amphiphilic fullerenes for biomedical and optoelectronical applications

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Transcript of amphiphilic fullerenes for biomedical and optoelectronical applications

  • AMPHIPHILIC FULLERENES FOR

    BIOMEDICAL AND OPTOELECTRONICAL

    APPLICATIONS

    Den Naturwissenschaftlichen Fakultten

    der Friedrich-Alexander-Universitt Erlangen-Nrnberg

    zur

    Erlangung des Doktorgrades

    vorgelegt von

    Patrick Witte

    aus Nrnberg

  • Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultten der

    Universitt Erlangen-Nrnberg

    Tag der mndlichen Prfung: 25.04.2008

    Vorsitzender

    der Prfungskommission: Prof. Dr. Eberhard Bnsch

    Erstberichterstatter: Prof. Dr. Andreas Hirsch

    Zweitberichterstatter: Prof. Dr. Tim Clark

  • Meinem Doktorvater, Prof. Dr. A. Hirsch, gilt mein besonderer Dank fr sein reges

    Interesse am Fortgang dieser Arbeit sowie fr seine Anregungen und die Diskussionen

    mit ihm.

    Die vorliegende Arbeit wurde in der Zeit zwischen Dezember 2003 bis Dezember

    2007 am Institut fr Organische Chemie der Friedrich-Alexander-Universitt Erlangen-

    Nrnberg durchgefhrt.

  • Dedication

    - Science is facts;just as houses are made of stones, so is science made of facts;

    but a pile of stones is not a house and a collection of facts is not necessarily science

    Henri Poincare (1854 - 1912)

    For my Parents and Kati

  • Index of Abbreviations

    tBu . . . . . . . . . . . . . . . . . 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 Nanostructured Materials . . . . . . . . . . . . . . . . . . . . . . . . . . 1

    1.2 The Discovery of Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . 4

    1.3 The Structure of Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . 5

    1.4 Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

    1.4.2 Solubility of C60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

    1.5 Spectroscopic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . 9

    1.5.1 UV/Vis-Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . 9

    1.5.2 Mass Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 10

    1.5.3 NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 11

    1.5.3.1 3He and 1H Spectroscopy . . . . . . . . . . . . . . . . . 11

    1.5.3.2 13C Spectroscopy . . . . . . . . . . . . . . . . . . . . . 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

    Unsaturated Fatty Acid . . . . . . . . . . . . . . . . . . . . . . . . 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 SWCNTs with a Fullerene-

    Monocarboxylic Acid Derivative . . . . . . . . . . . . . . . . . . . 103

    3.3.2 Non-Covalent Functionalization of SWCNTs with a Fullerene-

    Pyrene Dyad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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-

    Hexakisadduct Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 Chemicals and Instrumentation . . . . . . . . . . . . . . . . . . . . . . . 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 com-

    parable 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 occur-

    ring on this length scale are of interest to chemists, physicists, biologists, electrical

    and mechanical engineers, and computer scientists, making research in nanotech-

    nology a frontier activity in materials science. Nanomaterials, which can be classi-

    fied 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 tech-

    nology (nanophotonics, solar energy conversion, opto-electronics) and chemical syn-

    thesis (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 nano-

    materials that perform exactly as desired.

    3

  • Chapter 1 Introduction

    1.2 The Discovery of Fullerenes

    Figure 1.2: Leonardo da Vincis

    "Truncated Icosahedron".[2]

    The discovery of C60 has a long and very in-

    teresting 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 1960s, scientists were increas-

    ingly interested in non-planar aromatic struc-

    ture, and thereafter the bowl-shaped corannu-

    lene was synthesized.[4] In 1970, EIJI OSAWA

    realized that a molecule made up of sp2 hy-

    bridized carbons could have a spherical struc-

    ture. He therefore made the first proposal for

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

    dependently proposed the C60 structure, the

    paper published by BOCHVAR and GALPERN

    in 1973 not only predicted some properties of

    C60, but also of C20 (the smallest fullerene) as well.[6] The first spectroscopic evidence

    for C60 and other fullerenes was published in 1984 by ROHLFING and coworkers.[7]

