Research Collection

Doctoral Thesis

Synthesis and Characterization of Fullerene and Metallofullerene Derivatives by Prato Reaction

Author(s): Tiu, Elisha Gabrielle V.

Publication Date: 2017

Permanent Link: https://doi.org/10.3929/ethz-b-000228807

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Diss. ETH NO. 24504

Synthesis and Characterization of Fullerene and Metallofullerene Derivatives by Prato Reaction

A thesis submitted to attain the degree of

DOCTOR OF SCIENCES of ETH Zurich

(Dr. Sc. ETH Zurich)

presented by

Elisha Gabrielle V. Tiu

Master of Science in Chemistry, Osaka University, Osaka, Japan born January 22nd 1990 citizen of Philippines

accepted on the recommendation of

Prof. Dr. Yoko Yamakoshi, examiner Prof. Dr. Carlo Thilgen, co-examiner

2017

Acknowledgements

I would like to express my gratitude towards the people who supported me throughout my study: First of all, I would like to thank Prof. Dr. Yoko Yamakoshi for giving me the opportunity to work in ETH Zurich. I truly appreciate her guidance and help in refining me to become the person that I am today. I am deeply indebted to her for the training she gave me; she always had my best interest in mind, especially for my future career. I really thank her for all the things I have learned during my stay in her laboratory. Although challenging, I was truly interested in the projects I worked on, and I learned a lot. I would like to thank Prof. Dr. Carlo Thilgen for being the co-examiner of my thesis. During my study, he was always available to discuss with. He was always willing to help me in however way he can and I really appreciated that. I would also like to thank Prof. Dr. François Diederich and Prof. Dr. Jeffrey Bode for allowing me to attend their group meetings and for letting me to use their group equipment. I also appreciated the good friendships I formed with the members of their groups. I also extend my gratitude to Prof. Dr. Massimo Morbidelli for providing access to the DLS machine and to Prof. Lucio Isa for providing access to the tensiometer. Furthermore, I would like to thank to our group members: Sean Oriana, Safwan Aroua, Rakesh Kumar, Masayuki Nasuda, Alessandro Fracassi, Dac Ngan Nguyen-Giang, Ankita Ray, Korinne Liosi, Gabriele Zirpoli for the fun, laughter, and memorable times we enjoyed together. I would also like to thank my collaborators Dr. Silvia Osuna and Dr. Marc Garcia-Borràs for the meaningful discussions and the work we have done together. I would also like to thank Dr. Alla Sologubenko from ScopeM for taking extra time to help me in my STEM measurements. I also appreciate the help of Dr. Takashi Ishikawa for his expertise in cryoTEM, for his insights and discussions with him.

i I would like to especially thank NMR services, particularly, Mr. René Arnold, for all his efficiency and patience in measuring my samples in the smallest quantities and sensitive conditions. I would also like to extend my gratitude to Mr. Stephan Burkhardt and Mr. Rainer Frankenstein for their extensive support. I would like to specially thank Mass services group, in particular Dr. Xiangyang Zhang, for his advice and his mentoring when I had problems in my mass spectra. He was very efficient in measuring my samples. I also would like to thank the crystallographers, in particular Dr. Nils Trapp and Mr. Michael Solar for their efforts in measuring many of my crystal samples. They provided me with many meaningful advices. I also would like to thank Mr. Mario Kessinger and Ms. Helen Kaufmann-Baumgartner for their administrative help. They were very efficient and helpful. Finally, I would like to thank my grandmother for the inspiration she gave me to finish the race. Her strength, wisdom, and courage will always and forever be an inspiration for me. I would like to thank my friends, my relatives, my sister, my father, my mother, and my husband for the unwavering support they showered me throughout the years I spent abroad for my higher education. They were always there during the most difficult times. Truly, there is no distance that can separate us.

ii List of Publications and Presentations

Publications 1. E.G.V. Tiu, K. Liosi, S. Aroua, Y. Yamakoshi, J. Mater. Chem. B 2017, 5, 6676 – 6680. Micelle vs Vesicle Formation Controlled by Distal

Functionalization of C60-PEG Conjugates. 2. S. Aroua, E.G.V. Tiu, T. Ishikawa, Y. Yamakoshi, Helv. Chim. Acta

2016, 99, 805 – 813. Well-Defined Amphiphilic C60-PEG Conjugates: Water-Soluble and Thermoresponsive Materials. 3. R. Kumar, E. Gleißner, E.G.V. Tiu, and Y. Yamakoshi, Org. Lett., 2016,

18, 184 – 187. C70 as a Photocatalyst for Oxidation of Secondary Benzylamines to Imines. 4. S. Aroua, E.G.V. Tiu, M. Ayer, T. Ishikawa, Y. Yamakoshi Polym. Chem. 2015, 6, 2616. RAFT Synthesis of Poly(vinylpyrrolidone) Amine

and Preparation of a Water-Soluble C60-PVP Conjugate.

Oral Presentation E.G.V. Tiu, S. Aroua, and Y. Yamakoshi “Synthesis of a Well-Defined, Water- th Soluble C60-PVP Conjugate,” The 229 Electrochemical Society Meeting. San Diego, California, USA (2016).

Poster Presentations • E.G.V. Tiu, S. Aroua, T. Ishikawa, and Y. Yamakoshi “Synthesis of a

Well-Defined, Water-Soluble C60-PVP Polymer: a Potential PDT Agent,” The 8th Symposium SSCI Scholarship Fund of the Swiss Chemical Industry. Zurich, Switzerland (2016).

• E.G.V. Tiu, S. Aroua, T. Ishikawa, and Y. Yamakoshi “Synthesis of a

Well-Defined, Water-Soluble C60-PVP Polymer: a Potential PDT Agent,” The 16th International Symposium on Novel Aromatic Compounds. Madrid, Spain (2015).

iii

iv Table of Contents

List of Common Terms and Abbreviations ...... ix Abstract ...... xiii Résumé ...... xvii Chapter 1: General Introduction ...... 1 1.1 Fullerenes: Discovery and Production ...... 3 1.2 The Structures of Fullerenes ...... 4 1.2.1 C60 Fullerene ...... 5 1.2.2 C70 Fullerene ...... 6 1.2.3 Metallofullerenes ...... 7 1.3 The Properties of Fullerenes ...... 8 1.3.1 C60 Fullerene ...... 8 1.3.2 C70 Fullerene ...... 10 1.3.3 Metallofullerenes ...... 11 1.3.3.1 Electronic Properties ...... 11 1.3.3.2 Metal Cluster Dynamics ...... 12 1.3.3.3 Geometry of the Metal Cluster ...... 13 1.4 Purification of Fullerenes ...... 14 1.5 Chemical Reactions of Fullerenes ...... 16 1.5.1 Reduction Reactions ...... 16 1.5.2 Oxidation Reactions ...... 16 1.5.3 Halogenations ...... 17 1.5.4 Radical Reaction ...... 18 1.5.5 Nucleophilic Additions ...... 18 1.5.6 Cyclopropanation: The Bingel-Hirsch Addition ...... 20 1.5.7 Cycloaddition Reactions ...... 20 1.5.7.1 [1+2] Cycloaddition ...... 21 1.5.7.2 [2+2] Cycloaddition ...... 23 1.5.7.3 [4+2] Cycloaddition: Diels-Alder reaction ...... 26 1.5.7.4 [3+2] Cycloaddition ...... 27 1.5.7.4.1 Prato Reaction (1,3-Dipolar Cycloaddition of Azomethine Ylides) on C60 and C70 ...... 28 1.5.7.4.2 Mono-functionalization on M3N@C80 by Prato reaction ...... 29 1.5.7.4.3 Prato Bis-functionalization on M3N@C80 ...... 30 1.6 Applications of Fullerenes ...... 31 1.6.1 Organic Photovoltaics ...... 31 1.6.2 Hydrogen Gas Storage ...... 32 1.6.3 Fullerenes as Biological Materials ...... 33 1.6.3.1 Photodynamic Therapy ...... 33 1.6.3.2 MRI Contrast Agent: Reported MRI-CA Fullerene Based Materials ...... 35 1.7 Outline of Dissertation ...... 37

v Chapter 2: Synthesis of Water-Soluble C60-PEG Conjugates and Their Self- Assembly ...... 39 2.1 Background ...... 41

2.2 Synthesis of C60-PEG Conjugates 7, 8, and 9 ...... 45

2.3 Characterization of the C60-PEG Conjugates 7, 8, and 9 ...... 53 2.3.1 Surface Tension ...... 53 2.3.2 Dynamic Light Scattering ...... 54 2.3.3 Cloud Point ...... 57 2.3.4 STEM Measurements ...... 60 2.4 Conclusions ...... 65

Chapter 3: Funtionalization of C70: Water-Soluble C70 Derivative for Biomaterial Application ...... 67 3.1 Background ...... 69

3.2 Synthesis and Isolation of the Three Isomers of C70 Prato Monoadducts 72 3.3 Characterization by 1H- and 13C-NMR and Vis-NIR Measurements of the Three Isomers of C70 Prato Monoadducts ...... 74

3.4 Conjugation of C70 with a Monodispersed Polyethylene Glycol Derivative ...... 80 3.5 Conclusions ...... 81

Chapter 4: 1,3-Dipolar Cycloaddition of Y3N@C80 and Gd3N@C80 and Formation of Trisadducts ...... 83 4.1 Background ...... 85 4.2 Synthesis and Isolation of the N-Ethyl Fulleropyrrolidine Trisadducts of M3N@C80 (M = Gd, Y) ...... 86

4.3 Isomerization Study of the Trisadducts of Y3N@C80 and Gd3N@C80 ...... 90 4.3.1. Isomerization of the Trisadducts of Gd3N@C80 ...... 90 4.3.2. Isomerization of the Trisadducts of Y3N@C80 ...... 91 4.4 Characterization of Trisadducts ...... 92 4.5 Possible Structure Elucidation of Trisadducts by Computational Chemistry ...... 97 4.5.1 Theoretical Considerations for Gd3N@C80 ...... 97 4.5.2 Theoretical Considerations for Y3N@C80 ...... 100 4.6 Preliminary X-ray Analyses of N-ethyl Fulleropyrrolidine Bisadduct of Gd3N@C80 ...... 103 4.7 Conclusions ...... 105 Chapter 5: Conclusions and Outlook ...... 107

5.1 Preparation of Amphiphilic C60 Derivatives and Their Self-Assembly .... 109

5.2 Synthesis of C70 Monoadduct Isomers and Preparation of Water-soluble C70 derivative ...... 110

vi 5.3 Trisadducts of M3N@C80 (M = Gd, Y): Towards the Preparation of Water- Soluble Gd3N@C80 Derivatives as MRI-Contrast Agents ...... 111 Chapter 6: Experimental Section ...... 115 6.1. General ...... 117 6.2. Chapter 2 ...... 118 6.2.1. Synthesis ...... 118 6.2.2 Characterization of 7, 8, and 9 ...... 146 6.2.2.1 Tensiometry ...... 146 6.2.2.2 Dynamic Light Scattering (DLS) ...... 146 6.2.2.3 Scanning Transmission Electron Microscopy (STEM) ...... 146 6.2.2.4 Thermoresponsivity Tests and Cloud point experiments ...... 148 6.3. Chapter 3 ...... 149 6.4. Chapter 4 ...... 165 6.4.1 Synthesis ...... 165 6.4.2 Isomerization Studies of Trisadducts of M3N@C80 (M = Gd, Y) ...... 179 6.4.2.1 Isomerization Observed by HPLC ...... 179 6.4.2.2 Isomerization Observed by 1H-NMR ...... 179 Chapter 7: References ...... 181

vii

viii List of Common Terms and Abbreviations

Å Angstrom (1 Å = 10-10 m) br Broad C Celsius ca. circa cal calories CCA cyano-4-hydroxycinnamic acid COSY homonuclear correlation spectroscopy cryo-TEM cryogenic transmission electron microscopy d doublet DBU 1,8-diazabicyclo[5.4.0]undec-7-ene dd doublet of doublet DCTB trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-

propenylidene]malononitrile deg. degree DFT density functional theory DIPEA N,N-diisopropylethylamine DHB 2,5-dihydroxybenzoic acid DLS dynamic light scattering DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DTPA diethylenetriaminepentaacetic acid e.g. exempli gratia (for example) EG ethylene glycol eV electron volt EPR enhanced permeability and retention

ES singlet energy level ESI electrospray ionization ESR electron spin resonance Eq. equation

ix equiv equivalents

ET triplet energy level Et ethyl et al. et alia Gd Gadolinium h hour(s) HAADF high angle annular dark field HBTU O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate HOMO highest occupied molecular orbital HPLC high performance liquid chromatography HRMS high-resolution mass spectroscopy HSQC heteronuclear single quantum coherence spectroscopy i.e. id est (it is) IFT interfacial tension

Ih icosahedral IPR isolated pentagon rule IR infrared ISC intersystem crossing J coupling constant K Kelvin kcal/mol kilocalorie/mole Lu Lutetium LUMO lowest occupied molecular orbital m multiplet m-CPBA meta-chloroperoxybenzoic acid mg milligram MHz megahertz ml milliliter m/z mass to charge ratio MALDI matrix-assisted laser desorption/ionization mbar millibar

x Me methyl min minutes mN/m millinewton per meter mM-1 s-1 per millimolar per second MRI-CA magnetic resonance imaging contrast agent MS mass spectroscopy Nd:YAG Neodymium-doped Yttrium Aluminum Garnet NHS N-hydroxysuccinimide nm nanometer NMR nuclear magnetic resonance NSF nephrogenic systemic fibrosis NVP N-vinylpyrrolidone o-DCB ortho-dichlorobenzene

OD optical density PBB pentabromobenzyl PDA photodiode array PDT photodynamic therapy PEG polyethylene glycol PHHJ pentagon-hexagon-hexagon junction PhMe toluene POAV p-orbital axis vector ppm parts per million PVP poly(vinylpyrrolidone) PYE pyrenylethyl P3HT poly(3-hexylthiophene) q quartet quint. quintet ROS reactive oxygen species RT room temperature s singlet Sc Scandium STEM scanning transmission electron microscopy

xi tBu tert-butyl TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THF tetrahydrofuran THJ triple hexagon junction TLC thin-layer chromatography TNT-EMF trimetallic nitride template endohedral metallofullerenes UV/vis/NIR ultraviolet/visible/near infrared Voc Voltage open circuit VT variable temperature Y Yttrium Δ heat δ chemical shift (ppm)

θP pyramidalization angle

λmax wavelength of maximum absorbance μ micro (10-6) τ triplet lifetime Φ quantum yield

xii Abstract

Fullerenes, the third allotropic form of carbon after diamond and graphite, have been a subject of interest for many researchers. Their interesting photophysical properties have made them potential candidates for biomedical applications (e.g. photodynamic therapy, magnetic resonance contrast imaging). However, these promising potentials of fullerenes are often hampered by their limited solubility in many polar solvents. In order to solve this problem, fullerenes have been derivatized with various solubilizing groups such as water-soluble polymers. In this thesis, the [3+2] cycloaddition of azomethine ylides to fullerenes, known as the Prato reaction, was carried out on C60, C70, and M3N@C80 (M = Gd, Y) in order to functionalize them. In the previous study in our group by Dr. Safwan Aroua, water-soluble

C60-PEG and Gd3N@C80-PEG derivatives were synthesized and presumed to form micelles in aqueous solution (Fig. A). Such micelle formation may prevent water molecules from interacting with the gadolinium atoms of

Gd3N@C80, and thereby can lower the relaxivity and weaken its potential as a magnetic resonance imaging contrast agent (MRI-CA). In an attempt to disrupt micelle formations, C60-PEG derivative was functionalized with one or two positively charged pyrrolidine moieties on its hydrophobic C60 surface (Fig. A). As a result, whereas addition of one charge did not affect the micelle formation of C60-PEG conjugate, addition of two charges on the C60 surface showed a clear influence in their assembly behavior to show another kind of assembly, presumably a vesicle-type of structure.

xiii O H H O N N O O O O 20 20 O N O

Hydrophilic tail


H2O







Hydrophobic head

O H H O O H H O tBu N N tBu t O O O O Bu N N tBu O O O O 20 O O 20 N 20 O N O 20

N N I N I I

Figure A. Micelle formation of C60-PEG derivative and the addition of charged moieties.

The ellipsoidal C70 fullerene is the second most abundant empty cage fullerene. Similar to C60, it also generates a high yield of reactive oxygen species. Consequently, the medicinal application of the C70 fullerene is also a common topic of interest. In this work, it was found that Prato mono-addition of a bisester glycine derivative on C70 generated three isomers that were successfully isolated and characterized. Further functionalization of C70 with polyethylene glycol demonstrated the solubility of this C70 derivative in water (Fig. B).

O O H tBu N NH tBu O O O O 20 O N O 20

water-soluble C70 derivative

Figure B. Water-soluble C70-PEG derivative from C70. Derivatization of trimetallic nitride template endohedral metallofullerenes (TNT-EMFs), in particular, Gd3N@C80, is often a topic of interest because of their potential application as MRI contrast agents. A large effort was given by many research groups to understand the reactivity of TNT- EMFs and it was shown that the encapsulated metal clusters have large effect on the regioselectivity of the product. Previously, the bisadducts of M3N@C80

xiv (M = Gd, Y) were characterized and reported by Dr. Aroua. In this study, the generation of Prato M3N@C80 (M = Gd, Y) trisadducts was observed by HPLC and ESI-MS (Fig. C). NMR spectroscopy and theoretical calculations were also employed in order to investigate the possible tris-addition sites in

M3N@C80 (M = Gd, Y). The trisadduct isomers were found to be similar in energy and easily isomerize to each other at room temperature.

N

HN 3 O

O OH H H

M = Gd, Y

Figure C. Prato reaction on M3N@C80 (M = Gd, Y).

xv

xvi Résumé

Les fullerènes, la troisième forme connue du carbone après le diamant et le graphite a été le sujet de nombreuses études depuis leur découverte. Leurs propriétés photophysiques intéressantes les ont révélés comme de possibles candidats pour des applications biomédicales et matérielles (par exemple thérapie photodynamique, agents de contraste pour IRM). Toutefois, le potentiel prometteur des fullerènes est souvent entravé par leur solubilité très limitée dans les solvants polaires. Dans le but de régler ce problème, les fullerènes sont souvent fonctionnalisés avec différents groupes, tels que des polymères hydrosolubles, qui vont aider à améliorer la solubilité. Dans cette thèse, différents types de fullerènes (C60, C70, et M3N@C80 (M= Gd, Y)) ont été fonctionnalisés en utilisant une réaction de cycloaddition [3+2] connue sous le nom de réaction de Prato.

Précédemment, des dérivés C60-PEG, solubles dans l'eau, ont été synthétisés et il a été démontré que ses composés probablement forment des micelles en milieu aqueux. La formation de micelles peut limiter les interactions entre les molécules d’eaux et les atomes de gadolinium dans

Gd3N@C80 et ainsi limiter la relaxivité, ce qui pourrait altérer le potentiel de ces molécules en tant qu’agent de contraste. Dans le but de perturber la formation de micelles, le composé modèle C60-PEG20 a été fonctionnalisé avec un ou deux groupes de pyrrolidinium quaternaires. Alors que l’addition d’une charge à la surface du fullerène n’a pas d’effet sur la formation de micelles, l’ajout de deux ammoniums démontre un clair changement. De façon intéressante, cette molécule forme un autre arrangement qui peut être assimilé à une structure de type vésicule.

xvii O H H O N N O O O O 20 20 O N O

HydrophilicPartie hydrophile tail


H2O







HydrophobicPartie hydrophobe head

O H H O O H H O tBu N N tBu t O O O O Bu N N tBu O O O O 20 O O 20 N 20 O N O 20

N N I N I I

Figure A. Formation de micelles avec des dérivés C60-PEG et addition de groupes de pyrrolidinium quaternaires.

Le C70 est le second type de fullerène le plus abondant. Comme le C60, le C70 a le potentiel de générer des dérivés réactifs de l’oxygène. Par conséquent, l'utilisation du C70 en biomédecine est également un sujet d'intérêt. Dans cette thèse, il a été démontré que la mono-substitution du C70 par réaction de Prato permet l’obtention de trois isomères qui ont été isolés et caractérisés avec succès. Une fonctionnalisation supplémentaire du C70 avec du polyéthylène glycol a démontré la solubilité de ce dérivé en solution aqueuse.

O O H tBu N NH tBu O O O O 20 O N O 20

Dérivés du C70 soluble dans l’eau

Figure B. Synthèse de dérivés du C70 solubles dan l’eau. Les modifications des métallofullerènes (TNT-EMF), en particulier du

Gd3N@C80, sont souvent un sujet d'intérêt en raison de leur potentielle utilisation en tant qu'agents de contraste pour IRM. En raison de la difficulté à isoler ces fullerènes, leur réactivité est souvent moins étudiée que leurs analogues avec la cage vide. Un grand effort a été accordé à la

xviii compréhension de la réactivité de ces espèces, et il a été démontré que les métaux encapsulés ont une influence sur le contrôle de la régiosélectivité des produits obtenus. Auparavant, des produits de bis-addition sur M3N@C80 (M = Gd, Y) ont été reportés et caractérisés par Dr Aroua. Pour la première fois, dans cette étude, la réaction de Prato a été réalisée sur M3N@C80 (M = Gd, Y) afin d’obtenir des produits de triple addition. Ces produits ont été synthétisés, isolés et caractérisés. Des analyses par RMN et l’apport de calculs théoriques ont également été utilisés afin d'étudier la réactivité et la regiosélectivité de la réaction donnant accès aux différents produits de triple addition avec M3N@C80 (M = Gd, Y). Il a été constaté que les isomères obtenus étaient très proches en énergie et isomérisaient facilement à température ambiante.

N

HN 3 O

O OH H H

M = Gd, Y Figure C. Réaction de Prato sur M3N@C80 (M = Gd, Y).

xix

xx

CHAPTER 1

GENERAL INTRODUCTION

1

2 1.1 Fullerenes: Discovery and Production

Kroto, Curl, and Smalley won the 1996 Nobel Prize in Chemistry for their discovery in 1985 of the third allotropic form of carbon (after diamond and graphite) known as “fullerenes.” The discovery led to various breakthroughs in science, particularly in the fundamental studies on their intrinsic properties such as the photoinduced electron transfers in fullerenes, and their potential applications as photodynamic therapy agents or as components of photovoltaic cells. Fullerenes are carbon clusters which were first produced by vaporization of graphite using a pulsed Nd:YAG

(Neodymium-doped Yttrium Aluminum Garnet Nd:Y3Al5O12) laser irradiation at 532 nm. Using time-of-flight mass spectrometric analysis, laser vaporization of graphite provided a remarkably prominent 720 mass peak, followed by an 840 1 mass peak, corresponding to C60 and C70, respectively (Fig. 1).

Figure 1. The structure of a C60 and C70 fullerene.
Following the discovery of this new class of molecules, a technique to produce macroscopic quantities of fullerenes was developed in 1990. Krätschmer and Huffman produced soot that contained fullerenes via resistive heating of graphite.2 A schematic diagram of the original Krätschmer-Huffman reactor is shown in Figure 2a. Two graphite rods (one with a sharpened end and the other one with a flat end) held by two copper electrodes, are kept in contact. The reactor is also equipped with a pump and a helium gas inlet, enabling the chamber to be purged and filled with helium gas. When an electric current is passed through the rods, heat is produced and the generated vapor is collected in the bell jar. The amassed soot is then extracted by toluene or benzene to obtain a crude mixture of fullerenes.2

3 a. Bell jar b.

Graphite base electrode Graphite rod Graphite rod Cu electrode Cu electrode Graphite rods

electrode electrode pump

Gas inlet Figure 2. (a) Schematic diagram of the arc reactor used by Krätschmer (resistive heating of graphite) to produce fullerenes, adapted from literature.2 (b) Schematic diagram of the reactor used by Smalley (arc vaporization of graphite), adapted from literature.3

Smalley et al. also reported arc heating of graphite as an alternative way to obtain fullerenes. In this case, the sharpened graphite rod is kept close to the other flattened graphite disk, but not directly in contact with each other (Fig. 2b), thus coining the term “contact arc.” The effect is that the energy is dissipated in the arc rather than in Ohmic heating of the rod. This technique generates a mixture of primarily C60 and C70 fullerenes, with a yield of up to 10% of the original graphitic soot.3 Soon after, Parker and colleagues reported further improvements to this technique, obtaining a mixture of extractable fullerenes with yields of up to 44% of the original graphitic soot.4

1.2 The Structures of Fullerenes

The macroscale production of fullerenes was certainly a noteworthy contribution in the field. It unlocked answers about the structures, properties, and functions of fullerenes. The structure of fullerenes consists of fused hexagons and pentagons. The pentagons provide the curvature in order to form the spherical shape in contrast to the case of graphene with planar fused hexagons. The bonds in fullerenes are commonly described as either a [5,6] bond (a bond between a pentagon and a hexagon) or a [6,6] bond (a bond between two hexagons (Fig. 3)). These fullerenes can either have a hollow interior (empty-caged), or have encapsulated atoms or clusters (endohedral fullerenes). There are many different kinds of fullerenes that can form.

4 Possible structures of fullerenes can be determined by following a simple formula (Eq. 1).

Equation 1. Formula reflecting the general structure of fullerenes. N is the number of carbon atoms in a fullerene.5

Fullerene CN is composed of 12 pentagons + ((N/2)-10) hexagons N ≥ 20

For example, the C60 fullerene has a total of 12 pentagons and 20 hexagons.

On the other hand, the C70 fullerene has a total of 12 pentagons and 25 hexagons. This formula can be applied to other fullerenes as well.

Figure 3. The [6,6] bond (red) and [5,6] bond (blue) in C60.

1.2.1 C60 Fullerene
The most abundant fullerene is the C60 fullerene (Fig. 3). It is also known as “buckminsterfullerene,” named after the architect Buckminster Fuller (1895 - 1983), who designed geodesic domes, which is similar to the structure 2 of C60. Composed of 60 sp hybridized carbon atoms, it is entirely spherical with a hollow interior. All of the 12 pentagons of C60 are completely surrounded by its hexagons, perfectly obeying the Isolated Pentagon Rule (IPR).6

The C60 fullerene is the smallest possible fullerene to obey the IPR.

According to the X-ray crystal structure of C60, the bond length of [5,6]-bond in 7 Ih-C60 is 1.467 Å and [6,6]-bond length is about 1.355 Å (Table 1). Bond lengths obtained by electron diffraction using a high-energy electron beam intersecting a vapor of C60 are 1.458 Å for [5,6]-bonds and 1.401 Å for [6,6]- bonds.8 X-ray crystallographic and NMR spectroscopic methods of measuring the bond lengths were also reported.9, 10 However, in all cases, the length of a

5 C60 [6,6]-bond is shorter than the length of its [5,6]-bond, suggesting that [6,6]- bonds have higher electron density and higher double bond character than

[5,6]-bonds. The mean diameter of C60 was calculated to be ca. 7.10 ± 0.07 9, 11 Å, based on the carbon-carbon bond lengths in C60 obtained by magnetic dipolar coupling during NMR measurements. Upon considering the π-electron cloud surrounding the C60 fullerene, the outer diameter of C60 ball is 7.10 ± 3.35 = 10.34 Å, where 3.35 is the estimated thickness of the surrounding π- electron cloud.11

Table 1. Structural properties of C60 and C70 fullerenes. Fullerenes

C60 C70

Symmetry Ih D5h Number of Hexagons 12 12 Number of Pentagons 20 25 Bond lengths [6,6] 1.355 Å a 1.372 to 1.463 Åb or Bond lengths [5,6] 1.467 Å a 1.378 to 1.543 Åc a data taken from reference 7 b 12 data taken from reference (C70-Iridium complex) c 13 data taken from reference (C70 · 6 (S8))

1.2.2 C70 Fullerene

Along with the discovery of C60 in 1985 by Kroto, Smalley, and Curl, the

C70 fullerene was also detected and identified. C70 is the next smallest fullerene following the IPR rule. Compared with C60, D5h-C70 has five extra fused hexagons at the equatorial belt, to provide 25 hexagons in total. The 13 C-NMR spectrum of C70 displays five peaks with intensity ratios of

10/20/10/20/10, indicating that C70 has five different types of carbon atoms assigned to be A/C/B/D/E (Fig. 4).14,15, 16

6 A B C D E

Figure 4. The structure of a C70 fullerene. A, B, C, D, and E represent the five different types of carbon atoms observed. Bonds of the equatorial belt of the five extra hexagons are in bold.

