Amphiphilic Fullerenes for Biomedical and Optoelectronical Applications”

AMPHIPHILIC FULLERENES FOR

BIOMEDICAL AND OPTOELECTRONICAL

APPLICATIONS

Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades

vorgelegt von Patrick Witte aus Nürnberg Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 25.04.2008

Vorsitzender der Prüfungskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Andreas Hirsch Zweitberichterstatter: Prof. Dr. Tim Clark Meinem Doktorvater, Prof. Dr. A. Hirsch, gilt mein besonderer Dank für sein reges Interesse am Fortgang dieser Arbeit sowie für seine Anregungen und die Diskussionen mit ihm.

Die vorliegende Arbeit wurde in der Zeit zwischen Dezember 2003 bis Dezember 2007 am Institut für Organische Chemie der Friedrich-Alexander-Universität Erlangen- Nürnberg durchgeführt. Dedication

FormyParentsandKati

-Scienceisfacts; justashousesaremadeofstones,soissciencemadeoffacts; butapileofstonesisnotahouseandacollectionoffactsisnotnecessarilyscience

HenriPoincare(1854-1912) Index of Abbreviations

t Bu ...... tert-Butyl BAM ...... Brewster Angle Microscopy Boc ...... tert-Butoxycarbonyl CV ...... Cyclic Voltammetry DBU ...... 1,8-Diaza-bicyclo[5.4.0]undecen-7-en DCE ...... 1,2-Dichloroethane DCU ...... Dicyclohexylurea DMA ...... 9,10-Dimethylanthracene DMAP ...... 4-Dimethylaminopyridine DMSO ...... Dimethyl Sulfoxide dpf ...... Days Post Fertilization EA ...... Elemental Analysis EDC ...... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride eq ...... Equivalent FAB ...... Fast Atom Bombardment FC ...... Flash Column Chromatography HOBt ...... 1-Hydroxybenzotriazole hpf ...... Hours Post Fertilization HPLC ...... High Performance Liquid Chromatography IPR ...... Isolated Pentagon Rule IR ...... Infrared Spectroscopy LB ...... Langmuir-Blodgett

I MW ...... Molecular Weight NBA ...... 3-Nitrobenzylalcohol NMR ...... Nuclear Magnetic Resonance PBS ...... Phosphate Buffered Saline

PCBM ...... [6,6]-Phenyl-C61 Butyric Acid Methyl Ester pf ...... Post Fertilization ppm ...... Parts per Million ROS ...... Reactive Oxygen Species RT ...... Room Temperature STM ...... Scanning Tunneling Microscopy SWCNT ...... Single Walled Carbon Nanotube TFA ...... Triﬂuoroacetic Acid THF ...... Tetrahydrofuran TLC ...... Thin Layer Chromatography UV/Vis ...... Ultraviolet-Visible Spectroscopy XPS ...... X-ray Photoelectron Spectroscopy

II Table of Contents

1 Introduction 1 1.1 NanostructuredMaterials ...... 1 1.2 TheDiscoveryofFullerenes...... 4 1.3 TheStructureofFullerenes ...... 5 1.4 PhysicalProperties...... 7

1.4.1 Thermodynamic and Kinetic Stability of C60 ...... 7

1.4.2 Solubility of C60 ...... 7 1.5 SpectroscopicProperties ...... 9 1.5.1 UV/Vis-Spectroscopy ...... 9 1.5.2 MassSpectroscopy ...... 10 1.5.3 NMRSpectroscopy ...... 11 1.5.3.1 3He and 1HSpectroscopy...... 11 1.5.3.2 13CSpectroscopy ...... 13 1.6 Electronic Structure and Reactivity of Fullerenes ...... 14

1.7 Spherical Aromaticity of C60 ...... 15

1.8 Chemistry of C60 ...... 16

2 Proposal 20

3 Results and Discussion 22 3.1 Water-soluble Amphiphilic Fullerene-Monoadducts ...... 22 3.1.1 Synthesis of Anionic Amphiphilic Monoadducts ...... 24

III Table of Contents

3.1.2 Synthesis of an Anionic Amphiphilic Monoadduct Carrying an UnsaturatedFattyAcid...... 33 3.1.3 Synthesis of a Cationic Amphiphilic Monoadduct ...... 39 3.1.4 Amphiphilic Fullerenes as Potential Drug Candidates ...... 44 3.1.4.1 Introduction and Background ...... 44 3.1.4.2 Antioxidant Activity ...... 47 3.1.4.3 Cytochrome C Binding ...... 52 3.1.4.4 In vivo Studies of the Amphiphilic Fullerenes using Ze- braﬁsh (Danio Rerio) Embryos as Model System . . . . 57 3.1.5 Mechanistic Aspects of the Reaction of Fullerenes with Superoxide 69 3.1.5.1 Cyclic Voltammetry Measurements of Amphiphilic Mono- adducts...... 70 3.1.5.2 Kinetic Measurements of Amphiphilic Monoadducts . . 73 3.1.6 Amphiphilic Fullerenes in Material Science Applications . . . . . 77

3.1.6.1 Formation of LANGMUIR-Films with Amphiphilic Fullerene- Monoadducts...... 79 3.1.6.2 Incorporation of the Amphiphilic Fullerene-Monoadducts in Organic Solar Cell Devices ...... 85 3.2 Triazole Dendrimers Based Fullerenes via "Click Chemistry" ...... 89 3.2.1 Synthesis of Novel Dendritic Triazol-Fullerenes ...... 92 3.3 Synthesis of Novel Fullerene-SWCNT Hybrids ...... 102 3.3.1 Covalent Sidewall Functionalization of SWCNT’s with a Fullerene- Monocarboxylic Acid Derivative ...... 103 3.3.2 Non-Covalent Functionalization of SWCNT’s with a Fullerene- PyreneDyad ...... 106

3.4 Supramolecular Approach for the Formation of C60-Bisadducts . . . . . 116 3.4.1 Metallomacrocycles as Tethers for Regioselective Cyclopropana- tion ...... 117 3.4.2 Hydrogen-bonded Dimers as Tethers for Regioselective Cyclo- propanation...... 120

IV Table of Contents

3.5 Synthesis of Novel Multiple Fullerene Arrays Consisting of Mixed C60- HexakisadductUnits ...... 125 3.5.1 Synthesis of Bisfunctionalized Janus-Type Fullerene-Dimers . . 127 3.5.2 Synthesis of a Fullerene-Rich Nanocluster ...... 137

4 Summary 142

4 Zusammenfassung 146

5 Experimental Part 151 5.1 ChemicalsandInstrumentation ...... 151 5.2 Synthetic Procedures and Spectroscopic Data ...... 154

Appendices 224 A Materials and Methods for the Determination of Biological Activity in vivo 224 B Materials and Methods for the Preparation and Examination of SWCNT- Fullerene-Hybrid ...... 229

References 231

V CHAPTER 1

1 Introduction

1.1 Nanostructured Materials

Although the idea of carrying on manipulations at smaller and smaller scales has been around for quite some time the birth of nanotechnology, at least on an ideological level, is usually traced back to a speech by RICHARD FEYNMAN at the December 1959 meet- ing of the American Physical Society. In his speech, he challenged his fellow scientists to find ways by which to create manufacturing, storage, and retrieval systems that are as efficient as DNA and to contain such systems in a submicroscopic, self-contained unit with the size of a cell. It would be over two decades before the first recognized paper on molecular nanotechnology was published.[1] The challenge in nanoscience is to understand how materials behave when sample sizes are close to atomic dimensions. Figure 1.1 for example shows an overview of artificial nanostructures, being of the same size as biological entities, which allows them to interact with biomolecules on the surface of the cell and inside it. When the characteristic length scale of the structure is in the 1- 100 nm range, it becomes comparable with the critical length scales of physical phenomena, resulting in the so-called "size and shape effects". This leads to unique properties and the opportunity to use

1 Chapter 1 Introduction

Figure 1.1: Artificial (top) and biological (bottom) nanostructures. such nanostructured materials in novel applications and devices. Phenomena occurring on this length scale are of interest to chemists, physicists, biologists, electrical and mechanical engineers, and computer scientists, making research in nanotechnology a frontier activity in materials science. Nanomaterials, which can be classified as carbon-based nanomaterials, nanocomposites, biological nanomaterials, nano- polymers, nano-glasses and nano-ceramics find and promise applications in a wide range of fields such as medicine (therapeutic agent, sensors, labelling), device technology (nanophotonics, solar energy conversion, opto-electronics) and chemical synthesis (catalysis). This thesis deals with the design and synthesis of functionalized carbon-based nanomaterials, to get more insight in structure-function relationships and

2 Chapter 1 Introduction to provide a predictive mechanism that will allow chemists to efﬁciently design nanomaterials that perform exactly as desired.

3 Chapter 1 Introduction

1.2 The Discovery of Fullerenes

The discovery of C60 has a long and very interesting history.[3] The structure of truncated icosahedron was already known about more

than 500 years ago. ARCHIMEDES is credited

for discovering the structure and LEONARDO

DA VINCI included it in one of his drawings. At the end of 1960’s, scientists were increasingly interested in non-planar aromatic structure, and thereafter the bowl-shaped corannu-

lene was synthesized.[4] In 1970, EIJI OSAWA realized that a molecule made up of sp2 hybridized carbons could have a spherical structure. He therefore made the ﬁrst proposal for

[5] C60. Then, a group of Russian scientists in-

dependently proposed the C60 structure, the Figure 1.2: Leonardo da Vinci‘s paper published by BOCHVAR and GAL’PERN "Truncated Icosahedron".[2] in 1973 not only predicted some properties of

[6] C60, but also of C20 (the smallest fullerene) as well. The ﬁrst spectroscopic evidence [7] for C60 and other fullerenes was published in 1984 by ROHLFING and coworkers.

Eventually in 1985 ROBERT CURL and RICHARD SMALLEY from Rice University, and

HAROLD KROTO from the University of Sussex discovered the fullerenes while doing experiments with a laser-vaporization supersonic cluster beam apparatus developed by

SMALLEY. Upon vaporizing graphite from the disk with high-power laser pulses, they found in their data, to their surprise, an indication of what appeared to be a cluster consisting of 60 carbon atoms. After furiously debating, building models, and consulting the literature, they theorized that the new 60-carbon structure had the form of a sphere comprising 20 hexagons and 12 pentagons, known to mathematicians as the before mentioned truncated icosahedron and familiar to the form of a soccer ball. The struc-

4 Chapter 1 Introduction ture reminded KROTO of the geodesic dome in Montreal, so the group decided to name the new molecule buckminsterfullerene after the architect R. BUCKMINSTER FULLER, who popularized the geodesic dome. Eleven years later, in 1996, they were awarded for the NOBEL PRIZE in chemistry for their accomplishments. However, the evidence for the existence of these molecules remained indirect until 1990, when the researchers

KRÄTSCHMER and HUFFMAN at the Max Planck Institute for Nuclear Physics in HEIDEL-

BERG,GERMANY used a carbon-arc plasma to produce the ﬁrst macroscopic quantities of them.[8] Since then fullerenes were extensively investigated and are constantly attracting great amounts of attention. In 1991, SCIENCE MAGAZINE named C60 "Molecule of the Year", professing it "the discovery most likely to shape the course of scientiﬁc research in the years ahead".

1.3 The Structure of Fullerenes

Fullerenes are all-carbon molecules which have the form of hollow, closed nets composed of 12 pentagons and n hexagons and the composition C20+2n (Euler‘s Theo- rem).[9] A second empirical rule that governs fullerene-type structures is the Isolated Pentagon Rule (IPR). This rule, based on both steric and electronic considerations, states that two pentagons may never share a common edge. Indeed, among the 1812 distinct fullerene isomers of buckminsterfullerene, only Ih-C60 containing 12 pentagons isolated by 20 hexagons (soccer ball structure) is formed in accordance with the IPR. The precise geometric structure of this isomer was determined by X-Ray analysis of

[10–12] [13] [14,15] pristine C60 at low temperature ,C60 derivatives ,C60 solvates , and solid- state 13C NMR measurements [16]. Such experimental findings definitively confirmed the postulated Ih-symmetry with a mean diameter of ∼ 7.1 Å for the sphere. Its VAN DER

WAALS diameter is ∼ 10.4 Å, and the distance across the cavity is ∼ 3.5 Å. As a result of the presence of both ﬁve- and sixmembered rings within the structure of C60, there are two types of bonds – namely bonds at the junction between two sixmembered rings ([6,6-bonds], mean distance = 1.391 Å), and bonds at the junction between a ﬁve- and a six-membered ring ([6,5-bonds], mean distance = 1.449 Å).[10, 17] The electronic

5 Chapter 1 Introduction structure [18–20] of the fullerenes is such that bonds at [6,6]-ring junctions have much double bond character, while bonds at [6,5]-ring junctions are essentially single bonds.

This arrangement results in C60 having a strongly bond-alternated structure which can be best described as a spherical tessellation of [5]radialene and 1,3,5-cyclohexatriene subunits (ﬁgure 1.3).

(a) (b) (c)

Figure 1.3: Illustration of (a) a [6,6]-bond; (b) a [6,5]-bond; and (c) the [5]radialene and 1,3,5- cyclohexatriene substructures of C60

The chemical behavior of C60 mainly depends on these structural properties:

• The 30 bonds at the junctions of two hexagons ([6,6]-bonds) are shorter than the 60 bonds at the junctions of a hexagon and a pentagon ([5,6]-bonds).

3 • The highly pyramidalized sp C-Atoms in C60 cause a large amount of strain en-

ergy within the molecule. The pyramidalization angle, deﬁned by HADDON and

[21,22] RAGHAVACHARI, of carbon atom orbitals in C60 structure is 11.6, an angle between sp2 and sp3 hybridization, which are 0 and 19.47 respectively (see ﬁg- ure 1.4).

6 Chapter 1 Introduction

θ σπ = 90° θ σπ = 109.47°

sp2 sp3

Figure 1.4: Pyramidalization of the carbon atom orbitals in C60. The angle of pyramidalization 2 3 χ = θ σπ - 90°is between the angles for sp and sp hybridization, which causes the high strain energy in C60.

1.4 Physical Properties

1.4.1 Thermodynamic and Kinetic Stability of C60

The heat of formation of pristine C60 have been determined theoretically and experimentally by calorimetry to be 10.16 kcal mol−1 per C-atom (relative to graphite with 0.0 kcal mol−1 per C-atom).[23] Also in the comparison to diamond (0.4 kcal mol−1 per C- atom), C60 is a energy-rich carbon allotrope. This comparatively high energy content is 2 originated in the high strain energy of C60 due to pyramidalization of the sp orbitals and [24] accounts for about 80 % of the heat of formation. This makes C60 to one of the most strained molecule, which is stable under standard conditions. In addition, the fullerene- cluster gets over the extremely high temperatures within the fullerene production. Un- der the conditions of ion beam experiments, other stable aromatic molecules like ben-

[25] zene instantly decompose. This extraordinary kinetic stability of C60 can also be seen in the FAB-mass-spectroscopic characterization of fullerene-derivatives, where the fragmentation is leading to the dominating molecular peak of unfunctionalized C60.

1.4.2 Solubility of C60

The solubility of C60 in organic solvents is important to enable puriﬁcation and chemical modiﬁcation. In general, the solubility in the majority of solvents is very low, because

C60 exhibit a high tendency for aggregation. On the other hand the interaction between

7 Chapter 1 Introduction

the solvent molecules and C60 is usually very weak, since the fullerene is a nonpolar molecule, which is hardly polarizable due to the large HOMO-LUMO gap. In summary, the solubility of C60 in polar solvents such as methanol and water is nearly zero. This low solubility can also be seen in the case of alkanes as solvents, whereas the solubility in chloroalkanes is slightly better. The best solubilities are obtained in aromatic solvents and carbon disulﬁde, which makes these solvents to the standard solvents for preparative use. Table 1.1 summarizes the solubilities of C60 in the most common solvents.[26–29]

Solvents Solubility [mg/mL]

acetonitrile 0.000 THF 0.00 methanol 3.5 · 10−5 water 1.3 · 10−11 n-pentane 0.005 cyclohexane 0.036 chloroform 0.16 dichloromethane 0.26 tetrachloromethane 0.32 pyridine 0.89 benzene 1.70 toluene 2.80 1,1,2,2-tetrachloroethane 5.30 anisole 5.60 chinoline 7.20 carbon disulphide 7.90 1,2,4-trichlorobenzene 8.50 1,2-dichlorobenzene 27.00 1-chloronaphthalene 51.00

[30] Table 1.1: Solubilities of C60 in the most common solvents.

8 Chapter 1 Introduction

1.5 Spectroscopic Properties

1.5.1 UV/Vis-Spectroscopy

The UV/Vis spectrum of C60 shows intense absorption bands between 190 and 410 nm (maxima at 326, 253 and 208 nm). These bands are due to symmetry-allowed singlet- singlet transitions from the HOMO to the LUMO+1 (see figure 1.8). In the visible region, the spectrum is characterized by a weak broad band between 440 and 620 nm with two maxima located at 598 and 543 nm [31–33] which correspond to symmetry-forbidden singlet-singlet transitions from the HOMO to the LUMO and LUMO+1. Chemical functionalization of C60 modifies the electronic structure of the fullerene chromophore. This is strongly reflected in the UV-Vis spectra of its derivatives. The degree of variation is dependent on i) the number of addends, ii) the geometric addition pattern in multi- adducts, and iii) the electronic structure of the functional group.[31,34] The derivatization of the fullerene core reduces its symmetry, thereby enhancing transition probabilities.

Consequently, C60 derivatives show stronger absorptions in the visible region with respect to pristine C60. The absorptions at 257 and 329 nm are hardly shifted as a result of the functionalization but less intense, which is consistent with the transition from a 60- to a 58-π- electron system.[31] Very characteristic for all [6-6]-closed (see chapter 1.8) monoadducts is the absorption at ∼ 425 nm.[35–37] The inﬂuence of subsequent functionalization can be nicely followed by the characteristic color of the corresponding derivatives in solution. The introduction of additional groups reduces the π- system of the C60-core, which changes the color from red (monoadducts) over orange (pentaadducts) to yellow (hexaadducts). In the case of hexaadducts the remaining π- system is signiﬁcantly reduced, whereas the remaining π-electrons are located within a cubic, cyclophane-like substructure of eight benzenoid rings.[38]

9 Chapter 1 Introduction

absorbance

400 500 600 700 800

200 300 400 500 600 700 800 wavelenght [nm]

Figure 1.5: UV/Vis spectrum of C60 in heptane. Inset: Region between 420 - 470 nm.

1.5.2 Mass Spectroscopy

Mass spectrometric characterization of fullerenes has been vital since the ﬁrst carbon clusters were produced.[39,40] Mass spectrometry has played a key role in the discovery of fullerenes, and continues to reveal the structures and properties of these unique molecules.

In mass spectroscopy , C60 and its derivatives can be measured with different ionization methods (EI, FAB, MALDI) depending on the nature of the addends. The molecular peak of C60 can be easily identiﬁed at m/z = 720 by the accompanied series of frag- ment ions (M+-24, M+-48, M+-72, etc.) due to so-called "shrink wrapping", where the

[41,42] molecule subsequently loses C2 units.

10 Chapter 1 Introduction

1.5.3 NMR Spectroscopy

1.5.3.1 3He and 1H Spectroscopy

Already early in the discovery history of the fullerenes, the question about the aromaticity of these new molecules appeared. While in former times aromaticity was defined by the smell and later by the reactivity of certain molecular structures, today physical characteristics are consulted for the definition of aromaticity. Next to structural parameters like delocalized atomic bonds, magnetic characteristics serve to classify a molecule as aromatic. Cyclic aromatic molecules exhibit a diamagnetic ring current, which manifests itself in high magnetic susceptibilities and "abnormal" chemical shifts in the NMR spectrum. The first forecasts of KROTO, SMALLEY, CURL and coworkers

[43] predicted C60 as an aromatic molecule, whose π-electrons would move freely on [44,45] the fullerene surface. As a consequence C60 would be highly diamagnetic and would have to exhibit a particularly high magnetic susceptibility, which could not be

[46,47] conﬁrmed experimentally. The determined magnetic susceptibility χ of C60 with χ = -260 CGS ppm mol−1 (=b -4.3 CGS ppm per mol of C) is smaller than those from diamond with χ = -5.5 CGS ppm per mol of C.

[48,49] Nevertheless there are ring currents present in C60. The working groups HADDON as well as ZAMESI and BOWLER [50] could show that in the pentagons strong paramagnetic ring currents are present, while weaker diamagnetic ring currents circulate over larger areas of the fullerene surface (ﬁgure 1.6).

Figure 1.6: Left: Diamagnetic (blue) and paramagnetic (red) ring current in the corannulene [50] substructure of C60. Right: Ring current contour map of C60.

The paramagnetic and diamagnetic ring currents neutralize each other almost, which

[48] explains the small macroscopic magnetic susceptibility of C60. For the experimen-

11 Chapter 1 Introduction tal proof of the ring currents a method had to be found, which can measure the local magnetic properties. For this purpose NMR spectroscopy is a useful tool. Fullerenes have internal cavities, large enough to encapsulate atoms [51,52] and so it is possible to achieve noble-gas doped endohedral compounds. Because 3He has a spin of 1/2 and is an excellent NMR nucleus, it can be used as a probe for the magnetic shielding envi- ronment inside the fullerene cavity.[53] It was found, that the 3He nucleus encapsulated

3 in C60 is shielded by δ = -6.36 ppm, relative to free He. This chemical shift is a result of the ring currents in the π-system and the shielding effects of the sigma framework. Measurements on helium complexes of fullerene derivatives showed, that the chemical shift of the helium atom in homofullerenes with [5,6]-opened structure is almost the same shift as obtained with pure C60. In derivatives with [6,6]-closed structure, the He- signal is shifted highﬁeld for about 3 ppm, with the exception of methanofullerenes. The reason for the different relative shifts is to be seen in the respective electronic structures of the fullerene derivatives. While the electronic structure of a homofullerene agrees to a large extent with that of unfunctionalized C60, the π-electronic structure in a [6,6]-closed structure is distinctly disturbed in relation to C60. Methanofullerenes with [6,6]-closed structure stand structurally between the homofullerenes and the other [6,6]-closed structures. Through to addition of a cyclopropane ring, whose bondings possess partial double bond character, the 60 π-electron structure of C60 is disturbed, but the single bond located between the hexagons still possesses partial double bond character and locates the methanofullerenes between a 60 π-electron and a 58 π- electron system.

3.32 ppm 6.79 ppm

O O H H OEt EtO

1 2

Figure 1.7: Chemical Shifts for protons in homofullerenes.[54]

12 Chapter 1 Introduction

Beside helium atoms in the inside of the fullerene cage, also hydrogens attached close to the fullerene surface can serve as probe for local ring currents (ﬁgure 1.7). In the case of the homofullerenes 1 and 2 the π-electron system is hardly disturbed in relation to C60, what can be directly seen in the UV/Vis-, NMR-spectra and the electrochem- istry of the 3He doped complexes. In compound 1 the hydrogen atom is located over a hexagon and calculations indicate, that, because of the diamagnetic ring currents in the hexagon, the signal for the proton is shifted high-ﬁeld, as it is known for the [5]paracyclophane. On the other hand, paramagnetic ring currents, that are present in the pentagons, should magnetically shield the hydrogen atom in 2, which was indeed experimentally proven by different groups.[54–56]

1.5.3.2 13C Spectroscopy

In pure C60 all C-atoms are chemically and magnetically equivalent and therefore only one resonance signal is to be expected. In deuterated benzene, the resonance signal appears at 143.2 ppm and is at the low end of the range, where resonances for quaternary C-atoms of unsubstituted polycyclic aromatics are expected. The qualitative estimation of the chemical shift for 13C-NMR-signals in fullerenes is not simple, since apart from the local curvature also ring current effects are responsible for the observed chemical shift.[57,58] As general trend it can be stated however that a pyramidalization of the unsaturated system leads to a low field shift.[59] This trend is also recognizable in the case of the aromatics corannulene, fluoranthene and pyracylene, where the C-atom of the curved corannulene shows a remarkable low-field shift, compared to the planar molecules like fluoranthene and pyracylene. With this strong dependence of the chemical shifts from local parameters, the 13C-NMR-spectroscopy is an important method for the characterization and determination of the symmetry of fullerene derivatives.

13 Chapter 1 Introduction

1.6 Electronic Structure and Reactivity of Fullerenes

C60 exhibits highly interesting electrochemical properties which are related to its electronic conﬁguration. The before mentioned rehybridization is in part responsible for the high electron afﬁnity of C60 since it considerably reduces the energy of the lowest unoccupied molecular orbital (LUMO). A complete picture of the electronic structure of

C60 is obtained by Hückel Molecular Orbital (HMO) theory which predicts an electronic conﬁguration with a ﬁve-fold degenerate HOMO (hu) and the three-fold degenerate low-lying molecular orbitals LUMO (t 1u) and LUMO+1 (t 2u) separated by a moderate energy gap of 0.757 β.[60–62]

e 1 2u LUMO (+1)

0.5 t 2u LUMO+1 (0.38) t 1u LUMO (0.14) 0 2.00 β

Energy( )β 0.757 β

-0.5 h u HOMO (-0.62)

e -1 1u HOMO (-1)

Figure 1.8: Hückel Molecular Orbital (HMO) diagram of C60 and benzene.

In accordance with the degeneration of the LUMO level, the redox chemistry demonstrates the ability of [60]fullerene to accept up to six electrons.[63] The systematic proof for the triple degeneracy of the LUMO level of C60 came with the detection of fullerene n− anions C60 (n = 1 - 6), which can be generated by subsequent reversible one-electron reductions. The separation between any two successive reductions is relatively constant and amounts to ∼ 450 ± 50 mV.[64] This correlates well with the triple degeneracy

14 Chapter 1 Introduction

of the LUMO level. Considering the HMO diagram (ﬁgure 1.8), the oxidation of C60 consists in the removal of an electron from the low-lying HOMO, leading to an important destabilization of the π-electron system. Correspondingly, the ﬁrst one-electron oxida-

+ tion of C60 occurs at a highly positive potential, 1.26 V vs Fc/Fc in tetrachloroethane.

The difference in potential between the ﬁrst oxidation and the ﬁrst reduction of C60 1/ 2 1/ 2 (Eox − Eox = 2.32 V) is a good measure of the HOMO-LUMO gap in solution and correlates well with the calculated value (1.5 - 2.0 eV).[65]

Since C60 exhibits a highly rigid framework in the ground state as well as in the excited state, the reorganization energies are very low.[66] The reducibility of fullerenes together with their small reorganization energy associated with nearly all their reactions, make them especially interesting building blocks.

1.7 Spherical Aromaticity of C60

The magnetic properties of fullerenes clearly show, that the delocalized character of the conjugated π-electrons can cause the establishment of diamagnetic or paramagnetic ring currents within the loops of the hexagons and pentagons. Neutral C60, for instance, exhibits no pronounced overall aromaticity since it contains diatropic hexagons and paratropic pentagons and was labeled ambiguously aromatic. BÜHL and HIRSCH have showed that the treatment of the π-electrons system as a spherical electron gas allows to determine the nature of three-dimensional aromaticity of fullerenes and other cage compounds.[61] The aromaticity of two-dimensional annulenes in singlet ground states follows the Hückel rule, i. e. annulenes with 4N+2 π-electrons are aromatic, those with 4N π-electrons are antiaromatic. Due to their closed-shell structures, annulenes with 4N + 2 π-electrons are not distorted (Dnh-symmetry) and show strong diamagnetic ring currents. The rule is reversed for triplet open-shell analogues.

The aromaticity of icosahedral fullerenes (C20,C60 and C80) and their cluster distortions depends on the number of delocalized electrons in the valence shell. The maximum of spherical aromaticity of a cluster can only be achieved with 2(N + 1)2 electrons ﬁlling the shell completely. The 2(N +1)2 rule represents the spherical analogue of the Hückel

15 Chapter 1 Introduction rule for planar annulenes.

1.8 Chemistry of C60

Since the discovery of fullerenes, a broad variety of chemical modifications with C60 were performed. In general these modifications can be classified in five different topics (figure 1.9):[67–73]

Figure 1.9: Overview of the possible modiﬁcations of C60. a) Exohedral functionalization; b) Heterofullerenes; c) Endohedral functionalization; d) Cage-opening modiﬁcations and d) Alkali metal fullerides.

• a) Exohedral addition reactions, including nucleophilic- and radical additions, cycloadditions, hydrogenations, oxygenation and halogenation

• b) Substitution of carbon atoms in the fullerene framework with different atoms, e.g. boron or nitrogen, leading to heterofullerenes

16 Chapter 1 Introduction

• c) Encapsulation of one or more atoms inside the fullerene cage, yielding endohedral fullerenes

• d) Ring-opening and fragmentation reactions, which could be used for subsequent endohedral functionalization

• e) Reduction reactions with electropositive metals, e.g. alkali- and alkaline earth metals, yielding alkali metal fullerides

Considering the previously mentioned properties of C60, the following general reactivity patterns can be emerged:

• Due to the low-lying degenerate LUMO level the chemical reactivity of the formal double bonds is close to that of a strained, electron deﬁcient polyoleﬁn.

• The relief of strain due to a change in hybridization at the reacting carbons on the spherical surface is the driving force for addition reactions.

• The regiochemistry of all addition reactions is governed by the minimization of [5,6]-double-bonds within the fullerene cage. The relocation of each double-bond into the [5,6]-bond costs about 8.5 kcal/mol. Therefore addition reactions normally take place at [6,6]-bonds, whereby 1,2-additions (preferred addition) introduce eclipsing interactions between the addends. For a combination of sterically demanding addends, [1,4]- and [1,6]-addition can take place.

[6,5]-open [6,5]-closed [6,6]-open [6,6]-closed

R R R R R R R R

not a minimum + 6 kcal/mol + 21 kcal/mol energy structure 0 kcal/mol

Figure 1.10: Possible isomeric methanofullerenes and their relative energies from PM3 calculations (methanoannulene-type subunits highlighted in red).[74]

17 Chapter 1 Introduction

The following itemization lists the mainly used reactions for the chemical exohedral functionalization of C60:

• Cyclopropanation with carbon nucleophiles [75,76]

• DIELS-ALDER-type [4+2]-cycloadditions [77–79]

• Photochemical [2+2]-cycloaddition of α, β-unsaturated ketones [80,81]

• [3+2]-cycloaddition using diazo compounds, azomethine ylides and azides [82–89]

• Nucleophilic addition of Grignard or organolithium compounds

• Complexation of transition metal complexes [90]

• Oxygenation, osmylation, halogenation and hydrogenation

Since the work described in this thesis is mainly based on methanofullerenes, a brief account of the synthetic approaches toward methanofullerenes is given in the following section. For more details about the other reactions, as well as the different modiﬁcation possibilities in ﬁgure 1.9, the reader is referred to the following reviews [67,91–93] and books.[68,94,95]

O O 4 O O O O O O Br EtOOC Br Br 3 COOEt

NaH or DBU toluene

COOEt COOEt

Scheme 1.1: Cyclopropanation of C60 by nucleophilic addition of α-bromomalonate carbanions (BINGEL-reaction).

18 Chapter 1 Introduction

The cyclopropanation of C60 with carbon nucleophiles is one of the most commonly used reaction in fullerene derivatization due to the high yields and variability. The orig- inal conditions for the BINGEL reaction involve the treatment of bromomalonates with

[75] NaH in the presence of C60. From a mechanistic point of view a two-step process is involved at this type of reaction. Deprotonation of the α-bromomalonate 3 affords the α-bromo carbanion 4 that adds to C60, giving the anionic fullerene intermediate 5. In a second step, the displacement of bromide by an intramolecular nucleophilic substitution gives the methanofullerene 6 (scheme 1.1). Since the synthesis of more complex bromomalonate precursors is often rather difﬁcult, HIRSCH modiﬁed the reaction protocol by preparing the α-halomalonate in situ from the corresponding malonate in the presence of CBr4 and a non-nucleophilic base (DBU) (so called BINGEL-

HIRSCH reaction).[96] In some cases the use of iodine as halogenation reagent can be preferable.[97] These reactions proceed under mild conditions and can be adopted for a wide range of functional groups. The corresponding methanofullerenes are in general thermally stable, with a well-deﬁned directionality of the ester groups due to the highly rigid cyclopropane ring.

19 CHAPTER 2

2 Proposal

The aim of this work is the synthesis and characterization of novel fullerene architectures, which are accessible through exohedral functionalization of parent C60. The application of these compounds in material science and biomedicine should be considered, and the derivatives should be speciﬁcally designed to provide properties, which make the implementation possible. In the ﬁrst instance a series of anionic and cationic amphiphilic fullerene derivatives (amphifullerenes) should be synthesized. To minimize the disruption of the fullerene

π-system with increasing number of addends, these amphiphiles should be C60-monoadducts. Therefore the amphiphilic character has to be introduced by the attachment of an asymmetric malonate, which carries a hydrophilic (dendritic) and a lipophilic (alkyl chain) part. This concept enables the ﬁne-tuning of the ratio between hydrophilic and lipophilic part within the molecule, which is of importance for the bioavailability of these substances. In cooperation with C-SIXTY Inc. and PHYLONIX Pharmaceuticals Inc., the amphifullerenes should be investigated concerning their antioxidant activity against reactive oxygen species (ROS) in vitro and their ability to protect cells and tissue from oxidative injury and cell death in vivo. Furthermore the amphifullerenes should be utilized for the construction of nanostructured ﬁlms by the Langmuir-Blodgett (LB) technique.

20 Chapter 2 Proposal

In this context, the ability to prepare homogeneous layers with control over molecular organization, film thickness and architecture should be studied. In the second part of this work a novel concept for the efficient synthesis of neutral and charged water-soluble fullerene structures should be developed. Particularly the copper(I)-catalyzed HUISGEN 1,3-dipolar cycloaddition should be examined to its ap- plicability in fullerene chemistry and the influence of the multiple triazole linkages on the water-solubility should be studied. In line of this work novel hybrid nanomaterials should be prepared, consisting of fullerenes covalently and non-covalently attached to the outside surface of single wall carbon nanotubes (SWCNTs). This should be accomplished by the introduction of suitable functionalities onto the fullerene sphere, which allows to immobilize these derivatives onto the SWCNT surface. The structural investigation of these hybrids should include microscopic techniques, to determine the degree of functionalization and the constitution of the supramolecular hybrids.

The regioselective bisfunctionalization of C60 is generally accomplished by the use of macrocyclic or open-chain malonates as tethers for the subsequent nucleophilic cyclopropanation. In a new approach the preorganization of the malonates should be obtained by complexation with different metall ions and by dimerization via hydrogen- bonding. Furthermore a new synthetic strategy should be developed, which enables the synthesis of fullerene-rich macromolecules with different functionalities within the same molecule. This should lead to aesthetically pleasing architectures, where two ore more

C60 cages are covalently connected by bridging organic addends.

21 CHAPTER 3

3 Results and Discussion

3.1 Water-soluble Amphiphilic Fullerene-Monoadducts

Amphiphile is a term describing a chemical compound possessing both hydrophilic and hydrophobic properties. Such a compound is called amphiphilic or amphipathic. This forms the basis for a number of areas of research in chemistry and biochemistry, notably that of lipid polymorphism. As mentioned before, to obtain amphiphilic properties, the structure has to consist of two different functional groups. The hydrophobic group is typically a large hydrocarbon moiety, such as long saturated or polyunsaturated alkyl chains. The hydrophilic group falls into one of the following categories:

• charged groups, such as anionic groups (carboxylates, sulfates, sulfonates, phos- phates) and cationic groups (ammonium and pyridinium salts)

• polar, uncharged groups, such as polyalcohols or polyethers

• amphoteric groups

In the last few years HIRSCH and coworkers succeeded in synthesizing a broad variety of amphiphilic [60]fullerene derivatives, which differ in the number and the addition

22 Chapter 3 Results and Discussion pattern of the attached functional groups. Exemplary representatives of these derivatives are for example [5:1] hexakisadducts with an octahedral addition pattern, using a Newkome-type amide dendron as a hydrophilic addend and ﬁve didodecyl malonates as lipophilic addends. This globular amphiphile dissolves in water, forming unilamellar vesicles with diameters typically between 100 and 400 nm, and reveals a very small critical micelle concentration (cmc).[98,99] Latest results deal with the synthesis of amphiphilic [3:3] hexakisadducts, using a trisadduct precursor with an e,e,e-addition pattern as starting material. For the completion of the octahedral addition pattern, DMA template-mediated cyclopropanation was accomplished with different polar and dendritic ionic malonates. These new compounds were examined in detail for their tendency to form micelles and to build stable monolayers.[100]

All these examples use C60 as structure-forming core, systematically functionalized with different addends. It is well known, that the chemical reactivity and physical properties change, when the conjugated π-electron chromophore of the fullerene is reduced as a result of increasing functionalization. It has been shown, that in the series of mono- through hexakisadducts reductions become increasingly difﬁcult and more irreversible with increasing number of addends. Hence, with incremental functionalization of the fullerene, the LUMO of the remaining conjugated framework is raised in energy. [101]

In order to almost retain the unique properties of pristine C60, but to improve the water- solubility and bioavailability, the ﬁrst part of this thesis deals with the synthesis of a series of amphiphilic [60]fullerene monoadducts.

23 Chapter 3 Results and Discussion

3.1.1 Synthesis of Anionic Amphiphilic Monoadducts

In the ﬁrst instance anionic amphiphiles were synthesized, which carry alkyl chains, differing in number and length, as apolar addends. NEWKOME-type dendrons of different generations provide the polar part and serve for the solubility in aqueous solutions. The crucial point in the synthesis of amphiphilic monoadducts is the facile generation of unsymmetrical malonate precursors that serve as addends. Previously, unsymmetrical malonates have been prepared by the successive esteriﬁcation of malonic acid with suitable alcohols (scheme 3.1).[102,103]

O O HO OH

O O 2 eq HO HO 1 eq O O 7 DCC, DCC, 7 DMAP DMAP

O O O O O O O O O HO O O 8 O 12 O

TFA 1 DCC, R OH DMAP

O O O O HO OH O O O R1O O O 9 O 13 O

1 DCC, TFA R OH DMAP

O O O O R1O OH OH O O R1O O O 10 O 14 O

2 DCC, 2 DCC, R OH DMAP R OH DMAP

O O O O R1O OR2 OR2 O O R1O O O 11 O 15 O

Scheme 3.1: Different pathways for the synthesis of unsymmetrical malonates.

24 Chapter 3 Results and Discussion

In principle, the asymmetry can be obtained in different states of the reaction sequence. One possibility, as described in scheme 3.1 (left side), is the formation of a symmetric malonate 9, which can be monofunctionalized by a stoichiometrical controlled reaction with alcohols or amines. The sequence on the right side in scheme 3.1 starts with the formation of the asymmetric malonic derivative 12, which can be further bisfunctionalized by sequential reaction with different alcohols or amines. However, these approaches led in a number of cases to separation problems and unsatisfactory yields. In a new approach, MELDRUM’S acid (2,2-dimethyl-1,3-dioxane-4,6-dione) was allowed to react with a long chain alcohol to give the monoalkyl malonate (see scheme 3.2). This reaction can be carried out in large scale with almost quantitative yields and without extensive puriﬁcation.

OO 130 °C, 3 h O O + R1OH O O R1O OH

1 1 R = (CH2)5CH3 R = (CH2)5CH3 17 1 1 R = (CH2)17CH3 R = (CH2)17CH3 18 1 1 R = CH((CH2)7CH3)2 16 R = CH((CH2)7CH3)2 19

Scheme 3.2: New approach for the synthesis of unsymmetrical malonates, using MELDRUM’S acid.

It is important to notice, that these monoalkyl malonates can only be obtained by the use of primary and secondary alcohols. Tertiary alcohols do not react with MELDRUM’S acid, presumably to their decreased nucleophilicity. As the second terminus, an alcohol is required that contains a protected carboxylic group in its periphery serving as anchor point for the introduction of the dendritic building blocks. This alcohol also serves as a spacer, by increasing the distance between the malonate and the dendritic group.

This is an essential requirement to obtain adequate yields in the subsequent BINGEL-

HIRSCH reaction. The synthesis of the spacer was accomplished using a modiﬁed literature method [104] by treating ε-caprolactone with benzyl bromide leading to the formation of 20 (scheme 3.3). The protection of the terminal carboxylic groups was carried out by reaction with isobutene to give the tert-butyl ester 21. After removal of the benzyl group by catalytic hydrogenation with Pd/C as catalyst, the deprotected alcohol 22 was obtained in 71

25 Chapter 3 Results and Discussion

O R = H 20 O KOH, BzBr OR H2SO4, O t O R = Bu 21

Pd/C, H2

O HO 22 O

DCC, DMAP 17, 18, 19

1 O O R = (CH2)5CH3 t 23, 24, 25 1 OR R = Bu R = (CH2)17CH3 R1O O TFA 1 R = H 26, 27, 28 R = CH((CH2)7CH3)2 O

EDC, DMAP, EDC, DMAP, 1-HOBt 1-HOBt O 29 30 O O O O O O HN O O O O O H O O H O 1 N N H R O O O R1O O N O O O O O O 31, 32, 33 O O 34, 35, 36 O NH O O O O O O O O

C60, CBr4, DBU C60, CBr4, DBU

RO O O OR RO O OR HN OR O O O O H O O H O N N H R1O O OR R1O O N O OR O O O O O OR O NH O O OR RO OR O O OR R = tBu 37, 38, 39 R = tBu 40, 41, 42 TFA TFA R = H 43, 44, 45 R = H 46, 47, 48

Scheme 3.3: Optimized multistep synthesis of the amphiphilic monoadducts 1st generation 43, 44, 45 and 2nd generation 46, 47, 48.

26 Chapter 3 Results and Discussion

% overall yield. The coupling of the spacer unit 22 with the monoalkyl malonates

17, 18, 19 via the STEGLICH coupling procedure [105–107] gave the protected unsymmetrical malonates 23, 24, 25 in reasonable yields. After deprotection of the tert-butyl protection group with TFA in chloroform, the corresponding carboxylic acids 26, 27, 28 were coupled with the dendritic building blocks 29 and 30 via the in situ activation with EDC.[108] The dendrimers are based on the iterative architecture principle of the branch- ing of branches and represent NEWKOME-type dendrimers, which were synthesized following literature procedures.[109,110] The use of EDC instead of DCC as activation reagent for the formation of the first generation dendritic malonates 31, 32, 33 and the second generation malonates 34, 35, 36 turned out to be more effective. The higher yields (about 10 % more yield) of the coupling reaction with EDC and the saving of time during the purification progress justifies the higher price of EDC compared to DCC. The monoadducts 37, 38, 39 and 40, 41, 42 can be obtained via the BINGEL-HIRSCH reaction with CBr4 in good yields. However the use of iodine as halogenation reagent was not appropriate in this case, leading to decreased conversion rates and increased formation of side products. For the deprotection of the peripheral tert-butyl esters the monoadducts 37, 38, 39, 40, 41, 42 were dissolved in formic acid and stirred at rt for 48 h. Purification of the crude deprotected monoadducts can be obtained by reprecipitation from THF/diethyl ether in the case of 43, 44, 45 and from methanol/diethyl ether in the case of 46, 47, 48 (scheme 3.3). All monoadducts were isolated as red brownish solids. Surprisingly the amphifullerenes 43, 44, 45, where the hydrophilic part is represented by the 1st generation dendron, consisting of three carboxylic acids, showed no significant water solubility at pH = 7.2 or higher pH values. In previously synthesized trisadducts, where carboxylic acids were introduced by a tether controlled synthesis, three ionic groups were sufficient enough to promote a remarkable water-solubility.[111] This leads to the conclusion, that not only the number of carboxylic groups is important, but also the arrangement over the fullerene core plays an essential role for the solubility. However the water-solubility can be promoted by the use of DMSO as co- solvent. For the aqueous solutions the monoadducts were dissolved in a small amount of DMSO followed by the addition of water (pH = 7.2). Such solutions are remarkable

27 Chapter 3 Results and Discussion

100 2107 4.6E3

80 3.7E3

60 2.7E3

40 1.8E3

20 9.1E2 1902 1970 21612214 2273 2387 2483 2548 261226762734 2858 0 0.0E0 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800

Figure 3.1: FAB mass spectrum of 2nd generation amphiphile 47. stable and can be stored for several weeks. The 2nd generation analogs 46, 47, 48 on the other hand showed very good solubility in water at pH = 7.2. The characterization of the amphifullerenes was carried out by means of 1H- and 13C-NMR, by mass spectrometry and UV/Vis spectroscopy. Due to the similar spectroscopic properties of the amphiphiles the following figures show the characterization of 44, representative for the 1st generation amphiphiles and 47 for the 2nd generation amphiphiles. Figure 3.1 shows the FAB mass spectrum of 47. The dominating molecular peak at 2107 and the absence of fragments with higher molecular weight clearly indicates the complete deprotection. The quantitative conversion into the corresponding carboxylic acids can also be followed in the 1H-NMR spectrum by the disappearance of the characteristic signal for the tert-butyl groups at about 1.41 ppm. The 13C-NMR spectra in figure 3.2 show the spectroscopic changes within the conversion of the malonate 32, via the monoadduct 38 to the amphifullerene 44. The splitting of the signals for the carbonyl-C-atoms (3,5) at 164 ppm clarifies the asymmetry of the molecules. The comparison of the spectra of 38 and 44 show an abundantly clear low-field shift for the carbon atom 4. The sp2-region of 38 exhibits 21 carbon signals, whereas 9 signals show double intensity. This is in accordance with the 31 expected signals for a monoadduct with Cs-symmetry. In the case of the Cs-symmetrical amphifullerene 44, the sp2-region contains 28 signals, whereas 3 signals show double

28 Chapter 3 Results and Discussion

14 13 * 14 11 12 2 4 6 7 8 C 10 -H-2 1 3 5 9 32

10 11 3 5 13 12

2,6 4 1 167.2167.1 167.0 9 8 7

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 14 13 * 11 12 14 2 4 6 7 8 10 9 1 3 5 C -H-2 3 5

11 12 164.3 164.1 163.9 38 13 10 sp2 -C

1 sp3 -C 9 8 2,6 4 7

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

12 * * 2 4 6 7 8 10 11 9 C 1 3 5 -H-2

10 11

12 3 5 44 sp2 -C

164.2 164.1 164.0 9 1 7 8 sp3 -C 4

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ChemicalShift(ppm)

13 Figure 3.2: C NMR of malonate 32 (100.5 MHz, RT, CDCl3), protected monoadduct 38 (100.5 MHz, RT, CDCl3) and deprotected monoadduct 44 (100.5 MHz, RT, THF- d8).

29 Chapter 3 Results and Discussion intensity. The disappearance of the signals for the tert-butyl groups at 80.91 ppm and 28.31 ppm in the spectrum of 44 shows the complete cleavage of the ester groups. Figure 3.3 represents the spectroscopic changes within the conversion of the malonate 35, via the monoadduct 41 to the amphifullerene 47 in the case of the 2nd generation systems and show almost the same chemical shifts as observed in the 1st generation systems. The signals for the amide and ester carbon atoms are furthest shifted to low-ﬁeld and can be detected according to their number in the molecule in the intensity ratio 1:3:9. In contrast to the spectrum of 44, the spectrum of the deprotected monoadduct 47 shows a very bad signal to noise ratio, even at very high scan rates (≈ 20000 scans). Furthermore a broadening of the signals can be detected, indicating the aggregation tendency in polar solvents. Figure 3.4 shows the UV/Vis-spectra of the protected monoadduct 38 and deprotected monoadduct 44, exemplary for the 1st generation amphifullerenes. The UV/Vis spectrum of 38 reveals the unique features of a monoadduct, notably the small characteristic absorption peak at 425 nm. In the case of the deprotected monoadduct 44 the UV/Vis spectrum in DMSO indicates the formation of micellar organization in solution, whereas the slightly shifted absorption peak at 428 nm is still detectable. In buffered aqueous solution at pH = 7.2 (solubility was promoted by the addition of a small amount of DMSO), the aggregation behavior is clearly observable, which results in a characterless slope with a broadened maximum at 328 nm. As expected, the similar trend can be followed in the UV/Vis spectra of the 2nd generation amphifullerene 47 (ﬁgure 3.5). Contrary to the 1st generation analog even in polar-aprotic solvents the aggregation is highly facilitated, which can be seen in the almost identical slope of the spectrum in DMSO and buffered H2O, respectively. An effect of the different alkyl chains within the series of amphifullerenes could not be explored by UV/Vis spectroscopy, in fact the measured spectra show the identical characteristics as mentioned above. More sensitive methods, like cryo-transmission electron microscopy (cryo-TEM) would be useful to study the effect of the alkyl chain on the size and shape of these micellar structures.

