m, CF
2
CF
2
CF
2
), 72.3,
71.8, 70.5 – 70.2, 68.2 (t,
2
J
C,F
= 26.0 Hz, CF
2
CH
2
OCH
2
), 58.7 (s, CH
3
O), 42.1 (t,
2
J
C,F
=
22.1 Hz, CF
2
CH
2
NH
2
).
19
F NMR (470 MHz, CDCl
3
): δ = -75.78 (s, 3F, HOOCCF
3
), -117.86 (m, 2F), -119.32
(m, 2F), -127.35 (m, 2F).
FT-IR (cm
-1
, in CHCl
3
): ν = 3354−3015, 2921, 1684, 1457, 1204, 1150−1136, 755.
MALDI-TOF for C
15
H
27
F
6
N
3
O
5
[MH]
+
: calculated 444.19 g/mol, found 444.07 g/mol.

1
H (500 MHz, 55
o
C, CDCl
3
)

185
13
C (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

186
FT-IR (cm
-1
, in CHCl
3
)

187
PEG-Fluorinated Guanidinium Mono TFA Salts 2.9a

Compound 2.7a (1.00 g, 2.22 mmol) was added to a flame-dried 25 mL three-neck
round bottom flask via a syringe under nitrogen. To this flask were sequentially added
dry DMF (2.2 mL), Hünig’s base (773.3 µL, 4.44 mmol), and 1H-pyrazole-1-
carboxamidine hydrochloride (650.8 mg, 4.44 mmol) under nitrogen. Additions of
Hünig’s base (773.3 µL, 4.44 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride
(650.8 mg, 4.44 mmol) were made every 24 hours thereafter until a total of 8 additions
were reached. After 11 days, the solvent was removed under reduced pressure (35
o
C,
high vacuum) to obtain a yellow gum. The residual was suspended in H
2
O and passed
through a column packed with Amberlite IRN-78 ion exchange resin. The aqueous
fractions were collected, and the solvent was removed under a stream of nitrogen to yield
an orange, viscous liquid. The crude product was purified using reverse-phase
chromatography to obtain 2.9a as a yellowish, highly viscous liquid (1.00g, 92%).
1
H NMR (500 MHz, 55
o
C, CDCl
3
): δ = 8.28 (br, 1H, H
2
NCNHNH
2
), 7.34 (br, 4H,
H
2
NCNHNH
2
), 3.97 (t,
3
J
H,F
= 14.0 Hz, 2H, CH
2
OCH
2
CF
2
), 3.93 (NHCH
2
CF
2
), 3.74 –
3.73 (m, 2H), 3.66-3.60 (m, 12H), 3.54-3.52 (m, 2H), 3.33 (s, 3H, CH
3
O).
13
C NMR (125 MHz, CDCl
3
): δ = 158.72 (s, C=N), 117.82-108.96 (m,
CF
2
CF
2
CF
2
CF
2
), 72.21, 71.78, 70.50 – 70.13, 68.24 (t,
2
J
C,F
= 24.3 Hz, CF
2
CH
2
OCH
2
),
58.55 (s, CH
3
O), 41.84 (t,
2
J
C,F
= 23.54 Hz, CF
2
CH
2
NH
2
).
O
O
FF
4 4
2.9a
H
N
NH
NH
2
HO CF
3
O
188
19
F NMR (376 MHz, CDCl
3
): δ = -75.98 (s, 3F, HOOCCF
3
), -117.81 (m, 2F), -119.72
(m, 2F), -123.55 (m, 2F), -123.58 (m, 2F,).
FT-IR (cm
-1
, in CHCl
3
): ν = 3356−3014, 2920, 1684, 1457, 1177, 1132, 759.
MALDI-TOF for C
16
H
27
F
8
N
3
O
5
[MNa]
+
: calculated 494.19 g/mol, found 494.06 g/mol.

1
H (500 MHz, 55
o
C, CDCl
3
)

189
13
C (125 MHz, CDCl
3
)

19
F (376 MHz, CDCl
3
)

190
FT-IR (cm
-1
, in CHCl
3
)

191
PEG-Fluorinated Guanidinium Mono TFA Salts 2.9c

Compound 2.7c (200 mg, 0.363 mmol) was added to a flame-dried 25 mL three-neck
round bottom flask via a syringe under nitrogen. To this flask were sequentially added
dry DMF (0. 363 mL), Hünig’s base (126.4 µL, 0.726 mmol), and 1H-pyrazole-1-
carboxamidine hydrochloride (106.4 mg, 0.726 mmol) under nitrogen. The reaction was
stirred vigorously. Subsequent additions of Hünig’s base (290 µL, 1.67 mmol) and 1H-
pyrazole-1-carboxamidine hydrochloride (244.5 mg, 1.67 mmol) were made every 24
hours until a total of 8 additions were reached. The reaction reached completion after 13
days. The solvent was removed under reduced pressure overnight (35
o
C, high vacuum)
to yield the crude product as a yellow gum, which was suspended in H
2
O and passed
through a column packed with Amberlite IRN-78 ion exchange resin (Alfa Aesar). The
aqueous fractions were collected and the solvent was removed under a stream of nitrogen
to obtain a yellow, viscous liquid as the crude product. The crude was purified by
reverse-phase chromatography (H
2
O/MeOH, 0.1% TFA) to give a yellow, highly viscous
liquid (150.9 mg, 56%).
1
H NMR (500 MHz, 55
o
C, CDCl
3
): δ 8.34 (br, 1H, HOOCCF
3
), 7.41 (br, 4H,
H
2
NCNHNH
2
), 3.97 (t,
3
J
H,F
= 13.4 Hz, 2H, CH
2
OCH
2
CF
2
), 3.93 (NHCH
2
CF
2
), 3.75 –
3.73 (m, 2H), 3.65-3.59 (m, 12H), 3.53-3.51 (m, 2H), 3.33 (s, 3H, CH
3
O).
O
O
FF
6 4
2.9c
H
N
NH
NH
2
HO CF
3
O
192
13
C NMR (125 MHz, 55
o
C, CDCl
3
): δ = 158.88 (s, C=N), 118.15 - 108.56 (m,
CF
2
CF
2
CF
2
CF
2
CF
2
CF
2
), 72.31, 71.86, 70.61 – 70.26, 68.33 (t,
2
J
C,F
= 24.4 Hz,
CF
2
CH
2
OCH
2
), 58.60 (s, CH
3
O), 41.75 (t,
2
J
C,F
= 25.16 Hz, CF
2
CH
2
NH
2
).
19
F NMR (470 MHz, 55
o
C, CDCl
3
): δ = -76.07 (S, SCF
3
), -117.875 (m, 2F), -119.74
(m, 2F), -121.81 (m, 2F), -122.02 (m, 2F), -123.22 (m, 2F), -123.57 (m, 2F).
FT-IR (cm
-1
, in CHCl
3
): ν = 3360−3184, 3020, 2904, 1684, 1216−1144, 761.
MALDI-TOF for C
18
H
27
F
12
N
3
O
5
[MH]
+
: calculated 594.18 g/mol, found 593.99 g/mol.

1
H (500 MHz, 55
o
C, CDCl
3
)

193
13
C (125 MHz, 55
o
C, CDCl
3
)

19
F (470 MHz, 55
o
C, CDCl
3
)

194
FT-IR (cm
-1
, in CHCl
3
)

195
7.2.2 Preparation of Fluoroalkyl Compounds
Triflate 2.10

Diol 2.1a (5.0 g, 19.1 mmol) was added to a flame-dried 500 mL three-neck round
bottom flask under nitrogen. To this flask were sequentially added dry DCM (200 mL)
and trifluoromethanesulfonyl chloride (4.9 mL, 45.8 mmol) under nitrogen. The flask
was cooled in an ice bath, and triethylamine (10.7 mL, 76.3 mmol) was added to the
solution. The reaction was left to react overnight. The solvent was removed. The residual
was dissolved in ethyl acetate (200 mL) and washed with water (100 mL × 2). The
organic phase was washed sequentially with 1M HCl (200 mL), NaHCO
3
(200 mL),
brine (200 mL), and dried over MgSO
4
. The solvent was removed under reduced pressure
to give a yellow oil. It was then crystallized with 3:1 hexanes: ethyl acetate to yield 2.10
as clear crystals (9.4 g, 94%).
m.p. 55-57
o
C.
1
H NMR (400 MHz, CDCl
3
): δ = 4.82 (t,
3
J
H,F
= 12.1 Hz, 4H, CF
3
SO
2
OCH
2
CF
2
).
13
C NMR (150 MHz, CDCl
3
): δ = 118.42 (q,
1
J
C,F
= 320.1 Hz, CF
3
SO
2
), 115.5 (t, CF
2
),
113.6-112.6 (m, CF
2
), 111.3-109.9 (m, CF
2
), 108.0 (t,
2
J
C,F
= 32.9 Hz, CF
2
), 68.07 (t,
2
J
C,F
= 28.4 Hz, OCH
2
).
19
F NMR (376 MHz, CDCl
3
): δ = -123.35 (m, 4F, CF
2
CF
2
CH
2
), -120.1 (m, 4F,
CF
2
CH
2
O), -74.3 (s, 6F, CF
3
SO
2
).
FT-IR (cm
-1
, in CHCl
3
): ν = 3683, 3020, 2400, 1519, 1429, 1220, 931, 761.
Anal. Calc’d for C
8
H
4
F
14
O
4
S
2
: C, 18.26; H, 0.77. Found: C, 19.87; H, 0.77.
O O S S
O
O
CF
3
O
F
3
C
O FF
4
2.10
196
1
H (400 MHz, CDCl
3
)

13
C (150 MHz, CDCl
3
)

Ditriflate 10

TfO
OTf
F F
F F
F F
F F

1
H NMR

13
C NMR
!

Ditriflate 10

TfO
OTf
F F
F F
F F
F F

1
H NMR

13
C NMR
!

197
19
F (376 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

19
F NMR

198
Dibromide 2.11

Compound 2.10 (100 mg, 0.18 mmol) was added to a septum-sealed, oven-dried 8 dr.
vial. To this vial were sequentially added dry DMF (1.2 mL), potassium bromide (50.8
mg, 0.43 mmol), and 18-crown-6 (14.6 mg, 0.054 mmol) under nitrogen. To this solution
after 6 hours was added KBr (25 mg, 0.21 mmol). The reaction continued for another 26
hours. The solution was poured over H
2
O (3.6 mL), extracted with ether (2.4 mL × 3).
The combined ether fractions were washed with H
2
O (7.2 mL × 3) and dried over
MgSO
4
. Solvent was removed under reduced pressure to afford 2.11 as a clear, viscous
liquid (56.1 mg, 81%).
1
H NMR (500 MHz, CDCl
3
): δ = 3.76 (t,
3
J
H,F
= 15.6 Hz, 4H, BrCH
2
CF
2
).
13
C NMR (125 MHz, CDCl
3
): δ = 113.99 (tt,
1
J
C,F
= 257.25 Hz,
2
J
C,F
= 31.8 Hz,
CH
2
CF
2
), 110.91 (tq
1
J
C,F
= 268.01 Hz,
2
J
C,F
= 31.3 Hz, CH
2
CF
2
CF
2
), 26.05 (t,
2
J
C,F
=
25.4 Hz, BrCH
2
).
19
F NMR (470 MHz, CDCl
3
): δ = -113.10 (m, 4F, CF
2
CF
2
CH
2
), -122.03 (m, 4F,
CF
2
CF
2
CH
2
).
FT-IR (cm
-1
, in CHCl
3
): ν = 2987, 2961, 2932, 1727, 1428, 1287−1072, 875.
HR-ESI-MS for C
6
H
4
Br
2
F
8
[M]
+
: calculated 385.8552 g/mol, found 385.8544 g/mol.