The C70 fullerene is an ellipsoidal molecule with the length of 7.12 Å in its short axis and 7.96 Å in its long axis.17 It has a variety of bond lengths, spanning from 1.372 to 1.463 Å12 or 1.378 to 1.543 Å13 (Table 1).

1.2.3 Metallofullerenes

Endohedral metallofullerenes encapsulating metal clusters were detected soon after the discovery of the empty cage fullerenes (C60 and C70). The “@” symbol was incorporated in their nomenclature to denote that these chemical species are encapsulated inside the carbon cage (for example,

La@C82 means that a lanthanum atom is encapsulated inside a C82 fullerene). Metallofullerenes are classified into the five different types below. 18-20 (1) classical, monometallic fullerene (M@C2n) 21 (2) metallic carbide fullerenes (M2C2@C2n) (3) metallic nitride clusters or Trimetallic Nitride Template Endohedral 22,23,24, 25 Metallofullerenes TNT-EMFs (M3N@C2n) 26 (4) metallic oxides (M4O2@C2n) and 27 (5) metallic sulfides (M2S@C2n)

The third most abundant fullerene (after C60 and C70), by far, is Sc3N@C80, a prototype of the TNT-EMF class of metallofullerene (Fig. 5).28

Figure 5. The structure of a trimetallic nitride template endohedral metallofullerene (M3N@Ih-C80). The red (metals) and purple (nitrogen) colored structures inside the cage represent the metal cluster.

7 Scandium metallofullerenes were first observed by negative ion chemical ionization mass spectrometry as an unidentified peak at m/z 1109 from soot but no structural data could be obtained due to its low yields.29 Dorn and co- workers successfully increased the yields of TNT-EMFs by introducing a small amount of nitrogen inside the reactor. Analysis of the resulting fullerene soot revealed that the most abundant fullerene species (ca. 3 to 5% in soot) was

Sc3N@Ih-C80, with an icosahedrally symmetric C80 cage structure 22 encapsulating a Sc3N metal cluster. Currently, TNT-EMFs are generally produced by electric arc vaporization of a core-drilled graphite rod packed with metals or metal oxides that are to be encapsulated by the cage. Several techniques were developed in order to further increase the selectivity of the yields to TNT-EMFs. For example, Dunsch and coworkers reported that TNT- EMF amounts were increased when the atmosphere in the fullerene reactor 30 was enriched with the more reactive NH3 gas. Stevenson et al. introduced the “Chemically Adjusting Plasma Temperature, Energy, and Reactivity” (CAPTEAR) method wherein the graphite rods were packed with

Cu(NO3)2
2.5 H2O and Sc2O3. This approach increased the production of 31 TNT-EMFs, relative to empty cage C60 fullerene. Furthermore, the Stevenson group highlighted the importance of using copper in the production 32 of fullerenes and increased the yield of Sc3N@C80 by 3 to 5 times.

1.3 The Properties of Fullerenes

1.3.1 C60 Fullerene

2 The carbon atoms in C60 fullerene are all sp hybridized. Its molecular 33 orbitals are delocalized, which gives its aromatic character. The C60 fullerenes possess interesting photophysical properties (Table 2). The singlet energy level (ES) was calculated to be 1.99 eV. On the other hand, the triplet energy level was estimated to be 1.57 eV. The wavelengths of maximum absorbance (λmax) for singlet and triplet states were reported to be 920 and 747 nm, respectively. The triplet state lifetime in an argon-saturated solution of benzene was found to be 40 ± 4 μs. Lastly, the fluorescence and triplet quantum yields were very high. These were reported to be 1.0
10-4 and 0.96,

8 respectively. These results suggest that C60 can generate a high yield of 1 singlet oxygen ( O2) and that it can potentially be used in medicinal applications such as in photodynamic therapy.

34 Table 2. Photophysical properties of C60. All measurements were reported to be performed in benzene. ES stands for energy level of the singlet state and ET stands for energy level of the triplet state. λmax stands for wavelength of maximum absorbance. τ (triplet) stands for the triplet lifetime (in a benzene solution saturated with argon). Φ stands for quantum yield. Physical Property Value

E(singlet) 1.99 eV E(triplet) 1.57 eV λmax (singlet) 920 nm λmax (triplet) 747 nm τ (triplet) 40 ± 4 μs Φ (fluorescence) 1.0
10-4 Φ (triplet) 0.96

C60 fullerenes are capable of producing reactive oxygen species (ROS) upon photo-excitation.35 Through intersystem crossing (ISC), the singlet 1 3 fullerenyl excited state ( C60*) is converted to a triplet-excited state ( C60*) with high efficiency and with a quantum yield of near unity. Energy transfer from 3 1 C60* to molecular oxygen produces singlet oxygen ( O2), one of the reactive 36-38 3 oxygen species (Fig. 6). Alternatively, electron transfer reaction to C60* can occur in the presence of an electron donor to produce C60 radical anion

− (C60 ), which can react with molecular oxygen to generate superoxide radical

− anion (O2 ) (Fig. 6).

1 O2 3 O2 C60

hν ISC 1 3 C60 C60* C60*

e- donor C C60 60

3 O2 O2

Figure 6. Reactive oxygen species generation of photoexcited C60.

In terms of bioapplication, especially as a photosensitizer in photodynamic therapy (PDT), C60 has attractive properties such as long λmax and quantum yields in the generation of triplet state (Table 2). However,

9 solubility in many polar solvents is poor. C60 is soluble in toluene, benzene, o- dichlorobenzene, and carbon disulfide, but not soluble or slightly soluble in water, methanol, acetone, and n-pentane (Table 3).

Table 3. Solubilities of C60 in different solvents. a Solvent [C60] (μg/mL) Water 1.3
10-11 Methanol 0 Acetone 1 n-pentane 5 n-hexane 43 Dichloromethane 260 Benzene 1700 Toluene 2800 Carbon disulfide 7900 1,2-dichlorobenzene 27000 a Data obtained from literature.39, 40

1.3.2 C70 Fullerene

The C70 fullerene is the second most common fullerene. Both C60 and

C70 share similar electrochemical and photophysical properties (Table 4). The singlet energy level (ES) was calculated to be 1.90 eV and the triplet energy level (ET) was estimated to be 1.60 eV. The maximum absorbance wavelengths (λmax) for singlet and triplet states were reported to be 660 and 400 nm, respectively. The triplet state lifetime in an argon-saturated solution of benzene was found to be 130 ± 10 μs. The fluorescence and triplet quantum yields were reported to be 3.7
10-4 and 0.9, respectively. Similar to 1 41 C60, C70 can also generate a high yield of singlet oxygen ( O2), which suggests that C70 can also potentially be used in medicinal applications such as in photodynamic therapy.

10 34 Table 4. Physical properties of C70. All measurements were reported to be performed in benzene. ES stands for energy level of the singlet state and ET stands for energy level of the triplet state. λmax stands for wavelength of maximum absorbance. τ (triplet) stands for the triplet lifetime (in a benzene solution saturated with argon). Φ stands for quantum yield. Physical Property Value

ES 1.90 eV ET 1.60 eV λmax (singlet) 660 nm λmax (triplet) 400 nm τ (triplet) 130 ± 10 μs Φ (fluorescence) 3.7
10-4 Φ (triplet) 0.9

The solubility of C70 in polar solvents is also quite limited, similar to C60

(Table 5). C70 is soluble in toluene, benzene, o-dichlorobenzene, and carbon disulfide, but not soluble or slightly soluble in water, methanol, acetone, and n- pentane.

Table 5. Solubilities of C70 in different solvents. a Solvent [C70] (μg/mL) Water 1.1
10-13 Methanol 0.4 Acetone 1.9 n-pentane 2 n-hexane 13 Dichloromethane 80 Benzene 1300 Toluene 1406 Carbon disulfide 9875 1,2-dichlorobenzene 36210 a Data obtained from literature. 40, 42 1.3.3 Metallofullerenes

1.3.3.1 Electronic Properties

The C80 carbon cage has seven possible isomers that can satisfy the 43 IPR. These are, namely D2, D5d, C2v, C2v’, D3, D5h, and Ih. The most stable ones among these are the D2 and D5d isomers (close in energy). Both of them were calculated to be ca. 52 kcal/mol more stable than the Ih isomer, which is 44,45, 46 the least stable isomer. In fact, only the D2 and D5d isomers were obtained and characterized as empty cage molecules.47, 48 Analysis of the

HOMO-LUMO orbitals suggests that the instability in Ih-isomer arises from the fact that only two electrons fill the four-fold degenerate HOMO orbitals of the

11 carbon cage.47 The ionic model states that, to form an energetically stable and closed-shell structure, the encapsulated species must donate six more electrons to the empty HOMO orbitals of the cage (Fig. 7). This is the case for stable endofullerenes, for example, La2@C80 and Sc3N@C80, among others.

Ih-C80

N gu ag

6 e- gg

Figure 7. Formal transfer of six electrons from HOMO of the metal cluster to the lowest partially occupied orbitals of the Ih-C80.

In La2@Ih-C80 endofullerene, theoretical calculations showed that the two La atoms formally donate six electrons, thereby forming a very stable 45 La2@Ih-C80 fullerene. Similarly, theoretical calculations for Sc3N@Ih-C80 suggested that the three Sc3+ ions and N3- ion donate a total of six electrons to 22, 44, 46, 49 stabilize the Ih-C80 cage (Fig. 7).

1.3.3.2 Metal Cluster Dynamics

The motion of the encapsulated clusters inside Ih-C80 cage is also a common topic of interest. Data regarding their movement in the cage can immensely contribute to understanding the properties of endofullerenes. Provided evidence implies that the La atoms inside the endofullerene

La2@C80 are in motion with the calculated rotational energy barrier of ca. 5 kcal/mol.50 Furthermore, Akasaka et al. presented the first experimental NMR evidence of the circular motion of the metals inside La2@C80 at room 51 temperature. Electrostatic potential map of La2@C80 shows concentric rings with no clear minima, indicating the round Ih-C80 cage structure. This result also suggests that no specific positions for the metal inside the cage are 50 especially stable. NMR experiments on Sc3N@C80 suggested that the Sc3N metal cluster was also in motion inside the carbon cage.22 In the case of

12 Y3N@C80 with a larger metal cluster, NMR experiments suggested that the metal cluster is nearly freely isotropically rotating,52 with a relatively larger 53 rotational barrier than the smaller Sc3N@C80. The motional barrier of the metal cluster is highly related to the cluster size as also reported by Dorn.54

1.3.3.3 Geometry of the Metal Cluster

Crystal structures obtained for Sc3N@C80 show that the encapsulated 3+ 22 Sc3N metal cluster (ionic radius of Sc 0.75 Å) is planar. Slightly larger 3+ 55, 56 clusters Lu3N (ionic radius of Lu 0.85 Å) and ErSc2N are also planar. However, in case of larger clusters, a slight deviation from planarity was 3+ observed. For example, in the case of the Gd3N cluster (ionic radius of Gd 0.94 Å), the N atom is displaced from the plane by approximately 0.5 Å.57 The 3+ Y3N cluster (ionic radius of Y 0.90 Å), on the other hand, is slightly pyramidalized with the N atom displaced from the plane by approximately 0.13 Å.58 The differences in geometry may be due to the differences in sizes of the metal ions (Table 6).59

Table 6. Summary of the ionic sizes and geometry of metal cluster. Average POAV values of Sc3N@C80 and Y3N@C80. Sc3+ Lu3+ Y3+ Gd3+ Ionic radius 0.75 Å 0.85 Å 0.90 Å 0.94 Å Geometry Planar Planar Pyramidal Pyramidal Average POAV (PHHJ) 12.83° n.r. 14.37° n.r. Average POAV (THJ) 9.99° n.r. 11.68° n.r.

60 *n.r. stands for not reported in the reference. The p-orbital axis vector (POAV) analysis provides an accurate description about the electronic structure of conjugated, non-planar organic molecules. The POAV is the vector that indicates the angle between σ and π- orbitals and it denotes the sp3 character in the strained sp2 carbons in fullerenes. It creates equal angles (θσπ) with the three σ-bonds at a conjugated carbon atom (Fig. 8). The pyramidalization angle (θP) is obtained as θP = (θσπ - 90)°.61, 62

13 POAV

θσπ

θP = θσπ - 90º

Figure 8. POAV analysis diagram. The pyramidalization angle (θP) is defined as (θσπ - 90)°. The POAV values depend on the size of the encapsulated metal cluster. In the case of Sc3N@C80, the value of the POAV pyramidalization angle of a 5,6,6 junction (pentagon, hexagon, hexagon junction or PHHJ) is 12.83 and a 6,6,6 junction (triple hexagon junction or THJ) is 9.99 degrees. In the case of Y3N@C80, the value of the POAV pyramidalization angle of a PHHJ is 14.37 and that of a THJ is 11.68 degrees (Table 6).60

Metallofullerenes, in particular Gd3N@C80, can potentially be used as MRI contrast agents because each molecule contains three Gd atoms and they do not release the encapsulated metal clusters under in vivo conditions unlike metal chelates. However, these molecules are not as well studied as

C60 or C70 because production of sufficient amounts is still a challenge. Reactivity and function are still at the early stages of being understood.

1.4 Purification of Fullerenes

Although fullerenes can be obtained from the soot via previously mentioned methods (Chapter 1.1), the crude extracts are mixtures of fullerenes (C60, C70, and higher fullerenes). Further purification is therefore necessary to obtain a single class of pure fullerene. One approach to separate the endohedral fullerenes from the hollow fullerenes is by taking advantage of their reactivity differences. The Dorn group introduced this concept by immobilizing a cyclopentadiene moiety in a resin. Since TNT-EMFs have lower Diels-Alder reactivity than empty cage fullerenes, pure TNT-EMFs can be eluted in a facile way. The empty cage fullerenes bound to the resin could be recovered later on.63 Another method to isolate pure TNT-EMFs from soot

14 was reported by Stevenson et al.. They developed a method called “Stir and Filter Approach” (SAFA). In this method, the fullerene soot is mixed with a Lewis base, such as an amino-derivatized silica. The empty-cage fullerenes tend to bind to the reactive amino-silica, leaving behind the TNT-EMFs in solution. Using this method, the most inert Sc3N@Ih-C80 can be readily isolated in pure form simply through filtration.64, 65 Later on, the Stevenson group presented another method to efficiently purify TNT-EMFs. The fullerene soot was mixed with a Lewis acid (AlCl3 and

FeCl3). Insoluble complexes of the TNT-EMFs and metal chlorides crashed out as precipitates after stirring. Using this approach, isomerically pure 66 Sc3N@Ih-C80 was isolated. Another common technique used to purify fullerenes is by using high performance liquid chromatography (HPLC). A number of silica-based stationary phases were developed through the years in order to purify fullerenes. Currently, these stationary phases are more commonly used to purify functionalized fullerenes. Fig. 9 presents the summary of the structures of the different stationary phases that are commonly used.

O N 2 NO2

NO2

O O NO2

Br O NO2 Br Br O2N S Br Br

N O

Me Me Me Si Me Si Me Si Me Si Me Me

Buckyprep-M Buckyprep PBB Buckyclutcher

NO O2N 2

N

Me Me Me Si Me Si

Buckyprep-D PYE Figure 9. Commonly used silica-based columns for fullerene purification. First line (left to right): Buckyprep-M, Buckyprep, PentaBromoBenzyl (PBB), Buckyclutcher. Second line: Buckyprep-D and Pyrenylethyl (PYE).

15 1.5 Chemical Reactions of Fullerenes

Access to larger quantities of fullerenes made it possible to carry out the different reactions to study their reactivities. For empty cage fullerenes C60 and C70, the [6,6]-bonds are shorter and have higher double bond character than [5,6]-bonds. Therefore, majority of the reactions involving double bonds target [6,6]-bonds over [5,6] ones. On the other hand, the reactivities of metallofullerenes (M3N@C80) are highly dependent on the metal/metal clusters encapsulated inside the cage. In the following subchapters, the most common ways to functionalize

C60, C70, and M3N@Ih-C80 fullerenes are summarized.

1.5.1 Reduction Reactions

According to theoretical calculations, the LUMO and LUMO+1 of C60 are energetically low-lying and triply degenerated. Therefore, C60 was expected to be a fairly electronegative molecule, which is easily reducible by up to six electrons (hexa-anion).67 Cyclic voltammetry experiments have supported this to form stable hexa-anions in solution.68, 69 In a similar manner, the C70 fullerene also exhibited six reduction potentials and can also form stable hexa-anions in solution.69 Reduction reactions were the first chemical modifications carried out on fullerenes due to their pronounced electrophilicity. Fulleride anions have been prepared by reaction of fullerenes with electropositive metals (alkali and alkaline earth metals). Moreover, electrochemical and photochemical reductions have also been carried out on n- fullerenes. For example, C60 (n = 1 - 5) was generated in solution via the titration of a suspension of C60 in liquid ammonia with a solution of rubidium in 70 liquid ammonia. Reduction of C60 and C70 in THF using lithium metal with the aid of ultrasound has also been demonstrated.71

1.5.2 Oxidation Reactions

The oxidized fullerenes C60O and C70O were first extracted from fullerene soot (Fig. 10).72, 73 The formation of these derivatives was reported to be due to trace amounts of oxygen found in the fullerene reactor. Oxidation

16 reactions on fullerenes were achieved in a number of ways, via m-CPBA oxidation,74 reaction with dimethyldioxirane,75 ozonolysis reactions,76 among others.

O O O

C60 epoxide C70 epoxide 5,6-isomer C70 epoxide 1,2-isomer

Figure 10. C60 and C70 epoxides. 1.5.3 Halogenations

Fullerenes were reported to be reactive to halogenation reactions. Fluorine, chlorine, and bromine have previously been introduced to fullerenes (Fig. 11).77-79

(X)n

C60Xn X = F, Cl, Br

Figure 11. Halogenated C60 fullerene.

These derivatives are very useful for further fullerene derivatization. In fact, chlorinated fullerenes C60Cl6, C70Cl8 and C70Cl10 were used for further Friedel-Crafts arylation reactions (Fig. 12).80, 81

17 MeOOC

(a)

COOMe

MeOOC

COOMe Cl FeCl3 C60Cl6 C6H5NO2

COOMe

COOMe

C60Ar5Cl (b)

COOMe (CH2)3

(Cl)n 8

C6H5(CH2)3COOMe FeCl3

C6H5NO2

C70Cln n = 8 or 10

Figure 12. Friedel-Crafts Reactions carried out on chlorinated (a) C60 and (b) C70. 1.5.4 Radical Reaction

Radical addition to C60 is a fast process and C60 has been claimed to 82, 83 be a “radical sponge”. Radical additions to C70 have also been studied. Different photochemically generated radicals were added to fullerenes.84

Dibenzyl adducts of Sc3N@C80 and Lu3N@C80 were obtained by the reaction of fullerene and a photochemically generated benzyl radical (Fig. 13).85

Br

toluene irradiation (λ = 355 nm)

Sc3N@C80

Figure 13. A photochemically generated benzyl radical was added to Sc3N@C80. 1.5.5 Nucleophilic Additions

C60 is an electron-deficient molecule that can undergo reactions with nucleophiles. Reaction of C60 with NaCN generates a monoadduct anion, which can be quenched with various electrophiles (Fig. 14).86

18 CN H

CN CH3

TFAA

O CH O 3 S O F3C CN Na

NaCN o-DCB/DMF Br CN

O S O CN

CN CN

Figure 14. Quenching of C60 monoadduct anion with various electrophiles.
77, 87, 88 Amines easily undergo nucleophilic additions with C60 and C70. Nucleophilic addition of macromolecules such as polymers can also be used to functionalize fullerenes. For example, a nucleophilic surface is formed when diphenylmethyl functionalized polyethylene films are deprotonated and used to covalently bind C60 (Fig. 15a). In the same way, a living polystyrene 89, 90 carbanion was covalently attached to C60 (Fig. 15b). The dianion of

Lu3N@C80 reacts with PhCHBr2 to generate a methano derivative of

Lu3N@C80(CHC6H5). On the other hand, trianionic Sc3N@C80 reacts with 91, 92 PhCHBr2 to yield the Sc3N@C80(CHC6H5).
a. b.

CH Ph 3 [CH ] Polyethylene CH3 CH2 CH CH2 CH 3 n n Ph Ph

Figure 15. C60 conjugated to (a) polyethylene surface and (b) polystyrene units.

19 1.5.6 Cyclopropanation: The Bingel-Hirsch Addition

A fullerene cyclopropanation reaction was reported by Bingel et al..93 Using bromomalonate in the presence of a base, deprotonation occurs resulting in an α-halomalonate anion. This anion attacks the double bond of the fullerene. Subsequently, an intramolecular nucleophilic substitution reaction occurs to form the cyclopropanated product (Fig. 16). The Bingel- Hirsch reaction is considered as one of the most efficient and facile ways to functionalize fullerenes with mild conditions and relatively good yields.94-96

O O R R O O O O O O C R R base R R 60 O O O O Br Br R = alkyl group Figure 16. The Bingel-Hirsch reaction of fullerenes.

Among TNT-EMFs, the Bingel-Hirsch reaction was first reported on 97 Y3N@C80. The product was reported to be a [6,6]-open fulleroid and was stable under reflux for 21 hours. Bingel-Hirsch reactions on other TNT-EMFs such as Lu3N@C80 and Sc3N@C80 were also described in the literature, upon using DMF as solvent in the reaction. During a Bingel-Hirsch reaction, decarboxylation is prone to occur. Echegoyen and colleagues found that in the case of Bingel-Hirsch reaction between Lu3N@Ih-C80 and 2- bromodiethylmalonate, replacement of NaH by DBU in o-DCB/DMF limits the formation of decarboxylation product.98

1.5.7 Cycloaddition Reactions

Cycloaddition reactions are very efficient methods to functionalize fullerenes. Examples include Diels-Alder,99 1,3-dipolar cycloaddition (e.g. Prato reaction),100, 101 and carbene additions102 as shown in Fig. 17.103 Different cycloaddition reactions have been carried out using fullerenes to provide monoadducts, which have been characterized.

Two major factors can be considered to predict the reactivity of fullerenes in addition reactions. The first one is the release of strain by

20 transforming sp2-hybridized carbons to sp3-hybridized carbons. The fullerene sites with the highest degree of pyramidalization are most prone to react.104 The second one is the double bond character. The double bonds with shorter bond length and higher electron density tend to react faster.

R R

[1+2] cycloaddition

R R R R

[4+2] cycloaddition (Diels-Alder reaction) [2+2] cycloaddition

[3+2] cycloaddition (Prato reaction)

R N

Figure 17. Cycloaddition reactions of C60.

1.5.7.1 [1+2] Cycloaddition

Fullerenes undergo [1+2] cycloaddition reactions to form a three- membered (carbo- or hetero-) cyclic functional group on fullerenes. Theoretically, these reactions yield four possible monoadduct isomers from

21 C60 such as [5,6]-open, [6,6]-open, [5,6]-closed, and [6,6]-closed as shown in Fig. 18.

R R R R R R X X X R X R

[6,6]-Closed[6,6]-Open [5,6]-Closed [5,6]-Open

X = C, N, or Si Figure 18. Four possible isomeric products of [1+2] cycloaddition reactions on C60 fullerenes.

Nitrenes can be added to fullerenes, generating fulleroaziridines.105-108 Silylenes were also reported to react with fullerenes.109 Bis(2,6-diisopropyl- phenyl)silylene was added to either C60 or C70 to give the ring-closed 1,2- 110 bridged isomers. In the case of C70, the α (C1-2) and β (C5-6) isomers, located near the poles, were generated in 2 : 1 ratio, respectively (Fig. 19).111

Carbenes were also found to react with fullerenes. The reaction of C60 with O- benzyl- and O-pivaloyl protected sugar-derived diazirines in toluene yielded 1,2-methano-bridged fullerene-sugar monoadduct conjugates.112 Dichlorocarbene additions to fullerenes have also been reported.113

Furthermore, dimethoxycarbene addition to C60 has also been previously described.114 In all these cases, the products observed were additions occurring at the [6,6] ring junction. Since [5,6]-closed and [6,6]-open isomers are energetically unfavored, these are not observed experimentally.115

22 R R R R Si Si

α β

R =

Figure 19. The α- and β-isomers of silylated C70.

Carbene reactions performed on different M3N@C80 have been relatively well-studied. A [5,6] methano-adduct of Sc3N@C80 has been 116 isolated. Sc3N@C80 and Y3N@C80 metallofulleroids have also been successfully synthesized by reaction with methyl 4-benzoylbutyrate p- tosylhydrazone. In both cases, the same [6,6]-open phenyl-C81-butyric acid methyl ester adducts were isolated showing that the reactions occurred at the same [6,6] reactive site (Fig. 20).117

O

O

Figure 20. Structure of the [6,6]-open phenyl-C81-butyric acid methyl ester derivative of M3N@C80 (M = Sc, Y).

Finally, a Lu3N@C80 conjugated with the electron accepting perylenediimide was shown to have efficient electron transfer in the excited state. These developments can help realize optoelectronic devices.118

1.5.7.2 [2+2] Cycloaddition

The [2+2] cycloaddition reactions enable the synthesis of cyclobutane- fused fullerene derivatives. Benzyne, produced from anthranilic acid, reacts 119, 120 with fullerenes via a [2+2] cycloaddition reaction to C60 (Fig. 21).

23 NH 2 isoamyl nitrite C60

COOH

Figure 21. Benzyne addition to C60 via [2+2] cycloaddition reaction.

The reaction of C60 and 4,5-dimethoxybenzyne leads to a ring-closed [6,6] monoadduct.121 Higher adducts were shown to have multiple isomers (Fig. 22).122

MeO OMe

C60 MeO NH2 isoamyl nitrite toluene OMe MeO COOH

OMe Eight regioisomeric bis-adducts

Figure 22. Synthesis of C60-benzyne bisadducts.

On the other hand, benzyne addition to C70 is non-selective. Four monoadduct isomers were obtained, with a [6,6]-closed 1,2-isomer as the major product (Fig. 23).123-125 The [2+2] cycloaddition reaction of benzyne and metallofullerenes have also been demonstrated.126, 127 Particularly, in the case of Sc3N@C80, benzyne addition yields a mixture of [5,6]- and [6,6]-isomers (Fig. 24).128

1,2-isomer

Figure 23. Reaction of benzyne and C70 yielded several monoadduct isomers. The major isomer is a 1,2-isomer.

24

Sc3N@C80(C6H4) [6,6]-isomer Sc3N@C80(C6H4) [5,6]-isomer

Figure 24. Benzyne addition to Sc3N@C80 yielded two isomers: [6,6] and [5,6].
Fullerenes also undergo a [2+2] cycloaddition reactions with alkynes.

For C60, addition occurred at [6,6]-junction (Fig. 25). For C70, the addition occurred at the C1,2 bond, which is a [6,6] bond (Fig. 26).129, 130

CH2CH3 CH3 H C N 3 CH2CH3

N H3CH2C CH2CH3 hν, rt

Figure 25. C60 reaction with an alkyne.
H3CH2C CH2CH3 N Et2N NEt2

N H3CH2C CH2CH3

1,2-isomer
Figure 26. C70 reaction with an alkyne.
Enones can also undergo a photochemical [2+2] cycloaddition reaction with C60. Two [6,6] monoadduct isomers were obtained and assigned to be the cis and trans fused isomers (Fig. 27).131, 132

O O O H R H R

hν, benzene

cis trans

Figure 27. The cis and trans fused isomers of C60-enone conjugates.

