High catalytic activity of dendritic C60 monoadducts in metal-free superoxide dismutation

Article abstract of DOI:10.1002/anie.200800008

(Figure Presented) Fullerene antioxidants: A direct correlation between the redox and structural properties of water-soluble fullerenes (see picture) and their reactivity towards superoxide is demonstrated. Some of the C₆₀ monoadducts examined act as superoxide dismutase mimetics. The findings establish a guide for designing fullerene derivatives to catalytically decompose the superoxide radical anion.

Full text of DOI:10.1002/anie.200800008

Angewandte
Chemie
DOI: 10.1002/anie.200800008
Fullerene Dendrimers
High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free
Superoxide Dismutation**
ˇ
´
´
´
Gao-Feng Liu, Milos Filipovic, Ivana Ivanovic-Burmazovic,* Florian Beuerle, Patrick Witte, and
Andreas Hirsch*
[a]
Water-soluble fullerenes, in particular the tris-malonyl-C60
derivative 1 (so-called C3),[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
O2Cꢀ 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 super-
oxide dismutation mechanism instead of direct radical attack
on the C60 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
complexbetween C3 and O2Cꢀ.
Herein, we present for the first time clear and unambig-
uous evidence for a catalytic dismutation process, the key
steps of which are successive O2Cꢀ oxidation, within an outer-
sphere electron-transfer process, and fullerene-derivative-
mediated O2Cꢀ 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 C60.[2c,4] This led to the
identification of new lead compounds for neuroprotective
applications with significantly improved superoxide dismuta-
tion activity.
Table 1: Redox potentials, catalytic rate constants (kcat), and IC50 values
obtained by using direct stopped-flow measurements in DMSO (0.06%
water) and an indirect cytochrome c assay (kMcCF)
[b] in aqueous solution
(pH 7.8).
1
[c]
[c]
Fullerene E1/2
2E1/2
IC50
kMcCF ”106 kcat ”106 Modified
[V]
[V]
[mm]
[mꢀ1 sꢀ1
]
[mꢀ1 sꢀ1
]
NBT
assay[d]
2
3
4
5
ꢀ0.248 ꢀ0.614 2.11ꢁ0.02 1.3ꢁ0.2
ꢀ0.224 ꢀ0.647 1.86ꢁ0.01 1.9ꢁ0.3
ꢀ0.077 ꢀ0.521 0.31ꢁ0.02 8.7ꢁ0.3
ꢀ0.433 ꢀ0.726 2.85ꢁ0.03 0.9ꢁ0.2
2.64ꢁ
0.04
+
+
+
+
4.29ꢁ
0.06
12.02ꢁ
0.22
0.26ꢁ
0.02
[e]
[e]
6
7
ꢀ0.436 ꢀ0.732
ꢀ0.585 ꢀ1.094
[a]IC 50 is the concentration of putative SOD mimic that induces a 50%
inhibition of the reduction of cytochrome c. [b]McCF =McCord–Frido-
vich, reference [12]. [c] Calibrated by the Fc +/Fc couple (0.43 V vs. SCE).
[d]The NBT assay was qualitatively applied; NBT =nitroblue tetrazolium.
[e]A precipitate was formed.
To describe the structure–reactivity relationship with
respect to superoxide dismutase (SOD) activity, we first
present the redoxproperties of 2–7. Cyclic voltammetry
measurements[5] in DMSO have shown that in the potential
range from 0 to ꢀ1 V (vs. a saturated calomel electrode, SCE)
2–6 undergo two reversible reductions, whereas 7 exhibits one
reversible redoxcouple (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
Figure 1. Cyclic voltammograms of 2 in DMSO purged with nitrogen
(a) and oxygen (g), and of pure DMSO purged with oxygen
(c). Conditions: [2]=0.5”10ꢀ3 m, [Bu4NBF4]=0.1m, T=298 K, scan
rate=0.2 Vsꢀ1
.
