Uwe Hartnagel

Water solubility, antioxidant activity and cytochrome C binding of four families of exohedral adducts of C60 and C70

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.

Cooperativity and tunable excited state deactivation: modular self-assembly of depsipeptide dendrons on a Hamilton receptor modified porphyrin platform.

A series of novel supramolecular architectures were built around a tin tetraphenyl porphyrin platform 6--functionalized by a 2-fold 1-ethyl-3-3-(3-dimethylaminopropyl)carbodiimide (EDC) promoted condensation reaction--and chiral depsipeptide dendrons of different generations 1-4. Here, implementation of a Hamilton receptor provided the necessary means to keep the constituents together via strong hydrogen bonding. Characterization of all architectures has been performed, including 4 which is the fourth generation, on the basis of NMR and photophysical methods. In particular, several titration experiments were conducted suggesting positive cooperativity, an assessment that is based on association constants that tend to be higher for the second binding step than for the first step. Importantly, molecular modeling calculations reveal a significant deaggregation of the intermolecular network of 6 during the course of the first binding step. As a consequence, an improved accessibility of the second Hamilton receptor unit in 6 emerges and, in turn, facilitates the higher association constants. The features of the equilibrium, that is, the dynamic exchange of depsipeptide dendrons 1-4 with fullerene 5, was tested in photophysical reference experiments. These steady-state and time-resolved measurements showed the tunable excited-state deactivations of these complexes upon photoexcitation.

Testing Electron Transfer within Molecular Associates Built around Anionic C60 and C70 Dendrofullerenes and a Cationic Zinc Porphyrin

Mono- and bis-functionalized C(60) and C(70) fullerene derivatives (DF, 1-10) that carry one or two oligoanionic dendritic termini in their malonate addends and an oligocationic octapyridinium zinc porphyrin salt (ZnP) were found to self-assemble in buffered aqueous solution to yield a novel series of 1:1 and/or 1:2 electron transfer hybrid associates. Remarkably high association constants-typically on the order of 10(8) M(-1)-were derived that corroborate stable complex formations. A combination of electrostatic and charge-transfer interactions that are operative between the electron-accepting DF and the electron-donating ZnP is considered to contribute to the uniquely high complex stability. First insight into intracomplex excited state interactions came from steady-state and time-resolved fluorescence quenching experiments that were performed with the molecular ZnP/DF hybrid associates. Excited state quenching processes are, for example, evident in form of a bi-exponential fluorescence decay of ZnP-corresponding to a distribution of associated and non-associated ZnP. Unambiguous evidence for an intracomplex electron transfer quenching, namely, formation of ZnP(.+)/C(60) (.-) and ZnP(.+)/C(70) (.-) radical ion pairs, was gathered in time-resolved transient absorption measurements. Lifetimes of these radical ion-pairs range from nanoseconds to a few microseconds.

Cytoprotective activities of water-soluble fullerenes in zebrafish models

Using zebrafish (Danio rerio) embryos as a model system, we compared the antioxidant and cytoprotective effects of a series of new water-soluble fullerenes 1–12. Since zebrafish embryos are transparent during the first week of life, the effects of fullerenes on multiple organ systems, including CNS, PNS, and heart, could be assessed in situ. Both positively charged and negatively charged water-soluble fullerenes were added at concentrations between 1 and 500 µM to 96 well plates containing zebrafish embryos at 24–120 hours post fertilization (hpf). Direct toxicity of each fullerene compound was assessed by LC50. In addition, we assessed the ability of each fullerene to protect against toxicity induced by known chemical toxins in this system. Four different drug/chemical toxicity models were used in our study: (i) protection of neuromast hair cells from gentamicin-induced toxicity; (ii) protection of neuromast hair cells from cisplatinum-induced toxicity; (iii) protection of tyrosine hydroxylase-containing dopaminergic CNS neurons from 6-hydroxydopamine toxicity; and (iv) protection of total CNS neurons from 6-hydroxydopamine toxicity. Our results indicate that (i) positively charged water-soluble fullerenes tend to exhibit greater toxicity than negatively charged fullerenes with similar structures; (ii) toxicity varies considerably among negatively charged fullerenes from very low to moderate, depending on structural features; (iii) dendrofullerenes 2–7 (monoadducts of C60) show stronger protection against cisplatinum toxicity in neuromast hair cells while then the e,e,e-trismalonic acid 1 (so called C3) shows stronger protection against gentamicin-induced cytotoxicity in the same cells; (iv) C3 (1) is relatively unstable in all aqueous solvents tested and breaks down mainly through decarboxylation reactions to form penta, tetra and tris carboxylated forms, which exhibit increased toxicity in vivo compared with C3 (1). Our findings indicate that water-soluble fullerenes can protect against chemical toxin-induced apoptotic cell death in a vertebrate, whole-animal model that may be useful for predicting the efficacy and toxicity of these compounds in mammals. Furthermore, the relative potential for pharmacologic use of these compounds varies considerably with respect to stability.