30 Chapter 3 Results and Discussion

18 17 15 16 13 14 11 12 2 4 6 7 1 8 9 10 18 3 5 * 17 35 C -H-2

16 10,11 3 5 14,15 12 8 9,13 1 2,6 4 173.5 173.0 172.5 167.1 167.0 166.9 7

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 18 17 15 16 13 14 11 12 2 4 6 7 1 8 9 10 3 5 * 18

C -H-2 41 17 16 3 5 10,11 12 2 C 14,15 8 sp - 9,13 sp2 -C 1 173.4 173.0 164.2 163.8 2,6 4 7

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

15 16 13 14 * 14 16 11 12 2 4 6 7 1 8 9 10 10,11 15 3 5

C -H-2 47 9,13 sp2 -C sp3 -C 1 8,12 3,5 2,6 4 7

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 13 Figure 3.3: C NMR of malonate 35 (100.5 MHz, RT, CDCl3), protected monoadduct 41 (100.5 MHz, RT, CDCl3) and deprotected monoadduct 47 (100.5 MHz, RT, DMSO- d6).

31 Chapter 3 Results and Discussion

absorbance

38 in CH2Cl2 44 in DMSO 44 in H2O atpH = 7.2

300 350 400 450 500 550 600 wavelength[nm]

Figure 3.4: UV/Vis spectra of protected monoadduct 38 (black) and deprotected monoadduct 44 in DMSO (red) and H2O (pH = 7.2) (blue).

absorbance

41 in CH2Cl2 47 in DMSO 47 in H2O atpH = 7.2

300 350 400 450 500 550 600 wavelength[nm]

Figure 3.5: UV/Vis spectra of protected monoadduct 41 (black) and deprotected monoadduct 47 in DMSO (red) and H2O (pH = 7.2) (blue).

32 Chapter 3 Results and Discussion

3.1.2 Synthesis of an Anionic Amphiphilic Monoadduct Carrying an Unsaturated Fatty Acid

The blood-brain barrier (BBB) is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that allow relatively free exchange of substance across / between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. Thus the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain. Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of most brain disorders. Therapeutic molecules and genes that might otherwise be effective in diagnosis and therapy do not cross the BBB in adequate amounts. Given the benefits and substantial properties of C60 as therapeutic drugs, we considered that it may be feasible to increase the efficiency by modifying its access to brain target sites. As a strategy aimed at testing this hypothesis, we followed previous findings with ester or amide derivatives of fatty acids important in the composition of brain tissue, including some that are effectively transported into the central nervous system (CNS). Such derivatives can markedly enhance entry of some compounds into the brain, including for example hydrophilic agents such as GABA and dopamine.[112,113] Accordingly, we considered preparing derivatives of [60]fullerene and fatty acids that are abundant in brain tissue but must be supplied exogenously and transported through the blood-brain barrier. We hypothesized that such compounds might increase and prolong the uptake of water-soluble fullerenes into the brain. Docosahexaenoic acid (DHA) is a particularly interesting candidate for attachment to C60 because it is vigorously transported into the CNS and accounts for a high proportion of cerebral fatty acid content.[114,115] Moreover, in earlier work, DHA proved to be particularly effective in facilitating entry of hydrophilic compounds into the brain.[113] DHA is a 22-carbon chain, omega-3 unsaturated fatty acid, containing six cis-double bonds. It is present in neuronal tissue, nerve termi- nals and synapses, predominantly within membrane phospholipid constituents phos-

33 Chapter 3 Results and Discussion

=^ R1 EDC, DMAP OH + HO

OH O OH O O

49 50

O O O EDC, DMAP O O HO + O O HO OH HO O 22 51 O EDC, DMAP 50

O O R = tBu 52 R1 OR TFA O O R = H 53 O

EDC, DMAP, 30 1-HOBt O O O O O O HN O O O O H O 1 N H R N O O O 54 O O O O NH O O O O O O O C60, CBr4, DBU O

RO O O OR RO O HN OR O O O H O O N H O O N O OR O O O O NH O O OR RO OR O O OR R = tBu 55 TFA R = H 56

Scheme 3.4: Synthesis of the DHA functionalized monoadduct 56.

34 Chapter 3 Results and Discussion phatidylethanolamine, phosphatidylserine, and phosphatidylcholine.[115,116] The fatty acids are transported into the CNS by incompletely defined mechanisms that may include receptor-mediation.[115,117,118] DHA can be obtained by extraction from naturally occurring oils, such as marine animal oil and various vegetable oils. The resulting mixture of pure fatty acids is then subjected to separation by means of urea complex- ing to remove saturated fatty acids and most mono-unsaturated fatty acids. The urea is then removed from the filtrate which is subsequently subjected to low temperature fractional crystallization in the presence of an organic solvent such as acetone to give substantially pure polyunsaturated fatty acid.[119] Based on the preceding considerations, a water-soluble, DHA-functionalized fullerene monoadduct was synthesized. Scheme 3.4 shows the multistep synthesis of the DHA functionalized water-soluble monoadduct. In the first step the all-cis-docosahexaenoic acid was esterificated with glycol in order to receive the alcohol 50. Construction of the asymmetric malonate via the MELDRUM’S acid method failed in this case, leading to a decomposition of the starting material, probably by the thermal instability of the unsaturated fatty ester. To overcome this problem, the asymmetric malonate unit 51 was synthesized using the procedure described in scheme 3.1, followed by the coupling with 50 to yield the malonate 52. After deprotection of the tert-butyl ester with TFA in CH2Cl2, the corresponding carboxylic acid 53 was amidated with the 2nd generation dendron 30 by the use of EDC. The cyclopropanation of 54 with C60 gave the monoadduct 55 with traces of a less polar impurity (maybe side reactions with the DHA group). The repeated separation of these impurities with flash column chromatography failed in this case and made further purification by preparative HPLC necessary. Deprotection to 56 was accomplished in a TFA/CHCl3 mixture and was completed after 48 h in quantitative yield. The characterization of 55 is given in the following figures. The 1H NMR spectrum of 55 shows the characteristic resonances at 5.29 and 2.75 ppm for the polyunsaturated acid chain. The methylene groups 4 and 5 are shifted low-field by the fullerene core, resulting in the superimposed signal at 4.41 ppm for 3 and 5. This can be clearly identified by comparison of the spectra of the malonate 54 and the adduct 55. The signals for the dendritic branch and the spacer unit are in the typical range and are comparable

35 Chapter 3 Results and Discussion

2 4 5 7 9 3 6 8

2,9 4 3,5 1 * 6,8 7

8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 ChemicalShift(ppm)

1 Figure 3.6: H NMR of protected monoadduct 55 (300 MHz, RT, CDCl3). with the amphiphiles described in chapter 3.1.1. In the carbonyl region of the 13C NMR spectrum of 55 all non-equivalent C-atoms could be resolved as single signals. The resonances for the amide and ester carbon atoms are the furthest low-ﬁeld shifted signals and could be detected according to their quantity in the molecule in the intensity ratio 1:3:9. The splitting of the carbonyl C-atoms at 163.5 ppm into two signals clarify the asymmetry in the molecule. The sp2 area of the spectrum exhibits 26 of the 31 signals, as expected for the Cs-symmetry, whereas 5 signals show the double intensity (ﬁgure 3.7 (b)). The 12 C-atoms located at the double bonds of the fatty acid could be detected in the area from 127 to 132 ppm. The signals for the C-atoms located between the double bonds overlap to three different signals at about 25.7 ppm. The

OCH2 units from the ethylene glycol and spacer moiety are positioned between the sp3-signal at 71.5 ppm and the quaternary C-atoms of the dendron at 57.5 ppm. The signals for the dendritic branch can be taken from figure 3.7 (c). In analogy to chapter 3.1.1, 56 reveals a good solubility in buffered aqueous solution at pH = 7.2. The UV/Vis spectra of 56 follows the same trend as previously described. The amphiphilic character of the molecule leads to a significant line broadening in the absorption spectrum (see figure 3.8). Compared to the spectrum for 55, the characteristic small absorption peak at 425 nm is not identifiable any more (figure 3.8, inset).

36 Chapter 3 Results and Discussion

26 25 1 2 23 24 21 22 19 20 4 5 7 9 11 13 15 3 17 18 6 8 10 12 14 16

55 25 16,20,24 *

sp3 -C 17,21 3 2 1 8,10 11 7 6 9 15

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ChemicalShift(ppm) (a) 26

22,23

4 18,19

146 145 144 143 142 141 140 139 34 33 32 31 30 29 28 27 26 25 (b) (c)

13 Figure 3.7: C NMR of protected monoadduct 55 (75 MHz, RT, CDCl3) (a) and zoom into speciﬁc regions (b,c).

The investigation of 56 concerning the biological activity (chapter 3.1.4) is currently under progress. The ﬁndings will clarify, if the polyunsaturated fatty acid chain directly affects the transport properties of 56 to speciﬁc tissue regions.

37 Chapter 3 Results and Discussion

55 in CH2Cl2 56 in buffered H2O (pH = 7.2)

absorption

350 375 400 425 450 475 500

300 400 500 600 wavelength[nm]

Figure 3.8: UV/Vis spectra of protected precursor 55 (black) and deprotected water-soluble amphiphile 56 (red).

38 Chapter 3 Results and Discussion

3.1.3 Synthesis of a Cationic Amphiphilic Monoadduct

In the last years the research on anionic fullerene-derivatives proceeded rapidly and numerous compounds were tested for their efficiency as antioxidants and for their potential to inhibit neurodegenerative diseases. In the field of cationic fullerene-derivatives amazingly only moderate progress was made. From a synthetic point of view anionic water-soluble fullerene derivatives can be efficiently prepared by the unleash of carboxylic acids from the corresponding tert-butyl esters as described in the previous sections. As the structural motive, NEWKOME-type dendrons are an appropriate way to vary the numbers of carboxylic functions by simply adding higher generations. Never- theless, only a few cationic dendritic systems are literature known so far [120–123], probably by the missing of a suitable protection group and difficult purification procedures. In line with this thesis a cationic amphiphilic fullerene derivative should be prepared, which could be used as reference substances to the known anionic compounds, to give more detailed insight into the structure-efficiency relationship for the pharmaco- logical activity. These comparison measurements could help to clarify the question, if the biological activity of these compounds substantially depends on facts like addition pattern or the chemical nature of the addends. Another questions concerns, whether the charges only enable the water-solubility or if specific COULOMB-interactions with complementary charged proteins or other receptor-type molecules play an important role here. To avoid any pH-value problems, we decided to synthesize a permanently charged cationic derivative. Therefore an appropriate precursor had to be found, which could be transformed quantitatively into the corresponding charged derivative. This should be done in the last synthesis step, since charges complicate the purification rigorously. In scheme 3.5 the two possible synthetic pathways for the synthesis of the polycationic derivative 60 is shown. Pathway A takes advantage of the already synthesized amphiphilic carboxyfullerene 44 as starting material, which was esterificated with 2-bromoethanol in a threefold

STEGLICH-coupling. The introduction of the bromoethyl group displays a versatile tool for the initiation of cationic charges, as the halogen atom could be easily displaced by

39 Chapter 3 Results and Discussion

Pathway A Pathway B

OH OH O O H O O O H O N N R1O O OH R1O O OH O O O 57 O O OH O OH

DCC, DMAP, 1-HOBt, Br 44 O O O H O 1 N O R O O Br O O HO 58 DCC, DMAP, Br 1-HOBt, O O

Br Br CBr4, DBU, C O 60 O O H O 1 N O R O O Br O O O O

pyridine N

O O O H O 1 N O R O O N O O O O

1 R = (CH2)17CH3 N 3 Br 60

Scheme 3.5: Synthesis of the polycationic fullerene derivative 60. Pathway A: Synthesis via carboxyfullerene 44. Pathway B: Synthesis via bromo malonate 58.

40 Chapter 3 Results and Discussion substitution with a variety of different nucleophiles. As already mentioned, the coupling to the carboxy functionality under STEGLICH-conditions in the presence of the C60-core is often afflicted with intricateness. Also in this case the reaction did not lead to a satisfying result and even after long reaction times (120 h), remarkable amounts of mono and bis-esterificated byproducts could be detected. In order to obviate this problem, the malonate 57 was used as basic module for the synthesis via Pathway B. The deprotected malonate 57 could be easily achieved from the precursor 32 by acidic cleavage of the tert-butyl esters. Reaction with 2-bromoethanol gave the malonate 58, which exhibits three peripheral bromo groups. In contrast to the reaction described above the bromo malonate 58 could be obtained in relatively high yields (78 %) and only a small amount of fragmentary byproducts had to be separated by flash column chromatography. Subsequent cyclopropanation afforded the monoadduct 59, which should be expeditiously further converted, since decomposition could be observed. Quater- nization of the pyridine nitrogen via nucleophilic substitution reaction was achieved by stirring 59 in dry pyridine at 60°C and was completed after 48 h in quantit ative yield. The crude product 60 was purified by repeated precipitation from methanol with diethyl ether. The quantitative quaternization can be nicely followed in the 1H NMR spectrum (figure 3.9). The signals between 8.20 and 9.13 ppm correspond to the pyridinium rings and show the characteristic splitting pattern. The relation of the integral for the pyridinium rings and for the methyl group 1 with the ratio 5:1 verifies the complete quaternization. Com- pared to the spectrum of the bromo derivative 59, the introduction of the pyridinium groups results in obvious shifting of the resonances. Particularly strong is the effect in the case of the methylene groups 8 and 9, with a low-field shift of about 1 ppm.

41 Chapter 3 Results and Discussion

9 8 5 7 1 2 3 4 6

C -H-2 8 9

59 7 2,3 1 * 8 5 4

10 9.5 9 8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

9 8 5 7 * * 1 2 3 4 6

C -H-2

60 2,3,8 9 8 7 1 5 4

10 9.5 9 8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 ChemicalShift(ppm)

1 Figure 3.9: H NMR spectra of bromo-precursor 59 (400 MHz, RT, CDCl3) and cationic fullerene 60 (400 MHz, RT, DMSO-d6).

The UV/Vis spectra of 59 and 60 are shown in ﬁgure 3.10. In analogy to the anionic amphiphile 44 the clear tendency to form aggregates is also observed in the case of 60. Furthermore also the solubility in aqueous solution is similar to 44. For the measurement of the UV/Vis spectrum the solubility was promoted by adding a small amount of DMSO to the aqueous solution. As expected for the permantly present charges, the solubility of 44 is not dependent on the pH-value.

42 Chapter 3 Results and Discussion

absorbance

59 in CH2Cl2

60 in H2O

300 400 500 600 wavelength[nm]

Figure 3.10: UV/Vis spectra of bromo-precursor 59 and cationic fullerene 60.

43 Chapter 3 Results and Discussion

3.1.4 Amphiphilic Fullerenes as Potential Drug Candidates

3.1.4.1 Introduction and Background

Within a few years after the ﬁrst production of fullerenes in macroscopic quantities,[8] it was recognized that the extended conjugated π-system of C60 exhibits unusual potency for scavenging radicals including reactive oxygen species (ROS).[68] The major impediment for the development of fullerene-based biological antioxidants has been the relative insolubility of C60 in either aqueous or lipid-based solvents. Early attempts to modify the surface of the fullerene by polyhydroxylation and hexasulfobutylation produced water-soluble fullerenes with good biological distribution and cell penetration. [124–131] These early classes of water-soluble fullerenes exhibited surprisingly potent antioxidant and cytoprotective activities in vivo, including:

• signiﬁcant reduction of death and permanent tissue loss associated with severe ischemia/reperfusion injury resulting from complete blockage and subsequent re- opening of coronary [124] and carotid [125] vasculature

• protection of cultured neurons from glutamate excitotoxicity [126,127] and peroxide- induced injury to rat hippocampal slices [128]

• protection of small intestine from ischemia/reperfusion injury [129] and in intestinal grafts after transplantation [130]

• protection of pulmonary tissue from pulmonary hypertension induced by chronic hypoxia [131] and bronchoconstriction due to exsanguination and acute blood loss

One major drawback in the development of pharmaceutical applications for polyhydrox- ylated and hexasulfobutyl fullerenes is that they have highly heterogeneous structures with respect both to number of addends (polyhydroxyfullerenes) as well as the regioi- somerism of the addends attached to the fullerene core (both classes). From a pharmaceutical perspective, the most signiﬁcant advance in the development of fullerene- based antioxidants took place in the mid-1990s with the synthesis and characterization of methanofullerenes bearing terminal carboxy groups (carboxyfullerenes), a class of

44 Chapter 3 Results and Discussion exohedral fullerene adducts that could be synthesized in a highly puriﬁed and chemically homogeneous form.[109,132–135] Furthermore, since the carboxyfullerenes can be generated as single regioisomers, it is possible to design and synthesize a wide variety of different three-dimensional structures and charge distributions to optimize structure- function relationships and minimize "off target" binding and toxicity. So far different carboxyfullerenes have been synthesized and investigated with respect to their antioxidant properties: carboxyfullerenes C3-[e,e,e-C63(COOH)6] 61 and D3-[trans3,trans3,trans3- [134,135] C63(COOH)6] 62.

O OH HO O O OH HO O O O O O OH OH OH OH

61 62 HO OH HO OH O O O O

OH HO O O HO O O OH O OH HO O NH HN OH OH O O O O O H H O H N N H N O O N HO OH O O O O O O HN O O NH HO O O O O OH HO OH HO OH O O O O HO OH 63

Figure 3.11: C3-[e,e,e-C63(COOH)6] 61, D3-[trans3,trans3,trans3-C63(COOH)6] 62 and dendrofullerene 63.

Another prominent carboxyfullerene is the dendrofullerene 63, which is highly soluble in water and has been investigated with respect to a variety of biomedical properties.[109] In experiments similar to those described for fullerenols and hexasulfobutylated fullerenes, carboxyfullerenes 61 and 62 have been shown to exhibit the following therapeutic ben- eﬁts linked to their antioxidant activities:

• carboxyfullerenes locate preferentially to mitochondria [136] and can reconstitute

45 Chapter 3 Results and Discussion

mitochondrial superoxide dismutase protection from superoxide radicals in SOD2 (superoxide dismutase 2) genetically deﬁcient mice [137]

• carboxyfullerenes are highly potent neuroprotective agents, preventing cell death across a variety of different neuronal types in disease models as diverse as Parkinson’s, Alzheimer’s, ALS (amyotrophic lateral sclerosis), excitotoxicity, mac- ular degeneration and stroke [138–145]

• carboxyfullerenes protect cells from damaging effects of UV and gamma-irradiation

[146]

• carboxyfullerenes protect from morbidity and mortality in the presence of over- whelming infection with both, gram-positive and gram-negative organisms [147–150]

• carboxyfullerenes protect a variety of different cell types, including leukocytes, hepatocytes and renal tubular epithelial cells from oxidative injury associated with chemical and biological agents [151–153]

Although these surface-modified, water-soluble fullerenes have been shown to exhibit strong antioxidant activity against reactive oxygen species (ROS), the progress in developing fullerenes as bona fide drug candidates has been hampered by three development issues: 1) Lack of methods for scalable synthesis; 2) inability to produce highly-purified, single-species regioisomers compatible with pharmaceutical applications; and 3) inadequate understanding of structure-function relationships with respect to various surface modifications (e.g., anionic versus cationic versus charge-neutral polarity, degree of lipophilicity in the molecule). Also the stability of the derivatives is an important factor. In the case of the carboxyfullerenes 61, 62 for instance, it turned out, that these compounds are not stable in solution. HPLC analysis of the degradation products indicates that decarboxylation reactions of the malonyl adducts represents the major pathway for degradation of 61. Three major decomposition products, namely the mono-, bis- and tris-decarboxylation products 64, 65 and 66 could be identified (figure 3.12). The initial breakdown product is mostly 64, but with continued degradation, significant amounts of 65 and 66 are observed as well. In the presence of DMSO, complete degradation to 66 appears complete within 1-2 minutes at room temperature.

46 Chapter 3 Results and Discussion

O OH O O OH O O HO HO H HO O O O H H H OH OH OH

64 65 66 HO OH OH OH H H O O O O

Figure 3.12: Schematical representation of the C3-[e,e,e-C63(COOH)6] 61 decarboxylation products 64, 65 and 66. Only one stereoisomer each is represented although they were formed as mixtures of isomers (NMR).

Toxicological investigations disclosed, that these decomposition products exhibit a significantly increased toxicity, compared to the carboxyfullerene 61 (see chapter 3.1.4.4). On the other hand the dendrofullerene 63 showed no instability in solution; aqueous solutions of 63 were stable at room temperature for several weeks. In spite of the high stability and the good water-solubility of 63, the highly charged and voluminous dendritic branch, together with the absence of an additional lipophilic part, could be problematic concerning the membrane permeability. To combine the benefits of the dendrofullerene 63 with new structural advantages, concerning the lipophilicity, we decided to synthesize a series of novel amphiphilic fullerenes, which differ in the number and type of charge as well as in the lipophilicity (see chapter 3.1 and figure 3.13). The following chapters describe the examination of these compounds, with respect of their antioxidant activity against superoxide anions and the cytoprotective activity in zebrafish models.

3.1.4.2 Antioxidant Activity

Reactive oxygen species (ROS) are derived from the metabolism of molecular

[154] −· 1 oxygen. ROS include superoxide anion radical (O2 ), singlet oxygen ( O2), hydrogen peroxide (H2O2), and the highly reactive hydroxyl radical (·OH). The deleterious effects of oxygen results from its metabolic reduction to these highly reactive and toxic species.[155] In living cells, the major source of endogenous ROS are hydrogen peroxide and the superoxide anion, which are generated as by-products of cellular metabolism such as mitochondrial respiration.[156] Alternatively, hydrogen peroxide may be con-

47 Chapter 3 Results and Discussion

HO O O OH HO O OH HN OH O O O O H O O H O N N H R1O O OH R1O O N O OH O O O O O OH O NH O O OH HO OH O O OH

1 1 R = (CH2)5CH3 43 R = (CH2)5CH3 46 1 1 R = (CH2)17CH3 44 R = (CH2)17CH3 47 1 1 R = CH((CH2)7CH3)2 45 R = CH((CH2)7CH3)2 48

O 3 Br O O H O N O O O N O O O O

Figure 3.13: Overview over the amphiphilic monoadducts examined in chapter 3.1.4. verted into water by the enzymes catalase or glutathione peroxidase. Variability or inductive changes in the expression of these enzymes can significantly influence cellular redox potential. ROS can cause tissue damage by reacting with lipids in cellular membranes, nucleotides in DNA,[157] sulfhydryl groups in proteins [158] and cross- linking/fragmentation of ribonucleoproteins.[159] Oxidative stress is caused by an imbal- ance between the production of reactive oxygen and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. In the recent years it has been shown, that this oxidative stress plays a role in various clinical conditions such as malignant diseases, diabetes, atherosclerosis, chronic inflamma- tion, viral infection, and ischemia-reperfusion injury.[160–162] Diseases associated with

48 Chapter 3 Results and Discussion oxidative stress such as diabetes mellitus and cancer show a pro-oxidative shift in the redox state and impaired glucose clearance suggesting that muscle mitochondria is the major site of elevated ROS production. Because of its high metabolic rate and relatively reduced capacity for cellular regeneration, the brain is believed to be particularly susceptible to the damaging effects of ROS. In neurodegenerative diseases like Parkinson’s, Alzheimer’s and amyotrophic lateral sclerosis (ALS), ROS damage has been reported within the speciﬁc brain region that undergo selective neurodegen- eration. Protein oxidation has been reported in the hippocampus and neocortex of patients with Alzheimer’s disease, Lewy bodies in Parkinson’s disease and within the motor neurons in ALS.[163] An antioxidant is a molecule capable of deactivating these ROS. Well-known antioxidants include a number of enzymes (catalase, superoxide dismutase) and other substances such as vitamin C, vitamin E and beta-carotene (which is converted to vitamin A) that are capable of counteracting the damaging effects of oxidation. Fullerene- derivatives are able to permeate into cells and to scavenge ROS in a highly effective mechanism. The following properties are generally agreed to make fullerenes uniquely effective as intracellular antioxidants:

• The energy level of the LUMO fullerene orbital is comparable to the involved orbitals of superoxide radical anion

• The fullerene antioxidant quenching process appears to be an effective catalytic mechanism (see chapter 3.1.5)

• Fullerene antioxidants localize within cells to mitochondria and other sites where excess free radical production occurs

The standard assay to investigate the superoxide quenching ability of an antioxidant is based on xanthine/xanthine oxidase to generate superoxide causing the reduction of cytochrome C.[164] This process is followed photometrically by looking at the changes in intensity of the absorption band of reduced cytochrome C at 550 nm (see scheme 3.6).

49 Chapter 3 Results and Discussion

Xanthine 2O2 OD550nm XOD - H2O2 2O2 CytochromeC + Uricacid

Antioxidant

O2 +H2O2 Scheme 3.6: Schematic representation of reactions involved in the xanthine/xanthine oxidase assay.

Using this protocol we compared the new amphiphilic fullerene structures with trisadduct 61 as standard. The results are summarized in table 3.1 and allow to draw conclusions concerning a structure-function relationship.

fullerene derivatives IC50 [µM]

43 – 1st generation (anionic) 44 24.0 45 26.1 46 13.3 2nd generation (anionic) 47 15.4 47 14.7 1st generation (cationic) 60 3.1 61 18.5 Trisadduct 61 and decomposition 64 62 products 65 48 66 44 Dendrofullerene 63 11.0

Table 3.1: IC50 values for the superoxide quenching activities of fullerene derivatives in xanthine/xanthine oxidase assays.

Within the class of polycarboxyfullerenes, the best activities were reached for the dendrofullerene 63 with an IC50 value of 11.0 µM. Also the trisadduct 61 showed a remarkable good scavenging activity with an IC50 value of 18.5 µM. The decarboxylation

50 Chapter 3 Results and Discussion products themselves are less effective. With rising the degree of decarboxylation in 64, 65, 66, the IC50 values decrease from 62 to 44 µM. In the case of the amphiphilic monoadducts the second generation compounds 46, 47, 48 exhibit significant lower inhibition concentrations with IC50 between 13.3 and 15.5 µM than the first generation ones (44, 45), possessing values of 24.0 and 26.1 µM. A possible explanation for these observations may be the different aggregation states of these molecules in aqueous solutions due to their intrinsic structures. Whereas the dendrofullerene 63 is water-soluble in an almost monomeric form the amphiphilic strutures have a strong tendency to form aggregates in water as it is concerned by line broadening in the UV/Vis and NMR spectra. Building up such clusters decreases the actual concentration of active fullerene species in solution leading perhaps to lower quenching activities compared with monomers. Because of their considerable lower net charge and smaller hydrophilic head group together with a comparable lipophilic moiety and the essential presence of polar surfactants like DMSO the supramolecular superstructures of the first generation derivatives might be even greater resulting in lower fullerene availability comparing to the second generation derivatives. These considerations might be a possible explanation for the different superoxide scavenging activities. In the direct comparison the cationic compound 60 showed significant better antioxidant properties. This may result from the positive charge of the molecule, which is expected to be more likely to capture negatively charged superoxide anions.[165] Increasing the number of addends on the fullerene surface, which leads to an increased rupture of the conjugated π-system, causes a decreasing ability of superoxide quenching. For instance, the trisadduct 61 shows a substantially larger IC50 value. The decarboxylation products of 61 on their part are more effective than 61 by itself. With rising degree of the decarboxylation 64, 65, 66, the IC50 values decrease. These structure-function studies indicate that antioxidant properties of water-soluble fullerenes depend on a number of factors, not all of which are completely understood at present. However, within closely related structural families, the similarities of behavior are sufficient enough to adopt the following trends:

• Increasing the number of addends on the fullerene causes a decreasing ability of

51 Chapter 3 Results and Discussion

superoxide quenching.

• The antioxidant properties of water-soluble fullerenes depend on a number of factors, like the structure, size and shape of the addends. The empirical correlations of the IC50 values with the structures are not completely understood at present.

• The charge seems to play an important role for the activity.

3.1.4.3 Cytochrome C Binding

The problem of the antioxidant studies described above are various inﬂuences through additional components present in the assay matrix. In this regard it is important to ask whether the activity of the fullerene adducts against the xanthine/xanthine oxidase/cytochrome C assay is exclusively due to the ability of quenching superoxide or due to additional effects caused by the interplay of the components present in this matrix. One possible interaction, which may play a crucial role is electrostatic interaction between the charged fullerene derivatives and the positively charged cytochrome C.

Previous investigations in the HIRSCH group studied the electrostatic binding and pho- toinduced electron transfer between cytochrome C and 63.[166,167] In this case strong electrostatic binding with an association constant of Ks = 1.7 x 105 M−1 (pH = 6) was observed. To complement the aforementioned results of the assay the different fullerene derivatives were subjected to binding studies with cytochrome C in cooperation with the

GULDI group at the University Erlangen-Nürnberg. In the case of intact cytochrome C, the protein shell that surrounds the iron-(II) / iron-(III)-porphyrin hampers the direct electronic interactions that are readily observed, between fullerenes and unbound por- phyrins. To avoid this, the excited interactions with zinc-(II)-porphyrin chromophore rather than the iron-(II) / iron-(III)-porphyrin were studied. This was deemed necessary, because iron-(II) or iron-(III) centers tend to deactivate excited states efﬁciently. In the corresponding experiments, aqueous solutions of Zn-(II)-cytochrome C (i.e., 0.9 - 1.4 x 106 M) buffered at pH 7.2 were titrated with variable concentrations of the different amphiphilic fullerene derivatives (i.e., 0.7 - 22.4 x 106 M). Regardless of the strength of fullerene / Zn-(II)-cytochrome C interactions using other assays, the absorption spectra were not sufﬁciently sensitive to detect these interactions, and are best

52 Chapter 3 Results and Discussion described as the linear superimposition of the component spectra, namely, fullerene and Zn-(II)-cytochrome C. Fluorescence-quenching experiments provided a more sensitive and consistent assay for fullerene / Zn-(II)-cytochrome C interactions. For Zn-(II)- cytochrome C, recording the typical fluorescence pattern of a zinc-(II)-porphyrin with maxima at 585 and 644 nm and with high quantum yields of ca. 0.04, demonstrates the ability of this method to measure Zn-(II)-porphyrin photoactivity inside of the protein matrix. In the presence of the fullerene derivatives, a non-linear quenching of the Zn- (II)-cytochrome C centered fluorescence is clearly discernable (see figure 3.14). It is important to note that the fluorescence emission spectrum remains constant through- out the titrations, although the amplitude varies due to quenching by the fullerenes.

emission[a.u.]

wavelength[nm]

Figure 3.14: Room temperature ﬂuorescence spectra of 1.4 x 10−6 M Zn-(II)-Cyt C in aqueous solutions in the presence of variable concentrations of 47 – excitation wavelength: 420 nm.

53 Chapter 3 Results and Discussion

1.1

1.0

0.9

0.8

I/I

0.7

0.6

0.5 -6 -6 -6 -6 -5 -5 0.0 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10 1.2x10

Cfullerene[M]

Figure 3.15: A plot of the change in ﬂuorescence intensity as a ratio of I/I0 versus 47 concentration, the solid line represents the estimated curve-ﬁt obtained by non-linear least-squares analysis.

1.2

1.0

0.8 relative intensities of 0.6 fluorescing components 0.4

0.2

0.0 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6

Cfullerene[M] Figure 3.16: A plot of the change in relative ﬂuorescence intensities versus 47 concentration, the solid line represents the estimated curve-ﬁt obtained by non-linear least- squares analysis.

54 Chapter 3 Results and Discussion

This nicely mirrors the trend seen in the absorption spectra, where no notable impact due to fullerene binding was seen on the absorption spectra. It is important in this context to consider the mechanism of fluorescence quenching of Zn-(II)-cytochrome C in the presence of fullerenes: past work has provided thermodynamic, kinetic and spectroscopic evidence in support of an intra-ensemble charge separation that evolves between the photoexcited Zn-(II)-cytochrome C and the electron accepting fullerene 63.[166] In the next step, the non-linear quenching dependences were used to determine the binding constants (i.e., Ka) for forming Zn-(II)-cytochrome C / fullerene donor- acceptor ensembles held together by electrostatic forces (see figure 3.15). In table 3.2 the binding constants of in each case a representative of the different amphiphilic fullerenes are summarized. At first glance, the positively charged fullerene derivative 60 gives rise to binding constants below the detection limit of ca. 102 M−1. This is rationalized on the basis of repulsive interactions between equally charged donor and acceptor constituents. When comparing 44 with 47 a more than 20-fold enhancement is seen. This observation is in line with the expectations by the presence of eight positive charges that are located within the docking station of cytochrome C in general. Independent confirmation was gathered using time-resolved fluorescence experiments. The Zn-(II)-cytochrome C fluorescence lifetime was measured in the absence and presence of the electron accepting fullerene derivatives. When no fullerene is present the recorded fluorescence time profiles are well fitted by a mono-exponential decay function. A lifetime of 2.2 ± 0.2 ns was determined under such conditions.

I −1 II −1 Compound K a [M ] Fluorescence K a [M ] lifetimes [ns] 61 1.5 x 106 0.25 8.6 x 105 44 8.2 x 104 0.76 47 1.8 x 106 0.97 1.2 x 106

I Steady-state ﬂuorescence measurements.II Time-resolved ﬂuorescence measurements

Table 3.2: Binding constants and ﬂuorescence lifetimes of Zn(II)-cyt C/fullerene complexes with the amphifullerenes 44, 47 and the carboxyfullerene 61.

55 Chapter 3 Results and Discussion

In contrast, adding any of the studied fullerene derivatives led to fluorescence decays that were only reasonably fitted - with a χ2-value of close to 1 - by a bi-exponential function. Here, a long-lived component (i.e., 2.2 ± 0.2 ns) and a short-lived component were registered. Interestingly, the exact value of the short-lived component was found to be dependent on the structure of the fullerene derivative (see table 3.2). Through- out the titration experiments the two lifetimes remained constant. The only variable is the relative ratio between the long-lived and short-lived components. The long-lived component decreased continuously, while the short-lived component increased simul- taneously. Similar to the steady-state experiments, the differing ratios were used to derive the binding constants (see figure 3.16). Notably, an excellent agreement with the steady-state analysis is observed for the tested systems.

56 Chapter 3 Results and Discussion

3.1.4.4 In vivo Studies of the Amphiphilic Fullerenes using Zebraﬁsh (Danio Rerio) Embryos as Model System

The data from the xanthine/xanthine oxidase assay proved the fundamental potency of fullerenes to work as highly effective antioxidants. To further address the challenge of developing fullerene-based drugs, the evaluation of the toxicity and effectivity in the human organism is an important step. In this context the zebrafish embryo has become an important vertebrate model for the preclinical drug screening. It is well suited for studies in genetics, embryology, development, and cell biology. Zebrafish embryos exhibit unique characteristics, including the ability to absorb compounds through the water (ease of drug administration) and short reproductive cycles. Moreover, since zebrafish embryos are small (they can be studied qualitatively and quantitatively in a single well of a 96-well plate), and transparent up to ten days post fertilization, the toxic and cytoprotective effects of small molecules can be studied in situ during this period. Finally, since the morphological structure of zebrafish embryos has been mapped to the single-cell level, the toxic, apoptotic and cytoprotective effects on various organs can be tracked to approximately the single-cell level in situ.[168–171]

The following "zebraﬁsh-tests" were done in cooperation with PHYLONIX Pharmaceuti- cals Inc., Massachusetts. For the experimental details see appendix A.

3months

4h 24h 5days

Figure 3.17: Different stages of zebraﬁsh development.

57 Chapter 3 Results and Discussion

Toxicity of water-soluble fullerenes in zebraﬁsh embryos

LC50 values

To compare the overall toxicity of the amphiphilic fullerenes in zebraﬁsh embryos, the derivatives were added to the water in single-wells of a 96-well plate, each containing a single zebraﬁsh embryo at 24 hpf (hours post fertilisation). The lethality along with any observed morphologic abnormalities were scored at 120 hpf. In most cases, fullerenes were tested at varying concentrations up to 500 mM, but in some cases the maximum concentration tested was 250 mM due to solubility issues at higher concentrations. The LC50 was calculated as the fullerene concentration at which 50 % lethality is observed at 120 hpf. Table 3.3 summarizes the results for the amphiphilic fullerenes, with the trisadduct 61 and the decarboxylation products 64, 65, 66 as reference.

Compound Class LC 50 Zebraﬁsh [µM]

44 amphiphilic fullerene ≫ 250 (0 % lethality at 250 µM) 45 amphiphilic fullerene ND 46 amphiphilic fullerene ND 47 amphiphilic fullerene ND 48 amphiphilic fullerene ND 61 trisadduct 596 64 trisadduct 373 65 trisadduct 134 66 trisadduct 10 63 Dendrofullerene > 500 (20 % lethality at 250 µM)

Table 3.3: LC50 of amphiphilic fullerenes in zebraﬁsh.

For all of the amphiphilic fullerenes the LC50 are considerably high, reaching values up to 0 % lethality at the highest possible concentrations. The breakdown products of 61 represent an important exception with respect to the general low toxicity for the anionic fullerenes. With progressive decarboxylation steps, each resulting breakdown product shows increasing toxicity with respect to the parent trisadduct 61. The LC50 for 61 is 596 µM, followed by continuous increase in lethality to 10 µM for 66. The symmetrical dendrofullerene 63 is located in the range of 20 % lethality at 250 µM.

58 Chapter 3 Results and Discussion

Body Morphology

In specific cases morphological abnormalities such as shortened body length and abnormal body curvature were detected in embryos treated with fullerene concentrations at or near the LC50. In the case of 61 a shortened body length was observed at 250 µM (figure 3.18, panel B). Decarboxylation product 66, which caused an enhancement in the mortality of the zebrafish embryos, had no influences on the morphology during embryonic growth. Treatment of the zebrafish embryos with 500 µM of 63 resulted in a slight curvature of the body (panel C). In contrast, the amphiphilic fullerenes showed no observable body length or curvature abnormalities (figure 3.18 panel G,H,J,K,L; for the fullerene concentrations, see table 3.4). This together with the considerable high LC50 values elucidates the general low toxicity of the amphiphilic fullerenes.

Figure 3.18: Morphological body-length abnormalities caused by doses of fullerenes (for the compounds to the different entries see table 3.4. For comparison the corresponding free G-2 dendron 30 was investigated (panel I).

59 Chapter 3 Results and Discussion

Panel Compound Concentration [µM]

A control – B 61 250 C 63 500 D control – E 66 10 F control – G 45 250 H 44 250 I 67 J 46 500 K 47 500 L 48 500

Table 3.4: Fullerene concentrations used for the determination of morphological body-length abnormalities in ﬁgure 3.18.

Internal Organs

The optical transparency of zebrafish embryos permits the visualization of individual internal organs, which can be examined with high resolution microscopy. Therefore, compared with laborious histological examination in mouse, examining drug effects on internal organs in zebrafish is simple. The organ morphology in the development of liver, intestine and heart caused by doses of fullerenes is shown in figure 3.19. 250 µM 61-treated zebrafish exhibited unfolded cardiac chambers, underdeveloped liver and intestine. The 500 µM 63 treated zebrafish exhibited enlarged liver and intestine. For the amphiphilic fullerenes, 47 is exemplary shown. No obvious defects on liver, intestine, and heart were observed in this case and also for the other amphiphilic compounds 44, 45, 46, 47, 48. In general, for all examined fullerenes, no organ necrosis could be observed with any of the treatments.

Cardiotoxity

Cardiotoxicity is a major problem with numerous pharmaceutical agents and indus- trial chemicals. The use of zebraﬁsh as an animal model for cardiotoxicity testing can provide more accurate results compared to cell-based assays. Drug effects on heart

60 Chapter 3 Results and Discussion

control 250 µM 61 500 µM 63

(a) (b) (c) control 10 µM 66

(d) (e) control 250 µM 47

(f) (g)

Figure 3.19: Morphological organ abnormalities in the development of liver, intestine and heart caused by doses of carboxyfullerenes 61, 63, 66 and amphifullerene 47. The liver, intestine, heart are outlined by magenta, yellow and red lines respectively for easy visual assessment. rates, heart beat rhythm, and contractility can be directly assessed in the transparent zebrafish under a dissecting microscope, providing information on the pharmacologi- cal effects of drugs on cardiac function. Figure 3.20 displays the bradycardia values induced by the water soluble fullerenes. Figure 3.20 indicates that decarboxylation product 66 induces bradycardia at very low doses of 1 mM or less. In contrast, no significant bradycardia is observed for dendrofullerene 63 or carboxyfullerene 61 at concentrations of 100 mM. The extent of bradycardia and associated cardiac abnormalities is probably sufficient to account for the increased toxicity of the decarboxylation products of 61 in the LC50 assay. The observed effects on cardiac conduction could presumably be interpreted by the binding to the zERG potassium channel (zebrafish homolog

61 Chapter 3 Results and Discussion

100

%ofcontrol 80

75 control 61 66 44 45 47 46 48

4hours 24hours

Figure 3.20: Effects on heart rate induced by various fullerene derivatives, plotted vs. control (0.1 % DMSO) after 4 hours (blue bars) and 24 hours (red bars). to the hERG (Human Ether-a-go-go Related Gene) potassium channel in humans). Ion channels exhibit unique structures, including the pore that provides the physical pathway for ion movements across the plasma membrane and several charged domains that attract and/or repel ions. These characteristics make ion channels easy targets for external agents such as natural toxins and synthetic drugs that react with them by establishing electrochemical interactions. Figure 3.21 displays the possible pore oc- clusion mechanism. Pristine C60 with an average diameter of 0.7 nm can nicely fit into the entrance of the selectivity filter and stop the ion permeation. The decarboxylation products of 61 exhibit nearly the same diameter as pristine C60 and would therefore be suitable for docking to the ion channel. The voluminous shape of the dendritic branch of the amphifullerenes clearly disfavors the binding to the entrance of the ion channel. The experimental results approve this assumption (figure 3.20). No cardiac abnormalities were detected for all tested amphiphilic fullerenes, indicating that cardiac injury is not characteristic of fullerenes in general.

62 Chapter 3 Results and Discussion

Figure 3.21: Left: Crystal structure of the KscA K+ channel. The image was constructed from the ﬁle 1BL8 from the Protein Data Bank. Right: Docking simulation of a fullerene with an average diameter of 0.7 nm, which can block the entrance of the selectivity ﬁlter and stop the ion permeation.

Cytoprotective activities

Protection of zebraﬁsh embryos from CNS injury due to 6-hydroxydopamine (6-OHDA)

Programmed cell death is an essential regulator of normal development and homeosta- sis of the CNS.[172,173] Neuronal apoptosis is thought to occur to eliminate those neuronal precursors that fail to establish appropriate synaptic connections.[174] Inadequate apoptosis has been found in various human neurodegenerative diseases in which a massive neuronal loss occurs, such as Alzheimer’s disease or Parkinson’s disease. CNS dopaminergic neurons are particularly sensitive to the toxic effects of 6-OHDA. The apoptotic processes in zebrafish and mammals are very similar and therefore the ability of fullerenes to protect from 6-OHDA-induced apoptosis of CNS dopaminergic neurons in developing zebrafish was tested. Dopaminergic CNS neurons can be easily detected in live zebrafish embryos by the presence of tyrosine hydroxylase, which can be detected by immunohistochemistry. 6-OHDA crosses the blood-brain barrier and is taken up preferentially by dopaminergic CNS neurons, and to a lesser extent by non-dopaminergic CNS neurons. Intracellular 6-OHDA induces cellular apoptosis and death, at least in part through oxidative injury.[175] The amphiphilic fullerene 47 showed the highest levels of CNS neuroprotection against 6-OHDA injury, and was able to pro-

63 Chapter 3 Results and Discussion tect 60 % of tyrosine hydroxylase-positive dopaminergic neurons in the diencephalon. Figure 3.22 shows the results obtained with 47, in protecting CNS dopaminergic neurons against 6-OHDA-induced cell death.

control

6-OHDA

6-OHDA + 250 µM 47

Figure 3.22: Protection of CNS dopaminergic neurons from 6-OHDA-induced apoptosis by 47.

The untreated (top panels), 6-OHDA plus vehicle treatment (middle panels), and 6- OHDA plus 250 mM 47 (lower panels) zebrafish embryos were stained with anti-tyrosine hydroxylase antibody. At a time five images of each group were examined and the number of TH-immunoreactive cells clustered in the diencephalons (between the eyes) was detected. At untreated animals, 20-40 TH-cells could be observed (blue arrows). After 6-OHDA treatment, a decrease in the number of TH-immunoreactive cells was observed (1-17) (red arrows). After 250 mM 47 treatment, three of five 47-treated animals showed a normal number of TH-immunoreactive cells with normal morphology (black arrows).

Protection of zebraﬁsh embryos from mechanoreceptor hair cell injury and death induced by cis-platinum and gentamicin

In zebraﬁsh, the dorsal mechanoreceptor hair cells are responsible for sensing position, orientation, balance and movement and are biochemically and morphologically similar to outer hair cells of the inner ear in mammals. These hair cells could be easily examined by staining with 2-(4-dimethyl-aminostyryl)-N-ethyl pyridinium iodide (DASEPI), which speciﬁcally colorizes the mechanoreceptor cells.

64 Chapter 3 Results and Discussion

control

CDDP

Figure 3.23: Protection of hair cells from cis-platinum damage. Hair cells detected by DASEPI staining (black spots). For the different entries see table 3.5.

Entry Compound EC50 [µM]

A 61 ND (not detected) B 45 285 C 47 194 D 48 438

Table 3.5: EC50 values of the fullerene derivatives shown in ﬁgure 3.23.

65 Chapter 3 Results and Discussion

In this way, the fate of each lateral-line neuromast cell can be tracked in zebrafish embryos after exposure to chemical toxins which induce apoptosis in these cells.[176] Cis- platinum or cis-diamminedichloroplatinum(II) (CDDP) is a platinum-based chemother- apy drug, widely used to treat various types of cancers. The drawbacks of cis-platinum are the extensive side-effects, including damage to renal tubular cells and outer hair cells of the inner ear, leading to renal failure and loss of hearing acuity or deafness, respectively. The mechanism of cell damage, induced by CDDP is believed to be primarily direct DNA damage, although perturbation of cellular redox pathways may also play a role.[177] In zebrafish embryos, cisplatinum rapidly induces apoptotic cell death in all of the dorsal mechanoreceptor hair cells detectable by DASPEI staining. Figure 3.23 shows the ability to block cis-platinum induced mechanoreceptor cell apoptosis, by comparison of the amphiphilic fullerenes to carboxyfullerene 61. Fullerene 61, which was highly effective in the superoxide assay (see table 3.1), was almost completely unable to block apoptosis induced by cisplatinum, achieving only 5 % protection at 500 µM. In contrast to 61, the amphiphiles 45, 47, 48 show an efficient protection at quite low concentrations (table 3.5). To further evaluate the otoprotective activities of fullerenes, the protection of gentamicin treated zebrafish was tested. Gentamicin is an aminoglycoside antibiotic, and can treat many types of bacterial infections, particularly Gram-negative infection. All aminoglycosides are toxic to the sensory cells of the ear, but they vary greatly in their relative effects on hearing versus balance. Gentamicin is a vestibulotoxin, and can usually lead to permanent loss of equilibrioception, caused by damage to the vestibular apparatus of the inner ear.[178–181] In zebrafish embryos, exposure to gentamicin or the related antibiotic neomycin also induces apoptosis and complete loss of DASPEI-stained dorsal mechanoreceptor hair cells in a pattern indis- tinguishable for that described for cis-platinum. Surprisingly, the results for 61 contrast substantially with those observed in the same cells using cis-platinum-induced cell loss (see figure 3.23). In this case 61 is highly effective with an EC50 values of 34 µM (table 3.6). This indicates, that the protection ability of fullerenes from different chemical toxins could not be generalized and should be tested for each toxin separately.

66 Chapter 3 Results and Discussion

control

gentamicin

GSH

Figure 3.24: Protection of hair cells from gentamicin damage. Hair cells detected by DASEPI staining (black spots). For the different entries see table 3.6.

Entry Compound EC 50 [µM]

GSH glutathione 438 A 61 34 B 47 29 C 48 124 D 44 35 E 45 47

Table 3.6: EC50 values of the fullerene derivatives shown in ﬁgure 3.24.

67 Chapter 3 Results and Discussion

The tested amphiphiles 44, 45, 47, 48, which showed to be highly efficient against cis- platinum induced hair cell loss (table 3.5), represent moderate EC50 values, ranking between 29 for 47 and 124 µM for 48. In all cases, it should be mentioned that even the least effective fullerenes in this group are 3-4 fold more effective than glutathione (EC50 438 µM),[176,182,183] which has been used successfully in animal models to protect against aminoglycoside induced hearing loss and renal injury. The results described above, showed that water-soluble fullerenes are able to block apoptosis of specific cell types induced by chemical toxins and commonly used drugs in a highly accessible zebrafish embryo model system. It has been also shown, that the mechanism of protecting cells from apoptosis is not monolithic and depends both upon the specific mechanism of toxicity, even in a single cell type. The differing effectivities in the protection from gentamicin and cis-platinum may reflect, that the intracellular lo- calization of each fullerene, the chemical reactivity and the interaction with biological molecules, involved in apoptosis regulation, plays an important role. The introduction of a highly hydrophilic dendritic branch together with a lipophilic side chain, which should increase the cell tissue accessibility and biodistribution within lipid-rich regions, turned out to be an efficient tool to fulfill these issues. The overall high cytoprotective activity against both cis-platinum and gentamicin induced apoptosis may prove these amphiphiles to be useful as candidates for anti-apoptosis drugs.