Br Br
FF
4
2.11
199
1
H (500 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

200
19
F (470 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

201
Malonocycloheptane 2.12

Potassium hydride (26.8 mg, 0.67 mmol, in mineral oil) was dispensed into a flame-
dried 15 mL three-neck round bottom flask via a syringe, and the suspension was washed
with dry pentane (1 mL × 3). To this KH powder was added dry DMF (1.8 mL).
Dimethyl malonate (88.2 mg, 0.67 mmol) was added to this KH suspension in DMF
drop-wise via a syringe. Vigorous bubbling was observed. KH and dimethyl malonate
were allowed to react for 1 hour, after which compound 2.10 (150 mg, 0.27 mmol) was
added to the solution. To a septum-sealed, oven-dried 8 dr. vial was added potassium
hydride (26.8 mg, 0.67 mmol, in mineral oil) under nitrogen. The suspension was washed
with dry pentane (1 mL × 3). To this KH powder was added dry DMF. Dimethyl
malonate (88.2 mg, 0.67 mmol) was added to this KH suspension in DMF was drop-wise
via a syringe. The solution was stirred for 1 hour.
12 hours after compound 2.10 was added to the solution in the round bottom flask, the
enolate solution in the 8 dr. vial was cannula transferred to the round bottom flask. The
reaction was stirred for an additional 24 hours. The reaction was poured over H
2
O (3.6
mL). The aqueous phase was extracted with ether (3.6 mL × 3). The combined ether
fractions were washed with H
2
O (9 mL × 3) and dried over MgSO
4
. The solvent was
removed under reduced pressure to obtain the crude product as a clear liquid. The crude
product was purified using flash chromatography (benzene: ethyl acetate = 7 : 1, R
f
=
F
F
F
F F
F
F
F
O O
MeO OMe
2.12
202
0.74) to separate the unreacted starting material from the product. The product obtained
from the first chromatography was purified again using flash chromatography (hexanes:
ethyl acetate = 4 : 1, R
f
= 0.59) to obtain a clear liquid, which crystalizes on standing.
The crystal sublimes under room temperature. Yield: 54.3 mg, 57%.
m.p. 65-67
o
C.
1
H NMR (400 MHz, CDCl
3
): δ = 3.83 (s, 6H, CH
3
COC), 3.20 (t,
3
J
H,F
= 15.1 Hz, 4H,
CCH
2
CF
2
).
13
C NMR (125 MHz, CDCl
3
): δ = 168.42 (s, CH
3
COC), 117.78-113.72 (tt,
1
J
C,F
=
254.4 Hz,
2
J
C,F
= 27.9 Hz, CH
2
CF
2
), 111.38 -107.08 (tq,
1
J
C,F
= 229.8 Hz,
2
J
C,F
= 27.8 Hz,
CH
2
CF
2
CF
2
), 54.44 (s, CH
3
CO), 33.02 (t,
2
J
C,F
= 24.7 Hz, CH
2
CF
2
.
19
F NMR (470 MHz, CDCl
3
): δ = -111.41 (m, 4F, CF
2
CF
2
CH
2
), -120.1 (m, 4F,
CF
2
CH
2
O), -74.3 (s, 6F, CF
3
SO
2
).
FT-IR (cm
-1
, in CHCl
3
): ν = 3021, 2958, 1748, 1438, 1345, 1282, 1216, 1180, 1117,
1076, 1016, 932, 756, 668.
GC/MS for C
10
H
7
F
8
O
3
[M – OMe]
+
calculated 327.03, found 327.08 g/mol; C
9
H
7
F
8
O
2

[M – CO
2
Me]
+
calculated 299.03, found 299.08 g/mol.

203
1
H (400 MHz, CDCl
3
)

13
C (125 MHz, CDCl
3
)

204
19
F (470 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

205
Azide 2.13

Compound 2.10 (150 mg, 0.267 mmol) was added to a septum-sealed, oven-dried 8 dr.
vial. To this vial were added dry DMF (1.8 mL) and NaN
3
(41.6 mg, 0.64 mmol) under
nitrogen. The reaction was stirred for 3 hours. The reaction mixture was poured over H
2
O
(3.6 mL) and extracted with ether (3.6 mL × 3). The combined ether fractions were
washed with H
2
O (10 mL × 3) and dried over MgSO
4
. The solvent was removed under
reduced pressure to yield a colorless viscous liquid (83.3 mg, > 99%).
1
H NMR (500 MHz, CDCl
3
): δ = 3.77 (t,
3
J
H,H
= 14.7 Hz, 4H, CH
2
CF
2
CF
2
).
13
C NMR (150 MHz, CDCl
3
): δ = 115.73 (tt,
1
J
C,F
= 258.9 Hz,
2
J
C,F
= 30.1 Hz,
N
3
CH
2
CF
2
), 111.82 (tq,
1
J
C,F
= 265.87 Hz,
2
J
C,F
= 33.52 Hz, NH
2
CH
2
CF
2
CF
2
), 50.20 (t,
2
J
C,F
= 24.3 Hz, CH
2
N
3
).
19
F NMR (470 MHz, CDCl
3
): δ = -117.6 (m, CH
2
CF
2
), -123.3 (m, CF
2
CF
2
).
Data were consistent with a previously reported compound.
7

N
3
N
3
FF
4
2.13
206
1
H (500 MHz, CDCl
3
)

13
C (150 MHz, CDCl
3
)

207
19
F (470 MHz, CDCl
3
)

208
Phthalimide 2.14

Compound 2.10 (3.0 g, 5.7 mmol) was added into a 100 mL three-neck round bottom
flask. To this flask were sequentially added dry DMF (300 mL) and potassium
phthalimide (2.53 g, 13.7 mmol) under nitrogen. The solution was stirred at 85 °C
overnight. The reaction was cooled to room temperature and poured over brine (300 mL).
The product precipitated as a white solid that was collected by filtration (2.6 g, 89%).
M.p. 255-260
o
C.
1
H NMR (400 MHz, CDCl
3
): δ = 7.93 (q,
3
J
H,H
= 2.8 Hz, Ar, 4H), 7.78 (q,
3
J
H,H
= 2.8
Hz, Ar, 4H), 4.38 (t,
3
J
H,F
= 15.6 Hz, 4H, NCH
2
CF
2
).
13
C NMR (100 MHz, CDCl
3
): δ = 166.8 (s, Ar), 134.5 (s, Ar), 131.8 (s, Ar), 123.9 (s,
Ar), 37.6 (t,
3
J
C,F
= 25.1 Hz, NCH
2
).
19
F NMR (376 MHz, CDCl
3
): δ = -123.5- -123.6 (m, 4F, CF
2
CF
2
CH
2
), -116.4- -(116.6)
(m, 4F, CF
2
CH
2
N).
Anal. Calc’d for C
13
H
13
0
4
N: C, 50.78; H, 2.32; N, 5.38. Found: C, 50.73; H, 2.03; 5.05.
FT-IR (cm
-1
, in CHCl
3
): ν = 3019, 2400, 1522, 1424, 1216, 930, 759.

N N
FF
4
2.14
O
O
O
O
209
1
H (400 MHz, CDCl
3
)

13
C (100 MHz, CDCl
3
)

210
19
F (376 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

211
Diamine 2.15

Compound 2.14 (2.3 g, 4.4 mmol) was added into a 500 mL three-neck round bottom
flask. To this flask was added hydrazine (1.4 mL, 44.4 mmol) in absolute ethanol (300
mL). The reaction was placed in a 78
o
C oil bath overnight. The reaction was cooled to
the room temperature, and a white precipitate was removed via filtration. The filtrate was
collected and the solvent was removed under reduced pressure to obtain the crude
product. Chloroform (200 mL) was added to the crude product, and additional white
precipitate was removed by filtration. The solvent was removed by rotary evaporation to
afford the product as a white solid that was sublimated to obtain a clear crystal. Yield:
0.87 g, 76%.
M.p. 45.5-47
o
C.
Data are consistent with a previously-reported compound.
7,8

2.15 can also be prepared via direct hydrogenation using the following procedure:
Compound 2.13 (53 mg, 0.17 mmol), quinoline (0.8 mg), absolute EtOH (1.85 mL),
and palladium (5 wt% on calcium carbonate, poisoned by lead; 2.6 mg, 5% w/w) were
added to a 3 mL Schlenk flask. A balloon filled with H
2
was immediately attached to the
flask. The solution was stirred for 6 hour 45 min. The reaction was filtered over a pad of
celite. The filtrate was collected and poured over 1 mL of HCl. The aqueous phase was
washed with ether (2 mL × 1), and the pH was adjusted to basic with saturated NaOH
solution. The product was extracted with ether (2 mL × 3) from the basic aqueous phase.
The combined ether fractions were dried over MgSO
4
. The crude product was separated
H
2
N NH
2
FF
4
2.15
212
using flash-chromatography (chloroform : MeOH = 1:12, R
f
= 0.33) to give 2.15 as a
clear crystal which sublimes at room temperature. Yield: 26.2 mg, 59%.
1
H NMR (400 MHz, CDCl
3
): δ = 3.24 (t,
3
J
H,H
= 16.0 Hz, 4H, CH
2
CF
2
CF
2
), 1.35-1.18
(b, 4H, NH
2
CH
2
CF
2
).
13
C NMR (150 MHz, CDCl
3
): δ = 118.4-117.8 (m, CF
2
), 116.5-116.1 (m, CF
2
), 114.6
(t,
3
J
C,F
= 28.5 Hz, CF
2
), 113.4 (t,
3
J
C,F
= 35.1 Hz, CF
2
), 111.7 (q,
3
J
C,F
= 35.1 Hz, CF
2
),
109.9 (t,
3
J
C,F
= 35.1 Hz, CF
2
), 42.9 (t,
3
J
C,F
= 24.6 Hz, CH
2
NH
2
).
19
F NMR (376 MHz, CDCl
3
): δ = (-124.2)-(-124.4) (m, 4F, CF
2
CF
2
CH
2
), (-122.1)-(-
122.3) (m, 4F, CF
2
CH
2
NH
2
).
FT-IR (cm
-1
, in CHCl
3
): ν = 3020, 1215, 756.