    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 con-

    sisting 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

    KRTSCHMER 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 com-

    posed of 12 pentagons and n hexagons and the composition C20+2n (Eulers 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

    pristine C60 at low temperature [1012], C60 derivatives [13], C60 solvates [14,15], and solid-

    state 13C NMR measurements [16]. Such experimental findings definitively confirmed

    the postulated Ih-symmetry with a mean diameter of 7.1 for the sphere. Its VAN DER

    WAALS diameter is 10.4 , and the distance across the cavity is 3.5 . 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 ), and bonds at the junction between a five- and

    a six-membered ring ([6,5-bonds], mean distance = 1.449 ).[10, 17] The electronic

    5

  • Chapter 1 Introduction

    structure [1820] 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).

    The highly pyramidalized sp3 C-Atoms in C60 cause a large amount of strain en-

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

    RAGHAVACHARI, of carbon atom orbitals in C60 structure is 11.6,[21,22] an angle

    between sp2 and sp3 hybridization, which are 0 and 19.47 respectively (see fig-

    ure 1.4).

    6

  • Chapter 1 Introduction

    sp2 sp3

    = 90 = 109.47

    Figure 1.4: Pyramidalization of the carbon atom orbitals in C60. The angle of pyramidalization = - 90 is between the angles for sp2 and sp3 hybridization, which causesthe 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 experi-

    mentally by calorimetry to be 10.16 kcal mol1 per C-atom (relative to graphite with 0.0

    kcal mol1 per C-atom).[23] Also in the comparison to diamond (0.4 kcal mol1 per C-

    atom), C60 is a energy-rich carbon allotrope. This comparatively high energy content is

    originated in the high strain energy of C60 due to pyramidalization of the sp2 orbitals and

    accounts for about 80 % of the heat of formation.[24] 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-

    zene instantly decompose.[25] 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 solu-

    bility 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.[2629]

    Solvents Solubility [mg/mL]

    acetonitrile 0.000THF 0.00methanol 3.5 105

    water 1.3 1011

    n-pentane 0.005cyclohexane 0.036chloroform 0.16dichloromethane 0.26tetrachloromethane 0.32pyridine 0.89benzene 1.70toluene 2.801,1,2,2-tetrachloroethane 5.30anisole 5.60chinoline 7.20carbon disulphide 7.901,2,4-trichlorobenzene 8.501,2-dichlorobenzene 27.001-chloronaphthalene 51.00

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

    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 [3133] which correspond to symmetry-forbidden

    singlet-singlet transitions from the HOMO to the LUMO and LUMO+1. Chemical func-

    tionalization 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 re-

    spect 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-- electron system.[31] Very characteristic for all [6-6]-closed (see

    chapter 1.8) monoadducts is the absorption at 425 nm.[3537] The influence of subse-

    quent functionalization can be nicely followed by the characteristic color of the corre-

    sponding derivatives in solution. The introduction of additional groups reduces the -

    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 -

    system is significantly reduced, whereas the remaining -electrons are located within

    a cubic, cyclophane-like substructure of eight benzenoid rings.[38]

    9

  • Chapter 1 Introduction

    200 300 400 500 600 700 800

    abso

    rbance

    wavelenght [nm]

    400 500 600 700 800

    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 discov-

    ery 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 ioniza-

    tion 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

    molecule subsequently loses C2 units.[41,42]

    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 aromatic-

    ity 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 pa-

    rameters 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 -electrons would move freely on

    the fullerene surface. As a consequence C60 would be highly diamagnetic [44,45] and

    would have to exhibit a particularly high magnetic susceptibility, which could not be

    confirmed experimentally.[46,47] The determined magnetic susceptibility of C60 with

    = -260 CGS ppm mol1 (= -4.3 CGS ppm per mol of C) is smaller than those from

    diamond with = -5.5 CGS ppm per mol of C.