´
´
´
[*]M.Sc. G.-F. Liu, M. Filipovic , Dr. I. Ivanovic-Burmazovic
Department of Chemistry and Pharmacy
University of Erlangen-Nürnberg
dependence, especially for the first C60/C60Cꢀ redoxcouple,
with the positively charged derivative 4 being the strongest
electron acceptor. The observed redoxpotentials are consid-
erably higher than expected for fullerene mono- and trisad-
ducts.[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 C60–C60 interaction of 2–7 in solution, are responsible for
the positive shift of their redoxpotentials.
Egerlandstrasse 1, 91058 Erlangen (Germany)
Fax: (+49)9131-85-27387
E-mail: ivanovic@chemie.uni-erlangen.de
Dipl.-Chem. F. Beuerle, Dipl.-Chem. P. Witte, Prof. A. Hirsch
Department of Chemistry and Pharmacy & Interdisciplinary Center
for Molecular Materials (ICMM), University of Erlangen-Nürnberg
Henkestrasse 42, 91054 Erlangen (Germany)
E-mail: andreas.hirsch@chemie.uni-erlangen.de
[**]The authors gratefully acknowledge financial support from the DFG
within SFB 583.
Of special importance is the fact that the first reduction
potentials of 2–4 are much higher than the oxidation potential
of superoxide (ꢀ0.74 V vs. SCE in DMSO). This implies that
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Full þ O2 ! Fullꢀ þ O2
ð1Þ
ꢀ
DMSO, but also that it is possible in aqueous solution (E8(O2/
O2Cꢀ) = ꢀ0.4 V vs. SCE in water). Even in the case of the
more difficult to reduce trisadducts 5 and 6, this process could
be energetically feasible. Note that photoinduced C60Cꢀ is able
to reduce O2 in aqueous solution, thus generating O2Cꢀ in an
outer-sphere electron-transfer process, because its oxidation
potential is more negative (ꢀ0.56 V vs. SCE) than the
[7]
reduction potential of O2 and also more negative than the
corresponding potentials of 2–6 (Table 1).
The cyclic voltammograms of 2–5 in dioxygen-saturated
DMSO have revealed that in the presence of these fullerenes
the reoxidation wave of superoxide (electrochemically gen-
erated in situ) disappears. The current arising from reoxida-
tion of the fullerene anions diminishes, whereas the values of
corresponding fullerene anion reoxidation potentials remain
unaffected (Figure 1). This behavior clearly indicates that a
reaction between electrochemically generated superoxide
and the fullerene anions [Reaction (2)] takes place without
þHþ
Fullꢀ þ O2 ! ½Fullꢀ ꢂ O2ꢀꢃ
Full þ H O
ð2Þ
ꢀ
ƒƒ!
2
2
inducing chemical changes on the fullerenes. The superoxide
decomposition is also observed by applying much lower
(catalytic) concentrations of 2–5, whereas 6 and 7, independ-
ent of the applied concentrations, do not affect the superoxide
reoxidation. These experiments suggest not only that 2–5 and
their anions can react with O2Cꢀ, but also that the reaction has
a catalytic character.
To address this point, we further studied the reactions of
2–7 with a large excess of KO2 in DMSO containing a
controlled amount of water (0.06%), which was in excess of
the superoxide and fullerene concentrations.[5] Time-resolved
UV/Vis spectra (Figure 2) have shown that immediately after
mixing of a superoxide solution with a fullerene solution,
Figure 2. Time-resolved UV/Vis spectra for the reaction of 2
(5”10ꢀ5 m) with KO2 (1 mm) in DMSO at room temperature. A) Spec-
trum recorded before mixing (measurements in tandem cuvette).