1
H (400 MHz, CDCl
3
)

213
13
C (150 MHz, CDCl
3
)

214
19
F (376 MHz, CDCl
3
)

FT-IR (cm
-1
, in CHCl
3
)

215
Triazole 2.16

Compound 2.13 (100 mg, 0.32 mmol) was added to a septum-sealed 8 dr. vial via a
syringe. To this vial were sequentially added dry DMF (1 mL), phenylacetylene (78.4
mg, 0.768 mmol), and CuI (9.1 mg, 0.048 mmol) under nitrogen. The reaction was heated
at 70
º
C for 12 hours. The solution was cooled to room temperature. The solvent was
removed under reduced pressure to obtain a light green crude product. The crude product
was re-dissolved in a minimal amount of acetone. The acetone solution was pushed
through a plug of silica gel. The eluent was collected and solvent was removed under
reduced pressure to obtain a yellowish solid, which was triturated with ethyl acetate and
hexanes to obtain 2.16 as a white, fluffy solid, 147 mg, 89%.
M.p. 233-235
o
C.
1
H NMR (599.804 MHz, DMSO-D
6
): δ = 8.74 (s, 1H, NCHC), 7.91-7.90 (m, 2H, Ar,
CHCHCH), 7.48-7.49 (m, 2H, Ar, CHCHCHN), 7.38-7.36 (m, 1H, Ar, CHCHCH), 5.62
(t, 4H,
3
J
H,F
= 15.9 Hz, CH
2
CF
2
CF
2
).
13
C NMR (150.837 MHz, DMSO-D
6
): δ = 146.79 (s, CCNCH), 130.02 (s, CHCC),
128.98 (s, Ar, CHCHCH), 128.25 (s, Ar, CHCHCH), 125.34, (s, Ar, CHCHC), 123.50 (s,
CCHN), 116.3-112.9 (tm,
1
J
C,F
=260.5, CF
2
), 112.5-109.0 (tm,
1
J
C,F
= 270.3, CF
2
), 48.5
(t,
2
J
C,F
= 22.5 Hz, CH
2
N).

N N
FF
4
2.16
N
N N
N
Ph Ph
216
19
F NMR (470 MHz, DMSO): δ = -115.93 (m, 4F, CF
2
CF
2
CH
2
), -122.43 (m, 4F,
CF
2
CH
2
).
FT-IR (cm
-1
, in CH
2
Cl
2
): ν = 3052, 2988, 1419, 1265, 897, 737.
MALDI-TOF for C
22
H
17
F
8
N
6
[MH]
+
: calculated 517.14 g/mol, found 517.04 g/mol.

1
H (599.804 MHz, DMSO-D
6
)

217
13
C (150.837 MHz, DMSO-D
6
)

19
F (470 MHz, DMSO)

218
FT-IR (cm
-1
, in CH
2
Cl
2
)

219
7.3 Chapter 3 Experimental and Spectral Data
PEG-Fluorinated Malonyl Ester 3.4

Compound 2.5a was prepared as previously described. KH (0.24 mL, 6 mmol, in
mineral oil) was added to a septum-sealed, oven-dried 8 dr. vial via a syringe under
nitrogen. The KH suspension was washed with dry pentane (1 mL × 3) under nitrogen.
To this KH solid was added dry THF (4 mL) via a syringe under nitrogen to make a
suspension. Dimethyl malonate (823 mL, 9.5 mmol) was added drop-wise to the
suspension via a syringe under nitrogen. The solution was stirred for 1 hour. In a separate
oven-dried 8 dr. vial, 2.5a (0.5 g, 0.865 mmol) was dissolved in dry THF (2.8 mL) under
nitrogen. The 2.5a/THF solution was cannula transferred to the vial containing the
enolate solution under nitrogen. This vial was quickly capped with a green Teflon cap
and placed in an 80
o
C oil bath overnight. The solution was poured over 7 mL of H
2
O and
extracted with diethyl ether (7 mL × 2). The combined organic fractions were washed
with brine (7 mL × 1) and dried over MgSO
4
. The solvent was removed to afford the
crude product that was purified by flash chromatography (EtOAc : Hexanes = 2 : 1, R
f
=
0.35). 3.4a was obtained as a colorless oil (0.37g). Yield: 77.7%
1
H NMR (500 MHz, CDCl
3
): δ = 2.79 (dt,
3
J
H,F
= 19.3 Hz,
3
J
H,H
= 18.8, 2H,
CHCH
2
CF
2
), 3.34 (s, 3H, CH
3
O), 3.55-3.82 (m, 8H, OCH
2
CH
2
O, d, 1H,
CH2CH(CO
2
Me)
2
, s, 6H, CH
3
OCO), 3.98 (t,
3
J
H,F
= 14.3 Hz, 2H).
O
O
FF
4 4
3.4
O O
O
O
220
13
C NMR (150.837 MHz, CDCl
3
): δ = 30.61 (t,
3
J
C,F
= 25.8, CHCH
2
CF
2
), 44.59 (s,
CH
3
OCO), 53.40 (s, CHCH
2
CF
2
), 59.15 (s, CH
3
O), 68.46 (t,
3
J
C,F
= 30.5), 70.65-72.47
(OCH
2
CH
2
O), 168.205 (CH
3
OCO).
19
F NMR (470 MHz, CDCl
3
): δ = -113.98 (m, 2F, CF
2
CF
2
CH
2
), -120.80 (m, 2F,
CF
2
CH
2
), -123.41 (m, 2F, CF
2
), -124.00 (m, 2F, CF
2
).
FT-IR (cm
-1
, neat): ν = 2880.66, 1747.26, 1457.21, 1351.62, 1113.05, 953.70, 853.21,
761.48.
MALDI-TOF for C
18
H
26
F
8
O
8
[MH]
+
: calculated 523.16 g/mol, found 522.12 g/mol.

1
H NMR (500 MHz, CDCl
3
)

221
13
C NMR (150.837 MHz, CDCl
3
)

19
F NMR (470 MHz, CDCl
3
)

222
FT-IR (cm
-1
, neat)

223
PEG-Fluorinated Dicarboxylic Acid 3.5

Compound 3.4 (0.100 g, 0.177 mmol) was added to a Schlenk flask via a syringe under
air. To this flask was added a solution containing an equal volume of aqueous KOH
(50%, w/v) and EtOH. The flask was placed in a 100
o
C oil bath. After 4 hours of stirring,
the flask was placed in an ice bath. Conc. HCl (36.5%, ca. mL) was added drop-wise to
this flask until the pH reached ca. 2. The acidified solution was extracted with ether (2
mL × 3). The combined ether fractions were dried over MgSO
4
, and the solvent was
removed under reduced pressure to yield 3.4 as a light yellow oil (80 mg). The product
was used without purfication.

O
O
FF
4 4
3.5
OH O
O
OH
224
PEG-Fluorinated Monocarboxylic Acid 3.3

Compound 3.4 (0.52 g) was added to an 8 dr. vial. To this vial was added a mixture of
dioxane/H
2
O (7 : 3, 4.86 mL; containing 2% (v%) 36% HCl). The vial was placed in a
100
o
C oil bath for 2 hours. The solution was cooled to room temperature, and it was
extracted with ether (5 mL × 3). The combined ether fractions were washed with brine (5
mL × 1) and dried over MgSO
4
to obtain the crude product as a light yellow oil (0.44 g).
The crude product was purified by flash chromatography (manual column, chloroform :
MeOH = 8 : 1, R
f
= 0.34) to yield the product as a light yellow oil (128.7 mg). Two step
yield (including the previous step): 22.6%.
1
H NMR (599.804 MHz, CDCl
3
): δ = 2.47 (m, 2H, CF
2
CH
2
CH
2
), 2.68 (t,
3
J
H,H
= 7.4,
2H, CF
2
CH
2
CH
2
), 3.39 (s, CH
3
O), 3.55-3.57 (m, 2H, OCH
2
CH
2
O), 3.68-3.64 (m, 6H,
OCH
2
CH
2
O), 3.77-3.79 (m, 2H, OCH
2
CH
2
O), 4.02 (t,
3
J
H,F
= 14.7, OCH
2
CF
2
).
13
C NMR (150.837 MHz, CDCl
3
): δ = 26.69 (t,
3
J
C,F
= 21.4, CH
2
O), 58.90 (s, CH
3
O),
68.30 (t,
3
J
C,F
= 25.7, CH
2
CH
2
CF
2
), 70.35-70.65 (m, OCH
2
CH
2
O), 71.84 (s,
OCH
2
CH
2
O), 72.31 (s, OCH
2
CH
2
O), 109.10-119.89 (m, CF
2
), 174.73 (COOH).
19
F NMR (564 MHz, CDCl
3
): δ = -124.20 (m, 2F, OCH
2
CF
2
), -123.50 (m, 2F,
CH
2
CF
2
CF
2
), -120.09 (m, 2F, OCH
2
CF
2
), -113.99 (m, 2F, CF
2
CF
2
CH
2
CH
2
).
FT-IR (cm
-1
, neat): ν = 849.60, 951.00, 981.96, 1104.14, 1352.38, 1457.20, 1507.52,
1541.15, 1558.71, 1653.16, 1734.25, 2917.56.
MALDI-TOF for C
15
H
22
F
8
O
6
[MH]
+
: calculated 451.14 g/mol, found 450.03 g/mol.
O
O
FF
4 4
3.3
O
OH
225
1
H NMR (500 MHz, CDCl
3
)

13
C NMR (125 MHz, CDCl
3
)

226
19
F NMR (470 MHz, CDCl
3
)

FT-IR (cm
-1
, neat)

227
PEG-Fluorinated Benzyl Carbamate 3.6

To a septum-sealed, oven-dried 4 dr. vial were sequentially added ADMC (75.8 mg,
0.438 mmol) and dry CH
3
CN (0.88 mL) under nitrogen.
2
The vial was cooled to 0
o
C,
and sodium azide (28.5 mg, 0.44 mmol) was added to the cooled solution under nitrogen.
The solution was stirred for 30 min thereafter. Proton sponge (93.9 mg, 0.44 mmol) and
dry THF (1.6 mL) were added to a separate oven-dried 4 dr. vial. To this solution was
cannula transferred compound 3.3 (0.365 mmol, 180.8 mg) under nitrogen. The solution
was stirred for 30 min. The solution containing 3.3/THF was cannula transferred to the
vial containing ADMC and sodium azide. The formation of an intermediate acyl azide
was monitored with FT-IR (peak at 2153 cm
-1
), and the conversion reached completion
after 1 hour. The solution was quickly filtered over a pad of celite into an oven-dried 4 dr.
vial, and the solvent was removed under reduced pressure. During the solvent removal,
smooth effervescence was observed. Formation of an isocyanate was monitored using
FT-IR (peak at 2278 cm
-1
). Dry toluene (2.4 mL) and benzyl alcohol (0.045 mL) were
added to the residual under nitrogen. The vial was capped and placed in a 95
o
C oil bath
for 12 hours. Disappearance of the isocyanate was monitored using IR. The reaction was
poured over 2 mL H
2
O, and the aqueous phase was extracted with ether (2 mL × 2). The
combined ether fractions were dried over MgSO
4
, and the solvent was removed under
reduced pressure. The crude product was purified by flash chromatography (EtOAc :
hexanes = 2 : 1, R
f
= 0.55) to obtain 3.6 as a light yellow oil (108.9 mg). Yield: 50%.
O
O
FF
4 4
3.6
N
H
O
O
228
1
H NMR (600 MHz, CDCl
3
): 2.35 (m, 2H, CF
2
CH
2
CH
2
), 3.37 (s, CH
3
O), 3.55-3.72,
(m, 16H, OCH
2
CH
2
O, t, 2H, CH
2
CH
2
NH), 3.632-3.684 (m, 6H, OCH
2
CH
2
O), 3.77-3.79
(m, 2H, OCH
2
CH
2
O), 4.01 (t,
3
J
H,F
= 14.3, 2H, OCH
2
CF
2
), 5.01 (br, 1H, NH), 5.11 (s,
2H, COCH
2
C), 7.35 (m, 5H, Ar).
13
C NMR (150 MHz, CDCl
3
): δ = 31.61 (t, NHCH
2
CH
2
CF
2
), 33.79 (s, NHCH
2
CH
2
),
59.15 (s, CH
3
O), 67.10 (t, OCH
2
CF
2
), 70.66-72.47 (m, OCH
2
CH
2
O), 109.64-121.77 (m,
CF
2
), 128.38 (s, Ar, CH
2
), 128.70 (s, Ar, CH
2
), -136.35 (COCH
2
), -156.32 (COCH
2
).
19
F NMR (564 MHz, CDCl
3
): δ = -124.20 (m, 2F, OCH
2
CF
2
), -123.50 (m, 2F,
CH
2
CF
2
CF
2
), -120.09 (m, 2F, OCH
2
CF
2
), -113.99 (m, 2F, CF
2
CF
2
CH
2
CH
2
).
FT-IR (cm
-1
, neat): ν = 1457.07-1558.63, 1539.97, 1635.72-1733.79, 2159.95, 2923.12.
MALDI-TOF for C
22
H
29
F
8
NNaO
6
[MNa]
+
: calculated 578.18 g/mol, found 578.28
g/mol.