25 1.5.7.3 [4+2] Cycloaddition: Diels-Alder reaction

Diels-Alder reactions are very useful reactions for the functionalization of fullerenes. The [6,6] double bonds in C60 are dienophilic and can therefore participate in various Diels-Alder reactions. Several different dienes have been used. For example, C60-cyclopentadiene monoadduct can be formed at room temperature with relatively high yields (74%, Fig. 28).133, 134 On the other hand, to form C60-anthracene cycloadducts, an excess amount (10 equiv) of anthracene must be added and the reaction can proceed only by refluxing 134, 135 (Fig. 28). The Diels-Alder products of C60 with anthracene and cyclopentadiene were observed to be thermally unstable. At higher temperature, these cycloadducts tend to undergo retrocycloaddition.135, 136 In contrast, the cycloadducts of C60 with ortho-quinodimethanes (o-QDM) and isobenzofuran are thermally stable and do not undergo reversion.137, 138

cyclopentadiene anthracene toluene, r.t. toluene, reflux

Figure 28. [4+2] cycloaddition reaction of C60 with cyclopentadiene and anthracene.

Similar to C60, C70 has high reactivity in [4+2] cycloaddition reaction.

Diels-Alder bisadducts of C70 with anthracene were synthesized in relatively high yields (68%) and regioselectively provided the 12 o’clock isomer (Fig.

29). These α bonds are the [6,6] bonds located near the poles of C70 and are 139 therefore the most reactive bonds in C70. Potential organic photovoltaic components, indene-C70 mono and bisadduct isomers, were obtained from the

Diels-Alder reaction of C70 with isoindene.

Figure 29. Diels-Alder reaction of C70 with anthracene generated the 12 o’clock bisadduct isomer.

The first TNT-EMF derivative reported was a Diels-Alder product of

Sc3N@C80 using 10 equivalents of 6,7-dimethoxyisochromanone to generate

26 o-quinodimethane that acts as the diene (Fig. 30). NMR characterizations140 and X-ray structural analysis revealed that the addition occurred at the [5,6]- 141 junction. When Gd3N@C80 was subjected to the same reaction conditions, the generation of bisadducts of Gd3N@C80 was observed but structural 142 information was not reported. The same Diels-Alder reaction on C60 with ca. one equivalent of 6,7-dimethoxyisochromanone yielded 47% monoadduct and 22% bisadduct. These results suggested that the reactivity in Diels-Alder reactions of Gd3N@C80 was lower than C60 due to the effect of the endohedral metal cluster.143

O O

O O O O Δ -CO2

O

O 1,2,4-trichlorobenzene reflux

Figure 30. Diels-Alder reaction of Sc3N@C80 with an excess amount of 6,7-dimethoxyisochromanone.

1.5.7.4 [3+2] Cycloaddition

The [3+2] cycloaddition reactions were carried out with C60 fullerene using azides,144-147 nitrile oxides,148, 149 trimethylenemethanes,150, 151 diazomethanes,152,153 and azomethine ylides (Fig. 31). Since the main part of this thesis describes the [3+2] cycloaddition reaction of fullerenes with azomethine ylides (Prato reaction), it will be extensively discussed in this section.

27 N R R R N N N N

Δ +

N O R

[6,6]-closed [5,6]-open minor major H RN3 H

R N H N O N H

Δ CH hυ, N 2 2 R H [5,6]-Open N H R H hυ N H H H O O Prato reaction

O O OH H H

O O [6,6]-Closed

+

Figure 31. Examples of [3+2] cycloadditions on C60.

1.5.7.4.1 Prato Reaction (1,3-Dipolar Cycloaddition of Azomethine Ylides) on C60 and C70

The Prato reaction, which was initially introduced by Maggini and Prato, is now considered as one of the most widely used methods for the functionalization of fullerenes. The azomethine ylide used in the Prato reaction is generally formed by decarboxylation of the iminium formed by the reaction of formaldehyde and a glycine derivative. Subsequently, the azomethine ylide undergoes a [3+2] cycloaddition reaction with fullerene, forming a fulleropyrrolidine adduct (Fig. 32).

R H N H R’‘ R’ R O R R O O Δ C60 + H N H N H HN O -CO OH H R’’ -H2O 2 R’’ R’ R’’ R’ R’

Figure 32. The Prato reaction. Formation of the azomethine ylide and its subsequent [3+2] cycloaddition reaction with C60 fullerene.

28 Different substituents can be introduced in the pyrrolidine ring by using the corresponding amino acids and ketone/aldehyde as starting materials (Fig. 32).100 Some of the main advantages of the Prato reaction are (1) a single monoadduct can be obtained, (2) a wide range of addends are easily accessible, and (3) various substituent groups can be introduced in one step

(Fig. 32). The Prato reaction was first reported using C60 fullerene with a methyl group as a substituent (Fig. 33).100

Me H N H H H H O O C60 N + Me OH H H

Figure 33. Prato reaction to form N-methylfulleropyrrolidine derivative.

Later on, various functional groups were introduced to fullerenes by this reaction. For example, tetrathiafulvalenes,154-157 porphyrins,158, 159 and ferrocenes,156, 160 were conjugated to fullerenes to prepare charge transfer complexes and donor-acceptor systems. The conjugates of fullerenes with crown-ethers161 or calixarenes162, 163 were synthesized for the studies on molecular interactions and molecular recognition. Finally, functionalization of fullerenes with bioorganic molecules such as sugars164 helped in the development of fullerene biomaterials.

The Prato reaction can also be used for the functionalization of C70. 165 Wilson et al. observed three different C70 monoadduct isomers. Mono- functionalization using Prato reaction on empty cage fullerenes (C60 and C70) provides [6,6] monoadducts. Bis-additions using the Prato reaction typically generate a mixture of isomers (see Chapter 2.1). In order to control the bis- functionalization to empty-cage fullerenes, tethers are generally utilized.95, 166

1.5.7.4.2 Mono-functionalization on M3N@C80 by Prato reaction

Mono Prato additions to TNT-EMFs are rather well studied167-169 and used in various applications.170-173 Prato addition on TNT-EMFs can either yield a [5,6] or [6,6] monoadduct. It has been established that the endohedral metal cluster controls the reactivity of the TNT-EMF. For example, while Prato reaction of Sc3N@C80 and Lu3N@C80 initially occurred at the [6,6]-junction,

29 kinetic adducts thermally rearranged to the thermodynamically stable [5,6]- regioisomer.167, 174, 175 On the other hand, when the same reaction was carried out on the larger sized Y3N@C80 or Gd3N@C80, the reversible thermal isomerization between their [6,6]- and [5,6]-adducts occurred and was reported for the first time in our group by Dr. Aroua.176, 177 The isomerization and stability of the adducts are therefore highly likely to be correlated to the metal cluster size.178, 179 Further studies reveal that determination of the equilibrium between [6,6]-isomer and [5,6]-isomer was solely dependent on the size of the metal cluster inside the carbon cage and not the exohedral functional groups.177 The isomerization between the [6,6]- and [5,6]-adducts occurs via [1,5] sigmatropic rearrangement rather than a retrocycloaddition mechanism, proven by theoretical calculations and experimental results.177 The lowest energy structures of [5,6] and [6,6] monoadducts were studied by DFT calculations to estimate the most stable positions of the endohedral metal cluster inside the carbon cage. For [5,6]-adducts of

Sc3N@C80 and Lu3N@C80, the preferred position of the metal cluster is when the metals are pointing away from the addition site (0.0 kcal/mol, Fig. 34b). In the case of [5,6] monoadducts of Sc3N@C80, crystal structure described in the literature suggested that the metals are pointing away from the addition site.141, 180 On the other hand, for the [5,6] and [6,6] monoadducts of

Gd3N@C80 and Y3N@C80, the preferred position of the metal cluster is when the metals are pointing towards the addition sites (Fig. 34a).

a.
b.

Addition Site
Addition Site

Figure 34. Metals pointing (a) towards and (b) away from the addition site.

1.5.7.4.3 Prato Bis-functionalization on M3N@C80

Contrary to the case of the empty cage fullerenes, using tethers181, 182 to regioisomerically control bisfunctionalization on TNT-EMFs cages failed to work. Instead, surprisingly, bis-additions on these materials are highly

30 183, 184 regioselective. The reaction between M3N@C80 and N-ethylazomethine ylide yielded Prato bisadduct derivatives (Fig. 35). The bisadduct regioisomers are formed without difficulty, highlighting the important role of the metal cluster.

N

O

HN H H + o-DCB HO O 120 °C N M = Gd, Y bisadduct

Figure 35. Previously reported Prato reaction of M3N@C80. Mono and bisadducts were formed.

The bisadducts of Gd3N@C80 and Y3N@C80 were assigned to be [6,6],

[6,6] bisadducts. Subjecting the [6,6],[6,6] bisadducts of Y3N@C80 to heat converted it to a mixture of regioisomers which were not analyzed further. On the other hand the bisadduct of Gd3N@C80 was stable enough even under 184 thermal conditions. For Sc3N@C80, three bisadducts were isolated and 183 identified, and for Lu3N@C80, two new bisadduct isomers were formed.

1.6 Applications of Fullerenes

Fullerenes have been used in numerous fields, from medicinal and biological to material science applications.

1.6.1 Organic Photovoltaics

Common organic photovoltaic systems employ a photoinduced electron transfer from a donor to an acceptor. Since C60 is a strong electron acceptor (electrochemically accept up to 6 electrons) and produces stable reduced species,69 it is commonly paired with a thiophene-based semiconducting polymer donors to create efficient photovoltaic cells. The most widely used acceptors for bulk heterojunction solar cells are C60[PCBM] and C70[PCBM] 185 (C60[PCBM] stands for [6,6]-phenyl-C61-butyric methyl ester, Fig. 37). Improvements in the design of conjugated thiophene-based donor polymers increased the solar cell efficiency up to 10%.186, 187

31 Indene-fullerene derivatives are commonly used for photovoltaic material applications (Fig. 36). The power conversion efficiency for a polymer solar cell with poly(3-hexylthiophene) (P3HT) as a donor and indene-C70 188-190 bisadduct (IC70BA) as an acceptor was reported to be as high as 7.4%.

Its C60 counterpart (P3HT/IC60BA) had a power conversion efficiency of up to 7.5%.191

N n

IC60BA IC70BA P3HT

Figure 36. Structures of indene C70 bisadduct (IC70BA), poly(3-hexylthiophene) (P3HT), and indene C60 bisadduct IC60BA.

A high LUMO level of the acceptor is necessary for a high open circuit voltage (Voc), which translates to a higher solar cell efficiency. Since the

LUMO level of the TNT-EMF Lu3N@C80 is quite high and near the LUMO level of the polymer donor poly(3-hexyl)thiophene (P3HT), its derivative

Lu3N@C80[PCBH] was synthesized (Lu3N@C80[PCBH] stands for 1-(3- hexoxycarbonyl)propyl-1-phenyl-[6,6]-Lu3N@C81, Fig. 37). It was found that

P3HT/Lu3N@C80[PCBH] resulted into a higher overall power conversion efficiency.192

OCH3 OCH3 O O O O

[60]PCBM [70]PCBM [Lu3N@C80]PCBH

Figure 37. Structures of [60]PCBM, [70]PCBM, and [Lu3N@C80]PCBH. 1.6.2 Hydrogen Gas Storage

Hydrogenation of fullerenes transforms the C=C double bonds into C-H and C-C single bonds. Since C-H bonds (bond strength = 68 kcal/mol) are weaker than C-C bonds (bond strength = 83 kcal/mol), upon subjecting fullerene hydrides to heat, the C-H bonds tend to break before the C-C bonds,

32 thus preserving the fullerene structure.193 Using DFT, Sun et al. predicted that 194 C60 can be coated with twelve lithium atoms, enabling it to adsorb 60 H2. They also showed that lithium-coated and boron-doped heterofullerene 195 (Li12C48B12) could potentially store hydrogen up to 9 wt%. More recently, other designs of potential hydrogen gas storing heterofullerenes were suggested by Gao et al..196

1.6.3 Fullerenes as Biological Materials
Fullerene derivatives have been synthesized and reported to have efficient biological activities. Some examples of these derivatives include: C60-

PVP complex, C60-PVP copolymer, cyclodextrin bi-capped C60 and C70, C60 and C70 malonic acid carboxyfullerene derivatives. The structures and uses of these fullerene derivatives in biological applications are described in the next sections below.

1.6.3.1 Photodynamic Therapy

Photodynamic therapy (PDT) is one of the promising approaches for the treatment of cancer. There are two components used in photodynamic therapy (1) a photosensitizer and (2) light with specific wavelength which can activate the sensitizer. Ideally, a photosensitizer in PDT localizes in a target area in the body such as a tumor. Also, a candidate photosensitizer must be non-toxic and must generate reactive oxygen species in a high quantum yield (Fig. 38).

Activated photosensitizer

O2

Toxic effect ROS on tumor cells generation

Photosensitizer

Light

Figure 38. Schematic diagram of photodynamic therapy mechanism.

33 C60 has been reported to be non-toxic in a large variety of living 197 organisms. As known, C60 can generate a nearly quantitative yield of singlet 1 36-38 oxygen O2. This reactive oxygen species can cause damage to the tissues only in the area around the photosensitizer molecules that are exposed to light.198 Previously, Yamakoshi et al. prepared a water-soluble

C60/PVP complex by mixing C60 and commercially available poly(vinylpyrrolidone) (PVP). This was used to conduct a biological 199 haemolysis test. Later on, our group prepared a C60-PVP copolymer that generates superoxide radical anion upon photo-irradiation.200 In addition, Hamblin and co-workers reported N,N-dimethyl pyrrolidinium (Fig. 39a) functionalized cationic fullerenes, which induced apoptosis on three mouse 201 cancer cell lines. Cyclodextrin bi-capped C60 (Fig. 39b) also demonstrated high phototoxic properties when exposed to human lens epithelial cells.202 The photocytotoxicity of malonic acid-functionalized C60 (Fig. 39c) was also evaluated and was found to be toxic against HeLa cells under photoirradiation.203

OHn a. b. c. HO C CO H N 2 2 n n

OHn

Figure 39. The C60 (a) dimethylpyrrolidinium (b) cyclodextrin-bi-capped and (c) carboxyfullerene derivatives were reported for their photodynamic activity.

The physical properties of C70 are usually very similar to C60. Their yields in singlet oxygen generation are nearly quantitative. However, the 3 lifetime of the triplet excited state of C70 ( C70*) (130 ± 10 μs in benzene) is 38, 41 longer than that of C60 (40 ± 4 μs in benzene). Structurally, C70 has a more extended π-conjugate system. All of these attributes suggested that C70 can also be a potential sensitizer like C60. In fact, some studies have shown that C70 could potentially become an even better photosensitizer for photodynamic therapy. For example, cyclodextrin-bi-capped C70 (Fig. 40a)

34 was demonstrated to have better photosensitizer properties than its C60 204 counterparts. Doi et al. also incorporated C60 and C70 to lipid membranes.

After incorporating the C60 and C70 in the HeLa cells membrane in the dark, the medium was exposed to light for 60 minutes. Phototoxicity was assessed by fluorescence microscopy and it was found that the integrated C70 had 4.7 205 times higher photodynamic activity in HeLa cells than C60. Moreover, malonic acid C70 carboxyfullerene derivatives (Fig. 40b) were found to have 206, 207 better photodynamic activity than C60 carboxyfullerenes. Since both the

C60 and C70 fullerenes can generate reactive oxygen species under light irradiation, both of them are considered as a promising drug candidate for photodynamic therapy.

a. b. HO2C CO H 2 n

Figure 40. The C70 (a) cyclodextrin-bi-capped and (b) carboxyfullerene derivatives were reported for their photodynamic activity.

1.6.3.2 MRI Contrast Agent: Reported MRI-CA Fullerene Based Materials

MRI is a widely used tool to diagnose cancer, atherosclerosis, and other diseases. The intravenous injection of paramagnetic MRI contrast agents (CA) can improve the contrast in the images and therefore MRI-CAs are commonly used clinically. These contrast agents need to be biologically safe and ideally must also have a high relaxivity rx (x = 1, 2). Relaxivity (in units mM-1s-1), refers to the capability of a material to change water proton relaxation time. Paramagnetic ions like gadolinium cation (Gd3+) decrease the relaxation time of the water protons in their vicinity. When the difference in the relaxation times of protons in adjacent tissues increases, the magnetic resonance signal is enhanced. The different tissues in vivo, then, are more defined and distinguished. Thus, the higher the relaxivity is, the better the contrast is, and the amount of the required dose of agent for imaging can be

35 lowered.208

O O O O O O O O O N N O N N O N N O Gd Gd O Gd O O O N N N N OH O N N O O O O O OH Gadoteridol (ProHance) Gadoterate (Dotarem) Gadobutrol (Gadovist) Figure 41. Examples of MRI-CAs approved by U.S. Food and Drug Administration (FDA).

Currently, the most widely used contrast agents are the Gd3+ chelates (Fig. 41).209 However, a skin disorder known as nephrogenic systemic fibrosis (NSF), is associated to the exposure to Gd3+ ions from contrast agents, principally affecting patients suffering from renal failure. Consequently, there is a need for new contrast agents, with high relaxivity and low toxicity. Reports from Shinohara and Alford groups have established that monometallic Gd@C2n can be potentially used as new generation MRI- contrast agents (Fig. 42).210, 211 Since trimetallic nitride template endohedral fullerenes or TNT-EMFs, (in particular Gd3N@C80) possesses three paramagnetic atoms of Gd in a cluster instead of one Gd atom, it is possible that Gd3N@C80 can be a more efficient contrast agent. Moreover, since the 3+ Gd metals are encapsulated in a carbon cage in Gd3N@C80, there is a lower probability that the toxic metal ion will be released in vivo. Significant attention has therefore been drawn to Gd3N@C80 as a potential MRI-CA.

a. b. HO2C CO2H (OH)x 10 Gd@C2n Gd@C60

Figure 42. General structures of gadolinium metallofullerenes as MRI contrast agents. Gd metallofullerenes are commonly functionalized with (a) hydroxyl units or (b) bis-carboxylic acid units to achieve water-solubility.

Shinohara et al. demonstrated the higher relaxivity of water-soluble Gd3+ metallofullerenes or ‘gadofullerenes’ as it is commonly coined. They reported that Gd@C82(OH)40 had a significantly higher relaxivity compared to the corresponding Gd3+-DTPA (diethylenetriaminepentaacetic acid) chelate complexes (Fig. 42).210, 212 Furthermore, Gd@fullerene derivatives such as

Gd@C60(OH)x (x ≈ 27) and Gd@C60[C(COOH)2]10 provide relatively high

36 -1 -1 -1 -1 relaxivity values at 1.4 T (r1 at 83.2 – 97.7 mM s and 15 – 24 mM s , respectively, Fig. 42) compared to Gd3+-DTPA (3.9 mM-1 s-1 at 1 T).213 Dorn et al. functionalized a gadofullerene with PEG chains and hydroxylated it to make it water-soluble. The reported relaxivity of Gd3N@Ih-C80[DiPEG(OH)x] -1 -1 214,215 was quite high at 143 mM sec for r1 at 2.4 T. The attached hydroxyl groups increased the interaction of the hydrophobic carbon cage with the water molecules. Although Gd3N@Ih-C80[DiPEG(OH)x] gave a very high relaxivity, the structure of the compound was not well defined, since the used PEG was polydispersed and hydroxylation process was not controlled (Fig. 43).214

O O O O O O

(OH)x

Figure 43. Structure of the water-soluble Gd3N@Ih-C80[DiPEG(OH)x] which was reported to have a high -1 -1 relaxivity at 143 mM sec for r1 at 2.4 T.

1.7 Outline of Dissertation

Fullerenes, the third allotropic form of carbons after graphite and diamond, offer interesting photo-physical properties such as long wavelengths of absorption and high yields of reactive oxygen species. These molecules can potentially be used for medicinal applications. However, these molecules suffer from poor solubility in many polar solvents. Fullerenes have been functionalized by various cycloaddition reactions. The Prato reaction is a versatile reaction for fullerene functionalization with short reaction times and with reasonable yields. In this thesis, the Prato reaction was used to functionalize fullerenes.

Previously in our group, Dr. Aroua prepared water-soluble C60-PEG amphiphiles. These compounds were presumed to form micelle type aggregations in solution.216 The fullerene cage was functionalized by second and third cationic pyrrolidine moieties in order to disrupt the supposed micelle assembly (Chapter 2).

37 The C70 fullerenes can potentially be used for medicinal applications due to previously stated properties of C70 (see Chapter 1.3.2). C70 was functionalized via Prato reaction and was subsequently conjugated with a water-soluble polymer (Chapter 3).

Finally, the reactivity and property of M3N@C80 (M = Y, Gd) were investigated in detail using Prato reaction, with a focus on tris-addition. Previously, Dr. Aroua in our group described the synthesis of the bisadducts of M3N@C80 (M = Y, Gd). These adducts were obtained in a regioselective manner. In this thesis, the synthesis of the trisadducts of M3N@C80 (M = Y,

Gd) was described. One of the isomers of trisadducts of Y3N@C80 was characterized using experimental and theoretical methods (Chapter 4). The higher adducts of Gd3N@C80 fullerenes are of particular interest due to their potential application in MRI contrast imaging.

38

CHAPTER 2

SYNTHESIS OF WATER-SOLUBLE C60-

PEG CONJUGATES AND THEIR SELF-

ASSEMBLY

The work described in this chapter was a continuation of the previous work by Dr. Aroua and was carried out under his direct guidance. The molecular designs and synthetic plans were proposed by Dr. Aroua. Dynamic Light Scattering measurements were performed in collaboration with the group of Prof. Morbidelli. Tensiometry measurements were carried out in collaboration with the group of Prof. Isa and direct help from Mr. Vasudevan. Scanning transmission electron microscopy measurements were performed by Dr. Sologubenko at ScopeM in ETHZ. Ms. Liosi heavily helped in the synthesis of 8.

39

40 2.1 Background

It has been reported that amphiphilic C60 derivatives often self- assemble in solution. The formed morphologies vary depending on the experimental factors such as solvent conditions, counterion sizes, and the properties of the amphiphile. For example, Tour and co-workers reported that transmission electron microscopy (TEM) images of the Prato adduct C60- dimethylpyrrolidinium iodide 1 indicated the formation of vesicles upon sonication of its aqueous solution. Interestingly, the same amphiphile 1 formed nanorods when its DMSO solution was mixed with water and benzene (Fig. 44). Moreover, it was observed that the diameter sizes of the nanorods - - - 217 varied depending on the size of the counterion (NO3 , I , or Br ).

Me Me N I

1

ultrasonication

aq. soln.

water, benzeneDMSO

nanorod formation vesicle formation Figure 44. Self-assembly of 1 into nanorods or vesicles. 217

Another C60-Prato adduct 2 self-assembled into fibrous and disk-like microstructures with 10-12 nm thickness by the sonication of a film that was cast from a solution of 2 in hot methanol. X-ray diffraction studies suggested that 2 formed a bilayer structure, arising from a layer of hydrophobic n- decylpyrrolidine tails and another hydrophilic layer with charged ammonium cation end groups (Fig. 45). The aggregation sizes were in the range of ca. 150 to 400 nm.218

41 Cl NH3

O

Cl NH3 O Br CH3 CH3 N N CH3 N N CH3

2 3 4

Figure 45. Previous examples of C60 amphiphiles 2, 3, and 4 which self-assembled into different morphologies.

TEM images of another ionic fullerene Prato derivative 3 showed spherical vesicles when it was sonicated (Fig. 45). Nanorods with diameters of 4 nm and lengths of several microns were observed for PEGylated fullerene derivative 4 (Fig. 45).219 In these cases, the balance between two major non- covalent interactions are the driving force for the formation of more complex aggregations. One parameter is the π-stacking of fullerene moieties.220 Another parameter is the repulsive, ionic interaction among the charges. Other factors such as the length of the hydrophilic tail and the charged moieties clearly play a vital role in the morphologies formed. For example, although 3 and 4 both have a hydrophobic C60 core and terminal ammonium charged chains, altering the spacer types and lengths affected the type of self-assembled morphology.

Previously, Dr. Aroua in our group reported a series of PEGylated C60 derivatives 5, 6, and 7 (Fig. 46). These C60 derivatives have displayed water- solubility, presumably by forming micelles in aqueous solution due to their amphiphilic property as indicated by surface tension measurements and cryoTEM.216 It is important to study and identify the higher structures formed by such amphiphilic molecules. Such higher structures may cause aggregations, which can be a problem when used in vivo. These aggregates may cause coagulation inside the blood vessels, and consequently may result to vascular thrombosis.221

42

O H H O N N O O O O n n O N O

Hydrophilic tail


H2O







Hydrophobic head

5 (n = 12) 6 (n = 36) 7 (n = 20)

Figure 46. C60 derivatives 5, 6, and 7 presumably formed micelles in aqueous solution.

Since the hydrophobic interaction among the C60 moieties is one of the driving forces in micelle formation, introduction of more hydrophilic moieties on the surface of the C60 cages may affect the micelle formation of 5, 6, and 7.

Therefore, we planned the multi-functionalization at the surface of C60 by introducing cationic moieties. In this study, we aimed to synthesize the bis and trisadducts of C60 to evaluate the effect of the second and third functionalizations.

Since C60 has 30 reactive [6,6] bonds, poly-addition to C60 can yield a large number of products. While the Prato mono-addition provides only [6,6] monoadduct, bisaddition to C60 generates a mixture of isomers as shown in

Fig. 47. Nonetheless, well-defined and well-designed C60 polyadduct structures have a higher potential to be used for biological applications, since information about the structure of compounds can be especially useful to understand its mechanism of action. In order to synthesize compounds 8 and 9, the regioselectivity of the bis and trisadditions must be controlled. It is known that the bisaddition to C60 can yield up to eight possible isomers (cis-1, cis-2, cis-3, e, trans-1, trans-2, trans-3, and trans-4, see Fig. 47). These different isomers can be separated by multiple stages of purification by column chromatography and can be initially distinguished by their UV-Vis spectra (400 – 700 nm). Prato et al. successfully isolated all eight bisadducts and characterized them by ES-MS, UV, and NMR.222, 223

43 Me Me Me Me N N N N

cis-1 cis-2 cis-3 e

Me Me Me Me N N N N

trans-1 trans-2 trans-3 trans-4 Figure 47. The eight possible isomers of bisaddition on the N-methyl fulleropyrrolidine monoadduct of C60. The orange bonds correspond to the second addition sites. Therefore, in addition to the preparation of water-soluble products, the structures and further assembly of these materials must also be defined. In this study, well-defined C60 PEGylated adducts 7, 8, and 9 were designed and synthesized to analyze their aggregation behaviors in water (Fig. 48). Since our interest is on the potential biological application of these C60-PEG conjugates, we used a known biocompatible polymer PEG as the solubilizing polymer. One (8) or two (9) cationic pyrrolidine moieties were introduced in distal positions (the trans-1 and trans-3 positions) of C60 monoadduct. We expected that these charges could disperse micelle formation that was 216 observed in the C60-PEG monoadduct in a previous report.

44 O H H O O H H O tBu N N tBu tBu N N tBu O O O O O O O O 20 O N O 20 20 O N O 20

I N 7

8 O H H O t Bu N N tBu O O O O 20 O N O 20

N N I I 9

Figure 48. Structures of C60-PEG 7 and target compounds 8 and 9.

2.2 Synthesis of C60-PEG Conjugates 7, 8, and 9

Previously, a C60 bisester derivative 10 (Scheme 1) was synthesized by Dr. Aroua as a scaffold molecule for further fullerene functionalization.224 Compound 10 was synthesized using an N-glycine derivative and paraformaldehyde and was subsequently subjected to TFA deprotection.

