Electrostatic complexation and photoinduced electron transfer between Zn-Cytochrome c and polyanionic fullerene dendrimers

Article abstract of DOI:10.1002/chem.200204680

Two dendritic fullerene (DF) monoadducts, 2 and 3, which can carry up to 9 and 18 negative charges, respectively, were examined with respect to electrostatic complexation with Cytochrome c (Cytc). To facilitate comprehensive photophysical investigations,

Full text of DOI:10.1002/chem.200204680

FULL PAPER
Electrostatic Complexation and Photoinduced Electron Transfer between
Zn-Cytochromec and Polyanionic Fullerene Dendrimers
[
a]
[b]
[b]
[c]
[a]
Martin Braun, Stefan Atalick, Dirk M. Guldi,* Harald Lanig, Michael Brettreich,
[
a]
[a]
[a]
[d]
Stephan Burghardt, Maria Hatzimarinaki, Elena Ravanelli, Maurizio Prato,
Rudi van Eldik, and Andreas Hirsch*
[
e]
[a]
Abstract: Two dendritic fullerene (DF) monoadducts, 2 and 3, which can carry up to
and 18 negative charges, respectively, were examined with respect to electrostatic
9
complexation with Cytochromec (Cytc).To facilitate comprehensive photophysical
investigations, the zinc analogue of Cytc (ZnCytc) was prepared according to a novel,
modified procedure.The association of ZnCyt c and DF, and consequential photo-
induced electron transfer within ZnCytc-DF from the photoexcited protein to the
fullerene, was proven by fluorescence spectroscopy and transient absorption
spectroscopy.These findings were also supported by circular dichroism as well as
by extensive molecular dynamics (MD) simulations.
Keywords: charge separation
dendrimers ¥ fullerenes ¥ metallo-
proteins
¥
Introduction
Mitochondrial cytochromec (Cytc) is a polycationic redox
protein that serves as a mediator for electron-transfer
processes in biological systems.We recently discovered that
partially deprotonated and therefore negatively charged
monolayers of amphifullerene 1, generated at the air/water
interface, show a strong coupling to Cytc, present in the
subphase, and lead to the formation of a 3 nm-thick protein
layer underneath the amphifullerene layer.[ The Cytc/full-
erene coupling is provided by electrostatic interactions
between the two oppositely charged entities.This important
finding prompted us to investigate the association and
1]
[
a] Prof.Dr.A.Hirsch, Dipl.Chem.M.Braun, Dr.M.Brettreich,
S.Burghardt, Dr.M.Hatzimarinaki, Dipl.Chem.E.Ravanelli
Institut f¸r Organische Chemie
Universit‰t Erlangen-N¸rnberg
Henkestrasse 42, 91054 Erlangen (Germany)
Fax : (‡49)91318526864
E-mail: hirsch@organik.uni-erlangen.de
[
[
b] Dr.habil.D.M.Guldi, S.Atalick
Radiation Laboratory
University of Notre Dame (USA)
[
c] Dr.H.Lanig
Computer-Chemie-Centrum
Universit‰t Erlangen-N¸rnberg (Germany)
d] Prof.Dr.M.Prato
Dipartimento di Scienze Farmaceutiche
Universit a¡ degli Studi di Trieste (Italy)
electronic interaction of dendritic fullerenes (DF) 2[2] and 3
with Cytc in homogeneous solutions.In contrast to the
amphiphilic hexakisadduct 1, water-soluble systems 2 and 3
contain only a single addend covalently attached to the C60
[
e] Prof.Dr.R.van Eldik
Institut f¸r Anorganische Chemie
Universit‰t Erlangen-N¸rnberg (Germany)
Chem. Eur. J. 2003, 9, 3867 ± 3875
DOI: 10.1002/chem.200204680
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
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D.M.Guldi, A.Hirsch et al.
FULL PAPER
monoadducts 2 and 3 represent oligoelectrolytes, whose
number of negative charges (n), which is regulated by the
degree of deprotonation of the peripheral carboxylic acid
groups, is expected to depend on the pH value of the aqueous
solution.Conceptually, the n values can vary between 0 and 18
for 2, and 0 and 9 for 3.At a given pH value, the n values of
these adducts should be very different and lead to a different
interaction with Cytc, which carries eight positive charges at
pH 7.[
7]
For the analysis of the electrostatic interactions between
Cytc and 2 and 3, respectively, it is mandatory to determine
the number of negative charges, n, on the corresponding
adducts as a function of pH.We used pH titrations to analyze
the range of pK values of the carboxylic acid groups and, at
a
the same time, to deduce additional information on the
aggregation behavior of 2 and 3.[ We discovered that, at the
physiological pH of 7.2, adduct 2 carries approximately
10]
1
6 negative charges, whereas 3 carries about seven negative
charges.