1
H NMR (500 MHz, CDCl
3
)

229
13
C NMR (150 MHz, CDCl
3
)

19
F NMR (470 MHz, CDCl
3
)

230
PEG-Fluorinated Amine 3.2

Compound 3.6 (59.5 mg, 0.1 mmol) and ethanol (0.6 mL, containing 0.263% quinoline,
w/v) were added to a 3 mL Schlenk flask. To this solution was added Pd/C (5.95 mg,
10% w/w). A balloon filled with H
2
was attached to the flask, and the solution was stirred
for 1 hour 30 minutes. The reaction was filtered over a pad of celite. The filtrate was
poured over H
2
O (0.4 mL) and extracted with ether (0.4 mL × 3). The combined ether
fractions were dried over MgSO
4
. The solvent was removed under reduced pressure to
give the crude product which was separated using flash chromatography to afford 3.2 as a
yellow oil (33.6 mg). Yield: 72.7%.
1
H NMR (500 MHz, CDCl
3
): δ = 1.67 (2H, NH
2
), 2.24 (t, 2H, CF
2
CH
2
CH
2
), 3.04 (t,
2H, CF
2
CH
2
CH
2
), 3.37 (s, 3H, CH
3
O), 3.54-3.77 (m, 12H, OCH
2
CH
2
O), 4.01 (t, 2H,
OCH
2
CF
2
).
13
C NMR (125 MHz, CDCl
3
): δ = 34.55 (s, NHCH
2
CH
2
CF
2
), 34.96 (t,
NHCH
2
CH
2
CF
2
), 59.12 (s, CH
3
O), 68.47 (t, OCH
2
CF
2
), 70.84-72.45 (m, OCH
2
CH
2
O), ,
109.48-120.45 (m, CF
2
).
19
F (470 MHz, CDCl
3
): δ = -124.26 (2F, OCH
2
CF
2
CF
2
), -123.57 (2F,
CH
2
CH
2
CF
2
CF
2
), -120.13 (2F, OCH
2
CF
2
), -113.69 (2F, CH
2
CH
2
CF
2
CF
2
).
FT-IR (cm
-1
, neat): ν = 1120.66, 1457.02, 1457.37, 1558.96, 1653.10, 1700.29,
2361.10, 2880.32.
MALDI-TOF for C
14
H
24
F
8
NO
4
[MNa]
+
: calculated 422.16 g/mol, found 422.28 g/mol.
O
O
FF
4 4
3.2
NH
2
231
1
H NMR (500 MHz, CDCl
3
)

13
C NMR (125 MHz, CDCl
3
)

232
19
F (470 MHz, CDCl
3
)

FT-IR (cm
-1
, neat)

233
PEG-Fluorinated Guanidine 3.1 (as TFA salt)

Compound 3.6 (48.1 mg, 0.11 mmol) was added to a septum-sealed, oven-dried 2 dr.
vial under nitrogen. To this vial were sequentially added dry DMF (0.1 mL), 1H-
pyrazole-1-carboxamidine hydrochloride (31.4 mg, 0.21 mmol), and Hünig’s base (37.2
µL, 0.21 mmol) under nitrogen. After 24 hours, a second aliquot of 1H-pyrazole-1-
carboxamidine hydrochloride (31.4 mg, 0.21 mmol) and Hünig’s base (37.2 µL, 0.21
mmol) were added. After 48 hours, the reaction mixture was suspended in diethyl ether (3
mL) and filtered over a pad of celite. The filtrate was collected and the solvent was
removed under reduced pressure. The crude product was obtained as a yellow oil. The
crude product was purified on a reverse phase column (H
2
O/0.1% TFA, MeOH/0.1%
TFA) to obtain the guanidinium·triflate salt (46.5 mg) as a light yellow oil. Yield: 70%.
1
H NMR (500 MHz, CDCl
3
): δ = 2.56 (t, t, 4H, CF
2
CH
2
CH
2
), 3.50 (s, 3H, CH
3
O),
3.42-4.00 (m, 12H, OCH
2
CH
2
O), 4.13 (t, 2H, OCH
2
CF
2
), 7.41 (s, 2H, br, NH), 8.21 (s,
4H, br, NH).
13
C NMR (125 MHz, CDCl
3
): δ = 30.88 (t, NHCH
2
CH
2
CF
2
), 34.08 (s,
NHCH
2
CH
2
CF
2
), 58.81 (s, CH
3
O), 68.50 (t, OCH
2
CF
2
), 70.40-70.70 (s, OCH
2
CH
2
O),
71.99 (s, OCH
2
CH
2
O), 72.36 (s, OCH
2
CH
2
O), 111.72-120.07 (m, CF
2
), 157.99 (s, NCN).

O
O
FF
4 4
3.1
N
H
NH
2
NH
HO CF
3
O
234
19
F (470 MHz, CDCl
3
): δ = -124.03 (2F, OCH
2
CF
2
CF
2
), -123.43 (2F,
CH
2
CH
2
CF
2
CF
2
), -119.78 (2F, OCH
2
CF
2
), -113.80 (2F, CH
2
CH
2
CF
2
CF
2
).
FT-IR (cm
-1
, neat): ν = 1124.87, 1203.91, 1457.37, 1684.62, 2922.93.
MALDI-TOF for C
15
H
26
F
8
N
3
O
4
[MH]
+
: calculated 464.18 g/mol, found 464.24 g/mol.

1
H NMR (500 MHz, CDCl
3
)

235
13
C NMR (125 MHz, CDCl
3
)

19
F (470 MHz, CDCl
3
)

236
FT-IR (cm
-1
, neat)

237
7.4 Chapter 5 Experimental and Spectral Data
7.4.1 Synthesis of Ln-DOTP
5-
and Ln-DOTA
-
compounds
Na[Gd(DOTA)]·4H
2
O 5.1

Na[Gd-DOTA]·4H
2
O was prepared by Dr. Anna Dawsey according to a modified
literature procedure.
9 , 10
DOTA (24.3 mg, 0.06 mmol) was dissolved in distilled
Arrowhead water (3 mL). GdCl
3
·6H
2
O (22.3 mg, 0.06 mmol) was added to the ligand
solution while stirring. After the addition of GdCl
3
·6H
2
O was finished, the pH was
measured to be 2.12. The pH of the resulting reaction mixture was adjusted to be between
5.5 and 7.0 by adding 0.1 M NaOH, and the pH was continued to be adjusted until the pH
remained constant at a value between 5.5 and 7.0 for > 1 h. Upon equilibration the pH of
the reaction mixture was adjusted with 1 M NaOH to a final pH ≥ 11, and a white
precipitate was observed. The precipitate was filtered off using a 0.45 µm PTFE filter,
and the filtrate was lyophilized to dryness. The dried solid was purified using reverse
phase column chromatography (H
2
O/MeOH). Na[Gd-DOTA]·4H
2
O was produced as a
white solid (21.3 mg) in 54% yield.
M.p.: No sign of melting was observed at 250
o
C.
Elemental analysis: calc’d C, 29.44; H, 4.94; N, 8.58; found C, 29.73; H, 4.46, N, 8.41.

N
N
N
N
O
O O
O
O
Gd
O
O
O
Na H
2
O
238
Na
5
[Gd(DOTP)]·9H
2
O 5.2

Na
5
[Gd(DOTP)]·9H
2
O was prepared according to a modified literature procedure.
24

H
8
DOTP (150 mg, 0.274 mmol) was added into a 50 mL round bottom flask, and HPLC
grade H
2
O (27.4 mL) was added to dissolve the solid. This aqueous solution was heated
to 80
o
C, and the pH was adjusted to ca. 8-9. To this solution was added an aqueous
solution of GdCl
3
⋅6H
2
O (96.6 mg, 0.26 mmol in 13 mL HPLC grade H
2
O) at a speed of
20 sec/drop. The pH was maintained at ca. 8-9 by adjusting the pH with 0.05 M aqueous
NaOH solution or 1 M HCl solution. After the addition was finished, the solution was
stirred at 80
o
C for an additional 30 min. The solution was allowed to stand overnight at
room temperature, and it was lyophilized to obtain a white powder thereafter. The white
powder was dissolved in MeOH, and H
2
O was added drop-wise to the MeOH solution to
obtain a white precipitate. The resulting white precipitate was washed with a mixture of
MeOH and H
2
O (9:1 v/v) and filtered over a fine fritted funnel. The precipitate was dried
under reduced pressure to obtain a white powder (277.8 mg). The white powder was
added to a 2 dr. vial and dissolved in 0.2 mL H
2
O. This 2 dr. vial was placed in a 2 oz.
bottle containing ca. 5 mL isopropanol. After the bottle capped, crystals were obtained in
5 days. The crystals were filtered from the solution and dried at 90
o
C under high vacuum
to obtain the final product as translucent, square-shaped solids. Yield: 195.3 mg, 77.1%.
M.p.: Slight decomposition at 197.5
o
C.
11

N
N
N
N
P
P
P
P
Gd
O
O
O
O
O O
O
O
O
O
O
O
5
5Na 9H
2
O
239
Elemental analysis: calc’d C, 14.79; H, 4.34; N, 5.75; Na, 11.79; found C, 14.75; H,
4.02; N, 5.36; Na, 11.88.
FT-IR (cm-
1
, KBr pellet) ν = 589.1, 985.0, 1060.9, 1133.7, 1472.8, 1653.3, 2863.7,
3421.5.

FT-IR

240
Synthesis of Na[Eu(DOTA)]·4H
2
O 5.3

Na[Eu(DOTA)]·4H
2
O was prepared by Dr. Anna Dawsey.
10
EuCl
3
· 6H
2
O (22.0 mg,
6.00 × 10
-2
mmol) was added to an 8 dr. vial, and distilled H
2
O (5 mL) was added to
dissolve the solid. H
4
DOTA (24.6 mg, 6.09 × 10
-2
mmol) was added, and the pH of the
resulting solution was adjusted to > 5. No more adjustments were needed after the pH of
the solution was steady. The solution was stirred for 23 hours 45 min. The solution was
lyophilized thereafter to give a white powder. The content of the metals were determined
using ICP-OES priory to using them in the experiments.