PEGylation using a monodispersed PEG provided the C60-PEG conjugate 7. The length of the PEG chain of 7 was known to be long enough to gain water- solubility as demonstrated in the previous study.216 Scheme 1. Synthesis scheme of monoadduct 7. 216

O H H O O O HO OH tBu N N tBu tBu tBu O O O O O O O N O N 20 O N O 20 t NH2-PEG20CO2 Bu TFA HBTU, DIPEA DMF, RT, overnight, 83%

10 11 7

To obtain 8 and 9, C60 bisester 10 was subjected to a second Prato reaction to obtain bisadducts. It is known that Prato reaction generally provides various bisadduct isomers. Those products have previously been characterized in reports by Wilson et al. and Leach et al..222, 225 Referring to

45 these studies, several bisadduct isomers were obtained. However, only the trans-1 12a (Scheme 2) and trans-3 bisadducts 12b (Scheme 3) were isolated and fully characterized for our purposes. The crude mixture of the Prato reaction was subjected to silica gel column chromatography to remove the unreacted C60 monoadduct 10. The mixture of bisadduct isomers was then subjected to a second stage purification using HPLC. The trans-1 and trans-3 isomers were identified by their UV-vis traces, and then subsequently characterized by NMR. 1H-NMR of 12a showed two singlets at 4.65 and 4.77 ppm (pyrrolidine protons, four protons each, peaks labeled with orange and blue colored dots) and a quintet at 4.31 ppm (J = 6.6 Hz, (CH2)2-CH, peak labeled with a pink colored dot) in Fig. 49. Due to the symmetry in 12a, the two methylenes of each pyrrolidine are equivalent, thus only one singlet can be observed for each set of pyrrolidine protons (Fig. 49). The 13C-NMR spectrum of 12a shows thirteen carbon peaks in the aromatic region (130 – 155 ppm), including the eight vicinal carbons to the pyrrolidines. Nine carbon peaks were found in the region 28 – 82 ppm corresponding to the sp3 carbons. The two carbons at the attachment point of the bisester pyrrolidine are shown as a single peak at 68.0 ppm (brown dot in Fig. 50). Similarly, the two carbons at the attachment point of the N-methylpyrrolidine are shown as a single peak at 69.3 ppm (black dot in Fig. 50). The methylene carbons of each pyrrolidine also appear as two single peaks at 63.6 and 70.5 ppm, respectively (green dots in Fig. 50). The observation of only one signal for the sp3 attachment carbon of each pyrrolidine and one signal for the methylene groups of each pyrrolidine confirm the synthesis of a symmetrical bisadduct corresponding to the trans-1 isomer.

46 H O O tBu tBu O N O H H H H

bis-ester CH2 protons

H H // H N H

CH3

12a

Figure 49. 1H-NMR of 12a.

a.






















 b.


  Figure 50. 13C-NMR of 12a.

For the analysis of the 1H-NMR spectra of 12b, a series of multiplets and doublets can be observed in the region between 4.00 – 4.50 ppm. The number of protons in the spectrum in total is 32 protons, which is what is expected of a bisadduct. A singlet at 2.92 ppm integrating three protons corresponded to the methyl protons of the N-methyl pyrrolidine (red dot in Fig. 51), indicating that a single isomer was isolated. A multiplet at 4.04 – 4.17 ppm integrating four protons (one (CH2)2-CH proton and three pyrrolidine

47 protons), a doublet at 4.26 ppm (J = 8.8 Hz) with an integration of one proton (one pyrrolidine proton), another doublet at 4.31 ppm (J = 9.2 Hz) integrating one proton (one pyrrolidine proton), a multiplet at 4.44 – 4.36 ppm with an integration of two protons (two pyrrolidine protons), and finally a doublet at 4.51 ppm (J = 8.8 Hz), integrating one proton (one pyrrolidine proton) were observed in the spectra (Fig. 51). This pattern showed that all methylene protons are non-equivalent leading to the conclusion that the regioisomer obtained is unsymmetrical. Further analysis of the 13C-NMR spectrum shows a total of 13 carbon peaks in the region between 28 – 82 ppm corresponding to the sp3 carbons. This includes four distinct peaks for the four sp3 carbons of the fullerene cage (69.0, 69.5, 70.31 and 70.6 ppm, black colored dots in Fig. 52) and four single peaks for the methylene carbons of the pyrrolidine moieties (62.5, 63.3, 69.3 and 70.33 ppm, green colored dots in Fig. 52). The complex NMR spectral patterns are indicative of an unsymmetric molecule. Based on all these data, it was confirmed that 12b was the trans-3 isomer.

bis-ester CH2 protons

O O tBu tBu O N O //

N CH3

12b Figure 51. 1H-NMR of 12b.

48     
 

  
    
  





Figure 52. 13C-NMR of 12b.

The tert-butyl groups in purified compound 12a were then removed by

TFA to obtain bis-carboxylic acid 13. The monodispersed PEG20-amine was then conjugated with 13 using HBTU mediated amide coupling to yield the PEGylated bisadduct 14. Finally, compound 8 was successfully obtained by methylation of the trans-1 pyrrolidine unit of 14 (Scheme 2).

49 Scheme 2. Synthesis of 8.

O O tBu tBu O O tBu tBu O N O O N O N-methyl glycine paraformaldehyde TFA, DCM toluene, reflux, 3.5 h, 1% RT, 4 h, >99%

N

10 12a

O H H O HO OH tBu N N tBu O O O O O O N 20 O O 20 t N NH2-PEG20CO2 Bu HBTU, DIPEA

DMF, RT, 24 h, 33%

CF COO N 3 H N

13 14

O H H O tBu N N tBu O O O O 20 O N O 20

CH3I DMF, RT, 1 h, 87%

N I

8

On the other hand, trans-3 bisadduct 12b was subjected to a third Prato reaction to produce a mixture of trisadducts, among which the symmetric trisadduct 15 was isolated (Scheme 3).

50

Scheme 3. Synthesis of 9.

O O O O tBu tBu tBu tBu O N O O N O

N-methyl glycine N-methyl glycine paraformaldehyde paraformaldehyde

toluene, reflux, 3 h, 8% toluene, reflux, 2.5 h, 7%

N

10 12b

O O HO OH tBu tBu O N O O N O

TFA

DCM, RT, 2 h, >99%

H H N N N N

CF3COO CF3COO 15 16

O H H O t Bu N N tBu O O O O 20 O N O 20

t NH2-PEG20CO2 Bu HBTU, DIPEA

DMF, RT, 24 h, 27%

N N

17

O H H O t Bu N N tBu O O O O 20 O N O 20

CH3I DMF, RT, 4 h, 88%

N N I I 9 Compound 15 was characterized by 1H- and 13C-NMR. Due to the symmetry in 15, the 1H-NMR showed a singlet at 4.14 ppm integrating four protons (blue colored dots) corresponding to the methylene protons of the bisester pyrrolidine. Similarly, the methylene protons of the N-

51 methylpyrrolidines are all equivalent and appeared as a singlet at 4.02 ppm integrating eight protons in Fig. 53 (orange dots). A quintet integrating to one proton appeared at 3.98 ppm ((CH2)2-CH, pink dot). Finally, the methyl protons of the two N-methylpyrrolidines are equivalent and are shown as a singlet, integrating six protons at 2.81 ppm (red dot, Fig. 53).

H O O tBu tBu O N O H H H H

H H H H

N N H3C H CH3 H H H 15

Figure 53. 1H-NMR of 15.

The analysis of the sp3 region on the 13C-NMR confirmed the formation of the symmetric adduct 15. In fact, the two methylene carbons on the bis- ester pyrrolidine moiety and the four methylene carbons of the N- methylpyrrolidines are shown as two single peaks at 62.4 and 69.8 ppm (green colored dots). Furthermore, the carbons of the two methyl groups of the N-methylpyrrolidines are also shown as a single peak at 41.7 ppm (red colored dot). Finally, the two carbons at the attachment point of the bisester pyrrolidine and the four carbons at the attachment point of the N- methylpyrrolidines are shown as single peaks at 68.4 and 69.5 ppm (black colored dots, Fig. 54). All these data suggested that this compound is the symmetric trisadduct 15.

52

Figure 54. 13C-NMR of 15.

The tert-butyl groups of 15 were deprotected to obtain bis-carboxylic adduct 16. Conjugation of the PEG20-amine to the carboxylic acid groups of

16 generated C60-PEG 17. Finally, methylation of the pyrrolidine moieties in 17 was carried out to obtain the target product 9 (Scheme 3). It was confirmed that compounds 7, 8, and 9 were highly water-soluble, giving clear and colored aqueous solutions and were potentially suitable as fullerene-based therapeutic agents.

2.3 Characterization of the C60-PEG Conjugates 7, 8, and 9

In order to determine the self-assembled structure of compounds 7, 8, and 9, a series of experiments such as tensiometry, dynamic light scattering (DLS), and scanning transmission electron microscopy (STEM) measurements was performed.

2.3.1 Surface Tension
Surface tension data generally provides information on the micelle formation of the molecules in solution. The surface tension data of the aqueous solutions of 7, 8, and 9 were recorded at 25 °C at air-water interface using pendant drop method. In the pendant drop method, the balance between gravity and surface forces determines the profile of a drop of a liquid suspended in another medium. The drop profile is then related to the interfacial tension using the Laplace equation.226 Aqueous solutions of 7, 8, and 9 with varied concentrations (3x10-7 M to 1x10-3 M) were prepared and subjected to the measurements as shown in Fig. 55.

53 70

7 65 8 9

60

Surface Tension (mN/m) Surface Tension 55

50

-6 -5 -4 -3 10 10 10 10 Concentration [M] Figure 55. Surface tension data of compounds 7, 8 and 9 at air/water interface at 25 °C.

Both compounds 7 and 8 displayed similar interfacial tension (IFT) trends, showing an abrupt decrease in surface tension upon increase of the concentration between 3 x 10-7 and 1 x 10-5 M, indicating that micelles of 7 and 8 were formed above 1 x 10-5 M. In contrast, a significant difference in the IFT curve for 9 was observed. The IFT trend of 9 was moderately decreasing, implying that a different type of assembly may have been formed. Based on these results, surface tension data implied that addition of only one cationic charge at the C60 surface did not alter the assembly formed by the reference molecule 7. On the contrary, tensiometry data of 9 suggested that addition of two charged pyrrolidine moieties on the carbon cage has an influence on the formation of self-assembly.

2.3.2 Dynamic Light Scattering
Dynamic Light Scattering (DLS) measurements were also performed using 1 mM aqueous solutions of compounds 7, 8, and 9 to determine their particle sizes at a range of temperatures (5 to 80 °C).

54 Figures 56a and 56b shows the DLS data obtained from compounds 7, 8, and 9 at 20 °C and 80 °C, respectively. The mean of the particle size of 7 at 20 °C was 8 nm. The particle size of 8 was slightly larger with a mean of 13 nm. Interestingly, the DLS data of 9 at 20 °C showed polydispersed sizes compared to 7 and 8. At 20 °C, two distribution peaks were observed (Fig. 56a) with mean particle size of 91 nm and 450 nm. These DLS results revealed that compound 9 has a different self-assembly pattern as compared to compounds 7 and 8. Increase of size of the aggregation in aqueous solution suggested that a larger self-assembly was formed.

20 a. 20 ºC

15 7 8

10

volume [%] 5 9

0 0 1 2 3 4 10 10 10 10 10 particle size [nm]

b. 30 80 °C 25 7 20 8 9 15

volume [%] 10

5

0

0 1 2 3 4 10 10 10 10 10 particle size [nm] Figure 56. DLS data of the 1 mM aqueous solutions of 7, 8 and 9 (a) at 20 °C with particle size mean (width) [nm]: 8 (3) for 7, 13 (4) for 8, and 91 (49) and 450 (265) for 9 and (b) at 80 °C with particle size mean (width) [nm]: 1400 (363) for 7, 1900 (485) for 8, and 300 (84) for 9.

At higher temperatures (80 °C), the particle sizes of compounds 7, 8, and 9 increased drastically. This increase in size can be attributed to the behavior of PEG in solution at higher temperatures (Fig. 56b). The mean particle sizes of 7, 8, and 9 was 1400, 1900, and 300 nm at 80 °C. The particle sizes of 9 at 80 °C was significantly smaller than the ones of 7 and 8. Based on these results, the addition of two charged moieties presumably

55 increased the solubility and affected the agglomeration at higher temperatures. Figure 57 shows the analysis of the particle size in DLS over a range of temperatures from 5 to 80 °C.

80 °C 150 a. 70 °C 60 °C

100 50 °C 40 °C

% volume 30 °C 50 20 °C 10 °C 0 5 ºC 0 1 2 3 4 10 10 10 10 10 particle size [nm]

80 ˚C 150 b. 70 ˚C 60 ˚C 100 50 ˚C 40 ˚C

% volume 30 ˚C 50 20 ˚C 10 ˚C 0 5 ˚C 0 1 2 3 4 10 10 10 10 10 particle size [nm]

80˚C 150 70˚C c. 60˚C 100 50˚C 40˚C

% volume 30˚C 50 20˚C 10˚C 0 5˚C 0 1 2 3 4 10 10 10 10 10 particle size [nm] Figure 57. Variable Temperature-DLS of the aqueous solutions of (a) 7, (b) 8, and (c) 9 at 1 mM.

56 For compound 7, the change in particle size was abrupt. The transition occurred after 50 °C (Fig. 57a). In contrast, for compound 8, the change in particle size was less abrupt and started at temperatures higher than 40 °C. At lower temperatures (5 – 30 °C), a non-uniform size distribution can be observed (Fig. 57b). For compound 9, at lower temperatures (5 – 50 °C), varied size distributions were observed. The change in particle size started to appear at 60 °C to give a monodispersed size distribution (Fig. 57c). These results demonstrated that adding charged pyrrolidine moieties in compound 7 affected the particle size upon a change in temperature.

2.3.3 Cloud Point
In the course of solvation in water, the oxygen atoms in PEG can be hydrated by forming hydrogen bonds with water molecules. At an elevated temperature, these hydrogen bonds are disrupted, and the interaction among the hydrophobic groups becomes greater than the hydrogen bonding. This temperature is known as the “cloud point.” At the cloud point, the interactions among PEG moieties are promoted, consequently inducing phase separation and hence resulting to the turbidity of the solution.216, 227 In this experiment, the thermoresponsivity of 7, 8, and 9 were evaluated in order to assess their potential as thermoresponsive materials (Fig. 59). To observe the cloudiness, absorbances at 800 nm were recorded at 5 to 80 °C using aqueous solutions of 7, 8, and 9 (1 mM). As a control, 2 mM aqueous solution of PEG20-amine 18 was used (Fig. 58).

O

t Bu NH2 O O 20 18 t Figure 58. Structure of NH2-PEG20- Bu-ester 18.

57 1.5

7 8 9

NH2-PEG20-tBu-ester 18, 2 mM 1.0 800

A

0.5

0.0

20 40 60 80 Temperature [ºC] Figure 59. Thermoresponsivity of 7, 8, and 9 and control over a range of temperature.

The solution of PEG20-amine 18 did not change at all for the entire temperature range. The aqueous solutions of 7, 8, and 9 started to become turbid at 56, 40, and 40 °C, respectively. These results exhibited the significant effect of C60 on the cloud point of PEG.

58 7 80 °C a. 2.0

1.5 800 1.0 OD

0.5

0.0 25 °C

5 10 15 20 Cycles

b. 8 80 °C

1.5

800 1.0 OD

0.5

25 °C 0.0

5 10 15 20 Cycles

c. 1.4 9 80 °C 1.2

1.0

0.8 800

OD 0.6

0.4

0.2 25 °C 0.0

5 10 15 20 Cycles Figure 60. Reversibility of the thermoresponsivity of 7, 8, and 9. 1 mM aqueous solutions of 7, 8, and 9 were subjected to multiple rounds of heating (80 °C) and cooling (25 °C).

The OD800 of aqueous solutions of 7, 8, and 9 were measured at 20 and 80 °C repeatedly to see the reversibility of their thermoresponsive behavior. The solutions of 7, 8, and 9 were subjected to ten rounds of being heated (80 °C) and cooled down (25 °C). Results showed that this observed turbidity was reversible (Fig. 60). The formation of the aggregation can be repeatedly observed for ten cycles. These results imply that the thermoresponsive reversibility exhibited by 7, 8, and 9 were independent of the C60 head functionalization. This temperature-controlled agglomeration can potentially be used as a temperature-responsive compound for material applications.

59 2.3.4 STEM Measurements
Scanning transmission electron microscopy (STEM) is an imaging method, which entails a beam of electrons passing through a thin film of sample in order to form an image. The main advantage of using STEM compared to broad-beam illumination methods is the fact that it allows the use of a high angle annular dark field (HAADF) detector during imaging, which gives different levels of contrast depending on the chemical composition of the sample.228 STEM images of compounds 7, 8, and 9 were obtained at room temperature by preparing 3 mM aqueous solutions of each compound and subsequently drying them. The images of the samples were taken from random areas of the substrate. Each sample was subjected to the following conditions: (1) the sample was air-dried before measurement and (2) the sample was treated under vacuum (10-2 mbar) before the measurement.

(a) (b)

Figure 61. STEM images of 7. Aqueous solutions of 7 (3 mM) was (a) air-dried and (b) vacuum-dried before measurement. The sample was brought onto a conventional graphene support foil.

For compound 7, small bright particles were observed as shown in Fig. 61 and Figs. 139 and 140 (Chapter 6). Histogram analysis of both the air- dried sample (Fig. 62a) and the vacuum-dried sample (Fig. 62b) showed that the distribution mean of particle sizes were about 2 nm. These observed particles were comparable to the size of the C60 cores of the micelles of compound 7.

60 a. b. 25 35

20 30 7, air-dried 7, vacuum-dried mean= 1.9 ± 0.053 nm 25 2.3 ± 0.0326 nm 15 20

10 15

frequency frequency 10 5 5

0 0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 particle sizes [nm] particle sizes [nm] Figure 62. Histograms of the particle diameter of 7 obtained in STEM image (see Fig. 61). (a) air-dried and (b) vacuum-dried samples.

In the STEM images of compound 8, small bright particles similar to 7 were observed (Fig. 63 and Figs. 141 and 142). Both the air-dried samples (Fig. 63a) and the vacuum-dried samples (Fig. 63b) showed that the particle sizes were about 2 nm (Fig. 64). Again, these observed particles were comparable to the size of the C60 cores of the micelles of compound 8.

(a) (b)

Figure 63. STEM images of 8. Aqueous solutions of 8 (3 mM) were (a) air-dried and (b) vacuum-dried before measurement. The sample was brought onto a conventional graphene support foil.

STEM analysis also showed that 7 and 8 behaved similarly. The data were in agreement with the surface tension results where 7 and 8 both showed similar IFT curves and micelle formation above 1 x 10-5 M. These results suggested that the addition of one cationic charge did not disturb the aggregation pattern observed with 7.

61 a. b.

12

16 10 14 8, vacuum-dried 1.6 ± 0.058 nm 12 8, air-dried 8 mean= 2.0 ± 0.033 nm 10 6

8 frequency

frequency 6 4

4 2 2

0 0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 particle sizes [nm] particle size [nm] Figure 64. Histograms of the particle diameter of 8 obtained in STEM image (see Fig. 63). (a) air-dried and (b) vacuum-dried samples.

In contrast to the ones found for 7 and 8, clusters with different sizes were observed in the STEM images of compound 9 (Fig. 65). Spherically shaped structures with sizes varying from 20 to 300 nm were observed, resulting in a highly dispersed sample and the impossibility to calculate a mean size of particles (Fig. 66).

(a)

(b)

Figure 65. STEM images of 9. Aqueous solutions of 9 (3 mM) was (a) air-dried and (b) vacuum-dried before measurement. The sample was brought onto a conventional graphene support foil.

62 a. b. 8 120

100 6 9, air-dried 9, vacuum-dried 80

4 60

frequency

frequency 40 2 20

0 0 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500 particle size [nm] particle size [nm] Figure 66.Histograms of the particle diameter of 8 obtained in STEM image (see Fig. 65). (a) air-dried and (b) vacuum-dried samples. Each bar represents the total population of the particles in the corresponding interval.

While the STEM images of 7 and 8 did not show any clear large objects, the images obtained for 9 showed relatively larger particles, presumably corresponding to the self-assembly of the trisadduct. Addition of two charged pyrrolidine moieties on the C60 surface of C60-PEG 7 caused the formation of larger particles. Elemental mapping on the STEM images of 9 was performed in order to confirm the elemental composition of the observed clusters. Figure 67 shows that the distribution of green signals was concentrated particularly on the areas where the clusters were located. These green signals correspond to oxygen atoms (Fig. 67b and d). The detected oxygen atoms in the elemental distribution maps were presumably due to the PEG groups in 9. These results implied that the observed clusters indeed correspond to the supramolecular structure formed by compound 9.

63 a. b.

c. d.

Figure 67.(a) and (c) HAADF STEM micrographs of compound 9 in 3 mM aqueous solution and (b) and (d) their corresponding elemental distribution maps (green: oxygen, red: carbon).

STEM images of the vacuum-dried samples showed that compound 9 formed distinct shapes in our study. Upon subjecting the sample to vacuum drying (Fig. 65b), the large clusters seemed to have collapsed into several smaller particles. This led us to think that the large clusters of 9 are possibly related to a vesicle-type structure, whose assembly was disrupted after vacuum drying due to the removal of residual water from its core. The electrostatic interactions between the iodide ion and the ammonium cation may have driven particles of compound 9 to self-assemble in a higher

64 structure and disturbed the formation of micelles. Further studies are needed regarding the interesting self-assembly structures of 9.

2.4 Conclusions

In summary, the self-assembled structures of amphiphilic C60-PEG molecules 7, 8, and 9 were studied by tensiometry, DLS and STEM measurements. The C60 bisester derivative was bis-functionalized via Prato reaction to generate a mixture of isomers, of which trans-1 isomer 12a and trans-3 isomer 12b were isolated in 1% and 8% yield, respectively. Further synthesis steps successfully provided compounds 8 and 9. Surface tension, DLS, and STEM results did not show significant differences between 7 and 8, suggesting that addition of one charged pyrrolidine moiety in 8 did not affect the supposed micelle formation of 7. In contrast, more polydispersed particle sizes were observed in the case of 9. Tensiometry analysis of 9 did not give a similar curve as 7 and 8, suggesting that the micelle formation was disturbed and a different structure was formed by self-assembly in aqueous media. Differences between 7, 8, and 9 were also observed by DLS. Indeed, while at lower temperatures, 7 and 8 have narrow particle size distributions with mean particle sizes of about 8 nm for 7 and 13 nm for 8, 9 showed two main distributions with mean sizes of about 91 and 450 nm. These obtained DLS results gave another proof that a different type of self-assembly was observed upon the addition of two charged pyrrolidines on the surface of the C60-PEG. Finally, STEM showed very different images for 9 compared to 7 and 8. While the observed particle size of 7 and 8 was ca. 2 nm, images of 9 gave structures with sizes greater than 100 nm. All these observations showed that the addition of two charged moieties on the C60-PEG formed a different kind of morphology, possibly related to a vesicle-like assembly. On the other hand, cloud point analysis showed that, at higher temperatures, aqueous solutions of compounds 7, 8, and 9 became cloudy and turbid. Particle sizes at this temperature became significantly larger, perhaps forming an agglomerate due to the properties of PEG. The thermoresponsive behaviors of 7, 8, and 9 were also studied and shown to be

65 highly reversible. These results can help design other fullerene-PEG compounds for medicinal applications.

66

CHAPTER 3

FUNCTIONALIZATION OF C70: WATER-

SOLUBLE C70 DERIVATIVE FOR BIOMATERIAL APPLICATION

The synthesis and structural characterization of C70 derivatives described in this chapter is based on initial work done by Mr. Nasuda. Ms. Ray and Ms. Liosi greatly supported on the synthesis of 19.

67

68 3.1 Background

The C70 fullerene is the second most abundant fullerene after C60. It 229, 230 exhibits chemical properties that are closely related to those of C60. For example, additions occurring at the [6,6]-bond junctions are preferred in most cases. One major point of difference observed in D5h-C70 is its less symmetrical cage as compared to Ih-C60. The lower symmetry of D5h-C70 leads to a generation of several monoadduct isomers, in contrast to Ih-C60, where only one monoadduct isomer was obtained.

Due to its interesting properties, C70 can be used in a wide range of applications. For example, recently, C70 was reported to be more efficient than

C60 or tetraphenylporphyrin to oxidize benzylamines to corresponding imines upon light irradiation. This is due to the high efficiency of C70 as a source of 231 ROS. Because of its capability to generate ROS efficiently, C70, similar to

C60, is also a reported photosensitizer and can thus be used as a potential photodynamic therapy agent. A number of studies have investigated the medicinal properties of C70. For example, C70 fullerenes capped with cyclodextrins have been investigated for their photodynamic therapy properties. It was found that the photodynamic activity from treatment of the

HeLa cells with the C70-cyclodextrin complex was higher than that of its C60 204, 205 counterpart. In another study, C70-carboxyfullerene derivatives were reported to be better photosensitizers than C60-carboxyfullerenes under light irradiation (400 – 700 nm), possibly due to the extended π-system of C70, 206 which facilitated light absorption. Treating cancer cells with C70- carboxyfullerenes led to cell necrosis possibly due to the fact that the elevated amounts of ROS disrupted the organelles.

Analogous to C60, C70 fullerenes are also being used for the optimization of materials such as polymer solar cells. Li et al. used C70-indene bisadducts (IC70BA) as acceptor in combination with the polymer poly(3- hexylthiophene) (P3HT) as donor. Upon addition of 1-chloronapthalene as an additive, this system yielded a high power conversion efficiency (PCE) of 7.40%, which is the one of the highest reported PCE values for P3HT based polymer solar cells.188

69 In order to be able to utilize C70 as a biomaterial, water-soluble C70 derivatives must be prepared. Similar to C60, C70 suffers from poor solubility in polar solvents. C70 has been functionalized by a variety of reactions to provide 73, 74 232 C70 derivatives. These include epoxidation, hydrogenation, nucleophilic addition,233 cycloaddition,229, 230 among others. Diederich et al. introduced a qualitative method to predict the addition sites in C70 and higher fullerenes using a Diels-Alder reaction between C70 and o-quinodimethane as a model.234 In a fullerene surface, the curvature is induced by the insertion of pentagons into a network of hexagons. Hence, the [6,6]-bonds which have more pentagons surrounding them are more curved and are considered to be more reactive. The most strained bonds are therefore located at the center of a pyracylene structure (type α in Fig. 68). The type α bonds surrounded by type β bonds can be found at the poles of

C70. The flatter equatorial areas comprise less curved bonds (Fig. 69).

1 5 26

α β

Figure 68. Examples of the types of bonds found in C70.

Theoretical calculations have shown that the lowest energy addition products occur at the type α C1 – 2 bond (bond a in Fig. 69) and type β C5 – 6 (bond b in Fig. 69), implying that these two bonds are the most reactive 235 ones. Both [6,6] bonds are located closest to the poles of C70 (Fig. 69). These calculations support and confirm the experimental results obtained.

b a c b a d

Figure 69. C70 most reactive bonds in the order of a > b > c > d. The red colored bonds generally have a higher reactivity than the blue colored bonds.

70 On the other hand, the isomers C7 – 21 (bond c in Fig. 69) and C7 – 8 (bond d in Fig. 69) have relatively low local curvature and are therefore isolated in practice as minor adducts. The C7 – 8 isomer results from an addition occurring at the junction between a pentagon and a hexagon (a [5,6]- junction). The C7 – 8 isomer could exist due to the relatively high double bond character of the [5,6]-junction, underlining the importance of the Kekulé resonance structure of C70 wherein benzenoid hexagons can be drawn at the equatorial belt. (Fig. 70).123

Figure 70. An important Kekulé resonance structure of C70. The benzenoid character of the equatorial hexagons is illustrated.

Experimentally, mono-functionalization (e.g. cycloaddition reactions) of 124, 234, 236 C70 generally produces C1 – 2 products and C5 – 6 products.