    Nevertheless there are ring currents present in C60. The working groups HADDON [48,49]

    as well as ZAMESI and BOWLER [50] could show that in the pentagons strong paramag-

    netic 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 corannulenesubstructure of C60. Right: Ring current contour map of C60.[50]

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

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

    in C60 is shielded by = -6.36 ppm, relative to free 3He. This chemical shift is a result

    of the ring currents in the -system and the shielding effects of the sigma framework.

    Measurements on helium complexes of fullerene derivatives showed, that the chemi-

    cal 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 struc-

    tures of the fullerene derivatives. While the electronic structure of a homofullerene

    agrees to a large extent with that of unfunctionalized C60, the -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 -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 -electron and a 58 -

    electron system.

    H OEt

    OH

    O

    EtO

    3.32 ppm 6.79 ppm

    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 -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.[5456]

    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 qua-

    ternary 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 pyracy-

    lene, 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 elec-

    tronic 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 Hckel 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 (t1u) and LUMO+1 (t2u) separated by a moderate

    energy gap of 0.757 .[6062]

    0

    -1

    LUMO

    HOMO

    HOMO

    LUMO

    LUMO+1

    hu

    t1u

    e2u

    En

    erg

    y (

    )

    (0.38)

    (0.14)

    (-0.62)

    (+1)

    (-1)

    -0.5

    0.5

    1

    e1u

    t2u

    0.757

    2.00

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

    In accordance with the degeneration of the LUMO level, the redox chemistry demon-

    strates 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

    anions Cn60 (n = 1 - 6), which can be generated by subsequent reversible one-electron

    reductions. The separation between any two successive reductions is relatively con-

    stant 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 con-

    sists in the removal of an electron from the low-lying HOMO, leading to an important

    destabilization of the -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

    (E1/ 2ox E1/ 2ox = 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 -electrons can cause the establishment of diamagnetic or paramag-

    netic ring currents within the loops of the hexagons and pentagons. Neutral C60, for in-

    stance, exhibits no pronounced overall aromaticity since it contains diatropic hexagons

    and paratropic pentagons and was labeled ambiguously aromatic. BHL and HIRSCH

    have showed that the treatment of the -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 Hckel

    rule, i. e. annulenes with 4N+2 -electrons are aromatic, those with 4N -electrons are

    antiaromatic. Due to their closed-shell structures, annulenes with 4N + 2 -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 Hckel

    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):[6773]

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

    a) Exohedral addition reactions, including nucleophilic- and radical additions, cy-

    cloadditions, 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 endo-

    hedral fullerenes

    d) Ring-opening and fragmentation reactions, which could be used for subse-

    quent 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 nor-

    mally take place at [6,6]-bonds, whereby 1,2-additions (preferred addition) intro-

    duce eclipsing interactions between the addends. For a combination of sterically

    demanding addends, [1,4]- and [1,6]-addition can take place.

    RR

    RR R

    RR

    R

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

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

    Figure 1.10: Possible isomeric methanofullerenes and their relative energies from PM3 calcu-lations (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 [7779]

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

    [3+2]-cycloaddition using diazo compounds, azomethine ylides and azides [8289]

    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 [67,9193] and

    books.[68,94,95]

    O O

    O O

    Br

    NaH or DBUtoluene

    O O

    O O

    Br

    COOEt

    BrEtOOC

    COOEt

    COOEt

    3

    4

    5

    6

    Scheme 1.1: Cyclopropanation of C60 by nucleophilic addition of -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