N
N
N
N
O
O O
O
O
Eu
O
O
O
Na H
2
O
241
Synthesis of Na
5
[Eu(DOTP)]·9H
2
O 5.4

Eu-DOTP
5-
was prepared using a modified literature procedure.
12
H
8
DOTP (30 mg,
0.0547 mmol) was weighed into an 8 dr. vial, and HPLC grade H
2
O (5.47 mL) was added
to dissolve the solid to obtain an aqueous solution. The solution was heated to 80
o
C, and
the pH was adjusted to ca. 8-9. To this solution was added drop-wise an aqueous solution
of EuCl
3
⋅6H
2
O (18.3 mg, 0.0500 mmol in 2.5 mL HPLC grade H
2
O) at a speed of 20
sec/drop. The pH was maintained at ca. 8-9 by adjusting the pH with 0.05 M aqueous
NaOH solution or 1 M HCl solution. After the addition was finished, the solution was
stirred at 80
o
C for an additional 1 hour 30 min. The solution was allowed to stand at
room temperature overnight, and it was lyophilized to obtain a white powder thereafter.
The white powder was dissolved in MeOH, and H
2
O was added drop-wise to the MeOH
solution to obtain a white precipitate. The resulting white precipitate was washed with a
mixture of MeOH and H
2
O (9:1 v/v) and filtered over a fine fritted funnel. The
precipitate was dried under reduced pressure to obtain a white powder (23.2 mg). Yield:
46%. Before the luminescence decay experiment, the metal content in the sample was
determined by inductively coupled plasma optical emission spectroscopy (ICP–OES).
FT-IR (cm
-1
, KBr pellet) ν = 586.5, 801.9, 986.1, 1061.2, 1129.0, 1233.0, 1298.2,
1468.4, 1653.1, 2865.7, 3406.9, 3425.1, 3440.0, 3459.2, 3474.9.

N
N
N
N
P
P
P
P
Eu
O
O
O
O
O O
O
O
O
O
O
O
5
5Na 9H
2
O
242
FT-IR

243
Synthesis of and Na[Y(DOTA)]·4H
2
O 5.5

Na[Y(DOTA)]·4H
2
O was prepared by Dr. Anna Dawsey.
10
YCl
3
· 6H
2
O (12.8 mg,
6.00 × 10
-2
mmol) was added to an 8 dr. vial, and distilled H
2
O (5 mL) was added to
dissolve the solid. H
4
DOTA (24.6 mg, 6.09 × 10
-2
mmol) was added, and the pH of the
resulting solution was adjusted to > 5. No more adjustments were needed after the pH of
the solution was steady. The solution was stirred for 23 hours 45 min. The solution was
lyophilized thereafter to give a white powder. The content of the metals were determined
using ICP-OES priory to using them in the experiments.

N
N
N
N
O
O O
O
O
Y
O
O
O
Na H
2
O
244
Na
5
[Y(DOTP)]·9H
2
O 5.6

Na
5
[Y(DOTP)]·9H
2
O was synthesized according to a literature procedure.
13
To an 8 dr.
vial were added YCl
3
⋅6H
2
O solution (0.5 mL, 0.1 M, in HPLC grade H
2
O) and NaOH
solution (0.15 mL, 1 M). The solution was diluted by H
2
O (HPLC grade, 5.4 mL) and
stirred vigorously. H
8
DOTP (30.0 mg, 0.0525 mmol) was added, and a murky solution
formed. The pH of the solution was adjusted with 0.05 M NaOH to ca. 5. The vial was
placed in a 40
o
C oil bath for 24 hours, and the pH of the solution was adjusted to 9 using
0.05 M NaOH thereafter. The solution was filtered and lyophilized to afford a white
powder. The metal content was determined using ICP-OES priory to the VT
17
O NMR
experiment.
1
H NMR (500 MHz, D
2
O): δ = 2.49-2.61 (-NCH
2
CH
2
N-, -NCH
2
-PO
3
Na
2
), 3.60 (-
NCH
2
CH
2
N-), 3.81 (-NCH
2
-PO
3
Na
2
), 4.28 (-NCH
2
CH
2
N-)
13
C NMR (125 MHz, D
2
O): δ = 51.94 (s, -NCH
2
CH
2
N-), 51.98 (s, -NCH
2
CH
2
N-),
52.11 (s, -NCH
2
CH
2
N-), 54.23 (d, -NCH
2
-PO
3
Na
2
,
1
J
C-P
= 137.5).
31
P NMR (202 MHz, D
2
O): δ = 18.17 (s, -PO
3
Na
2
).
FT-IR (cm
-1
, KBr pellet) ν = 803.91, 987.87, 1072.54, 1262.07, 1652.99, 2863.53,
2964.02, 3458.00.

N
N
N
N
P
P
P
P
Y
O
O
O
O
O O
O
O
O
O
O
O
5
5Na 9H
2
O
245
1
H NMR (500 MHz, D
2
O)

246
13
C NMR (125 MHz, D
2
O)

247
31
P NMR (202 MHz, D
2
O)

248
FT-IR (cm
-1
, KBr pellet)

249
7.4.2 Relaxivity (r
1
) Measurements at 9.4 T
7.4.2.1 Relaxivity (r
1
) Measurement Procedures at 9.4 T
For Gd-DOTP
5-
system:
The samples were prepared according to a procedure previously described by Dr. Anna
Dawsey.
10
Two stock solutions were prepared: A stock solution of Gd-DOTP
5-
and a
stock solution of 2.9a. The stock solution of Gd-DOTP
5-
(100 mM) was prepared by
weighing out the appropriate amount of the contrast agent into a tared 1 dram vial and
dissolving it in an appropriate amount of distilled H
2
O. This stock solution was diluted to
make 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM stock solutions. To prepare a stock
solution of 2.9a, a methanol solution of 2.9a was dispensed into a tared 1 dr. vial. The
vial was placed under high vacuum overnight to remove the methanol. 2.9a was
redissolved in an appropriate amount of HPLC grade H
2
O to make a 100 mM solution.
T
1
measurement on samples containing Gd-DOTP
5-
only: The Gd-DOTP
5-
stock
solution (0.25 mM, 0.5 mM, 0.75 mM, or 1 mM) was dispensed into the coaxial inserts
which are placed inside 5 mm NMR tubes as described in section 7.1.4. This sample was
ready for T
1
measurements.
T
1
measurement on samples containing Gd-DOTP
5-
and 2.9a (using 4 equiv. as a
example): An aliquot of the 2.9a stock solution (1 µL, 100 mM) corresponding to 4
equiv. of the contrast agent (100 µL, 0.25 mM) was added to a 0.5 dram vial and the vial
was lyophilized. 2.9a was then rinsed out of the 0.5 dram vial using 100 µL of 0.25 mM
contrast agent solution and added to an external coaxial insert NMR tube as described in
section 7.1.4. This sample was ready for T
1
measurement. Subsequently, 100 µL of the
Gd-DOTP
5-
of corresponding concentrations (0.25, 0.5, 0.75, or 1.0 mM Gd-DOTP
5-
in
250
HPLC H
2
O) was used to rinse the respective vials of 2.9a in order to prepare the sample
containing 2.9a and Gd-DOTP
5-
for T
1
measurement. When an amount of 2.9a other than
4 equiv. is needed, the stock solution of 2.9a dispensed into the vial can be adjusted
accordingly.
To prepare a sample containing Gd-DOTP
5-
, 4 equiv. of 2.9a, and urea: An aliquot of
the 2.9a stock solution (1 µL, 100 mM) corresponding to 4 equiv. of the contrast agent
(100 µL, 0.25 mM) was added to a 0.5 dram vial and lyophilized. 100 µL of 0.25 mM
contrast agent solution was added to this vial containing 2.9a. The vial was left to stand
for 10 min, and urea (0.9 mg, final concentration 150 mM) was added to the vial. The
solution was added to an external coaxial insert NMR tube as described in section 7.1.4,
and this sample was run immediately. Subsequently, 100 µL of the Gd-DOTP
5-
of
corresponding concentrations (0.25, 0.5, 0.75, or 1.0 mM Gd-DOTP
5-
in HPLC H
2
O) was
added to the respective vials of 2.9a in order to prepare the sample containing 2.9a and
Gd-DOTP
5-
for T
1
measurement. These vials were also let stand for 10 min before urea
(0.9 mg, 150 mM final concentration) was added.
To prepare a sample containing Gd-DOTP
5-
, 4 equiv. of 2.9a, urea, and sonicated:
Samples prepared as described in the paragraph above were placed in a bench top
sonicator for 1s × 3 and then 3s × 1. This sample was run immediately.
The coaxial inserts were placed in NMR tubes containing D
2
O for T
1
measurements,
and
1
H T
1
measurements were taken on a 400MR NMR instrument using an inversion
recovery method.
1
H T
1
relaxation times obtained from these samples were given by
VnmrJ program. The 1/T
1
(s
-1
) values were plotted against the concentration of the
contrast agent (mM) to obtain r
1
.
251
For Gd-DOTA
-
system:
The samples were prepared according to a procedure previously described by Dr. Anna
Dawsey.
10
Two stock solutions were prepared: A stock solution of Gd-DOTA
-
and a
stock solution of 2.9a. The stock solution of Gd-DOTA
-
(100 mM) was prepared by
weighing out the appropriate amount of the contrast agent into a tared 1 dram vial and
dissolving it in an appropriate amount of distilled H
2
O. This stock solution was diluted to
make 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM stock solutions. To prepare a stock
solution of 2.9a, 2.9a was dissolved in an appropriate amount of distilled H
2
O to make a
100 mM solution.
T
1
measurement on samples containing Gd-DOTA
-
only: The Gd-DOTA
-
stock solution
(0.25 mM, 0.5 mM, 0.75 mM, or 1 mM) was dispensed into the coaxial inserts which
were placed inside 5 mm NMR tubes as described in section 7.1.4. This sample was
ready for T
1
measurements.
T
1
measurement on samples containing Gd-DOTA
-
and 2.9a (using 4 equiv. as a
example): An aliquot of the 2.9a stock solution (1 µL, 100 mM) corresponding to 4
equiv. of the contrast agent (100 µL, 0.25 mM) was added to a 0.5 dram vial and the vial
was lyophilized. 2.9a was then rinsed out of the 0.5 dram vial using 100 µL of 0.25 mM
contrast agent solution and added to an external coaxial insert NMR tube as described in
section 7.1.4. This sample was ready for T
1
measurement. Subsequently, 100 µL of the
Gd-DOTA
-
of corresponding concentrations (0.25, 0.5, 0.75, or 1.0 mM Gd-DOTA
-
in
HPLC H
2
O) was used to rinse the respective vials of 2.9a in order to prepare the sample
containing 2.9a and Gd-DOTA
-
for T
1
measurement. When an amount of 2.9a other than
252
4 equiv. is needed, the stock solution of 2.9a dispensed into the vial can be adjusted
accordingly.
To prepare a sample containing Gd-DOTA
-
, 4 equiv. of 2.9a, and urea (using 4 equiv.
as a example): An aliquot of the 2.9a stock solution (1 µL, 100 mM) corresponding to 4
equiv. of the contrast agent (100 µL, 0.25 mM) was added to a 0.5 dram vial and
lyophilized. 100 µL of 0.25 mM contrast agent solution was added to this vial containing
2.9a. The vial was left to stand for 10 min, and urea (0.9 mg, final concentration 150
mM) was added to the vial. The solution was added to an external coaxial insert NMR
tube as described in section 7.1.4, and this sample was run immediately. Subsequently,
100 µL of the Gd-DOTA
-
of corresponding concentrations (0.25, 0.5, 0.75, or 1.0 mM
Gd-DOTA
-
in HPLC H
2
O) was added to the respective vials of 2.9a in order to prepare
the sample containing 2.9a and Gd-DOTA
-
for T
1
measurement. These vials were also let
stand for 10 min before urea (0.9 mg, 150 mM final concentration) was added.
To prepare a sample containing Gd-DOTA
-
, 4 equiv. of 2.9a, urea, and sonicated:
Tubes prepared as described in the previous paragraph was placed in a bench top
sonicator for 1s × 3 and then 3s × 1. This sample was run immediately.
The coaxial inserts were placed in NMR tubes containing D
2
O for T
1
measurements,
and
1
H T
1
measurements were taken on a 400MR NMR instrument using an inversion
recovery method.
1
H T
1
relaxation times obtained from these samples were given by
VnmrJ program. The 1/T
1
(s
-1
) values were plotted against the concentration of the
contrast agent (mM) to obtain r
1
.