By virtue of electrostatic attractions, supramolecular Cytc-
DF complexes are assembled in buffered solutions upon
simple mixing of Cytc and DF components, namely 2 or 3.An
accurate and widely applied method to investigate such
complexes is fluorescence spectroscopy coupled with optical
transient absorption spectroscopy.The iron center in the
heme group of Cytc, however, exerts a significant deactivation
on the excited states of the Cytc porphyrin and, therefore,
necessitates replacement, for example, by zinc.In addition,
iron ions contain partially filled 3d orbitals and undergo
thermal redox reactions.This is in contrast to zinc( ii) ions
1
0
which have a 3d configuration.The inactivity of zinc-
substituted hemes in the electronic ground state, as well as
its properties in the excited state, renders ZnCytc a very
suitable candidate for fluorescence investigations.
ZnCytc is a previously reported derivative of Cytc.It is
widely investigated within the scope of conformational
fluctuation studies[
11]
and of complexation studies with
core.In monoadducts of C 60, the characteristic electron-
accepting behavior of the parent system is largely retained,
[12±15]
porphyrins and other metalloproteins.
The preparation
method of ZnCytc is based on the first method published by
while in hexakisadducts, such as 1, significant alterations of
[16, 17]
Vanderkooi et al.
Although several minor modifications
the fullerene character occur.[
3, 4]
As a consequence, the
of this method concerning buffer, workup, and purification
fullerene core within 2 and 3 provides a more sensitive probe
for the investigation of electrostatic interactions between Cytc
and anionic oligoelectrolytes.Hybridization of redox proteins
with molecules that modify their electron-transfer proper-
[11, 14, 18, 19]
are reported,
the crucial step of demetalation of the
heme group with anhydrous hydrogen fluoride (AHF), which
is difficult to handle, was always performed as described in the
[16, 20]
original literature.
To avoid these handling difficulties, a
[
5]
[6, 7]
ties and conformational behavior,
tant topic within bioorganic chemistry.
is currently an impor-
new method to remove the iron ion was developed that used
pyridinium-bound poly(hydrogen fluoride) which is both
commercially available and easy to handle.[
21, 22]
The two-step
preparation of ZnCytc starts with the demetalation of
III-
Results and Discussion
commercially available horse-heart ferricytochromec (Fe
Cytc) with pyridinium poly(hydrogen fluoride).In the second
step, zinc was inserted into the heme group of the cytochro-
mec.The thus-obtained ZnCyt c was characterized as descri-
bed in the literature.[
In principle, formation of 1:1 and 2:1 complexes may evolve
from the association of ZnCytc and 2.Considering a 2:1
complex for ZnCytc/2–see modeling below–we ran parallel
assays with ZnCytc/3, whose structure precludes a 2:1
stoichiometry.
The synthesis of the new water-soluble adduct 3 was
performed according to Scheme 1.Starting with the formation
of the mono-spacer malonate 8 by a new protective-group
17, 18]
[8]
strategy and subsequent cyclopropanation of C , the final
6
0
coupling of the polyamide dendron 11[ yielded the dendro-
fullerene precursor 12.Cleavage of the tert-butyl groups leads
to the target compound 3.The monoadduct 2 was synthesized
according to literature procedures.[ Both dendrofullerene
9]
2]
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Electrostatic Complexation between Fullerene Dendrimers and Cytochromec
3867±3875
Scheme 1.Synthesis of a new water-soluble dendrofullerene 3.DBU ˆ 1,8-diazabicyclo[5.4.0]undec-7-ene, DCC ˆ dicyclohexylcarbodiimide, HOBTˆ 1-
hydroxy-1H-benzotriazole, TFA ˆ trifluoroacetic acid.