Electrophoresis of electrostatically assembled fullerene–porphyrin conjugates

The formation of electrostatically coupled assemblies of a series of anionic dendritic fullerene derivatives and cationic porphyrins in buffered aqueous media was studied with gel electrophoresis. Of central interest in these investigations was the variation of the amount of charge carried by the molecules, their size, shape and self-aggregation. Ferric cytochrome c 1 and the more rigid zinc porphyrin served as octacationic species. The two new anionic fullerene derivates 3 and 6 were synthesized. The formation of electrostatic complexes of the fullerene polyelectrolytes 3 and 4 with 1and 2 was clearly evident in the gel electrophoresis experiment. Compounds and showed a similar behaviour towards 2. The electrophoresis experiments confirmed previous results obtained with other techniques on a qualitative level and gave new insights into aggregation phenomena.

Reciprocal principle of molecular recognition in supramolecular chromatography—highly selective analytical separation of cyclodextrin congeners on a silica-bonded [60]fullerene stationary phase

The supramolecular pair fullerene/cyclodextrin represents a new example for the concept of reciprocal molecular recognition in liquid chromatography. Through inverting their roles as selectand and selector, small-to-large-ring cyclodextrins were chromatographed on silica-bonded [60]fullerene, whereas [60]- and [70]fullerenes were previously separated on a silica-bonded β-cyclodextrin. The recognition of cyclodextrins of intermediate ring size on silica-bonded [60]fullerene is highly selective. Thus, CD8, CD9 and CD10 are separated both from lower (CD6 and CD7) and higher (CD11–CD25) congeners with a remarkable and unprecedented retention window. By using the retention-increment method, employing a reactor column (with selector) and a reference column (without selector), apparent relative complexation constants Krel of CD6–CD12 and [60]fullerene were determined by supramolecular liquid chromatography. The historical development of the reciprocal principle of molecular recognition in chromatography is reported in the introduction.

A chiral dumbbell shaped bis(fullerene) oligoelectrolyte

The first icosacationic bis(fullerene) whose water solubility is provided by twenty peripheral ammonium groups has a rigid, almost cylindrical shape and was synthesized by connecting two fullerene cores with a chiral cyclo-bis(malonate), followed by the regioselective addition of ten amino-terminated malonates into the octahedral positions of the fullerene moieties and subsequent cleavage of the Boc-protecting groups.

Spreading behaviour of a series of amphiphilic [3 : 3] [60]fullerene hexakisadducts: a systematic study

The spreading behaviour at the air–water interface of a series of [60]fullerene [3 : 3] hexakisadducts has been studied as a function of the hydrophobic–hydrophilic balance of the amphiphiles. More precisely, by changing both the nature and the size of the polar headgroup, by varying the nature of the chemical linker and/or spacer, by modifying the nature and/or the length of the spacer, it has been possible to perform a systematic study. The latter was carried out not only according to the pH of the aqueous subphase but also to its temperature highlighted various behaviours which in particular allowed to us to conclude that the optimal chemical design for this type of amphiphilic molecules is reached with the couples amine polar head/ester linker (Hexa-4) or carboxylic acid polar head/amide linker (Hexa-5).

Carbon nanotube nanocomposites: quasi 1-dimensional structures for electron transfer

We integrated single wall carbon nanotubes (SWNT), several water-soluble pyrene derivatives, which either bear negatively (pyrene-) or positively charged (pyrene+) charged ionic headgroups, and a series of water-soluble metalloporphyrins (MP8+/MP8-) into functional nanohybrids through a combination of associative van-der-Waals and electrostatic interactions. The resulting SWNT / pyrene and SWNT / pyrene / MP were characterized by spectroscopic and microscopic means and were found to form stable nanohybrid structures in aqueous media. When the nanohybrid systems are photoexcited with visible light, a rapid intrahybrid charge separation causes the reduction of the electron accepting SWNT and, simultaneously, the oxidation of the electron donating MP. Beneficial are charge recombination dynamics that are located in the Marcus-inverted region.