B) First spectrum obtained after mixing (using a stopped-flow module)
followed by spectra recorded at time intervals of 10 s (total observation
time 30 min). Inset: control reaction without addition of the fullerene
followed over 2.5 h.
the electron transfer from O2Cꢀ to those fullerenes [Reac-
tion (1)] is not only strongly driven thermodynamically in
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rapid decomposition of O2Cꢀ (decrease of the absorbance in
the range of 240–330 nm within the dead time of the stopped-
flow instrument) is observed in the case of 2–5.[8] The products
of superoxide disproportionation, O2 and H2O2, were qual-
itatively detected in all four experiments.[5] The corresponding
unattached malonate addends themselves, as well as 6 and 7,
do not induce superoxide decomposition.[9]
interaction between O2Cꢀ and the functionalized region of the
C60 cage, which could include H binding with the three
OH groups of the malonate addends. Such an interaction,
however, could be essential for superoxide reduction and
stabilization of the resulting peroxide in the second step
[Reaktion (2)] of the SOD cycle (as was observed in the case
of metal-based SOD mimetics[5,11]).
The rapid process was quantified by following the
corresponding absorbance decrease at 270 nm in a series of
stopped-flow measurements, in which the catalytic concen-
tration of the studied fullerenes was varied. Application of a
microcuvette accessory, which reduced the dead time of the
instrument to 0.4 ms, enabled the observation of the fast
disappearance of the 270-nm absorption. This behavior could
best be fitted as a first-order process to obtain the character-
istic value of the rate constant kobs (sꢀ1). A good linear
Although widely used indirect SOD assays are not very
reliable in the case of enzyme mimetics (the direct stopped-
flow method is a better probe for SOD activity even though it
requires a nonaqueous medium),[5,10] for comparison we also
applied cytochrome c (cyt c)[12] (Figure 4a) and modified
NBTassays[5] to investigate the SOD activity of our fullerenes
correlation between kobs and the fullerene concentration was
[5,10]
observed, and the catalytic rate constants (kcat
)
were
determined from the slopes of the corresponding plots
(Figure 3, Table 1). This is the first time that the catalytic
SOD activity of fullerenes has been detected by using a direct
method. Interference with additional components that are
always present in indirect assays can also be ruled out.
Figure 4. a) Kinetics of the reduction of ferricytochrome c (550 nm)
without and with the fullerenes at room temperature. b) Production of
H2O2 by SOD (25 mm) and fullerenes (25 mm) from KO2 (500 mm;
pH 7.8; 378C).
Figure 3. kobs as a function of fullerene concentration for the reaction
between fullerenes and saturated KO2 in DMSO solution at room
temperature.
in an aqueous buffer in a manner utilized in the literature. The
trisadducts 6 and 7 show no SOD activity, whereas 2–5[13]
[5]
Importantly, our results show a clear dependence of the
SOD activity (kcat) of the fullerenes on their reduction
potentials, namely, the higher the reduction potential (ability
to be reduced by O2Cꢀ) the higher the SOD activity (Table 1).
This finding suggests that the electron transfer from O2Cꢀ to
the fullerene plays a key role in the overall catalytic
dismutation of superoxide. At the same time, although 6 has
almost the same reduction potential as 5, it does not induce
catalytic decomposition of O2Cꢀ, which additionally implies
that the architecture of fullerene derivatives plays an
important role, too. In contrast to 5, the capped structure of
6 most probably does not allow for a favorable attractive
exhibit activity with the catalytic rate constants (kMcCF)
reflecting the same trend as that determined by the direct
stopped-flow measurements (Table 1). None of the fullerenes
interfered with the cyt c assay by reoxidizing cyt cII to
cyt cIII.[14]
A lower solubility of 2–4 in water caused the somewhat
smaller kMcCF compared with kcat determined in DMSO. Upon
using an excess of superoxide over the putative SOD mimetic,
we also determined the formation of H2O2.[15] The SOD-
active fullerenes 2–5 yield more H2O2 as compared with the
amount produced by spontaneous dismutation of superoxide
in the aqueous buffer, in a similar fashion to the native
Angew. Chem. Int. Ed. 2008, 47, 3991 –3994
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3993

Communications
Lebovitz, A. Savouchkina, S. Sali, D. Guldi, N. Chronakis, A.
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enzyme (Figure 4b). The amount of H2O2 produced corre-
lated with the fullerene SOD activity. The capped trisadducts
6 and 7 had no effect on the formation of H2O2, which again
proves their lack of SOD activity.