253
7.4.2.2 Relaxivity (r
1)
of the Gd-DOTP
5-
System at 9.4 T.
Relaxivity (r
1
) of Aqueous Solutions of Gd-DOTP
5-
Titrated by 2.9a. (25
o
C, 9.4T)
1/T
1
(s
-1
)
[Gd
3+
] conc.
(mM)
0 equiv. 1 equiv. 2 equiv. 4 equiv. 8 equiv.
0.00 1.09 1.09 1.09 1.09 1.09
0.25 1.64 1.59 1.75 1.51 1.76
0.50 2.36 2.35 2.55 2.50 2.67
0.75 3.26 3.30 3.56 3.58 3.81
1.0 4.08 4.27 4.59 4.64 4.90
r
1
(mM
-1
s
-1
) 3.04 3.22 3.52 3.67 3.86
error 0.16 0.23 0.19 0.30 0.22

Relaxivity (r
1
) of Aqueous Solutions of Gd-DOTP
5-
Treated with 2.9a + urea, and 2.9a +
urea + sonication. (25
o
C, 9.4T)
1/T
1
(s
-1
)
[Gd
3+
] conc.
(mM)
4 equiv. 2.9a + urea
4 equiv. 2.9 a + urea +
sonication
0.00 1.09 1.09
0.25 1.63 1.59
0.50 2.57 2.60
0.75 3.54 3.56
1.0 4.60 4.63
r
1
(mM
-1
s
-1
) 3.56 3.62
error 0.22 0.24

254
Gd-DOTP
5-
only Gd-DOTP
5-
+ 1 equiv. 2.9a

Gd-DOTP
5-
+ 2 equiv. 2.9a Gd-DOTP
5-
+ 4 equiv. 2.9a

255
Gd-DOTP
5-
+ 8 equiv. 2.9a Gd-DOTP
5-
+ 4 equiv. 2.9a + urea

Gd-DOTP
5-
+ 4 equiv. 2.9a + urea +
sonication

256
7.4.2.3 Relaxivity (r
1
) of Gd-DOTA
-
at 9.4 T
Relaxivity (r
1
) of aqueous Solutions of Gd-DOTA
-
Treated with 2.9a, Urea, or Urea +
Sonication. (25
o
C, 9.4T)
1/T
1
(s
-1
)
[Gd
3+
] conc.
(mM)
0 equiv. 2.9a 4 equiv. 2.9a
4 equiv. 2.9a +
urea
4 equiv. 2.9a + urea
+ sonication
0.00 1.09 1.09 1.09 1.09
0.25 1.64 1.51 1.63 1.59
0.50 2.36 2.50 2.57 2.60
0.75 3.26 3.58 3.54 3.56
1.0 4.08 4.64 4.60 4.63
r
1
(mM
-1
s
-1
) 3.04 3.67 3.56 3.62
error 0.22 0.24 0.22 0.24

Gd-DOTA
-
only Gd-DOTA
-
+ 4 equiv. 2.9a

257
Gd-DOTA
-
+ 4 equiv. 2.9a + urea
Gd-DOTA
-
+ 4 equiv. 2.9a + urea +
sonication

258
7.4.3 Relaxivity (r
1
) Measurements at 1.4 T
7.4.3.1 Procedures
Na
5
[Gd(DOTP)]·9H
2
O and Na[Gd-DOTA]·4H
2
O samples were synthesized at USC
and mailed to Wayne State University. Relaxivity measurements were carried out by Dr.
Buddhima Siriwardena-Mahanama at Wayne State University. The content of Gd
3+
was
determined by ICP-OES priory to the T
1
measurements. Relaxation times (T
1
) were
obtained using a Bruker mq 60 NMR Analyzer (1.4 T) at 37 °C. The measurements were
triplicated using three independently prepared samples. Relaxivities were obtained from
the slopes of the linear plots of 1/T
1
versus Gd
3+
concentration. Errors were generated as
the standard errors between the three measurements.

259
7.4.3.2 Relaxivity (r
1
) of the Gd-DOTP
5-
system at 1.4 T
Gd-DOTP
5-
Only
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.296 3.38
0.487 0.502 1.99
0.243 0.816 1.22
0.122 1.24 0.807
0.000 3.83 0.261

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.294 3.41
0.487 0.502 1.99
0.243 0.905 1.10
0.122 1.32 0.756
0.000 3.83 0.261

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.296 3.37
0.487 0.513 1.95
0.243 0.913 1.10
0.122 1.35 0.739
0.000 3.83 0.261

y = 3.14x + 0.389
R² = 0.994
0
1
2
3
4
0 0.2 0.4 0.6 0.8 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.21x + 0.332
R² = 0.997
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.18x + 0.324
R² = 0.998
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
260
Gd-DOTP
5–
with 4 equiv of shell
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.263 3.81
0.487 0.495 2.02
0.243 0.909 1.10
0.122 1.44 0.696
0.000 3.70 0.270

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.263 3.80
0.487 0.495 2.02
0.243 0.907 1.10
0.122 1.44 0.697
0.000 3.69 0.271

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.263 3.81
0.487 0.495 2.02
0.243 0.908 1.10
0.122 1.43 0.698
0.000 3.68 0.272

y = 3.65x + 0.247
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.64x + 0.250
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.65x + 0.249
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
261
Gd-DOTP
5–
with 4 equiv of shell in urea (150 mM)
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.256 3.91
0.487 0.492 2.03
0.243 0.870 1.15
0.122 1.45 0.689
0.000 3.69 0.271

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.255 3.92
0.487 0.493 2.03
0.243 0.894 1.12
0.122 1.44 0.694
0.000 3.69 0.271

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.256 3.91
0.487 0.491 2.04
0.243 0.886 1.13
0.122 1.43 0.700
0.000 3.69 0.271

y = 3.74x + 0.243
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.77x + 0.232
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.75x + 0.241
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
262
Gd-DOTP
5–
with 4 equiv of shell in urea (150 mM) after sonication
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.255 3.92
0.487 0.492 2.03
0.243 0.872 1.15
0.122 1.44 0.694
0.000 3.71 0.269

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.257 3.89
0.487 0.492 2.03
0.243 0.894 1.12
0.122 1.45 0.688
0.000 3.71 0.269

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.973 0.257 3.90
0.487 0.492 2.03
0.243 0.891 1.12
0.122 1.43 0.697
0.000 3.71 0.269

y = 3.76x + 0.240
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.74x + 0.235
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.74x + 0.239
R² = 0.999
0
4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
263
7.4.2.3 Relaxivity (r
1
) of the Gd-DOTA
-
System at 1.4 T
Gd-DOTA
-
only
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.301 3.32
0.494 0.567 1.76
0.247 1.07 0.932
0.124 1.57 0.636
0 3.83 0.261

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.301 3.31
0.494 0.573 1.74
0.247 1.05 0.953
0.124 1.57 0.637
0 3.83 0.261

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.301 3.32
0.494 0.568 1.76
0.247 1.015 0.985
0.124 1.61 0.622
0.000 3.83 0.261

y = 3.11x + 0.230
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.10x + 0.234
R² = 0.999
0
3.4
0 1
1/T
1
(s–1)
Gd concentration (mM)
y = 3.11x + 0.2397
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
264
Gd-DOTA
–
with 4 equiv of shell

Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.305 3.28
0.494 0.557 1.79
0.247 0.983 1.02
0.124 1.57 0.636
0.000 3.70 0.270

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.306 3.273
0.494 0.556 1.798
0.247 0.982 1.02
0.124 1.571 0.637
0.000 3.69 0.271

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.305 3.28
0.494 0.557 1.80
0.247 0.984 1.02
0.124 1.576 0.635
0.000 3.68 0.272

y = 3.06x + 0.2685
R² = 0.999
0
3.4
0.00 1.00
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.270
R² = 0.999
0.00
3.40
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.268
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
265
GdDOTA
–
with 4 equiv of shell in urea (150 mM)
Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.306 3.27
0.494 0.559 1.79
0.247 0.99 1.01
0.124 1.57 0.637
0 3.69 0.271

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.305 3.28
0.494 0.559 1.79
0.247 0.992 1.01
0.124 1.57 0.636
0 3.71 0.269

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.306 3.27
0.494 0.56 1.79
0.247 0.992 1.01
0.124 1.57 0.635
0 3.71 0.269

y = 3.05x + 0.267
R² = 0.999
0
3.4
0
1

1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.267
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.268
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concnetration (mM)
266
GdDOTA
–
with 4 equiv. of shell in urea (150 mM), after sonication

Trial 1

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.306 3.26
0.494 0.560 1.79
0.247 0.990 1.01
0.124 1.58 0.635
0.000 3.71 0.269

Trial 2

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.305 3.28
0.494 0.559 1.79
0.247 0.992 1.01
0.124 1.57 0.636
0.000 3.71 0.269

Trial 3

Concn T
1
(s) 1/T
1
(s
–1
)
0.988 0.306 3.27
0.494 0.560 1.79
0.247 0.992 1.01
0.124 1.57 0.635
0.000 3.71 0.269

y = 3.04x + 0.267
R² = 0.999
0
3.4
0.00 1.00
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.265
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
y = 3.05x + 0.265
R² = 0.999
0
3.4
0 1
1/T
1
(s
–1
)
Gd concentration (mM)
267
7.4.4 Luminescence Decay Measurements
Eu-DOTP
5–
and Eu-DOTA
–
were synthesized at University of Southern California.
Luminescence decay measurements were acquired by Dr. Buddhima Siriwardena-
Mahanama at Wayne State University. Preparation of samples for Gd-DOTP
5-
and Gd-
DOTA
-
systems were carried out according to procedures described in section 7.4.2.1.
The data were obtained on a HORIBA Jobin Yvon Fluoromax-4 spectrofluorometer in
decay by delay scan mode using the phosphorescence lifetime setting. Excitation and
emission wavelengths of 393 and 596 nm were used, respectively while the other
parameters were kept constant: excitation and emission slit widths (5 nm), flash count
(100), initial delay (0.01 ms), maximum delay (2 ms for solutions in H
2
O and 8 ms for
solutions in D
2
O), and delay increment (0.01 ms). All measurements were repeated using
three independently prepared samples. The number of coordinated water molecules, q,
was determined using the method developed by Horrocks and coworkers.
8