    NaH in the presence of C60.[75] From a mechanistic point of view a two-step process

    is involved at this type of reaction. Deprotonation of the -bromomalonate 3 affords

    the -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 re-

    action protocol by preparing the -halomalonate in situ from the corresponding mal-

    onate 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 archi-

    tectures, which are accessible through exohedral functionalization of parent C60. The

    application of these compounds in material science and biomedicine should be consid-

    ered, 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

    -system with increasing number of addends, these amphiphiles should be C60-mono-

    adducts. 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 re-

    active oxygen species (ROS) in vitro and their ability to protect cells and tissue from ox-

    idative 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 suit-

    able functionalities onto the fullerene sphere, which allows to immobilize these deriva-

    tives 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 cy-

    clopropanation. 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 syn-

    thesis 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, no-

    tably 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 vari-

    ety 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 deriva-

    tives 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 am-

    phiphilic [3:3] hexakisadducts, using a trisadduct precursor with an e,e,e-addition pat-

    tern as starting material. For the completion of the octahedral addition pattern, DMA

    template-mediated cyclopropanation was accomplished with different polar and den-

    dritic ionic malonates. These new compounds were examined in detail for their ten-

    dency 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 prop-

    erties change, when the conjugated -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 differ-

    ent 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]

    HO OH

    O O

    DCC,DMAP

    O O OO O

    OO

    TFA

    O O OHO O

    O

    HO

    O

    O O OHO O

    O

    R1O

    O

    HO O OO O

    O

    R1O O OO O

    O

    DCC,DMAP

    R1O O OHO O

    O

    R1O O OR2

    O O

    OO O OR

    2O O

    O

    R1O

    O

    DCC,DMAP

    2 eq 1 eq

    R1OH

    TFADCC,DMAPR1OH

    DCC,DMAPR

    2OHDCC,DMAPR

    2OH

    O

    OHO O

    OHO

    O

    77

    8

    9

    10

    11

    12

    13

    14

    15

    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 bisfunc-

    tionalized by sequential reaction with different alcohols or amines. However, these ap-

    proaches led in a number of cases to separation problems and unsatisfactory yields. In

    a new approach, MELDRUMS 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.

    O O

    O O+ R1OH

    R1O OH

    O O

    R1 = (CH2)5CH3R1 = (CH2)17CH3R1 = CH((CH2)7CH3)2

    R1 = (CH2)5CH3 R1 = (CH2)17CH3 R1 = CH((CH2)7CH3)2

    130 C, 3 h

    16

    171819

    Scheme 3.2: New approach for the synthesis of unsymmetrical malonates, using MELDRUMSacid.

    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 MELDRUMS

    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 -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 iso-

    butene 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

    O

    O OR

    OKOH, BzBr R = H

    R = tBuH2SO4,

    Pd/C,H2

    HO O

    O

    DCC,DMAP

    O OR

    OR1O

    OOR1 = (CH2)5CH3R1 = (CH2)17CH3R1 = CH((CH2)7CH3)2

    R = tBuR = H

    TFA

    OO

    R1O

    OOO

    OR1O

    OO HN

    O

    O

    O

    O

    O O

    EDC, DMAP, 1-HOBt

    EDC, DMAP, 1-HOBt

    HN

    HN

    OHN

    O

    O

    O

    O

    O

    O

    OONH

    OO

    OO

    O

    O

    OO

    O O O

    O

    C60, CBr4, DBU C60, CBr4, DBU

    OO

    R1O

    OO HN

    OR

    O

    OR

    O

    O OR

    OO

    R1O

    OO HN

    HN

    OHN

    O

    O

    OR

    O

    OR

    O

    ORONH

    ORO

    ORO

    RO

    O

    ROO

    RO O OR

    O

    R = tBuR = H

    TFAR = tBuR = H

    TFA

    2021

    22

    17, 18, 19

    23, 24, 2526, 27, 28

    29 30

    31, 32, 33 34, 35, 36

    37, 38, 39 40, 41, 4243, 44, 45 46, 47, 48

    Scheme 3.3: Optimized multistep synthesis of the amphiphilic monoadducts 1st generation43, 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 [105107] gave the protected unsym-