68 Chapter 3 Results and Discussion

3.1.5 Mechanistic Aspects of the Reaction of Fullerenes with Superoxide

Chapter 3.1.4 describes the capability of a series of functionalized fullerenes to serve as highly efficient antioxidants. However the mechanism behind the reaction of C60 with reactive oxygen species is still not definitely clarified. On the one hand it is well known, that irridation of solubilized [60]fullerene and its derivatives generate super- ·− oxide (O2 ) in aqueous solutions, under aerobic conditions and in the presence of [184–187] 1 reducing agents. This, besides the production of singlet oxygen ( O2), hydroxyl · radical ( OH) and hydrogen peroxide (H2O2), may be the primary factor for the photo- toxicity of fullerenes,[185,188–191] on which their potential application as photosensitizers in the photodynamic therapy is based. The generation of superoxide is based on the outer-sphere electron transfer from reduced C60 to O2 (eq. 3.1).

·− ·− C60 + O2 −→ C60 + O2 (3.1)

On the other hand the antioxidant activity of fullerenes by scavenging superoxide anion radicals could be clearly demonstrated (chapter 3.1.4.2). From a mechanistic point of view, DUGAN et al. offered the evidence for a catalytic superoxide dismutation mecha-

[137] nism, instead of direct radical attack on the C60 moiety. She found that trisadduct 61 treatment of SOD2 −/ − mice, which lack expression of mitochondrial manganese superoxide dismutase (MnSOD), significantly increased their life span, by the replacement of MnSOD. The catalytic promoted dismutation was proposed to result from complexation ·− between C60 and O2 , which electrostatically guide and stabilize the superoxide. The observed high antioxidant efficiencies also suggests, that the reaction with superoxide is not based on stoichiometric scavenging. To further determine the mechanistic pathway, we studied in cooperation with the IVANOVIC-BURMAZOVIC group the redox properties of the amphiphilic fullerene derivatives (chapter 3.1.1, 3.1.3 and figure 3.25) and their reactivity towards superoxide in DMSO and aqueous solutions.

69 Chapter 3 Results and Discussion

OH O O O H HO 1 N O R O O OH O OH O O HO O O OH HN OH O O O H O N H 1 N R O O OH O O 44 N O O NH O OH O O O O H O HO OH N O O R1O O O N OH O 47 O O O

N 1 3 Br R = (CH2)17CH3 60

Figure 3.25: Overview over the amphiphilic monoadducts examined in chapter 3.1.5.

3.1.5.1 Cyclic Voltammetry Measurements of Amphiphilic Monoadducts

A consequence of the unique spherical structure of C60 is the electronic configuration, which exhibits a five-fold degenerated HOMO (hu) and a triply degenerated LUMO (t1u) level (figure 1.8). In accord with the degeneration of the lowest unoccupied molecular orbital, electrochemical investigations, namely cyclic voltammetry studies, demonstrated unambiguously the ability to reduce C60 up to the corresponding hexaanion, by reversible one-electron reduction steps. As demonstrated in figure 3.26 (a) the first reduction potential is located at -0.98 V vs. Fc/Fc+ with PhMe/MeCN as solvent mixture. The subsequent reduction steps follow in the typical range of 0.4 and 0.5 V.[63,64] The cyclic voltammetry measurements [192] of an assortment of amphiphilic monoadducts (chapter 3.1.1, 3.1.3) have been carried out in DMSO as solvent, which allows the direct comparison of the data with the results observed for the kinetic studies (chapter 3.1.5.2). Two reversible electro-reductions in the potential range from 0 to -1 V (vs. SCE) could be observed in all cases (figure 3.26). Table 3.7 summarizes the measured redox potentials of amphiphile 44, 47 and 60, exposing the much higher first reduction potentials for 44, 47 and 60, compared to the oxidation poten-

70 Chapter 3 Results and Discussion

-16

-12

-8

[µA] -4

I 0 47

-0,5 -1,0 -1,5 -2,0 -2,5 -3,0 -3,5 0,2 0,0 -0,2 -0,4 -0,6 -0,8 -1,0 E[V]vs.Fc/Fc+ E[V]vs. Ag/AgCl (a) (b)

-15 -40 -12 -30 -9 -6 -20

[µA] -3 [µA]

I I -10 0 44 0 3 60 6 10

0,0 -0,2 -0,4 -0,6 -0,8 -1,0 0,2 0,0 -0,2 -0,4 -0,6 -0,8 -1,0 E [V]vs Ag/AgCl E [V]vs Ag/AgCl (c) (d)

[64] Figure 3.26: Cyclic voltammogramms of C60(a), 47 (b), 44 (c) and 60 (d) in DMSO purged −4 with nitrogen. Conditions: [F] = 5 x 10 M, [Bu4NBF4] = 0.1 M, T = 298 K, scan rates = 0.2 V/s.

1 2 Entry Fullerene E 1/ 2 [V] E 1/ 2 [V]

A 47 -0.248 -0.614 B 44 -0.224 -0.647 A 60 -0.077 -0.521

Table 3.7: Redox potentials of 44,47 and 60 in DMSO vs. SCE calibrated by the Fc/Fc+ couple (0.43 V vs. SCE). tial of superoxide (-0.74 V vs. SCE in DMSO). Noteworthy in the case of the oppo- sitely charged fullerenes 44 and 60 a prominent charge dependence for the ﬁrst F/F ·− (fullerene/fullerene radical anion) redox couple can be followed. The positively charged

71 Chapter 3 Results and Discussion

·− 1 60 exhibits the most positive F/F redox potential with E1/ 2 = -0.077 V, compared to 1 E1/ 2 = -0.224 V for the anionic equivalent 44. The data suggests, that the electron ·− transfer from O2 to these fullerenes is not only strongly thermodynamically driven in ·− DMSO, but it is also possible in aqueous solutions [E° (O 2/O2 ) = -0.4 V vs. SCE in

H2O]. The effects of the fullerenes on the re-oxidation of superoxide can be nicely followed by saturating the DMSO solution with dioxygen. In this process the superoxide is electrochemically generated in situ. In the presence of the fullerene derivatives the re-oxidation wave of superoxide disappears in line with a diminished current for the re-oxidation of the fullerene anions. The values of corresponding fullerene anions re- oxidation potentials remain unaffected (see ﬁgure 3.27). These changes indicate that a reaction between electrochemically generated superoxide and the fullerene anions takes place, without inducing chemical changes on the fullerenes. The decomposition of superoxide could also be observed with low (catalytic) concentrations of the amphiphiles. The effect on the superoxide re-oxidation were found to be independent

4 DMSO purged with O2

47 in DMSO purged with O2

2 47 in DMSO purged with N2

[µA] I -2

-4

-6

-1,5 -1,0 -0,5 0,0 E [V]vs Ag/AgCl Figure 3.27: Cyclic voltammogramms of 47 purged with oxygen and nitrogen, and pure DMSO −3 purged with oxygen as reference. Conditions: [47] = 0.5x 10 M, [Bu4NBF4] = 0.1 M, T = 298 K, scan rates = 0.2 V/s.

72 Chapter 3 Results and Discussion from the applied concentrations, which supports the proposed catalytic mechanism.

3.1.5.2 Kinetic Measurements of Amphiphilic Monoadducts

An exclusive method to follow rapid reactions is the stopped-flow spectrophotometry. In a stopped-flow spectrophotometer, two syringes rapidly inject equal small volumes of reactant solutions through a mixer and an observation cell, into another syringe. When this receiving syringe is filled completely, the flow stops and a trigger activates the computer to acquire data. The photomultiplier output is recorded in digitized form at an acquisition rate set by the user. Depending on the instrument, mixing is essentially complete within a matter of a few milliseconds, and reactions that are complete within a range of milliseconds are amenable to this technique. Compared with the MCCORD-

FRIDOVICH assay, which was used in chapter 3.1.4.2 to determine the antioxidant effectivities, this method allows the direct measurement of superoxide decomposition without any possible side effects through matrix components, present in the assays. Using the stopped-ﬂow method we studied the reactions of 44, 47 and 60 with a large excess of KO2 in DMSO containing a controlled amount of water (0.06 %), which was in excess over the superoxide and fullerene concentrations.[192]

2.5 2.5 A

2.0 2.0

B 1.5 1.5

1.0 1.0

Absorbance

Absorbance 0.5 0.5

0.0 0.0 300 400 500 600 700 800 300 400 500 600 700 800 Wavelength[nm] Wavelength[nm] (a) (b)

Figure 3.28: (a) Time resolved UV/vis spectra recorded for the reaction of 47 (5 x 10−5 M) with 1 mM KO2 in DMSO at rt. A: spectrum before mixing; B: spectra obtained after mixing (using a stopped-ﬂow module) with time intervals of 10 s (total observation time 30 min). (b) Control reaction without addition of 47 followed over 2.5 h.

73 Chapter 3 Results and Discussion

Time-resolved UV/vis spectra using a stopped-flow module (figure 3.28) showed the ·− rapid decomposition of O2 , after mixing a superoxide solution with the fullerene solution (decrease of the absorbance in the range of 270 nm). Less prominent spectral changes at the wavelengths higher than 350 nm, in the case of 44 and 47 were found to be caused by KOH which is inevitably present in KO2. By using an electrochemically generated superoxide solution these spectral changes were not observed. The products of superoxide disproportionation, O2 and H2O2, were qualitatively detected in all four experiments.[192] To verify the effect of the dendritic branches as structural parts of the amphiphiles, the dendrimere 30 was measured as reference. For 30 itself, no induced superoxide decomposition could be observed. To access the rate constants of the rapid reaction, the decrease in the absorbance at 270 nm in the presence of catalytic concentrations of the fullerenes was followed by stopped-flow measurements (figure 3.29). Application of a microcuvette accessory (which reduced the dead time of the instrument down to 0.4 ms) enabled observation of the fast disappearance of the 270 nm absorption, which could be fitted as a first-order process to obtain the char-

−1 acteristic k obs (s ) value. The plots of k obs vs. the fullerene concentrations showed the desired linearity, from which the catalytic rate constants k cat can be determined (see ﬁgure 3.30). The summarized data in table 3.8 show a clear dependence of the fullerenes SOD activity (k cat ) on their redox potentials, namely, the higher the redox ·− potential (ability to be reduced by O2 ) the higher is the SOD activity. Contrary to [193–195] ·− the literature, this suggests that the electron transfer from O2 to the fullerene indeed is possible and plays an important role for the efﬁciency in the catalytic dismutation of superoxide.

1 2 6 Entry Fullerene E 1/ 2 [V] E 1/ 2 [V] k cat x 10 [M−1s−1]

A 44 -0.224 -0.647 4.29 ± 0.06 B 47 -0.248 -0.614 2.64 ± 0.04 C 60 -0.077 -0.521 12.02 ± 0.22

Table 3.8: Redox potentials and catalytic rate constants of anionic amphifullerenes 44, 47 and cationic amphifullerene 60 in DMSO obtained by using direct stopped-ﬂow measurements (k cat ) in DMSO (0.06 % water).

74 Chapter 3 Results and Discussion

1.00 1.30 0.98 1.28

0.96 1.26

1.24 0.94 1.22 0.92 1.20

Absorbance

Absorbance 0.90 1.18

0.88 1.16 1.14 0.86 0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10 Time[s] Time[s] (a) (b) 1.14

1.12

1.10

1.08

1.06

1.04

Absorbance 1.02

1.00

0.98

0.96 0.00 0.02 0.04 Time[s] (c)

Figure 3.29: Stopped-ﬂow kinetics of the reaction of 2nd generation anionic amphifullerene 47 (a), 1st generation anionic amphifullerene 44 (b) and cationic amphifullerene 60 (c) with superoxide at 270 nm (298 K).

The results in this chapter from electrochemical, spectrophotometrical and submillisecond mixing UV/vis stopped-flow measurements, approved that amphiphilic fullerenes act as metal free SOD mimetics. The direct measurement using stopped-flow-technique excluded any possible interferences through additional matrix components and ratified a catalytic superoxide dismutation mechanism (see figure 3.31). We could also demonstrate by examining a series of amphiphiles (differing in charge and hydrophilicity) that the redox and structural properties of the studied fullerenes directly influences their SOD activity.

75 Chapter 3 Results and Discussion

47 600 44 60 500

] 400

-1

obs

k 300

200

100

0 0.0 2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-5 1.0x10-4 c(fullerene)[M]

Figure 3.30: Plots of k obs versus [fullerene] for the reaction between fullerenes and saturated KO2 in DMSO at 298 K.

- H2O2 O2

C60

+ H O2

- ⋅ - - [ C60 O2 ] C60

- O2

Figure 3.31: Proposed catalytic cycle for the superoxide dismutation mechanism of fullerenes.

76 Chapter 3 Results and Discussion

3.1.6 Amphiphilic Fullerenes in Material Science Applications

With the cost of fossil fuels rising, attention is turning to organic solar cells, which can produce electricity much more cheaply than conventional silicon based solar cells. However, organic solar cells have to date been plagued by low efficiency, typically con- verting the sun’s energy to electricity in the range of 4-5 percent.[196,197] The advantage of these photovoltaic cells when compared to electro-chemical cells is predominantly the absence of a liquid electrolyte, which generates problems with sealing against air, but also the prospect of using flexible substrates. In recent years, polymer-fullerene composites turned out to be one of the most effective and therefore extensively studied concepts in device design for organic solar cells.[198,199] In these composites, the photon absorption takes place in the polymer and forms bound electron-hole pairs that separate due to the electron-donating nature of conjugated polymers and the electron-accepting nature of C60. As it is well known, the precise microstructural features of the films are of crucial importance for the op- timal device performance, where photogeneration of carriers must be accompanied by its efficient transport and collection.[200] Although blend structures exhibit the maximum interface between donor and acceptor molecules, paths for charge percolation to the electrodes and ohmic contacts at the film/electrode interfaces become limiting factors to the device performance. The bilayer structure, on the other hand, has limited interfacial area, however charge transport through the appropriate phase and charge collection at the right electrode are intuitively less complicated. Various strategies have been followed to optimize the blend structures and to control the weak compatibility of conjugated polymers and C60, including:

• chemical modiﬁcation of C60

• modiﬁcation of the conjugated polymer

• addition of plasticizer components

In the typical composite devices, poly(3,4-ethylenedioxythiophene)-poly(styrene- sulfonate) (PEDOT-PSS) is broadly used to form the ohmic contact with several metals

77 Chapter 3 Results and Discussion and indium tin oxide (ITO) coated glass substrates. Since it is a water-soluble poly- thiophene derivative, its polar groups could cause the segregation of the hydrophobic molecules of fullerene derivatives present in the active bulk layer of the device. An amphiphilic molecular layer, with the proper electrical and optical properties, seems adequate to solve this problem.

OH O O H O 1 N R = (CH2)17CH3 R1O O OH O O O OH

HO O O OH HO O 44 N HN OH O O O H O O O O H O H 1 N N R O O N R1O O O OH N O O O O O O NH O O O O OH HO OH N O O 3 Br OH 47 60

HO O O OH HO O HN OH O O O H O N H O N O O OH O O O O O NH O O OH HO OH O O OH 56

Figure 3.32: Overview over the amphiphilic monoadducts examined in chapter 3.1.6.

78 Chapter 3 Results and Discussion

3.1.6.1 Formation of LANGMUIR-Films with Amphiphilic Fullerene-Monoadducts

A possible approach towards structurally ordered fullerene assemblies is the preparation of LANGMUIR ﬁlms at the air-water interface and their subsequent transfer onto solid substrates.[99,201,202] Typically a monomolecular layer is conveniently formed when a solution of surface-active material (amphiphilic compound), dissolved in a water- insoluble, volatile organic solvent, is placed drop by drop onto a clean water surface. The hydrophobic tail of the amphiphiles makes this part of the molecule insoluble in water which supports the formation of a monolayer, while the hydrophilic head serves to orient the molecules at the water-air interface. The drops spread and the solvent evaporates, leaving behind a monomolecular layer of the dissolved material which can be compressed by moving barriers on the surface. The monolayer surface pressure π [mNm−1] can be deﬁned by the change in surface tension of the water surface caused by the addition of the monolayer, where γ0 is the surface tension of the clean water surface, and γm is the surface tension of the monolayer-covered surface.

π = γ0 − γm (3.2)

It is important to note, that π is a twodimensional pressure, which is generally a function of many variables such as pH of the water, temperature, type of dissolved compound, etc. It also varies with the surface concentration of the monolayer forming molecules, making it possible to plot isotherms of surface pressure versus the average surface area occupied per molecule in the monolayer. These isotherms describe the different phase states of the monomolecular ﬁlms during the compression. In the uncompressed condition no intermolecular interactions are present, resulting in a gaseous-like state. Continuous compression of the surface leads to an almost solid-like behaviour arising from tight molecular packing in the monolayer, by increased intermolecular interactions. When a monolayer is compressed into this tightly packed, coherent state it is possible, by passing a carefully prepared solid substrate through it, to transfer the monolayer intact from the liquid to solid surfaces. Depending on the nature of the solid material, either the hydrophilic or hydrophobic parts of the amphiphiles adhere on the surface.

79 Chapter 3 Results and Discussion

In cooperation with Dr. GALLANI in Strasbourg, we examined the amphiphilic monoadducts (chapter 3.1.1, 3.1.3) for their ability to build LB-films (LANGMUIR-BLODGETT- films) and the possibility to transfer them onto substrates. The anionic 1st generation monoadduct 44 forms LANGMUIR films quite easily.

Figure 3.33: BAM-images of LB-ﬁlms from 1st generation anionic amphifullerene 44 at 200 Å2 (top left), 130 Å2 (top right), 65 Å2 (bottom left) and collapsus (bottom right).

The Brewster-Angle-Microscopy (BAM) images of the film formation (figure 3.33), shows that the final film at 53 mNm−1 is perfect and solid-like. The examined molecular area at this pressure is 65 Å2. Continuing compression results in the collapse of the monomolecular film (long wrinkles), which comes from the evasion of the molecules into the third dimension. The cationic 1st generation monoadduct 60 again nicely form films at the air-water interface. The final molecular area is almost pH independent, with 130 Å2 at pH = 7, 110 Å2 at pH = 3.2 and 150 Å2 at pH = 11.7 (see figure 3.34 (a)). The finally formed film showed a perfect molecular layer under the Brewster-Angle-Microscope (see figure 3.34 (b)).

80 Chapter 3 Results and Discussion

(a) (b)

Figure 3.34: pH dependence of the molecular area of the cationic 1st generation amphifullerene 60.

The reversibility of the isotherms on the other hand is much more pH-dependent (see figure 3.35). Going to higher pH-values significantly reduces the reversibility, which is nearly lost at pH = 11.7. The reason for this observation is not completely understood yet. The pH value should not have an influence on the solubility of the permanently charged 60. Maybe the exchange of the counterions with buffer-ions or the saponification of the esters play an important role here. Further experiments will help to clarify this issue. For the anionic 2nd generation monoadducts, the amphiphiles 47 and 56 were examined. In both cases the solubility in CHCl3 was not sufficient enough and therefore dissolution was improved with the adjunction of a small amount of ethanol. This comes together with a small error in the measurements of the molecular area, because some of the compound may dissolve into the water subphase upon spreading. Nevertheless the obtained values seem to be reasonable in view of the molecular shape. For 47 the final molecular area at neutral pH is 100 Å2, which would be exactly what is expected if the compression of the fullerene core is the limiting factor. Since the reversibility is quite good, the limiting factor cannot be the compression of the fullerene core since C60-C60 interactions would lead to irreversible aggregation (see figure 3.36 (a)). Moreover the films are stable in the sense that the surface pressure stays constant when the compression is paused. This means that the molecules do not dissolve or spontaneously form supramolecular architectures. Brewster-Angle-Microscopy (BAM) observations

81 Chapter 3 Results and Discussion

pH=3.2 pH=6.8

30 30

20 20

10 10 Surface pressure [mN/m] Surface pressure [mN/m]

0 0

100 150 200 100 150 200

2 2

Molecular area [Å ] Molecular area [Å ]

(a) (b)

pH=11.7

10 Surface pressure [mN/m]

100 150 200

Molecular area [Å ]

(c)

Figure 3.35: pH dependence on the reversibility of the isotherms for the cationic amphifullerene 60. show that the molecules experience intermolecular attraction forces (filament-like structures in the first pictures), but eventually the film is homogeneous and quite nice, without defects. No obvious sign of a collapse, even at the very end of the compression, could be detected (see figure 3.37). Change of the pH value directly influences the collapse pressure and the reproducibility. At higher pH values the pressure decreases significantly to ca. 13 mNm−1, corresponding to the increased solubility by ionization of the polar head group. At pH = 10.1 the reversibility is obviously lost, meaning that the water-solubility at this pH value accelerates the dipping into the subphase (figure 3.36 (b,c)). Interestingly this behavior is directly influenced by the nature of the base (NaOH, KOH) used for changing the pH. Figure 3.36 (d) displays the effects of the base on the surface pressure. In the case of NaOH as the base, the isotherm is significantly

82 Chapter 3 Results and Discussion

60 pH=10.5 pH=10.1 pH=9.7 othercurves:pH4.4->9.3

Surfacepressure[mN/m] 0 50 100 150 200 250 2 Moleculararea[Å ] (a) (b) 40 50 purewater

40 30

30 pH=10.7KOH 20 pH=10.5NaOH 20

10 pH=10.1 10 pH=9.3

Surfacepressure[mN/m]

Surfacepressure[mN/m] 0 0 100 150 200 250 0 100 200 300 400 2 Moleculararea[Å ] Moleculararea[Å 2] (c) (d)

Figure 3.36: Reversibility of 2nd generation anionic amphifullerene 47 in water – 3 compression expansion cycles with pressure limited to 35 mNm−1 (a); isotherms and reversibility as a function of the pH in a NaOH subphase (b,c); inﬂuence of the ionic content of the subphase (d).

flattened resulting in a decreased surface pressure at nearly the same pH. The reason for this behavior is not clarified yet, but maybe results from a degradation of the compound, which is directly influenced by the nature of the base. The observed pH dependent tendency can also be seen in the case of the fatty acid functionalized anionic amphifullerene 56. The final molecular area for 56 ranges between 87 Å2 at pH = 3.2 to 7 and 140 Å2 at pH = 11.2. The shape of the isotherms nicely corresponds to the isotherms of 47, although a difference in the final molecular area can be observed. Possibly the increased rigidity of the unsaturated fatty acid chain effects a more rod-like structure in the case of 56. In contrast the saturated chain in 44 is more flexible and therefore tend to increase the molecular area.

83 Chapter 3 Results and Discussion

Figure 3.37: BAM-images of LB-ﬁlms from anionic 2nd generation amphifullerene 47 at 200 Å2 (top left), 150 Å2 (top right), 125 Å2 (bottom left) and 87 Å2 (bottom right).

For compound 47 the ability to transfer LB films onto hydrophilic silicon was tested. It is possible to transfer at least up to five layers with good quality. The quality of the LB films can be assessed with grazing incidence X-ray reflectivity. The results are summarized in Figure 3.38. The left spectra shows the diffracted intensity at different numbers of monomolecular layers. Although the quality of the films is very good, the

1E12 1E8 80 70 1E10 5layers 60 3layers 1E6 50 1E8 1layer 40 30 20 1E6 electronicdensity 1E4 10 0 0 5 10 15 20 25 1E4 distancefromsubstrate 100 100

Diffractedintensity[a.u.]

Diffractedintensity[a.u.] 1 1 0 2 4 6 8 10 12 0 2 4 6 8 10 2θ[degrees] 2θ[degrees] Figure 3.38: X-ray reflectivity of the LB films of 47 at different numbers of layers (left); X-ray scan and "best" fit for 5 layers. Inset is the profile of the electronic density for one layer.

84 Chapter 3 Results and Discussion thickness of each sublayer stays not constant, ranging from 30 Å2 for the first layer to 125 Å2 for 5 layers. The right graph in figure 3.38 shows the spectrum of a 5-layer-thick LB film on silicon. We tried to fit the data with a box model, where each molecular layer has been divided into three sublayers, corresponding to polar head, C60 and alkyl chain. Each sublayer could further be ascribed with a specific thickness, electronic density, and roughness. With this model, the fit of the data for 47 was not acceptable. If it was assumed, that each molecular layer is not a perfect mono-molecular and has some "defects", e.g. supramolecular architectures, the fit quality was significantly improved. Nevertheless the data so far can not elucidate if aggregation is the reason for this behavior, since the quality of the observed LB film is actually immaculate. The investigation of additional parameters is still under progress and will help to understand this issue.

3.1.6.2 Incorporation of the Amphiphilic Fullerene-Monoadducts in Organic Solar Cell Devices

In the past view years the preparation of photovoltaic devices, made from composite ﬁlms of π-conjugated polymers and fullerene derivatives attracted considerable attention. As a concept a bulk heterojunction is used, which consists of a three dimensional interpenetrating donor-acceptor network, sandwiched between two electrodes with different work functions to generate an electric ﬁeld across the organic layer. From photo- physical studies it has been demonstrated that after absorption of a photon, ultra-fast electron transfer takes place from the excited state of a conducting polymer to acceptor

[203,204] molecules such as C60, with a quantum efﬁciency close to unity. Subsequently, the separated charge carriers are transported via the interpenetrating network to the electrodes. The photogenerated current is directly governed by the charge carrier mobility, alongside the number of photoexcited charge carriers. For the understanding of the opto-electronic properties of these photovoltaic devices, knowledge about the charge transport properties of the individual components is indispensable.

In cooperation with KONARKA Technologies GmbH [205] the amphiphiles 44 and 47 have been incorporated in organic ﬁeld-effect transistors (OFETs) to derive a value for

85 Chapter 3 Results and Discussion

Drain Source

Semiconductor

Insulator

Siliconsubstrate

Gate PCBM Figure 3.39: Schematic representation of a top contact organic field-effect transistor (left). [6,6]-phenyl C61-butyric acid methyl ester (PCBM) (right). the mobility of the charge carriers.[206] The field effect transistors were prepared using highly doped p-Si wafers as the gate electrode. A layer of thermally grown SiO2, with a thickness of 100-150 nm, served as the gate insulator. After applying the fullerene layer to the wafer, the source and drain electrodes (LiF/Al) were deposited onto the semiconducting layer. The structure of a typical top contact OFET is shown in figure 3.39 (left). The mobilities can be determined from the transfer characteristics of the OFET (Ids vs. Vgs for a low, [207] constant Vd s) according to the gradual channel approximation, with the equation

∂Ids WCi Vds = µFE (3.3) ∂Vgs L where Ids is the current ﬂowing in the channel between the drain and source contacts,

Vgs the gate voltage, µFE the field effect mobility, W the width of the channel, Ci the capacitance of the insulating layer, Vds the voltage between the drain and source contacts, and L the length of the channel. In the OFET containing amphiphile 44 the electron mobilities determined according to eq. 3.3 were found to be in the range of 5 x 10−4 cm2/Vs. The mobility of the OFET with 47 was quite low and could not be determined exactly (experimental resolution limited to 10−6 cm2/Vs). This large difference in the mobilities maybe results from the larger hydrophilic head group of amphiphile 47. Presumably the larger dendritic branch directly influences the morphology of the device films with an increased C60-C60 distance, which affects the charge transport in the blend. Compared to PCBM ([6,6]-phenyl C61-

86 Chapter 3 Results and Discussion butyric acid methyl ester) which is widely used as "standard" fullerene derivative in polymer electronics and photovoltaics, the mobility value for 44 is only about 1 magni- tude lower (7 x 10−3 cm2/Vs for PCBM). This is one of the highest values observed for this type of fullerenes, so far. We also studied the influence of amphiphile 44 on the morphology in blends with terthiophene polymers.[205] Polyterthiophenes are attractive conducting polymers for use in photoelectric conversion devices mainly due to their exceptional stability in air and high charge carrier mobility values.[208] Poly(3,3"-dihexyl-2,2’:5’2"-terthiophene) (C6-TT) can be synthesized by using Stille copolymerization [209] with an average molecular weight of 42000 g/mol. Pristine C6-TT shows a distinctive shoulder at 600 nm indicative for the aggregation of the thiophene backbone. This behavior can be used to determine the morphology properties in corresponding composites via optical absorption spectroscopy. The polymer was utilized to build up devices, containing 44 as electron-accepting moiety which were examined concerning the blend morphology. Figure 3.40 (a) shows the UV/vis spectrum of the terthiophene in the presence of 44. The characteristic signal at 600 nm indicates the packaging of the polyterthiophene backbone in presence of the fullerene. In contrast blends containing PCBM/C6-TT as active layer, the aggregation is clearly hindered (figure 3.40 (b)). This suggests, that PCBM could fill the side-chain spacing of the C6-TT backbone, as suggested in figure 3.40 (c), which would suppress the cofacial packing of the parallel chains. The asymmetric monoadduct 44 is too big to fit nicely in this spacing, which leads to ad- vantageous morphology forming properties. Further investigations are currently being carried out and will verify the efficiency of this promising class of materials for photovoltaic operation.

87 Chapter 3 Results and Discussion

beforetreatment 1min140°C C6-TT 4min140°C C6-TT ppt 4min165°C 1.0 1.0 C6-TT/PCBM 4min190°C C6-TT/PCBMppt 0.8 0.8

0.6 0.6

OD[a.u.] 0.4 OD[a.u.] 0.4

0.2 0.2

0.0 0.0 400 600 800 1000 400 500 600 700 Wavelenght[nm] Wavelenght[nm] (a) (b)

(c)

Figure 3.40: (a) Absorption spectrum of 44/C6-TT blend. (b) Absorption spectrum of a PCBM/C6-TT blend. (c) Comparison of the side-chain distance with the diam- [210] eter of C60. Pristine C60 would ﬁt nicely into this gap (as well as PCBM).

88 Chapter 3 Results and Discussion

3.2 Triazole Dendrimers Based Fullerenes via "Click Chemistry"

"Click chemistry" is a new approach that takes advantage of chemical building blocks with built-in high-energy content to drive a spontaneous and irreversible linkage reaction with appropriate complementary sites in other blocks. SHARPLESS and coworkers deﬁned what makes a click reaction as one that is wide in scope and easy to perform, uses only readily available reagents, and is insensitive to oxygen and water.[211] In fact, in several instances water is the ideal reaction solvent, providing the best yields and highest rates. Reaction work-up and puriﬁcation uses benign solvents and avoids chromatography. Carbon-heteroatom bond forming reactions comprise the most common examples for this type of reaction, including the following classes of chemical transformations:

• cycloadditions of unsaturated species, especially 1,3-dipolar cycloaddition reactions, but also the Diels-Alder family of transformations [212]

• nucleophilic substitution chemistry, particularly ring-opening reactions of strained heterocyclic electrophiles such as epoxides, aziridines, aziridiniumions, and episul- foniumions [213]

• carbonyl chemistry of the "non-aldol" type, such as formation of ureas, thioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides

• additions to carbon-carbon multiple bonds, especially oxidative cases such as epoxidation, dihydroxylation, aziridination, and sulfenyl halide addition, but also

MICHAEL additions of Nu-H reactants [214]

These click reactions achieve their required characteristics by having a high thermodynamic driving force, usually greater than 20 kcal mol−1.

Of the reactions comprising the click universe, the "perfect" example is the HUISGEN 1,3-dipolar cycloaddition of alkynes to azides, which gives acess to 1,2,3-triazoles as

89 Chapter 3 Results and Discussion

R1 80 - 120 °C 1 2 N N 1 R + R N3 + R N N N N R2 R2

1,4-regioisomer 1,5-regioisomer

Scheme 3.7: HUISGEN cyclization of azides and alkynes. a mixture of 1,4- and 1,5-regioisomers, which can be explained by the similarity in activation energies for both processes (see scheme 3.7).[215,216] Such intrinsic features of the HUISGEN cycloaddition make it unsuitable for being considered as a click reaction. Nevertheless, the observation that copper(I) salts promote faster (up to 107 times) and regiospeciﬁc couplings between terminal alkynes and azides allowed for the rapid development of this reaction; these results were reported independently by the groups of SHARPLESS [217] and MELDAL.[218] The mechanistic outline of the Cu[I]- catalyzed reaction is depicted in scheme 3.8. The stepwise cycloaddition catalyzed by a monomeric Cu[I] species lowers the activation barrier relative to a uncatalyzed process by as much as 11 kcal/mol, which explains the incredible rate enhancement observed under CuI catalysis.[219] The catalytic cycle begins with the formation of a Cu[I] acetylide species 70 via the π-complex 68, which is proposed based on earlier results of CuI insertion into terminal alkynes [220,221] and the fact, that internal alkynes show no activity in this type of reaction.[217,218]. This active species reacts further by the azide displacement of one ligand, generating a copper acetylide-azide complex, such as the dicopper species 71. The following metallocycle 72 is generated by the nucleophilic attack of the acetylide carbon to the azide, which is activated therefore by copper complexation.[222,223] In this way the bound azide is perfectly situated for the subsequent ring contraction by a transannular association of the nitrogen lone pair of electrons with the C-Cu π∗orbital.[219] Protonation of triazole-copper derivative 73 followed by dissociation of the product ends the reaction and regenerates the catalyst.

90 Chapter 3 Results and Discussion

CumLn R1 H

1 B R H 68 + B-H LnCu [LnCu]2 Cu catalyst 2 69 LnCu2 R

1 N R N N B

H R2 B-H 2 70 LnCu2 R 2 1 N Cu acetylide R N N 74 2 LnCu2 R 1 R N3

1 N R N N 73 R2 N R2 N L Cu R2 Cu n 2 N N L L 1 R L N Cu N Cu Cu R2 R1 L 72 71

Scheme 3.8: Proposed outline of species involved in the catalyzed cycle of the copper catalyzed HUISGEN cyclization.

In supramolecular chemistry dendrimeric materials are an extremely attractive class of substances. SHARPLESS and coworkers introduced a highly efﬁcient and prepara- tively simple approach for the generation of diverse dendritic structures of high purity and in excellent yield.[224] The topological and electronic similarities of 1,2,3-triazoles

mimicked by O N N R2 R1 N R1 N R2 H R1 to R2 distance: R1 to R2 distance: 3.9 Å 5.0 Å

Figure 3.41: Topological and electronic similarities of amides and 1,2,3-triazoles

91 Chapter 3 Results and Discussion to amides (ﬁgure 3.41), together with the fact, that the susceptibility toward hydrolytic cleavage is considerably decreased, make them promising candidates for biological

[225–227] [228] [229] applications. Anti-HIV activity, selective β3 adrenergic receptor inhibition, anti-bacterial activity,[230] and potent anti-histamine activity [231,232] are examples reported in the literature so far.

3.2.1 Synthesis of Novel Dendritic Triazol-Fullerenes

To combine the unique properties of 1,2,3-triazoles and [60]fullerene, we decided to synthesize triazole dendrimers by the copper(I)-catalyzed ligation of azides and alkynes and attach these moieties to the C60 core. An additional advantage of the triazole scaffold is a reasonable solubility in alcohols and even aqueous mixtures and could be therefore a perfect building block for the structural modification of fullerenes, to produce charged and primarily neutral water-soluble derivatives. For the topic of fullerenes in drug design (see chapter 3.1), such neutral compounds could exhibit to all intents and purposes perspicuous advantages, compared to charged systems. Neu- tral fullerene derivatives should increase the bioavailability and pharmacokinetics, by a demanding membrane permeability. Preexaminations on polyether and oligohydroxy functionalized fullerenes showed, that these polar groups were not sufficient to promote an appropriate water-solubility.[233] Through the introduction of the triazole functionality, combined with polar groups like polyethers we expected to obtain a significantly higher water-solubility. For the synthesis of the dendritic triazoles, the divergent approach was utilized (scheme

[234,235] 3.9). The AB2 monomer unit 77 can be easily synthesized by etheriﬁcation of methyl 3,5-dihydroxybenzoate 75 with propargyl bromide, yielding the benzoate 76.

Reduction of the methyl ester using LiAlH4 gave the benzyl alcohol 77 in 83 % yield.

For the attachment to the C60-core a convenient functional group has to be introduced.

In order to deploy the advantages of the BINGEL-HIRSCH reaction (high yields, high reaction rates) the benzyl alcohol 77 was further allowed to react with malonyl dichloride to give the benzyl malonate 78, which was puriﬁed by ﬂash column chromatography. For the synthesis of dendritic triazol-fullerene derivatives, in principle two different syn-

92 Chapter 3 Results and Discussion

Br HO OH O O O O K2CO3, LiAlH2 18-crown-6 77 O O O O OH 75 76 malonyl dichloride, pyridine

O O O O O O 78 O O Pathway A Pathway B

O N C60, CBr4, 3 N O 81 5 mol % CuSO4, DBU H 10 mol % sodium ascorbat N O 3 O 82

O O O O R1 N N O O N N R1 N O O N O O O O O O O O N N N N N N 1 R1 R O 1 83 R : N O 79 H

2 O R : O 84

O N 3 N O 81 5 mol % CuSO , H 4 10 mol % sodium ascorbat N O 3 O 82 C60, CBr4, DBU

R1 N N O O N N R1 N O O N O O

O O N N N N N N 1 R1 R

O 1 R : N O 85 H

2 O R : O 86

Scheme 3.9: Synthesis of the dendritic triazol-fullerenes 85 and 86 via 1,3-dipolar cycloaddition of azides on alkyne-malonates (Pathway A) and alkyne-monoadducts (Path- way B).

93 Chapter 3 Results and Discussion thetic pathways can be adopted. Pathway A starts with the cyclopropanation of the alkyne malonate 78 with C60, followed by the 1,3-dipolar cycloaddition of monoadduct 79 with azides. This pathway would provide the beneﬁt of using 79 as general building block, which can subsequently be modiﬁed with a variety of azides. Unfortunately it turned out, that the reaction conditions for the copper catalyzed HUISGEN cycloaddition is not suitable in the case of fullerene derivatives. Table 3.9 reviews the different reaction conditions tested for this type of reaction.

Conditions Entry Base or Reducing Catalyst Solvent T[°C ] Time Yield[%] Agent

1 sodium ascorbate CuSO4 4:1 THF/water 25 18 h 0 2 sodium ascorbate CuSO4 4:1 THF/water 80 18 h 0 3 Hünig Base CuI THF 25 18 h ≤5 4 Hünig Base CuI THF 65 18 h ≤5 5DBU CuI THF 6518h 0 6 DBU CuI toluene 25 18 h 0 7 Et3N CuI toluene/MeCN 75 18 h 0 8 Hünig Base (Ph3P)3CuBr THF 65 18 h ≤5 9 DBU (Ph3P)3CuBr THF 65 18 h ≤5 10 DBU (Ph3P)3CuBr THF/CH2Cl2 60 18 h ≤5

Table 3.9: Reaction conditions used for the copper catalyzed Huisgen cycloaddition of 79 with azides.

In Pathway B the triazol scaffold is build up first, followed by the derivatization with C60. For the synthesis of an uncharged triazol-fullerene derivative, the copper(I)-catalyzed reaction of malonate 78 with azide 82 was carried out in the presence of CuSO4 (2-5 mol%) and sodium ascorbate (5-10 mol%) in a 1:1 mixture of water and tert-butyl alcohol at room temperature to generate the triazole 84. By reason of the low solubility of 78 in the used solvent system, it was first suspended in the tert-butyl alcohol/water mixture and gently warmed, till complete dissolution was achieved. In contrast to the results reported above, this reaction proceeds very fast (TLC control showed the complete conversion of the starting material after 45 min) and in near-quantitative yields. Owing to the high degree of efficiency, the reaction could be conducted with a stoichiometric amount (4.0 equiv) of the azide. The literature work-up procedure,[224] using

94 Chapter 3 Results and Discussion

1081 100 1.2E6

80 9.7E5

60 7.3E5

40 4.9E5

20 1143 2.4E5 201 276 489 898 391 621 715 791 121312901384 1539 0 0.0E0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

Figure 3.42: FAB mass spectrum of the crude reaction mixture of 84 after 45 min. ammonium hydroxide for the removal of trace amounts of copper salts, could not be adopted in this case, since saponification of the malonic esters occured. Therefore the crude product was washed several times with a highly diluted ammonium hydroxide/citrate aqueous buffer. Purifications were greatly simplified due to the absence of side products. In the case of the triazol dendron 84 purification was obtained via flash column chromatography through a short silica plug. Figure 3.42 shows the FAB-MS of 84 recorded from the crude reaction mixture after 45 min reaction time. The dominating peak at 1081 belongs to the molecular peak of 84, whereas the missing molecular peak for the starting material 78 shows the high reaction rates for this conversion. The 1H NMR spectroscopy is an especially useful tool for illustrating the growth of the dendrimer generation, as shown from the stacked plots in Figure 3.43. The new resonances at 7.7 ppm correspond to the unique triazol protons and are in the typical range for 1,4-disubstituted 1,2,3-triazoles. Missing of a high-field shifted signal at 7.4 ppm (typical proton signal for 1,5-disubstituted 1,2,3-triazoles), confirms the exclusive formation of the 1,4-isomer. The splitting of the aromatic protons 3 and 4 with the integration ratio of 2:1 in the case of the alkyne malonate 78 could not be obtained in the case of the triazol dendron 84, resulting in a superimposed broad signal. The signals for the polyether chain are shifted low-field, whereas the signals for the methyl groups next to the triazol ring show the largest shift. The solubility of dendron 84 was expectable high in polar solvents, like THF, DMSO and alcoholic solutions as well as in chlorinated hydrocarbons. Unfortunately the solubility in aromatic organic solvents

95 Chapter 3 Results and Discussion

1 3

2,3 1

8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5

1 2 78 1

* *

8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 ChemicalShift(ppm)

1 Figure 3.43: H NMR spectra of alkyne-terminated malonate 78 (300 MHz, RT, CDCl3) (bottom) and polyether-terminated triazole dendron 1st generation 84 (400 MHz, RT, CDCl3) (top). was insufficient. This required a modification of the subsequent BINGEL-HIRSCH reaction, which is generally performed in toluene or other suitable chloroaromatics as solvent. A binary solvent mixture was found to be the best way to explore solubility for the triazole as well as for C60. Table 3.10 illustrates the different solvent systems, used for the reaction with C60 under the BINGEL-HIRSCH conditions. The most efficient solvent system tested, was a toluene/DMSO mixture. The in this way obtained triazole fullerene 86 could be purified by flash column chromatography. Although the separation of the polar products from higher adducts has been found to be difficult, the pure monoadduct 86 could be obtained by stepwise increase of the polarity gradient, using dichloromethane/methanol as mobile solvent system. Figure 3.44 represents the 13C NMR spectrum of 86. The unique signal for the triazole at 141 ppm is located in the

96 Chapter 3 Results and Discussion

Conditions Entry Solvent Base Brominating Agent Yield[%]

1 toluene DBU CBr4 ≤5 2 toluene/CH2Cl2 = 2:1 DBU CBr4 5 3 toluene/CH2Cl2 = 1:1 DBU CBr4 5 4 toluene/THF = 3:1 DBU CBr4 8 5 toluene/DMSO = 3:1 DBU CBr4 32

Table 3.10: Tested binary solvent mixtures for the cyclopropanation of triazole dendron 84 with C60 at rt after 24 h. range of the fullerene sp2 signals, but could be clearly associated through the C-H correlated HETCOR-spectrum. The signal for the methylene group 8 could be detected at 62.1 ppm and shows the typical high-ﬁeld shift, caused by the electron-rich triazole

13 15 12 14 1 3 4 5 11 2 6 9 10 8 14 7 13 15 12 11 86

10 8

sp2 -C 6 5 sp3 -C 1 3 9 7 4 2

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ChemicalShift(ppm)

Figure 3.44: 13C NMR spectrum of polyether-terminated triazole monoadduct 86 (100.5 MHz, RT, CDCl3).

97 Chapter 3 Results and Discussion

O O O O HN N N O O N N NH N O O N O O

N N O O N N N N O O NH HN O O 85

TFA, CH2Cl2

H3N N N O O N N NH3 N O O N O O

N N N O O N 4 CF3COO N N

H3N NH3

Scheme 3.10: Synthesis of charged triazole-fullerene 87 through acidic cleavage of the carbamate groups in triazole-fullerene 85. unit. The signals for the peripheral polyether-groups remain nearly unaffected from the

C60-core and show no obvious shifts, compared to the malonate 84. Triazole-fullerene 86 exhibited solubility in alcoholic solutions, THF and chlorinated hydrocarbons. Dis- solution in water could not be obtained. Since the solubility of 86 did not fulﬁll the expectations, we decided to introduce terminal amino groups to elevate the water-solubility. Although there are a variety of different amino-fullerenes known in the literature,[236,237] these compounds combine the property of only being soluble in acidic aqueous solutions. Therefore the incorporation of triazole linkages could promote the solubility over an extended pH-range. The basic chemical 2-aminoethanol was used for the synthesis of the precursor. The nucleophilic amino-functionality of 2-aminoethanol was protected with BOC-anhydride as carbamate 80,[238] followed by the nucleophilic substitution with sodium azide to obtain the corresponding azide 81.[224] The 1,3-dipolar cycloaddition with the acetylenic

98 Chapter 3 Results and Discussion malonate 78, as depicted in scheme 3.9 (Pathway B), gave the corresponding triazole dendron 83 by following the synthetic procedure described for 84. The reaction succeeded with the same reaction rates and in nearly quantitative yield, as mentioned before in the case of 84. Triazole dendron 83 could be easily purified by recrystallization from methanol. The yield of monoadduct 85 after nucleophilic cyclopropanation could be again clearly increased by the use of a toluene/DMSO mixture as solvent. The purification via flash column chromatography emerged to be facile in this case.

Stirring of 85 in a TFA/CH2Cl2-mixture resulted in the complete cleavage of the carbamates (scheme 3.10). The adduct 87 could be isolated after reprecipitation in nearly quantitative yield in the form of the corresponding ammonium salts. The purity of the precursors and the adducts 85, 87 could be conﬁrmed by the means of FAB-MS, 1H and 13C NMR-spectroscopy. The observed spectra show the same characteristica as reported for 86. As expected, the introduction of the triazole groups results in a pH independent solubility behavior of 87 in aqueous solutions.

absorbance pH = 11.0 pH = 7.0 pH = 4.0

250 300 350 400 450 500 550 600 wavelength[nm]

Figure 3.45: UV/Vis spectra of amino-terminated triazole-fullerene 87 at pH = 4.0 (red), pH = 7.0 (grey) and pH = 11.0 (blue).

99 Chapter 3 Results and Discussion

The stacked graphs in ﬁgure 3.45 represent the UV/Vis spectra of 87 at different pH values. In buffered alkaline and neutral solution the typical ﬂattened spectra for ag- gregated monomers can be observed. Although the slope of the spectrum at pH = 4 does not differ substantially from the spectra at higher pH values, the appearance of the characteristic monoadduct absorption at about 428 nm indicates the decreased aggregation tendency in acidic solution. Since all amine groups are completely protonated at this pH value, this result is consistent with repulsive interactions between the corresponding ammonium groups, which should clearly disfavor the formation of supramolecular aggregates.

O O SOCl2 O O

OH 77 Cl 88

Cu(I), base 1 R -N3

N N N N N N N N 1 1 1 1 R N O O N R R N O O N R NaN3

N3 90 Cl 89

Cu(I), base 78

N R1 R1 N N N N N

O O N N O O N N N N N O O N N R1 N N O O N R1 O O R1 N N R1 N N O O N N

O N N O N N NN

O O

1 N N 91 N N 1 R N N R

Scheme 3.11: Possible synthetic strategy for the preparation of higher generation triazole- dendrons.

100 Chapter 3 Results and Discussion

The near-perfect reliability of the copper(I) catalyzed synthesis of 1,2,3-triazoles from azides and alkynes should facilitate the synthesis of higher generation triazole dendrimers. A possible synthetic strategy, as outlined in scheme 3.11, could be the introduction of a chloromethyl group in the AB2 monomer 77, which allows the facile activation of the focalpoint group during dendrimer construction by the convergent-growth approach. The attachment of higher generation dendrons to the fullerene core could enhance the solubility in aqueous solutions and can be considered in future investigations.