Luminescence Dacay Rate Data of Gd-DOTP
5-
System
Sample
τ
−1
H2O
τ
−1
D2O

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0 equiv. of 2.9a 0.853 0.853 0.850 0.433 0.433 0.430
4 equiv. of 2.9a 0.856 0.862 0.848 0.450 0.456 0.456
4 equiv. of 2.9a and urea 0.875 0.867 0.873 0.455 0.455 0.456
4 equiv. of 2.9a and urea, after
sonication 0.880 0.865 0.870 0.453 0.455 0.456

268
Luminescence Dacay Rate Data of Gd-DOTA
-
System
Sample
τ
−1
H2O
τ
−1
D2O

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0 equiv. of 2.9a 1.49 1.50 1.50 0.399 0.400 0.400
4 equiv of 2.9a 1.49 1.50 1.49 0.400 0.411 0.405
4 equiv of 2.9a and urea 1.49 1.48 1.48 0.408 0.407 0.409
4 equiv of 2.9a and urea, after
sonication 1.50 1.48 1.48 0.406 0.408 0.408

269
7.4.5 Variable-Temperature
17
O-NMR Data
7.4.5.1 Procedures
Gd-DOTP
5–
, Y-DOTP
5–
, Gd-DOTA
–
and Y-DOTA
–
were synthesized at University of
Southern California. Variable Temperature
17
O-NMR measurements were carried out by
Dr. Buddhima Siriwardena-Mahanama at Wayne State University. Variable temperature
17
O NMR measurements of Gd-DOTP
5–
(25 mM) and Gd-DOTA
–
(4.4 mM) systems and
their diamagnetic Y
3+
analogues, Y-DOTP
5–
(25 mM) and Y-DOTA
–
(4.4 mM) systems
in 1%
17
O-enriched water starting from 10%
17
O-enriched water (Cambridge Isotope
Laboratories, Inc.) were carried out on a Varian-500S (9.4 T) spectrometer. Line widths
at half height of the bulk water peaks were measured at 15, 20, 30, 40, 50, 60, and 70 °C.
A/ħ and ΔE were fixed to –3.8 × 10
–6
rad/s and 2.5 × 10
–11
J/mol, respectively, for the
Gd-DOTP
5–
and Gd-DOTA
–
systems. Water-coordination numbers, q, were set to the
values obtained from luminescence-decay measurements. The least-squares fits of the
17
O NMR relaxation data were calculated using origin software (8.0951 B951) following
a previously published procedure
16b,c
to obtain the water-exchange rates.

270
7.4.5.2 Gd-DOTP
5–
Systems
Gd-DOTP
5–
only

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 80.9 77.1
293 70.9 67.2
303 55.7 53.1
313 45.8 44.2
323 38.8 38.0
333 33.8 33.5
343 30.3 29.9

Gd-DOTP
5–
+ 4 equiv. 2.9a
Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 94.9 87.3
293 83.2 75.5
303 63.6 58.6
313 51.6 47.7
323 43.2 40.2
333 36.9 35.0
343 32.5 31.0

Gd-DOTP
5–
+ 4 equiv. 2.9a + 150 mM urea
Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 94.5 87.1
293 83.0 75.6
303 64.6 58.6
313 52.1 47.7
323 43.5 40.3
333 37.0 35.0
343 32.6 31.0

271
Gd-DOTP
5–
+ 4 equiv. 2.9a + 150 mM urea, after sonication

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 94.5 87.3
293 82.6 75.3
303 64.3 58.4
313 51.8 47.6
323 43.3 40.4
333 37.2 35.1
343 32.5 31.2

7.4.5.3 Gd-DOTA
–
Systems

Gd-DOTA
–
Only

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 185 151
293 180. 128
303 158 87.3
313 133 63.8
323 103 48.2
333 76.4 38.7
343 56.9 32.8

Gd-DOTA
–
+ 4 equiv. 2.9a

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 119 92.2
293 116 79.4
303 112 61.2
313 101 49.4
323 82.8 41.1
333 64.7 35.4
343 49.9 31.2
272
Gd-DOTA
–
+ 4 equiv 2.9a + 150 mM urea

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 141 97.3
293 133 83.5
303 118 63.1
313 105 50.3
323 85.2 42.0
333 66.1 36.1
343 51.8 32.2

Gd-DOTA
–
+ 4 equiv. 2.9a + 150 mM urea, after sonication

Temperature (K) Line width at half height of the bulk H
2
17
O peak

Gd Y
288 173 105
293 163 88.3
303 145 66.8
313 123 53.0
323
97.2 43.8
333 74.1 37.8
343 55.1 34.3

273
7.4.6 EPR Spectroscopy
7.4.6.1 EPR spectra on Gd-DOTP
5-
System
Gd-DOTP
5-
EPR spectra were taken at 25
o
C on a Bruker EMX instrument at 9.34 T,
at University of Southern California. The Gd-DOTP
5-
solutions for EPR measurements
were prepared as follows. To an 2 mL Schlenk flask were sequentially added
Na
5
[Gd(DOTP)]·9H
2
O (2.9 mg, 2.98 × 10
-3
mmol) and HPLC grade H
2
O (119.2 µL). To
this solution was added H
4
EDTA (0.9 mg, 3.08 × 10
-3
mmol), and 1 M NaOH (3 µL).
The solution was placed in a 100
o
C oil bath for 16 hours. The solution was cool to room
temperature thereafter. This Na
4
EDTA-buffered Gd-DOTP
5-
solution is ready for EPR
measurements.
To a separated 1 dr. vial, a 100 mM stock solution of 2.9a was prepared in HPLC H
2
O.
To a 0.5 dr. vial was added the 100 mM 2.9a stock solution (40 µL). The moles of 2.9a in
this vial is equal to 8 times of the moles of Gd-DOTP
5-
in 20 µL of 25 mM Na
4
EDTA-
buffered Gd-DOTP
5-
solution. The vial containing 2.9a was lyophilized to dryness, and
the residual 2.9a formed a thin membrane on the wall of the vial. This membrane was
rinsed with Na
4
EDTA-buffered Gd-DOTP
5-
solution (20 µL, 25 mM), and the rinse was
transferred into a borosilicate glass tube (0.6 I.D. × 0.84 O.D.) via a plastic micropipette.
This borosilicate glass tube is now ready for EPR measurement.
Subsequently, vials containing the amounts of 2.9a equal to 25 and 50 equiv. of Gd-
DOTP
5-
in 20 µL of 25 mM Na
4
EDTA-buffered Gd-DOTP
5-
solution were respectively
prepared and lyophilized. And 20 µL of 25 mM Na
4
EDTA-buffered Gd-DOTP
5-
solution
were used to rinse the vials to prepare samples containing 25 equiv. of 2.9a and 50 equiv.
of 2.9a.
274
7.4.6.2 EPR Spectra on Gd-DOTA
–
Systems
EPR measurements of the Gd-DOTA
–
systems (1 mM) were performed on a Bruker
EMX X-band spectrometer by Dr. Budhima, Siriwardena-Mahanama at Wayne State
University.
From the EPR spectra the electronic Landé g factor, g
L
,

peak-to-peak line width, ΔH
pp
,
and transverse electronic relaxation rates, 1/T
2e
, were obtained according to a previously
reported method.
1b14

EPR spectrum for a sample containing Gd-DOTA
–
only (–196 °C, 5 mM)

EPR spectrum for a sample containing Gd-DOTA
–
+ 4 equiv. of 2.9a (–196 °C, 5 mM)

-400
400
2800 3800
Intensity
Field strength (G)
-3000
3000
2800 3800
Intensity
Field strength (G)
275
EPR spectrum for a sample containing Gd-DOTA
–
+ 4 equiv. of 2.9a + urea (150 mM) (–
196 °C, 5 mM)

EPR spectrum for a sample containing Gd-DOTA
–
+ 4 equiv. of 2.9a + urea (150 mM),
after sonication (–196 °C, 5 mM)

-6000
6000
2800 3800
Intensity
Field strength (G)
-600
800
2800 3800
Intensity
Field strength (G)
276
7.4.7 ICP-OES
ICP–OES measurements were performed on a HORIBA Jobin Yvon ULTIMA
spectrometer. Concentrations of [Gd
3+
] in samples used for T
1
measurements, Gd
3+
and
Y
3+
in samples used for variable-temperature
17
O NMR measurements, and Gd
3+
in
samples used for EPR spectroscopy were verified by ICP–OES. Samples for ICP–OES
were diluted with nitric acid (2% v/v, aqueous), and standards were prepared by serial
dilution of Gd, Eu, and Y standards (High-Purity Standards).

277
7.5 Chapter 6 Experimental and Spectral Data
7.5.1 Synthesis of Phosphonate-Coated Gold Nanoparticles (1.5 nm Core in
Diameter)
The phosphine-stabilized gold nanoparticles (1 mg) were weighted into an 8 dr. vial,
and the particles were dissolved in dichloromethane (1 mL). To this DCM solution was
added (12-mercaptododecyl)phosphonic acid (1 mg) in H
2
O (1 mL). After 24 hours of
stirring at room temperature, the DCM layer was removed using a Pasteur pipette. The
pH of the aqueous layer was adjusted to ca. 2 with 1 M HCl, and a resulting black
precipitate was filtered on a fine-porosity fritted funnel. The precipitate was washed on
the funnel with 10 mL of DCM : MeOH = 1 : 1 mixture. The particles were dialyzed in a
dialysis bag (3.5 kDa MW cut off) in basic H
2
O (the pH was ca. 8, adjusted with 1 M
NaOH) for 24 hours, with solvent change every 8 hours. The dialyzed particle solution
was lyophilized to a black powder.
To a 8 dr. vial were added phosphonate-coated gold nanoparticles (4.5 mg) and HPLC
grade H
2
O (10 mL) to make a brown solution. The solution was acidified to pH ca. 2
using 1M HCl. A resulting black precipitate was centrifuged into a pellet that was
lyophilized to obtain a black solid (4.3 mg). This solid is now ready for TGA analysis.
TGA was carried out on a TA Q50 instrument. The lyophilized solid particles were added
to a ceramic crucible before being loaded to the pan.

278
DLS histogram of the phosphonic acid-coated gold nanoparticles

TGA graph of the phosphonate-coated gold nanoparticles

279
UV-Vis Spectrum of the phosphonate-coated gold nanoparticles

DLS histogram of the phosphonic acid-coated gold nanoparticles in the presence of 2.9a

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)
280
7.5.2 BODIPY Encapsulation in the Surfactant-Coated Nanoparticles
BODIPY was a generous gift from Dr. Mark E. Thompson’s at University of Southern
California. BODIPY encapsulation was carried out according to literature procedures.
15

To an 8 dr. vial were added acetone (7.2 mL) and BODIPY (5.6 mg) to make a green
fluorescent solution. To a separate 8 dr. vial were added phosphonate-coated gold
nanoparticles (2.8 mg) and HPLC grade H
2
O (1.4 mL) to make a brown solution. To the
BODIPY in acetone solution was added the phosphonate-coated gold nanoparticles
aqueous solution. Acetone was removed under reduced pressure to induce the
precipitation of BODIPY, which was separated on a fine-porosity fritted funnel. The
filtrate was lyophilized to dryness to obtain the loaded particles.