    metrical 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 re-

    action 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 reprecipi-

    tation 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 repre-

    sented 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 impor-

    tant, 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

    80

    60

    40

    20

    0

    4.6E3

    3.7E3

    2.7E3

    1.8E3

    9.1E2

    0.0E0

    1900 2000 2100 2200 2300 2400 2500 2600 2700 2800

    1902 1970

    2107

    21612214 2273

    2387 2483 2548 261226762734 2858

    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 char-

    acteristic 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 conver-

    sion 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 am-

    phifullerene 44, the sp2-region contains 28 signals, whereas 3 signals show double

    28

  • Chapter 3 Results and Discussion

    0102030405060708090100110120130140150160170180

    0102030405060708090100110120130140150160170180

    0102030405060708090100110120130140150160170180

    Chemical Shift (ppm)

    12

    8

    13

    2,69 4

    7

    1

    12

    8

    13

    2,6

    9

    4 7

    10

    1

    11

    sp -2 C

    sp -3 C

    - H -C 2

    14

    14

    10 11

    12

    8

    9

    4

    71

    sp -C2

    sp -3 C

    - H -C 2

    10 11

    - H -C 21

    2

    3

    4

    5

    6 7 89

    10

    11 12

    1314

    1

    2

    3

    4

    5

    6 7 89

    10

    11 12

    1314

    3 5

    3 5

    3 5

    * *

    *

    *

    167.2 167.0

    164.3 164.1 163.9

    164.2 164.1 164.0

    167.1

    1

    2

    3

    4

    5

    6 7 8910 11

    12

    32

    38

    44

    Figure 3.2: 13C 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 genera-

    tion 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 in-

    tensity 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

    0102030405060708090100110120130140150160170180

    0102030405060708090100110120130140150160170180

    0102030405060708090100110120130140150160170180

    173.5 173.0 172.5 167.1 167.0 166.9

    173.4 173.0 164.2 163.8

    sp -2 C

    812

    163 5

    17

    2,6

    9,13

    47

    10,11

    14,15

    - H -C 2

    1

    18

    *

    812

    163 5

    17

    2,6

    9,13

    4 7

    - H -C 2

    1

    18*

    sp -2 C

    12

    3

    4

    5

    6 7 8 9 10

    11 1213 14

    15 16

    sp -2 C

    2,6

    9,13

    4 7

    1sp -3 C

    16

    8,12 3,5

    - H -C 2

    10,11

    14,15

    10,11

    14*

    12

    3

    4

    5

    6 7 8 9 10

    11 1213 14

    15 16

    1718

    12

    3

    4

    5

    6 7 8 9 10

    11 1213 14

    15 16

    1718

    15

    35

    41

    47

    Figure 3.3: 13C 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

    300 350 400 450 500 550 600

    ab

    sorb

    an

    ce

    wavelength [nm]

    44

    38 in CH2Cl244 in DMSO44 in H2O at pH = 7.2

    Figure 3.4: UV/Vis spectra of protected monoadduct 38 (black) and deprotected monoadduct44 in DMSO (red) and H2O (pH = 7.2) (blue).

    300 350 400 450 500 550 600

    ab

    sorb

    an

    ce

    wavelength [nm]

    47

    41 in CH2Cl247 in DMSO47 in H2O at pH = 7.2

    Figure 3.5: UV/Vis spectra of protected monoadduct 41 (black) and deprotected monoadduct47 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 (en-

    zymes) barriers. Thus the BBB is often the rate-limiting factor in determining perme-

    ation of therapeutic drugs into the brain. Overcoming the difficulty of delivering thera-