101 Chapter 3 Results and Discussion

3.3 Synthesis of Novel Fullerene-SWCNT Hybrids

Carbon nanotubes (CNTs) are an exciting allotrope of carbon. A single-walled carbon nanotube is a one-atom thick sheet of graphite (called graphene) rolled up into a seam- less cylinder with a diameter in the order of a nanometer. This results in a nanostructure where the length-to-diameter ratio exceeds 10,000. Such cylindrical carbon molecules have exciting novel physical properties [239–242] that make them potentially useful in many applications in nanotechnology, electronics, op- tics and other ﬁelds of materials science.[243–249] They exhibit extraordinary strength and unique electrical properties, and are efﬁcient conductors of heat. Nanotubes are members of the fullerene structural family, which also includes fullerenes. Whereas

C60 is spherical in shape, a nanotube is a cylindrical and hollow tube shape carbon material. Their name is derived from their size, since the diameter of a nanotube is in the order of a few nanometers (approximately 50,000 times smaller than the width of a human hair), while they can be up to several millimeters in length. There are two main types of nanotubes: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). The nature of the bonding of a nanotube is described by applied quantum chemistry, speciﬁcally orbital hybridization. The chemical bonding of nanotubes is composed entirely of sp2 bonds, similar to those of graphite. This bonding structure, which is stronger than the sp3 bonds found in diamond, provides the molecules with their unique strength. Nanotubes naturally align themselves into "ropes" held together by van der Waals forces. Under high pressure, nanotubes can merge together, trading some sp2 bonds for sp3 bonds, giving great possibility for pro- ducing strong, unlimited-length wires through high-pressure nanotube linking.[250] In the last years the functionalization of carbon nanotubes became a topic of interests for chemists and material researchers. Due to the insolubility of carbon nanotubes in both water and organic solvents, the enhancement of the solubility [251] is a major task for the application in materials and devices. Additionally the chemical functionalization allows the unique properties of SWCNT´s to be coupled to those of other types of materials. Figure 3.46 shows the four different approaches for the chemical functionalization

102 Chapter 3 Results and Discussion

SWCNT

D C Figure 3.46: Schematic representation of the different possibilities for the functionalization of carbon nanotubes. A) endohedral functionalization; B) defect functionalization; C) non covalent functionalization; D) covalent sidewall functionalization. of carbon nanotubes such as, endohedral functionalization (A), defect functionalization (B), non covalent functionalization (C) and covalent sidewall functionalization (D). The following chapters describe the synthesis and characterzation of novel [60]fullerene- SWCNT hybrids via the covalent and non-covalent pathway.

3.3.1 Covalent Sidewall Functionalization of SWCNT’s with a Fullerene-Monocarboxylic Acid Derivative

With the chemical sidewall functionalization it is possible to link a broad variety of functional groups onto the sidewall of the nanotube. It is accompanied by a change of hybridization from sp2 to sp3 and a simultaneous loss of conjugation. Due to the lower reactivity of CNT’s compared to fullerenes,[252] the functionalization requires very re-

103 Chapter 3 Results and Discussion active reagents, like elemental ﬂuorine,[253] GRIGNARD-reagents,[254] radicals [255] or nitrenes.[256] Recently the HIRSCH group succeeded in the synthesis of hydroxy functionalized SWCNT’s [257] through reductive alkylation. For this purpose, the SWCNTs were ﬁrst reduced using sodium and naphthalene in absolute THF. The following alkylation was accomplished by the use of 2-(3-bromopropoxy)-tetrahydropyran as alkyl halide. The cleavage of the ether moieties could be obtained with a THF/HClconc. solution in quantitative yields. In order to functionalize the remaining hydroxy groups with suitable fullerene-derivatives, we had to apply methods leading to a complete conversion, since the separation of a reaction mixture is impossible because of the presence of multiple addends on a molecular SWCNT. We therefore decided to synthesize a sterically unhindered fullerene monoadduct, which holds a carboxylic acid functionality that can easily be transferred to the corresponding acid chloride. In scheme 3.12 the synthetic pathway for the carboxyfullerene 94 is shown. To avoid cross-linking between the fullerene and the SWCNTs the asymmetric malonate 92 was obtained by reaction of 22 with methylmalonyl chloride. The following cyclopropanation via the

BINGEL/HIRSCH reaction yielded monoadduct 93 in high yields. Deprotection following standard procedures with TFA in toluene gave 94 in quantitative yields. By reason of the impossibility to isolate the corresponding acyl chloride, it was generated in situ, with the use of SOCl2 in a mixture of TCE and CHCl3 at 60°C followed by the addition of the hydroxy-SWCNTs [257] in TCE. The reaction mixture was stirred for additional 4 d at rt. After evaporation of the solvents under reduced pressure, the crude product 95 was resuspended in a mixture of 50 ml water and 50 ml ethanol. The suspension was filtered through a 0.2 µm PTFE membrane filter, washed with ethanol and THF. The resulting black solid was dried in a vacuum oven at 50°C overnig ht. The characterization of 95 by standard spectroscopic methods fails in this case, therefore X-ray photoelectron spectroscopy (XPS) [258,259] was used to investigate the atomic composition of the material and to determine the degree of functionalization. The measured XPS spectra showed a significant increase of the amount of oxygen in the survey spectrum of 95 compared to the starting material. This increase can be assigned to the introduction of the fullerene-malonate moiety by esterification. Recorded

104 Chapter 3 Results and Discussion

O O MeO Cl O O O O HO O O O pyridine O 22 92

C60, CBr4, DBU

O O O O OH O O O O O O O

TFA

94 93

1. SOCl2, TCE/CHCl3, 60 °C 2. Hydroxy-SWCNT, rt

O O O O O

Scheme 3.12: Synthetic pathway for the fullerene/SWCNT-hybrid 95.

TEM images of the fullerene-SWCNTs allowed the direct proof of the presence of fullerene molecules on the sidewalls of the SWCNT’s.[257] Figure 3.47 represents the comparison of the hydroxy-SWCNTs and the fullerene-SWCNT-hybrids, showing the unique structure of the new compound 95. Through the combination of the outstanding properties of fullerenes and SWCNTs, this new compound may show novel electron transfer properties and would be a interesting candidate for new material applications.

105 Chapter 3 Results and Discussion

(a) (b)

Figure 3.47: TEM images of 3-hydroxypropyl-SWCNTs (a) and fullerene-SWCNT-hybrid 95 (b).

3.3.2 Non-Covalent Functionalization of SWCNT’s with a Fullerene-Pyrene Dyad

The extended π-system of the carbon nanotubes sidewall allows the functionalization with various groups through π-π-stacking interactions. This has for example a special interest for the non destructive puriﬁcation process of carbon nanotubes by increased solubilization. In this context the pyrene-group showed to be a promising candidate for the effective assembling to the graphitic sidewall via π-stacking interactions.[260,261] The pyrene moiety was therefore speciﬁcally designed in such a way that a variety of molecules with intriguing properties could be further coupled via amide linkages.[260] With this aproach, the immobilization of proteins, DNA, and other smaller biomolecules on the side-walls of SWNT’s could be achieved. Similarly, pyrene-carrying ammonium ions were used to help solubilize CNT’s in water. This chapter reports on a new type of [60]fullerene-hybridized SWCNT material, in which a symmetrically covalently linked [60]fullerene-pyrene conjugate was immobilized onto the surface of SWCNTs by using a noncovalent method. The preparation of the conjugate 99 is depicted in scheme 3.13

106 Chapter 3 Results and Discussion

OH HO OH OH O EDC, DMAP O O

96O O 97 Cl Cl pyridine

O O O O O O O 98 O

C60, CBr4, DBU

O O O O O O O O

Scheme 3.13: Synthesis of the pyrene-fullerene-conjugate 99. and starts with the reaction of 1-pyrenebutyric acid 96 with 1,2-ethanediol. The incorporation of this C2-unit should increase the ﬂexibility of the conjugate and therefore a higher reciprocal effect with the sidewall of the SWCNT’s is expected. The esteriﬁca- tion of the alcohol 97 with malonyl dichloride yielded the symmetric malonate 98 in 67

% yield. Coupling of the malonate 98 to C60 was sucessfully obtained by the use CBr4 and DBU. Separation of the monoadduct 99 from higher adducts was accomplished by flash column chromatography using toluene/ethyl acetate = 10/1 to 8/1 as eluent. To prevent any impurities (e.g remaining silica) the conjugate 99 was further purified by HPLC (Nucleosil 5 µm) using CH2Cl2/methanol = 98/2 as eluent. Monoadduct 99 could be characterized by the standard methods. In figure 3.48 the 1H-NMR spectra is shown. The protons of the C2-spacer unit (marked green) are shifted lowfield, due to the influence of the fullerene core. The remaining signals could be detected in almost unaltered position in the spectrum. The zoom into the pyrene region of 98 and

107 Chapter 3 Results and Discussion

a a 1 b b 1 98

a b * *

8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 *

a a

b b

a b

8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 ChemicalShift (ppm) (a)

8.25 8.15 8.05 7.95 7.85 7.75 7.65 8.25 8.15 8.05 7.95 7.85 7.75 7.65 ChemicalShift (ppm) ChemicalShift (ppm) (b)

1 Figure 3.48: (a) H-NMR spectra of pyrene-malonate 98 (400 MHz, RT, CDCl3) and pyrene- fullerene-conjugate 99 (400 MHz, RT, CDCl3); (b) zoom into the pyrene region of 98 (left) and 99 (right).

108 Chapter 3 Results and Discussion

99 (see figure 3.48 (b)) show no significant changes in the observed chemical shifts, and indicates no significant backbinding of the pyrene-units to the fullerene-core via

π-π-interactions, which can be explained on the basis of the spherical shape of C60. [262] Prior investigations on the interaction of a water-soluble pyrene derivative with C60 restated this result. For the cylindrical shape of the SWCNTs we expected much higher interactions. The UV/Vis spectrum of 99 exhibits the characteristic slope of a pyrene derivative. The characteristic absorptions of the C60 moiety signiﬁcantly increases the intensity in the UV region between 260 and 400 nm. The absorption maximum at 425 nm shows the characteristic feature of a C60 monoadduct (ﬁgure 3.49).

200

180

160 99

] 140

3 120

x 10

-1 100 98

-1 80

[M ε 60

40 98 99 20

0 300 350 400 450 500 wavelength[nm]

Figure 3.49: UV/Vis spectra of bispyrenylmalonate 98 and bispyrenylmonoadduct 99.

For the preparation of the fullerene-SWCNT-hybrid, a solution of 99 in 1,2-dichloroethane (DCE) was added to a suspension of SWCNT’s in DCE. The suspension was stirred for several minutes, till decolorization of the supernatant occurred (for experimental details see appendix B). The disappearance of the typical reddish color for the

109 Chapter 3 Results and Discussion

Figure 3.50: Images of conjugate 99 in DCE (left), after addition of SWCNT’s in DCE and stirring (right) and after sedimentation of the suspension (middle). monoadduct 99 gave the first indication of the successful immobilization of conjugate 99 on the SWCNT surface (figure 3.50). To verify the degree of functionalization, we decided to use the Scanning Tunneling Microscopy (STM) technique.[263,264] The STM has been shown to be a valuable tool for viewing the surface morphology down to the atomic scale. Against the expectations, the STM measurements indicate, that the SWCNTs reveal very low amount of functional moieties. Figure 3.51 left shows an unfunctionalized SWCNT. This individual SWCNT crosses a monoatomic step of the substrate surface. As expected for the π-stacking functionalization, the SWCNT sidewall remained intact, there are no additional defects introduced by the functionalization. The herringbone reconstruction appearing on the substrate surface surrounding this SWCNT indicates that the surface did not suffer from contamination, but remained mainly clean on the atomic scale. We expect this result as the preparation method is carried out in ultra high vacuum. Due to their little corrugation, the reconstruction is not visible in the current color scale. However, no bundles of SWCNTs were observed, which on the other hand indicates a debundeling effect through the functionalization process, as well as the preparation method via dry transfer printing which is believed to peel the SWCNTs out of bundles. The relatively big particle reflects the content of residual catalyst material from the synthesis process. Figure 3.51 right shows an STM image of typically protruding functional molecules on

110 Chapter 3 Results and Discussion

5nm

2.0

1.5

1.0 Height [nm]

0.5

0.0 0 5 10 15 20 25 30 35 Position [nm]

Figure 3.51: Left: STM topograph of an individual unfunctionalized SWCNT. The surrounding Au surface is not further contaminated, the herringbone reconstruction conserved. Right top: STM image of a functionalized SWCNT. The fullerene derivative 99 is placed on top of the SWCNT. Right bottom: Line profile over the SWCNT and the moiety. the SWCNTs. The height of this protrusion with respect to the SWCNT reaches up to 9 Å. The diameter of the moiety appears 5 nm which seems to be surprisingly big. But as the STM topography resembles the convolution of sample and tip geometry, the lateral size thus is expected to be overestimated. An additional enlarging effect is that the functional molecule could rotate around the SWCNT during sampling. This might be induced by the attractive VAN DER WAALS interaction with the STM tip. As conjugate 99 is bound to the SWCNT via the relatively weak π-stacking interaction, this force could be sufficient to make the molecule hopping to neighboring SWCNT lattice sites. Therefore, the lateral dimensions of the moiety clearly are difficult for quantitative iden- tification purposes. We can only conclude rotational symmetry reliably. Fortunately, the height information does not suffer from the mentioned effects and a height of 9 Å nicely fits the assumption of a π-stacking bound 99 molecule on top of the SWCNT (figure 3.52). But although the geometry fits nicely, of course these protrusions have not necessarily to be fullerene-derivatives. Catalyst particles could fit the size as well and are present in every SWCNT material. For further identifaction, it is compulsory to have a look on the molecular substructure of the moieties. Figure 3.53 shows the atomically resolved

111 Chapter 3 Results and Discussion

Figure 3.52: Representation of the possible π complex between 99 and the SWCNT.

3nm 3nm

(a) (b)

Figure 3.53: STM topography (a) and current (b) image of 99 functionalized SWCNT’s. The SWCNT conﬁguration is irregular, there are various noisy regions indicating mobile particles or insufﬁciently stabilized SWCNT’s. Markedisa 99 molecule which is suspected to functionalize the SWCNT.

STM topography (a) and current image (b) of functionalized SWCNT’s. Here several SWCNTs are alinged near each other. Only one relatively dense layer of SWCNT’s appears, it is no bundle as it might appear on the ﬁrst look. The marked monoadduct 99 has ﬂipped beside so that it is not only bound to the corresponding SWCNT but also is mechanically supported by the neighboring SWCNT as well as by the substrate sur-

112 Chapter 3 Results and Discussion

3 SWCNT PTFE @ SWCNT 2

1 dI/dV [arb. units]

-1 0 1 Bias Voltage [V]

Figure 3.54: STS measurement on 99 functionalized SWCNT. face. This allows to resolve it atomically. The revealed rich substructure seems to be nearly spherical and resembles that of fullerenes. Although 99 is a fullerene derivate, it has not necessarily to reveal fullerene substructure. One clearly can see something which can be associated with the additional pyrene sidegroup pointing to the SWCNT. To verify that a chemical interaction is formed and the 99 molecules are not only located occasionally nearby the SWCNTs, scanning tunneling spectroscopy (STS) [265] measurements on the functional molecules of which figure 3.54 shows an example, were carried out. Three different measuring points were performed at one time. The surface state of Au(111) has been measured to line up the measuring setup, mainly the tip configuration concerning adsorbates and particles. Figure 3.54 illustrates the measurements of the DOS (Electronic Density of States) of the SWCNT a few nm beside the moiety (solid line) and on the functional molecule itself (dotted line). The SWCNT DOS reflects a metallic SWCNT and even reveals the pseudo gap arising from the bending of the graphene states. The VAN HOOVE singularities however appear more steplike. In the case of the functional molecule, a variety of additional states arise and also the underlying SWCNT DOS is modified. After having proven that indeed functionalization of the SWCNT’s occured, it had to be tested, whether functional molecules can be removed from the surface. Interest- ingly, the substrate surface nearby the SWCNT’s not always but usually reveals the coverage with removed functional molecules of which one is marked with an arrow in

113 Chapter 3 Results and Discussion

5nm

(a) (b)

Figure 3.55: STM topography (a) and current (b) image of connected SWCNT fragments and 99 molecules on the neighboring substrate surface.

10nm 3nm

(a) (b)

Figure 3.56: (a): C60 nanowire at a relatively large scan size. The unaffected monoatomic gold step edges indicate clearly, that the nanowires assembly is not substrate mediated. (b): close-up of a C60 nanowire. Little interruptions makes evident that there is no nanotube underneath the fullerene chain.

ﬁgure 3.55. Especially in the current image, there are several fullerene-like spheres which seem to stand on two sail-like aligned and ﬂat protrusions pointing towards the gold surface. The SWCNT itself seem to consist of several fragments. Surprisingly, the removed 99 molecules seemed to be in a kind preorientated, using the SWCNT as a template. Figure 3.56 shows this result. On the left, a large section of a nanowire

114 Chapter 3 Results and Discussion is shown. The length of those wires reach up to 300 nm or even more (larger scans were not performed). As the surface area provides a variety of different step edges and different orientations of them, it can be excluded that the wire simply follows one of the step edges. This can also be seen from the fact that the wire is not exactly straight but somehow bent. The typical height of those wires is in the range of 4 Å, so we can exclude the presence of a SWCNT under the fullerenes to possibly cause the wirelike structure. Figure 3.56 right shows a close up of a nanowire. Clearly the wire consists of aligned spherical particles, the functional fullerenes. The height of the wire measures 0.4 nm. This can be attributed to the lower transmission coefficient of electron tunneling through the wire. Nearby the wire, there are contaminations of the gold surface visible, e.g. residual solvents or mechanically crushed 99 molecules could be possible. To handle, operate and manipulate nanoscopic devices, selforganization of molecular modules on solid surfaces and interfaces, is one of the main challenges in nanotechnology and nanoscience. The results demonstrate the ability to fine-tune interfacial and intermolecular interactions, which permits the building of well-defined, ordered structures over large areas, which could be visualized and characterized by scanning tunneling spectroscopy. By using SWCNTs as templates, it is possible to transfer preor- ganized fullerene structures to suitable substrates in well-defined strands of molecules by stamping-like printing onto a surface. These one-dimensional nanostructures of organic semiconductors can be attractive candidates as active components in electronic nanodevices.

115 Chapter 3 Results and Discussion

3.4 Supramolecular Approach for the Formation of

C60-Bisadducts

Chemical functionalization of fullerenes is the first step to investigate their intriguing properties and applications. With higher adducts of C60 it is possible to generate unique architectures, in which the fullerene core can be modified with different functional groups. By controlling the degree of addition and the addition patterns, it is possible to adjust the physical and chemical properties directly to possible applications in nanomaterials. In contrast to the mono-functionalization of C60, which is straightforward, the multiple functionalization is afflicted with many problems. The stepwise multiple additions to C60 yield a mixture of geometric isomers with low yield and low selectivity for respective isomers, and tedious chromatographic separations are required. A general solution to overcome these problems is to find a generic linker which can direct the following additions to expected positions on the C60 surface. DIEDERICH and coworkers reported the first important approach in this content, by the tether di- rected functionalization of C60 with open-chain bismalonates. With the subsequent cyclopropanation of these sterically rigid and predefined malonates with C60, a broad variety of different bisadducts with defined addition pattern have been synthesized so far.[97,266–268] (For a detailed description of the possible regioisomeric bisadducts see [68,269]). Although this method represents a particularly suitable method for the regioselective cyclopropanation, it takes disadvantage from poor yields, extensive synthetic procedures and the lack of possible further functionalization. To circumvent this diffi- culties, HIRSCH and coworkers introduced a modified concept. Thereby macrocyclic cyclo-[n]-malonates are used, to build the suitable tether. Variation of the length of the flexible alkylchain between the malonate groups allows to tune the regioselectivity of the tether. In contrast to the method described above, the regioselectivity is not based on steric preorganization, but on the avoidance of unequal strain in the alkyl chains. Within this approach it was possible to synthesize a diversity of highly regioselective

[132,269–271] bis-, tris- and tetrakisadducts of C60. Although the selectivity and yields of the cyclopropanation with this cyclic tethers is more than satisfying, the corresponding

116 Chapter 3 Results and Discussion

Tether

O O O O O O O O C , DBU, CBr 60 4 O O O Tether O O O toluene O O

(CH2)n O O

O (CH2)n O O O O C60, DBU, CBr4 O O O toluene O O (CH2)n O O

O O (CH2)n

Scheme 3.14: Regioselective bisfunctionalization of C60 with open-chain malonate tethers (top) and macrocyclic malonate tethers (bottom). malonates are often hardly accessible. The standard method for their preparation is the reaction of malonyl dichloride with alkanediols. This reaction can only be accomplished by the dropwise addition of malonyl dichloride to a highly diluted solution of the diol.[132] Nevertheless side products like higher macrocycles and polymeric material require an extensive chromatographic puriﬁcation process, which is aggravated by the similar polarities of product and side products. The following chapter describes a new approach for multiple C60 adducts, where the malonates are linked via supramolecular interactions.

3.4.1 Metallomacrocycles as Tethers for Regioselective Cyclopropanation

The strategy to ligate metal ions as building blocks for the construction of cyclic malonate structures is depicted in scheme 3.15. The symmetric malonate 100 can be easily obtained by reaction of malonyl dichloride with an 2-fold excess of the alcohol 22. Deprotection of the tert-butyl esters with TFA in chloroform yielded the dicarboxylic malonate 101 in quantitative yield. For the preparation of the bridged dinuclear

117 Chapter 3 Results and Discussion

O O Cl Cl

O pyridine HO 22 O

O O R = tBu 100 RO OR TFA O O R = H 101 O O

M: metal L: co-ligand

O O O O O O O O L M M L O O O O O O O O

Scheme 3.15: Concept for the metal ion mediated cyclization of dicarboxylic malonates. metal complexes, the dicarboxylate ligand 101 was generally treated with an equimolar amount of the metal salt in the presence of a suitable co-ligand. The following table 3.11 summarizes the different reagents and reaction conditions used for this type of dimerization.

Entry Metal salt Co-ligand Reaction conditions

A Zn(OH)2 none hexane, reﬂux, 3 h B MnCl2 ·4H2O none H2O, pH = 5.5, reﬂux, 8 h C CuCl2 ·2H2O 1,10-phenanthroline MeOH/H2O = 1/1, 65°C , 3 monohydrate days

Table 3.11: Reagents and reaction conditions for the metallomacrocyclization of dicarboxylic malonate 101.

118 Chapter 3 Results and Discussion

Complexation with Zn [272]

For the complexation with Zn, Zn(OH)2 was freshly precipitated from a ZnSO4 solution with NaOH, extensively washed (to avoid any NaOH residues, which could lead to the decomposition of the malonesters) and dried. Zn(OH)2 was further refluxed with an equimolar amount of 101 in hexane. After 3 h a transparent solution was obtained. The reaction mixture was allowed slowly to cool to room temperature, whereas the precipitation of a white solid could be observed. The solid material was filtered off, washed with hexane and dried in vacuum. Although the dissolution of the malonate indicates the neutralization reaction between Zn(OH)2 and 101, IR and NMR studies showed no evidence for the formation of the bis(carboxylato)zinc complex. However, most of the precipitate was identified as unconverted starting material 101.

Complexation with Mn [273]

For the complexation with Mn, MnCl2 ·4H2O was reﬂuxed with an equimolar amount of 101 in destilled water at pH = 5.5 for 8 h. The reaction mixture was allowed to cool to room temperature, whereas no precipitation of solid material could be observed. Evap- oration of the solvent and subsequent IR and NMR studies again showed no evidence for the formation of the bis(carboxylato)manganese complex.

Complexation with Cu [274]

For the complexation with Cu, CuCl2 ·2H2O was added to an equimolar amount of

101 in a mixture of MeOH/H2O = 1/1. Treatment with an equimolar amount of 1,10- phenanthroline monohydrate (C12H8N2 ·H2O) as co-ligand resulted in a blue solution, which was filtered. Na2CO3 (1 M) was dropwise added, leading to a fine, pale-blue precipitate which was separated. The filtrate (pH = 5.5) was kept at 65°C for 3 days and after cooling to room temperature, blue crystals could be obtained. X-ray analysis of the crystals showed not the formation of the azelaato-bridged dinuclear copper(II) complex. Instead of, the aqua-(malonato-O,O’)-(1,10-phenanthroline-N,N’)-copper(II)- complex 102 was identified, indicating the metal induced hydrolysis of the malonesters (figure 3.57). The X-ray sample contained two different crystal species, which ex-

119 Chapter 3 Results and Discussion

Figure 3.57: Crystal structure of (malonato-O,O’)-(1,10-phenanthroline-N,N’)-copper(II) 102 (thermal ellipsoids at 50 % probability level).

hibited the complex 102 in the two different packing motives 2[Cu(H2O)(mal)(phen)] [275] [Cu(mal)(phen)] x 11 H2O and [Cu(H2O)(mal)(phen)] x 1.5 H2O.

3.4.2 Hydrogen-bonded Dimers as Tethers for Regioselective Cyclopropanation

Hydrogen bonding displays a very attractive interaction for the formation and organization of supramolecular architectures. One of the most astonishing example is offered by nature, where multiple hydrogen bonds and hydrophobic interactions form the exceptionally stable double-stranded DNA. Such biological examples have provided a power- ful incentive for researchers to explore molecular association more broadly, and a variety of hydrogen bonding motifs were discovered by different groups so far. In ﬁgure 3.58 the most relevant hydrogen-bonding arrays are summarized. In the case of the simplest

−1 donor-acceptor (DA) arrays, association constants (K a) of around 10 M could be observed in CHCl3. Subsequent increase of the number of donor-acceptor units to triple hydrogen bonding and quadruple hydrogen bonding (ﬁgure 3.58 b) and c)), signiﬁcantly

3 −1 5 −1 [276–278] increases the K a values to 10 M and 10 M (CHCl3), respectively. If hydrogen bonds are combined with other noncovalent interactions, such as complementary amidinium-carboxylate bridges (ﬁgure 3.58 a)), association constants greater than 106

120 Chapter 3 Results and Discussion

R1 H R2 R2 O H N N N N H H N H N N Et N N O O Bu O H N H R3 b) a)

R2 H N N N O O N H O N N N R1 N H O H N H H 1 2 H H R O N R R1 N N N O H N N H O O N N 2 O N H H R N

c) O d)

Figure 3.58: Relevant hydrogen-bonding motifs with high association constants.

M−1 can be obtained.[279,280] One of the most stable arrays discovered so far, is the

HAMILTON motif (figure 3.58 d)), where a receptor, which contains 2,6-diaminopyridine units could efficiently bind targets like barbiturates and their derivatives through six hydrogen bonds.[281] This motif was assimilated by HIRSCH and coworkers for the self- assembly of extremely stable supramolecular structures.[282–284] By taking advantage of the unique features of hydrogen-bonding we decided to utilize them for the building of novel tether systems for multiple functionalization of C60. Thereby the tether is primarily formed by dimerization through multiple hydrogen- bonding, followed by the subsequent reaction with C60 (see scheme 3.16). The dependence of the association constant upon the solvent, allows directly to influence the hydrogen bond equilibria between the monomeric and dimeric constitution. As bonding motif we decided to use a system, similar to the array described in figure 3.58 c), composed from self-complementary 2-ureido-4-pyrimidinones units which have a

121 Chapter 3 Results and Discussion

O O R1 O O (CH ) 2 n (CH2)n

(CH2)n O O O O O O O O R1 R1

(CH2)n

C60, CBr4, DBU O O O

(CH2)n O R1 O O O O R1

Scheme 3.16: Schematic representation for the building of hydrogen-bonded tethers. donor donor acceptor acceptor (DDAA) hydrogen bonding motif. This motif gives rise

7 to a very high dimerization constant (K d ≥ 6·10M ), as a result of attractive secondary interactions.[285,286] To overcome solubility problems of the sparely soluble ureidopyrimidines, the asymmetric carboxy malonate 27 was used. The lipophilic alkyl chain in 27 should provide sufﬁcient solubility in most organic solvents. The carboxylic acid 27 was converted into the corresponding acid chloride 103, after which reaction with sodium azide yielded the acyl azide 104. Heating 104 in the presence of 6-methylisocytosine at 85°C afforded 106 in good yield (76 %, four steps, starting from 27 via isocyanate 105, scheme 3.17).

122 Chapter 3 Results and Discussion

R1 = COOH 27 O O C Cl O R2 2 2 2 O O R1 R1 = COCl 103 NaN3 1 R = CON3 104 ∆ N 100 °C, R1 = NCO 105 pyridine HO N NH2

O O O HN R2 O O N N N O H H 106 2 R = (CH2)17CH3

O O O HN R2 O O N N N O H H 106 H H O N N N O O R2 NH O O O

C60, I2, DBU O HN N O N O HN H O N N N N H H H O N H H O N H N N O O N N H H O O N O NH O O O 2O R R2O O O O O O O O 2 O R R2

107 107

Scheme 3.17: Reaction sequence for bisadduct 107 via dimeric hydrogen-bonded malonate 106.

1 The H NMR shows the deﬁnite evidence for the dimeric structure of 106. In CDCl3 large lowﬁeld shifts were found for the protons of the hydrogen-bonding motif. The signals of the urea NH protons are observed at δ = 11.79 and 10.31 ppm and the intramolecularly chelated pyrimidinone NH at δ = 13.23 ppm. This observation is

123 Chapter 3 Results and Discussion

6 7 1 3

2 4 5 10 106 8 9

-CH2 -

3 9 1 * 2,4

10 5 8 7 6

13 12 11 10 9 8 7 6 5 4 3 2 1 0 ChemicalShift(ppm)

1 Figure 3.59: H NMR of the malonate-dimer 106 (400 MHz, RT, CDCl3). fully consistent with four DDAA hydrogen bonds in the supramolecular malonate-dimer structure. Reaction of the malonate 106 with C60 in the presence of DBU and CBr4 was accomplished under diluted conditions, analog to the method described for macrocyclic malonates.[269] The formation of the bisadducts could be verified by mass spectrometry. Beside the molecular peak at 1901, only the always observed C60 peak at m/z = 720 could be detected. To proof the selectivity of the formation of bisadducts, the same reaction was carried out by using a 2-fold excess of C60. Even under these conditions no traces of monoadducts could be detected in the mass spectra. The isolation of the different regioisomers of the bisadducts via flash column chromatography as well as by repeated preparative HPLC did not lead to success in this case. The relatively high polarity and smearing property of the substance, made it impossible to separate the product bands on the column. For a complete characterization of the bisadduct by NMR and UV/Vis the separation of the different regioisomers is inevitable. Therefore new purification methods have to be found, which allow the separation and isolation of the regioisomeric bisadducts.

124 Chapter 3 Results and Discussion

3.5 Synthesis of Novel Multiple Fullerene Arrays

Consisting of Mixed C60-Hexakisadduct Units

Multiple additions to the C60 core enable the facile access to highly symmetrical, poly- functional macromolecules. By controlling the addition pattern, these three-dimensional nanostructures with deﬁned symmetry and shape could target interesting topics in supramolecular chemistry and advanced materials. One of the most important reactions in this context is the template mediated sixth-fold cyclopropanation using 9,10- dimethylanthracene (DMA).[287,288] The thermal reversibility of the Diels-Alder reaction between C60 and 9,10-dimethylanthracene creates an equilibrium mixture containing e,e-C60(DMA)2 and e,e,e-C60(DMA)3 templates that direct subsequent cycloadditions to the remaining equatorial sites.[289] The method allows the synthesis of a number of

BINGEL-type Th symmetrical hexakisadducts, C66(COOR)12, in yields up to 50 %. This mediation strategy was also adopted for the synthesis of mixed hexakisadducts. These structures contain two different types of addends in octahedral positions, which allows the synthesis of new bifunctional macromolecules, with unique properties.[290] In principle two different synthetic strategies can be used for the synthesis of [5,1]- hexakisadducts. The best accessible one starts with the synthesis of the monoadduct 108, followed by the formation of the hexakisadduct 109 via the DMA templated reaction sequence (Pathway A, sheme 3.18).[98,291,292] The alternative route starts with the synthesis of the triazoline monoadduct 111, followed by the template-mediated cyclopropanation to the mixed triazoline hexakisadduct 112. The hexakisadduct could further be converted into the pentakisadduct 113, by thermally induced [3+2] cyclore- version of the labile triazoline group. The following cyclopropanation to the mixed hexakisadducts proceeds with quantitative regioselectivity and high yields. The major drawback of this strategy is the thermal decomposition of 112, which also leads to the formation of the open [5,6]- and closed [6,6]-azahomofullerene as side products by expulsion of a nitrogen atom. The overall yield for 113 is comparatively low and puriﬁcation process of the involved intermediates is laborious.[293]

1 Substitution of the malonate H2(COOR )2 with a bis(functional) addend, makes it pos-

125 Chapter 3 Results and Discussion

Pathway A Pathway B

1 H2C(COOR )2, CBr , DBU 4 O O N3 110

MEM N O O R2 R2 R1 R1 N MEM NN O O N N O O O O DMA, O O 2 H2C(COOR )2 2 R2 O O R O O CBr4, DBU R2 R2 O O O O OO O 2 O 2 2 R 2 R R 108 111 R O O 112 DMA, 2 H2C(COOR )2, CBr4, DBU ∆

R1 R1 O O R2 R2 R2 R2 O O O OO O O O O O H C(COOR1) , O O O 2 2 O 2 CBr4, DBU O R2 R2 O O R R2 O O O O O R2 R2 R2 R2 O O O O O O O O O O O O O O O 2 O 2 2 R2 2 R2 R 2 R R 2 R R O O R O O

109 113

Scheme 3.18: Different strategies for the synthesis of [5,1]-hexakisadducts, using template mediation with DMA.

sible to achieve fullerene-rich molecular architectures, by the direct coupling of two C60 units. With this method HIRSCH and coworkers recently synthesized a water soluble bis(fullerene) icosacation, where the fullerenes are connected via a chiral C4-spacer unit.[294] Since the synthesis of this dumbbell shaped molecule starts with the generation of a bis(fullerene), followed by the completion of the octahedral addition pattern

126 Chapter 3 Results and Discussion

1 R1 R1 1 R 1 1 R 1 O O R R O O 1 R O O R O O O O O O O O O O O O O O 1 O O O O 1 R Spacer R R1 O O O O R1 O O OO O O OO

O OO O O OO O R1 O O R1 R1 O O R1 R1 R1 R1 R1

(a)

1 R1 R2 2 R 1 2 R 1 O O R R O O 2 R O O R O O O O O O O O O O O O O O 1 O O O O 2 R Spacer R R1 O O O O R2 O O OO O O OO

O OO O O OO O R1 O O R1 R2 O O R2 R1 R1 R2 R2

(b)

Figure 3.60: Illustration of a symmetrical (a) and asymmetrical (b) functionalized fullerene dimer.

(scheme 3.18, pathway A), only symmetrical functionalized fullerene dimers are accessible (see ﬁgure 3.60 (a)). For the synthesis of asymmetric fullerene dimers (ﬁgure 3.60 (b)) a new concept had to be found, which will be described in the following chapter.

3.5.1 Synthesis of Bisfunctionalized Janus-Type Fullerene-Dimers

In principle both synthetic strategies for mixed [5,1]hexakisadducts can be adopted for the synthesis of Janus-type (Janus: Roman good of gates and doors, represented with a double-faced head) fullerene-dimers. The preparation of two different pentakisadducts should enable the combination of two totally different functionalities into one dimer molecule, by direct bridging of the two fullerene moieties with a bis(functional) addend. Since the preparation of pentakisadducts via the triazoline route, introduced in scheme 3.18, is afﬂicted with laborious synthetic procedures and low yields, we decided to utilize the retro-cycloaddition reaction of pyrrolidinofullerenes, recently discovered by MARTIN et al., for the synthesis of fullerene pentakisadducts.[295] Pyrrolidi- nofullerenes are easily accessible by the 1,3-dipolar cycloaddition reaction of in situ

127 Chapter 3 Results and Discussion

generated azomethine ylides to C60. The pyrrolidine ring can be efﬁciently removed by a thermal reaction in the presence of an excess of a highly efﬁcient dipolarophile. In some cases the addition of a metal lewis acid catalyzed this reaction, presumably by coordination to the nitrogen atom. The pyrrolidinofullerene 114 was synthesized

[296] following literature procedure by the reaction of C60 with benzaldehyde and N- methylglycine in boiling toluene. The condensation product of the aromatic aldehyde and N-methylglycine successively undergoes dehydration and decarboxylation to give the corresponding ylide. Nucleophilic attack by the latter on C60 followed by intramolecular ring closure leads to the fused pyrrolidinofullerene system. The template-mediated DMA strategy was further used to obtain the mixed hexakisadduct 116 by the nucleophilic cyclopropanation with diethylmalonate 115. The yield in this reaction step was relatively low, supposably through the different regioselectivity of the pyrrolidine ring in directing the malonates for the subsequent additions. Compared to methanofullerenes, where the second attack of a nucleophile is specified at the preferred e and trans- 3 positions,[297] the bisaddition on pyrrolidinofullerenes generally forms the regioisomers with no significant favoritism.[298] Despite the DMA mediation, which should ac- tivate the equatorial positions, the reaction yield could not be improved. The retro- cycloaddition reaction was carried out using standard conditions.[295] Hexakisadduct 116 was refluxed in o-DCB with the presence of an excess of maleic anhydride, in order to trap the corresponding ylide that results from the thermal retro-cycloaddition reaction. After 12 h, reaction control via TLC and mass spectrometry showed no conversion of the starting material. Also longer reaction periods gave no evidence for the formation of the corresponding pentakisadduct. The addition of 1 eq. CuTf2 (copper(II)trifluoromethylsulfonate) as a metal lewis acid, did not improve the efficiency of the removal of the pyrrolidine ring. In fact, the hydrolysis of the malonesters could be observed, resulting in a decomposition product, which could not be purified and characterized further more. Since it has been shown, that the retro-cycloaddition reaction of pyrrolidinofullerenes suffers from its incompatibility with various functional groups, we decided to use alternatively a synthetic procedure similar to pathway A described in scheme 3.18. In

128 Chapter 3 Results and Discussion

N N R1 PhR1 Ph O O O O benzaldehyde, DMA, O O N-methylglycine CBr , DBU 1 4 R1 O O R ∆ O O O O R1 R1 O O O O O O O O O O 115 R1 1 R1 114 1 R R O O

116

R1 R1 O O O O O O 1 R1 O O R 1 O O R = CH2CH3 R1 R1 O O O O OO O 1 O 1 1 R 1 R R R O O

117

Figure 3.61: Synthesis of the [5,0]pentakisadduct 117 via the retro-cycloaddition reaction of pyrrolidinofullerenes. order to have the possibility to introduce different functionalities by this method, a new hexakisadduct precursor had to be found, which allows the attachment of an additional

C60 molecule that can be itself further functionalized. Therefore the bifunctional macro- cycle 118 [132] was used for the synthesis of the monofunctionalized fullerene 119, which still exhibits a free malonic group for further functionalization (scheme 3.19). cyclo-[2]-Octylmalonate 118 is a suitable building block for this reaction, because the speciﬁc length of the spacer units is not appropriate to the formation of a strain-free bisadduct, which is normally the competitive reaction pathway. The nucleophilic cyclopropanation of 118 was performed with an equimolar amount of C60, in the presence of CBr4 and DBU. Considering the possible side reactions of the bifunctional malonate, including the formation of DBU-salts and polymerization, the reaction proceeded with reasonable yield (28 %). For the synthesis of the corresponding [5,1]hexakisadduct, different reaction conditions and malonates were applied and tested for their efﬁciency in this type of reaction. All reactions were carried out under DMA- mediation conditions and the results are summarized in table 3.12. However the use

129 Chapter 3 Results and Discussion

O O O O O O O O O O O O O O C60, CBr4, DBU O O 119 118

DMA, CBr4, H2C(COO(CH2)6NHBoc)2 120 P1-base

1 R1 R 1 1 O O R R O O O O O O O O O O R1 O O R1 = (CH ) NHBoc 121 O 2 6 R1 O O O O OO O

O OO O R1 O O R1 R1 R1 C60, CBr4, DBU 1 R1 R 1 1 O O R R O O O O O O O O O O 1 O 1 R O R = (CH2)6NHBoc 122 O R1 O O O O OO O

O OO O R1 O O R1 R1 R1

DMA, t H2C(COO(CH2)5COO Bu)2 100 CBr4, P1-base

R2 2 1 R1 2 R R 1 R O O R2 1 O O R O R O O O O O O O O O O O O O O O O O R2 R1 O O 2 1 O O R R O O O O O OO OO O O O O O 2 OO 2 1 OO 1 R O O R R O O R R2 R2 R1 R1

1 2 t R = (CH2)6NHBoc R = (CH2)5COO Bu 123 TFA 1 + - 2 R = (CH2)6NH3 CF3COO R = (CH2)5COOH 124

Scheme 3.19: Multistep synthesis of the Janus-type fullerene-dimer 124.

130 Chapter 3 Results and Discussion

Entry Malonate Base Halogenating Yield reagent

A 10 eq. 120 10 eq. DBU CBr4 ≤ 5% t B 10 eq. 120 10 eq. P1- Bu CBr4 11 % C 10 eq. diethyl 10 eq. DBU — — α-bromomalonate 125 t D 10 eq. diethyl 10 eq. P1- Bu — ≤ 5% α-bromomalonate 125 E 20 eq. 120 10 eq. DBU CBr4 14 % t F 20 eq. 120 10 eq. P1- Bu CBr4 25 %

Table 3.12: Reaction conditions for the synthesis of mixed [5,1]hexakisadduct precursor 121. of α-bromomalonic esters, which should suspend possible side reactions on the free malonic group through the absence of a halogenating reagent, were not sufﬁcient in this case. The best result could be obtained by the use of a 20-fold excess of malonate

t 120 in the presence of the phosphazene base P1- Bu. Non converted malonate 120 could be recovered by preliminary flash column chromatography. Final purification of the [5,1]hexakisadduct 121 was achieved by preparative HPLC. The attachment of an additional C60 molecule to the free malonic group in 121 was accomplished by the cy- t clopropanation with a 1.5-fold excess of C60 in the presence of CBr4 and P1- Bu-base. The isolation of the [5,1]hexakis-monoadduct 122 via flash column chromatography was straight forward and no additional purification via preparative HPLC had to be applied. The UV/Vis spectrum of 122 can be interpreted as a superposition of the individual spectra of its components. Figure 3.62 shows the spectra of monoadduct 119, [5,1]hexakisadduct 121, the parent [5,1]hexakis-monoadduct 122 and the calculated product spectrum, which was obtained by addition of the monoadduct spectrum and the [5,1]hexakisadduct spectrum. The UV/Vis spectra of 119 and 121 itself show the typical characteristic slope of a C60-monoadduct and a C60-hexakisadduct respectively. The measured spectrum of [5,1]hexakis-monoadduct 122 fits the calculated prediction quite well, whereas the typical monoadduct maximum at 425 nm is clearly recognizable (figure 3.62, inset). The presence of a less functionalized C60 hemisphere in the [5,1]hexakis-monoadduct 122 opens the possibility to introduce further functional

131 Chapter 3 Results and Discussion

200

180 119 121 160 122 calculated (119 + 121) 140

3 120

x10

-1 100

cm -1 80

ε 60

20 400 425 450

0 250 300 350 400 450 500 550 600 wavelength[nm]

Figure 3.62: UV/Vis spectra of monoadduct 119 (black), [5,1]hexakisadduct 121 (red), [5,1]hexakis-monoadduct 122 (blue) and the calculated spectrum for 122 (green). groups into the molecule. Similar to the reaction described for the synthesis of 121, the completion of the octahedral addition pattern with malonate 100 results in the dimeric structure 123, whereas two different functionalized [5,1]hexakisadducts are bridged via a macrocyclic addend. Compared to the above mentioned reaction, the synthesis of

t 123 only requires a 10-fold excess of malonate 100 in the presence of P1- Bu. Also in this case the separation of 123 from lower adducts via preparative HPLC was inevitable. Figure 3.63 shows the comparison of the 13C NMR spectra of [5,1]hexakisadduct 121,

[5,1]hexakis-monoadduct 122 and [5,1]hexakis-dimer 123. The resonances of the C60 2 sp -carbons in the case of 121 can be clearly assigned to a pseudo Th symmetrical hexakisadduct. The resonance at 42.42 ppm and the signals for the malonester carbons at 166.63 ppm, indicates the existence of the free malonic group, which was further manifested by the C-H correlated HETCOR-spectrum. The conversion of 121 into the

132 Chapter 3 Results and Discussion

* 23

22 20 21 18 16 14 19,18, 19 17 1 17,16 15 13 1210 8 6 4 2 11 9 7 5 3 4,5,6, 121 7,8,9 sp3 -C 21 sp2 -C 11,14 22 10,15 20 2 3 12,13 1

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 * 23

22 20 21 18 16 14 19 17 10 8 6 4 1 19,18, 15 13 12 17,16 2 11 9 7 5 3 4,5,6, 122 7,8,9 sp3 -C (mono) sp2 -C (hexa) 3 C 21 sp - (hexa) sp2 -C (mono) 20 14,11,2 22 3,10,15 1 12,13

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

* 23 23 33 33 20 31 22 18 29 21 16 25 27 32 14 28 30 19 17 1 26 15 13 1210 8 6 4 24 19,18, 9 7 5 3 2 11 17,16 4,5,6, 123 7,8,9 27,28, 22 29

31 sp3 -C 30 1,11, 3,10, 14,25 21 sp2 -C 32 1,12, 20 15,26 13,24

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ChemicalShift(ppm)

Figure 3.63: Comparison of the 13C NMR spectra of [5,1]hexakisadduct 121 (100.5 MHz, RT, CDCl3), [5,1]hexakis-monoadduct 122 (75 MHz, RT, CDCl3) and [5,1]hexakis- dimer 123 (75 MHz, RT, CDCl3).

133 Chapter 3 Results and Discussion corresponding [5,1]hexakis-monoadduct 122 results in a spectrum, which in principle shows the characteristics of a hexakisadduct as well as a monoadduct. This can be clearly seen in the sp2-carbon region of 122. Beside the dominating signals at 145.92 and 141.32 ppm for the hexa-substituted molecule part, the additional resonances between 145.45 and 139.32 ppm can be clearly allocated to a mono-substituted C60 molecule. Also the sp3-carbon signal at 71.79 ppm exhibits the typical low-ﬁeld shift

3 ofaC60-monoadduct, compared to the signal at 69.28 ppm for the hexakisadduct sp - carbons. This trend can be further followed for the malonate bridge carbon atoms, whereas the chemical shift amounts to almost 7 ppm. The 13C NMR spectrum of protected [5,1]hexakis-dimer 123 is similar to the spectrum observed for 121. Due to the large distance of the functional groups in the malonate addends 100 and 120 to the

C60 cores, no influence on the chemical shift can be detected. On account of this, the high local symmetry of both fullerene moieties results in only two signals for all 96 sp2 carbon atoms. Furthermore, all 24 sp3-carbons can be detected as one signal at 69.28 ppm. The additional signals compared to the spectrum of 122 can be clearly assigned to the malonate addend 100. In order to achieve water solubility, the deprotection was carried out by treatment of 123 with pure TFA (scheme 3.19). Next to the quantitative conversion of the Boc-groups into the corresponding ammonium salts, the simultaneous cleavage of the acid labile tert-butyl esters took place as demonstrated by the absence of any signals for both functional groups in the 1H- and 13C NMR spectra of 123. Beside pure water, the ammonium salt of the asymmetric dumbbell shaped bis(fullerene) 123 is very soluble in methanol. Due to the presence of ten amino groups and ten carboxylic acid groups within the structure of 124, it can be best described as amphoteric oligoelectrolyte. Contrary to the natural occurring polyampholytes (proteins), the potential charge carriers in macromolecule 124 are allocated and separated over the two C60hemispheres in a well-defined way, comparable to a Janus medal. The pH dependent solubility behavior of 124 in aqueous solutions is mainly influenced by this amphoteric character. At a lower pH value (pH = 3), both the carboxylates and amine functions are protonated, resulting in a positive net charge and a good water-solubility. At higher pH value (pH

134 Chapter 3 Results and Discussion

= 10), the amines exist as neutral bases and the carboxylic acids as the conjugate bases, giving the molecule a negative net charge and a good water-solubility. At both mentioned pH values, a clear yellow aqueous solution was obtained (figure 3.65, left and right). The UV/Vis spectra of dimer 124 in acidic and basic aqueous solution are almost similar (figure 3.64). However, compared to the spectrum of the corresponding ammonium salt in methanol, the distinct shoulders at about 317 and 335 nm flatten out. Supposably the bolaamphiphilic character of dimer 124 causes the agglomeration of the hydrophobic bridging moieties, which could facilitate the formation of oligomers with a characterless slope of the spectrum in this region.

absorbance

124 in methanol 124 in water (pH = 3) 124 in water (pH = 10)

250 300 350 400 450 500 550 600 wavelength [nm]

Figure 3.64: UV/Vis spectra of 124 as ammonium salt in methanol (black),

At intermediate pH the zwitterion concentration increases, and at a characteristic pH, called the isoelectric point (IEP), the negatively and positively charged molecular species are present in equal concentration. In the case of 124 the IEP ranges from approximately 6 - 6.5. In the space of this pH-region, dimer 124 is not soluble in water any

135 Chapter 3 Results and Discussion

Figure 3.65: Aqueous solutions of deprotected asymmetric [5,1,1,5]-dimer 124 at pH = 3 (left), pH = 6.5 (middle) and pH = 10 (right). more, which causes the precipitation as pale orange solid, which is completely insoluble in methanol (ﬁgure 3.65, middle). These observations indicate the formation of highly stable supramolecular aggregates through attractive electrostatic interactions between the charged terminal groups near the IEP.