Previously, a Diels-Alder reaction between C70 and o-quinodimethane reportedly yielded C1 – 2, C5 – 6, and C7 – 21 isomers.229, 230, 234 Wilson et al. reported that a [3+2] cycloaddition reaction of N-methylazomethine ylide with

C70 produced three monoadduct isomers, with additions at the C1 – 2, C5 – 6, and C7 – 21 bonds. These were isomerically assigned by 1H-NMR 165 spectroscopy. Benzyne addition to C70 reportedly generated four monoadduct isomers, namely the C1 – 2, C5 – 6, C7 – 21 and the C7 – 8 adducts.123 In all these cases, the C1 – 2 and C5 – 6 are the major isomers. The other isomers such as C7 – 21 and C7 – 8 are generated only in low yields.

Our group previously reported functionalization of C60 using a bisester derivative as an excellent scaffold to access water-soluble, biocompatible C60- PEG derivatives.216 This scaffold has previously been shown to be a versatile 216, 224 and facile moiety for further fullerene functionalization. Since C70 also exhibits very attractive properties for medicinal and material applications, it was interesting to functionalize C70 with the same bisester scaffold and prepare water-soluble C70 polymer conjugates. In this chapter, C70 Prato bis-

71 ester monoadducts were synthesized, isolated, and characterized. A new, water-soluble C70-PEG conjugate was also successfully synthesized.

3.2 Synthesis and Isolation of the Three Isomers of C70 Prato Monoadducts

C70 was subjected to a Prato reaction using 1.3 equivalents of N- glycine derivative 19 and paraformaldehyde under reflux for 1 h to obtain a mixture of C70 Prato monoadducts (Scheme 4).

Scheme 4. Prato reaction of C70 with N-glycine derivative 19 and paraformaldehyde.

O O O O NH O O O O O O O N O O N O O HO O O N O

19 H H + + toluene, 1 h, 120 °C

C5-6 isomer, 16%C1-2 isomer, 13% C7-8 isomer, 2% 20 21 22

Figure 71 shows the HPLC trace of the C70 Prato reaction. The C70 Prato monoadducts were purified in three stages. For the first step of purification, a normal silica gel column chromatography was performed using toluene as eluent. Pristine C70 was eluted first, followed by the C70 Prato monoadduct.

300

250

200

3

x10 C70 150 mono-adduct

100 polyadducts

50

0 0 2 4 6 8 10 12 retention time [min] Figure 71. The crude reaction mixture of the Prato reaction of C70. Column: Buckyprep-M (Ø 4.6 x 250 mm), eluent: toluene, flow rate: 1 ml/min, 390 nm.

After the Prato monoadducts were isolated, they were re-purified by HPLC using a Silica Shiseido column (Ø 4.6 x 250 mm) with toluene as the

72 eluent for the second stage purification. Two peaks, isomer 20 and peak 2 were separated as shown in Fig. 72.

50

40 peak 2

30

3 20 x10 20

10

0 0 10 20 30 40 retention time [min] Figure 72. Second stage purification. The mixture of monoadducts isolated from the first stage purification was re-injected to Silica Shiseido column Ø 4.6 x 250 mm, toluene, 1.0 mL/min. Two peaks, peak 1 and peak 2 were successfully observed.

Finally, peak 2, which still contained a mixture of two C70 monoadducts, was re-injected into the BuckyPrep column for the third stage purification (Fig. 73). Two peaks (21 and 22) were separated. The MALDI mass spectra of three isolated isomers (isomers 20, 21, and 22) confirmed that these are indeed N-pyrrolidine monoadducts of C70. High Resolution Mass Spectrometry (HRMS) results revealed that isomers 20, 21, and 22 had m/z peaks of 1148.1828 [M+Na+], 1148.1831 [M+Na+], and 1148.1828 [M+Na+], respectively.

20

15

21

10 3

x10 22

5

0

0 5 10 15 20 retention time [min] Figure 73. Third stage purification. Peak 2 (in Fig. 57) was re-injected in Buckyprep column Ø 4.6 x 250 mm, toluene/acetonitrile 3:1, 1.0 mL/min. Two separate peaks 21 and 22 were successfully separated.

Isomers 20 and 21 were the major products, with yields of 16% and 13% respectively. On the other hand, isomer 22 was the minor product and was isolated with only 2% yield. The assignment of structures of the three isomers is discussed in the next section.

73 3.3 Characterization by 1H- and 13C-NMR and Vis-NIR

Measurements of the Three Isomers of C70 Prato Monoadducts

The structures of the isomers 20, 21, and 22 were assigned using symmetry considerations and by matching these considerations with their obtained 1H-NMR spectra (Fig. 74). The expected number of carbons for each isomer was also considered in their 13C-NMR spectra. Furthermore, the vis- NIR spectra are characteristic of the addition patterns. The vis-NIR spectra of the three isomers 20, 21, and 22 were obtained and compared with previously reported isomers.124, 234, 236

C5-6 isomer C1-2 isomer C7-8 isomer

Figure 74. Representative illustration of the addition sites of the different monoadduct isomers of C70. Isomer 20: C5 – 6 isomer The 1H-NMR of isomer 20 showed a doublet at 3.53 ppm with an integration of 2 and another doublet overlapped with a multiplet at 3.76 ppm with an integration of 3 (Fig. 75). These doublets correspond to the pyrrolidine protons, which are non-equivalent in this case. Considering the symmetry of a C5 – 6 isomer (Fig. 74), it possesses two equivalent methylenes with two non- equivalent protons. The 1H-NMR spectral pattern of 20 was identified to be that of a C5 – 6 monoadduct isomer, an isomer belonging to the Cs point group.

74 O O H O N O H H H

bis-ester CH2 protons

20
 
 
        
    





 

1 Figure 75. H-NMR of 20 in CDCl3. The peaks labeled with orange and pink dots represent the two non- equivalent protons on the two equivalent methylenes of the pyrrolidine ring.

(a)




































(b)

*

        

  

13 2 3
Figure 76. C-NMR of 20 in CDCl3. (a) the sp region. (b) the sp region. The peak labelled with a red dot is the sp3 carbon from the fullerene cage. Solvent contaminant is marked with an *.

Further evidence that 20 is a C5 – 6 isomer can be found in its 13C- NMR spectrum (Fig. 76). In the aromatic region (126 - 156 ppm) of the 13C- NMR spectrum of compound 20, a total of 28 double intensity sp2 carbon fullerene peaks and four single intensity sp2 peaks and two quadruple intensity sp2 (two overlapping double intensity peaks) are observed. This is in addition to the six sp3 carbon peaks (including 1 sp3 fullerene carbon peak with double intensity at 59 ppm, carbon labeled with a red dot in Fig. 76b)

75 observed at 28 - 82 ppm. These data indicate that the molecule is symmetric and both the 1H-NMR and 13C-NMR spectra of 20 are consistent with that of a

C70 C5-6 monoadduct isomer.

1.2

1.0 20

0.8

0.6

Absorbance 0.4

0.2

0.0 400 500 600 700 800 900 wavelength Figure 77. Absorption spectra of 20. Spectrum was obtained from the PDA detector equipped on the HPLC.

Lastly, the vis-NIR data of 20 was also obtained. For a C5 – 6 isomer, characteristic bands typically occur at 410, 450 nm and 590 nm. Indeed, isomer 20 displayed these exact characteristic bands appearing at 410 nm, 450 nm, and 590 nm (Fig. 77). Again, NMR and vis-NIR data are in agreement, allowing us to conclude that isomer 20 is a C70 C5 – 6 monoadduct isomer.

Isomer 21: C1 – 2 isomer R H H N H H O O R = O O

bis-ester CH2 protons 21
  



         
 



 

1 Figure 78. H-NMR of 21 in CDCl3. The peaks labeled with orange and pink dots represent the two equivalent protons on the two non-equivalent methylenes of the pyrrolidine ring.

76 The 1H-NMR of the isolated isomer 21 showed two distinct singlets (one singlet was at 3.67 ppm and another singlet at 4.01 ppm) in a ratio of 1:1 (Fig. 78). These singlets correspond to the pyrrolidine protons. This pattern corresponds to an addition occurring at the C1 – 2 bond. Two methylenes are non-equivalent, but the two protons on each methylene are equivalent (Fig. 74). The 1H-NMR spectral pattern of 21 was identified to be that of a C1 – 2 monoadduct isomer, with Cs symmetry.

(a)
































 
 
 (b)

 
 
 
 
  *






13 2 3 Figure 79. C-NMR of 21 in CDCl3. (a) the sp region. (b) the sp region. The peak labelled with red dots are the sp3 carbons from the fullerene cage. Inset: Zoomed in version of the two carbons at 63 ppm. Solvent contaminant is marked with an *.

Analysis of the 13C-NMR spectrum of 21 further confirmed that it is a C1 – 2 isomer (Fig. 79). In the aromatic region (130 - 160 ppm) of the 13C- NMR spectrum of compound 21, a total of 35 lines or 33 double intensity sp2 carbon fullerene peaks and 2 single intensity sp2 carbon fullerene peaks were observed. In addition, eight sp3 carbon peaks in the sp3 region (28 - 82 ppm) were also observed (including 2 sp3 fullerene carbon peaks at 61 ppm and 62.97 ppm, carbon labeled with red dots in Fig. 79b).

77 2.0

21 1.5

1.0

Absorbance

0.5

0.0 400 500 600 700 800 900 wavelength Figure 80. Absorption spectra of 21. Spectrum was obtained from the PDA detector equipped on the HPLC.

On the other hand, for previously reported C1 – 2 monoadduct isomer, Vis bands were shown to appear at 400 nm, 470 nm and 540 nm.123 In the same way, isomer 21 exhibited these characteristic bands at 400 nm, 480 nm and 530 nm (Fig. 80), which was in perfect agreement with the previously reported absorption spectra. The obtained NMR and vis-NIR data are fitting to a C70 C1 – 2 monoadduct isomer. Isomer 22: C7 – 8 isomer

The 1H-NMR of the isolated isomer 22 exhibited two doublets of two protons each (2.50 ppm and 4.03 ppm). These doublets correspond to the two pyrrolidine protons. The two methylenes are equivalent, but the two protons of 1 each CH2 group are non-equivalent (Fig. 74). The H-NMR spectral pattern of 22 was identified to be that of a C7 – 8 monoadduct isomer, belonging to the

Cs point group.

78 O

O O H N O bis-ester CH2 protons H H H

22
  

          
 





 

1 Figure 81. H-NMR of 22 in CDCl3. The peaks labeled with orange and pink dots represent the two non- equivalent protons on the two equivalent methylenes of the pyrrolidine ring.


















(b) *






13 2 3 Figure 82. C-NMR of 22 in CDCl3. (a) the sp region. (b) the sp region. The peak labelled with a red dot is the sp3 carbon from the fullerene cage. Solvent contaminant is marked with an *.

Further evidence that 22 is a C7 – 8 isomer can be found in its 13C- NMR spectrum (Fig. 82). In the aromatic region (126 - 156 ppm) of the 13C- NMR spectrum of compound 22, a total of 35 carbon peaks were counted. This is almost exactly equal to the 36 carbon peaks expected of a C7-8 isomer. The 35 sp2 peaks included three single intensity peaks were observed at 127.21, 154.15 and 154.98 ppm. It is possible that the fourth single carbon

79 peak might have been overlapped with a larger intensity carbon peak around the sp2 region. In addition, six sp3 carbon peaks (including 1 sp3 fullerene carbon peak with double intensity at 62 ppm, carbon labeled with a red dot in Fig. 82b) observed at 28 - 82 ppm. These data indicate that the molecule is symmetric.

1.2

1.0 22

0.8

0.6

Absorbance 0.4

0.2

0.0 400 500 600 700 800 900 wavelength Figure 83. Absorption spectra of 22. Spectrum was obtained from the PDA detector equipped on the HPLC.

The vis-NIR spectrum of 22 showed bands at 400, 510, and 620 nm

(Fig. 83). These bands are consistent with previously reported C7 – 8 C70 monoadduct isomer,123 which had bands at 400, 510, and 630 nm. The 1H- NMR, 13C-NMR and vis-NIR spectra of 22 are consistent with a structure that is a C7 – 8 monoadduct isomer. Taking the NMR and vis-NIR data altogether into consideration, the three isomers 20, 21, and 22 were characterized to be the C1 – 2, C5 – 6, and C7 – 8 adducts, respectively.

Since C70 was successfully functionalized with the bisester functionality, we were curious to see if it can be useful for further biological applications by making it water-soluble. As a model compound, we conjugated compound 20 with a water-soluble polymer.

3.4 Conjugation of C70 with a Monodispersed Polyethylene Glycol Derivative

Since C70 has similar photophysical properties as C60, it can also be potentially used for medicinal applications. However, similar to C60, C70 also

80 suffers from poor solubility in polar solvents. Therefore, it is interesting to make them water-soluble. A biocompatible, monodispersed polymer PEG was conjugated to C70 to make it water-soluble (Scheme 5).

Scheme 5. Preparation of water-soluble C70 derivative 24.

O

O O HO OH t Bu NH2 O O O N O O N O 20 18 TFA HBTU/ DIPEA DCM distilled DMF >99% overnight, RT

5, 6-isomer 20 23

O O H tBu N NH tBu O O O O 20 O N O 20

24

As a model substrate, the isomer 20 was made water-soluble via PEGylation. The tBu group of 20 was removed using TFA to yield the corresponding bis-acid derivative 23 in quantitative yield. Subsequently, bis- acid 23 was subjected to a coupling reaction in the presence of HBTU, DIPEA and a monodispersed PEG-amine to provide C70 monoadduct 24 with a yield of 20%. Compound 24 proved to be water-soluble at 3 mM. Since 24 proved to be water-soluble, it could be interesting for a future study to investigate its reactive oxygen species generation, in order to evaluate it for its potential photodynamic therapy applications. Additional experiments are now ongoing.

3.5 Conclusions

Since C70 is non-toxic, has long wavelength of absorption, and can generate reactive oxygen species in high yield, it can be a potential candidate as a photodynamic therapy drug. Therefore, C70 was successfully

81 functionalized via Prato reaction to provide three bisester monoadduct derivatives 20, 21, and 22. The three isomers were isolated and characterized. Isomer 21 corresponds to an addition that occurred at bond C1 – 2. On the other hand, the structure of isomer 20 was found to match that of an addition that occurred at bond C5 – 6. The 1H-NMR pattern of isomer 22 was shown to agree with that of an addition at bond C7 – 8. Water-solubility is an issue with fullerene compounds. Compound 20 was functionalized with PEG to form water-soluble C70 Prato monoadduct 24 (water-soluble at 3 mM). This material can be useful for medicinal applications. The well-defined, water-soluble C70 derivative 24 could potentially be used as a photodynamic therapy drug upon further conjugation with a cancer-targeting ligand, e.g. folic acid. It is suggested to do ESR investigations on compound 24. Further studies are now underway.

82

CHAPTER 4

1,3-DIPOLAR CYCLOADDITION OF

Y3N@C80 AND GD3N@C80 AND

FORMATION OF TRISADDUCTS

The work described in this chapter was carried out based on the previous results of Dr. Aroua on the Prato mono and bisadducts of M3N@C80 (M = Sc, Lu, Y, Gd). He provided a direct guidance on the synthesis, isolation, and structural characterization of the trisadducts in this chapter. Mr. Arnold from the NMR service of the LOC at ETH helped in high resolution NMR analysis of the trisadducts. Dr. Garcia-Borràs and Dr. Osuna from the University of Girona performed the computational calculations. Dr. Trapp and Mr. Solar from SMoCC at ETH provided the X-ray crystallography data.

83

84 4.1 Background

Since the first report on the endohedral metallofullerenes (EMFs) in 1985,237 they have been a topic of interest for many researchers. EMFs can be future materials for nanoscale devices because of their relatively small band gaps, which are dependent on the size of the fullerene cage and the kind of encapsulated metal atoms. Among them, Trimetallic Nitride Template Endohedral Metallofullerenes (TNT-EMFs) are a very interesting class of compounds due to their potential in the application for organic photovoltaic 214 devices and MRI-contrast agents, in particular, Gd3N@C80. TNT-EMFs are a class of fullerenes consisting of a trimetallic nitride cluster encapsulated inside a host fullerene cage (M3N@C2n, n = 34-55, M = Sc, Y, or lanthanides).22, 103 In order to maximize the use of these materials, it is important to develop the controlled chemical functionalizations of TNT-EMFs. So far, a variety of chemical reactions of TNT-EMFs were reported (Chapter 1.5). Especially, the regioselectivity of the functionalization reactions of these fullerenes have been intensively studied.167, 174-176, 179, 238 Previously Dr. Aroua reported the use of Prato reaction to functionalize

M3N@C80 (M = Gd, Y, Sc, Lu) and yielded their corresponding fulleropyrrolidine derivatives (Scheme 6).

Scheme 6. Previously reported mono and bisadducts.176, 184

O O

O N O

O O O

+ O HN O H H o-DCB HO O 120 °C

19 M = Gd, Y, Sc, Lu mono-adduct

N

O

HN H H + o-DCB HO O 120 °C N M = Gd, Y bis-adduct

85 The regio-ratios of the Prato products ([6,6] or [5,6]) were dependent 176, 177 on the size of the metal cluster inside the C80 carbon cage. Prato bis- additions on M3N@C80 (M = Gd, Y) were also carried out and surprisingly yielded highly regioselective products.183 This is in contrast to bis-additions on empty-cage fullerenes, which requires the use of tether templates to give 95, 166, 239, 240 regioselective additions. The bisadduct of Y3N@C80 was isolated by HPLC and assigned to be a single isomer with [6,6],[6,6] bis-addition due to the effect of the metal which directed the second addition.184 However, the higher additions (trisadducts and polyadducts) on TNT-EMFs remain unexplored. In this study, the synthesis of the trisadducts of M3N@C80 (M = Y, Gd) was performed in a quest to further understand the reactivity and regioselectivity of these compounds. The synthesis, purification, and characterization of Prato trisadducts of M3N@C80 (M = Gd and Y) are discussed in this section. Density Functional Theory (DFT) calculations were also employed by our collaborator to discuss the possible structures of the Prato trisadduct.

4.2 Synthesis and Isolation of the N-Ethyl Fulleropyrrolidine

Trisadducts of M3N@C80 (M = Gd, Y)

Prato reactions of M3N@C80 (M = Gd, Y) were carried out in the presence of N-ethylglycine and formaldehyde in o-dichlorobenzene (o-DCB) at 120 °C for 15 minutes (Scheme 7). Previously, N-ethyl fulleropyrrolidine mono and bisadducts of M3N@C80 (M = Gd, Y) were synthesized in the same way using 8.5 equiv of N-ethylglycine by Dr. Aroua. It was expected that using a larger excess of N-ethylglycine will yield the higher adducts. By screening a series of conditions, it was found that using 50 equiv of N-ethylglycine and excess amount of formaldehyde provided the optimum amount of trisadducts as shown in Fig. 84. In fact, based on these HPLC traces, increasing the amount of N-ethylglycine to 100 or 200 equiv provided less of the desired trisadduct peak. Likewise, reducing the amount of N-ethylglycine to 20 equiv resulted to the formation of more of the lower adducts (bis and monoadducts).

86

1.0

0.8 50 equiv. N-ethyl glycine

0.66

x10

0.4 100 equiv. N-ethyl glycine

0.2

200 equiv. N-ethyl glycine 0.0 0 5 10 15 time [min]

Figure 84. Second stage purification. HPLC traces of the reaction mixtures of the reaction of Y3N@C80 with 50 equiv, 100 equiv, and 200 equiv of N-ethylglycine at 120 °C for 15 mins. Trisadduct peaks is enclosed in boxes. The highest amount of trisadduct peak can be obtained when 50 equiv. was used. Conditions: Buckyprep, Ø 10 x 250 mm toluene-acetonitrile 2:1, 3 ml/ min, 390 nm.

The highest amount of trisadduct was obtained when 50 equiv of N- ethylglycine and 400 equiv of formaldehyde was used and the reaction time was kept at 15 min at 120 °C.

Scheme 7. Reaction scheme of the Prato reaction on M3N@C80 (M = Gd, Y) to yield trisadducts.

HN 50 equiv. N O OH 3 O

400 equiv. H H

o-DCB, 120 °C, 15 mins

M = Gd, Y

87 The crude reaction mixture was first subjected to HPLC separation using Buckyprep as column and toluene as an eluent. A mixture of polyadducts was observed as shown in Fig. 85a. A mixture of polyadducts was collected and re-injected for the second stage HPLC purification using the same column (Buckyprep) and a mixture of toluene-acetonitrile 2:1 as the eluent. The mixture of polyadducts revealed a major peak at a retention time of about 13 minutes (Fig. 85b). This peak was collected and further subjected to the third stage HPLC purification, using PBB as a column and toluene with 1% pyridine as an eluent. Two peaks were observed, a minor peak A and a major peak B (the combined yield of peak A and peak B for Gd3N@C80 was 11%, Fig. 85). Interestingly, both peak A and peak B (in the case of

Gd3N@C80) showed the m/z corresponding to trisadducts by ESI-MS (Chapter 6, Figs. 160 and 161). This suggested that at least two N-ethyl fulleropyrrolidine trisadduct isomers of Gd3N@C80 were obtained under the conditions used.

(a) Buckyprep, toluene 10

5

3 0 x10

-5

-10 0 10 20 30 40

80 60(b) Buckyprep, toluene:ACN (2:1) 40

3 20 x10 0

-20

0 5 10 15 20

80 Peak B 60

3 (c) PBB, toluene - pyridine (99:1) 40 x10 Peak A 20

0 0 5 10 15 20 retention time [min] Figure 85. HPLC traces of the first, second, and third stage purification of the crude reaction mixture of Gd3N@C80. Collected fractions are the peaks enclosed in a box. (a) Buckyprep, Ø 4.6 x 250 mm, toluene, 1 ml/min, 390 nm. (b) Buckyprep, Ø 4.6 x 250 mm, toluene-acetonitrile 2:1, 1 ml/min, 390 nm. (c) PBB, Ø 4.6 x 250 mm toluene-pyridine 99:1, 1 ml/min, 390 nm.

88 Due to the paramagnetic character of Gd3+, the 1H- and 13C-NMR study of Gd3N@C80 trisadducts was difficult and the exact structures and number of isomers could not be determined. In order to try to elucidate the structure of these trisadducts, the same reaction was carried out using Y3N@C80.

Reaction performed under the same conditions for Y3N@C80 yielded similar results. The crude reaction mixture was also purified by three stages

(the combined yield of peak C and peak D for Y3N@C80 was 15%, Fig. 86). However, the obtained peak D contained a clear shoulder (Fig. 86c). This suggested that more than two trisadduct isomers were obtained in the case of

Y3N@C80. Both isolated peaks C and D from Y3N@C80 also displayed the same m/z for the trisadduct of Y3N@C80 (Chapter 6, Figs. 162 and 163).

(a) Buckyprep, toluene 5

0 3 x10 -5

-10

0 5 10 15 20 25 30

50

40 (b) buckyprep, toluene - ACN (2:1)

3 30

x10 20

10

0 0 5 10 15 20

50

40

30 Peak D

3 (c) PBB, toluene - pyridine (99:1) Peak C x10 20

10

0 0 5 10 15 20 retention time [min]

Figure 86. HPLC traces of the first, second, and third stage purification of the crude reaction mixture of Y3N@C80. Collected fractions are the peaks enclosed in a box. (a) Buckyprep, Ø 4.6 x 250 mm, toluene, 1 ml/min, 390 nm. (b) Buckyprep, Ø 4.6 x 250 mm, toluene-acetonitrile 2:1, 1 ml/min, 390 nm. (c) PBB, Ø 4.6 x 250 mm toluene-pyridine 99:1, 1 ml/min, 390 nm.

Attempts to separate the isomers contained in peak D were performed. However, even after trials using various HPLC columns (PBB, Buckyprep-M, Buckyprep, Buckyprep-D, Buckyclutcher, PYE, silica) and several solvent systems (toluene-acetonitrile (99:1), toluene-pyridine (99:1), toluene-

89 acetonitrile (2:1), toluene-acetonitrile (9:1), toluene-methanol (99:1), toluene- THF (1:1), toluene-ethyl acetate (1:1)), no good condition was found for the separation of peak D.

4.3 Isomerization Study of the Trisadducts of Y3N@C80 and

Gd3N@C80

184 Similar to the Prato bisadduct of Y3N@C80, trisadducts of M3N@C80 (M = Gd, Y) peaks A and C isomerized to peaks B and D, respectively and vice versa at room temperature. Isomerization studies were conducted as shown below.

4.3.1. Isomerization of the Trisadducts of Gd3N@C80
Peaks A and B were separated by HPLC. Each peak was collected in separate vials and was left in toluene-pyridine (99:1) at room temperature. Aliquots (10 μL) were taken after 0, 24, 72, 144, 216, 360 hours and analyzed by HPLC (Buckyprep, toluene) to observe the isomerization. In both cases (peak A and peak B), the other isomer started to be detectable after 24 h at room temperature. Since isomerization occurred easily at room temperature, the activation barriers among these isomers were expectedly low. Isomerization continued until peak A and peak B reached equilibrium (Fig. 87).

Figure 87. Isomerization of the trisadduct isomers of Gd3N@C80. Conditions used: PBB, Ø 4.6 x 250 mm toluene-pyridine 99:1, 1 ml/min, 390 nm. Left: peak B to peak A. Right: peak A to peak B.

90 4.3.2. Isomerization of the Trisadducts of Y3N@C80
In a similar manner, peaks C and D were collected in separate vials and left in toluene-pyridine (99:1) at room temperature. Aliquots (10 μL) were taken after 0, 1, 3, 5, 7, 24, 48 hours and analyzed by HPLC (Buckyprep, toluene) to observe the isomerization (Fig. 88). Interestingly, the appearance of the other isomer was relatively quicker. Just one hour after the separation, the other isomer already appeared. The isomerization was completed within 24 h. Since isomerization occurred easily at room temperature, the activation barriers among these isomers were expectedly low. Also, isomerization between peak C and peak D for Y3N@C80 was faster compared to Gd3N@C80. This phenomenon was also observed for a [6,6],[6,6] bisadduct wherein upon heating, the formed Y3N@C80 bisadduct isomerized whereas the Gd3N@C80 bisadduct remained intact. This difference in isomerization rate can possibly be related to the fact that the size of Gd metal is larger than the size of the Y metal. We can suppose that the larger metal cluster presumably stabilizes the trisadduct.184

Figure 88. Isomerization of the trisadduct isomers of Y3N@C80. Conditions used: PBB, Ø 4.6 x 250 mm toluene-pyridine 99:1, 1 ml/min, 390 nm. Left: peak D to peak C. Right: peak C to peak D.

Noticeably, peak C of Y3N@C80 trisadduct is directly isomerizing to the mixture of isomers in peak D with the same ratio that was obtained right after the reaction. This suggests that the isomers in peak D are near in energy.

91 4.4 Characterization of Trisadducts

One of the most useful characterization methods of the higher adducts of M3N@C80 (M = Gd, Y) is by comparison of their absorption traces, especially since Gd3+ is paramagnetic. The vis-NIR spectra of peaks A and C of M3N@C80 (M = Gd, Y) are shown in Fig. 89. As a standard, previously reported Prato [6,6] monoadduct and [6,6],[6,6] bisadducts of M3N@C80 (M = Gd, Y) are also shown.128, 176, 184

0.35a. b. 0.5 Y N@C 0.30 Gd3N@C80 3 80

0.4 0.25 tris (peak A) tris (peak C)

0.20 [6,6][6,6]-bis 0.3 [6,6][6,6]-bis 0.15 Absorbance Absorbance 0.2 0.10 0.1 0.05 [6,6]-mono [6,6]-mono

600 650 700 750 800 850 900 600 650 700 750 800 850 900 wavelength [nm] wavelength [nm]

c.

