Over the past decade, surface-modified, water soluble fullerenes have been shown by many different investigators to exhibit strong antioxidant activity against reactive oxygen species (ROS) in vitro and to protect cells and tissues from oxidative injury and cell death in vivo. Nevertheless, 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). To address these challenges, we have designed and synthesized more than a dozen novel water soluble fullerenes that can be purified as single isomers and which we believe can be manufactured to scale at reasonable cost. These compounds differ in addition pattern, lipophilicity and number and type of charge and were examined for their water solubility, antioxidant activity against superoxide anions and binding of cytochrome C. Our results indicate that dendritic water soluble fullerene[60] monoadducts exhibit the highest degree of antioxidant activity against superoxide anions in vitro as compared with trismalonate-derived anionic fullerenes as well as cationic fullerenes of similar overall structure. Among the higher adducts, anionic derivatives have a higher antioxidant activity than comparable cationic compounds. To achieve sufficient water solubility without the aid of a surfactant or co-solvent at least three charges on the addends are required. Significantly, anionic in contrast to cationic fullerene adducts bind with high affinity to cytochrome C.

Water-soluble fullerenes, in particular the tris-malonyl-C 60 derivative 1 (so-called C 3 ), [1] have been shown to exhibit strong antioxidant activity against reactive oxygen species in vitro and to protect cells and tissue from oxidative injury and cell death in vivo.[2] The ability to destroy the toxic superoxide O 2 C À was suggested to be responsible for fullerene antioxidant activity, [3] although its mechanism is still not clear. Dugan and co-workers offered evidence in support of a catalytic superoxide dismutation mechanism instead of direct radical attack on the C 60 moiety of 1, thus showing that it could act as a metal-free mitochondrial manganese superoxide dismutase (MnSOD) mimetic.[3a] They proposed the formation of a complex between C 3 and O 2 C À . Herein, we present for the first time clear and unambiguous evidence for a catalytic dismutation process, the key steps of which are successive O 2 C À oxidation, within an outersphere electron-transfer process, and fullerene-derivativemediated O 2 C À reduction. At the same time we are able to rationalize structure-property relationships by the systematic investigation of a series of stable, readily accessible, and nontoxic mono-and trisadducts 2-7 of C 60 . [2c, 4] This led to the identification of new lead compounds for neuroprotective applications with significantly improved superoxide dismutation activity.To describe the structure-reactivity relationship with respect to superoxide dismutase (SOD) activity, we first present the redox properties of 2-7. Cyclic voltammetry measurements [5] in DMSO have shown that in the potential range from 0 to À1 V (vs. a saturated calomel electrode, SCE) 2-6 undergo two reversible reductions, whereas 7 exhibits one reversible redox couple (Table 1, Figure 1, Figure S1 in the Supporting Information). The corresponding reduction potentials of the monoadducts 2-4 are significantly higher than those of trisadducts 5-7 and show a prominent charge dependence, especially for the first C 60 /C 60 C À redox couple, with the positively charged derivative 4 being the strongest electron acceptor. The observed redox potentials are considerably higher than expected for fullerene mono-and trisadducts.[6a] It seems that solvent effects (predominantly solvent polarity) [6b] and the amphiphilic nature of the attached addends, which facilitate micellar organization and therefore close C 60 -C 60 interaction of 2-7 in solution, are responsible for the positive shift of their redox potentials.Of special importance is the fact that the first reduction potentials of 2-4 are much higher than the oxidation potential of superoxide (À0.74 V vs. SCE in DMSO). This implies that [a] values obtained by using direct stopped-flow measurements in DMSO (0.06 % water) and an indirect cytochrome c assay (k McCF ) [b] in aqueous solution (pH 7.8).

We report here on the synthesis of three new prototypes (types I-III) of very large fullerene-based polyelectrolytes which can carry up to 60 charges on their periphery. All fullerene moieties incorporated in these macromolecular structures have an octahedral hexakisaddition pattern. Dumbbell-shaped icosacarboxylate 5 (type I), which can accumulate up to twenty negative charges, is very soluble in methanol as well as in neutral and basic water. On the other hand, Janus dumbbell 13 (type II) contains both positively and negatively chargeable fullerene building blocks and is very soluble in acidic and basic media. However, in the region of the isoelectric point at pH 6.0-6.5 it precipitates as a pale orange solid due to pronounced intermolecular Coulomb interactions. Giant heptafullerene 15 (type III) can store up to 60 positive charges in its periphery and is the largest molecular polyelectrolyte with defined three-dimensional structure.

Water-soluble fullerenes, in particular the tris-malonyl-C 60 derivative 1 (so-called C 3 ), [1] have been shown to exhibit strong antioxidant activity against reactive oxygen species in vitro and to protect cells and tissue from oxidative injury and cell death in vivo.[2] The ability to destroy the toxic superoxide O 2 C À was suggested to be responsible for fullerene antioxidant activity, [3] although its mechanism is still not clear. Dugan and co-workers offered evidence in support of a catalytic superoxide dismutation mechanism instead of direct radical attack on the C 60 moiety of 1, thus showing that it could act as a metal-free mitochondrial manganese superoxide dismutase (MnSOD) mimetic.[3a] They proposed the formation of a complex between C 3 and O 2 C À . Herein, we present for the first time clear and unambiguous evidence for a catalytic dismutation process, the key steps of which are successive O 2 C À oxidation, within an outersphere electron-transfer process, and fullerene-derivativemediated O 2 C À reduction. At the same time we are able to rationalize structure-property relationships by the systematic investigation of a series of stable, readily accessible, and nontoxic mono-and trisadducts 2-7 of C 60 . [2c, 4] This led to the identification of new lead compounds for neuroprotective applications with significantly improved superoxide dismutation activity.To describe the structure-reactivity relationship with respect to superoxide dismutase (SOD) activity, we first present the redox properties of 2-7. Cyclic voltammetry measurements [5] in DMSO have shown that in the potential range from 0 to À1 V (vs. a saturated calomel electrode, SCE) 2-6 undergo two reversible reductions, whereas 7 exhibits one reversible redox couple (Table 1, Figure 1, Figure S1 in the Supporting Information). The corresponding reduction potentials of the monoadducts 2-4 are significantly higher than those of trisadducts 5-7 and show a prominent charge dependence, especially for the first C 60 /C 60 C À redox couple, with the positively charged derivative 4 being the strongest electron acceptor. The observed redox potentials are considerably higher than expected for fullerene mono-and trisadducts.[6a] It seems that solvent effects (predominantly solvent polarity) [6b] and the amphiphilic nature of the attached addends, which facilitate micellar organization and therefore close C 60 -C 60 interaction of 2-7 in solution, are responsible for the positive shift of their redox potentials.Of special importance is the fact that the first reduction potentials of 2-4 are much higher than the oxidation potential of superoxide (À0.74 V vs. SCE in DMSO). This implies that [a] values obtained by using direct stopped-flow measurements in DMSO (0.06 % water) and an indirect cytochrome c assay (k McCF ) [b] in aqueous solution (pH 7.8).

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