136 Chapter 3 Results and Discussion

3.5.2 Synthesis of a Fullerene-Rich Nanocluster

In the last years only a few fullerene-rich nanostructures have been reported so far, which were prepared as dendritic structures with fullerene units at their surface or with

[299–301] C60 spheres in the dendritic branches. This is mainly associated with the diffi- culties related to the synthesis of such fullerene-rich macromolecules.[302] The major problems for the preparation are the low solubility of C60 and its chemical reactivity limiting the range of reactions that can be used for the synthesis of branched structures bearing multiple C60 units. In a new approach we decided to synthesize a monodisperse fullerene-rich macromolecule with a C60 core as well as peripheral C60 subunits. The [5,1]hexakisadduct 121 could be therefore used as precursor, which provides high solubility in most organic solvents. Treatment of 121 with pure C60 in the presence of DMA, CBr4 and phosphazene base in toluene gave the highly substituted macromolecular structure 126. In this star-type architecture the octahedral substituted C60-core ([6,0]hexakisadduct) is surrounded by six [5,1]hexakisadducts. The major difficulty in the purification of 126 is the similar polarity of adducts with lower addition degree and the overall high polarity of 126. Therefore it was necessary to pre-clean compound 126 twice via flash column chromatography, followed by purification via preparative HPLC. The high molecular weight of 126 (22622.26 g/mol) made it impossible to characterize the macromolecule by FAB mass spectrometry. Additionally, attempts for MALDI-TOF mass spectrometry with 126 gave no evidence for the molecule ion peak. The laser bombardment in MALDI-MS caused the fragmentation of 126, with detectable ions in the region of 2840- 4330 mass/charge, which could not be clearly allocated to specific molecule fragments. Despite of the missing mass spectrum, the NMR spectroscopy shows the evidence for the formation of 126. The 13C NMR spectrum is greatly simplified through the high symmetry (pseudo-Th) of 126. As would be expected, two resonances are observed for the carbonyl C atoms (164.06 and 156.25 ppm), two for the tert-butyl units (79.13 and 28.68 ppm), one for the OCH2 units (67.11 ppm) and five for the CH2 groups of the Nboc-malonates (40.71, 30.18, 28.57, 26.61 and 25.80 ppm). The signals of the cyclo-[2]-octylmalonates show less intensities, whereas two of the three expected signals

137 Chapter 3 Results and Discussion

R R R O O R O OO O O O O O O O R O O 121 + R O O O O O O O O

O OO O R O O R R R

H DMA, R = N O CBr , O 4 P1-base

R R R R R R O O R O O R O O O OO O O O O O O O O O O O R O R O O R O R R O R R O O R O O O O O O O O O O O OO O O O O O O O R O R R R

R O R R R O R O O O R O O O R R O O O O OO O O O O O O O O O O O O O O O O O O O R O O O R O O O R R O O O O O O O O O O OO O O O O O O O O O O OO O O O OO R O O R R O O R R R R R

R R R O R O O O OO O O O O OO O O O O O O O O O R O O R R O R R O R O R R O O O O O O O O O O O OO O O R O O O O O O R R R R O R R R

H R = N O 126 O TFA + - R = NH3 CF3COO 127

Scheme 3.20: Synthesis of the fullerene-rich nanocluster 127 by sixfold addition of the [5,1]hexakisadduct precursor 121 to parent C60.

138 Chapter 3 Results and Discussion

23 20 2 18 16 1110 8 6 4 22 21 14 19 17 15 13 12 9 7 5 3 1

126

sp3 -C 21 20 2,11,14 22 3,10,15 * 1,12,13 * *

180 160 140 120 100 80 60 40 20 0 ChemicalShift(ppm)

23,5,8

2 2 C sp -C sp - 19 18 17 16

4,9 6,7

150 148 146 144 142 140 138 31.5 30.5 29.5 28.5 27.5 26.5 25.5 24.5 Figure 3.66: 13C NMR spectrum of [5,1]hexakisfullerenyl-[6,0]hexakisadduct 126 (100.5 MHz, RT, CDCl3) (* = solvent + toluene impurities). could be clearly identiﬁed (29.79 and 26.28 ppm). The missing signal is located under

2 the tert-butyl resonance. The C60-sp region of the spectrum simply consists of two signals (145.93 and 141.34 ppm), which is characteristic for a pseudo-Th-symmetrical addition pattern (broadening of these typical signals would indicate the presence of

3 adducts with lower addition degree). Furthermore all C60-sp carbon atoms could be detected as one signal at 69.29 ppm. Beside the 13C NMR spectrum, also the UV/Vis spectrum exhibits the characteristic slope of a hexakisadduct. The distinct shoulders at 316 and 335 nm do not ﬂatten out, which would indicate the presence of pentakisadduct impurities. The cleavage of the carbamates in 126 could be obtained in pure TFA. The

139 Chapter 3 Results and Discussion

900

800

700

3 600

x10 500

-1

-1 400

ε 300

200

100

0 250 300 350 400 450 500 550 600 wavelength[nm]

Figure 3.67: UV/Vis spectrum of [5,1]hexakisfullerenyl-[6,0]hexakisadduct 127 in H2O (pH = 4).

NMR spectra of 127 after reprecipitation from MeOH/Et2O exhibited no carbamate resonances any more. The water-solubility of 127 strongly depends on the pH value of the solution. In neutral and slightly acidic solution, the solubility of 127 is comparatively low. If the solutions are further acidified, a significant increase of the solubility can be observed, resulting in a bright yellow solution. Comparable to Nboc-protected structure 126, the UV/Vis spectrum of 127 shows the typical distinct double-bands at 271 nm, 280 nm and 316 nm, 335 nm. The sum of seven hexasubstituted C60-cores within the structure of 127 accounts for a remarkable extinction coefficient of approximately 550000 M−1cm−1 at 245 nm (figure 3.67). The geometry optimized (MMFF) gas phase structure shown in figure 3.68 (left) illustrates the highly symmetric star-shaped structure of 127 in the neutral constitution with peripheral amino groups. The side chains of the malonate units are thereby parallel aligned to each other with a distance of the amino group to the opposite one of approximately 68 Å. The protonation of the amino

140 Chapter 3 Results and Discussion

Figure 3.68: MMFF [303] geometry optimized gas phase structure of [5,1]hexakisfullerenyl- [6,0]hexakisadduct 126 in the neutral (left) and charged (right) constitution. groups disrupt the compact alignment and the repulsion of the corresponding charged ammonium end groups results in a more spread out structure (figure 3.68 (right)). Beside the aesthetically pleasing architecture of 127, the presence of sixty amino groups in a well-defined, monodisperse macromolecule could make it a promising vector for nonviral gene delivery. The spherical shape of 127 would be able to mimic the globular shape of the natural DNA-histone complex, and fits the size-range of the natural protein complex (around 8 nm) quite well.[304]

141 CHAPTER 4

4 Summary

Fullerenes have an enormous potential in applications to physics and biology. Specif- ically [60]fullerene with its unique electronic, optical and structural properties has attracted considerable attention for its application in biomedical materials and optoelec- tronic devices. In this context the selective functionalization of C60, which allows to combine the parent properties with new attributes like water-solubility or amphiphilicity is still a challenging topic for the synthetic chemist. In the ﬁrst part of the present work a series of water-soluble anionic and cationic amphiphilic fullerene monoadducts 43, 44, 45, 46, 47, 48, 56, 60 were synthesized. The amphifullerenes are basically composed of a hydrophilic dendritic branch and a lipophilic moiety, e.g. alkyl chains or unsaturated fatty acids. By the ﬁne tuning of the lipophilic side chain and the size of the dendritic part, a better cell tissue accessibility and biodistribution within lipid rich regions or easier crossing of the blood-brain barrier is expected. The synthesis of these amphifullerenes starts with the reaction of primary and secondary alcohols with MELDRUM’S acid to the corresponding mono carboxylic malonates, which are the precursor compounds for the asymmetric malonates. Unlike previous reports on the synthesis of asymmetric malonates, this reaction proceeds in almost quantitative yield and can be easily scaled up. By using these building-blocks,

142 Chapter 4 Summary a straightforward synthesis procedure could be evaluated, which allows the synthesis of the anionic amphifullerenes 43, 44, 45, 46, 47, 48 and the cationic amphifullerene 60 in gram scale. The availability of larger amounts enabled detailed examinations of these amphiphilic fullerene structures as bioactive materials. In cooperation with

C-SIXTY Inc. and PHYLONIX Pharmaceuticals Inc. the antioxidative activity could be determined applying the xanthine/xanthine oxidase/cytochrome c assay. The overall low IC50 values for all tested amphifullerenes showed the pronounced ability of these structures to efficiently deactivate reactive oxygen species. In line with this cooperation zebrafish embryos as a model system were applied to investigate the toxicity and the cytoprotective activities of the new compounds. Unlike previous results on specific carboxyfullerenes, it could be demonstrated, that none of the amphifullerenes exhibited significant toxicity even at very high concentrations. By the comparison of the cytoprotective activities in the series of amphifullerenes, it could be shown, that the anionic amphiphilic derivative 47 exhibit cytoprotective activity against both cisplatinum and gentamicin- induced apoptosis and represents a promising platform for the development of new potent anti-apoptosis drugs.

In cooperation with the IVANOVIC-BURMAZOVIC group the ability of the amphifullerenes to act as metal free SOD mimetics was investigated by electrochemical, spectrophotometrical and submillisecond mixing UV/Vis stopped-flow measurements. For the first time it could be demonstrated that there is a direct correlation between SOD activity of the water soluble fullerenes and molecular properties such as i) reduction potential, ii) charge and iii) molecular structure. The positively charged monoadduct 60 could be identified as very active new lead structure, which approaches the performance of natural Mn- and Fe-SODs. Furthermore the capability of amphifullerenes 44, 47, 56, 60 as materials for the formation of monomolecular films was examined. The LANGMUIR films were characterized by film balance experiments and BAM microscopy. The 1st generation amphiphilic monoadducts 44, 60 as well as the 2nd generation amphiphilic monoadducts 47, 56 were able to form stable monolayers. In all cases a dependence of the reversibility for the film formation on the pH value could be detected. In addition, up to five layers made

143 Chapter 4 Summary from amphifullerene 47 could be successfully transferred onto hydrophilic silicon.

In the second part of this work the copper(I)-catalyzed HUISGEN 1,3-dipolar cycloaddition was adopted for the synthesis of novel neutral and charged fullerene structures containing multiple triazol moieties. It could be demonstrated that this reaction is very efficient for the synthesis of triazol malonates, which can be easily modified with different azides. By modification of the solvent system for the nucleophilic cyclopropanation reaction, the novel dendritic triazol-fullerenes 86 and 87 have been prepared. The amine terminated triazol-fullerene 87 exhibits a pronounced solubility in water. Con- trary to other literature known amine functionalized fullerenes the solubility of 87 is not limited to acidic aqueous solutions and can be observed over the complete range from pH = 4 to 11. The third part describes the covalent and non-covalent sidewall functionalization of SWCNTs with different fullerene derivatives. For the covalent functionalization the monocarboxylic fullerene derivative 94 was synthesized. Monoadduct 94 was further esterificated with functional groups on the SWCNT surface, which were introduced via reductive alkylation. This transformation resulted in carbon nanotubes decorated with fullerene molecules on the surface and makes it possible to combine the properties of both carbon allotrops in new functional materials. Among the available non-covalent functionalization approaches, the π − π stacking of aromatic compounds on the nanotube surface is found to be a viable approach. Since the spherical shape of C60 is not suitable for the formation of strong π−π interactions, the pyrene-fullerene conjugate 99 was synthesized. STM investigations could show that the π − π interactions between the SWCNT and the pyrene moiety govern the association of 99 with the sidewalls of the SWCNT. Furthermore it is possible to remove the pyrene-fullerenes from the SWCNT surface, which results in structures of aligned functional fullerenes. Thereby the SWCNTs act as templates, which preorientate the fullerene molecules and make it possible to transfer these supramolecular structures to substrates in well-defined strands of aligned molecules. Chapter 3.4 was mainly focused on the synthesis of new tether-based malonate systems for the regioselective bisfunctionalization of C60. Therefor it was tried to coordi-

144 Chapter 4 Summary nate the dicarboxylic malonate 101 to different metal ions, which however did not lead to the desired metallomacrocycles. In a new approach the ureido-pyrimidinone derivative 106 was synthesized from the monocarboxylic malonate 27. Through the self- complementary quadruple hydrogen bonding between the 2-ureido-4(1H)-pyrimidinone moieties dimeric structures are formed, which were utilized as tethers for the bisfunctionalization of C60. Although it could be demonstrated that the reaction of 106 with C60 almost exclusively led to the formation of C60-bisadducts, the separation of the different regioisomers could not be achieved. In the last part of this work a new concept for the synthesis of fullerene-rich nanostructures is described. For this purpose the cyclo-[2]-octylmalonate monoadduct 119 was synthesized and successfully transferred into the corresponding [5,1]hexakisadduct 121, applying the DMA mediation method. Due to the presence of a free malonic group within the hexakisadduct 121, this structure serves as perfect building block for the synthesis of multiple C60-arrays. By means of this precursor compound it was possible to accomplish the synthesis of the dimeric fullerene structure 124, where two different functional groups are allocated and separated over two C60-spheres in a well-deﬁned way. Beside the unique properties of this amphoteric oligoelectrolyte, this structure is to the best of our knowledge the only example for an asymmetric fullerene dimer so far. Furthermore precursor 121 was used for the synthesis of a star-shaped fullerene-rich nanocluster. The linkage of six [5,1]hexaksiaddducts to one C60-core gave the nanocluster 126, which combines a globular structure with an exceptionally high charge density.

145 CHAPTER 4

4 Zusammenfassung

Fullerene, die neben Graphit und Diamant die dritte Verbindungsgruppe sind, zu der sich reiner Kohlenstoff zusammenschließen kann, besitzen enormes Potential für An- wendungen im Bereich der Physik und Biologie. Speziell Buckminster-Fulleren C60 mit seinen einzigartigen elektronischen, optischen und strukturellen Eigenschaften sorgt für hohe Aufmerksamkeit in biomedizinischen Materialien und optoelektronischen Ap- plikationen. In diesem Zusammenhang spielt die selektive Funktionalisierung von C60 immer noch eine große Rolle, die es erlaubt, neue Attribute wie Wasserlöslichkeit oder

Amphiphilie mit den ursprünglichen Eigenschaften von C60 zu verbinden. Im ersten Teil dieser Arbeit wurde eine Serie wasserlöslicher, anionischer und kation- ischer amphiphiler Fulleren Monoaddukte 43, 44, 45, 46, 47, 48, 56, 60 synthetisiert. Diese Amphifullerene bestehen generell aus einem hydrophilen dendritischen Teil und einer lipophilen Einheit, wie z.B. Alkylketten oder ungesättigte Fettsäuren. Durch die stufenweise Modulierung der lipophilen Gruppe, sollte es möglich sein die Biokom- patibilät dieser Verbindungen zu steigern, um auch schwer passierbare physiologis- che Barrieren wie die Blut-Hirn-Schranke zu durchdringen. Die Synthese der Am- phifullerene startet mit der Umsetzung von primären und sekundären Alkoholen mit

MELDRUM’S Säure zu den entsprechenden Mono-Carbonsäure Malonaten, die als

146 Chapter 4 Zusammenfassung

Vorstufe für die unsymmetrischen Malonate dienen. Im Gegensatz zu früheren Meth- oden für die Synthese von unsymmetrischen Malonaten, erfolgt diese Reaktion mit nahezu quantitativer Ausbeute und kann leicht in großem Ansatz durchgeführt werden. Mit Hilfe dieser Bausteine konnte nun ein efﬁzientes Synthesekonzept entwickelt werden, was es erlaubte die anionischen Amphifullerene 43, 44, 45, 46, 47, 48 und das kationische Amphifulleren 60 im Gramm-Maßstab herzustellen. Durch die Verfügbarkeit von größeren Mengen, konnte somit die Untersuchung der Verbindun- gen auf deren biomedizinische Eigenschaften bewerkstelligt werden. Die Evaluierung der antioxidativen und neuroprotektiven Eigenschaften und die Toxizitätsuntersuchun- gen dieser Substanzen wurden dabei in Kooperation mit C-SIXTY Inc. und PHY-

LONIX Pharmaceuticals Inc. durchgeführt. Durch die Bestimmung der IC50 Werte mittels Xanthin/Xanthinoxidase/Cytochrom C - Assay, konnte gezeigt werden, dass alle Amphifullerene ausgeprägte antioxidative Eigenschaften besitzen. Weiterhin konnten Tierversuche an Zebraﬁschen zeigen, dass im Gegensatz zu früher untersuchten Verbindungen, keinerlei toxische oder organtoxische Nebenwirkungen zu beobachten sind. Im Hinblick auf die neuroprotektiven Eigenschaften stellte sich heraus, dass das anionische Amphifulleren 47 äußerst wirkungsvoll sowohl Cisplatin als auch Gen- tamizin induzierte Apoptose verhindern kann. Um zu prüfen, ob die Amphifullerene das katalytische Potenzial von SOD-Mimetika besitzen, wurde in Kooperation mit dem Arbeitskreis IVANOVIC-BURMAZOVIC elektro- chemische, spektrophotometrische und Stopped-Flow Untersuchungen durchgeführt. Zum ersten Mal konnte dadurch gezeigt werden, dass ein direkter Zusammenhang zwischen der SOD Aktivität der wasserlöslichen Amphifullerene und den Moleküleigen- schaften, wie i) Reduktionspotenzial, ii) Ladung und iii) Molekülstruktur besteht. Das positiv geladene Monoaddukt 60 stellte sich in diesem Zusammenhang als äußerst wirkungsvolle Verbindung heraus, die nahezu die katalytischen Umsätze von natür- lichen Mn- und Fe-SOD-Enzymen erreicht. Im Folgenden wurde die Eignung der Amphifullerene 44, 47, 56, 60 als Materalien für die Bildung von monomolekularen Filmen untersucht. Die LANGMUIR-Filme wurden durch Filmwaagenexperimente und Brewster-Angle-Microscopy näher charakterisiert.

147 Chapter 4 Zusammenfassung

Sowohl die amphiphilen Monoaddukte erster Generation 44, 128 als auch die amphiphilen Monoaddukte zweiter Generation 47, 56 bilden dabei stabile Monomoleku- lare Filme aus. In allen Fällen wurde dabei eine pH Abhängigkeit der Reversibilität der Filmbildung detektiert. Zudem lassen sich im Fall des Amphifullerens 47 durch die

LANGMUIR-BLODGETT-Methode diese Filme wiederholt auf hydrophiles Silizium über- tragen, so dass dieses mit bis zu fünf Schichten überzogen werden konnte.

Im zweiten Kapitel wurde mittels der Kupfer(I) katalysierten HUISGEN 1,3-dipolaren Cycloaddition neuartige neutrale und geladene Fulleren Strukturen synthetisiert, die mehrere Triazol Einheiten als Strukturmerkmal aufweisen. Es konnte dabei gezeigt werden, dass mit dieser Reaktion äußerst effizient Triazol-Malonate dargestellt werden können und diese durch Variation der verwendeten Azide leicht modifiziert werden können. Durch eine Änderung des Lösungsmittelsystems bei der nukleophilen cyclo- propanierung mit C60, konnten somit die Triazol-Fullerene 86 und 87 hergestellt werden. Durch die terminalen Aminogruppen im Triazol-Fulleren 87 konnte zudem eine Löslichkeit in Wasser erreicht werden, die im Gegensatz zu bisher bekannten Amino- fullerenen nahezu pH unabhängig ist. Der dritte Teil dieser Arbeit fokussiert auf die kovalente und nicht-kovalente Funk- tionalisierung von SWCNTs mit verschiedenen Fulleren Derivaten. Um eine kovalente Verknüpfung mit den SWCNTs zu erreichen, wurde dafür das Monocarbonsäure- Fulleren 94 synthetisiert, welches erfolgreich mit funktionellen Gruppen auf der SWCNT Oberfläche umgesetzt werden konnte, die zuvor über reduktive Alkylierung eingeführt wurden. Auf diese Weise wurden SWCNT-Derivate dargestellt, an deren Seitenwände Fullerenmoleküle kovalent gebunden sind, was es ermöglicht, die Eigenschaften bei- der Kohlenstoff Allotrope in neuen funktionalen Materialien zu vereinigen. Unter den veschiedenen Möglichkeiten der nicht-kovalenten Funktionalisierung, stellen vor allem π−π-Wechselwirkungen zwischen aromatischen Verbindungen und der SWCNT Ober- fläche einen brauchbaren Ansatz dar. Da die spherische, dreidimensionale Struktur von C60 nicht für die Ausbildung starker π − π-Wechselwirkungen geeignet ist, wurde als Modifikation das Pyren-Fulleren Konjugat 99 dargestellt. STM Untersuchungen zeigten dabei, dass aufgrund der aromatischen Wechselwirkungen zwischen Pyren

148 Chapter 4 Zusammenfassung

Einheit und SWCNT, die Immobilisierung des Fullerens 99 auf der SWCNT Oberﬂäche erfolgte. Weiterhin konnte gezeigt werden, dass ein Entfernen der Fulleren Moleküle von der Oberﬂäche möglich war und sich dadurch perfekt angeordnete Fulleren- Struk- turen realisieren lassen. Dabei agieren die SWCNTs als Strukturtemplate, die eine Vororientierung der Fulleren Moleküle ermöglichen. Die entstehenden supramolekularen Überstrukturen können anschließend auf Substrate übertragen werden, was zur Bildung von hochgeordneten "Fulleren-Drähten" führt. Das vierte Kapitel der vorliegenden Arbeit beschäftigt sich mit der Synthese von neuar- tigen Malonat-Systemen, die als Template für die regioselektive Bisfunktionalisierung von C60 dienen. Dabei wurde zunächst untersucht, ob durch die Koordination des Dicarbonsäure Malonats 101 an verschiedene Metallionen, die Bildung von Metall Makrozyklen zu realisieren ist, was nicht zu dem erwünschten Ergebnis führte. Des- halb wurde in einem neuen Ansatz, ausgehend von dem unsymmetrischen Malonat 27 das Ureido-Pyrimidon Derivat 106 dargestellt. Durch die vier Wasserstoffbrücken- bindungen vermittelte Selbstkomplementarität der Ureido-Pyrimidon Einheiten, bilden sich dabei äußerst stabile dimere Strukturen aus, die als Template für die Bisfunktion- alisierung von C60 eingesetzt wurden. Obwohl bewiesen werden konnte, dass sich bei der Umsetzung von 106 mit C60 fast ausschließlich C60-Bisaddukte bilden, konnte eine Trennung der verschiedenen Regioisomere nicht erreicht werden. Im letzten Teil dieser Arbeit wurde ein neues Konzept für die Synthese von Nanostruk- turen mit hohem C60-Gehalt entwickelt. Zu diesem Zweck wurde ausgehend von Cy- clo-[2]-octylmalonat, das entsprechende Monoaddukt 119 synthetisiert, welches erfolgreich mittels DMA zu dem entsprechenden [5,1]Hexakisaddukt 121 umgesetzt werden konnte. Durch die Anwesenheit einer freien Malonat Gruppe in Verbindung 121 eröffnet dieser Baustein die Möglichkeit, Strukturen mit mehreren C60-Kernen herzustellen. Somit war es möglich, die dimere Fulleren Struktur 124 darzustellen, in der zwei un- terschiedlich funktionalisierte C60 Sphären miteinander verbrückt sind. Diese Struktur, die die Eigenschaften eines amphoteren Oligoelektrolyten besitzt, ist nach unserem Wissen bisher die einzige hochsubstituierte, unsymmetrische, dimere Fulleren Struk- tur. Desweiteren wurde ausgehend von Verbindung 121 der "sternförmige" Fulleren-

149 Chapter 4 Zusammenfassung

Nanocluster 126 synthetisiert. In dieser Struktur sind sechs [5,1]Hexakisaddukte mit einem C60 Kern verbunden, was zu einer äußerst sphärischen Struktur mit extrem ho- her Ladungsdichte führt.

150 CHAPTER 5

5 Experimental Part

5.1 Chemicals and Instrumentation

Chemicals: Most reagents were purchased from Aldrich, Fluka and Acros Organics and, if not otherwise noted, used as purchased. C60 was obtained by the Hoechst AG/AVENTIS and separated from higher fullerenes by a plug ﬁltration.[54,305] All analytical-reagent grade solvents were puriﬁed by distillation.

Thin Layer Chromatography (TLC):

Riedel-de-Haën silica gel F254 and Merck silica gel 60 F254, 0.2 mm on aluminum foil. Detection: UV lamp (254 or 366 nm), H3[P(Mo3O10)4]/Ce(SO4)2/H2SO4/H2O bath,

KMnO4/H2O bath or iodine chamber.

Flash Chromatography (FC): MERCK silica gel 60 (230 - 400 mesh, 0.04 - 0.063 nm). ICN silica gel 32 - 63, 60 Å.

151 Chapter 5 Experimental Part

Analytical High Performance Liquid Chromatography (HPLC): Shimadzu Class-LC10: Liquid Chromatograph LC-10AT, Communication Bus Module CBM-10A, Diode Array Detector SPD-M10A, Auto Injector SIL-10A, Refractive Index Detector RID-10A and Selection Valve FCV-10AL. Columns: Nucleosil 200 x 4 mm, 5 µm, Nucleosil 200 x 4 mm, 3 µm, Macherey-Nagel; Solvents were purchased in HPLC grade from Acros Organics.

Preparative High Performance Liquid Chromatography (HPLC): Shimadzu Class-LC10: System Controller SCL-10AVP, Preparative Liquid Chromato- graph LC-8A, Communication Bus Module CBM-10A, UV/Vis Detector SPD-10A, Auto Injector SIL-10A and Fraction Collector FRC-10A. Columns: Nucleosil 250 x 21 mm, 5 µm, Macherey-Nagel; Solvents were puriﬁed by distillation.

UV/Vis Spectroscopy: Shimadzu UV-3102 PC, UV-VIS-NIR Scanning Spectrophotometer. The absorption

−1 −1 maxima λmax are given in [nm], the extinction coefﬁcients ǫ in [M cm ], shoulders are indicated as sh.

IR Spectroscopy: FT-IR Vector 22 from Bruker Analytische Messtechnik GmbH. The spectra were measured as KBr pellets. React-IRTM from ASI Applied Systems with ATR DiComp-Detector. The spectra were measured as powders or liquid ﬁlms. Absorptions are given in wavenumbersν ˜ [cm−1].

Mass Spectrometry: Micromass Zabspec FAB+ mode (3-Nitrobenzylalcohol as matrix).

NMR Spectroscopy: Jeol JNM EX 400 and Jeol JNM GX 400 (1H: 400MHz, 13C: 100,5 MHz), Bruker Avance 300 (1H: 300 MHz, 13C: 75,4 MHz), Bruker Avance 400 (1H: 400 MHz, 13C: 100,5 MHz).

152 Chapter 5 Experimental Part

The chemical shifts are given in [ppm]. The resonance multiplicities are indicated as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet), broad resonances as br. The raw data were processed using the program MestReNova 5.1.0 from Mestrelab Research S.L.

Elementary Analysis: EA 1110 CHNS from CE Instruments. The results are given as percentage.

153 Chapter 5 Experimental Part

5.2 Synthetic Procedures and Spectroscopic Data

Remarks The compound names are generated with the algorithm used in the program Chem- DrawUltra 8.0 from CambridgeSoft. The following compounds have been synthesized according to the literature. The synthetic procedures and the characterization of these compounds are not described in detail in this section.

• 9-Heptadecanol 16 [306]

• Newkome-type dendrimers [G-1] 29 and [G-2] 30 [109,110]

• (all cis-4,7,10,13,16,19) Docosahexaenoic acid 49 [307]

• Methyl 3,5-bis(propargyloxy)benzoate 76 [224]

• 3,5-Bis(propargyloxy)benzyl alcohol 77 [224]

• 1-Azido-2-(2-methoxyethoxy)ethane 82 [30]

• N-(tert-Butoxycarbonyl)-2-hydroxyethylamine 80 [308]

• N-(2-Azidoethyl)-N-(tert-butoxycarbonyl)amine 81 [309]

• cyclo-[2]-Octylmalonate 118 [132]

• bis(6-(tert-butoxycarbonylamino)hexyl) malonate 129 [310]

General Procedures for Ester- and Amide-formation a) Formation of esters and amides by reaction of the corresponding acyl chlorides (GP 1):

The reaction is carried out under inert gas conditions and with dried glassware.

A solution of the alcohol/amine (1 eq.) in an adequate solvent (normally dry CH2Cl2) and dry pyridine (1 eq.) were cooled in an ice bath. The acyl chloride (1 eq.) was

154 Chapter 5 Experimental Part

diluted in dry CH2Cl2 and added dropwise over a period of 1h via a dropping funnel. [In very small-scale reactions the diluted acyl chloride (less than 1 mL) was added slowly via a syringe over a period of 15 minutes.] The reaction mixture was stirred for 20 h at room temperature and then washed with saturated NH4Cl-solution and H2O. After drying over MgSO4 and rotary evaporation of the solvent, FC on silica yielded the desired product. b) Formation of ester and amide bonds via activation of the carboxyl group with DCC

(STEGLICH -reaction) (GP 2):

The reaction is carried out under inert gas conditions and with dried glassware. A solution of the carboxylic acid (1 eq.) and the alcohol/amine (1 eq.) in an adequate dry solvent were cooled in an ice bath. DMAP (20 mol%, 0.2 eq.) and DCC (1.1 eq.) were added subsequently. After stirring the solution for 2 h at 0°C and 20 h at room temperature, the resulting dicyclohexylurea as side product precipitated during the reaction and was ﬁltered off. Traces of the urea which remained in solution, were removed by repeated precipitation from ethyl acetate. Puriﬁcation by FC on silica yielded the desired product. The rearrangement to the unreactive N-acylurea as an undesirable side-reaction (notably when DMF is used as solvent) can be prevented by cooling and by the addition of DMAP and 1-HOBt, which accelerate the respective alcoholysis or aminolysis of the intermediate O-acylisourea formed by DCC and the carboxyl group, by forming stabile active esters. 1-HOBt also prevents racemization in peptide synthesis. c) Formation of ester and amide bonds via activation of the carboxyl group with EDC

(STEGLICH -reaction) (GP 3):

A solution of the carboxylic acid (1 eq.) and the alcohol/amine (1 eq.) in an adequate solvent were cooled in an ice bath. DMAP (20 mol%, 0.2 eq.), 1-HOBt (1 eq.) and EDC (1.2 eq.) were added subsequently. After stirring the solution for 2h at 0 °C and

20 h at room temperature, the reaction mixture was washed with H2O and saturated

NaCl-solution. After drying over MgSO4 and rotary evaporation of the solvent, FC on

155 Chapter 5 Experimental Part silica yielded the desired product. The advantage of this method is the facile separation of the urea side product, which can be easily extracted with water.

General Procedure for the Formation of C60 Monoadducts (GP 4)

The synthesis of monoadducts of C60 was performed according to the modiﬁed BIN-

GEL reaction.[96] However, to obtain higher yields of the desired monoadduct and less multiple adducts an excess of C60 is applied. The unreacted C60 can be recovered easily by FC on silica with toluene. C60 (1.5 eq. up to 2 eq.) was dissolved in dry, degassed toluene (ca. 0.5 mL toluene per mg C60) resulting in a dark purple solution. For reasons of solubility of the malonate or the desired monoadduct other solvents like DMSO/toluene mixtures were also used. The solution was stirred until complete dissolution was obtained. Afterwards, CBr4 (1.1 eq.) and the malonate (1 eq.) were added. DBU (1.1 eq.) was diluted in toluene and added dropwise over a period of 1 h to the stirred solution at room temperature. After the solution was stirred for 2 h TLC control showed the remaining C60, the monoadduct and traces of bis- and trisadducts. When unreacted malonate was detected, additional CBr4 and diluted DBU were added. If the retention factor of the product in pure toluene was small, the reaction mixture was transferred directly to the FC column. It has been shown, that preliminary rotary evaporation of the solvent induced a decrease of the yield. The yield of the pure monoadduct was typically 35-50 % depending on the attached malonate. The monoadducts were typically obtained as black solids or black, highly viscous oils.

General Procedure for the Deprotection of tert-Butylesters (GP 5)

The tert-butyl protected carboxylic acid (1 eq.) was dissolved in CH2Cl2 and treated with TFA (10-20 eq.) under vigorous stirring at room temperature, till TLC control indicated the complete deprotection of the starting material. Excess of TFA was removed in vacuo, followed by repeated coevaporation with appropriate solvents like CHCl3 or toluene. When the starting material contains substantial amounts of tert-butylesters, pure formic acid should be preferred, to avoid precipitation of the partially deprotected compound.

156 Chapter 5 Experimental Part

General Procedure for the Formation of Mono-Carboxylic Malonates (GP 6)

A mixture of the alcohol (1 eq.) and MELDRUM’s acid (1 eq.) was heated at 115°C under vigorous stirring for 3 h. After cooling to room temperature, the product was washed three times with pentane and dried in vacuo to afford the desired product.

General Procedure for the Formation of [5,1] and [6,0]-Hexakisadducts of C60 (GP 7)

The starting material (C60 or monoadducts of C60) determines the addition pattern ([6,0] or [5,1]) of the obtained hexakisadducts. C60 (1 eq.) was dissolved completely in thoroughly degassed, dry toluene under nitrogen (in the case of monoadducts dry CH2Cl2 was used as well). High dilutions should be avoided. An excess of DMA (10 -12 eq.) was added to the solution and stirred for 2 h at ambient temperature. Subsequently the malonate (10 - 20 eq.) and CBr4 (10 - 12 eq.) was added and stirred to allow com- t plete dissolution. The base (10 - 12 eq.) (in this work the P1- Bu-base turned out to be more efficiently than DBU) was diluted in the used dry solvent and added dropwise over a period of 1 h. The solution was stirred for 2 - 6 days at room temperature under N2, till TLC control remains unchanged (to exclude light, the flasks were wrapped with aluminum foil). The unpolar DMA and highly polar by-products were separated by preliminary flash column chromatography, followed by purification via preparative

HPLC. The essential characteristics for C60 hexakisadducts can be found in the UV/Vis spectrum and 13C NMR spectrum. The UV/Vis spectra exhibit typical absorptions at approximately 245, 271, 281, 316 and 335 nm and are unaffected by the addition pattern. The 13C NMR spectra exhibit typical resonances between 146 and 141 ppm (all hexakisadducts in this work exhibit two characteristic signals for a pseudo Th symmetrical hexakisadduct).

157 Chapter 5 Experimental Part

Malonic acid monohexylester (17)

Compound 17 was synthesized according to general proce- O O dure GP 6 from 1-hexanol (1.42 g, 13.9 mmol) and MEL- O OH 17 DRUM’s acid (2 g, 13.9 mmol) and puriﬁed by elutriation with pentane.

Yield: 2.54 g, 13.5 mmol, 97 %, light yellow oil.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 11.30 (s, br, 1H, COOH), 4.08 (t, J = 6.8 Hz, 2H, 3 OCH2), 3.36 (s, 2H, OCCH2CO), 1.57 (m, 2H, CH2), 1.22 (m, 6H, CH2), 0.80 (t, J =

6.9 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 171.53 (1C, CO), 166.57 (1C, CO), 65.72 (1C,

OCH2), 40.78 (1C, OCCH2CO), 31.06, 28.06, 25.12, 22.21 (4C, CH2), 13.63 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 189 [M+H] ; MWcalc.: 188.22 g/mol.

IR (ATR): ν˜ = 3146, 2901, 2733, 1723, 1693, 1468, 1431, 1384, 1234, 1187, 1058, 936, 693 cm−1.

EA: C9H16O4: calcd. C 57.43, H 8.57, O 34.00; found C 57.09, H 8.63.

Malonic acid monooctadecylester (18)

Compound 18 was synthesized accord- O O O OH ing to general procedure GP 6 from 1- 18 Octadecanol (4.69 g, 17.3 mmol) and

MELDRUM’s acid (2.5 g, 17.3 mmol) and puriﬁed by elutriation with pentane.

Yield: 6.11 g, 17.1 mmol, 99 %, white solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 10.08 (s, br, 1H, COOH), 4.18 (t, J = 6.8 Hz, 2H, 3 OCH2), 3.44 (s, 2H, OCCH2CO), 1.66 (m, 2H, CH2), 1.26 (m, 30H, CH2), 0.88 (t, J =

6.8 Hz, 3H, CH3) ppm.

158 Chapter 5 Experimental Part

13 C-NMR (75 MHz, RT, CDCl3): δ = 170.65 (1C, CO), 167.34 (1C, CO), 66.22 (1C,

OCH2), 40.47 (1C, OCCH2CO), 31.91 (1C, CH2), 29.69 (5C, CH2), 29.65 (2C, CH2),

29.63, 29.55, 29.48, 29.35, 29.16, 28.36, 25.73, 22.68, (8C, CH2), 14.11 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 357 [M+H] ; MWcalc.: 356.54 g/mol.

IR (ATR): ν˜ = 3126, 2920, 2850, 2742, 1746, 1692, 1468, 1429, 1344, 1282, 1244, 1166, 1058, 927, 726, 680 cm−1.

EA: C21H40O4: calcd. C 70.74, H 11.31, O 17.95; found C 70.55, H 11.59.

Malonic acid mono(1-octylnonyl)ester (19)

Compound 19 was synthesized according to general procedure GP 6 from heptadecan-9-ol (3 g, 11.7 mmol) and O O O OH MELDRUM’s acid (1.69 g, 11.7 mmol) and puriﬁed by elu- 19 triation with pentane.

Yield: 3.85 g, 11.2 mmol, 96 %, white solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 9.01 (s, br, 1H, COOH), 4.96 (quin, J = 6.7 Hz,

1H, OCH), 3.42 (s, 2H, OCCH2CO), 1.54 (m, 4H, CHCH2), 1.25 (m, 24H, CH2), 0.88 3 (t, J = 6.7 Hz, 6H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 171.07 (1C, CO), 167.08 (1C, CO), 76.78 (1C,

CH), 40.70 (1C, OCCH2CO), 33.85 (2C, CH2), 31.82 (2C, CH2), 29.42 (4C, CH2),

29.20 (2C, CH2), 25.15 (2C, CH2), 22.63 (2C, CH2), 14.09 (2C, CH3) ppm.

+ MS (FAB, NBA): m/z = 343 [M+H] ; MWcalc.: 342.51 g/mol.

IR (ATR): ν˜ = 3113, 2958, 2919, 2850, 1730, 1684, 1468, 1413, 1321, 1274, 1182, 1128, 1081, 1004, 958, 888, 757 cm−1.

EA: C20H38O4: calcd. C 70.13, H 11.18, O 18.68; found C 69.88, H 11.21.

159 Chapter 5 Experimental Part

6-(Benzyloxy)hexanoic acid (20)

Freshly crushed 85 % KOH (49.2 g, 0.745 mol) was added O O to a solution of benzyl bromide (120 g, 0.7 mol) and ǫ- OH 20 caprolactone (20.06 g, 0.176 mol) in 300 mL toluene. The mixture was heated at reﬂux for 48 h using a Dean-Stark apparatus, then diluted with 200 mL of Et2O and washed with 300 mL of H2O. The aqueous layer was extracted twice with 200 mL Et2O. The combined organic layers were concentrated in vacuo to 150 mL. The aqueous layer was cooled in an ice bath and 2M H2SO4 (180 mL) was added. The turbid aqueous solution was extracted with

CH2Cl2 (3 x 200 mL), dried over anhydrous MgSO4 and concentrated in vacuo to afford

20 as a pale yellow oil. To the residue of the toluene/Et2O layers NaOH (16 g, 0.4 mol) and H2O (80 mL) was added and the resulting mixture was heated at reﬂux for 24 h.

The aqueous layer was separated, diluted to 250 mL with H2O and washed with Et2O

(3 x 100 mL). The aqueous layer was acidiﬁed with a slurry of 25 mL conc. H2SO4 in

100 mL of ice and extracted with CH2Cl2 (3 x 100 mL). The organic layers were dried over anhydrous MgSO4 and the solvent was removed in vacuo to afford 20 as a pale yellow oil.

Yield: 36.3 g, 0.16 mmol, 92.7 %, pale yellow oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 10.41 (s, br, 1H, COOH), 7.34 (m, 5H, arom.H), 3 3 4.51 (s, 2H, ArCH2O), 3.48 (t, J = 6.5 Hz, 2H, OCH2), 2.37 (t, J = 7.6 Hz, 2H,

CH2COOH), 1.66 (m, 4H, CH2), 1.45 (m, 2H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 179.02 (1C, COOH), 138.47 (1C, ArC), 128.31

(2C, ArC), 127.59 (2C, ArC), 127.47 (1C, ArC), 72.91 (1C, ArCH2O), 70.06 (1C, OCH2),

33.89 (1C, CH2COOH), 29.42 (1C, CH2CH2COOH), 25.75 (1C, CH2), 24.54 (1C, CH2).

+ MS (FAB, NBA): m/z = 223 [M+H] ; MWcalc.: 222.28 g/mol.

IR (ATR): ν˜ = 3065, 2933, 2876, 1717, 1614, 1598, 1467, 1443, 1365, 1244, 1201, 1145, 1098, 987, 902, 865, 748, 718 cm−1.

EA: C13H18O3: calcd. C 70.24, H 8.16, O 21.59; found: C 70.09, H 8.18.

160 Chapter 5 Experimental Part tert-Butyl 6-(benzyloxy)hexanoate (21)

A solution of 20 (30 g, 0.135 mol) in dry CH2Cl2 (30 mL) O O was treated with condensed isobutene (100 mL at -60°C) O 21 and conc. H2SO4 (2 mL). The resulting solution was stirred for three days at room temperature, then neutral- ized with a solution of K2CO3 (25 g) in H2O (300 mL). The organic layer was washed with K2CO3 (saturated solution), citric acid (10 wt % in H2O), and H2O. After the mixture was dried over anhydrous MgSO4, ﬁltered, and concentrated, a pale yellow oil was obtained.

Yield: 33.6 g, 0.12 mmol, 89.3 %, pale yellow oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.34 (m, 5H, arom.H), 4.51 (s, 2H, ArCH2O), 3.48 3 3 t (t, J = 6.6 Hz, 2H, OCH2), 2.22 (t, J = 7.5 Hz, 2H, CH2COO Bu), 1.62 (m, 4H, CH2),

1.45 (s, 9H, C(CH3)3), 1.42 (m, 2H, CH2) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.07 (1C, COO Bu), 138.54 (1C, ArC),

128.28 (2C, ArC), 127.54 (2C, ArC), 127.43 (1C, ArC), 79.97 (1C, C(CH3)3), 72.86 (1C, t t ArCH2O), 70.19 (1C, OCH2), 35.55 (1C, CH2COO Bu), 29.48 (1C, CH2CH2COO Bu),

28.15 (3C, C(CH3)3), 25.75 (1C, CH2), 24.97 (1C, CH2).

+ MS (FAB, NBA): m/z = 278 [M] ; MWcalc.: 278.39 g/mol.

IR (ATR): ν˜ = 3033, 2986, 2901, 2865, 1745, 1463, 1364, 1254, 1165, 1121, 863, 768, 689 cm−1.

EA: C17H26O3: calcd. C 73.34, H 9.41, O 17.24; found: C 73.14, H 9.45. tert-Butyl 6-hydroxyhexanoate (22)

A solution of 21 (26 g, 93 mmol) in dry methanol (300 mL) was O HO treated with 10% palladium on carbon (3 g) and H at room O 2 22 temperature. After 48 h, the suspension was ﬁltered through celite and concentrated under reduced pressure to give 22 as an oily product in quantitative yield.

161 Chapter 5 Experimental Part

Yield: 17.6 g, 93 mmol, 100 %, pale yellow oil.

1 3 3 H-NMR (400 MHz, RT, CDCl3): δ = 3.63 (t, J = 6.5 Hz, 2H, OCH2), 2.22 (t, J = 7.4 t Hz, 2H, CH2COO Bu), 1.81 (s, br, 1H, OH), 1.58 (m, 4H, CH2), 1.43 (s, 9H, C(CH3)3),

1.40 (m, 2H, CH2) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.19 (1C, COO Bu), 80.06 (1C, C(CH3)3), t 62.54 (1C, HOCH2), 35.41 (1C, CH2COO Bu), 32.29 (1C, CH2CH2OH), 28.05 (3C,

C(CH3)3), 25.15 (1C, CH2), 24.68 (1C, CH2).

+ MS (FAB, NBA): m/z = 188 [M] ; MWcalc.: 188.26 g/mol.

IR (ATR): ν˜ = 2998, 2941, 2876, 1793, 1743, 1465, 1432, 1387, 1264, 1161, 1065, 1028, 989, 963, 901, 874, 799, 735 cm−1.

EA: C10H20O3: calcd. 63.80, H 10.71, O 25.50; found: C 63.53, H 10.59.

5-(tert-Butoxycarbonyl)pentyl hexyl malonate (23)

Compound 23 was synthesized according to O O O general procedure GP 2 from 17 (1 g, 5.31 O O O 23 mmol), 22 (1 g, 5.31 mmol), DMAP (64.9 mg, 0.53 mmol) and DCC (1.1 g, 5.31 mmol). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 100:30) to give 23 as a colorless oil.

Yield: 1.43 g, 3.98 mmol, 75 %, colorless oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 3.89 (m, 4H, OCH2), 3.12 (s, 2H, OCCH2CO), 3 1.97 (t, J = 7.5 Hz, 2H, CH2CO), 1.41 (m, 6H, CH2), 1.20 (s, 9H, CH3), 1.08 (m, 8H, 3 CH2), 0.66 (t, J = 6.4 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 172.08 (1C, CO), 165.99 (2C, CO), 79.18 (1C,

C(CH3)3), 64.81 (1C, OCH2), 64.52 (1C, OCH2), 40.89 (1C, OCCH2CO), 34.59 (1C,

CH2CO), 30.78, 27.84, 27.60 (3C, CH2), 27.38 (3C, C(CH3)3), 24.84, 24.70, 24.00,

21.88 (4C, CH2), 13.29 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 358 [M] ; MWcalc.: 358.47 g/mol.

162 Chapter 5 Experimental Part

IR (ATR): ν˜ = 2935, 2866, 1730, 1460, 1367, 1329, 1259, 1151, 1043, 950, 896, 850, 757, 687 cm−1.

EA: C19H34O6: calcd. C 63.66, H 9.56, O 26.78; found: C 63.91, H 9.78.

5-(tert-Butoxycarbonyl)pentyl octadecyl malonate (24)

Compound 24 was synthe- O O O sized according to general O O O 24 procedure GP 2 from 18 (3.79 g, 10.64 mmol), 22 (2.00 g, 10.64 mmol), DMAP (130 mg, 1.06 mmol) and DCC (2.20 g, 10.64 mmol).

The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 60:15) to give 24 as a colorless oil.

Yield: 4.54 g, 8.62 mmol, 81 %, colorless oil.

1 3 3 H-NMR (300 MHz, RT, CDCl3): δ = 4.13 (t, J = 6.7 Hz, 2H, OCH2), 4.12 (t, J = 6.8 3 Hz, 2H, OCH2), 3.35 (s, 2H, OCCH2CO), 2.20 (t, J = 7.4 Hz, 2H, CH2CO), 1.63 (m, 3 6H, CH2), 1.43 (s, 9H, CH3), 1.25 (m, 32H, CH2), 0.87 (t, J = 6.4 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 172.77 (1C, CO), 166.57 (2C, CO), 80.00 (1C,

C(CH3)3), 65.62 (1C, OCH2), 65.27 (1C, OCH2), 41.58 (1C, OCCH2CO), 35.29 (1C,

CH2CO), 31.88, 29.65, 29.61, 29.53, 29.47, 29.31, 29.16, 28.42, 28.15 (15C, CH2),

28.05 (3C, C(CH3)3), 25.74, 25.27, 24.60, 22.64 (4C, CH2), 14.06 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 526 [M] ; MWcalc.: 526.79 g/mol.

IR (ATR): ν˜ = 2927, 2858, 1738, 1460, 1367, 1329, 1259, 1151, 1043, 896, 850, 726, 687 cm−1.

EA: C31H58O6: C 70.68, H 11.10, O 18.22. found: C 70.32, H 11.31.