[3] a) S. S. Ali , J. I. Hardt, K. L. Quick, J. S. Kim-Han, B. F.
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3676 – 3678; b) for the synthesis and characterization of 4, see
the Supporting Information.
In conclusion, we have shown by electrochemical, spec-
trophotometric, and submillisecond mixing UV/Vis stopped-
flow measurements, as well as by indirect SOD assays and
H2O2 detection, that 2–5 act as SOD mimetics. For the first
time we have demonstrated that there is a direct correlation
between the SOD activity of the water-soluble fullerenes and
molecular properties such as 1) reduction potential, 2) charge,
and 3) molecular structure. Monoadducts 2–4 of C60 have
been identified as very active new lead structures. In
particular, the positively charged monoadduct 4 is more
active than C3 (1) by one order of magnitude and approaches
the performance of natural Mn- and FeSODs.[3a] Its activity is
comparable to that of the highly active metal-containing SOD
mimics.[5] The high SOD activity of 4 can be explained by its
[5] For the experimental details regarding solution preparations,
instrumentation techniques, and cyt c and NBTassays, see: G.-F.
´
´
´
Liu, M. Filipovic, F. W. Heinemann, I. Ivanovic-Burmazovic,
Inorg. Chem. 2007, 46, 8825 – 8835.
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2003, 125, 12803 – 12809.
[8] Less prominent spectral changes at wavelengths higher than
350 nm, in the case of 2 and 3, were found to be caused by KOH
which is inevitably present in KO2. By using an electrochemically
generated superoxide solution these spectral changes were not
observed.
[9] At a much longer timescale the pyridinium groups of two
cationic dendrimers and 7 react stoichiometrically with KO2,
which results in a product with strong absorbance at 273 nm
(Figure S2). The same product is formed upon reaction with
KOH.
[10] a) D. P. Riley, W. J. Rivers, R. H. Weiss, Anal. Biochem. 1991,
196, 344 – 349; b) R. H. Weiss, A. G. Flickinger, W. J. Rivers,
M. M. Hardy, K. W. Aston, U. S. Ryanll, D. P. Riley, J. Biol.
Chem. 1993, 268, 23049.
[11] D. T. Sawyer, J. S. Valentine, Acc. Chem. Res. 1981, 14, 393 – 400.
[12] J. M. McCord, I. Fridovich, J. Biol. Chem. 1969, 244, 6049 – 6055.
[13] 5 has quite low, but still detectable, SOD activity.
[14] The time-resolved UV/Vis spectra for the reaction between
electrochemically reduced cyt c and the fullerenes under an
argon atmosphere were monitored over 30 min and 12 h. No
change in the redoxstate of cyt cII was observed. However, in the
entire spectral range a slow shift of the cyt c spectrum was
observed in the case of 5 and 6, which suggests that these
fullerenes induce precipitation of cyt c from the solution
(Figure S3). This finding is attributed to the somewhat higher
value of apparent kcat for 5 in comparison to the value obtained
in the stopped-flow experiment, by causing an additional
decrease of the absorbance at 550 nm (Figure S3).[5,11]
[15] Experimental details are given in the Supporting Information.
high redoxpotential, which facilitates its reduction by O Cꢀ
2
[Reaction (1)], and its positive charge, which through electro-
static interactions favors a closer contact between O2Cꢀ and
the C60 cage assisting in the superoxide reduction [Reac-
tion (2)].
In addition to the high activity, the monoadducts have a
number of advantages over C3 (1), as they are: 1) more stable,
2) considerably less toxic,[2d] and 3) can be readily produced in
large quantities. Knowing that upon irradiation solubilized
C60, its aggregates, and some water-soluble derivatives can
generate superoxide[7] (on which their potential application in
photodynamic therapy is based), it is crucial to understand
how fullerene derivatives should be designed in order to
either decompose or generate O2Cꢀ, depending on the
direction of their biomedical application. In that light our
results will certainly be of fundamental importance.
Received: January 2, 2008
Published online: April 14, 2008
Keywords: dendrimers · disproportionation · electron transfer ·
.
fullerenes · radical ions
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