[5,6] mono-adduct of Y3N@C80

[5,6] mono-adduct of Gd3N@C80 Absorbance

wavelength [nm] Figure 89. (a) Vis-NIR spectra of Gd3N@C80 trisadduct peak A compared with [6,6] monoadduct and [6,6],[6,6] bisadducts of Gd3N@C80. (b) Vis-NIR spectra of Y3N@C80 trisadduct peak C compared with [6,6] monoadduct and [6,6],[6,6] bisadducts of Gd3N@C80. Most prominent bands are at 650 and 800 nm. (c) Vis-NIR spectra of [5,6] monoadducts of Gd3N@C80 and Y3N@C80. Most prominent band is at 850 nm. The spectra were obtained using an HPLC with a PDA detector.

The spectral pattern of the [6,6] monoadducts and [6,6],[6,6] bisadducts (for both Gd3N@C80 and Y3N@C80) were compared with the spectral patterns obtained for trisadduct peaks A and C (Fig. 89a and b). As shown in the figure above, the bands around 650 nm and 800 nm are the most prominent ones for [6,6] mono and [6,6],[6,6] bisadducts with both metals. On the other hand, the previously isolated and characterized Gd and

92 Y [5,6] monoadducts showed only one band at 850 nm (Fig. 89c).176 By comparison of the absorption spectra, the peak A trisadduct of Gd3N@C80 showed two maxima at 660 and 780 nm. Likewise, the absorption spectra of the peak C trisadducts of Y3N@C80 gave a similar pattern, with 660 and 780 nm bands as their maxima. The general appearance of these absorption spectra matched the absorption spectra obtained for the previously assigned [6,6] mono and [6,6],[6,6] bisadducts. Based on this pattern, it is suggested that the obtained peak A and peak C are [6,6],[6,6],[6,6] trisadducts.

Further structure elucidation of the trisadducts of Y3N@C80 was carried out using 1H- and 13C-NMR spectroscopy. Due to the small amounts of trisadducts obtained, NMR analysis proved to be tedious. NMR measurements had to be done as quickly as possible and low temperature was employed in carrying out extensive NMR analysis in order to limit the thermal isomerization. In order to record 1H-NMR, 13C-NMR, COSY, and HSQC spectra of peak C, it was separated from peak D and was quickly stored at -20 °C to prevent thermal isomerization. The NMR spectrum was recorded at 4 °C as shown in Fig. 90.

H1B N H2A H1A H2B

H6A H6B H3A N H3B 5B H5A H H4B N H4A

methylene protons

1 Figure 90. H-NMR of peak C measured at 4 °C, CDCl3.

93 Three quartets integrating for 2 protons were observed around 2.8 - 3.1 ppm. These 3 signals correspond to the methylene groups of the ethyl moiety. Moreover, six pairs of methylene protons from the pyrrolidine moieties (twelve doublets, H1-4A to H1-4B) were observed. This result suggested that the isolated 13 peak C was a single C1-symmetric trisadduct isomer. The C-NMR spectrum also supported this result as shown below.

H1A N H2B H1B H2A

H 6A Y N 3 H3B H6B H3A N N H H5A 5B H H4A 4B III II I

13 Figure 91. C-NMR of peak C measured at 4 °C, CDCl3. Based on 13C-NMR and HSQC measurements, it was shown that carbons at 69, 70, and 72 ppm (labeled with green dots) each correlate with two protons in the region of 3.6 to 4.4 ppm. Likewise, a second set of three carbons situated between 64 and 66 ppm (labeled with green dots) each correlates with 2 protons (Figs. 91 and 92). These six carbon peaks labeled with green dots were assigned to the CH2 carbons of the pyrrolidine moieties. On the other hand, the three carbons between 66 and 69 ppm (set I, carbons labeled with red dots) did not show correlation with any proton in HSQC, meaning that these carbons are quaternary and they have no protons attached to them. These carbon peaks were labeled with red dots and assigned as the sp3 carbons on the carbon cage (Fig. 91). The other set of sp3 quaternary carbons was also identified between 53 and 57 ppm (set II, carbons labeled with red dots) and assigned to the three remaining sp3 carbons of the fullerene cage. Interestingly, these two sets (sets I and II) of

94 peaks were divided into two, with three carbons more downfield shifted at around 66 - 69 ppm (set I) and 53 - 57 ppm (set II). This peak pattern was also observed for the previously characterized [6,6] monoadduct.176 These upfield, sp3 carbons (set II) correspond to the carbons located in a pentagon-hexagon- hexagon junction and the downfield shifted ones (set I) generally correspond to the carbons at a triple-hexagon junction. In total, six carbons correlating with two protons were assigned to the methylenes of the pyrrolidine moieties. Six carbons with no HSQC correlation were observed and attributed to the sp3 carbon of the cage. This pattern can be expected of a trisadduct structure with C1-symmetry.

Figure 92. HSQC of peak C measured at 4 °C, CDCl3. The peaks labeled with green dots are the carbons which are correlated with the pyrrolidine protons. The peaks labeled with red dots are the quaternary carbons which are found at the addition sites.

There are also exactly six carbon peaks (set III) between 100 and 125 ppm (carbons labeled with brown dots, Figs. 91 and 93). These signals correspond to the carbons adjacent to the sp3 carbons on the addition sites. By analogy with the previously reported N-ethyl [6,6],[6,6] bisadduct results,184 the other set of six carbon peaks is presumably downfield-shifted to the region between 130 and 145 ppm.

95 74 peaks of the C80 cage III





















13 2 Figure 93. C-NMR of peak C in the sp region measured at 4 °C, CDCl3.

Further information on the structure of peak C can be found in the aromatic region of the 13C-NMR spectrum of peak C (Fig. 93). If the structure of peak C is an unsymmetric [6,6],[6,6],[6,6] trisadduct, then the total number of carbons in the sp2 region of its 13C-NMR spectrum should be a total of 74 peaks. Peak C exhibited exactly 74 carbon peaks in its sp2 region (including five double intensity peaks), in support of the conclusion that it is an unsymmetric trisadduct. All these data (HPLC, MS, vis-NIR and NMR) suggested that peak C was isolated as a single trisadduct isomer. The appearance of UV-Vis bands at 660 and 780, along with the conclusions obtained from 1H-, 13C-NMR

COSY, and HSQC, allowed us to conclude that peak C is most likely a C1- symmetric [6,6],[6,6],[6,6] trisadduct. The pattern observed by 1H- and 13C- NMR, with distinct peaks for all pyrrolidine methylenes, sp3 carbons at the addition sites, and sp2 carbons of the fullerene cage, also demonstrated that the isolated [6,6],[6,6],[6,6] trisadduct of peak C is unsymmetrical.

The HPLC traces for the trisadducts of M3N@C80 (M = Gd, Y) were very similar, with peak A at a lesser amount than peak B and peak C at a lesser amount than peak D. Also, based on the vis-NIR analysis of peak A, two peaks at 660 and 780 nm were also observed, as in the case of peak C.

Since the metal sizes of M3N@C80 (M = Gd, Y) are similar (ionic radius of Gd3+ is 0.94 Å, ionic radius of Y3+ is 0.90 Å), their reactivities could be compared. Considering that the HPLC and vis-NIR data obtained for peaks A

96 and C were similar, it can also be understood that the trisadduct peak A of

Gd3N@C80 is a [6,6],[6,6],[6,6] trisadduct. Finally, NMR data (1H and 13C) for peak D were also obtained (Chapter 6, Figs. 168 and 169), clearly showing that peak D consists of a mixture of isomers, agreeing with the obtained HPLC trace shown above (Fig. 86c). The analysis of these NMR spectra did not allow us to conclude on the structure of these regioisomers due to complex NMR spectra resulting from overlapping signals.

4.5 Possible Structure Elucidation of Trisadducts by Computational Chemistry

The spectroscopic data suggested that peak C was an unsymmetric [6,6],[6,6],[6,6] trisadduct. However, there was not enough information to determine the exact tris-addition site. DFT at the BP86-D2/TZP//BP86- D2/DZP level using Amsterdam Density Functional (ADF) program was employed in order to estimate the thermodynamic stabilities of different trisadducts of M3N@C80 (M = Gd, Y). These predictions revealed that the most stable tris-addition sites also present high pyramidalization angles in C80 cage.

For both M3N@C80 (M = Gd, Y), three possible bisadducts were considered as starting points for the computational analysis based on previous published work.184 Based on the calculated pyramidalization angles for the carbon atoms in the bisadducts, different trisadducts were optimized. Several possible trisadducts could form. In the following figures, the numbers in orange and purple (Figs. 94 and 96) represent the relative stabilities of the

Gd3N@C80 trisadducts. The numbers in orange represent the trisadducts with the lowest relative energies (in kcal/mol).

4.5.1 Theoretical Considerations for Gd3N@C80
A first possibility is that the first and second addition occurred at [6,6] bonds. The bonds 79 – 80 and 42 – 43 are those where the first and second

97 additions occurred. In this case, seven tris-addition sites were considered (Fig. 94a).

a. b.

c.

Figure 94. Theoretical considerations for the tris-addition sites in Gd3N@C80. (a). First theoretical consideration of tris-addition site: mono-addition and bis-addition both occurred at [6,6]-junctions. The bonds 79 – 80 and 42 – 43 are where the first and second additions occurred. (b) Second theoretical consideration of tris-addition site: mono-addition occurred at a [6,6]-junction and bis-addition occurred at a [5,6]-junction. The bonds 79 – 80 and 1 – 5 are where the first and second additions occurred. (c) Third theoretical consideration of tris-addition site: mono-addition and bis-addition both occurred at [6,6]- junctions. The bonds 79 – 80 and 7 – 8 are where the first and second additions occurred. The numbers in orange and purple represent the relative stabilities of the different trisadducts (in kcal/mol).

For the tris-addition, the most pyramidalized carbons are 5, 7, and 59 with pyramidalization angles of 13.42°, 11.74°, and 12.14°, respectively. Expectedly, the lowest energy addition sites are also localized in the same region. There are three possible tris-addition sites, i.e. bonds 1 – 5 (0.8 kcal/mol), 5 – 59 (0.9 kcal/mol) and 5 – 7 (1.0 kcal/mol). Bond 5 – 7 is a [6,6]- junction, whereas both bonds 1 – 5 and 5 – 59 are [5,6]-junctions. Since their

98 energies are relatively close with less than 1 kcal/mol difference, it is highly possible that isomerization can occur as previously found for bisadducts.184 The second possible scenario is when the first addition occurred at a [6,6] bond and second addition occurred at a [5,6] bond (Fig. 94b). The bonds 79 – 80 and 1 – 5 are where the first and second additions occurred based on previously reported literature.184 In this case, eleven possible tris-addition sites were considered. The most pyramidalized carbons are 43 and 47 with pyramidalization angles of 13.35° and 13.26°, respectively, and this is exactly where the lowest energy trisadducts are found. Four lowest energy trisadducts were considered, namely: 43 – 37 (0.2 kcal/mol, [5,6] junction), 47 – 35 (0.3 kcal/mol, [5,6] junction), 43 – 47 (1.0 kcal/mol, [5,6] junction) and 42 – 43 (1.0 kcal/mol, [6,6] junction). The energies are relatively close to each other, as in the first case, therefore, isomerization can be expected when the activation barriers are low enough. This second possibility however, is unlikely to be the case, because the second addition site was previously shown to be regioselectively occurring to a [6,6] bond.184 A third possibility is that first and second addition both occurred at a [6,6] bond (Fig. 94c). The bonds 79 – 80 and 7 – 8 are where the first and second additions occurred based on previously reported literature.184 In this case, eleven possible tris-addition sites were again considered. The most pyramidalized carbons are 43 and 47 with pyramidalization angles of 13.62° and 13.17°, respectively. The lowest energy trisadducts are as follows: bond 43 – 37 (0.0 kcal/mol, [5,6]-junction), 42 – 43 (1.1 kcal/mol, [6,6]-junction), 43 – 47 (1.5 kcal/mol, [5,6]-junction), and 47 – 35 (1.7 kcal/mol, [5,6]-junction). Energies are relatively similar, as in the first two cases, therefore, isomerization is very much possible when the activation barriers are low enough. Fig. 95 shows the optimized structure for the lowest energy trisadduct 0.0 kcal/mol (addition at a [5,6]-junction). Combining these results with the experimental data, it is highly likely that peak A is a [6,6],[6,6],[6,6] trisadduct, isomerizing to peak B.

99

Figure 95. Optimized structure of lowest energy trisadduct of Gd3N@C80. Metal positions are pointing towards the addition sites.

4.5.2 Theoretical Considerations for Y3N@C80
First possibility is that the first addition occurred at a [6,6] bond and second addition occurred at a [5,6] bond. The bonds 79 – 80 and 1 – 5 are where the first and second additions occurred based on previously reported literature.184 In this case, twelve tris-addition sites were considered (Fig. 96). For the tris-addition, the most pyramidalized carbons are 20, 28, 43, and 47 with pyramidalization angles of 11.02°, 11.47°, 13.33°, 13.23°, respectively. The lowest energy trisadduct at bond 24 – 28 (0.0 kcal/mol, a [5,6]-junction) has the pyrrolidine moiety opposite from the location of the metal, in contrast to all the other calculated low energy trisadducts. Other lower energy trisadducts are localized around bond 43 – 47 (ca. 3.0 kcal/ mol) (Fig. 96a and 97). The second possible case is that first and second addition both occurred at a [6,6] bond (Fig. 96b). The bonds 79 – 80 and 7 – 8 are where the first and second additions occurred based on previously reported literature.184 In this case, eleven possible tris-addition sites were considered. The most pyramidalized carbons are 37 and 43 with pyramidalization angles

100 of 13.26° and 13.04°, respectively, and this is exactly where the lowest energy addition sites are found. The three lowest energy trisadducts were identified, namely: 43 – 37 (3.3 kcal/mol, [5,6] junction), 43 – 47 (4.5 kcal/mol, [5,6]- junction), 42 – 43 (4.4 kcal/mol, [6,6] junction). The energies again differ by only about 1.0 kcal/mol, therefore isomerization can be expected when the activation barriers among the trisadducts are low enough.

a. b.

c.

Figure 96. Theoretical considerations for the tris-addition sites in Y3N@C80. (a) First theoretical consideration of tris-addition site: mono-addition occurred at a [6,6]-junction and bis-addition occurred at a [5,6]-junction. The bonds 79 – 80 and 1 – 5 are where the first and second additions occurred. (b) Second theoretical consideration of tris-addition site: mono-addition and bis-addition both occurred at [6,6]-junctions. The bonds 79 – 80 and 7 – 8 are where the first and second additions occurred. (c) Third theoretical consideration of tris-addition site: mono-addition and bis-addition both occurred at [6,6]- junctions. The bonds 79 – 80 and 42 – 43 are where the first and second additions occurred. The numbers in orange and purple represent the relative stabilities of the different trisadducts (in kcal/mol).

101

Figure 97. Optimized structure of lowest energy trisadduct of Y3N@C80.

Finally, the last considered possibility is that the first and second additions take place at [6,6],[6,6]-based bonds (Fig. 96c). The bonds 79 – 80 and 42 – 43 are where the first and second additions occurred based on previously reported literature.184 In this case, seven possible tris-addition sites were considered. The most pyramidalized carbons are 5 and 59, with pyramidalization angles of 13.22° and 12.09°, respectively. The lowest energy trisadducts are as follows: bond 1 – 5 (3.5 kcal/mol, [5,6]-junction), 5 – 59 (4.0 kcal/mol, [6,6]-junction), 5 – 7 (4.5 kcal/mol, [6,6]-junction). Energies are similar, therefore, isomerization is very much possible when the activation barriers among the trisadducts are low enough. In summary, it can be seen that the trisadducts with the lowest energies for both Gd and Y correspond to additions in similar locations. Tables 8 and 9 review the theoretical considerations carried out on the tris- addition sites of M3N@C80 (M = Gd, Y).

Table 7. Summary of the theoretical considerations for the possible tris-addition sites of Gd3N@C80: Lowest energy 3rd 1st addition site 2nd addition site addition site 1st consideration 79 – 80 42 – 43 1 – 5 2nd consideration 79 – 80 1 – 5 43 – 47 3rd consideration 79 – 80 7 – 8 43 – 47

102

Table 8. Summary of the theoretical considerations for the possible tris-addition sites of Y3N@C80: Lowest energy 3rd 1st addition site 2nd addition site addition site 1st consideration 79 – 80 1 – 5 24 – 28 2nd consideration 79 – 80 7 – 8 43 – 47 3rd consideration 79 – 80 42 – 43 1 – 5

It can be observed that when 42 – 43 was considered as second addition site, the trisadduct with the lowest energy was localized at bond 1 – 5, which is the location of the most pyramidalized carbons. Conversely, when bonds 7 – 8 and 1 – 5 (both in the same region and nearby each other) were considered as the second addition sites, the lowest energy trisadduct was bond 43 – 47, a bond which is in the same region as bond 42 – 43 (location of the most pyramidalized carbons). This trend was observed for all theoretical considerations in both metals, except for one case. In the first theoretical consideration for the tris-addition site of Y3N@C80, it can be observed that when the second addition site was considered to be bond 1 – 5, the lowest energy trisadduct is when the third addition occurred at bond 24 – 28. These results imply that the reactivity of Gd and Y towards the third addition was comparable. Moreover, these results also highlight the influence of carbon cage pyramidalization on the reactivity of these fullerenes. Finally, these results demonstrate that the lowest energy trisadducts are very close in their relative energies. In these cases, when the activation barriers among the different trisadducts are low, isomerization is likely to occur between the experimentally formed isomers.

4.6 Preliminary X-ray Analyses of N-ethyl Fulleropyrrolidine

Bisadduct of Gd3N@C80

Information regarding the orientation of the metal cluster is considered to be crucial towards understanding the reactivity and isomerization of these molecules. X-ray crystallography is an efficient technique in determining the metal positions in the cage and identifying the addition site on the C80 cage. For example, reports in literature suggest that the position of the Sc metal

103 cluster in the monoadducts of Sc3N@C80 orientates in such a way wherein the Sc atoms point away from the site of the first addition (Fig. 98).141, 180 Furthermore, an X-ray single crystal structure will be helpful for DFT calculations, since this will provide a more accurate starting point for the computations. X-ray data can give an exact basis of the first and second addition sites and will give a better prediction on the sites of higher addition.

Addition Site

Figure 98. Metals pointing away from addition site.

As a model compound, we used the previously reported N-ethyl

[6,6],[6,6] bisadduct of Gd3N@C80, since the trisadducts isomerized even at room temperature. The preparation of the bisadduct is shown below in

Scheme 8. The [6,6],[6,6] bisadduct of Gd3N@C80 was synthesized and purified according to reported procedures.184

Scheme 8. Synthesis of N-ethyl bisadduct of Gd3N@C80.

N

8.5 equiv. N-ethyl glycine 45 equiv. paraformaldehyde o-DCB, reflux, 1 h

N M = Gd [6,6][6,6] bis-adduct yield: 10%

Crystallization of the bisadduct was achieved using CS2-hexane 1:1 at room temperature and yielded a crystal which diffracted well. However, it was highly disordered and no direct conclusions could be obtained from the measurement. The disorder could arise from the possible movement of the metal cluster inside the cage. More conditions are now being tried to obtain a single crystal that is less disordered.

104 4.7 Conclusions

Since the discovery of metallofullerenes, a lot of interest was focused on their synthesis and functionalization. In this study, trisadducts of M3N@C80 (M = Gd, Y) were synthesized, isolated and characterized by MS, NMR and vis-NIR. Under the optimized Prato reaction conditions using 50 equiv of N-ethyl glycine and 400 equiv of paraformaldehyde at 120 °C for 15 mins, at least three trisadduct isomers of Y3N@C80 were formed with a combined yield of

15%. In the case of Gd3N@C80, at least two trisadduct isomers were generated, with a combined yield of 11%. In order to isolate these compounds, a three-stage purification was performed using two different types of HPLC columns. NMR and vis-NIR analysis suggested that the peaks A and C were [6,6],[6,6],[6,6] trisadducts. Results from the computational calculations show that the lowest energy trisadduct isomers of M3N@C80 (M = Gd, Y) were close in their relative energies and isomerization was likely to occur between these isomers. This isomerization was experimentally observed since in case of Gd3N@C80 and Y3N@C80, isolated trisadducts peak A isomerized to peak B (peak A to B and peak B to A in 24 h, see Fig. 87, Chapter 4.3.1) and peak C isomerized to peak D at room temperature within a few hours (peak C to D and peak D to C in 1 h, see Fig. 88, Chapter 4.3.2). Isomerization of isolated peak B or D reached equilibrium with peak A or C under the same conditions. Since isomerization occurred easily at room temperature, the activation barriers among these isomers were expectedly low. To get further information on the structures of the trisadducts of

M3N@C80 (M = Gd, Y), crystallization of a bisadduct of Gd3N@C80 was tried and provided a crystal with good diffraction. However, the crystal was highly disordered and did not allow us to obtain an image. In future work, it would be interesting to further functionalize these trisadducts, for example with PEG groups, in order to make them suitable as MRI agents.

105

106

CHAPTER 5

CONCLUSIONS AND OUTLOOK

107

108 5.1 Preparation of Amphiphilic C60 Derivatives and Their Self- Assembly

In a previous study, C60-PEG conjugates were synthesized and were suggested to self-assemble into micelles.216 In this study, charged pyrrolidine moieties were introduced to the hydrophobic C60 cage surface of C60-PEG in order to disrupt the formation of the micelle and C60 derivatives 7, 8 and 9 with respectively zero, one, or two cationic charges were obtained (Fig. 99). These three compounds were characterized by tensiometry, DLS, and STEM in aqueous media. As results, addition of one charged moiety did not have significant changes on the morphology. Compared to the parent molecule 7, compound 8 showed a very similar surface tension curve. Similarly, STEM and DLS indicated that the particle size distributions of 7 and 8 were comparable. However, adding two charged pyrrolidines indeed affected the morphology. As shown by tensiometry experiments, the obtained surface tension curve for 9 was different from those of 7 and 8. Also, STEM and DLS of 9 showed larger particle sizes compared with 7 and 8. With all these data taken into account, it can be concluded that a different type of assembly was formed with 9, possibly related to a vesicle- type morphology. The photophysical properties of these materials will be interesting. For example, it is interesting to test the ability of these materials to generate reactive oxygen species (ROS) upon light irradiation to evaluate their potential as photodynamic therapy agents.

109 O H H O O H H O tBu N N tBu tBu N N tBu O O O O O O O O 20 O N O 20 20 O N O 20

I N 7

8 O H H O tBu N N tBu O O O O 20 O N O 20

N N I I 9

Figure 99.Synthesized C60-PEG amphiphiles 7, 8, 9.

5.2 Synthesis of C70 Monoadduct Isomers and Preparation of

Water-soluble C70 derivative

Due to the high quantum yield in the generation of reactive oxygen species, C60 has been considered to be a very good candidate as a photodynamic therapy agent. C70 exhibits similar photophysical properties to

C60, and therefore is an interesting material for photodynamic therapy. In this study, C70 was functionalized with a bisester 19 via the Prato reaction. Three monoadduct isomers were isolated. The addition sites of two major isomers were C1-2 and C5-6. A third minor isomer C7-8 was also isolated. One of the two major isomers (C5-6) was conjugated with polyethylene glycol unit, in order to make it water-soluble.

Compound 24 was a water-soluble, well-defined C70 derivative (Fig. 100). Study of the ROS generation under photoirradiation will be interesting to evaluate their capability as a photodynamic therapy drug.

110 O O H tBu N NH tBu O O O O 20 O N O 20

24

Figure 100. Synthesized compound 24. A water-soluble, C70-PEG derivative.

Compound 24 can be further conjugated with cancer-targeting ligands, such as folic acid (Fig. 101). Cancer cells sometimes overexpress folate receptors on their surfaces, and folic acid is therefore considered as a good targeting ligand for tumors. 241, 242

O O H   
 
N NH   
 
O O O O n n O N O

25

Figure 101. C70- PEG conjugated to disease-targeting ligands.

5.3 Trisadducts of M3N@C80 (M = Gd, Y): Towards the

Preparation of Water-Soluble Gd3N@C80 Derivatives as MRI- Contrast Agents

In the last few years, production and functionalization of metallofullerenes attracted a lot of attention. Due to their unique property with metals encapsulated in a carbon cage, these molecules are particularly interesting for medical application. In order to produce these materials, development of functionalization methods is of great interest.

In this study, the trisadducts of Gd3N@C80 and Y3N@C80 were synthesized by the Prato reaction and characterized. More than one tris- isomer was isolated for each metal. The trisadduct isomers of Y3N@C80 were

111 isolated and characterized. Peak C isomerized to peak D and peak D isomerized to peak C at room temperature. Peak D contained at least two isomers, which were found to be inseparable. A similar situation was observed for the trisadducts of Gd3N@C80 (peaks A and B). Vis-NIR and NMR data suggested that the minor isomers of the trisadducts of M3N@C80 (M = Gd, Y) were [6,6],[6,6],[6,6] trisadducts. DFT calculations showed that energies of possible trisadduct isomers were very similar (energy differences were ca. 1.0 kcal/mol or less) and were therefore expected to easily isomerize. Since these isomers were experimentally observed to isomerize to each other at room temperature, it could be expected that their activation barriers are low. Gadolinium chelates are commonly used as MRI contrast agents. However, de-chelation of the Gd3+ remains a drawback in using these for medicinal purposes. On the other hand, in the case of Gd3N@C80, the Gd atoms are encapsulated in a carbon cage, making them relatively more stable. 3+ Another advantage of Gd3N@C80 is that it has three Gd ions that can perhaps enhance imaging, in contrast to only one Gd atom per chelate. Therefore, aside from the minimal toxic effects, a higher relaxivity value can potentially be achieved upon using Gd3N@C80 as MRI contrast agent. A major limitation of utilizing fullerenes in medical applications is that they are poorly soluble in most polar solvents. Addition of different functional groups or polymers is a key technique in solubilizing them. However, one potential problem observed was that mono-functionalized fullerene-polymer 216 conjugates (C2n-PEG derivatives) presumably formed micelles in solution. Micelle formation could prevent water molecules from interacting with the Gd atoms, and thereby lower the relaxivity.

112 Water-soluble polymer NH

O N NH O Water-soluble polymer

Water-soluble polymer

HN O N O
O NH N NH O Water-soluble polymer

NH Water-soluble polymer

Water-soluble polymer 26

Figure 102. M3N@C80 trisadduct conjugated with water-soluble polymers.

As shown in Chapter 2, trisadducts of C60 with charged pyrrolidine moieties formed a different type of assembly in solution, possibly due to the repulsion between the charges. Tris-functionalization of Gd3N@C80 with water-soluble polymers in distal positions can potentially increase the water- solubility even more and in turn also increase the relaxivity. Prato reaction on

Gd3N@C80 and the bisester scaffold 19 can provide access to six areas in the molecule where a water-soluble polymer can be attached. Attaching water- soluble polymers all around Gd3N@C80 will promote interaction of Gd metals with water, without having to form micelles (Fig. 102). In summary, fullerenes are molecules with fascinating electronic and chemical properties that can be useful for biomedical and material applications. Primarily, fullerenes are functionalized to improve their solubility. In the process of aiming to make them more water-soluble, their assembly behaviors and interactions in solution are increasingly understood. Additionally, techniques in isolation and identification of the fullerene isomers produced are continually being developed. In order to be able to use these fullerenes to their potential, it is undoubtedly essential to broaden our current understanding on the reactivity of fullerenes.