163 Chapter 5 Experimental Part

5-(tert-Butoxycarbonyl)pentyl 1-octylnonyl malonate (25)

Compound 25 was synthesized according to general procedure GP 2 from 19 (2.25 g, O O O O O 6.57 mmol), 22 (1.24 g, 6.57 mmol), DMAP O 25 (81 mg, 0.66 mmol) and DCC (1.36 g, 6.57 mmol). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 90:12) to give 25 as a colorless oil.

Yield: 2.84 g, 5.39 mmol, 82 %, colorless oil.

1 3 3 H-NMR (300 MHz, RT, CDCl3): δ = 4.91 (quin, J = 6.3 Hz, 1H, OCH), 4.14 (t, J = 3 6.7 Hz, 2H, OCH2), 3.35 (s, 2H, OCCH2CO), 2.22 (t, J = 7.4 Hz, 2H, CH2CO), 1.64 3 (m, 6H, CH2), 1.55 (m, 2H, CH2), 1.45 (s, 9H, CH3), 1.26 (m, 26H, CH2), 0.81 (t, J =

6.6 Hz, 6H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 172.86 (1C, CO), 166.74 (1C, CO), 166.35 (1C,

CO), 80.08 (1C, C(CH3)3), 75.97 (1C, CH), 65.28 (1C, OCH2), 41.94 (1C, OCCH2CO),

35.33 (1C, CH2CO), 33.93 (2C, CH(CH2)2), 31.84, 29.48, 29.47, 29.23, 28.20 (9C,

CH2), 28.09 (3C, C(CH3)3), 25.30, 25.18 (3C, CH2), 24.65 (1C, CH2CH2CO), 22.65

(2C, CH2CH3), 14.10 (2C, CH3) ppm.

+ MS (FAB, NBA): m/z = 512 [M] ; MWcalc.: 512.76 g/mol.

IR (ATR): ν˜ = 2927, 2858, 1738, 1460, 1367, 1321, 1259, 1151, 1043, 989, 896, 850, 803, 726, 680 cm−1.

EA: C30H56O6: calcd. C 70.27, H 11.01, O 18.72; found: C 69.89, H 11.10.

164 Chapter 5 Experimental Part

Hexanoic acid hexyl malonate (26)

Compound 26 was synthesized according to gen- O O OH eral procedure GP 5 from 23 (1.1 g, 3.07 mmol) O O O 26 and triﬂuoroacetic acid (2.4 mL, 31.0 mmol) in 50

mL CH2Cl2.

Yield: 919 mg, 3.04 mmol, 99 %, white solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 7.91 (s, br, 1H, COOH), 4.15 (t, J = 6.7 Hz, 2H, 3 3 OCH2), 4.14 (t, J = 6.8 Hz, 2H, OCH2), 3.38 (s, 2H, OCCH2CO), 2.37 (t, J = 7.4 Hz, 3 2H, CH2CO), 1.65 (m, 6H, CH2), 1.43 (m, 2H, CH2), 1.30 (m, 6H, CH2), 0.89 (t, J =

6.9 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 179.42 (1C, COOH), 166.75 (1C, CO), 166.72

(1C, CO), 65.74 (1C, OCH2), 65.23 (1C, OCH2), 41.58 (1C, OCCH2CO), 33.76 (1C,

CH2CO), 31.32, 28.36, 28.07, 25.40, 25.24, 24.15, 22.48 (7C, CH2), 13.94 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 303 [M+H] ; MWcalc.: 302.36 g/mol.

IR (ATR): ν˜ = 2935, 2866, 1730, 1460, 1413, 1320, 1274, 1151, 1043, 1004, 950, 904, 796, 734 cm−1.

EA: C15H26O6: calcd. C 59.58, H 8.67, O 31.75. found: C 59.17, H 8.96.

Hexanoic acid octadecyl malonate (27)

Compound 27 was synthe- O O OH sized according to general O O O 27 procedure GP 5 from 24 (2.00 g, 3.80 mmol) and triﬂuo- roacetic acid (2.9 mL, 38.0 mmol) in 100 mL CH2Cl2.

Yield: 1.77 g, 3.76 mmol, 99 %, white solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 10.10 (s, br, 1H, COOH), 4.15 (t, J = 6.6 Hz, 2H, 3 3 OCH2), 4.13 (t, J = 6.7 Hz, 2H, OCH2), 3.37 (s, 2H, OCCH2CO), 2.37 (t, J = 7.4 Hz,

165 Chapter 5 Experimental Part

3 2H, CH2CO), 1.66 (m, 6H, CH2), 1.42 (m, 2H, CH2), 1.26 (m, 30H, CH2), 0.87 (t, J =

6.7 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 179.49 (1C, COOH), 166.66 (1C, CO), 166.65

(1C, CO), 65.71 (1C, OCH2), 65.19 (1C, OCH2), 41.58 (1C, OCCH2CO), 33.77 (1C,

CH2CO), 31.90, 29.67, 29.65, 29.64, 29.62, 29.56, 29.49, 29.34, 29.19, 28.43, 28.10,

25.76, 25.26 (17C, CH2), 24.17 (1C, CH2CH2CO), 22.67 (1C, CH2), 14.09 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 471 [M+H] ; MWcalc.: 470.68 g/mol.

IR (ATR): ν˜ = 2920, 2850, 1746, 1707, 1468, 1406, 1352, 1313, 1259, 1182, 1050, 1004, 942, 726, 680 cm−1.

EA: C27H50O6: calcd. C 68.90, H 10.71, O 20.40; found: C 69.07, H 10.77.

Hexanoic acid 1-octylnonyl malonate (28)

Compound 28 was synthesized according to general procedure GP 5 from 25 (2.00 g, 3.80 O O OH O O mmol) and triﬂuoroacetic acid (2.9 mL, 38.0 O 28 mmol) in 100 mL of CH2Cl2.

Yield: 1.70 g, 3.72 mmol, 98 %, white solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 10.82 (s, br, 1H, COOH), 4.91 (quin, J = 6.3 Hz, 3 3 1H, OCH), 4.15 (t, J = 6.7 Hz, 2H, OCH2), 3.36 (s, 2H, OCCH2CO), 2.37 (t, J = 7.4

Hz, 2H, CH2CO), 1.68 (m, 4H, CH(CH2)2), 1.53 (m, 4H, CH2), 1.43 (m, 2H, CH2), 1.26 3 (m, 24H, CH2), 0.88 (t, J = 6.8 Hz, 6H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 178.90 (1C, COOH), 166.75 (1C, CO), 166.38

(1C, CO), 76.03 (1C, CH), 65.14 (1C, OCH2), 41.93 (1C, OCCH2CO), 33.93 (2C,

CH(CH2)2), 33.68 (1C, CH2CO), 31.84, 29.49, 29.48, 29.23, 28.14, 25.27, 25.19 (12C,

CH2), 24.20 (1C, CH2CH2CO), 22.65 (2C, CH2CH3), 14.09 (2C, CH3) ppm.

+ MS (FAB, NBA): m/z = 457 [M+H] ; MWcalc.: 456.66 g/mol.

166 Chapter 5 Experimental Part

IR (ATR): ν˜ = 2927, 2858, 1730, 1460, 1413, 1321, 1274, 1159, 1043, 981, 896, 842, 796, 726 cm−1.

EA: C26H48O6: calcd. C 68.38, H 10.59, O 21.02; found: C 68.36, H 10.80.

Asymmetric dendritic malonate [C6-G1] (31)

Compound 31 was synthesized accord-

O ing to general procedure GP 3 from 26 O O H O (500 mg, 1.65 mmol), 29 (688 mg, 1.65 N O O O O mmol), DMAP (41 mg, 0.33 mmol), 1- 31 O O O HOBt (223 mg, 1.65 mmol) and EDC

(317 mg, 1.65 mmol) in dry CH2Cl2/THF

= 1/1 (100 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 20:15) to give 31 as a colorless oil.

Yield: 716 mg, 1.02 mmol, 62 %, colorless oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 6.01 (s, br, 1H, CONH), 4.16 (t, J = 6.8 Hz, 2H, 3 3 OCH2), 4.14 (t, J = 6.9 Hz, 2H, OCH2), 3.32 (s, 2H, OCCH2CO), 2.34 (t, J = 7.5 t 3 3 Hz, 6H, CH2COO Bu), 2.18 (t, J = 7.2 Hz, 2H, CH2CO), 1.99 (t, J = 7.6 Hz, 6H,

NHC(CH2)3), 1.62 (m, 6H, CH2), 1.45 (m, 2H, CH2), 1.41 (s, 27H, C(CH3)3), 1.28 (m, 3 6H, CH2), 0.89 (t, J = 6.5 Hz, 3H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 172.98 (3C, COO Bu), 172.14 (1C, CONH),

166.75 (1C, CO), 166.69 (1C, CO), 80.44 (3C, C(CH3)3), 65.77 (1C, OCH2), 65.25

(1C, OCH2), 57.34 (1C, NHC(CH2)3), 41.58 (1C, OCCH2CO), 36.99 (1C, CH2CO), t 31.33 (1C, CH2), 29.87 (3C, NHC(CH2)3), 29.63 (3C, CH2COO Bu), 28.36, 28.07 (2C,

CH2), 28.01 (9C, C(CH3)3), 25.98 (1C, CH2CH2CO), 25.40, 24.19, 22.55 (3C, CH2),

13.95 (1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 722 [M+Na] , 699 [M] , 532 [M+H-3C4H8] ; MWcalc.: 699.91 g/mol.

IR (ATR): ν˜ = 3349, 3001, 2936, 2886, 1716, 1654, 1563, 1501, 1476, 1432, 1401, 1366, 1318, 1253, 1114, 987, 865, 807, 759, 723, 695 cm−1.

167 Chapter 5 Experimental Part

EA: C37H65NO11: calcd. C 63.49, H 9.36, N 2.00, O 25.15; found: C 63.12, H 9.44, N 2.03.

Asymmetric dendritic malonate [C18-G1] (32)

Compound 32 was

O synthesized accord- O O H O ing to general pro- N O O O O cedure GP 3 from 32 O O O 27 (1.5 g, 3.19 mmol), 29 (1.32 g, 3.19 mmol), DMAP (78 mg, 0.64 mmol), 1-HOBt (431 mg, 3.19 mmol) and EDC (673 mg, 3.51 mmol) in dry CH2Cl2/THF = 1/1 (150 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 15:10) to give 32 as a colorless oil.

Yield: 1.88 g, 2.17 mmol, 68 %, colorless oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.90 (s, br, 1H, CONH), 4.13 (t, J = 6.6 Hz, 2H, 3 3 OCH2), 4.12 (t, J = 6.8 Hz, 2H, OCH2), 3.36 (s, 2H, OCCH2CO), 2.22 (t, J = 7.7 t 3 3 Hz, 6H, CH2COO Bu), 2.11 (t, J = 7.4 Hz, 2H, CH2CO), 1.95 (t, J = 7.8 Hz, 6H,

NHC(CH2)3), 1.63 (m, 6H, CH2), 1.43 (s, 27H, C(CH3)3), 1.25 (m, 32H, CH2), 0.87 (t, 3 J = 6.7 Hz, 3H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 172.87 (3C, COO Bu), 172.07 (1C, CONH),

166.66 (1C, CO), 166.61 (1C, CO), 80.63 (3C, C(CH3)3), 65.67 (1C, OCH2), 65.28 (1C,

OCH2), 57.28 (1C, NHC(CH2)3), 41.56 (1C, OCCH2CO), 37.22 (1C, CH2CO), 31.89 t (1C, CH2), 29.94 (3C, NHC(CH2)3), 29.79 (3C, CH2COO Bu), 29.66, 29.64, 29.62,

29.55, 29.48, 29.33, 29.18, 28.43, 28.18 (14C, CH2), 28.03 (9C, C(CH3)3), 25.75 (1C,

CH2), 25.48 (1C, CH2CH2CO), 25.23, 22.65 (2C, CH2), 14.09 (1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 891 [M+Na] , 868 [M] , 700 [M+H-3C4H8] ; MWcalc.: 868.23 g/mol.

IR (ATR): ν˜ = 3321, 2920, 2858, 1730, 1645, 1545, 1460, 1421, 1367, 1321, 1251,

168 Chapter 5 Experimental Part

1151, 1020, 950, 850, 803, 757, 718, 680 cm−1.

EA: C49H89NO11: calcd. C 67.78, H 10.33, N 1.61, O 20.27; found: C 67.93, H 10.58, N 1.64.

Asymmetric dendritic malonate [(C8)2-G1] (33)

Compound 33 was synthesized ac-

O cording to general procedure GP O O H O 3 from 28 (1 g, 2.19 mmol), 29 N O O O O (0.91 g, 2.19 mmol), DMAP (54 33 O O O mg, 0.44 mmol), 1-HOBt (296 mg, 2.19 mmol) and EDC (462 mg, 2.41 mmol) in dry CH2Cl2/THF = 1/1 (100 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 20:10 to 15/10) to give 33 as a colorless oil.

Yield: 1.20 g, 1.40 mmol, 64 %, colorless oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.91 (s, br, 1H, CONH), 4.91 (quin, J = 6.2 3 3 Hz,1H, OCH), 4.13 (t, J = 6.7 Hz, 2H, OCH2), 3.36 (s, 2H, OCCH2CO), 2.22 (t, J = t 3 3 7.8 Hz, 6H, CH2COO Bu), 2.11 (t, J = 7.6 Hz, 2H, CH2CO), 1.97 (t, J = 7.9 Hz, 6H,

NHC(CH2)3), 1.64 (m, 4H, CHCH2), 1.52 (m, 2H, CH2), 1.43 (s, 27H, C(CH3)3), 1.41 3 (m, 2H, CH2), 1.26 (m, 26H, CH2), 0.88 (t, J = 6.8 Hz, 6H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.40 (3C, COO Bu), 172.59 (1C, CONH),

167.20 (1C, CO), 166.85 (1C, CO), 80.69 (3C, C(CH3)3), 75.96 (1C, CH), 65.19 (1C,

OCH2), 57.20 (1C, NHC(CH2)3), 41.72 (1C, OCCH2CO), 37.04 (1C, CH2CO), 33.70

(2C, CH(CH2)2), 31.61, 30.04 (3C, CH2), 29.71 (3C, NHC(CH2)3), 29.55 (3C, t CH2COO Bu), 29.43, 29.24, 29.22, 28.98, 27.98 (6C, CH2), 27.80 (9C, C(CH3)3),

25.26 (1C, CH2CH2CO), 25.00, 24.91, 22.37 (5C, CH2), 13.78 (2C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 877 [M+Na] , 854 [M] , 686 [M+H-3C4H8] ; MWcalc.: 854.20 g/mol.

IR (ATR): ν˜ = 3383, 3329, 2935, 2858, 1730, 1661, 1537, 1460, 1367, 1321, 1259,

169 Chapter 5 Experimental Part

1159, 1043, 966, 850, 803, 734 cm−1.

EA: C48H87NO11: calcd. C 67.49, H 10.27, N 1.64, O 20.60; found: C 67.20, H 10.17, N 1.64.

Asymmetric dendritic malonate [C6-G2] (34)

Compound 34 was synthe-

O sized according to general O O O procedure GP 3 from 26 (330 O O HN mg, 1.09 mmol), 30 (1.57 g, O O O O H O 1.09 mmol), DMAP (27 mg, N H N O O O O O 0.22 mmol), 1-HOBt (147 mg, 34 O O NH 1.09 mmol) and EDC (230 mg, O O O O O 1.20 mmol) in dry CH2Cl2/THF O O O = 1/1 (100 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 10:10 to 10:15) to give 34 as a colorless oil.

Yield: 1.38 g, 0.80 mmol, 73 %, colorless oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.50 (s, br, 1H, CONH), 6.15 (s, br, 3H, CONH), 3 3 3.98 (t, J = 6.5 Hz, 2H, OCH2), 3.96 (t, J = 6.7 Hz, 2H, OCH2), 3.24 (s, 2H, t OCCH2CO), 2.06 (m, 26H, CH2COO Bu, OCCH2), 1.81 (m, 24H, NHC(CH2)3), 1.50

(m, 6H, CH2), 1.38 (m, 2H, CH2), 1.30 (m, 83H, C(CH3)3, CH2), 1.17 (m, 4H, CH2), 3 0.71 (t, J = 6.8 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 172.87 (1C, CONH), 172.75 (3C, CONH), t 172.32 (9C, COO Bu), 166.38 (1C, CO), 166.35 (1C, CO), 80.09 (9C, C(CH3)3), 65.27

(1C, OCH2), 65.04 (1C, OCH2), 57.24 (1C, NHC(CH2)3), 57.07 (3C, NHC(CH2)3),

41.23 (1C, OCCH2CO), 36.76 (1C, CH2CO), 31.53 (3C, NHC(CH2)3), 31.34 (3C, t CH2CON), 31.06 (1C, CH2), 29.49 (9C, NHC(CH2)3), 29.37 (9C, CH2COO Bu), 28.73,

28.54 (2C, CH2), 27.72 (27C, C(CH3)3), 25.27, 25.15 (2C, CH2), 24.97 (1C,

170 Chapter 5 Experimental Part

CH2CH2CO), 22.27 (1C, CH2), 13.85 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 1723 [M] ; MWcalc.: 1724.24 g/mol.

IR (ATR): ν˜ = 3314, 2974, 2935, 2873, 1730, 1653, 1537, 1460, 1367, 1313, 1251, 1151, 1035, 950, 850, 803, 757, 680 cm−1.

EA: C91H158N4O26: calcd. C 63.39, H 9.24, N 3.25, O 24.13; found: C 63.01, H 8.98, N 3.38.

Asymmetric dendritic malonate [C18-G2] (35)

O O O O O O HN O O O O H O N H N O O O O O 35 O O NH O O O O O O O O

Compound 35 was synthesized according to general procedure GP 3 from 27 (500 mg, 1.06 mmol), 30 (1.53 g, 1.06 mmol), DMAP (30 mg, 0.21 mmol), 1-HOBt (143 mg, 1.06 mmol) and EDC (224 mg, 1.17 mmol) in dry CH2Cl2/THF = 1/1 (100 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 20:10 to 13:10) to give 35 as a colorless oil.

Yield: 1.48 g, 0.78 mmol, 74 %, colorless oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.61 (s, br, 1H, CONH), 6.09 (s, br, 3H, CONH), 3 3 4.05 (t, J = 6.6 Hz, 2H, OCH2), 4.03 (t, J = 6.6 Hz, 2H, OCH2), 3.27 (s, 2H, t OCCH2CO), 2.09 (m, 26H, CH2COO Bu, OCCH2), 1.84 (m, 24H, NHC(CH2)3), 1.54 3 (m, 6H, CH2), 1.33 (m, 83H, C(CH3)3,CH2), 1.15 (m, 30H, CH2), 0.77 (t, J = 6.6 Hz,

3H, CH3) ppm.

171 Chapter 5 Experimental Part

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 172.88 (1C, CONH), 172.66 (3C, CONH), t 172.38 (9C, COO Bu), 166.46 (1C, CO), 166.43 (1C, CO), 80.25 (9C, C(CH3)3), 65.39

(1C, OCH2), 65.13 (1C, OCH2), 57.26 (1C, NHC(CH2)3), 57.16 (3C, NHC(CH2)3),

41.30 (1C, OCCH2CO), 36.85 (1C, CH2CO), 31.65 (4C, NHC(CH2)3, CH2), 31.47 (3C, t CH2CON), 29.54 (1C, CH2), 29.52 (9C, NHC(CH2)3), 29.42 (9C, CH2COO Bu), 29.41,

29.38, 29.32, 29.25, 29.09, 28.96, 28.21, 27.99 (13C, CH2), 27.83 (27C, C(CH3)3),

25.53 (1C, CH2), 25.32 (1C, CH2CH2CO), 25.02, 22.42 (2C, CH2), 13.88 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 1891 [M] ; MWcalc.: 1892.56 g/mol.

IR (ATR): ν˜ = 3321, 2981, 2927, 2857, 1730, 1653, 1545, 1460, 1367, 1313, 1251, 1213, 1151, 1035, 958, 850, 757, 680 cm−1.

EA: C103H182N4O26: calcd. C 65.37, H 9.69, N 2.96, O 21.98; found: C 65.47, H 9.79, N 3.04.

172 Chapter 5 Experimental Part

Asymmetric dendritic malonate [(C8)2-G2] (36)

Compound 36 was synthe-

O sized according to general O O O procedure GP 3 from 28 O O HN (350 mg, 0.77 mmol), 30 O O O O H O (1.10 g, 0.77 mmol), DMAP N H O O N O (19 mg, 0.15 mmol), 1- O O 36 O O NH HOBt (104 mg, 0.77 mmol) O O O O O and EDC (162 mg, 0.85 O O O mmol) in dry CH2Cl2/THF = 1/1 (100 mL). The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 13:10 to 10:10) to give 36 as a colorless oil.

Yield: 998 mg, 0.53 mmol, 69 %, colorless oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.57 (s, br, 1H, CONH), 6.09 (s, br, 3H, CONH), 3 3 4.84 (quin, J = 6.3 Hz, 1H, CH), 4.08 (t, J = 6.7 Hz, 2H, OCH2), 3.29 (s, 2H, t OCCH2CO), 2.13 (m, 26H, CH2COO Bu, OCCH2), 1.88 (m, 24H, NHC(CH2)3), 1.60

(m, 4H, CH(CH2)2), 1.46 (m, 4H, CH2), 1.37 (m, 83H, C(CH3)3, CH2), 1.19 (m, 24H, 3 CH2), 0.81 (t, J = 6.8 Hz, 6H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.11 (1C, CONH), 172.90 (3C, CONH), t 172.64 (9C, COO Bu), 166.75 (1C, CO), 166.36 (1C, CO), 80.40 (9C, C(CH3)3), 75.72

(1C, CH), 65.18 (1C, OCH2), 57.28 (1C, NHC(CH2)3), 57.23 (3C, NHC(CH2)3), 41.64

(1C, OCCH2CO), 36.88 (1C, CH2CO), 33.70 (2C, CH2), 31.66 (3C, NHC(CH2)3), 31.62 t (2C, CH2), 31.53 (3C, CH2CON), 29.60 (9C, NHC(CH2)3), 29.56 (9C, CH2COO Bu),

29.25, 29.23, 29.00, 28.06 (7C, CH2), 27.85 (27C, C(CH3)3), 25.36 (1C, CH2CH2CO),

25.06, 24.94, 22.41 (5C, CH2), 13.87 (2C, CH3) ppm.

+ MS (FAB, NBA): m/z = 1877 [M] ; MWcalc.: 1878.53 g/mol.

IR (ATR): ν˜ = 3306, 2981, 2935, 2866, 1730, 1661, 1537, 1460, 1367, 1313, 1259, 1151, 1105, 1035, 958, 850, 803, 734 cm−1.

173 Chapter 5 Experimental Part

EA: C102H180N4O26: calcd. C 65.22, H 9.66, N 2.98, O 22.14; found: C 64.83, H 9.77, N 3.10.

Asymmetric dendritic monoadduct [C6-G1] (37)

Compound 37 was synthesized accord-

O ing to general procedure GP 4 from O O O H malonate 31 (400 mg, 0.57 mmol), C60 N O O O O (494 mg, 0.69 mmol), CBr (209 mg, O 4 O O 0.63 mmol) and DBU (94 µL, 0.63 mmol) in 250 mL toluene. The crude

37 product was puriﬁed by column chro-

matography (SiO2; toluene/ethyl acetate, 80:15 to 80:30) to give 37 as a red brownish solid.

Yield: 396 mg, 0.28 mmol, 49 %, red brownish solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.99 (s, br, 3H, CONH), 4.54 (t, J = 6.7 Hz, 2H, 3 3 t OCH2), 4.52 (t, J = 6.8 Hz, 2H, OCH2), 2.36 (t, J = 7.5 Hz, 6H, CH2COO Bu), 2.22 3 3 (t, J = 7.1 Hz, 2H, CH2), 2.00 (t, J = 7.5 Hz, 6H, NHC(CH2)3), 1.86 (m, 2H, CH2),

1.67 (m, 4H, CH2), 1.49 (m, 2H, CH2), 1.41 (s, 27H, C(CH3)3), 1.28 (m, 6H, CH2), 0.91 3 (t, J = 6.5 Hz, 3H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 172.93 (3C, COO Bu), 172.03 (1C, CONH), 163.66 (1C, CO), 163.57 (1C, CO), 145.15, 145.14, 145.12, 145.07, 145.05, 145.03, 145.01, 144.76, 144.57, 144.53, 144.52, 144.49, 144.48, 143.75, 142.93, 142.92, 142.88, 142.85, 142.09, 142.05, 141.79, 141.71, 140.86, 140.75, 139.05, 138.89

2 3 (58C, C60-sp ), 80.49 (3C, C(CH3)3), 71.78 (2C, C60-sp ), 67.66 (1C, OCH2), 67.28

(1C, OCH2), 57.30 (1C, NHC(CH2)3), 51.38 (1C, OCCCO), 37.09 (1C, CH2CO), 31.29 t (1C, CH2), 29.85 (3C, NHC(CH2)3), 29.65 (3C, CH2COO Bu), 28.36, 28.12 (2C, CH2),

28.02 (9C, C(CH3)3), 25.96 (1C, CH2CH2CO), 25.41, 24.20, 22.43 (3C, CH2), 13.95

(1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 1440 [M+Na] , 1417 [M] , 720 [C60] ; MWcalc.: 1418.54 g/mol.

174 Chapter 5 Experimental Part

IR (ATR): ν˜ = 2989, 2945, 1801, 1732, 1664, 1535, 1456, 1418, 1396, 1356, 1254, 1201, 1165, 1101, 1065, 989, 963, 841, 787, 723, 701, 687, 645 cm−1.

UV/Vis (CH2Cl2): λmax = 254, 323, 425, 475 nm.

Asymmetric dendritic monoadduct [C18-G1] (38)

Compound 38 was

O synthesized accord- O O H O ing to general pro- N O O O O cedure GP 4 from O O O malonate 32 (500 mg, 0.58 mmol), C (498 mg, 0.69 38 60

mmol), CBr4 (212 mg, 0.64 mmol) and DBU (96 µL, 0.64 mmol) in 250 mL toluene. The crude product was puriﬁed by column chromatography (SiO2; toluene/ethyl acetate, 80:10 to 80:15) to give 38 as a red brownish solid.

Yield: 488 mg, 0.31 mmol, 53 %, red brownish solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.90 (s, br, 1H, CONH), 4.50 (t, J = 6.6 Hz, 3 3 t 2H, OCH2), 4.49 (t, J = 6.7 Hz, 2H, OCH2), 2.22 (t, J = 7.8 Hz, 6H, CH2COO Bu), 3 3 2.13 (t, J = 7.6 Hz, 2H, CH2CO), 1.98 (t, J = 7.8 Hz, 6H, NHC(CH2)3), 1.85 (m, 4H, 3 OCH2CH2), 1.70 (m, 2H, CH2), 1.44 (s, 27H, C(CH3)3), 1.25 (m, 32H, CH2), 0.88 (t, J

= 6.8 Hz, 3H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 172.88 (3C, COO Bu), 171.96 (1C, CONH), 163.67 (1C, CO), 163.59 (1C, CO), 145.37, 145.33, 145.25, 145.16, 145.14, 144.67, 144.63, 144.59, 143.86, 143.09, 143.06, 143.07, 143.00, 142.97, 142.19, 141.88,

2 3 140.93, 138.98, 138.93 (58C, C60-sp ), 80.69 (3C, C(CH3)3), 71.65 (2C, C60-sp ), 67.50

(1C, OCH2), 67.13 (1C, OCH2), 57.34 (1C, NHC(CH2)3), 49.87 (1C, OCCCO), 37.22 t (1C, CH2CO), 31.92 (1C, CH2), 30.00 (3C, NHC(CH2)3), 29.83 (3C, CH2COO Bu),

29.72, 29.70, 29.67, 29.62, 29.37, 29.22, 28.60, 28.35 (14C, CH2), 28.06 (9C, C(CH3)3),

175 Chapter 5 Experimental Part

26.00 (1C, CH2), 25.64 (1C, CH2CH2CO), 25.24, 22.69 (2C, CH2), 14.14 (1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 1609 [M+Na] , 1586 [M] , 720 [C60] ; MWcalc.: 1586.86 g/mol.

IR (ATR): ν˜ = 3001, 2953, 1823, 1801, 1743, 1674, 1553, 1467, 1421, 1388, 1327, 1299, 1207, 1163, 1089, 1045, 993, 942, 868, 734, 712, 679, 663 cm−1.

UV/Vis (CH2Cl2): λmax = 254, 323, 425, 475 nm.

Asymmetric dendritic monoadduct [(C8)2-G1] (39)

Compound 39 was synthesized ac-

O cording to general procedure GP 4 O O H O from malonate 33 (300 mg, 0.35 N O O O O mmol), C (304 mg, 0.42 mmol), O 60

O O CBr4 (130 mg, 0.39 mmol) and DBU (59 µL, 0.39 mmol) in 175 mL

39 toluene. The crude product was puriﬁed by column chromatography

(SiO2; toluene/ethyl acetate, 90:10 to 70:10) to give 39 as a red brownish solid.

Yield: 310 mg, 0.20 mmol, 56 %, red brownish solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 5.89 (s, br, 1H, CONH), 5.25 (m,1H, OCH), 4.48 3 3 t 3 (t, J = 6.8 Hz, 2H, OCH2), 2.22 (t, J = 7.8 Hz, 6H, CH2COO Bu), 2.13 (t, J = 7.7 Hz, 3 2H, CH2CO), 1.97 (t, J = 7.8 Hz, 6H, NHC(CH2)3), 1.87 (m, 2H, CH2), 1.78 (m, 2H,

CH2), 1.68 (m, 6H, CHCH2,CH2), 1.44 (m, 29H, C(CH3)3,CH2), 1.27 (m, 22H, CH2), 3 0.88 (t, J = 6.8 Hz, 6H, CH3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.01 (3C, COO Bu), 172.08 (1C, CONH), 163.70 (1C, CO), 163.29 (1C, CO), 145.61, 145.46, 145.33, 145.25, 144.93, 144.77, 144.75, 144.65, 143.95, 143.94, 143.18, 143.09, 143.05, 142.28, 141.98, 141.00,

2 139.11, 138.89 (58C, C60-sp ), 80.69 (3C, C(CH3)3), 78.56 (1C, CH), 71.79 (2C, C60- 3 sp ), 67.12 (1C, OCH2), 57.31 (1C, NHC(CH2)3), 52.74 (1C, OCCCO), 37.16 (1C,

CH2CO), 33.87 (2C, CH(CH2)2), 31.83, 30.11 (3C, CH2), 29.92 (3C, NHC(CH2)3),

176 Chapter 5 Experimental Part

t 29.76 (3C, CH2COO Bu), 29.63, 29.55, 29.48, 29.21, 28.35 (6C, CH2), 28.01 (9C,

C(CH3)3), 25.59 (1C, CH2CH2CO), 25.32, 25.18, 22.60 (5C, CH2), 14.08 (2C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 1595 [M+Na] , 1572 [M] , 720 [C60] ; MWcalc.: 1572.83 g/mol.

IR (ATR): ν˜ = 2996, 2932, 1866, 1813, 1796, 1732, 1671, 1543, 1446, 1432, 1388, 1346, 1263, 1197, 1134, 1073, 1036, 988, 916, 763, 703, 683, 655 cm−1.

UV/Vis (CH2Cl2): λmax = 254, 324, 425, 475 nm.

Asymmetric dendritic monoadduct [C6-G2] (40)

Compound 40 was synthe-

O sized according to general O O O procedure GP 4 from mal- O O HN onate 34 (260 mg, 0.15 mmol), O O O O H O C60 (144 mg, 0.2 mmol), N H N O O O O O CBr4 (56 mg, 0.17 mmol) and O O NH DBU (25 µL, 0.17 mmol) in O O O O O 100 mL toluene. The crude O O O 40 product was puriﬁed by col-

umn chromatography (SiO2; toluene/ethyl acetate, 30:10 to 15:10) to give 40 as a red brownish solid.

Yield: 171 mg, 0.07 mmol, 47 %, red brownish solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.57 (s, br, 1H, CONH), 6.14 (s, br, 3H, CONH), 3 3 4.50 (t, J = 6.5 Hz, 2H, OCH2), 4.49 (t, J = 6.4 Hz, 2H, OCH2), 2.19 (m, 26H, t CH2COO Bu, OCCH2), 1.95 (m, 24H, NHC(CH2)3), 1.84 (m, 4H, CH2), 1.69 (m, 2H, 3 CH2), 1.47 (m, 2H, CH2), 1.44 (m, 83H, C(CH3)3,CH2), 1.26 (m, 4H, CH2), 0.85 (t, J

= 6.7 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.29 (1C, CONH), 173.15 (3C, CONH), 172.80 (9C, COOtBu), 163.81 (1C, CO), 163.74 (1C, CO), 145.67, 145.53, 145.43, 145.32, 145.26, 145.12, 144.95, 144.83, 144.76, 144.68, 144.65, 143.95, 143.91,

177 Chapter 5 Experimental Part

143.53, 143.13, 143.05, 142.26, 141.98, 141.00, 139.12, 139.03, 139.00 (58C, C60- 2 3 sp ), 80.59 (9C, C(CH3)3), 71.65 (2C, C60-sp ), 67.47 (1C, OCH2), 67.33 (1C, OCH2),

57.58 (1C, NHC(CH2)3), 57.42 (3C, NHC(CH2)3), 52.36 (1C, OCCCO), 37.01 (1C,

CH2CO), 31.68 (3C, NHC(CH2)3), 31.30 (3C, CH2CON), 30.75 (1C, CH2), 29.72 (9C, t NHC(CH2)3), 29.61 (9C, CH2COO Bu), 28.47, 28.32 (2C, CH2), 28.01 (27C, C(CH3)3),

25.55, 25.23 (2C, CH2), 24.38 (1C, CH2CH2CO), 22.53 (1C, CH2), 14.00 (1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 2466 [M+Na] , 2443 [M] , 720 [C60] ; MWcalc.: 2442.87 g/mol.

IR (ATR): ν˜ = 3278, 3265, 3002, 2945, 2937, 1801, 1768, 1586, 1467, 1413, 1395, 1346, 1274, 1201, 1166, 1114, 1065, 1004, 901, 857, 765, 694 cm−1.

UV/Vis (CH2Cl2): λmax = 254.5, 324, 424.5, 476 nm.

Asymmetric dendritic monoadduct [C18-G2] (41)

O O O O O O HN O O O O H O N H N O O O O O O O NH O O O O O O O O 41

Compound 41 was synthesized according to general procedure GP 4 from malonate

35 (600 mg, 0.32 mmol), C60 (302 mg, 0.42 mmol), CBr4 (117 mg, 0.35 mmol) and DBU (52 µL, 0.35 mmol) in 150 mL toluene. The crude product was puriﬁed by column chromatography (SiO2; toluene/ethyl acetate, 70:20 to 70:50) to give 41 as a red brownish solid.

Yield: 366 mg, 0.14 mmol, 44 %, red brownish solid.

178 Chapter 5 Experimental Part

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.63 (s, br, 1H, CONH), 6.04 (s, br, 3H, CONH), 3 3 4.51 (t, J = 6.4 Hz, 2H, OCH2), 4.50 (t, J = 6.8 Hz, 2H, OCH2), 2.20 (m, 26H, t CH2COO Bu, OCCH2), 1.96 (m, 24H, NHC(CH2)3), 1.83 (m, 4H, CH2), 1.71 (m, 2H, 3 CH2), 1.44 (m, 83H, C(CH3)3,CH2), 1.25 (m, 30H, CH2), 0.88 (t, J = 6.8 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.07 (1C, CONH), 172.99 (3C, CONH), 172.77 (9C, COOtBu), 163.75 (1C, CO), 163.68 (1C, CO), 145.48, 145.26, 145.19, 145.16, 145.11, 145.09, 144.80, 144.63, 144.62, 144.59, 144.55, 144.53, 143.83, 143.81, 143.02, 142.95, 142.93, 142.90, 142.15, 142.13, 141.85, 140.88, 139.16,

2 3 139.10, 138.75 (58C, C60-sp ), 80.43 (9C, C(CH3)3), 71.66 (2C, C60-sp ), 67.44 (1C,

OCH2), 67.32 (1C, OCH2), 57.57 (1C, NHC(CH2)3), 57.39 (3C, NHC(CH2)3), 52.42

(1C, OCCCO), 37.02 (1C, CH2CO), 31.84 (3C, NHC(CH2)3), 31.73 (1C, CH2), 31.63

(1C, CH2), 31.62 (3C, CH2CON), 29.74 (3C, CH2), 29.71 (9C, NHC(CH2)3), 29.64 t (9C, CH2COO Bu), 29.61, 29.59, 29.56, 29.55, 29.29, 29.27, 29.14, 28.53, 28.28 (9C,

CH2), 28.02 (27C, C(CH3)3), 27.97, 25.51 (2C, CH2), 25.23 (1C, CH2CH2CO), 24.84,

22.61 (2C, CH2), 14.04 (1C, CH3) ppm.

+ + + MS (FAB, NBA): m/z = 2634 [M+Na] , 2611 [M] , 720 [C60] ; MWcalc.: 2611.18 g/mol.

IR (ATR): ν˜ = 3337, 3267, 2974, 2927, 2858, 1730, 1661, 1537, 1460, 1423, 1367, 1305, 1244, 1151, 1105, 950, 850, 757, 711, 684 cm−1.

UV/Vis (CH2Cl2): λmax = 254, 324, 424, 476 nm.

179 Chapter 5 Experimental Part

Asymmetric dendritic monoadduct [(C8)2-G2] (42)

Compound 42 was synthe-

O sized according to general O O O procedure GP 4 from mal- O O HN onate 36 (250 mg, 0.13 O O O O H O mmol), C60 (122 mg, 0.17 N H O O N O mmol), CBr (48 mg, 0.14 O O 4 O O NH mmol) and DBU (22 µL, O O O O O 0.14 mmol) in 100 mL O O O toluene. The crude prod- 42 uct was puriﬁed by column chromatography (SiO2; toluene/ethyl acetate, 70:20 to 70:50) to give 42 as a red brownish solid.

Yield: 159 mg, 0.06 mmol, 47 %, red brownish solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.59 (s, br, 1H, CONH), 6.04 (s, br, 3H, CONH), 3 3 5.24 (quin, J = 6.4 Hz, 1H, CH), 4.48 (t, J = 7.1 Hz, 2H, OCH2), 2.19 (m, 26H, t CH2COO Bu, OCCH2), 1.95 (m, 24H, NHC(CH2)3), 1.69 (m, 4H, CH(CH2)2), 1.59 (m, 3 4H, CH2), 1.43 (m, 83H, C(CH3)3,CH2), 1.25 (m, 24H, CH2), 0.87 (t, J = 6.9 Hz, 6H,

CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 172.82 (3C, CONH), 172.74 (1C, CONH), 172.62 (9C, COOtBu), 163.54 (1C, CO), 163.12 (1C, CO), 145.62, 145.36, 145.24, 145.20, 145.15, 145.13, 145.10, 144.80, 144.68, 144.64, 144.56, 144.53, 143.85, 143.82, 143.02, 142.98, 142.96, 142.94, 142.91, 142.17, 141.86, 140.89, 139.10,

2 138.72 (58C, C60-sp ), 80.55 (9C, C(CH3)3), 77.20 (1C, CH), 67.32 (1C, OCH2), 57.42

(3C, NHC(CH2)3), 57.38 (1C, NHC(CH2)3), 49.08 (1C, OCCCO), 37.02 (1C, CH2CO),

33.91 (2C, CH2), 31.88 (2C, CH2), 31.85 (3C, NHC(CH2)3), 31.70 (3C, CH2CON), t 29.79 (9C, NHC(CH2)3), 29.76 (9C, CH2COO Bu), 29.65, 29.57, 29.48, 29.23, 28.44

(8C, CH2), 28.07 (27C, C(CH3)3), 25.58 (1C, CH2CH2CO), 25.34, 24.91, 22.64 (6C,

CH2), 14.13 (2C, CH3) ppm.

180 Chapter 5 Experimental Part

+ + + MS (FAB, NBA): m/z = 2620 [M+Na] , 2597 [M] , 720 [C60] ; MWcalc.: 2597.16 g/mol.

IR (ATR): ν˜ = 3301, 3293, 2974, 2864, 2803, 1763, 1702, 1678, 1565, 1487, 1443, 1353, 1312, 1266, 1207, 1168, 1121, 963, 882, 757, 703, 693 cm−1.

UV/Vis (CH2Cl2): λmax = 254.5, 324, 424.5, 476 nm.

Deprotected asymmetric dendritic monoadduct [C6-G1] (43)

Compound 43 was synthesized accord- OH ing to general procedure GP 5 from O O H O N O O OH monoadduct 37 (200 mg, 0.14 mmol) in O O formic acid (20 mL). The crude prod- O OH uct was puriﬁed by reprecipitation from

THF/Et2O to give 43 as a red brownish 43 solid.

Yield: 170 mg, 0.14 mmol, 97 %, red brownish solid.

1 H-NMR (400 MHz, RT, THF-d8): δ = 11.09 (s, br, 3H, COOH), 7.01 (s, br, 1H,

CONH), 4.49 (m, br, 4H, OCH2), 2.05 (m, br, 6H, CH2COOH, CH2), 1.79 (m, br, 8H,

NHC(CH2)3,CH2), 1.51 (m, br, 2H, CH2), 1.39 (m, br, 8H, CH2), 1.19 (m, br, 4H, CH2),

0.88 (m, br, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, THF-d8): δ = 174.97 (3C, COOH), 171.95 (1C, CONH), 162.87 (1C, CO), 162.79 (1C, CO), 145.48, 145.01, 144.79, 144.26, 143.56, 143.01,

2 142.59, 141.71, 141.43, 140.69, 140.42, 139.12, 137.91 (58C, C60-sp ), 71.59 (2C, 3 C60-sp ), 67.53 (2C, OCH2), 56.99 (1C, NHC(CH2)3), 52.03 (1C, OCCCO), 37.43 (1C,

CH2CO), 31.01 (1C, CH2), 30.01 (3C, NHC(CH2)3), 29.78 (3C, CH2COOH), 28.44,

28.22 (2C, CH2), 26.01 (1C, CH2CH2CO), 25.44, 24.26, 22.39 (3C, CH2), 13.98 (1C,

CH3) ppm.

+ + MS (FAB, NBA): m/z = 1250 [M] , 720 [C60] ; MWcalc.: 1250.22 g/mol.

IR (ATR): ν˜ = 3231, 2989, 2673, 1756, 1674, 1635, 1555, 1432, 1401, 1265, 1144, 1087, 1001, 836, 796, 712, 674 cm−1.

181 Chapter 5 Experimental Part

UV/Vis (DMSO): λmax = 252, 322, 424 nm.

Deprotected asymmetric dendritic monoadduct [C18-G1] (44)

Compound 44 was OH synthesized accord- O O H O N O O OH ing to general pro- O O cedure GP 5 from O OH monoadduct 38 (300 mg, 0.19 mmol) in 44 formic acid (25 mL).

The crude product was puriﬁed by reprecipitation from THF/Et2O to give 44 as a red brownish solid.

Yield: 259 mg, 0.18 mmol, 96 %, red brownish solid.

1 H-NMR (400 MHz, RT, THF-d8): δ = 12.05 (s, br, 3H, COOH), 7.14 (s, br, 1H,

CONH), 4.39 (m, br, 4H, OCH2), 2.09 (m, br, 8H, CH2COOH, CH2CO), 1.82 (m, br,

8H, NHC(CH2)3, CH2), 1.51 (m, br, 4H, OCH2CH2), 1.36 (m, br, 32H, CH2), 0.85 (m, br, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, THF-d8): δ = 174.42 (3C, COOH), 171.81 (1C, CONH), 163.63 (1C, CO), 163.52 (1C, CO), 144.71, 144.13, 143.40, 142.53, 141.69, 141.30,

2 3 140.61, 140.35, 139.10, 138.85 (58C, C60-sp ), 71.42 (2C, C60-sp ), 67.12 (1C, OCH2),

67.02 (1C, OCH2), 56.34 (1C, NHC(CH2)3), 52.32 (1C, OCCCO), 38.13 (1C, CH2CO),

31.73 (1C, CH2), 29.97 (3C, NHC(CH2)3), 29.88 (3C, CH2COOH), 29.69, 29.70, 29.60,

29.62, 29.33, 29.28, 28.54, 28.37 (14C, CH2), 25.99 (1C, CH2), 25.42 (1C, CH2CH2CO),

25.06, 22.57 (2C, CH2), 14.23 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 1419 [M] , 720 [C60] ; MWcalc.: 1418.54 g/mol. IR (ATR): ν˜ = 3221, 2920, 2850, 2647, 1692, 1537, 1452, 1228, 1182, 1105, 1058, 881, 788, 703, 664 cm−1.

UV/Vis (DMSO): λmax = 254, 323.5, 424.5 nm.

182 Chapter 5 Experimental Part

Deprotected asymmetric dendritic monoadduct [(C8)2-G1] (45)

Compound 45 was synthesized ac- OH cording to general procedure GP 5 O O H O N O O OH from monoadduct 39 (250 mg, 0.16 O O mmol) in formic acid (25 mL). The O OH crude product was puriﬁed by repre-

cipitation from THF/Et2O to give 45 as 45 a red brownish solid.

Yield: 218 mg, 0.16 mmol, 97 %, red brownish solid.

1 H-NMR (400 MHz, RT, THF-d8): δ = 11.89 (s, br, 3H, COOH), 7.04 (s, br, 1H, CONH),

5.18 (m, br, 1H, OCH), 4.41 (m, br, 2H, OCH2), 2.18 (m, br, 8H, CH2COOH, CH2CO),

1.91 (m, br, 8H, NHC(CH2)3, CH2), 1.65 (m, br, 8H, CHCH2, CH2), 1.41 (m, br, 2H,

CH2), 1.21 (m, br, 22H, CH2), 0.85 (m, br, 6H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, THF-d8): δ = 175.02 (3C, COOH), 172.00 (1C, CONH), 163.59 (1C, CO), 163.51 (1C, CO), 144.88, 144.76, 144.36, 144.01, 143.76, 143.35,

2 143.08, 142.28, 141.88, 141.00, 139.11, 138.79 (58C, C60-sp ), 78.01 (1C, CH), 71.59 3 (2C, C60-sp ), 67.54 (1C, OCH2), 56.69 (1C, NHC(CH2)3), 52.56 (1C, OCCCO), 37.99

(1C, CH2CO), 33.98 (2C, CH(CH2)2), 31.89, 30.13 (3C, CH2), 29.89 (3C, NHC(CH2)3),

29.71 (3C, CH2COOH), 29.63, 29.48, 29.01, 28.37 (6C, CH2), 25.61 (1C, CH2CH2CO),

25.37, 22.60 (5C, CH2), 14.11 (2C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 1405 [M] , 720 [C60] ; MWcalc.: 1404.51 g/mol.

IR (ATR): ν˜ = 3199, 2974, 2603, 1815, 1767, 1656, 1599, 1531, 1433, 1403, 1301, 1265, 1141, 1078, 967, 844, 797, 703, 688 cm−1.

UV/Vis (DMSO): λmax = 254, 323.5, 424.5 nm.

183 Chapter 5 Experimental Part

Deprotected asymmetric dendritic monoadduct [C6-G2] (46)

Compound 46 was synthesized HO O O OH according to general procedure HO O GP 5 from monoadduct 40 (150 HN OH O mg, 0.06 mmol) in formic acid O O H O N H N O O OH (35 mL). The crude product was O O O puriﬁed by reprecipitation from O NH O O OH MeOH/Et2O to give 46 as a red HO OH O O brownish solid. OH 46

Yield: 112 mg, 0.06 mmol, 97 %, red brownish solid.

1 H-NMR (400 MHz, RT, DMSO-d6): δ = 12.01 (s, br, 9H, COOH), 8.05 (s, br, 1H,

CONH), 7.16 (s, br, 3H, CONH), 4.51 (m, br, 4H, OCH2), 2.11 (m, br, 26H, CH2COOH,

CH2CO), 1.91 (m, br, 28H, NHC(CH2)3, CH2), 1.51 (m, br, 6H, CH2), 1.26 (m, br, 4H,

CH2), 0.84 (m, br, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, DMSO-d6): δ = 174.97 (9C, COOH), 172.39 (4C, CONH), 163.31, 163.28 (2C, CO), 145.55, 145.46, 145.26, 145.09, 144.78, 144.72, 144.65, 144.62, 144.31, 144.21, 143.96, 143.78, 143.39, 143.163, 142.50, 142.47, 141.99,

2 3 141.77, 141.05, 139.78, 139.41, 139.19, 138.58 (58C, C60-sp ), 71.34 (2C, C60-sp ),

67.43, 67.39 (2C, OCH2), 56.67 (4C, NHC(CH2)3), 52.91 (1C, OCCCO), 36.84 (1C,

CH2CO), 30.51 (6C, NHC(CH2)3,CH2CON), 30.68, (1C, CH2), 29.33 ((9C, NHC(CH2)3),

28.65, 28.19 (2C, CH2), 28.24 (9C, CH2COOH), 25.98, 25.55, 24.50, 22.19 (4C, CH2),

14.11 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 1938 [M+H] , 720 [C60] ; MWcalc.: 1937.91 g/mol.