281
7.5.3 BODIPY Loading Capacity
The molar extinction coefficient (ε) of BODIPY (λ
max
= 506 nm) in THF:H
2
O = 1:1
(v/v) solution was calculated by measuring the absorbance of solutions of various
concentrations, and plotting the absorbance against the concentration. ε was determined
to be 119.4 mM
-1
. The loaded particles (3.3 mg) was dissolved in THF:H
2
O = 1:1 (2 mL,
v/v) mixture in a cuvette. Sodium cyanide (10 mg) was added to the solution, which was
stirred for 24 hours. The molarity of the gold nanoparticle was calculated using literature
procedures assuming the diameter of the phosphine-stabilized gold nanoparticle to be 1.5
nm.
16
The UV-Vis absorbance of the solution containing the NaCN-degraded particles
was recorded. The amount of BODIPY in the degraded solution was calculated based on
the molar extinction coefficient.

282
7.5.4 BODIPY Release in a Biphasic System
To obtain the rate of BODIPY release from the gold nanoparticles while 2.9a is not
present:
A BODIPY-loaded phosphonate-coated gold nanoparticle solution (2.2 mg, in 0.3 mL
HPLC grade H
2
O) was added to a quartz cuvette (10 mm, 3.5 mL) fitted with a stir-bar.
On top of the aqueous layer was added toluene (2 mL). The UV-Vis absorbance in the
toluene phase was monitored over time.
To obtain the rate of BODIPY release from the gold nanoparticles while 1 equiv. of
2.9a is present:
A methanol solution of 2.9a (20µL, 0.25 mg/mL, 8.23 × 10
-6
mmol) was added to the
cuvette (10 mm, 3.5 mL) fitted with a stir bar, and methanol was removed under a stream
of nitrogen. The moles of 2.9a in this methanol solution is equal to ca. 1 equiv. of the
phosphonate thiols on the surface of the gold nanoparticles (1.9 mg of BODIPY-loaded
gold nanoparticles). To this 2.9a in the cuvette were sequentially added HPLC grade H
2
O
(0.25 mL) and BODIPY-loaded gold nanoparticles (1.9 mg). To this particle solution was
added toluene (2 mL) on top. The UV-Vis absorbance of the toluene layer was
monitored. The absorbance at 510 nm was plotted against time.
To obtain the rate of BODIPY release from the gold nanoparticles while 2.5 equiv. of
2.9a is present:
A methanol solution of 2.9a (50µL, 0.25 mg/mL, 2.96 × 10
-5
mmol) was added to the
cuvette (10 mm, 3.5 mL) fitted with a stir bar, and methanol was removed under a stream
of nitrogen. The moles of 2.9a in this methanol solution is equal to ca. 2.5 equiv. of the
phosphonate thiols on the surface of the gold nanoparticles (1.9 mg of BODIPY-loaded
283
gold nanoparticles). To this 2.9a in the cuvette were sequentially added HPLC grade H
2
O
(0.25 mL) and BODIPY-loaded gold nanoparticles (1.9 mg). To this particle solution was
added toluene (2 mL) on top. The UV-Vis absorbance of the toluene layer was
monitored. The absorbance at 510 nm was plotted against time.

284
7.6 References

1
Sherry, A. D.; Malloy, C. R.; Jeffrey, M. H.; Cacheris, W. P.; Geraldes, C. F. G. C.
“Dy(DOTP)5-: A New, Stable
23
Na Shift Reagent” J. Magn. Reson. 1988, 76, 528-533.
2
Bell, J. R.; Luo, H.; Dai, S. “Superbase-Derived Protic Ionic Liquids with Chelating
Fluorinated Anions” Tetrahedron Lett. 2011, 52, 3723-3725.
3
Weare, W. W.; Reed, S. M.; Warner, M. G.; Hutchison, J. E. “Improved Synthesis of
Small (d
CORE
≈ 1.5 nm) Phosphine-Stabilized Gold Nanoparticles” J. Am. Chem. Soc.
2000, 122, 12890-12891.
4
Wang, R.; Zheng, Z.; Koknat, F. W.; Marko, D. J.; Müller, A.; Das, S. K.; Krickemeyer,
E.; Kuhlmann, C.; Therrien, B.; Plasseraud, L.; Süss-Fink, G.; Pasquale, A. D.; Lei, X.;
Fehlner, T. P.; Diz, E. L.; Haak, S.; Cariati, E.; Dragonetti, C.; Lucenti, E.; Roberto, D.;
Lee, C. Y.; Song, H.; Lee, K.; Park, B. K.; Park, J. T.; Hutchison, J. E.; Foster, E. W.;
Warner, M. G.; Reed, S. M.; Weare, W. W. Cluster and Polynuclear Compounds. In
Inorganic Syntheses; Shapley, J. R., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA,
2004; Vol. 34, pp 228-234.
5
Li, V. The Use of Non-Covalent Interactions for the Modification of Nanoparticle
Surface and MRI Contrast Agents. Ph.D. Thesis, University of Southern California,
August 2014.
6
Gentilini, C.; Boccalon, M.; Pasquato, L. “Straightforward Synthesis of Fluorinated
Amphiphilic Thiols” Eur. J. Org. Chem. 2008, 2008, 3308-3313.
7
Greenwald, R. B. “A Facile Preparation of Highly Fluorinated Diamines” J. Org. Chem.
1976, 41, 1469-1470.
285

8
Velez-Herrera, P.; Ishida, H. “Synthesis and Characterization of Highly Fluorinated
Diamines and Benzoxazines Derived therefrom” J. Fluorine Chem. 2009, 130, 573-580.
9
Averill, D. J.; Garcia, J.; Siriwardena-Mahanama, B. N.; Vithanarachchi, S. M.; Allen,
M. J. “Preparation, Purification, and Characterization of Lanthanide Complexes for Use
and Contrast Agents for Magnetic Resonance Imaging” J. Vis. Exp. 2011, 53, e2844.
10
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, August 2013.
11
The compound turned slightly yellow at 197.5
o
C, however, the color did not darken
even after it was heated to 230
o
C.
12
Sherry, A. D.; Malloy, C. R.; Jeffrey, M. H.; Cacheris, W. P.; Geraldes, C. F. G. C.
“Dy(DOTP)5-: A New, Stable
23
Na Shift Reagent” J. Magn. Reson. 1988, 76, 528-533.
13
Merritt, M.; Harrison, C.; Kovacs, Z.; Kshirsagar, P.; Malloy, C. R.; Sherry, A. D.
“Hyperpolarized
89
Y Offers the Potential of Direct Imaging of Metal Ions in Biological
Systems by Magnetic Resonance” J. Am. Chem. Soc. 2007, 129, 12942-12943.
14
Powell, H. P.; Ni Dhubhghai, O. M.; Pubanz, D.; Helm, L.; Levedev, Y. S.; Schlaepfer,
W.; Merbach, A. E. “Structural and Dynamic Parameters Obtained from
17
O NMR, EPR,
and NMRD Studies of Monomeric and Dimeric Gd
3+
Complexes of Interest in Magnetic
Resonance Imaging: An Integrated and Theoretically Self-Consistent Approach” J. Am.
Chem. Soc. 1996, 118, 9333-9346.
15
Kim, C. K.; Ghosh, P.; Pagliuca, C.; Zhu, Z.-J.; Menichetti, S.; Rotello, V. M.
“Entrapment of Hydrophobic Drugs in Nanoparticle Monolayers with Efficient Release
into Cancer Cells” J. Am. Chem. Soc. 2009, 131, 1360-1361.
286

16
Liu, X.; Atwater, M.; Wang, J.; Hou, Q. “Extinction Coefficient of Gold Nanoparticles
with Different Sizes and Different Capping Ligands” Colloids Surf. B 2007, 58, 3-7.

287

Abstract (if available)

Abstract The reversible activation of medical imaging agents continues to interest the MRI contrast agent chemists. We have envisioned that by bringing self‐associating fluorocarbons close to a gadolinium(III)‐based small molecule contrast agent, q and τm (number and residence lifetime of the water molecules in the inner‐sphere, respectively) modulation can be achieved, thus enabling the design and optimization of a new class of responsive contrast agents. The synthesis of a tri‐segmented fluorous amphiphile consisting of a guanidinium head, a fluorocarbon middle segment and a polyethylene glycol tail was developed as a small molecule platform for this technology. ❧ We have since developed the methodology to synthesize a novel class of fluorous amphiphiles fulfilling the aforementioned requirements. The route described herein is facile and applicable on a gram scale. The synthesis involves a versatile triflate intermediate that enabled us to install a series of functionalities β‐ to the fluorocarbon. The synthesis was concluded with guanylation of an extremely electron‐deficient primary amine with satisfying yield. Curious as to how varying length of methylene spacers would affect the pKₐ value of the fluorous guanidinium, a new synthesis route placing a two‐methylene spacer between the guanidinium and fluorocarbon was devised. It was concluded that an extra methylene spacer increases the pKₐ by 0.4 units. ❧ Using Gd‐DOTP⁵⁻ as the study subject, it was demonstrated that the fluorous amphiphile is capable of augmenting the r₁ of Gd‐DOTP⁵⁻ in an aqueous solution. Mechanistic studies showed that although τm was marginally attenuated, an enhancement in τR (rotational correlation time) was primarily accountable for the accentuated relaxivity. Additionally, ¹⁹F T₁ evidence complies with a model in which a non‐covalent conjugate is formed between Gd‐DOTP⁵⁻ and the amphiphile. ❧ The last portion of the project describes a drug delivery project in which the primary objective is to decrease the diffusion rate of an encapsulated organic molecule from a nanoparticle carrier by decorating the particles with the fluorous amphiphiles. Boron‐dipyrrolemethene (BODIPY) molecules were encapsulated in an alkyl‐phosphonic acid coated gold nanoparticle in order to probe these experimental parameters. No change in the diffusion rate was observed upon addition of the amphiphile. Weak association energy between the fluoroalkyls and high hydrophobicity of the fluorocarbon may account for this observation.

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Asset Metadata

Creator Wu, Xinping (author)

Core Title Syntheses of a series of fluorous amphiphiles and modulation of the relaxivity of gadolinium(III)-based contrast agents

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/10/2014

Defense Date 06/17/2014

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

Tag contrast agents,drug delivery,fluorous,gold nanoparticles,magnetic resonance imaging,OAI-PMH Harvest,synthesis

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 angelawu.usc@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-435477

Unique identifier UC11287529

Identifier etd-WuXinping-2645.pdf (filename),usctheses-c3-435477 (legacy record id)

Legacy Identifier etd-WuXinping-2645.pdf

Dmrecord 435477

Document Type Dissertation

Format application/pdf (imt)

Rights Wu, Xinping

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