113

114

CHAPTER 6

EXPERIMENTAL SECTION

115

116 6.1. General

NMR spectra were recorded on Varian 300 spectrometer (Varian Inc., CA, USA), Bruker 400 spectrometer, or Bruker 600 spectrometer equipped with a CryoProbe (Bruker BioSpin GmbH, Rheinstetten, Germany). MALDI-MS spectra were recorded on a Bruker MALDI-TOF (Bruker Daltonics GmbH, Bremen, Germany) and HRMS analyses were by Bruker Ultraflex II LRF200 MALDI-TOF with MALDI-FT ion spec Ultima and Bruker maXis ESI (Bruker Daltonics). ESI-MS were recorded on a Bruker maXis ESI (Bruker Daltonics). FT-IR spectra were recorded on PerkinElmer Spectrum One FT-IR Spectrometer with Universal ATR Sampling Accessory (PerkinElmer Inc., MA, USA). HPLC analyses were carried out by JASCO PU-2080 Plus HPLC pump, JASCO MD-2018 Plus detector, and ChromNAV Chromatography Data System (JASCO Co., Tokyo, Japan) using Buckyprep column (analytical: Ø 4.6 x 250 mm and semi-preparative: Ø 10 x 250 mm), PBB (analytical: Ø 4.6 x 250 mm) from Nacalai Tesque, Kyoto, Japan, BuckyprepM (analytical: Ø 4.6 x 250 mm) from Nacalai Tesque, Silica (SG80 analytical: Ø 4.6 x 250 mm and preparative: Ø 30 x 250 mm from Shiseido, Tokyo, Japan) and C4 column (Phenomenex Ø 10 x 250 mm) from Phenomenex Inc., CA, USA. Column chromatograpy and analytical TLC were performed on SILICYCLE SilicaFlash® F60 (230 – 400 mesh) and silica gel 60 F254 TLC (Merck KGaA, Darmstadt, Germany), respectively. Dynamic light scattering was recorded on Zetasizer Nano S (Malvern Instruments Ltd., Worcestershire, UK). Interfacial tension was recorded on DSA100 Drop Shape Analyzer (Krüss GmbH, Hamburg, Germany). All the solvents used are HPLC grade and were purchased from Acros Organics (Thermo Fischer Scientific Inc., Geel, Belgium). All the reagents were purchased from corresponding suppliers and purified as described when needed. All C60 and C70 fullerenes were purchased

(SES Research, Texas, USA). All M3N@C80 were purchased (Luna Innovations, VA, USA).

117 6.2. Chapter 2

6.2.1. Synthesis

Compounds 12a and 12b

O O tBu tBu O O tBu tBu O N O O N O

N N

12a 12b

To a solution of C60 bisester monoadduct 10 (2.11 g, 2.09 mmol) in toluene (650 mL), sarcosine (317 mg, 3.56 mmol, 1.7 equiv) and paraformaldehyde (189 mg, 6.28 mmol, 3.0 equiv) were added. The reaction mixture was sonicated for about 1.5 h and subsequently refluxed for 3 h. The solvent was removed in vacuo and the obtained crude mixture was purified by silica gel column chromatography. The monoadduct (starting material), trans-1 12a, and trans-3 12b were eluted with toluene, toluene - EtOAc 99 : 1, and 96 : 4, respectively. Additional purification of trans-1 12a was carried out by preparative HPLC (column: SHISEIDO Silica SG80, size: Ø 30 x 250 mm, solvent: toluene - EtOAc (99 : 1), flow rate: 10 mL/min, detection: 390 nm, rt of 12a: 20 min) to provide trans-1 bis-adduct 12a (25.0 mg, 0.024 mmol, 1%); IR –1 (ATR) νmax (cm ): 2963 (w), 2776 (w), 1724 (m), 1471 (w), 1453 (w), 1425 (w), 1390 (w), 1365 (w), 1340 (w), 1319 (w), 1258 (m), 1146 (m), 1087 (s), 1012 (s), 861 (w), 793 (s), 766 (m), 742 (m), 732 (m), 705 (m), 662 (w), 646 1 (w); H-NMR (400 MHz, in CDCl3) δ 1.61 (s, 18H, C(CH3)3), 2.87 (dd, J = 6.5 t Hz, 15.0 Hz, 2H, CH–CH2–CO2 Bu), 3.07 (dd, J = 6.9, 15.0 Hz, 2H, CH–CH2– t CO2 Bu), 3.15 (s, 3H, N–CH3), 4.31 (quint, J = 6.6 Hz, 1H, (CH2)2–CH–N), 13 4.65 (s, 4H, C–CH2–N), 4.77 (s, 4H, C–CH2–N); C-NMR (100 MHz, in t CDCl3) δ 28.7 (C(CH3)3), 38.8 (CH–CH2–CO2 Bu), 42.1 (N–CH3), 54.9

((CH2)2–CH–N), 63.6 (C–CH2–N), 70.5 (C–CH2–N), 81.5 (C(CH3)3), 171.5 t (CH2–CO2 Bu); carbon cage: 68.0 (C–CH2–N), 69.3 (C–CH2–N), 136.8 (4C),

118 137.04 (4C), 141.1 (8C), 142.7 (4C), 144.5 (4C), 145.6 (4C), 145.8 (8C), 146.5 (4C), 146.6 (4C), 148.0 (2C), 148.1 (2C), 153.8 (2C), 153.84 (6C); + - HRMS (MALDI , matrix: DCTB) m/z calcd. for C78H34N2O4 : 1062.2524, found 1062.2524 [M]-. Additional purification of trans-3 12b was carried out by preparative HPLC (column: SHISEIDO Silica SG80, size: Ø 30 x 250 mm, solvent: toluene - EtOAc (99 : 4), flow rate: 10 mL/min, detection: 390 nm, rt of 12b: 38 min), to provide trans-3 bis-adduct 12b (187 mg, 0.176 mmol, 8%); IR –1 (ATR) νmax (cm ): 2972 (w), 2936 (w), 2776 (w), 1722 (s), 1470 (w), 1454 (w), 1425 (w), 1389 (w), 1364 (m), 1339 (m), 1248 (m), 1136 (s), 1117 (s), 1092 (m), 1029 (m), 949 (w), 884 (w), 840 (w), 768 (m), 751 (m), 729 (m), 702 (w), 1 665 (w); H-NMR (400 MHz, CDCl3) δ 1.53 (s, C(CH3)3, 18H), 2.71 (m, CH– t t CH2–CO2 Bu, 2H), 2.94 – 2.85 (m, CH–CH2–CO2 Bu and N–CH3, 5H), 4.04 -

4.17 (m, (CH2)2–CH–N and C–CH2–N, 4H), 4.26 (d, J = 8.8 Hz, C–CH2–N,

1H), 4.31 (d, J = 9.2 Hz, C–CH2–N, 1H), 4.44 – 4.36 (m, C–CH2–N, 2H), 4.51 13 (d, J = 8.8 Hz, C–CH2–N, 1H); C-NMR (100 MHz, CDCl3) δ 28.4 (C(CH3)3), t 38.4 (CH–CH2–CO2 Bu), 41.8 (N–CH3), 54.6 ((CH2)2–CH–N), 62.5 (C–CH2–

N), 63.3 (C–CH2–N), 69.0 (C–CH2–N), 69.3 (C–CH2–N), 81.2 (C(CH3)3), 171.1 t (CH2–CO2 Bu); carbon cage: 69.5 (C–CH2–N), 70.31 (C–CH2–N), 70.33 (C–

CH2–N), 70.6 (C–CH2–N), 135.7, 135.9, 136.6, 136.8, 139.90, 139.94, 141.2, 141.39, 141.42, 141.6, 141.8, 142.0, 142.7, 143.76, 143.79, 144.1, 144.7, 144.8, 145.0, 145.1, 145.26, 145.32, 145.34, 145.36, 145.46, 145.49, 145.51, 146.8, 146.9, 148.4, 148.5, 148.96, 149.02, 149.09, 149.10, 149.2, 149.3, 155.1, 155.7, 155.8, 158.4; HRMS (MALDI+, matrix: DCTB) m/z calcd. for + + C78H34N2O4Na : 1085.2411, found 1085.2412 [M+Na] .

119 
          
 

 
 
            
 

 
 
    

 
 
  


  

  

    









 
 

1 Figure 103. H-NMR of trans-1 12a (400 MHz, in CDCl3).

 
         
        
    
    
     
 



 

 
 











 











  












13 Figure 104. C-NMR of trans-1 12a (100 MHz, in CDCl3).

120
Figure 105. HRMS spectrum of 12a

Figure 106. FT-IR spectrum of 12a

121 


  
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Figure 107. H-NMR H-NMR of 12b (400 MHz, in CDCl3).




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13 Figure 108. C-NMR spectrum of 12b (100 MHz, in CDCl3).

122 Figure 109. HRMS spectrum of 12b

Figure 110. FT-IR spectrum of 12b

123 Compound 13

HO OH

O N O

CF3COO

N H

13

To a solution of 12a (14.0 mg, 13.2 μmol) in CH2Cl2 (3.5 mL), TFA (3.5 mL) was added dropwise. The solution was stirred at room temperature for 4 h. The solvents were removed by flushing nitrogen gas. The residue was washed with hexane and the product was dried under vacuum to provide 13 –1 (14 mg, 13.1 μmol, quant); IR (ATR) νmax (cm ): 2924 (w), 1718 (m), 1619 (m), 1406 (w), 1135 (s), 894 (w), 834 (w), 795 (w), 766 (w), 719 (m), 623 (w); 1 H-NMR (400 MHz, DMF-d7) δ 3.04 (dd, J = 15.4, 6.7 Hz, CH–CH2–CO2H,

2H), 3.33 (dd, J = 15.4, 6.7 Hz, CH–CH2–CO2H,
2H), 3.66 (s, N–CH3,
3H),

4.46 (quint, J = 6.7 Hz, (CH2)2–CH–N, 1H), 4.97 (s, C–CH2–N,
4H), 5.54 (s, 13 C–CH2–N, 4H); C-NMR (100 MHz, DMF-d7) δ 36.9 (CH–CH2–CO2H), 40.3

(N–CH3), 54.3 ((CH2)2–CH–N), 62.8 (C–CH2–N), 67.2 (C–CH2–N), 173.5

(CH2–CO2H); carbon cage: 68.1 (C–CH2–N), 68.2 (C–CH2–N), 136.8 (4C), 136.9 (4C), 140.5 (4C), 140.7 (4C), 142.3 (4C), 143.9 (4C), 144.4 (4C), 145.3 (4C), 145.6 (4C), 146.1 (4C), 146.7 (4C), 147.76 (2C), 147.80 (2C), 152.4 − - (2C), 154.4 (6C); (MALDI , matrix: DCTB) m/z calcd. for C70H18N2O4 : - 950.1272, found 950.1273 [M-H] .

124  

  
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1 Figure 111. H-NMR of 13 (400 MHz, DMFd7).


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13 Figure 112. C-NMR of 13 (100 MHz, DMF-d7).

125
Figure 113. HRMS spectrum of 13

Figure 114. FT-IR spectrum of 13

126 Compound 14

O H H O tBu N N tBu O O O O 20 O N O 20

N

14 To a solution of 13 (15.0 mg, 14.1 μmol) in distilled DMF (1.25 mL), HBTU

(CHEM-IMPEX international, 42.7 mg, 112.8 μmol, 8.0 equiv), Amino-dPEG20- tBu ester (Quanta Biodesign, 43.4 mg, 42.3 μmol, 3.0 equiv) and DIPEA (18.5 mg, 141 μmol, 10 equiv) were added. Reaction mixture was stirred 24 h at room temperature. The solvent was removed under vacuum and subsequently water was added. The crude mixture was purified by HPLC (column:

Phenomenex C4 Ø 10 x 250 mm, solvent: isocratic CH3CN/H2O 45:55, flow rate: 3 mL/min, detection: 370 nm, retention time of 14: 15.0 min) and collected fraction was freeze-dried to provide a brown sticky solid 14 (13.8 –1 mg, 4.7 μmol, 33%); IR (ATR) νmax (cm ): 3300 (w), 2868 (m), 1725 (w), 1669 (m), 1547 (w), 1453 (w), 1349 (w), 1294 (w), 1248 (w), 1200 (w), 1094 (s), 946 (m), 843 (m), 798 (w), 767 (w), 719 (w), 678 (w); 1H-NMR (600 MHz, t CDCl3) δ 1.44 (s, C(CH3)3,
18H), 2.51, (t, J = 6.4 Hz, CH2–CH2–CO2 Bu, 4H), 13 3.61 – 3.69 (m, CH2–CH2–O, 160 H); C-NMR (150 MHz, CDCl3) δ 28.2 t t (C(CH3)3), 36.4 (CH2–CH2–CO2 Bu), 67.0 (O–CH2–CH2–CO2 Bu), 70.2 - 70.4

(CH2–CH2–O), 80.7 (C(CH3)3), 171.14 (CH2–CONH–CH2), 171.15 (CH2– t CO2 Bu), carbon cage: 136.6 (4C), 136.7 (4C), 140.91 (4C), 140.95 (4C), 142.5 (4C), 144.4 (8C), 145.64 (4C), 145.65 (4C), 146.2 (4C), 146.3 (4C), 147.79 (2C), 147.84 (2C), 153.3 (2C), 153.4 (6C); HRMS (MALDI+, matrix: + DCTB) m/z calcd. for C164H204N4O46Na : 2988.3639, found 2988.3642 [M+Na]+.

127 
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1 Figure 115. H-NMR of 14 (600 MHz, in CDCl3).

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13 Figure 116. C-NMR of 14 (150 MHz, in CDCl3).

128
Figure 117. HRMS spectrum of 14

Figure 118. FT-IR spectrum of 14

129 Compound 8

O H H O tBu N N tBu O O O O 20 O N O 20

N I

8

To a solution of 14 (13.8 mg, 4.7 μmol) in dry DMF (0.5 mL) CH3I (0.5 mL, 8.0 mmol, excess equiv) was added. The reaction mixture was stirred at room temperature for 1 h. The solvent was removed by rotary evaporator, the residue was washed with hexane and diethyl ether, and the product was dried under vacuum to give 8 as a brown sticky solid (12.7 mg, 4.1 μmol, 87%); IR –1 (ATR) νmax (cm ): 3430 (w), 2921 (m), 1721 (m), 1663 (m), 1454 (w), 1350 (w), 1248 (w), 1090 (s), 947 (m), 842 (m), 766 (w), 720 (w); 1H-NMR (600

MHz, DMF-d7) δ 1.43 (s, C(CH3)3,
18H), 2.47 (t, J = 6.2 Hz, CH2–CH2– t CO2 Bu, 4H), 2.78 (s, N(CH3), 3H), 2.81 (brs, CONH-CH2 and CH2–CH2– t 13 CO2 Bu, 8H), 2.95 (s, N(CH3), 3H), 3.52 - 3.64 (m, CH2–CH2–O, 160H). C- t NMR (151 MHz, DMF-d7) δ 28.6 (C(CH3)3), 37.2 (CH2–CH2–CO2 Bu), 40.2 t (N–CH3), 53.6 (N–CH3), 67.7 (O–CH2–CH2–CO2 Bu), 63.5 (C–CH2–N), 70.8

(C–CH2–N), 71.4 (CH2–CH2–O), 80.8 (C(CH3)3), 171.7 (CH2–CONH–CH2), t 172.4 (CH2–CO2 Bu); carbon cage: 68.7 (C–CH2–N), 69.3 (C–CH2–N), 137.4 (4C), 137.8 (4C), 141.3 (4C), 141.6 (4C), 143.3 (4C), 144.5 (4C), 145.4 (4C), 146.2 (4C), 146.5 (4C), 146.7 (4C), 147.7 (4C), 148.6 (2C), 148.8 (2C), 152.4 (4C), 155.8 (4C). HRMS (MALDI+, matrix: DCTB) m/z calcd. for + + C165H207N4O46 : 2980.3976, found 2980.3970 [M] .

130    
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1 Figure 119. H-NMR of 8 (600 MHz, in DMF-d7).


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13 Figure 120. C-NMR of 8 (150 MHz, in DMF-d7).

131

Figure 121. HRMS spectrum of 8

Figure 122. FT-IR spectrum of 8

132 Compound 15

O O tBu tBu O N O

N N

15

To a solution of trans-3 12b (109 mg, 103 μmol) in toluene (46 mL), sarcosine (12.8 mg, 144 μmol, 1.4 equiv) and paraformaldehyde (8 mg, 267 μmol, 2.6 equiv) were added. The mixture was sonicated for 30 min and the reaction mixture was refluxed for 2.5 h. The solvent was removed in vacuo and the crude mixture was subjected to a plug of SiO2 eluted with toluene - EtOAc (93:7). The solvent was removed and the residue was purified by a preparative HPLC (column: SHISEIDO Silica SG80 (Ø 30 x 250 mm), solvent: toluene - EtOAc (93:7), flow rate: 10 mL/min, detection: 390 nm). The solvent was removed to give trans-3 trans-3 trans-3 15 (8 mg, 7.14 μmol, 7%); IR –1 (ATR) νmax (cm ): 2968 (w), 2936 (w), 2777 (w), 1724 (m), 1471 (w), 1446 (w), 1388 (w), 1364 (w), 1335 (w), 1255 (w), 1137 (m), 1026 (w), 904 (w), 839 1 (w), 764 (w), 728 (w); H-NMR (400 MHz, CDCl3) δ 1.50 (s, C(CH3)3, 18H), t 2.64 (dd, J = 6.5 Hz, 15.0 Hz, CH–CHH–CO2 Bu, 2H), 2.80 (dd, J = 6.9, 15.0 t Hz, CH–CHH–CO2 Bu, 2H), 2.81 (s, 2(N–CH3), 6H), 3.98 (quint, J = 6.6 Hz, 13 (CH2)2–CH–N, 1H), 4.02 (s, 2(C–CH2–N), 8H), 4.14 (s, C–CH2–N, 4H); C- t NMR (101 MHz, CDCl3) δ 28.4 (C(CH3)3), 38.3 (CH–CH2–CO2 Bu), 41.7 (N–

CH3), 54.5 ((CH2)2–CH–N), 62.4 (C–CH2–N), 69.8 (C–CH2–N), 81.1 (C(CH3)3), t 171.1 (CH2–CO2 Bu); carbon cage: 68.4 (C–CH2–N), 69.5 (C–CH2–N), 125.4 (2C), 128.4 (2C), 129.2 (2C), 140.4 (2C), 140.5 (2C), 140.7 (2C), 141.8 (4C), 142.0 (2C), 143.15 (4C), 144.7 (4C), 144.8 (2C), 148.8 (2C), 148.9 (2C), 149.0 (2C), 149.44 (2C), 149.46 (2C), 149.51 (2C), 152.4 (4C), 156.0 (2C), 156.1 (2C), 158.5 (2C), 158.6 (2C), 158.7 (2C); HRMS (MALDI+, matrix: + + DCTB) m/z calcd. For C81H41N3O4Na : 1142.2989, found 1142.2987 [M+Na] .

133

1 Figure 123. H-NMR of 15 (400 MHz, in CDCl3).

134

13 Figure 124. C-NMR of 15 (101 MHz, in CDCl3).

Figure 125. HRMS spectrum of 15

135
Figure 126. FT-IR spectrum of 15

Compound 16

HO OH O N O

H H N N

CF3COO CF3COO 16

To a solution of 15 (8.8 mg, 7.9 μmol) in CH2Cl2 (2 mL), TFA (2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The solvent and reagent were removed by flushing nitrogen, and obtained residue was washed with hexane and subsequently dried under vacuum to –1 give 16 (8 mg, 7.9 μmol, quant); IR (ATR) νmax (cm ): 2925 (w), 2800 (w), 1667 (s), 1469 (w), 1414 (w), 1172 (s), 1123 (s), 1058 (m), 1019 (m), 963 (m), 947 (m), 828 (m), 796 (m), 771 (m), 718 (m); 1H-NMR (400 MHz, DMF-d7) δ

136 2.81 (dd, J = 15.4, 6.6 Hz, CH–CH2–CO2H,
2H), 3.06 (dd, J = 15.3, 6.9 Hz,

CH–CH2–CO2H,
2H), 3.23 (s, 2(N–CH3),
6H), 4.13 (quint, J = 6.6 Hz, (CH2)2– 13 CH–N,
1H), 4.33 (s, C–CH2–N,
4H), 4.71 (s, 2(C–CH2–N),
8H); C-NMR (101

MHz, DMF-d7) δ 36.7 (CH–CH2–CO2H), 40.3 (N–CH3), 54.2 ((CH2)2–CH–N),

62.0 (2(C–CH2–N)), 69.1 (C–CH2–N)), 173.5 (CH2–CO2H); carbon cage: 67.0

(C–CH2–N), 68.96 (C–CH2–N), 68.99 (C–CH2–N), 140.6 (2C), 140.8 (2C), 140.9 (2C), 142.0 (2C), 142.2 (2C), 142.3 (2C), 142.8 (2C), 142.9 (2C), 143.1 (2C), 144.5 (2C), 144.6 (2C), 144.8 (2C), 148.6 (2C), 148.7 (2C), 149.1 (2C), 149.4 (2C), 149.5 (2C), 149.9 (2C), 152.3 (4C), 152.4 (4C), 157.0 (2C), 159.2 (2C), 159.5 (4C), 159.6 (2C); HRMS (MALDI+, matrix: DCTB) m/z calcd. for + + C73H26N3O4 : 1008.1918, found 1008.1922 [M+H] .



  


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1 Figure 127. H-NMR of 16 (400 MHz, in DMF-d7).

137 


  
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13 Figure 128. C-NMR of 16 (101 MHz, DMF-d7).

Figure 129. HRMS spectrum of 16

138
Figure 130. FT-IR spectrum of 16

Compound 17

O H H O t Bu N N tBu O O O O 20 O N O 20

N N

17

To a solution of 16 (3.7 mg, 3.0 μmol) in DMF (distilled, 0.28 mL), HBTU (9.3 t mg, 24.5 μmol, 8.2 equiv), Amino-dPEG20- Bu ester (9.6 mg, 9.3 μmol, 3.1 equiv), DIPEA (5.6 mg, 7.5 μL, 43 μmol, 14 equiv) were added and reaction mixture was 24 h at room temperature. The solvent was removed under vacuum and water (2.5 mL) was added. The crude mixture was purified by

HPLC (column: Phenomenex C4 Ø 10 x 250 mm, solvent: CH3CN-H2O (5 : 95 to 88 : 12 (5 min), 88 : 12 to 95 : 5 (11 min), 95 : 5 to 5 : 95 (3 min)), flow rate: 3 mL/min, detection: 370 nm, retention time of 17: 9 min) the collected fraction

139 was freeze-dried to provide a sticky red material 17 (2.4 mg, 0.81 μmol, 27%); –1 IR (ATR) νmax (cm ): 3210 (m), 2879 (w), 1678 (s), 1469 (m), 1427 (m), 1348 (m), 1197 (s), 1128 (s), 949 (w), 831 (w), 799 (m), 720 (m), 674 (w), 647 (w); 1 H-NMR (400 MHz, CDCl3) δ 1.44 (s, C(CH3)3,
18H), 2.49 (t, J = 6.6 Hz, CH2– t 13 CH2–CO2 Bu, 4H), 3.63 (m, CH2–CH2–O, 160H); C-NMR (101 MHz, CDCl3) t δ 28.2 (C(CH3)3), 36.4 (CH2–CH2–CO2 Bu), 40.1 (N–CH3), 45.1 ((CH2)2–CH– t N), 67.0 (O–CH2–CH2–CO2 Bu), 70.7 (CH2–CH2–O), 80.6 (C(CH3)3), 171.0 t (CH2–CONH–CH2 and CH2–CO2 Bu); carbon cage: 68.3 (C–CH2–N), 69.7 (C–

CH2–N), 70.3 (C–CH2–N), 140.8 (4C), 142.1 (2C), 142.8 (4C), 143.0 (4C), 143.9 (2C), 144.4 (2C), 144.5 (2C), 148.2 (4C), 148.8 (2C), 149.01 (2C), 149.05 (2C), 149.15 (2C), 149.2 (2C), 149.6 (2C), 152.4 (4C), 152.5 (2C), 161.2 (2C), 161.5 (2C), 161.9 (2C), 163.1 (6C); HRMS (MALDI+, DCTB) m/z + + calcd. for C167H211N5O46Na : 3045.4217, found: 3045.4204 [M+Na] .




  
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1 Figure 131. H-NMR of 17 (400 MHz, in CDCl3).

140  

  
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13 Figure 132. C-NMR of 17 (100 MHz, in CDCl3).

Figure 133. HRMS spectrum of 17

141
Figure 134. FT-IR spectrum of 17

Compound 9

O H H O t Bu N N tBu O O O O 20 O N O 20

N N I I 9

To a solution of 17 (2.5 mg, 0.8 μmol) in dry DMF (1.0 mL) CH3I (1.0 mL) was added. The reaction mixture was stirred at room temperature for 4 h. Subsequently the solvent and reagent were removed by rotary evaporator, and the residue was washed with hexane and diethyl ether and the product was dried under vacuum to give a red material 9 (2.5 mg, 0.7 μmol, 88%); IR –1 (ATR) νmax (cm ): 2873 (m), 1725 (w), 1266 (w), 1101 (s), 949 (w), 730 (m); 1 H-NMR (600 MHz, DMF-d7) δ 1.44 (s, C(CH3)3,
18H), 2.47 (t, J = 6.2 Hz, t CH2–CH2–CO2 Bu, 4H), 2.87 (m, J = 5.2 Hz, CONH-CH2 and CH2–CH2– t 13 CO2 Bu, 8H), 3.08 (s, N-CH3), 12H), 3.58 (m, CH2–CH2–O, 160H); C-NMR t (150 MHz, DMF-d7) δ 28.6 (C(CH3)3), 37.2 (CH2–CH2–CO2 Bu), 40.5 (N–CH3),

45.8 ((CH2)2–CH–N), 53.6 (N–CH3), 62.6 (C–CH2–N), 70.3 (2 C–CH2–N), 67.7 t (O–CH2–CH2–CO2 Bu), 71.1-71.4 (CH2–CH2–O), 80.8 (C(CH3)3), 171.7 (CH2–

142 t CONH–CH2), 172.1 (CH2–CO2 Bu); carbon cage: 67.9 (C–CH2–N), 68.97 (C–

CH2–N), 69.02 (C–CH2–N), 141.2 (2C), 141.7 (2C), 141.9 (2C), 142.7 (2C), 143.00 (2C), 143.03 (2C), 143.21 (2C), 143.23 (2C), 143.7 (2C), 144.6 (2C), 145.0 (2C), 145.5 (2C), 148.7 (2C), 148.9 (2C), 149.7 (2C), 149.9 (2C), 150.1 (2C), 151.0 (2C), 153.1 (4C), 153.4 (2C), 154.2 (2C), 154.6 (2C), 156.4 (2C), 157.2 (2C), 158.4 (2C), 160.6 (2C); HRMS (MALDI+, matrix: CCA) m/z + + calculated for C169H218O46N5 : 3053.4868, found: 3053.4867 [M+H] .


 
     
      
       
        
 

    



     








 
 

  

1 Figure 135. H-NMR of 9 (600 MHz, DMF-d7).

143  
 
 




 










  
 
 
 













 



   
 
 
 

                 






 
 











 













  












13 Figure 136. C-NMR of 9 (150 MHz, in DMF-d7).

Figure 137. HRMS spectrum of 9

144
Figure 138. FT-IR spectrum of 9

145 6.2.2 Characterization of 7, 8, and 9

6.2.2.1 Tensiometry
Aqueous solutions of 7, 8, and 9 in a series of concentrations (1x10-3 M to 3x10-7 M) were prepared. A pendant drop device DSA100 (Krüss GmbH) was used to measure the interfacial tension values of these solutions. A water/air interface was made at the tip of an inverted J-shaped, stainless steel needle with a diameter of 1.451 mm. A drop shape analysis system imaged with a CCD camera as a function of time was used to perform the measurements. The droplet profile was automatically detected using the analysis software package DSA3 (Krüss GmbH) by fitting the Laplace–Young equation to the profile to obtain interfacial tension values as a function of time.

6.2.2.2 Dynamic Light Scattering (DLS)
Aqueous solutions of 7, 8, and 9 at 1 mM concentration were prepared, filtered and measured using a glass cuvette (l = 1 cm).