UV/Vis (DMSO): λmax = 264, 319 nm.

184 Chapter 5 Experimental Part

Deprotected asymmetric dendritic monoadduct [C18-G2] (47)

HO O O OH HO O HN OH O O O H O N H N O O OH O O O O NH O O OH HO OH O O OH 47

Compound 47 was synthesized according to general procedure GP 5 from monoadduct 41 (300 mg, 0.11 mmol) in formic acid (50 mL). The crude product was puriﬁed by reprecipitation from MeOH/Et2O to give 47 as a red brownish solid.

Yield: 237 mg, 0.11 mmol, 98 %, red brownish solid.

1 H-NMR (400 MHz, RT, DMSO-d6): δ = 12.03 (s, br, 9H, COOH), 8.12 (s, br, 1H,

CONH), 7.20 (s, br, 3H, CONH), 4.46 (m, br, 4H, OCH2), 2.09 (m, br, 26H, CH2COOH,

CH2CO), 1.81 (m, br, 30H, NHC(CH2)3,CH2), 1.21 (m, br, 32H, CH2), 0.82 (m, br, 3H,

CH3) ppm.

13 C-NMR (100.5 MHz, RT, DMSO-d6): δ = 174.66 (9C, COOH), 172.49 (4C, CONH), 163.28 (2C, CO), 145.67, 145.61, 145.14, 144.94, 144.85, 144.77, 144.46, 144.38, 144.25, 143.56, 143.48, 142.74, 142.67, 141.89, 141.81, 141.52, 140.66, 140.60,

2 3 139.20, 138.14 (58C, C60-sp ), 71.56 (2C, C60-sp ), 67.42 (2C, OCH2), 56.34 (4C,

NHC(CH2)3), 52.86 (1C, OCCCO), 36.05 (1C, CH2CO), 31.45, 30.76, 30.26 (5C, CH2),

29.22 (6C, NHC(CH2)3, CH2CON), 29.07 ((9C, NHC(CH2)3), 28.89, 28.66 (5C, CH2),

28.09 (9C, CH2COOH), 25.70, 25.34, 25.17, 24.50, 22.25 (9C, CH2), 14.06 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 2106 [M+H] , 720 [C60] ; MWcalc.: 2106.23 g/mol.

IR (ATR): ν˜ = 3356, 3101, 2968, 2865, 2664, 1714, 1654, 1552, 1464, 1424, 1278, 1231, 1201, 1104, 912, 878, 801, 769, 703, 685 cm−1.

185 Chapter 5 Experimental Part

UV/Vis (DMSO): λmax = 263, 320 nm.

Deprotected asymmetric dendritic monoadduct [(C8)2-G2] (48)

Compound 48 was synthe- HO O O OH sized according to general HO O procedure GP 5 from mono- HN OH adduct 42 (150 mg, 0.06 O O O H O N H N O O OH mmol) in formic acid (35 mL). O O O The crude product was pu- O NH O OH O riﬁed by reprecipitation from HO OH O O MeOH/Et2O to give 48 as a OH 48 red brownish solid.

Yield: 117 mg, 0.06 mmol, 97 %, red brownish solid.

1 H-NMR (400 MHz, RT, DMSO-d6): δ = 12.11 (s, br, 9H, COOH), 8.07 (s, br, 1H,

CONH), 7.01 (s, br, 3H, CONH), 5.20 (m, br, 1H, CH), 4.41 (m, br, 2H, OCH2), 2.13

(m, br, 26H, CH2COOH, CH2CO), 1.89 (m, 28H, NHC(CH2)3, CH(CH2)2), 1.51 (m, br,

6H, CH2), 1.259 (m, br, 24H, CH2), 0.87 (m, br, 6H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, DMSO-d6): δ = 174.71 (9C, COOH), 172.44 (4C, CONH), 163.31, 163.01 (2C, CO), 145.63, 145.59, 145.24, 145.19, 145.01, 144.97, 144.80, 144.69, 144.64, 144.41, 144.22, 143.87, 143.82, 143.01, 142.77, 142.69, 141.86,

2 3 141.44, 140.89, 139.11, 138.73 (58C, C60-sp ), 77.55 (1C, CH), 71.66 (2C, C60-sp ),

67.39 (1C, OCH2), 56.46 (4C, NHC(CH2)3), 52.01 (1C, OCCCO), 36.44 (1C, CH2CO),

33.87, 32.01 (4C, CH2), 29.63 (6C, NHC(CH2)3, CH2CON), 29.24 ((9C, NHC(CH2)3),

29.78, 29.14, 29.06, 29.01, 28.94 (6C, CH2), 28.22 (9C, CH2COOH), 25.11 (1C,

CH2CH2CO), 24.99, 24.89, 22.66 (6C, CH2), 14.21 (2C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 2092 [M+H] , 720 [C60] ; MWcalc.: 2092.20 g/mol.

IR (ATR): ν˜ = 3343, 3153, 2987, 2878, 2666, 1801, 1732, 1701, 1634, 1549, 1501, 1498, 1433, 1263, 1203, 1144, 1103, 987, 953, 871, 771, 734, 691 cm−1.

UV/Vis (DMSO): λmax = 264, 318 nm.

186 Chapter 5 Experimental Part

2-Hydroxyethyl all-cis-docosa-4,7,10,13,16,19-hexaenoate (50)

Compound 50 was synthesized O OH according to general procedure O 50 GP 3 from all-cis-docosa- 4,7,10,13,16,19-hexaenoic acid 49 (2.5 g, 7.61 mmol), ethane-1,2-diol (710 mg, 11.42 mmol), EDC (1.90 g, 9.89 mmol) and DMAP (140 mg, 1.14 mmol) in dry CH2Cl2. The crude product was puriﬁed by

ﬂash column chromatography (SiO2; hexane/ethyl acetate, 50:10 to 50:30) to give 50 as yellow oil.

Yield: 2.51 g, 6.74 mmol, 89 %, yellow oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.33 (m, 12H, CH), 4.16 (t, J = 4.8 Hz, 2H, 3 OCH2), 3.75 (t, J = 4.8 Hz, 2H, CH2OH), 2.80 (m, 10H, CH2), 2.41 (s, br, 1H, OH), 3 2.36 (m, 4H, CH2)), 2.03 (m, CH2CH3), 0.92 (t, J = 7.5 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.50 (1C, CO), 132.07, 129.50, 128.62,

128.37, 128.33, 128.31, 128.06, 127.94, 127.86, 127.09 (12C, CH), 66.07 (1C, OCH2),

61.12 (1C, CH2OH), 34.14 (1C, CH2CO), 25.72, 25.67, 25.62 (5C, CH2), 22.82

(CH2CH2CO), 20.64 (1C, CH2CH3), 14.35 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 373 [M+H] ; MWcalc.: 372,54 g/mol.

IR (ATR): ν˜ = 3360, 2981, 2935, 1722, 1676, 1537, 1452, 1367, 1321, 1251, 1151, 1043, 958, 850, 757 cm−1.

EA: C24H36O3: calcd. C 77.38, H 9.74, O 12.88; found: C 76.58, H 9.53.

187 Chapter 5 Experimental Part

3-(6-tert-Butoxy-6-oxohexyloxy)-3-oxopropanoic acid (51)

Malonic acid (1.11 g, 10.62 mmol) and 22 (2.0 g, 10.62 O O O mmol) were dissolved in dry CH Cl and cooled to 0°C. HO O 2 2 O 51 DCC (2.19 g, 10.62 mmol) was dissolved in dry CH2Cl2 and added dropwise over a period of 2 h via a dropping funnel. After stirring the solution for additional 1 h at 0°C and 8 h at rt, TLC control showed the complete conversion of the starting material. The resulting dicyclohexylurea was filtered off and traces which remained in solution were removed by repeated precipitation from ethyl acetate. The filtrate was extracted with saturated NaHCO3- solution (3 x 200 mL), followed by acidification with 5 N HCl. The aqueous solution was extracted with Et2O (3 x 200 mL) and dried over anhydrous MgSO4. Removal of the solvent under reduced pressure gave 51 as pale yellow oil.

Yield: 1.01 g, 3.68 mmol, 35 %, pale yellow oil.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 11.19 (s, br, 1H, COOH), 4.13 (t, J = 6.4 Hz,2H, 3 OCH2), 3.38 (s, 2H, OCCH2CO), 2.19 (t, J = 7.1 Hz, 2H, CH2CO), 1.59 (m, 4H, CH2),

1.40 (s, 9H, C(CH3)3), 1.34 (m, 2H, CH2) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 173.53 (1C, CO), 170.89 (1C, CO), 167.09 (1C,

CO), 80.59 (1C, C(CH3)3), 65.90 (1C, OCH2), 41.05 (1C, OCCH2CO), 35.53 (1C,

CH2CO), 28.25 (3C, C(CH3)3), 25.47, 24.77 (3C, CH2) ppm.

+ t MS (FAB, NBA): m/z = 274 [M] , 217 [M- Bu]; MWcalc.: 274.31 g/mol.

EA: C13H22O6: calcd. C 56.92, H 8.08, O 35.00; found: C 56.56, H 7.98.

188 Chapter 5 Experimental Part

6-tert-Butoxy-6-oxohexyl(2-all-cis-docosa-4,7,10,13,16,19-hexaenoyloxy)ethyl malonate (52)

O O O O O O O O 52

Compound 52 was synthesized according to general procedure GP 3 from 50 (2.26 g, 6.07 mmol), 51 (1.51 g, 5.52 mmol), EDC (1.38 g, 7.17 mmol) and DMAP (101 mg, 0.83 mmol) in dry CH2Cl2. The crude product was puriﬁed by ﬂash column chromatography

(SiO2; hexane/ethyl acetate, 65:10) to give 52 as yellow oil.

Yield: 2.52 g, 4.01 mmol, 66 %, yellow oil.

1 H-NMR (300 MHz, RT, CDCl3): δ = 5.34 (m, 12H, CH), 4.28 (m, 4H, OCH2, CH2O), 3 4.11 (t, J = 6.7 Hz, 2H, OCH2), 3.36 (s, 2H, OCCH2CO), 2.81 (m, 10H, CH2), 2.36 (m, 3 4H, CH2)), 2.18 (t, J = 7.4 Hz, 2H, CH2CO), 2.04 (m, CH2CH3), 1.60 (m, 4H, CH2), 3 1.40 (s, 9H, C(CH3)3), 1.34 (m, 2H, CH2), 0.94 (t, J = 7.5 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 172.99 (1C, CO), 172.91 (1C, CO), 166.50 (1C, CO), 166.43 (1C, CO), 132.18, 129.57, 128.73, 128.46, 128.44, 128.41, 128.26, 128.24,

128.19, 128.03, 127.91, 127.18 (12C, CH), 80.25 (1C, C(CH3)3), 65.62 (1C, OCH2),

63.29 (1C, OCH2), 61.94 (1C, CH2O), 41.48 (1C, OCCH2CO), 35.49 (1C, CH2CO),

34.10 (1C, CH2CO), 28.35 (1C, CH2), 28.28 (3C, C(CH3)3), 25.80, 25.76, 25.71 (5C,

CH2), 24.80, 22.81 (2C, CH2), 20.72 (1C, CH2CH3), 14.43 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 629 [M] ; MWcalc.: 628,84 g/mol.

IR (ATR): ν˜ = 3165, 2976, 2932, 2868, 1731, 1654, 1458, 1404, 1399, 1367, 1301, 1256, 1203, 1152, 1101, 1055, 989, 956, 887, 848, 763, 701 cm−1.

EA: C37H56O8: calcd. C 70.67, H 8.98, O 20.35; found: C 70.17, H 8.57.

189 Chapter 5 Experimental Part

6-(3-(2-(all-cis-Docosa-4,7,10,13,16,19-hexaenoyloxy)ethoxy)-3- oxopropanoyloxy)hexanoic acid (53)

O O O OH O O O O 53

Compound 53 was synthesized according to general procedure GP 5 from 53 (1.5 g,

2.39 mmol) and triﬂuoroacetic acid (1.8 mL, 24 mmol) in CHCl3.

Yield: 1.36 g, 2.37 mmol, 99 %, yellow oil.

1 H-NMR (300 MHz, RT, CDCl3): δ = 9.76 (s, br, 1H, COOH), 5.34 (m, 12H, CH), 4.28 3 (m, 4H, OCH2,CH2O), 4.11 (t, J = 6.9 Hz, 2H, OCH2), 3.37 (s, 2H, OCCH2CO), 2.81

(m, 10H, CH2), 2.34 (m, 4H, CH2), 2.01 (m, CH2CH3), 1.63 (m, 4H, CH2), 1.37 (m, 2H, 3 CH2), 0.93 (t, J = 7.5 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 179.65 (1C, CO), 173.32 (1C, CO), 166.84 (1C, CO), 166.78 (1C, CO), 132.41, 129.81, 128.94, 128.68, 128.65, 128.63, 128.47,

128.45, 128.40, 128.25, 128.09, 127.39 (12C, CH), 65.78 (1C, OCH2), 63.55 (1C,

OCH2), 62.21 (1C, CH2O), 41.69 (1C, OCCH2CO), 34.32 (1C, CH2CO), 34.16 (1C,

CH2CO), 28.49 (1C, CH2), 26.02, 25.97, 25.93 (5C, CH2), 24.58, 23.01 (2C, CH2),

20.95 (1C, CH2CH3), 14.69 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 573 [M+H] ; MWcalc.: 572,73 g/mol.

190 Chapter 5 Experimental Part

Asymmetric dendritic malonate [DHA-G2] (54)

Compound 54 was

O synthesized according O O O to general procedure O O HN GP 3 from 53 (1.31 O O O O H O g, 2.29 mmol), 30 O N H O O N O (3.95 g, 2.74 mmol), O O O 54 O O NH EDC (526 mg, 2.74 O O O O O mmol), DMAP (140 O O O mg, 1.14 mmol) and 1-HOBt (371 mg, 2.74 mmol) in dry CH2Cl2. The crude product was puriﬁed by ﬂash column chromatography

(SiO2; hexane/ethyl acetate, 50:40) to give 54 as yellow oil.

Yield: 2.42 g, 1.21 mmol, 53 %, yellow oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.53 (s, br, 1H, CONH), 6.00 (s, br, 3H, CONH), 3 5.25 (m, 12H, CH), 4.21 (m, 4H, OCH2, CH2O), 4.03 (t, J = 6.7 Hz, 2H, OCH2),

3.29 (s, 2H, OCCH2CO), 2.72 (m, 10H, CH2), 2.28 (m, 4H, CH2), 2.07 (m, 24H, t CH2COO Bu), 1.84 (m, 24H, NHC(CH2)3), 1.54 (m, 4H, CH2), 1.31 (m, 83H, C(CH3)3, 3 CH2), 0.85 (t, J = 7.5 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.09 (1C, CONH), 172.90 (3C, CONH), 172.63 (9C, COOt Bu), 166.41 (1C, CO), 166.32 (1C, CO), 131.96, 129.35, 128.52, 128.24, 128.23, 128.20, 128.07, 128.05, 128.01, 127.84, 127.75, 127.00 (12C, CH),

80.51 (9C, C(CH3)3), 65.50 (1C, OCH2), 63.10 (1C, OCH2), 61.82 (1C, CH2O), 57.49

(1C, NHC(CH2)3), 57.42 (3C, NHC(CH2)3), 41.27 (1C, OCCH2CO), 37.10 (1C, CH2CO),

33.91 (1C, CH2CO), 31.88 (3C, NHC(CH2)3), 31.73 (3C, CH2CON), 29.82 (9C, t NHC(CH2)3), 29.78 (9C, CH2COO Bu), 28.23 (1C, CH2), 28.08 (27C, C(CH3)3), 25.62,

25.57, 25.52 (5C, CH2), 25.26, 22.63 (2C, CH2), 20.54 (1C, CH2CH3), 14.27 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 1993 [M] , 2016[M+Na] ; MWcalc.: 1994.61 g/mol.

191 Chapter 5 Experimental Part

IR (ATR): ν˜ = 3244, 3165, 2976, 1766, 1701, 1653, 1488, 1399, 1363, 1356, 1301, 1278, 1213, 1188, 1094, 1046, 976, 914, 855, 8418, 743, 684 cm−1.

EA: C109H180N4O28 · 1/3 C4H8O2: calcd. C 65.47, H 9.10, N 2.77, O 22.66; found: C 65.09, H 8.99, N 2.54.

Asymmetric dendritic monoadduct [DHA-G2] (55)

Compound 55 was syn-

O thesized according to O O O general procedure GP O O HN 4 from malonate 54 O O O O H O (866 mg, 0.43 mmol), O N H O O N O C (376 mg, 0.52 O O O 60 O O NH mmol), CBr4 (173 mg, O O O O O 0.52 mmol) and DBU O O O (78 µL, 0.52 mmol) in 55 175 mL toluene. Pre- cleaning by ﬂash column chromatography (SiO2; toluene/ethyl acetate, 70:40 to 60:40) and subsequent puriﬁcation by preparative HPLC (Nucleosil 5 µm; toluene/ethyl acetate 68:32) gave 55 as a red brownish solid.

Yield: 443 mg, 0.16 mmol, 38 %, yellow oil.

1 H-NMR (300 MHz, RT, CDCl3): δ = 7.61 (s, br, 1H, CONH), 6.02 (s, br, 3H, CONH),

5.29 (m, 12H, CH), 4.65 (m, 2H, CH2O), 4.41 (m, 4H, OCH2), 2.75 (m, 10H, CH2), t 2.30 (m, 4H, CH2), 2.12 (m, 24H, CH2COO Bu), 1.90 (m, 24H, NHC(CH2)3), 1.63 (m, 3 4H, CH2), 1.36 (m, 83H, C(CH3)3,CH2), 0.89 (t, J = 7.4 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 173.03 (1C, CONH), 172.98 (3C, CONH), 172.71 (9C, COOt Bu), 163.52 (1C, CO), 163.38 (1C, CO), 145.44, 145.31, 145.24, 145.21, 145.16, 145.08, 144.94, 144.74, 144.72, 144.69, 144.65, 144.61, 143.95, 143.90, 143.13, 143.08, 143.06, 142.97, 142.25, 142.22, 141.90, 141.02, 140.96, 139.39,

2 138.75 (58C, C60-sp ), 132.06, 129.50, 128.60, 128.35, 128.31, 128.28, 128.16, 128.13,

192 Chapter 5 Experimental Part

3 128.09, 127.92, 127.77, 127.07 (12C, CH), 80.46 (9C, C(CH3)3), 71.49 (2C, C60-sp ),

67.50 (1C, OCH2), 64.77 (1C, CH2O), 61.88 (1C, OCH2), 57.50 (4C, NHC(CH2)3),

52.08 (1C, OCCCO), 37.14 (1C, CH2CO), 33.97 (1C, CH2CO), 31.80 (6C, NHC(CH2)3, t CH2CON), 29.82 (18C, NHC(CH2)3, CH2COO Bu), 28.40 (1C, CH2), 28.16 (27C,

C(CH3)3), 25.71, 25.61, 25.38 (5C, CH2), 22.69 (1C, CH2), 20.64 (1C, CH2CH3), 14.41

(1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 2712 [M] , 720 [C60] ; MWcalc.: 2713.23 g/mol.

IR (ATR): ν˜ = 3320, 2981, 2943, 1722, 1668, 1529, 1460, 1421, 1367, 1313, 1267, 1151, 1105, 958, 896, 850, 734 cm−1.

UV/Vis (CH2Cl2): λmax = 258, 325, 425, 495 nm.

Deprotected asymmetric dendritic monoadduct [DHA-G2] (56)

Compound 56 was syn- HO O O OH thesized according to HO O general procedure GP HN OH 5 from monoadduct 55 O O O H O N H O N O O OH (250 mg, 0.09 mmol) in O O O O formic acid (15 mL). The O NH O OH O crude product was pu- HO OH O O riﬁed by reprecipitation OH 56 from MeOH/Et2O to give 56 as a red brownish solid.

Yield: 185 mg, 0.08 mmol, 91 %, red brownish solid.

1 H-NMR (400 MHz, RT, DMSO-d6): δ = 11.99 (s, br, COOH), 7.97 (s, br, 3H, CONH),

6.84 (s, br, 1H, CONH), 5.44 (m, br, 12H, CH), 4.43 (m, br, 2H, CH2O), 4.33 (m, br, 4H,

OCH2), 2.81 (m, br, 10H, CH2), 2.22 (m, br, 4H, CH2), 2.09 (m, br, 24H, CH2COOH),

1.86 (m, br, 24H, NHC(CH2)3), 1.55 (m, br, 4H, CH2), 1.29 (m, br, 2H,CH2), 0.83 (m, br, 3H, CH3) ppm.

193 Chapter 5 Experimental Part

13C-NMR: the spectrum was of low quality, due to the broadening of the signals through aggregation. Nevertheless no signals for the tert-butyl groups were observed.

+ + MS (FAB, NBA): m/z = 2208 [M+H] , 720 [C60] ; MWcalc.: 2208.28 g/mol.

UV/Vis (H2O pH = 7.2): λmax = 257, 325 nm.

Deprotected asymmetric dendritic malonate [C18-G1] (57)

Compound 57 was OH synthesized accord- O O H O N O O OH ing to general pro- O O 57 cedure GP 5 from O OH malonate 32 (1.37 g,

1.58 mmol) in formic acid (25 mL). The product was repeated evaporated with CHCl3 as entraining solvent under reduced pressure to give 57 as white solid.

Yield: 1.09 g, 1.58 mmol, 99 %, white solid.

1 H-NMR (300 MHz, RT, THF-d8): δ = 10.33 (s, br, 3H, COOH), 6.21 (s, br, 1H, 3 3 CONH), 4.17 (t, J = 6.6 Hz, 2H, OCH2), 4.14 (t, J = 6.8 Hz, 2H, OCH2), 3.32 (s, 2H, 3 3 OCCH2CO), 2.22 (t, J = 7.7 Hz, 6H, CH2COOH), 2.11 (t, J = 7.4 Hz, 2H, OCCH2), 3 1.98 (t, J = 7.8 Hz, 6H, NHC(CH2)3), 1.67 (m, 6H, CH2), 1.25 (m, 32H, CH2), 0.91 (t, 3 J = 6.7 Hz, 3H, CH3) ppm.

13 C-NMR (75 MHz, RT, THF-d8): δ = 174.88 (3C, COOH), 172.12 (1C, CONH), 166.87

(1C, CO), 166.79 (1C, CO), 65.66 (1C, OCH2), 65.31 (1C, OCH2), 57.19 (1C,

NHC(CH2)3), 41.44 (1C, OCCH2CO), 37.19 (1C, CH2CO), 31.86 (1C, CH2), 29.89 (3C,

NHC(CH2)3), 29.68, 29.64, 29.61, 29.49, 29.48, 29.37, 29.19 (10C, CH2), 28.71 (3C,

CH2COOH), 28.39, 28.18, 25.71 (5C, CH2), 25.48 (1C, CH2CH2CO), 25.18, 22.66 (2C,

CH2), 14.12 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 699 [M] ; MWcalc.: 699.91 g/mol.

IR (ATR): ν˜ = 3402, 2963, 2745, 2612, 2545, 1871, 1648, 1507, 1482, 1467, 1457, 1432, 1324, 1266, 1117, 950, 791, 686, 624 cm−1.

194 Chapter 5 Experimental Part

EA: C37H65NO11 ·C2HF3O2: calcd. C 57.55, H 8.17, F 7.00, N 1.72, O 25.55; found: C 57.99, H 8.52, N 2.01.

Asymmetric dendritic malonate [C18-G1-(Br)3] (58)

O O O H O N O O O Br O 58 O O O

Compound 58 was synthesized according to general procedure GP 2 from malonate 57 (1.09 g, 1.56 mmol), 2-bromoethanol (0.79 g, 6.32 mmol), DMAP (193 mg, 1.58 mmol), 1-HOBt (747 mg, 5.53 mmol) and DCC (1.14 g, 5.53 mmol) in dry THF (150 mL). The crude product was puriﬁed by column chromatography (SiO2; dichloromethane/ethyl acetate, 15:1 to 5:1) to give 58 as pale yellow oil.

Yield: 1.22 g, 1.20 mmol, 76 %, pale yellow oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.68 (s, br, 1H, CONH), 4.39 (t, J = 6.2 Hz, 6H, 3 3 CH2CH2Br), 4.15 (t, J = 6.7 Hz, 2H, OCH2), 4.13 (t, J = 6.7 Hz, 2H, OCH2), 3.52 (t, 3 3 J = 6.1 Hz, 6H, CH2Br), 3.37 (s, 2H, OCCH2CO), 2.36 (t, J = 7.8 Hz, 6H, CH2COO), 3 3 2.14 (t, J = 7.6 Hz, 2H, OCCH2), 2.06 (t, J = 7.2 Hz, 6H, NHC(CH2)3), 1.64 (m, 6H, 3 CH2), 1.25 (m, 32H, CH2), 0.88 (t, J = 6.9 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 172.73 (3C, COO), 172.35 (1C, CONH),

166.69 (1C, CO), 166.62 (1C, CO), 65.69 (1C, OCH2), 65.21 (1C, OCH2), 64.01 (3C,

CH2CH2Br), 57.24 (1C, NHC(CH2)3), 41.54 (1C, OCCH2CO), 37.11 (1C, CH2CO),

31.86 (1C, CH2), 29.81 (7C, NHC(CH2)3, CH2), 29.64, 29.47, 29.34 (6C, CH2), 28.77

(3C, CH2COO), 28.58 (3C, CH2Br), 28.53, 28.29 (4C, CH2), 25.91 (1C, CH2CH2CO),

25.63, 25.29, 22.80 (3C, CH2), 14.22 (1C, CH3) ppm.

+ MS (FAB, NBA): m/z = 1020 [M] ; MWcalc.: 1020.76 g/mol.

195 Chapter 5 Experimental Part

IR (ATR): ν˜ = 3379, 2972, 1832, 1501, 1455, 1432, 1281, 1268, 1102, 950, 799, 791, 697, 686, 624 cm−1.

EA: C43H74Br3NO11: calcd. C 50.60, H 7.31, Br 23.48, N 1.37, O 17.24; found: C 59.87, H 7.48, N 1.52.

Asymmetric dendritic monoadduct [C18-G1-(Br)3] (59)

O O O H O N O O O Br O O O O

Compound 59 was synthesized according to general procedure GP 4 from malonate 58

(750 mg, 0.73 mmol), C60 (741 mg, 1.03 mmol), CBr4 (342 mg, 1.03 mmol) and DBU (153 µL, 1.03 mmol) in 300 mL toluene. The crude product was puriﬁed by column chromatography (SiO2; toluene/ethyl acetate, 80:5 to 80:25) to give 59 as red brownish solid.

Yield: 406 mg, 0.28 mmol, 32 %, red brownish solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 5.59 (s, br, 1H, CONH), 4.47 (t, J = 6.6 Hz, 4H, 3 3 OCH2), 4.36 (t, J = 6.1 Hz, 6H, CH2CH2Br), 3.49 (t, J = 6.1 Hz, 6H, CH2Br), 2.33 (t, 3 3 3 J = 7.6 Hz, 6H, CH2COO), 2.12 (t, J = 7.5 Hz, 2H, OCCH2), 2.03 (t, J = 7.3 Hz, 6H, 3 NHC(CH2)3), 1.83 (m, 4H, CH2), 1.67 (m, 2H, CH2), 1.25 (m, 30H, CH2), 0.85 (t, J =

7.0 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.08 (3C, COO), 172.48 (1C, CONH), 164.00 (1C, CO), 163.92 (1C, CO), 145.65, 145.64, 145.55, 145.48, 145.47, 145.42, 145.17, 144.99, 144.96, 144.93, 144.89, 144.16, 143.40, 143.38, 143.31, 143.28, 143.26,

2 3 142.49, 142.15, 141.25, 141.22, 139.24, 138.14 (58C, C60-sp ), 71.84 (2C, C60-sp ),

196 Chapter 5 Experimental Part

67.74 (1C, OCH2), 67.28 (1C, OCH2), 64.25 (3C, CH2CH2Br), 57.46 (1C, NHC(CH2)3),

52.61 (1C, OCCCO), 37.32 (1C, CH2CO), 32.08 (1C, CH2), 29.87 (5C, NHC(CH2)3,

CH2), 29.82, 29.78, 29.77, 29.52, 29.38 (7C, CH2), 28.85 (3C, CH2Br), 28.75 (2C,

CH2), 28.56 (3C, CH2COO), 28.47 (2C, CH2), 26.15 (1C, CH2CH2CO), 25.81, 25.27,

22.84, 21.59 (4C, CH2), 14.28 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 1739 [M] , 720 [C60] ; MWcalc.: 1739.39 g/mol.

IR (ATR): ν˜ = 3332, 3062, 2992, 1832, 1766, 1703, 1654, 1603, 1533, 1478, 1403, 1281, 1235, 1107, 1033, 799, 787, 653 cm−1.

UV/Vis (CH2Cl2): λmax = 325.5, 425.5, 492 nm.

+ − Cationic asymmetric dendritic monoadduct [C18-G1-(C5H5N Br )3] (60)

3 Br N

O O O H O N O O O N O O O O

A solution of 38 (150 mg, 0.086 mmol) in 10 mL of dry pyridine was stirred for two days at 60 °C. After the addition of 10 mL of toluene, the reaction m ixture was ﬁltrated and the residue was suspended in toluene and distilled under vacuum for several times to remove traces of pyridine. Reprecipitation from methanol/diethyl ether gave 60 as red brownish solid.

Yield: 156 mg, 0.079 mmol, 92 %, red brownish solid.

1 3 3 H-NMR (400 MHz, RT, DMSO-d6): δ = 9.13 (d, J = 5.6 Hz, 6H, o-PyrH), 8.65 (t, J = 7.8 Hz, 3H, p-PyrH), 8.20 (dd, 3J = 6.0, 7.7 Hz, 6H, m-PyrH), 7.20 (s, br, 1H, CONH),

4.91 (m, 6H, CH2-Pyr), 4.51 (m, 10H, CH2CH2-Pyr, OCH2), 2.13 (m, 6H, CH2COO),

197 Chapter 5 Experimental Part

2.05 (m, 2H, OCCH2), 1.73 (m, 6H, NHC(CH2)3), 1.48 (m, 2H, CH2), 1.35 (m, 4H, 3 CH2), 1.17 (m, 32H, CH2), 0.84 (t, J = 6.2 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, DMSO-d6): δ = 172.59 (3C, COO), 172.56 (1C, CONH), 162.92 (1C, CO), 162.90 (1C, CO), 146.33 (3C, p-PyrC), 145.56 (6C, o-PyrC), 145.51, 145.16, 144.95, 144.94, 144.89, 144.86, 144.80, 144.73, 144.52, 144.36, 144.34, 144.30, 144.25, 143.54, 143.51, 142.81, 142.74, 142.73, 141.85, 141.84, 141.53,

2 141.46, 140.67, 138.74, 138.22 (58C, C60-sp ), 128.21 (6C, m-PyrC), 71.55 (2C, C60- 3 sp ), 67.37 (2C, OCH2), 62.56 (3C, CH2CH2-Pyr), 59.76 (3C, CH2-Pyr), 56.29 (1C,

NHC(CH2)3), 52.70 (1C, OCCCO), 35.84 (1C, CH2CO), 31.42 (6C, NHC(CH2)3, CH2),

29.18, 29.11, 29.01 (4C, CH2), 28.84 (3C, CH2COO), 28.65, 28.57, 28.13, 27.80, 27.67

(7C, CH2), 25.69 (1C, CH2CH2CO), 25.08, 24.97, 22.22 (4C, CH2), 14.06 (1C, CH3) ppm.

+ 2+ MS (FAB, NBA): m/z = 1897 [M] , 908 [M] ; MWcalc.: 1976.69 g/mol.

IR (ATR): ν˜ = 3368, 3020, 3001, 2651, 1799, 1765, 1654, 1613, 1546, 1434, 1406, 1256, 1249, 1237, 1111, 1023, 807, 753, 653, 603 cm−1.

UV/Vis (DMSO/H2O): λmax = 259, 325.5 nm.

3,5-Bis(propargyloxy)benzyl malonate (78)

Compound 78 was synthesized according to gen-

O O eral procedure GP 1 from 3,5-bis(propargyloxy)- O O O O benzyl alcohol 130, 131 (3.5 g, 16.19 mmol), 78 O O malonyl dichloride (787 µL, 8.09 mmol) and dry pyridine (1.31 mL, 16.19 mmol) in 100 mL dry

CH2Cl2. The crude product was puriﬁed by recrystallization from dichloromethane/hexane to give 78 as white solid.

Yield: 2.79 g, 5.58 mmol, 69 %, white solid.

1 4 H-NMR (300 MHz, RT, CDCl3): δ = 6.57 (m, 6H, ArH), 5.11 (4H, Ar-CH2), 4.63 (d, J 4 = 2.4 Hz, 8H, OCH2), 3.48 (s, 2H, OCCH2CO), 2.51 (t, J = 2.5 Hz, 4H, CH) ppm.

198 Chapter 5 Experimental Part

13 C-NMR (75 MHz, RT, CDCl3): δ = 166.27 (2C, CO), 159.01 (4C, ArC-O), 137.85 (2C,

ArC-CH2), 107.71 (4C, ArC), 102.37 (2C, ArC), 78.45 (4C, CCH), 76.00 (4C, CCH),

67.06 (2C, Ar-CH2), 56.16 (4C, OCH2), 41.66 (1C, OCCH2CO) ppm.

+ MS (FAB, NBA): m/z = 501 [M] ; MWcalc.: 500.50 g/mol.

IR (ATR): ν˜ = 2978, 1768, 1720, 1656, 1624, 1576, 1544, 1448, 1368, 1241, 1192, 1160, 1016, 888, 839, 776, 744, 681 cm−1.

EA: C29H24O8: calcd. C 69.59, H 4.83, O 25.57; found C 69.12, H 4.78.

1,2-((3,5-Bis(propargyloxy)benzyloxycarbonyl)-methano)-1,2-dihydro- [60]fullerene (79)

Compound 79 was synthesized according to general procedure GP 4 from malonate 78 (500 mg, O O O O O O 1.00 mmol), C60 (864 mg, 1.20 mmol), CBr4 (398

O O mg, 1.20 mmol) and DBU (179 µL, 1.20 mmol) in 400 mL toluene. The crude product was puriﬁed by

ﬂash column chromatography (SiO2; toluene/ethyl 79 acetate, 100:3) to give 79 as red brownish solid.

Yield: 475 mg, 0.39 mmol, 39 %, red brownish solid.

1 4 4 H-NMR (400 MHz, RT, CDCl3): δ = 6.67 (d, J = 2.3 Hz, 4H, ArH), 6.58 (t, J = 2.3 4 4 Hz, 2H, ArH), 5.42 (4H, Ar-CH2), 4.63 (d, J = 2.4 Hz, 8H, OCH2), 2.52 (t, (t, J = 2.4 Hz, 4H, CH) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 163.58 (2C, CO), 159.17 (4C, ArC-O), 145.54, 145.46, 145.36, 145.20, 145.15, 144.97, 144.88, 144.87, 144.16, 143.34, 143.30,

2 143.27, 142.48, 142.13, 141.22, 139.33 (58C, C60-sp ), 137.21 (2C, ArC-CH2), 108.37 3 (4C, ArC), 102.71 (2C, ArC), 78.41 (4C, CCH), 76.19 (4C, CCH), 71.53 (2C, C60-sp ),

68.73 (2C, Ar-CH2), 56.17 (4C, OCH2), 51.82 (1C, OCCCO) ppm.

+ + MS (FAB, NBA): m/z = 1219 [M] , 720 [C60] ; MWcalc.: 1219.12 g/mol.

IR (ATR): ν˜ = 3290, 1738, 1599, 1452, 1367, 1267, 1228, 1151, 1058, 996, 942, 826,

199 Chapter 5 Experimental Part

742, 672 cm−1.

UV/Vis (CH2Cl2): λmax = 233, 259, 326, 425, 495 nm.

Bis(3,5-bis((1-(2-(tert-butoxycarbonylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy) benzyl) malonate (83)

Malonate 78 (326 mg, 0.72 R N N O O N N R N O O N mmol) and azide 81 (536 mg, O O 83 2.88 mmol) were dissolved in 2 O O N N mL tert-butanol/H O =1/1 under N N N 2 N R R careful heat up. Subsequently, R = NHboc sodium ascorbate (24 mg, 0.12 mmol) and CuSO4 (18 mg, 0.07 mmol) were added and the reaction mixture was stirred at rt. After 45 min, TLC control showed almost complete conversion of the starting material. The light yellow mixture was stirred for additional 2 h, diluted with 2 mL ammonium hydroxide/citrate buffer (15 %) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (3 x 25 mL). The organic phase was washed with saturated NaCl (2 x 25 mL), dried over MgSO4 and evaporated under reduced pressure. The crude product was puriﬁed by recrystallization from dichloromethane/pentane to give 83 as white solid.

Yield: 825 mg, 0.66 mmol, 92 %, white solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.64 (s, 4H, triazoleCH), 6.51 (m, 6H, ArH), 5.34 3 (s, br, 4H, NH), 5.07 (s, 4H, OCH2), 5.04 (s, 8H, OCH2), 4.45 (t, J = 5.6 Hz, 8H,

NCH2), 3.60 (m, 8H, HNCH2), 3.49 (s, 2H, OCCH2CO), 1.38 (s, 36H, C(CH3)3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 166.46 (2C, CO), 159.74 (4C, (4C, ArC-O)), t 156.25 (4C, COO Bu), 143.86 (2C, ArC-CH2), 137.95 (4C, triazoleC), 124.08 (4C, tri- azoleC), 107.26 (4C, ArC), 102.03 (2C, ArC), 80.07 (4C, C(CH3)3), 67.02 (2C, OCH2),

62.01 (4C, OCH2), 50.22 (4C, NCH2), 41.83 (1C, OCCH2CO), 40.64 (4C, HNCH2),

28.47 (12C, C(CH3)3) ppm.

+ MS (FAB, NBA): m/z = 1245 [M+H] ; MWcalc.: 1244.59 g/mol.

200 Chapter 5 Experimental Part

IR (ATR): ν˜ = 3301, 3140, 2978, 2936, 1688, 1640, 1576, 1528, 1448, 1384, 1288, 1240, 1144, 1048, 984, 840, 761, 710, 664 cm−1.

UV/Vis (CH2Cl2): λmax = 239, 280 nm.

EA: C57H80N16O16 · 3 CH2Cl2: calcd. C 48.04, H 5.78, N 14.94, O 17.06 ; found C 47.65, H 6.01, N 17.17.

Bis(3,5-bis((1-((2-methoxyethoxy)methyl)-1H-1,2,3-triazol-4-yl)methoxy)benzyl) malonate (84)

Compound 84 was synthesized R N N O O N N R N O O N following the procedure described O O 84 for 83 from malonate 78 (400 O O N N mg, 0.80 mmol), azide 82 (464 N N N N R R mg, 3.20 mmol), sodium ascor- R = O(CH2)2OCH3 bate (28 mg, 0.14 mmol) and

CuSO4 (20 mg, 0.09 mmol) in 2.5 mL tert-butanol/H2O =1/1. The crude product was puriﬁed by column chromatography (SiO2; dichloromethane/methanol, 100:8) to give 84 as colorless oil.

Yield: 730 mg, 0.71 mmol, 89 %, colorless oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.73 (s, 4H, triazoleCH), 6.49 (m, 6H, ArH), 5.03 3 (s, 8H, OCH2), 5.01 (s, 4H, OCH2), 4.44 (t, J = 5.0 Hz, 8H, NCH2), 3.76 (m, 8H,

NCH2CH2), 3.47 (m, 8H, OCH2), 3.42 (s, 2H, OCCH2CO), 3.38 (m, 8H, OCH2), 3.22

(s, 12H, OCH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 166.10 (2C, CO), 159.52 (4C, ArC-O), 143.40

(2C, ArC-CH2), 137.63 (4C, triazoleC), 124.08 (4C, triazoleC), 106.97 (4C, ArC), 101.74

(2C, ArC), 71.62 (4C, OCH2), 70.38 (4C, OCH2), 69.29 (4C, OCH2), 66.77 (2C, OCH2),

61.93 (4C, OCH2), 58.86 (4C, OCH3), 50.17 (4C, NCH2), 41.35 (1C, OCCH2CO) ppm.

+ MS (FAB, NBA): m/z = 1081 [M+H] ; MWcalc.: 1081.14 g/mol.

IR (ATR): ν˜ = 2996, 2901, 2861, 1592, 1496, 1448, 1352, 1272, 1192, 1144, 1112,

201 Chapter 5 Experimental Part

1064, 984, 904, 856, 808, 776, 711, 668 cm−1.

UV/Vis (CH2Cl2): λmax = 239.5, 281 nm.

EA: C49H68N12O16 · 1.5 CH3OH: calcd. C 53.71, H 6.61, N 14.88, O 24.80 ; found C 53.24, H 6.63, N 15.07.

1,2-(Bis(3,5-bis((1-(2-(tert-butoxycarbonylamino)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)benzyloxycarbonyl)-methano)-1,2-dihydro[60]fullerene (85)

Compound 85 was synthesized R N N O O N N R N O O N according to general procedure O O GP 4 from malonate 83 (200 mg, O O N N N 0.16 mmol), C60 (1500 mg, 0.21 N N N R R mmol), CBr4 (59 mg, 0.18 mmol)

85 and DBU (27 µL, 0.18 mmol) in 100 mL toluene/DMSO = 3:1. R = NHboc The crude product was puriﬁed by ﬂash column chromatography (SiO2; toluene, ethyl acetate/ethanol, 80:15) to give 85 as red brownish solid.

Yield: 98 mg, 0.05 mmol, 29 %, red brownish solid.

1 H-NMR (300 MHz, RT, CDCl3): δ = 7.70 (s, 4H, triazoleCH), 6.57 (m, 6H, ArH), 5.55

(s, br, 4H, NH), 5.28 (s, 4H, OCH2), 5.04 (s, 8H, OCH2), 4.45 (m, 8H, NCH2), 3.60 (m,

8H, HNCH2), 1.39 (s, 36H, C(CH3)3) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 163.45 (2C, CO), 159.71 (4C, ArC-O), 156.21 (4C, COOt Bu), 145.47, 145.39, 145.28, 145.10, 145.06, 144.88, 144.82, 144.76,

144.05, 143.57, 143.27, 143.24, 143.18, 142.38, 142.05, 141.17, 139.28 (60C, C60- 2 sp , ArC-CH2), 137.05 (4C, triazoleC), 124.17 (4C, triazoleC), 107.69 (4C, ArC), 102.23 2 (2C, ArC), 80.02 (4C, C(CH3)3), 71.52 (2C, OCH2), 68.72 (2C, C60-sp ), 62.02 (4C,

OCH2), 53.66 (1C, OCCCO), 50.33 (4C, NCH2), 40.74 (4C, HNCH2), 28.57 (12C,

C(CH3)3) ppm.

+ + MS (FAB, NBA): m/z = 1964 [M+H] , 720 [C60] ; MWcalc.: 1963.97 g/mol.

202 Chapter 5 Experimental Part

IR (ATR): ν˜ = 3268, 3103, 2964, 2902, 1708, 1638, 1586, 1503, 1438, 1401, 1298, 1243, 1163, 1054, 991, 882, 763, 681 cm−1.

UV/Vis (CH2Cl2): λmax = 232, 257, 324.5, 425.5, 483 nm.

1,2-(Bis(3,5-bis((1-((2-methoxyethoxy)methyl)-1H-1,2,3-triazol-4-yl)methoxy) benzyloxycarbonyl)-methano)-1,2-dihydro[60]fullerene (86)

Compound 86 was synthesized R N N O O N N R N O O N according to general procedure O O GP 4 from malonate 84 (200 mg, O O N N N 0.19 mmol), C (200 mg, 0.28 N N 60 N R R mmol), CBr4 (74 mg, 0.22 mmol)

86 and DBU (33 µL, 0.22 mmol) in 100 mL toluene/DMSO = 3:1. R = O(CH2)2OCH3 The crude product was puriﬁed by ﬂash column chromatography (SiO2; toluene, dichloromethane/methanol, 100:6) to give 86 as red brownish solid.

Yield: 109 mg, 0.06 mmol, 32 %, red brownish solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 7.77 (s, 4H, triazoleCH), 6.60 (m, 6H, ArH), 5.11 3 3 (s, 8H, OCH2), 5.08 (s, 4H, OCH2), 4.50 (t, J = 5.0 Hz, 8H, NCH2), 3.82 (t, J = 4.6

Hz, 8H, NCH2CH2), 3.53 (m, 8H, OCH2), 3.45 (m, 8H, OCH2), 3.29 (s, 12H, OCH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 163.36 (2C, CO), 159.81, 159.69 (4C, ArC- O), 145.27, 145.21, 145.01, 144.76, 144.64, 144.12, 143.89, 143.56, 143.08, 142.28,

2 141.94, 141.00, 139.14 (60C, C60-sp , ArC-CH2), 137.10 (4C, triazoleC), 124.21 (4C, triazoleC), 107.88 (4C, ArC), 102.27 (2C, ArC), 71.84 (4C, OCH2), 70.62 (4C, OCH2), 2 69.53 (4C, OCH2), 68.77 (2C, C60-sp ), 65.00 (2C, OCH2), 62.15 (4C, OCH2), 59.13

(4C, OCH3), 52.23 (1C, OCCCO), 50.40 (4C, NCH2) ppm.

+ MS (FAB, NBA): m/z = 1081 [M+H] ; MWcalc.: 1081.14 g/mol.

IR (ATR): ν˜ = 3001, 2913, 2901, 2855, 1601, 1503, 1441, 1415, 1353, 1199, 1151,

203 Chapter 5 Experimental Part

1107, 1071, 1001, 9834, 904, 858, 773, 709, 671 cm−1.

UV/Vis (CH2Cl2): λmax = 232.5, 258, 325, 425, 486 nm.

Tetraammoniumtriﬂuoroacetate triazolfullerene (87)

Compound 87 was synthesized R N N O O N N R N O O N according to general procedure O O GP 5 from momoadduct 85 (60 O O N N N mg, 0.03 mmol) in formic acid N N N R R (15 mL). The crude product was

87 puriﬁed by reprecipitation from

MeOH/Et2O to give 87 as a red R = NH CF COO 3 3 brownish solid.

Yield: 36 mg, 0.03 mmol, 91 %, red brownish solid.

1 H-NMR (300 MHz, RT, MeOH-d4): δ = 8.10 (s, 4H, triazoleCH), 6.67 (m, 6H, ArH), + 5.04 (m, 12H, OCH2), 4.71 (m, 8H, NCH2), 3.49 (m, 8H, NH3NCH2) ppm.

13 C-NMR (75 MHz, RT, MeOH-d4): δ = 161.20 (2C, CO), 158.69 (4C, (4C, ArC-O)), 146.55, 146.53, 146.49, 146.36, 145.80, 145.40, 145.26, 145.14, 144.31, 143.51,

143.17, 142.22, 142.18, 141.47, 141.44, 140.33, 139.17, 139.09, 138.19 (60C, C60- 2 sp , ArC-CH2), 138.02 (4C, triazoleC), 126.33 (4C, triazoleC), 106.36 (4C, ArC), 101.89 3 (2C, ArC), 71.41 (2C, OCH2), 69.97 (2C, C60-sp ), 62.31 (4C, OCH2), 53.04 (1C,

OCCCO), 49.15 (4C, NCH2), 40.39 (4C, HNCH2) ppm.

+ + MS (FAB, NBA): m/z = 1564 [M] , 720 [C60] .

IR (ATR): ν˜ = 3314, 3098, 3091, 2971, 2914, 1715, 1651, 1642, 1579, 1511, 1439, 1402, 1301, 1263, 1157, 1101, 993, 969, 878, 755, 689 cm−1.

UV/Vis (DMSO): λmax = 263.5, 325.5, 428 nm.

204 Chapter 5 Experimental Part

6-tert-Butoxy-6-oxohexyl methyl malonate (92)

Compound 92 was synthesized according to general O O O procedure GP 1 from 22 (2 g, 10.62 mmol), methyl mal- O O O 92 onyl chloride (1.14 mL, 10.62 mmol) and dry pyridine

(857 µL, 10.62 mmol) in 150 mL dry CH2Cl2. The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 100:30) to give 92 as colorless oil.