In the associated ensembles (i.e., ZnCytc-DF) the strongly
fluorescent (F ꢀ 0.04) and energetically high-lying (E
ˆ
singlet
2
.1 eV) photoexcited ZnCytc state, is quenched by an
electron-transfer process involving the electron-accepting
C60 core in DF (vide infra).For example, at pH 72 . upon
À6
titration of a 2.6 Â 10 m solution of ZnCytc with DF (i.e.,
À5
either 2 or 3) in the concentration range 0.38 ± 3.5 Â 10 m, the
intensity of the ZnCytc emission decreases until a minimum
value is reached.Figure 1 documents the ZnCyt c emission
spectra for a few selected concentrations of 3.In the minimum
region the complexation of ZnCytc is assumed to be complete
with an effective ZnCytc-DF concentration of at least 95%.
In Figure 2 the I/I0 versus concentration relationship is
shown for an entire set of concentrations of 3.The fitting of
[
23]
such titration curves to a previously developed procedure
À6
Figure 1.Fluorescence spectra ( lexc ˆ 420 nm) of ZnCytc (2.6  10 m)
5
À1
5
À1
gave values of 1.7(Æ0.3) Â 10 m and 3.1(Æ0.4) Â 10 m for
the stability constants of ZnCytc-2 and ZnCytc-3, respective-
ly, at pH7.2. These values are in good agreement with
previously published stability constants of ZnCytc with fern
À5
and different concentrations of 3 (0.38 ± 3.5 Â 10 m) in H
2
O at room
2 4
temperature, pH 7.2, 0.05m Na HPO .Arrows indicate the decreased
fluorescence of ZnCytc with increasing concentration of 3.
[24]
[25]
cupriplastocyanin,
porphyrins.[
plastocyanin,
and several free-base
3, namely [ZnCytc] ꢁ [DF].These conditions are necessary to
guarantee a detectable fluorescence quenching, whereas
working at [ZnCytc] > [DF], does not lead to any observable
changes in the fluorescence spectra.Thus at [ZnCyt c] ꢁ [2],
26]
Note that in our fluorescence experiments the effective
ZnCytc concentrations are much smaller than those of 2 and
Chem. Eur. J. 2003, 9, 3867 ± 3875
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D.M.Guldi, A.Hirsch et al.
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This estimate is in good agreement with the value derived
9
À1
by Equation (2) (2.6 Â 10 s ), which uses the fluorescence
quantum yields F recorded for ZnCytc and ZnCytc-3.
‰
FꢂZnCytc† À FꢂZnCytc-C60†Š
k ˆ
(2)
‰
tꢂZnCytc†  FꢂZnCytc-C60†Š
Evidence for an electron-transfer scenario (ÀDG
ꢀ
ET
0
.9 eV), opposing to a less exothermic transduction of singlet
excited-state energy (ÀDG
ˆ 0.34 eV), was obtained
EnergyT
from transient absorption measurements with ZnCytc and
ZnCytc-DF, following 8 ns laser excitation at 532 nm.Differ-
ential absorption spectra of ZnCytc in an oxygen-free
aqueous solution reveal the growth of a near-infrared
absorption centered around 880 nm.Generally, a distinct
near-infrared absorption peak in this region is seen in the
triplet ± triplet absorption spectra of tetraphenylporphyrin-
based metalloporphyrins.Thus, we ascribe the transient to the
ZnCytc triplet state.In the visible region, additional maxima
at 575, 610, and 720 nm and minima at 540 and 580 nm
corresponding to ground-state bleaching of the zinc porphyrin
Q-band transitions, complete the transient characteristics.The
ZnCytc triplet transient decays by a first-order process with a
lifetime of 80 Æ 10 ms, regenerating the singlet ground state.
A different picture emerges for ZnCytc-DF.Immediately
following the short laser excitation into the ZnCytc×s Q bands
at 532 nm, spectral characteristics were monitored that
correspond to the attributes of the one-electron oxidized
form of ZnCytc and the one-electron reduced form of the C60
moiety of DF.Maxima in the visible (600 ± 700 nm) and the
near-infrared (1040 nm, see Figure 3) clearly attest to the
Figure 2. I/I
0
versus [3] relationship used to determine the association
2
constant.The fitted curve gives a c value of 0.00007.
formation of a 2:1 ZnCytc/2 complex is believed to be
precluded.Additional support for this assumption is obtained
from the I/I versus [2] relationship, for which we only found
satisfying fits (i.e., c at least 0.005) of the experimental data
0
2
by applying a 1:1 complex stoichiometry.