    peutic 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 ef-

    fective 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, includ-

    ing 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

    O

    OH

    + HO OH

    O

    O OH

    HO O

    O+

    HO OH

    OO

    O O

    OHO

    O O

    = R1

    R1 O O

    O OOR

    O

    OO

    O

    OO HN

    HN

    OHN

    O

    O

    O

    O

    O

    O

    OONH

    OO

    OO

    O

    O

    OO

    O O O

    O

    C60, CBr4, DBU

    OO

    O

    OO HN

    HN

    OHN

    O

    O

    OR

    O

    OR

    O

    ORONH

    ORO

    ORO

    RO

    O

    ROO

    RO O OR

    O

    O

    O

    R1

    R = tBuR = H

    TFA

    R = tBuR = H

    TFA

    EDC, DMAP, 1-HOBt

    EDC, DMAP

    EDC, DMAP

    EDC, DMAP

    49 50

    22 51

    50

    5253

    30

    54

    5556

    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 in-

    clude 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 considera-

    tions, 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 MELDRUMS 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 corre-

    sponding 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 sep-

    aration of these impurities with flash column chromatography failed in this case and

    made further purification by preparative HPLC necessary. Deprotection to 56 was ac-

    complished 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 iden-

    tified 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

    00.511.522.533.544.555.566.577.58

    4 3,5

    2,91

    Chemical Shift (ppm)

    *

    1

    2

    3

    4 5

    6

    7

    8

    9

    6,8 7

    55

    Figure 3.6: 1H 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 sig-

    nals 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 charac-

    teristic small absorption peak at 425 nm is not identifiable any more (figure 3.8, inset).

    36

  • Chapter 3 Results and Discussion

    0102030405060708090100110120130140150160170180

    Chemical Shift (ppm)

    16,20,24

    8,10

    sp -3 C

    6711

    17,21

    9 15123

    1

    2

    34

    6

    7

    8910

    11

    12

    13

    14

    15

    16

    5 17 18

    19 20

    21 22

    23 24

    2526

    25

    *

    55

    (a)

    139140141142143144145146

    (b)

    25262728293031323334

    4 18,19

    22,23

    26

    (c)

    Figure 3.7: 13C NMR of protected monoadduct 55 (75 MHz, RT, CDCl3) (a) and zoom intospecific 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

    300 400 500 600

    abso

    rptio

    n

    wavelength [nm]

    350 375 400 425 450 475 500

    55 in CH2Cl256 in buffered H2O (pH = 7.2)

    Figure 3.8: UV/Vis spectra of protected precursor 55 (black) and deprotected water-solubleamphiphile 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 po-

    tential 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 car-

    boxylic 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 [120123], prob-

    ably 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

    OO

    R1O

    OO HN

    OH

    O

    OH

    O

    OHO

    OO

    R1O

    OO HN

    O

    O

    O

    OH

    OH

    OH

    OO

    R1O

    OO HN

    O

    O

    O

    O

    OO

    Br

    Br

    Br

    OO

    R1O

    OO HN

    O

    O

    O

    O

    OO

    Br

    Br

    Br

    OO

    R1O

    OO HN

    O

    O

    O

    O

    OO

    N

    N

    N

    3 Br

    DCC, DMAP,1-HOBt,

    BrHODCC, DMAP,1-HOBt,

    CBr4, DBU,C60

    pyridine

    R1 = (CH2)17CH3

    Pathway A Pathway B

    44

    57

    58

    59

    60

    Scheme 3.5: Synthesis of the polycationic fullerene derivative 60. Pathway A: Synthesis viacarboxyfullerene 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 sat-

    isfying 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 depro-

    tected 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 chro-

    matography. 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 60C and was completed after 48 h in quantitative 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

    00.511.522.533.544.555.566.577.588.599.510

    00.511.522.533.544.555.566.577.588.599.510

    Chemical Shift (ppm)

    * *

    *

    1 2 3 4 67

    8

    9

    5

    12,3

    8 9

    4

    7

    8

    - H -C 2

    5

    1 2 3 4 67

    8

    9

    5

    1

    2,3,89

    4

    78

    5

    - H -C 2

    59

    60

    Figure 3.9: 1H NMR spectra of bromo-precursor 59 (400 MHz, RT, CDCl3) and cationicfullerene 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

    300 400 500 600

    abso

    rbance

    wavelength [nm]

    59 in CH2Cl260 in H2O

    60

    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 -system of C60 exhibits unusual po-

    tency 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 pro-

    duced water-soluble fullerenes with good biological distribution and cell penetration.