Yield: 2.35 g, 8.67 mmol, 82 %, colorless oil

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 4.12 (t, J = 6.6 Hz, 2H, OCH2), 3.72 (s, 3H, 3 OCH3), 3.35 (s, 2H, OCCH2CO), 2.19 (t, J = 7.4 Hz, 2H, CH2CO), 1.61 (m, 4H, CH2),

1.41 (s, 9H, C(CH3)3), 1.35 (m, 2H, CH2) ppm.

13 t C-NMR (75 MHz, RT, CDCl3): δ = 173.09 (1C, COO Bu), 167.21 (1C, CO), 166.73

(1C, CO), 80.31 (1C, C(CH3)3), 65.63 (1C, OCH2), 52.70 (1C, OCH3), 41.57 (1C,

OCCH2CO), 35.55 (1C, CH2CO), 28.37 (1C, CH2), 28.31 (3C, C(CH3)3), 25.51, 24.84

(2C, CH2) ppm.

+ t + MS (FAB, NBA): m/z = 288 [M] , 231 [M- Bu] ; MWcalc.: 288.34 g/mol.

IR (ATR): ν˜ = 3001, 2984, 1736, 1641, 1448, 1368, 1256, 1144, 1016, 936, 856, 776, 728, 696 cm−1.

EA: C14H24O6: calcd. C 58.32, H 8.39, O 33.29; found: C 57.66, H 8.31.

205 Chapter 5 Experimental Part

1,2-(Bis(6-tert-butoxy-6-oxohexyl-methyloxy)-methano)-1,2-dihydro [60]fullerene (93)

Compound 93 was synthesized according to general O O O procedure GP 4 from malonate 92 (395 mg, 1.37 O O O mmol), C60 (1.48 g, 2.06 mmol), CBr4 (500 mg, 1.51 mmol) and DBU (225 µL, 1.51 mmol) in 750 mL toluene. The crude product was puriﬁed by column chromatog-

93 raphy (SiO2; toluene/ethyl acetate, 100:5) to give 93 as a red brownish solid.

Yield: 736 mg, 0.73 mmol, 43 %, red brownish solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 4.48 (t, J = 6.5 Hz, 2H, OCH2), 4.07 (s, 3H, 3 OCH3), 2.22 (t, J = 7.4 Hz, 2H, CH2CO), 1.85 (m, 2H, CH2), 1.66 (m, 2H, CH2), 1.48

(m, 2H, CH2), 1.43 (s, 9H, C(CH3)3) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.14 (1C, COO Bu), 163.44 (1C, CO), 163.91 (1C, CO), 145.57, 145.49, 145.47, 145.41, 145.20, 145.00, 144.94, 144.19, 143.38,

143.32, 143.28, 142.50, 142.23, 142.19, 141.27, 141.26, 139.41, 139.22 (58C, C60- 2 3 sp ), 80.43 (1C, C(CH3)3), 71.72 (2C, C60-sp ), 67.41 (1C, OCH2), 54.21 (1C, OCH3),

52.26 (1C, OCCCO), 35.54 (1C, CH2CO), 28.46 (1C, CH2), 28.30 (3C, C(CH3)3),

25.61, 24.75 (2C, CH2) ppm.

+ t + + MS (FAB, NBA): m/z = 1006 [M] , 951 [M- Bu] , 720 [C60] ; MWcalc.: 1000.96 g/mol.

IR (ATR): ν˜ = 2943, 2866, 2016, 1977, 1730, 1537, 1429, 1367, 1236, 1151, 1097, 1058, 1004, 942, 896, 842, 734 cm−1.

UV/Vis (CH2Cl2): λmax = 259, 326, 426, 493 nm.

206 Chapter 5 Experimental Part

1,2-(Bis(6-(3-methoxy-3-oxopropanoyloxy)hexanoic acid)-methano)-1,2-dihydro [60]fullerene (94)

Compound 94 was synthesized according to general pro- O O OH cedure GP 5 from 93 (285 mg, 0.28 mmol) and triﬂuo- O O O roacetic acid (2.4 mL, 31.0 mmol) in 50 mL CH2Cl2.

Yield: 257 mg, 0.27 mmol, 96 %, red brownish solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 4.49 (t, J = 6.4 Hz, 2H, OCH2), 4.07 (s, 3H, 3 OCH3), 2.38 (t, J = 7.3 Hz, 2H, CH2CO), 1.86 (m, 2H, CH2), 1.71 (m, 2H, CH2), 1.52

(m, 2H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 179.02 (1C, COOH), 164.44 (1C, CO), 163.92 (1C, CO), 145.58, 145.50, 145.49, 145.46, 145.41, 145.39, 145.21, 145.00, 144.95, 144.94, 144.92, 144.19, 143.39, 143.33, 143.31, 143.28, 142.50, 142.22, 142.17,

2 3 141.28, 141.26, 139.44, 139.17 (58C, C60-sp ), 71.69 (2C, C60-sp ), 67.30 (1C, OCH2),

54.23 (1C, OCH3), 52.24 (1C, OCCCO), 33.85 (1C, CH2CO), 28.42 (1C, CH2), 25.64,

24.35 (2C, CH2) ppm.

+ + MS (FAB, NBA): m/z = 951 [M+H] , 720 [C60] ; MWcalc.: 950.86 g/mol.

IR (ATR): ν˜ = 2991, 2850, 2802, 2645, 2364, 1746, 1699, 1429, 1236, 1182, 1097, 1058, 996, 935, 796, 742, 703 cm−1.

UV/Vis (CH2Cl2): λmax = 259, 325.5, 425.5, 489 nm.

207 Chapter 5 Experimental Part

2-Hydroxyethyl 4-(pyren-1-yl)butanoate (97)

Compound 97 was synthesized according to general procedure GP 3 from ethane-1,2-diol (1.72 g, 27.75 mmol), 1-

O pyrenebutyric acid (2g, 6.94 mmol), EDC (2 g, 10.43 mmol), OH O DMAP (847 mg, 6.94 mmol) and 1-HOBt (1.41 g, 10.40 97 mmol) in dry THF/ CH2Cl2 = 2/ 1. The crude product was puriﬁed by column chromatography (SiO2; dichloromethane/acetone, 10:1) to give 97 as yellow oil.

Yield: 1.81 g, 5.45 mmol, 78 %, yellow oil.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 8.08 (m, 9H, ArH), 4.23 (t, J = 4.7 Hz, 2H, 3 3 OCOCH2), 3.82 (t, J = 4.7 Hz, 2H, CH2OH), 3.40 (t, J = 7.7 Hz, 2H, ArCH2), 2.50 (t, 3 J = 7.4 Hz, 2H, CH2CO), 2.22 (m, 2H, CH2), 1.26 (s, br, 1H, OH) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.78 (1C, CO), 135.50, 131.36, 130.82, 129.94, 128.70, 127.385, 126.70, 125.82, 125.04, 124.91, 124.75, 123.20 (16C, ArC),

65.98 (1C, CH2OH), 61.18 (1C, OCOCH2), 33.63 (1C, ArCH2), 32.62 (1C, CH2CO),

26.65 (1C, CH2) ppm.

+ MS (FAB, NBA): m/z = 332 [M] ; MWcalc.: 332.39 g/mol.

IR (ATR): ν˜ = 3461, 2984, 2901, 1736, 1654, 1448, 1368, 1272, 1224, 1160, 1064, 1016, 936, 872, 712, 648 cm−1.

UV/Vis (CH2Cl2): λmax = 235.5, 244.5, 266, 278, 300 (sh), 314, 328, 344 nm.

208 Chapter 5 Experimental Part

Bis(2-(4-(pyren-1-yl)butanoyloxy)ethyl)malonate (98)

Compound 98 was synthesized ac- O O O O cording to general procedure GP 1 O O O O 98 from 97 (750 mg, 2.26 mmol), malonyl dichloride (110 µL, 1.13 mmol) and dry pyridine (182 µL, 2.26 mmol) in dry CH2Cl2. The crude product was puriﬁed by column chromatography (SiO2; dichloromethane/acetone, 96:4) to give 98 as yellow oil.

Yield: 553 mg, 0.75 mmol, 67 %, yellow oil.

1 H-NMR (400 MHz, RT, CDCl3): δ = 8.06 (m, 18H, ArH), 4.32 (m, 4H, OCOCH2), 4.27 3 (m, 4H, CH2OCO), 3.44 (s, 2H, OCCH2CO), 3.35 (t, J = 7.7 Hz, 4H, ArCH2), 2.46 (t, 3 J = 7.3 Hz, 4H, CH2CO), 2.20 (m, 4H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.06 (2C, CO), 166.01 (2C, CO), 135.45, 131.27, 130.75, 129.85, 128.59, 127.35, 127.28, 127.18, 126.62, 125.74, 124.93,

124.83, 124.68, 123.12 (32C, ArC), 63.02 (2C, OCOCH2), 61.53 (2C, CH2OCO), 40.86

(1C, OCCH2CO), 33.32 (2C, ArCH2), 32.38 (2C, CH2CO), 26.35 (2C, CH2) ppm.

+ MS (FAB, NBA): m/z = 733 [M] ; MWcalc.: 732.82 g/mol.

IR (ATR): ν˜ = 2927, 2858, 2116, 1746, 1684, 1452, 1359, 1298, 1244, 1190, 1151, 1043, 950, 888, 842, 788, 718 cm−1.

UV/Vis (CH2Cl2): λmax = 234, 244, 266, 279, 301 (sh), 314, 327, 344.5 nm.

EA: C47H40O8: calcd. C 77.03, H 5.50, O 17.47; found C 76.77, H 5.63.

209 Chapter 5 Experimental Part

1,2-(Bis(2-(4-(pyren-1-yl)butanoyloxy)ethyloxy-carbonyl)-methano)-1,2-dihydro [60]fullerene (99)

Compound 99 was synthesized ac- O O O O cording to general procedure GP 4 O O O O from malonate 98 (475 mg, 0.65

mmol), C60 (700 mg, 0.97 mmol),

CBr4 (258 mg, 0.78 mmol) and DBU

99 (117 µL, 0.78 mmol) in 350 mL toluene. The crude product was purified by flash column chromatography (SiO2; toluene/ethyl acetate, 10:1 to 8:1) to give 99 as red brownish solid. To prevent any impurities that can distort the STM measurements (e.g. remaining silica in the probe), the product was additionally purified by preparative HPLC (Nucleosil, 5µm, dichloromethane/methanol = 98/2).

Yield: 452 mg, 0.31 mmol, 48 %, red brownish solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 8.02 (m, 18H, ArH), 4.64 (m, 4H, OCOCH2), 4.40 3 3 (m, 4H, CH2OCO), 3.35 (t, J = 7.7 Hz, 4H, ArCH2), 2.43 (t, J = 7.5 Hz, 4H, CH2CO),

2.16 (m, 4H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.15 (2C, CO), 163.43 (2C, CO), 145.01, 144.78, 144.66, 144.50, 144.36, 144.21, 143.59, 142.81, 142.68, 142.54, 141.89,

2 141.52, 140.60, 138.76 (58C, C60-sp ), 135.41, 131.49, 130.95, 130.09, 128.80, 127.56, 127.39, 126.86, 125.95, 125.11, 125.05, 124.90, 124.88, 123.25 (32C, ArC), 70.95

3 (2C, C60-sp ), 64.78 (2C, OCOCH2), 61.68 (2C, CH2OCO), 51.57 (1C, OCCCO), 33.65

(2C, ArCH2), 32.63 (2C, CH2CO), 26.48 (2C, CH2) ppm.

+ + MS (FAB, NBA): m/z = 1451 [M] , 720 [C60] ; MWcalc.: 1451.44 g/mol.

IR (ATR): ν˜ = 2934, 2901, 2861, 2109, 1746, 1708, 1693, 1453, 1362, 1297, 1244, 1203, 1188, 1103, 1044, 948, 843, 777, 716, 681 cm−1.

UV/Vis (CH2Cl2): λmax = 236, 245, 257.5, 266, 278, 315 (sh), 328.5, 345, 425, 495 nm.

The purity (99 %) was determined by HPLC (on Nucleosil MN, 5 µm,

210 Chapter 5 Experimental Part dichloromethane/methanol = 99/1).

Bis-(5-(tert-butoxycarbonyl)pentyl) malonate (100)

Compound 100 was synthesized accord- O O O O ing to general procedure GP 1 from 22 (7 O O O O 100 g, 37 mmol), malonyl dichloride (1.81 mL, 18.62 mmol) and dry pyridine (2.99 mL, 37 mmol) in 250 mL dry CH2Cl2. The crude product was puriﬁed by column chromatography (SiO2; hexane/ethyl acetate, 100:20) to give 100 as white solid.

Yield: 5.5 g, 12.48 mmol, 67 %, white solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 4.11 (t, J = 6.7 Hz, 4H, OCH2), 3.33 (s, 2H, 3 OCCH2CO), 2.19 (t, J = 7.5 Hz, 4H, CH2CO), 1.62 (m, 8H, CH2), 1.41 (s, 18H,

C(CH3)3), 1.34 (m, 4H, CH2) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 173.23 (2C, COO Bu), 166.96 (2C, CO), 80.33

(1C, C(CH3)3), 65.57 (2C, OCH2), 41.73 (1C, OCCH2CO), 35.50 (2C, CH2CO), 28.33

(2C, CH2), 28.24 (6C, C(CH3)3), 25.45, 24.78 (4C, CH2) ppm.

+ t + t + MS (FAB, NBA): m/z = 445 [M] , 389 [M- Bu] , 333 [M-2 Bu] ; MWcalc.: 444.56 g/mol.

IR (ATR): ν˜ = 2943, 2873, 1730, 1460, 1421, 1367, 1329, 1251, 1151, 1043, 950, 850, 734, 687 cm−1.

EA: C23H40O8 · 1/2 C4H8O2: calcd. C 61.45, H 9.08, O 29.47; found C 60.99, H 9.03.

Bis-(5-(pentylcarbonyl)) malonate (101)

Compound 101 was synthesized according to O O HO OH general procedure GP 5 from 100 (2 g, 4.50 O O O O 101 mmol) in formic acid (20 mL).

Yield: 1.45 g, 4.36 mmol, 97 %, white solid.

1 3 H-NMR (400 MHz, RT, CDCl3): δ = 10.11 (s, br, 2H, COOH), 4.11 (t, J = 6.6 Hz, 4H, 3 OCH2), 3.34 (s, 2H, OCCH2CO), 2.33 (t, J = 7.4 Hz, 4H, CH2CO), 1.63 (m, 8H, CH2),

211 Chapter 5 Experimental Part

1.39 (m, 4H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 179.87 (2C, COOH), 166.85 (2C, CO), 65.45

(2C, OCH2), 41.73 (1C, OCCH2CO), 33.93 (2C, CH2CO), 28.26 (2C, CH2), 25.43,

24.34 (4C, CH2) ppm.

+ MS (FAB, NBA): m/z = 333 [M+H] ; MWcalc.: 332.15 g/mol.

IR (ATR): ν˜ = 3347, 3065, 2963, 1912, 1766, 1761, 1686, 1578, 1563, 1415, 1263, 1193, 1146, 933, 867, 713 cm−1.

5-(3-(6-Methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)pentyl octadecyl malonate (106)

Compound 27 (1.00 g,

O O O HN 2.13 mmol) was dis- O O N N N O H H solved in 100 mL dry 106 CH2Cl2, followed by the addition of oxalyl chloride (365 µL, 4.25 mmol). The resulting mixture was heated under reﬂux for 2 h. After cooling to rt the volatiles were removed in vacuo. The remaining acid chloride 103 (1.03 g, 2.11 mmol) was redissolved in 15 mL dry DMF and sodium azide (275 mg, 4.23 mmol) was added. The resulting mixture was stirred at rt until TLC control showed complete conversion of the starting material. Subse- quently, 2-amino-4-hydroxy-6-methylpyrimidine (625 mg, 4.99 mmol) and dry pyridine (15 mL, 0.19 mol) was added and the reaction mixture was heated to 70 - 80°C, until TLC control showed complete conversion of the starting material. After cooling to rt the mixture was partitioned between 250 mL CH2Cl2 and 250 mL H2O. The organic phase was washed with aqueous HCl (10 %, 2 x 200 mL) and H2O (2 x 200 mL), dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was puriﬁed by recrystallization from methanol to give 106 as white solid.

Yield: 960 mg, 1.62 mmol, 76 %, white solid.

1 H-NMR (400 MHz, RT, CDCl3): δ = 13.08 (1H, NH), 11.84 (1H, NH), 10.18 (1H, NH), 3 3 5.79 (1H, CH), 4.11 (t, J = 6.5 Hz, 2H, OCH2), 4.09 (t, J = 6.5 Hz, 2H, OCH2), 3.33

212 Chapter 5 Experimental Part

(s, 2H, OCCH2CO), 3.22 (m, 2H, CH2NH), 2.21 (s, 3H, CH3), 1.64 (m, 8H, CH2), 1.39 3 (m, 2H, CH2), 1.22 (m, 28H, CH2), 0.85 (t, J = 6.8 Hz, 3H, CH3) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 173.39 (1C, C-Pyrimidine), 167.03 (1C, CO), 167.02 (1C, CO), 156.89 (1C, CO-Pyrimidine), 154.99 (1C, HNCONH), 148.57 (1C,

C-Pyrimidine), 106.96 (1C, CH-Pyrimidine), 65.90 (1C, OCH2), 65.68 (1C, OCH2),

41.78 (1C, OCCH2CO), 39.89 (1C, CH2NH), 32.08, 29.85, 29.83, 29.81, 29.74, 29.67,

29.51, 29.37, 29.24, 28.61, 28.26, 25.94, 23.35, 22.83 (19C, CH2), 19.08 (1C, CH3-

Pyrimidine), 14.26 (1C, CH3) ppm.

+ + MS (FAB, NBA): m/z = 594 [M+H] , 1188 [2M+2H] ; MWcalc.: 592.81 g/mol.

IR (ATR): ν˜ = 2920, 2850, 1730, 1668, 1583, 1529, 1468, 1406, 1344, 1259, 1182, 1020, 935, 881, 796, 742, 687 cm−1.

EA: C32H56N4O6: calcd. C 64.83, H 9.52, N 9.45, O 16.19; found C 65.40, H 10.01,N 9.58. cyclo-[2]-Octylmalonyl)-1,2-dihydro[60]fullerene (119)

Compound 119 was synthesized according to O general procedure GP 4 from malonate 118 (1 g, O O O O O O 2.33 mmol), C60 (1.34 g, 1.87 mmol), CBr4 (773 O mg, 2.33 mmol) and DBU (348 µL, 2.33 mmol) in 119 750 mL toluene. The crude product was puriﬁed

by ﬂash column chromatography (SiO2; toluene; dichloromethane) to give 119 as red brownish solid.

Yield: 615 mg, 0.54 mmol, 23 %, red brownish solid.

1 3 3 H-NMR (400 MHz, RT, CDCl3): δ = 4.45 (t, J = 6.7 Hz, 4H, OCH2), 4.15 (t, J = 6.6

Hz, 4H, OCH2), 3.36 (s, 2H, OCCH2CO), 1.84 (m, 4H, CH2), 1.66 (m, 4H, CH2), 1.49

(m, 4H, CH2), 1.38 (m, 12H, CH2) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 166.82 (2C, CO), 163.95 (2C, CO), 145.55, 145.50, 145.47, 145.18, 144.98, 144.95, 144.90, 144.18, 143.37, 143.31, 143.28,

213 Chapter 5 Experimental Part

2 3 142.50, 142.21, 141.23, 139.40 (58C, C60-sp ), 71.78 (2C, C60-sp ), 67.63 (2C, OCH2),

65.74 (2C, OCH2), 52.25 (1C, OCCCO), 42.42 (1C, OCCH2CO), 29.58 , 28.77, 28.73,

26.18, 26.09 (12C, CH2) ppm.

+ + MS (FAB, NBA): m/z = 1147 [M+H] , 720 [C60] ; MWcalc.: 1147.14 g/mol.

IR (ATR): ν˜ = 2930, 2856, 1741, 1463, 1428, 1390, 1305, 1287, 1235, 1208, 1185, 1154, 1101, 1075, 1019, 946, 884, 737, 706 cm−1.

UV/Vis (CH2Cl2): λmax = 258.5 (123200), 326 (38500), 425 (2700), 491 (1800) nm.

214 Chapter 5 Experimental Part

(Bis-(6-N-tert-butoxycarbonylamino-1-hexanol)-malonate-cyclo-[2]-octyl malonate)-[5,1]-hexakisadduct (121)

O O O O HN NH

O O NH HN O O

O O O O O O O O N O O O H O O O O O O O O O H O O N O O O 121 O OO O O O O

O O NH HN O O

HN NH O O O O

Compound 121 was synthesized according to general procedure GP 7 from monoadduct 119 (346 mg, 0.30 mmol), malonate 120 (3.04 g, 6.04 mmol), DMA (623 mg, 3.02 mmol), CBr4 (1.00 g, 3.02 mmol) and P1-base (768 µL, 3.02 mmol) in dry

CH2Cl2. Pre-cleaning by ﬂash column chromatography (SiO2; dichloromethane/ethyl acetate 65:35) and subsequent puriﬁcation by preparative HPLC (Nucleosil 5 µm; dichloromethane/ethyl acetate 76:24) gave 121 as yellow solid.

Yield: 256 mg, 0.07 mmol, 23 %, yellow solid.

1 H-NMR (300 MHz, RT, CDCl3): δ = 4.75 (s, br, 10H, NH), 4.21 (m, 24H, OCH2), 4.09

(m, 4H, OCH2), 3.32 (s, 2H, OCCH2CO), 3.04 (m, 20H, CH2NH), 1.65 (m, 28H, CH2),

1.39 (s, 90 H, C(CH3)3), 1.31 (m, 76H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, CDCl3): δ = 166.63 (2C, CO), 164.01 (12C, CO), 156.20

215 Chapter 5 Experimental Part

t 2 (10C, COO Bu), 145.88, 141.29 (48C, C60-sp ), 79.07 (10C, C(CH3)3), 69.26 (12C, 3 C60-sp ), 67.25 (2C, OCH2), 67.03 (10C, OCH2), 65.71 (2C, OCH2), 45.63 (6C,

OCCCO), 42.42 (1C, OCCH2CO), 40.66 (10C, CH2NH), 30.13. 29.57 (16C, CH2),

28.61 (30C, C(CH3)3), 28.52, 26.55, 26.12, 25.74 (36C, CH2) ppm.

+ + MS (FAB, NBA): m/z = 3649 [M] , 720 [C60] ; MWcalc.: 3650.28 g/mol.

IR (ATR): ν˜ = 3412, 3354, 2964, 2934, 2860, 1741, 1698, 1513, 1455, 1412, 1393, 1366, 1247, 1239, 1170, 1081, 1042, 1019, 865, 799, 733, 664 cm−1.

UV/Vis (CH2Cl2): λmax = 245.5 (86900), 271 (64200), 281 (66500), 315 (40400), 335 (30800) nm.

Boc-protected [5,1]-hexakisadduct-monoadduct (122)

O O O O HN NH

O O NH HN O O

O O O O O O O O N O O O H O O O O O O O O O H O O N O O O 122 O OO O O O O

O O NH HN O O

HN NH O O O O

Compound 122 was synthesized according to general procedure GP 4 from [5,1]- hexakisadduct 121 (205 mg, 0.056 mmol), C60 (61 mg, 0.084 mmol), CBr4 (24 mg,

0.073 mmol) and P1-base (16 µL, 0.073 mmol) in 75 mL dry toluene. The crude product was puriﬁed by ﬂash column chromatography (SiO2; toluene/ethyl acetate 75:35)

216 Chapter 5 Experimental Part to give 122 as yellow brownish solid.

Yield: 100 mg, 0.023 mmol, 41 %, yellow brownish solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 4.76 (s, br, 10H, NH), 4.46 (t, J = 6.4 Hz, 4H, 3 OCH2-mono), 4.23 (t, J = 6.5 Hz, 24H, OCH2-hexakis), 3.06 (m, 20H, CH2NH), 1.86

(m, 4H, CH2), 1.66 (m, 28H, CH2), 1.41, (m, 114H, C(CH3)3,CH2), 1.33 (m, 48H, CH2) ppm.

13 C-NMR (75 MHz, RT, CDCl3): δ = 164.05 (12C, CO-hexakis), 163.83 (2C, CO- t 2 mono), 156.23 (10C, COO Bu), 145.92, 141.32 (48C, [5,1]hexakis-C60-sp ), 146.01, 145.97, 145.89, 145.45, 145.41, 145.37, 145.08, 144.87, 144.85, 144.80, 144.08,

2 143.27, 143.20, 143.18, 142.40, 142.11, 141.25, 141.13, 139.32 (58C, mono-C60-sp ), 3 3 79.12 (10C, C(CH3)3), 71.79 (2C, mono-C60-sp ), 69.28 (12C, [5,1]hexakis-C60-sp ),

67.68 (2C, OCH2-mono), 67.09 (12C, OCH2-hexakis), 52.27 (1C, OCCCO-mono),

45.63 (6C, OCCCO-hexakis), 40.69 (10C, CH2NH), 30.17 (10C, CH2), 29.75 (4C,

CH2), 28.66 (34C, C(CH3)3, CH2), 28.55 (10C, CH2), 26.61 (10C, CH2), 26.32, 26.27

(4C, CH2), 25.79 (10C, CH2) ppm.

+ + MS (FAB, NBA): m/z = 4367 [M+H] , 720 [C60] ; MWcalc.: 4368.90 g/mol.

IR (ATR): ν˜ = 3412, 3350, 2943, 2926, 2856, 1741, 1695, 1517, 1459, 1393, 1366, 1243, 1216, 1170, 1081, 1042, 992, 892, 865, 780, 760, 737, 672 cm−1.

UV/Vis (CH2Cl2): λmax = 246 (185500), 257 (sh, 175200), 321 (74500), 426 (7500), 488 (5700).

217 Chapter 5 Experimental Part

Asymmetric (Boc-protected) (tert-butyl-protected) [5,1,1,5]-dimer (123)

2 1 R O OR2 R1O OR 2 O O O 1 R O O OR2 R1O O OR O O O O N O O O O O O H O O O O O O O O O O O H O O O O N O O O O R2O OR2 O R1O OR1 OO OO R2O OR2 O R1O OR1

R1 123 R2

Compound 123 was synthesized according to general procedure GP 7 from [5,1]- hexakisadduct-monoadduct 122 (80 mg, 0.018 mmol), malonate 100 (98 mg, 0.22 mmol), DMA (45 mg, 0.22 mmol), CBr4 (73 mg, 0.22 mmol) and P1-base (47 µL, 0.22 mmol) in dry CH2Cl2. Pre-cleaning by ﬂash column chromatography (SiO2; dichloromethane/ methanol 100:5) and subsequent puriﬁcation by preparative HPLC (Nucleosil 5 µm; dichloromethane/methanol 100:4) gave 123 as orange solid.

Yield: 36 mg, 0.0055 mmol, 31 %, orange solid.

1 3 H-NMR (300 MHz, RT, CDCl3): δ = 4.76 (s, br, 10H, NH), 4.22 (t, J = 5.9 Hz, 48H, 3 OCH2), 3.06 (m, 20H, CH2NH), 2.19 (t, J = 7.3 Hz, 20H, CH2CO), 1.66 (m, 68H,

CH2), 1.41 (m, 204H, C(CH3)3,CH2), 1.33 (m, 68H, CH2) ppm.

13 t C-NMR (75 MHz, RT, CDCl3): δ = 172.98 (10C, COO Bu), 164.05 (24C, CO), 156.23 t 2 (10C, COO Bu), 145.91, 141.26 (96C, C60-sp ), 80.25 (10C, CH2COOC(CH3)3), 79.11 3 (10C, HNCOOC(CH3)3), 69.28 (24C, C60-sp ), 67.08, 66.92 (24C, OCH2), 45.64 (12C,

OCCCO), 40.69 (10C, CH2NH), 35.53 (10C, CH2CO), 30.18 (10C, CH2), 29.78 (4C,

CH2), 28.67 (34C, HNCOOC(CH3)3, CH2), 28.56 (10C, CH2), 28.33 (40C,

CH2COOC(CH3)3, CH2), 26.61 (10C, CH2), 26.30 (4C, CH2), 25.80 (10C, CH2), 25.53

(10C, CH2), 24.85 (10C, CH2) ppm.

IR (ATR): ν˜ = 3416, 3370, 3057, 2976, 2937, 2864, 1714, 1513, 1459, 1393, 1366, 1239, 1216, 1154, 1081, 1042, 988, 957, 896, 849, 780, 733, 703 cm−1.

UV/Vis (CH2Cl2): λmax = 246 (169300), 271 (125300), 281.5 (126600), 316 (78900),

218 Chapter 5 Experimental Part

336 (60100).

Deprotected [5,1,1,5]-dimer (124)

2 1 R O OR2 R1O OR 2 O O 1 R O O OR2 R1O O OR O O OH H N O O O 3 O O O O O O CF3COO O O O O O O O O CF3COO O O O OO O OH H3N R2O OR2 O R1O OR1 OO OO R2O OR2 R1O OR1

R1 124 R2

(N-Boc-protected) (tert-butyl-protected) [5,1,1,5]-dimer 123 (20 mg, 0.003 mmol) was dissolved in 3 mL pure TFA and stirred at room temperature for 36 h. Excess TFA was removed in vacuo and coevaporated with methanol for several times to give 124 as pale orange solid.

Yield: 17 mg, 0.0055 mmol, 92 %, pale orange solid.

1 H-NMR (300 MHz, RT, MeOH-d4): δ = 4.30 (m, 48H, OCH2), 2.89 (m, 20H, CH2NH), 3 2.25 (t, J = 7.1 Hz, 20H, CH2CO), 1.69 (m, 48H, CH2), 1.61 (m, 40H, CH2), 1.38 (m,

76H, CH2) ppm.

13 C-NMR (75 MHz, RT, MeOH-d4): δ = 177.36 (10C, COOH), 164.77 (24C, CO), 2 3 146.77, 142.62 (96C, C60-sp ), 70.56 (24C, C60-sp ), 68.59, 68.23 (24C, OCH2), 47.59

(12C, OCCCO), 40.63 (10C, CH2NH), 34.80 (10C, CH2CO), 30.43 (4C, CH2), 29.67

(4C, CH2), 29.39 (20C, CH2), 28.46 (10C, CH2), 27.00 (14C, CH2), 26.73 (10C, CH2),

26.53 (10C, CH2), 25.67 (10C, CH2) ppm.

IR (ATR): ν˜ = 3308, 2945, 2833, 1745, 1683, 1451, 1420, 1254, 1204, 1139, 1023, 838, 799, 718, 686 cm−1.

UV/Vis (MeOH): λmax = 215.5, 243.5 (sh), 270, 280 (sh), 317.5, 335.5 nm.

219 Chapter 5 Experimental Part

R R R R R R O O R O O R O O O OO O O O O O O O O O O O R O R O O R O R R O R R O O R O O O O O O O O O O O OO O O O O O O O R O R R R

R O R R R O R O O O R O O O R R O O O O OO O O O O O O O O O O O O O O O O O O O R O O O R O O O R R O O O O O O O O O O OO O O O O O O O O O O OO O O O OO R O O R R O O R R R R R

R R R O R O O O OO O O O O OO O O O O O O O O O R O O R R O R R O R O R R O O O O O O O O O O O OO O O R O O O O O O R R R R O R R R

H R = N O 126 O

Boc-protected [5,1]-hexakisfullerenyl-[6,0]-hexakisadduct (126)

Compound 126 was synthesized according to general procedure GP 7 from [5,1]- hexakisadduct 121 (101 mg, 0.028 mmol), C60 (2 mg, 0.0028 mmol), DMA (6 mg,

0.028 mmol), CBr4 (10 mg, 0.028 mmol) and P1-base (11 µL, 0.028 mmol) in 10 mL dry toluene. Pre-cleaning by ﬂash column chromatography (SiO2; toluene/ethyl acetate 150:60 to 100:250) and subsequent puriﬁcation by preparative HPLC (Nucleosil 5 µm; toluene/ethyl acetate 60:40) gave 126 as yellow solid.

Yield: 18 mg, 0.0008 mmol, 28 %, yellow solid.

220 Chapter 5 Experimental Part

1 H-NMR (400 MHz, RT, CDCl3): δ = 4.78 (s, br, 60H, NH), 4.23 (m, 168H, OCH2),

3.06 (m, 120H, CH2NH), 1.67 (m, 168H, CH2), 1.44 (m, 168H, CH2), 1.41 (s, 540H,

C(CH3)3), 1.32 (m, 288H, CH2) ppm.

13 t C-NMR (100.5 MHz, RT, CDCl3): δ = 164.06 (84C, CO), 156.25 (60C, COO Bu), 2 3 145.93, 141.34 (336C, C60-sp ), 79.13 (60C, C(CH3)3), 69.29 (84C, C60-sp ), 67.11

(84C, OCH2), 45.67 (42C, OCCCO), 40.71 (60C, CH2NH), 30.18 (60C, CH2), 29.79

(24C, CH2), 28.68 (180C, C(CH3)3), 28.57 (84C, CH2), 26.61 (60C, CH2), 26.28 (24C,

CH2), 25.80 (60C, CH2) ppm.

IR (ATR): ν˜ = 3412, 3350, 2934, 2860, 1745, 1695, 1521, 1459, 1393, 1366, 1251, 1220, 1170, 1081, 1034, 922, 865, 826, 784, 760, 714, 697 cm−1.

UV/Vis (CH2Cl2): λmax = 246 (575600), 270 (425600), 280.5 (436600), 317 (282500), 335.5 (231200) nm.

221 Chapter 5 Experimental Part

Deprotected [5,1]-hexakisfullerenyl-[6,0]-hexakisadduct (127)

R R R R R R O O R O O R O O O OO O O O O O O O O O O O R O R O O R O R R O R R O O R O O O O O O O O O O O OO O O O O O O O R O R R R

R O R R R O R O O O R O O O O R R O O O OO O O O O O O O O O O O O O O O O O O O R O O O R O O O R R O O O O O O O O O O OO O O O O O O O O O O OO O O O OO R O O R R O O R R R R R

R R R O R O O O OO O O O O OO O O O O O O O O O R O O R R O R R O R O R R O O O O O O O O O O O OO O O R O O O O O O R R R R O R R R

+ - R = NH3 CF3COO 127

Boc-protected [5,1]-hexakisfullerenyl-[6,0]-hexakisadduct 126 (18 mg, 0.0008 mmol) was dissolved in 2 mL pure TFA and stirred at room temperature for 48 h. The crude product was puriﬁed by reprecipitation from MeOH/Et2O to give 127 as a yellow solid.

Yield: 13 mg, 0.0008 mmol, 98 %, yellow solid.

1 H-NMR (400 MHz, RT, MeOH-d4): δ = 4.31 (m, 168H, OCH2), 2.90 (m, 120H, CH2NH),

1.71 (m, 168H, CH2), 1.63 (m, 168H, CH2), 1.39 (m, 288H, CH2) ppm.

13 C-NMR (100.5 MHz, RT, MeOH-d4): δ = 164.93 (84C, CO), 146.93, 142.81 (336C,

222 Chapter 5 Experimental Part

2 3 C60-sp ), 68.40 (84C, C60-sp ), 67.06 (84C, OCH2), 45.19 (42C, OCCCO), 40.78 (60C,

CH2NH), 29.54 (84C, CH2), 28.62 (84C, CH2), 27.39 (24C, CH2), 27.16 (60C, CH2),

26.69 (60C, CH2) ppm.

IR (ATR): ν˜ = 3416, 3266, 3065, 2941, 2864, 2687, 1745, 1675, 1536, 1467, 1432, 1397, 1355, 1254, 1204, 1135, 1085, 1046, 996, 957, 888, 838, 799, 760, 722, 672 cm−1.

UV/Vis (H2O/pH 4): λmax = 215 (772100), 242.5 (551900), 271 (422900), 280 (416500), 316 (284700), 334 (228300) nm.

223 Appendices

A Materials and Methods for the Determination of Biological Activity in vivo

Standard procedures for embryo collection

Embryos were generated by natural pair-wise mating, as described in the Zebrafish Handbook.[311] Four to five pairs of adult zebrafish were set up for each mating, and, on average, 100 - 150 embryos per pair were generated. Embryos were maintained at 28°C in fish water (200 mg Instant Ocean Salt per liter of dei onized water; pH 6.6 - 7.0 maintained with 2.5 mg/liter Jungle pH Stabilizer (Jungle Laboratories Corpora- tion, Cibolo, TX); conductivity 670 - 760 µS). Embryos were cleaned (dead embryos removed) and sorted by developmental stage [312] at 6 and 24 hours post-fertilization (hpf). Because the embryo receives nourishment from an attached yolk sac, no feeding was required for seven days post fertilization (dpf).

A.1 Determination of LC50 and Organ Toxicity

Treatment of embryos Zebrafish embryos at 24 hpf were distributed into 96-well cell culture plates, one embryo per well in 100 ml fish water containing the compound. Embryos were exposed from day 1 to day 5 post-fertilization (pf). Typically, the fish water contains PBS (phosphate buffered saline) at a final concentration of 10 %. Treated embryos were compared with untreated and 0.1 % DMSO-treated controls.

224 Appendices

Measurement of LC50 and lethality curves Mortality was recorded every 24 hrs. At 120 hpf, total mortality was used to generate the concentration-response curves. The data were averaged from multiple experiments. Best-ﬁt concentration-response curves were generated using KaleidaGraph software (Synergy Software, Reading, PA, USA) using the equation:

M2 − M1 Y = M1 + X (5.1) 1 + ( )M4 M3 where M1 = maximum Y value (100 % in this case), M2 = minimum Y value (0 % in this case), M3 the concentration corresponding to the value midway between M1 and 2 M2 (LC50), M4 slope of the curve at M3 (best ﬁt, generating R closest to 1), X = concentration of compound, Y = percent lethality.

Visual toxicity assessment of developing embryos At 5 dpf, organs in 5 randomly selected embryos were inspected by light microscopy. Body morphology, liver, intestine, and heart were assessed. Since drug treatment was initiated at 24 hpf, which is after circulation and heart beat are present, but before the liver and intestine are developed, observations are for effects on developing organs.

Body morphology Defects in the development of midline structures, including notochord and ﬂoor plate, often result in abnormal body shape in zebraﬁsh. To assess the potential drug effects on midline development, abnormalities in body shape, including small body size, bent or missing tail were examined.

A.2 Determination of Otoprotective Activity

Hair cell assessment To damage inner hair cells, 5-day zebraﬁsh were treated with gentamicin (2.5 µg/ml) or cisplatinum (10 µM) for 24 hours to induce apoptosis in inner hair cells. To test drug effects on protection of hair cell damage, test compounds were administered at 0, 0.1, 1, 10, 100 and 250 µM concentrations with either the gentamicin or cisplatinum treat-

225 Appendices ment. The inner hair cells in the lateral neuromasts were examined by 2,4-dimethyl- aminostyryl-N-ethyl pyridinium iodide (DASEPI or 2,4 Di-Asp) staining.

DASPEI staining Zebrafish were incubated with DASPEI solution (1mM) for 2 hours, then rinsed thoroughly in fish water. Zebrafish were anaesthetized with MESAB (0.5 mM 3-aminobenzoic acid ethyl ester, 2 mM Na2HPO4), and mounted in methylcellulose in depression slide for observation.

Morphometric analysis Fluorescent signals (SS) were quantified as: SS = staining area x staining intensity of neuromasts by particle analysis (Scion Image, Scion Corporation, Frederick, Mary- land). Images of lateral sides in each animal were obtained by the same exposure time and fluorescent gain (anterior on the left; posterior on the right; dorsal on the top). The size of neuromasts were defined and specified for particle analysis. Five animals for each treatment were quantified and intensity of fluorescent signals was averaged.

Statistics All data were presented as mean ± standard deviation (SD). Student’s t-Test was used to compare vehicle-treated and drug treated zebrafish. Significance was defined as P < 0.05, n = 5.

A.3 Determination of General CNS Neuroprotective Activity Against 6-Hydroxdopamine (6-OHDA) Induced Neuronal Apoptosis

Microinjection of 6-OHDA 6-OHDA is extremely unstable in solution, it was prepared fresh for each injection. 3- day zebraﬁsh were anesthetized in MESAB (0.5 mM 3-aminobenzoic acid ethyl ester,

2 mM Na2HPO4) and 500 mM 6-OHDA was microinjected into the midbrain region. The estimated amount of injection was about 1-2 pmole per compound. Zebraﬁsh were rapidly transferred to fresh ﬁsh water after injection, allowed to recover for 30

226 Appendices minutes, and incubated with neuroprotective antioxidants. Water alone was injected into zebraﬁsh brain region as a vehicle control.

Treatment of embryos Embryos were exposed to water-soluble fullerenes at 1, 10, 100 and 250 µM for 24 hours, then microinjected with 6-OHDA. Twenty embryos were treated with each concentration of each compound.

Morphometric analysis Fluorescent signals (SS) were quantified as: SS = staining area x staining intensity of neuromasts by particle analysis (Scion Image, Scion Corporation, Frederick, Mary- land). Images of dorsal sides in each animal were obtained by the same exposure time and fluorescent gain. The positive staining was defined by size and fluorescent intensity evaluation, and specified for particle analysis. Five animals for each treatment were quantified and intensity of fluorescent signals was averaged.

Fluorescence microscopy and image analyses All fluorescence microscopy studies were performed using a Zeiss M2Bio fluorescence microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) equipped with a rhodamine cube and a green FITC filter (excitation: 488 nm, emission: 515 nm), and a chilled CCD camera (Axiocam MRM, Carl Zeiss Microimaging Inc., Thornwood, NY). Screens were routinely done using a 1.6X, 10X and 20X objective archromats and 10X eye pieces. The system was also equipped with a z-motorized stage, deconvolution software and 4-D reconstruction software (Axiovision, Carl Zeiss Microimaging Inc., Thornwood, NY) which permits reconstruction of 3-D objects and analyzes Z-stacks. Images were analyzed with Axiovision software Rel 4.0 (Carl Zeiss Microimaging Inc., Thornwood, NY), the Adobe Photoshop 6.0 computer program (Adobe, San Jose, CA) and NIH image software (Bethesda, MD). The patterns and the intensity of staining were recorded and quantitated.

227 Appendices

Acridine orange staining At 48, 72 and 120 hpf, five embryos were immersed in 0.5 µg/ml acridine orange (acridinium chloride hemi-[zinc chloride]) in PBS for 60 minutes and rinsed thoroughly twice in 10 ml of fresh fish water. Stained embryos are anesthetized with MESAB (0.5 mM 3-aminobenzoic acid ethyl ester, 2 mM Na2HPO4), and mounted in methycellulose in a depression slide for observation using fluorescent microscopy. Effects of water- soluble fullerenes apoptosis in the hatching glands, retina, lateral neuromasts, and olfactory pits were examined.

A.4 Determination of Dopaminergic CNS Neuroprotective Activity Against 6-Hydroxydopamine Induced Apoptosis

Treatment of embryos 2-day zebraﬁsh were treated with 1 % DMSO for vehicle control and treated with 1 % DMSO plus 250 µM 6-OHDA for DA-loss control. For compound testing, 2-day embryos were exposed to a mixture of 250 µM 6-OHDA and fullerenes for 72 hours.

Antibody staining Embryos were fixed in 4 % paraformaldehyde overnight at 4°C. F ixed embryos were permeabilized with cold acetone at -20°C for 20 minutes and r ehydrated in step-wise descending ethanol/PBS solutions (95, 79, 50, 25, 0 % ethanol/PBS mix, ten minutes for each step). Embryos were then stained with anti-tyrosine hydroxylase antibody (mouse anti-human, Sigma, St Louis, MO) at 4°C overni ght. The next day, samples were washed with PBS-T (0.1 % Tween 20), incubated with secondary antibody (goat anti-mouse) and color was developed using ABC reagent (Vector Labs, Burlingame, CA) according to manufacturer’s instructions. Stained embryos were further flat-mounted on a glass slide and five randomly selected embryos were examined for DA neuron loss.

Light microscopy and image analyses All microscopy studies were performed using a Zeiss light microscope (Carl Zeiss Mi- croimaging Inc., Thornwood, NY) equipped with a Spot camera (Diagnostic instru-

228 Appendices ments, Sterling, MI). The patterns and the intensity of staining were recorded and quantiﬁed.

Fluorescence microscopy All fluorescence microscopy studies were performed using a Nikon Eclipse E600 fluorescence microscope (Nikon Inc., Melville, NY) equipped with a 200 watt mercury/xenon lamp and a green FITC filter (excitation: 488 nm, emission: 515 nm), and a C5985 chilled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Images were analyzed with the Adobe Photoshop 6.0 computer program (Adobe, San Jose, CA).

B Materials and Methods for the Preparation and Examination of SWCNT-Fullerene-Hybrid

The single-walled carbon nanotubes (laser-ablation, University of Karlsruhe) were purified using an oxidative protocol described elsewhere.[313] The purified nanotubes were then suspended in 1,2-dichloroethane (DCE) by pulsed high-power ultrasound (Dr. Hielscher, UP400S, 400W, 24 kHz) to yield a black colored suspension. Bundles of agglomerated nanotubes were subsequently removed in a centrifugation step for 5 minutes at 5000 rpm. The supernatant was given to a solution of 99 in DCE and stirred thoroughly for 5 minutes. The fullerene-nanotube hybrid was subsequently removed from the suspension by centrifugation for 30 minutes at 10000 rpm. The supernatant was discarded and the precipitate was re-suspended in deionized water. The resulting suspension was filtered using a microporous membrane (cellulose nitrate, pore-size 0.2 µm) to yield a black-colored solid film (buckypaper). For the preparation of the fullerene nanowires, the buckypaper consistent of fullerene- nanotube hybrid is mounted onto a teflon stamp and fixed with a double-sided UHV compliant scotch tape. Then the configuration is brought into the load lock of the STM and pumped until the pressure falls below 10−7 mbar. The bucky paper is brought into contact with the gold substrate in the preparation chamber (10−9 mbar). Therefore simply the manipulator is lowered via micrometer screws until the bucky paper touches the surface. This in situ preparation procedure ensures that the surface does not suffer

229 Appendices from ambient contamination, e.g. hydrocarbons. Prior to STM investigation, the surface is gently annealed to allow possible adsorbates out of the bucky paper to desorb. We used temperatures of 75◦C to 150◦C applied for several hours. It turned out that the annealing does not improve the sample quality signiﬁcantly.

230 References

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High catalytic activity of dendritic C60 monoadducts in metal-free superoxide dismutation. Liu, Gao-Feng; Filipovic, Milos; Ivanovic-Burmazovic, Ivana; Beuerle, Florian; Witte, Patrick; Hirsch, Andreas. Angew. Chem. Int. Ed., 2008, 47(21), 3991–3994.

Water solubility, antioxidant activity and cytochrome C binding of four families of exohedral adducts of C60 and C70. Witte, Patrick; Beuerle, Florian; Hartnagel, Uwe; Lebovitz, Russell; Savouchkina, Anas- tasia; Sali, Sevda; Guldi, Dirk; Chronakis, Nikos; Hirsch, Andreas. Org. Biomol. Chem., 2007, 5(22), 3599–3613.

Cytoprotective activities of water-soluble fullerenes in zebraﬁsh models. Beuerle, Florian; Witte, Patrick; Hartnagel, Uwe; Lebovitz, Russell; Parng, Chuenlei; Hirsch, Andreas. J. Exp. Nanosci., 2007, 2, 147–170.

The transition from oxygen chemisorption to oxidation of ultra-thin Ni layers on Cu(111). Domnick, Ralph; Held, Georg; Witte, Patrick; Steinrück, Hans-Peter. J. Chem. Phys., 2001, 115, 1902–1908.

247 Curriculum Vitae

Personal Data

Name Patrick Sven Witte Date of birth 09.09.1977 Place of birth Nürnberg Nationality German

Education and Military Service

09/1983 – 07/1987 Elementary school in Chamerau 09/1987 – 06/1996 Grammar school in Cham 06/1996 School leaving examination (Abitur) 09/1996 – 09/1997 3./ Panzergrenadierbataillon 112, Regen

Higher Education

10/1997 – 09/1999 Basic studies: Chemistry (Diplom), University Regensburg 10/1999 – 02/2003 Main studies: Chemistry (Diplom), Friedrich-Alexander- University Erlangen-Nürnberg 04/2003 – 10/2003 Diploma thesis at the Institute of Inorganic Chemistry, Friedrich-Alexander-University Erlangen-Nürnberg Supervisor: Prof. Dr. R. van Eldik Title: “Synthese und Reaktivität modiﬁzierter Pt(II) Komplexe” (Synthesis and Reactivity of Modiﬁed Pt(II)-Complexes)

248 12/2004 – 11/2007 PhD studies at the Institute of Organic Chemistry, Friedrich-Alexander-University Erlangen-Nürnberg Supervisor: Prof. Dr. A. Hirsch Title: “Amphiphilic Fullerenes for Biomedical and Optoelectronical Applications”