6.2.2.3 Scanning Transmission Electron Microscopy (STEM)
The morphological and elemental distribution analyses of assemblies in different states were done by transmission electron microscopy (TEM) on a FEI Talos F200X instrument operated at 200kV in both, TEM and STEM, scanning transmission electron microscopy, modes. An atomic number sensitive, high angle annular dark field (HAADF) detector was used for STEM imaging. The elemental content analyses were carried out in an STEM mode with a probe size of about 0.8 nm by employing a SuperX EDX system consisting of 4 SDD detectors and used in the hypermap and line profile modes. Specimen preparation for TEM/STEM studies was performed immediately prior a TEM session. A drop of a particle suspension was placed on a graphene (monolayer) covered conventional Ni-grid. After that the suspension was left to dry out in air or in vacuum (10-2 mbar) for about an hour. The latter yielded considerably reduced contamination rate during scanning in STEM. Samples were prepared in ca. 3 mM aqueous solution.

146

Figure 139. STEM images of 7 (3 mM concentration). Sample was air-dried before measurement.

Figure 140. STEM images of 7 (3 mM concentration). Sample was vacuum-dried before measurement.

Figure 141. STEM images of 8 (3 mM concentration). Sample was air-dried before measurement.

Figure 142. STEM images of 8 (3 mM concentration). Sample was vacuum-dried before measurement.

147

Figure 143. STEM images of 9 (3 mM concentration). Sample was air-dried before measurement.

Figure 144. STEM images of 9 (3 mM concentration). Sample was vacuum-dried before measurement.

6.2.2.4 Thermoresponsivity Tests and Cloud point experiments
Reversibility tests and cloud points were measured in water at 1 mM concentration (for 7, 8, and 9) or 2 mM concentration (for CH3CONH-EGn- t CO Bu, because 2 EG chain in C60-PEG materials). The absorbance was monitored at 800 nm.

148 6.3. Chapter 3

General Procedure:
C70 (17.6 mg, 21 μmol), N-glycine derivative 19 (8.8 mg, 27.7 μmol, 1.3 equiv) and paraformaldehyde (1.89 mg, 63 μmol, 3.0 equiv) were dissolved in toluene (21.5 mL) inside a round bottom flask. The mixtre was sonicated for 30 mins and then was put to reflux for 1 h. The solvents were evaporated and the crude reaction mixture was purified by silica gel chromatography using toluene as the eluent. A mixture of C70 monoadduct isomers were obtained.

The combined mixture of C70 monoadducts was subjected to a second stage purification step using HPLC (column: SHISEIDO Silica SG80, size: Ø 30 x 250 mm, solvent: toluene, flow rate: 10 mL/min, detection: 390 nm, rt of 20: 80 min). C70 Prato monoadduct 20 (yield: 3.8 mg, 16%) and a mixture of two other C70 monoadduct isomers were separated. The latter was then re- injected into Buckyprep column using toluene-acetonitrile 3:1 as eluent. Isomers 21 (yield: 2.9 mg, 13%) and 22 (yield: 0.5 mg, 2%) were isolated.

Compound 20 (Isomer C5 – 6)

O O

O N O

C5-6 isomer 20 –1 IR (ATR) νmax (cm ): 2923 (m), 1726 (m), 1430 (w), 1365 (w), 1275 (m), 1260 1 (m), 1143 (w), 764 (s), 750 (s); H-NMR (600 MHz, CDCl3) δ 1.47 (s, C(CH3)3,

18H), 2.47 (dd, J = 15.0, 6.5 Hz, CH-(CH2)2, 2H), 2.58 (dd, J = 15.0, 6.9 Hz,

CH-(CH2)2, 2H), 3.53 (d, J = 9.3 Hz, C–CH2–N, 2H), 3.79 – 3.72 (m, C–CH2– 13 N, 2H and CH-(CH2)2, 1H). C-NMR (150 MHz, CDCl3) δ 28.3 (C(CH3)3), 38.1 t (CH–CH2–CO2 Bu), 54.0 ((CH2)2–CH–N), 61.9 (C–CH2–N), 81.1 (C(CH3)3),

170.8 (CH2–CO2tBu), carbon cage: 60.0 (C–CH2–N), 126.4 (2C), 131.50 (2C), 131.51 (2C), 132.2 (2C), 132.5 (2C), 141.0 (2C), 142.1 (2C), 142.6 (2C),

149 144.0 (2C), 144.4 (2C), 144.6 (4C), 145.0 (2C), 146.0 (2C), 146.1 (4C), 146.4 (2C), 146.8 (2C), 146.9 (1C), 147.0 (2C), 147.2 (2C), 147.3 (2C), 147.6 (2C), 148.1 (2C), 148.3 (2C), 148.4 (2C), 149.1 (2C), 149.2 (1C), 149.3 (2C), 149.5 (2C), 150.1 (2C), 150.7 (1C), 151.8 (2C), 152.1 (2C), 153.1 (2C), 154.9 (1C). + + HRMS (MALDI , matrix: DCTB) m/z calcd. for C85H27NO4Na : 1148.1832, found 1148.1828 [M+Na]+.

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1 Figure 145. H-NMR of 20 (600 MHz, CDCl3).

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13 Figure 146. C-NMR of 20 (150 MHz, CDCl3).

Figure 147. HRMS spectrum of 20.

151

Figure 148. FT-IR spectrum of 20.

Compound 21 (Isomer C1 – 2)

O O

O N O

C1-2 isomer 21 –1 1 IR (ATR) νmax (cm ): 2923 (m), 1729 (m), 1428 (w), 1366 (w), 1145 (w); H-

NMR (600 MHz, CDCl3) δ 1.53 (s, C(CH3)3, 18H), 2.59 (dd, J = 15.1, 6.5 Hz,

CH-(CH2)2, 2H), 2.73 (dd, J = 15.1, 7.0 Hz, CH-(CH2)2, 2H), 3.67 (s, C–CH2– 13 N, 2H), 3.91 (quint, J = 6.8 Hz, CH-(CH2)2, 1H), 4.01 (s, C–CH2–N, 2H). C- t NMR (150 MHz, CDCl3) δ 28.42 (C(CH3)3), 38.24 (CH–CH2–CO2 Bu), 54.08

((CH2)2–CH–N), 58.98 (C–CH2–N), 62.98 (C–CH2–N), 81.24 (C(CH3)3),

170.95 (CH2–CO2tBu), carbon cage: 61.40 (C–CH2–N), 62.97 (C–CH2–N),

152 131.36 (2C), 131.42 (2C), 131.76 (2C), 133.88 (2C), 133.97 (2C), 137.72 (2C), 140.64 (2C), 140.91 (2C), 142.92 (2C), 143.34 (2C), 143.37 (2C), 143.49 (2C), 145.91 (2C), 146.44 (2C), 147.01 (2C), 147.14 (2C), 147.16 (1C), 147.22 (2C), 147.25 (2C), 147.60 (2C), 148.94 (2C), 149.21 (2C), 149.50 (2C), 149.61 (2C), 149.85 (2C), 150.00 (2C), 150.39 (2C), 150.86 (2C), 150.94 (2C), 151.11 (2C), 151.36 (2C), 151.58 (1C), 151.61 (2C), 155.88 (2C), 158.35 (2C). HRMS (MALDI+, DCTB) m/z calcd. for + + C85H27NO4Na : 1148.1832, found 1148.1831 [M+Na] .

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13 Figure 150. C-NMR of 21 (150 MHz, CDCl3).

Figure 151. HRMS spectrum of 21.

154

Figure 152. FT-IR spectrum of 21.

Compound 22 (Isomer C7 – 8)

O O O N O

C7-8 isomer 22 –1 IR (ATR) νmax (cm ): 2923 (m), 1728 (m), 1275 (m), 1260 (m), 1143 (w), 764 1 (s), 750 (s); H-NMR (600 MHz, CDCl3) δ 1.46 (s, C(CH3)3, 18H), 2.40 (dd, J =

14.9, 6.5 Hz, CH-(CH2)2, 2H), 2.54 – 2.48 (m, CH-(CH2)2, 2H and C–CH2–N, 13 2H), 3.77 – 3.70 (m, CH-(CH2)2, 1H), 4.03 (d, J = 9.8 Hz, C–CH2–N, 2H). C- t NMR (150 MHz, CDCl3) δ 28.2 (C(CH3)3), 37.8 (CH2–CH2–CO2 Bu), 54.0

((CH2)2–CH–N), 66.0 (C–CH2–N), 81.0 (C(CH3)3), 170.7 (CH2–CO2tBu), carbon cage: 61.7 (C–CH2–N), 127.2 (1C), 129.0 (2C), 132.0 (2C), 132.7 (2C), 132.8 (2C), 133.9 (2C), 136.0 (2C), 139.3 (2C), 140.0 (2C), 141.0 (2C),

155 141.1 (2C), 143.5 (2C), 144.4 (2C), 145.4 (2C), 145.6 (2C), 145.8 (3C), 146.0 (2C), 146.01 (2C), 146.04 (2C), 146.1 (2C), 146.2 (2C), 146.7 (2C), 146.71 (2C), 146.74 (2C), 147.6 (2C), 147.7 (2C), 148.0 (2C), 148.37 (2C), 148.41 (2C), 149.3 (2C), 149.4 (2C), 149.8 (2C), 151.3 (2C), 154.2 (1C), 155.0 (1C). + + HRMS (MALDI , DCTB) m/z calcd. for C85H27NO4Na : 1148.1832, found 1148.1828 [M+Na]+.

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1 Figure 153. H-NMR of 22 (600 MHz, CDCl3).

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13 Figure 154. C-NMR of 22 (150 MHz, CDCl3).

Figure 155. HRMS spectrum of 22.

157

Figure 156. FT-IR spectrum of 22,

Compound 23

HO OH

O N O

23

To a solution of 20 (7.38 mg, 6.55 μmol) in CH2Cl2 (7 mL), TFA (7 mL) was added dropwise. The reaction mixture was stirred at room temperature for 4 h. The solvent and reagent were removed by flushing nitrogen, and obtained residue was washed with hexane and subsequently dried under vacuum to –1 give 23 in a quantitative yield; IR (ATR) νmax (cm ): 2957 (w), 2540 (w), 1717 (m), 1659 (w), 1427(w), 1180 (m), 1162 (m), 1150 (m), 1067 (w), 1040 (w), 1 992 (w), 874 (w), 795 (w); H-NMR (600 MHz, DMF-d7) δ 2.63 (dd, J = 15.1,

158 6.7 Hz, CH2–CH2–CO2H, 2H), 2.83 (dd, J = 15.3, 7.2 Hz, CH2–CO2H, 2H),

3.70 (d, J = 9.5 Hz, C–CH2–N, 2H), 3.92 – 3.87 (m, (CH2)2–CH–N, 1H), 3.94 13 (d, J = 9.5 Hz, C–CH2–N, 2H). C-NMR (150 MHz, DMF-d7) δ 36.6 (CH2–

CH2–CO2H), 53.6 ((CH2)2–CH–N), 59.9 (C–CH2–N), 173.2 (CH2–CO2H), carbon cage: 61.3 (C–CH2–N), 126.6 (2C), 131.5 (4C), 132.1 (2C), 132.4 (2C), 141.1 (2C), 142.0 (2C), 142.3 (2C), 143.7 (2C), 144.42 (2C), 144.44 (2C), 144.6 (2C), 144.9 (2C), 145.4 (2C), 146.0 (2C), 146.3 (2C), 146.69 (2C), 146.73 (1C), 146.9 (2C), 146.97 (2C), 147.04 (2C), 147.2 (2C), 147.6 (2C), 147.9 (2C), 148.27 (2C), 148.34 (2C), 149.0 (2C), 149.19 (2C), 149.22 (1C), 149.5 (2C), 150.2 (2C), 150.5 (1C), 151.7 (2C), 152.9 (2C), 153.9 (2C), 154.9 + + (1C). HRMS (MALDI , DCTB) m/z calcd. for C77H12NO4 : 1014.0761, found 1014.0746 [M+H]+.



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1 Figure 157. H-NMR of 23 (600 MHz, DMF-d7).

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13 Figure 158. C-NMR of 23 (150 MHz, DMF-d7).

Figure 159. HRMS spectrum of 23.

160

Figure 160. FT-IR spectrum of 23.

Compound 24

O O H tBu N NH tBu O O O O 20 O N O 20

24 To a solution of 23 (8.7 mg, 8.58 μmol) in distilled DMF (1.5 mL), HBTU t (11.39 mg, 30 μmol, 3.5 equiv), Amino-dPEG20- Bu ester (20.25 mg, 19.7 μmol, 2.3 equiv), DIPEA (4.93 μL, 28 μmol, 3.3 equiv) were added and reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and water (2 mL) was added. The crude mixture was purified by HPLC (column: Phenomenex C4 Ø 10 x 250 mm, solvent: CH3CN-

H2O (5 : 95 to 88 : 12 (5 min), 88 : 12 to 95 : 5 (11 min), 95 : 5 to 5 : 95 (3 min)), flow rate: 3 mL/min, detection: 370 nm, retention time of 24: 17 min) the collected fraction was freeze-dried to provide a brown material 24 (5.31 mg,

161 –1 1.75 μmol, 20%); IR (ATR) νmax (cm ): 2860 (m), 1725 (w), 1668 (w), 1544 (w), 1349 (w), 1289 (w), 1249 (w), 1095 (s), 949 (m), 847 (m); 1H-NMR (600 t MHz, CDCl3) δ 1.44 (s, C(CH3)3, 18H), 2.50 (t, J = 6.5 Hz, CH2–CH2–CO2 Bu, 13 4H), 3.64 (s, CH2–CH2–O, 160H). C-NMR (151 MHz, CDCl3) δ 28.2 t (C(CH3)3), 36.3 (CH2–CH2–CO2 Bu), 39.2 (CH–CH2–CO–NH), 59.5 ((CH2)2– t CH–N), 62.0 (C–CH2–N), 67.0 (O–CH2–CH2–CO2 Bu), 70.0-70.4 (CH2–CH2– t O), 80.8 (C(CH3)3), 171.2 (CH2–CONH–CH2), 173.0 (CH2–CO2 Bu), carbon cage: 126.2 (2C), 131.46 (2C), 131.49 (2C), 132.2 (2C), 132.5 (2C), 140.9 (2C), 142.0 (2C), 142.7 (2C), 144.0 (2C), 144.4 (2C), 144.6 (2C), 144.7 (2C), 145.0 (2C), 145.5 (2C), 146.1 (2C), 146.16 (2C), 146.17 (2C), 146.8 (2C), 146.9 (1C), 147.0 (2C), 147.3 (2C), 147.3 (2C), 147.5 (2C), 148.1 (2C), 148.3 (2C), 148.4 (2C), 149.1 (2C), 149.2 (1C), 149.3 (2C), 149.5 (2C), 150.1 (2C), 150.6 (1C), 151.7 (2C), 151.8 (2C), 152.8 (2C), 154.9 (1C). HRMS (MALDI+, + + DHB) m/z calcd. for C171H198N3O46Na : 3052.3139, found 3052.3098 [M+Na] .


        
   
     




 
   
   











 
 
  

1 Figure 161. H-NMR of 24 (600 MHz, CDCl3).

162  

       
   
       
  
                
  

   



   
    
 
 
   


   
  
       
      




  










  









 


13 Figure 162. C-NMR of 24 (150 MHz, CDCl3).

Figure 163. HRMS spectrum of 24.

163

Figure 164. FT-IR spectrum of 24.

164 6.4. Chapter 4

6.4.1 Synthesis
Peak A and Peak B

N 3

Peak A and Peak B

Gd3N@Ih-C80 (3.0 mg, 2.1 μmol), N-ethyl glycine (10 mg, 103 μmol, 50 equiv), and paraformaldehyde (25 mg, 828 μmol, 400 equiv) were dissolved in o-DCB (14 mL) and were treated under sonication for 10 min. Nitrogen gas was bubbled through the mixed solution for 10 min to remove oxygen. The reaction was carried out at 120 °C for 15 min. The mixture was cooled to rt and quickly concentrated in vacuo. For the first stage purification, the crude reaction mixture was purified by HPLC (column: Buckyprep, solvent: toluene). The collected mixture of polyadducts were purified for the second time using Buckyprep (solvent: toluene-acetonitrile 2 : 1). The major peak was isolated and was re-injected for the final stage purification (column: PBB, solvent: toluene with 1% pyridine). Two major tris-adducts peaks were separated (peak A and peak B). The solvent was removed and dried for 24 h under vacuum to provide a tris-adduct (0.37 mg, 0.22 μmol, isolated yield 11%). MS + (ESI-MS+,) m/z calcd for C92H28N4Gd3 : 1662.0032; found: (for peak A) + + 1662.0059 [M+H] found: (for peak B) C92H28N4Gd3: 1662.0074 [M+H] .

peak B peak A

Figure 165. HPLC trace of the separation of peak A and peak B.

165 Peak C and Peak D

N 3

Peak C and Peak D

Y3N@Ih-C80 (5.1 mg, 4.0 μmol, 1 equiv), N-ethyl glycine (22 mg, 0.20 mmol, 50 equiv), and paraformaldehyde (51 mg, 1.61 mmol, 400 equiv) were dissolved in o-DCB (26 mL) and were treated under sonication for 10 min. Nitrogen gas was bubbled through the mixed solution for 15 min to remove oxygen. The reaction was carried out at 120 °C for 15 min. The solvent o-DCB was quickly removed under pressure and the material was re-dissolved in toluene. For the first stage purification, the crude reaction mixture was purified by HPLC (column: Buckyprep, solvent: toluene). The collected mixture of polyadducts were purified for the second time using Buckyprep (solvent: toluene-acetonitrile 2 : 1). The major peak was collected and the solvent was removed, dried for 24 h under vacuum to provide a combined yield of tris- adduct isomers (0.9 mg, 0.62 μmol, isolated yield 15%). This mixture was re- injected for the final stage purification (column: PBB, solvent: toluene with 1% pyridine) to separate tris-adduct isomers peak C and peak D (peak D contains a mixture of isomers which could not be further separated).

To avoid the isomerization, the fraction containing peak C was quickly concentrated and the NMR was immediately recorded at 4 °C.

1 Peak C: H-NMR (600 MHz, CDCl3) δ 1.29 (t, J = 7.2 Hz, CH2-CH3, 3H), 1.40

(dt, J = 12.7, 7.2 Hz, CH2-CH3, 6H), 2.85 (q, J = 7.2 Hz, CH2-CH3, 2H,), 2.98

(q, J = 7.1 Hz, CH2-CH3, 2H,), 3.07 - 3.01 (q, J = 7.1 Hz, CH2-CH3, 2H), 3.63

(m, C-CH2-N, 2H), 3.83 (d, J = 10.2 Hz, C-CH2-N, 1H), 3.90 (d, J = 10.5 Hz,

C-CH2-N, 1H), 3.96 (m, C-CH2-N, 2H), 4.11 (m, C-CH2-N, 2H), 4.24 - 4.16 (m,

C-CH2-N, 2H), 4.28 (d, J = 10.2 Hz, C-CH2-N, 1H), 4.37 (d, J = 10.0 Hz, C- 13 CH2-N, 1H). C-NMR (150 MHz, CDCl3) δ 14.4 (−N−CH2−CH3), 48.1 (C−CH2-

N), 48.4 (C−CH2-N), 53.8 (C−CH2-N), 54.0 (C−CH2-N), 56.5 (C−CH2-N), 64.1

166 (C−CH2-N), 65.3 (C−CH2-N), 65.5 (C−CH2-N), 66.9 (C−CH2-N), 68.6 (C−CH2-

N), 68.9 (C−CH2-N), 69.2 (C−CH2-N), 69.8 (C−CH2-N), 72.0 (C−CH2-N), carbon cage: 103.3 (1C, adjacent to sp3), 115.5 (1C, adjacent to sp3), 119.4 (1C, adjacent to sp3), 120.20 (1C, adjacent to sp3), 120.22 (1C, adjacent to sp3), 121.1 (1C, adjacent to sp3), 127.2 (1C), 128.8 (1C), 132.56 (2C), 132.58 (1C), 132.78 (1C), 132.79 (1C), 133.3 (1C), 133.6 (1C), 133.7 (1C), 133.9 (1C), 134.0 (1C), 134.1 (1C), 134.77 (1C), 134.82 (1C), 134.9 (1C), 135.18 (1C), 135.21 (1C), 135.3 (1C), 135.59 (1C), 135.62 (1C), 135.7 (1C), 135.9 (1C), 136.0 (1C), 136.5 (1C), 136.9 (1C), 138.0 (1C), 138.3 (1C), 138.5 (1C), 138.8 (1C), 139.0 (1C), 139.3 (2C), 139.4 (1C), 139.46 (2C), 139.51 (1C), 140.16 (1C), 140.19 (1C), 140.35 (1C), 140.39 (1C), 140.9 (1C), 141.07 (1C), 141.13 (2C), 141.3 (1C), 141.60 (1C), 141.62 (1C), 142.3 (1C), 142.5 (1C), 142.7 (1C), 143.2 (1C), 143.4 (1C), 143.5 (1C), 143.7 (1C), 143.8 (1C), 143.9 (1C), 144.2 (1C), 144.7 (1C), 145.8 (1C), 146.2 (1C), 147.0 (1C), 148.3 (2C), 149.8 (1C), 150.3 (1C), 154.0 (1C), 159.7 (1C). HRMS (ESI-MS+,) m/z calcd + for C92H28N4Y3 : 1454.9484; found for C92H28N4Y3 (peak C): 1454.9491 [M+H]+.

To avoid the isomerization, the fraction containing peak D was quickly concentrated and the NMR was immediately recorded at 4 °C. Since peak D was a mixture of isomers, the 13C-NMR spectra could not be attributed to the corresponding carbons.

1 Peak D: H-NMR (600 MHz, CDCl3) δ 1.48 - 1.26 (m, CH2-CH3, 9H), 3.14 -

2.79 (m, CH2-CH3, 10H), 3.52 (dd, J = 32.1, 9.0 Hz, C-CH2-N, 2H), 3.78 - 3.61

(m, C-CH2-N, 4H), 3.87 (d, J = 9.1 Hz, C-CH2-N, 1H), 4.41 - 3.91 (m, C-CH2- 13 N, 12H). C-NMR (151 MHz, CDCl3) δ 14.02, 14.25, 14.28, 14.38, 14.48, 48.27, 48.49, 48.56, 48.60, 48.91, 49.14, 54.36, 55.27, 56.14, 57.40, 58.80, 63.72, 64.33, 64.85, 65.38, 65.46, 65.83, 67.22, 68.51, 69.33, 69.94, 70.14, 70.77, 70.88, 71.43, 72.05, 75.60, carbon cage: 106.40 (1C, adjacent to sp3), 107.21 (1C, adjacent to sp3), 110.19 (1C, adjacent to sp3), 112.38 (1C, adjacent to sp3), 113.02 (1C, adjacent to sp3), 114.88 (1C, adjacent to sp3), 115.97 (1C, adjacent to sp3), 116.62 (1C, adjacent to sp3), 117.59 (1C,

167 adjacent to sp3), 118.22 (1C, adjacent to sp3), 120.25 (1C, adjacent to sp3), 120.67 (1C, adjacent to sp3), 126.09, 128.07, 128.12, 128.91, 128.96, 128.98, 129.29, 129.33, 129.99, 130.18, 131.12, 131.31, 132.00, 132.36, 132.79, 132.89, 132.95, 133.04, 133.17, 133.29, 133.32, 133.50, 133.56, 133.78, 133.99, 134.04, 134.06, 134.18, 134.25, 134.34, 134.38, 134.53, 134.76, 134.78, 135.09, 135.21, 135.34, 135.67, 135.72, 135.75, 135.83, 135.93, 135.99, 136.07, 136.16, 136.35, 136.48, 136.67, 136.91, 137.11, 137.22, 137.57, 137.68, 137.77, 137.82, 138.24, 138.48, 138.63, 138.79, 138.83, 138.89, 138.94, 139.02, 139.15, 139.20, 139.31, 139.66, 139.69, 139.74, 139.76, 139.90, 139.96, 139.99, 140.02, 140.43, 140.58, 140.61, 140.97, 141.08, 141.13, 141.14, 141.26, 141.27, 141.37, 141.42, 141.83, 141.85, 142.04, 142.33, 142.41, 142.51, 142.82, 143.00, 143.06, 143.25, 143.29, 143.47, 143.65, 143.78, 144.27, 144.34, 145.47, 145.85, 145.93, 146.22, 146.52, 146.86, 147.43, 148.24, 148.93, 149.24, 149.30, 149.87, 151.44, + 152.01, 153.95, 154.38, 164.26. HRMS (ESI-MS+,) m/z calcd for C92H28N4Y3 : + 1454.9484; found for C92H28N4Y3 (peak D): 1454.9446 [M+H] .

peak D

peak C

PBB, toluene - pyridine (99:1)

0 5 10 15 20 retention time [min]

Figure 166. HPLC trace of the separation of peak C and peak D.

168 peak A peak B

Figure 167. Vis/NIR spectra of tris-adduct peak A and peak B (M = Gd) in toluene-pyridine (1%).

peak C peak D

Figure 168. Vis/NIR spectra of tris-adduct peak C and peak D (M = Y) in toluene-pyridine (1%).

169

Figure 169. ESI-MS of tris-adduct peak A (M = Gd). Calibrant peaks marked with an *

170

Figure 170. ESI-MS of tris-adduct peak B (M = Gd). Calibrant peaks marked with an *

171

Figure 171. ESI-MS of tris-adduct peak C (M = Y). Calibrant peaks marked with an *

172

Figure 172. ESI-MS of tris-adduct peak D (M = Y). Calibrant peaks marked with an *

173

1 Figure 173. H-NMR of tris-adduct peak C (M = Y, in CDCl3, 600 MHz, measured at 4 °C)

 




 




 







 













 


 
 


   

       
       

   

       
       



 

 
















 








   



  










13 Figure 174. C-NMR of tris-adduct peak C (M = Y, in CDCl3, 150 MHz, measured at 4 °C)

174 (a)

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Figure 175. (a) Zoomed out version and (b) zoomed in version of DQF COSY of tris-adduct peak C (M =

Y, in CDCl3, 150 MHz, measured at 4 °C).

175

Figure 176. HSQC spectrum of tris-adduct peak C (M = Y, in CDCl3, 600 MHz, measured at 4 °C)

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13 Figure 178. C-NMR of tris-adduct peak D (M = Y, in CDCl3, 600 MHz, measured at 4 °C)

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13 Figure 180. C-NMR of tris-adduct mixture of peak C and peak D (M = Y, in CDCl3, 600 MHz)

178 6.4.2 Isomerization Studies of Trisadducts of M3N@C80 (M = Gd, Y)

6.4.2.1 Isomerization Observed by HPLC
Isomerization of Gd tris-adduct peak A. A solution of purified tris-adduct A was let stand at room temperature in toluene-pyridine (1%). Aliquots (10 μL) were taken after 0, 24, 72, 144, 216, 360 hours and were analyzed by HPLC (column: Buckyprep, toluene). Isomerization of Gd tris-adduct peak B. A solution of purified tris-adduct B was let stand at room temperature in toluene-pyridine (1%). Aliquots (10 μL) were taken after 0, 24, 72, 144, 216, 360 hours and were analyzed by HPLC (column: Buckyprep, toluene). Isomerization of Y tris-adduct peak C. A solution of purified tris-adduct A was let stand at room temperature in toluene-pyridine (1%). Aliquots (10 μL) were taken after 0, 1, 3, 5, 7, 24, 48 hours and were analyzed by HPLC (column: Buckyprep, toluene). Isomerization of Y tris-adduct peak D. A solution of purified tris-adduct A was let stand at room temperature in toluene-pyridine (1%). Aliquots (10 μL) were taken after 0, 1, 3, 5, 7, 24, 48 hours and were analyzed by HPLC (column: Buckyprep, toluene).

6.4.2.2 Isomerization Observed by 1H-NMR
Peak C was separated from peak D; the fraction containing peak C was quickly concentrated. The sample was dissolved in CDCl3 and was measured every hour over the course of 25 h.

179

Figure 181. Isomerization of peak C observed in 1H-NMR.

Peak D was separated from peak C; the fraction containing peak D was quickly concentrated. The sample was dissolved in CDCl3 and was measured every hour over the course of 25 h.

Figure 182. Isomerization of peak D observed in 1H-NMR.

180

CHAPTER 7

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181

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