    [124131] 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 phar-

    maceutical 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 chem-

    ically homogeneous form.[109,132135] 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 car-

    boxyfullerenes 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-

    C63(COOH)6] 62.[134,135]

    HO

    OH

    O

    O

    OHHO

    O O

    OH

    OHO

    O

    HO

    OH

    O

    O

    OHHO

    O O

    OH

    OHO

    O

    O

    O

    O

    O

    O

    HN

    HN

    OHN

    O

    O

    OH

    O

    OH

    O

    OHONH

    OHO

    OHO

    HO

    O

    HOO

    HO O OH

    O

    O

    HN

    NH

    OHN

    O

    O

    OH

    O

    HO

    O

    HO O HN

    HOO

    HOO

    OH

    O

    OHO

    OHOHO

    O

    61 62

    63

    Figure 3.11: C3-[e,e,e-C63(COOH)6] 61, D3-[trans3,trans3,trans3-C63(COOH)6] 62 and den-drofullerene 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

    Parkinsons, Alzheimers, ALS (amyotrophic lateral sclerosis), excitotoxicity, mac-

    ular degeneration and stroke [138145]

    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 [147150]

    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 [151153]

    Although these surface-modified, water-soluble fullerenes have been shown to exhibit

    strong antioxidant activity against reactive oxygen species (ROS), the progress in de-

    veloping fullerenes as bona fide drug candidates has been hampered by three de-

    velopment issues: 1) Lack of methods for scalable synthesis; 2) inability to produce

    highly-purified, single-species regioisomers compatible with pharmaceutical applica-

    tions; and 3) inadequate understanding of structure-function relationships with respect

    to various surface modifications (e.g., anionic versus cationic versus charge-neutral po-

    larity, 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 degrada-

    tion, 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

    HO

    H

    O

    OHHO

    O O

    OH

    OHO

    O

    HO

    H

    O

    OHHO

    OH

    OHO

    O

    HO

    H

    O

    OHHO

    OH

    HO

    64 65 66

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

    Toxicological investigations disclosed, that these decomposition products exhibit a sig-

    nificantly 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 de-

    cided 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

    oxygen.[154] ROS include superoxide anion radical (O2 ), singlet oxygen (1O2), hydro-

    gen 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

    OO

    R1O

    OO HN

    OH

    O

    OH

    O

    O OH

    OO

    R1O

    OO HN

    HN

    OHN

    O

    O

    OH

    O

    OH

    O

    OHONH

    OHO

    OHO

    HO

    O

    HOO

    HO O OH

    O

    R1 = (CH2)5CH3

    R1 = (CH2)17CH3

    R1 = CH((CH2)7CH3)2

    R1 = (CH2)5CH3

    R1 = (CH2)17CH3

    R1 = CH((CH2)7CH3)2

    OO

    O

    OO HN

    O

    O

    O

    O

    O O

    N

    N

    N

    3 Br

    43

    44

    45

    46

    47

    48

    60

    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 cel-

    lular redox potential. ROS can cause tissue damage by reacting with lipids in cellu-

    lar 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 systems 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.[160162] 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 par-

    ticularly susceptible to the damaging effects of ROS. In neurodegenerative diseases

    like Parkinsons, Alzheimers 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 Alzheimers disease, Lewy bodies in Parkinsons disease and within the

    motor neurons in ALS.[163]

    An antioxidant is a molecule capable of deactivating these ROS. Well-known antioxi-

    dants include a number of enzymes (catalase, superoxide dismutase) and other sub-

    stances 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