Prior to the addition of DF, the fluorescence signal of
ZnCytc is well fitted by a monoexponential decay, for which a
lifetime of 2.98 ns was determined in deoxygenated aqueous
solutions at pH 7.2. On addition of 2 or 3, a double-
exponential decay is observed with lifetimes of ꢀ0.26 ns and
2.98 ns, respectively. The two lifetimes are maintained
throughout the titration assay, with, however, a relative
increase in statistical weight of the short-lived component
on increasing the concentration of DF until the plateau region
is reached.Addition of acid restores the original fluorescence
intensity, concomitant with the disappearance of the short-
lived component.
In summary, the decrease in fluorescence intensity in
ZnCytc on addition of DF, and the presence of only one
short lifetime (0.26 ns), which is invariant throughout the
titration assay, suggest a static quenching event inside the
well-defined ZnCytc-DF complexes.The restoration of the
original fluorescence and the disappearance of the short-lived
component upon addition of acid, on the other hand, result
from the protonation of the carboxylic groups and the
consequent dissociation of ZnCytc-DF into separate frag-
ments.
.
‡
.À
successful formation of ZnCytc -C60 , for which we deter-
mined a lifetime of 1.8 microseconds in deoxygenated aque-
ous solutions.The only appreciable changes seen upon
varying the concentration of 2 or 3 were different yields of
.
‡
.À
the ZnCytc -C60 radical pair.
In the ZnCytc-DF associate, the fluorescent state of
ZnCytc, which is a good electron donor (E ˆ 0.8 V versus
1
/2
NHE),[
27]
is quenched by a thermodynamically allowed
Figure 3.Transient absorption spectrum (NIR part) of ZnCyt c (2.6 Â
electron transfer (ÀDG ꢀ0.9 eV) to the electron-accepting
ET
À6
À5
1
0
m) and 3 (3.5 Â 10 m) in H
2
O at pH 7.2, recorded 50 ns after a 8 ns
C
moiety of DF (2: E ˆ À0.44 V versus NHE; 3: E ˆ
60
1/2
1/2
.À
laser pulse (lexc ˆ 532 nm), which shows the characteristic C60 fingerprint
with lmax ˆ 1040 nm.
[28]
À0.5 V versus NHE).
The rate constant for the electron-transfer quenching
process is obtained from Equation (1), where t and t are
0
the short-lived (0.26 ns) and long-lived (2.98 ns) fluorescence
An elegant means to control the ZnCytc-DF association is
to vary the pH and/or the ionic strength of the medium.At
pH 6 or 7.2 and by the use of the same C60 increments of DF,
the fluorescence is, for example, stronger relative to that seen
at pH 8.4. This implies less effective interactions between the
two redox-active moieties at more acidic pH values.This
components, respectively.The value of k was calculated to be
9
À1
3
.5 Â 10 s .
1
1
k ˆ
À
(1)
t
t
0
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Electrostatic Complexation between Fullerene Dendrimers and Cytochromec
3867±3875
assumption is further corroborated by a weaker stability
5
À1
constant (ZnCytc-3; pH 6: 1.7(Æ0.3) Â 10 m ).In contrast, at
a more alkaline pH, a higher stability constant (ZnCytc-3;
5
À1
pH 8.4: 3.9(Æ0.5) Â 10 m ) reflects the stronger interactions.
Similarly, ionic strengths of 0.1 ± 0.01m (i.e., Na HPO -
2
4
based solutions) effected the ZnCytc ± DF association.We
noticed differences in K as large as one order of magnitude
4
À1
(
ZnCytc-3; 0.05m Na HPO : 3.9(Æ0.1) Â 10 m ).Consider-
2
4
2À
ing that HPO4 , despite its double charge, possesses a large
radius, studies of smaller and single-charged anions were
deemed necessary.To explore the function of charge and size,
we probed solutions of 0.1m chloride and 0.1m bromide, while
À
keeping the cation (i.e., sodium) constant. The smaller Cl is
more effective in influencing the ZnCytc ± DF association,
Figure 4.a) Circular dichroism spectra of ZnCyt c, b) difference spectra of
ZnCytc-2 hybrids (complexed minus uncomplexed) with 3.6-fold excess of
2À
relative to what we determined for HPO4 : a factor of ꢀ2
was found at the same (0.1m) ionic strength.On the other
hand, the differences seen between chloride and bromide are
insignificant.
As far as the fluorescence lifetime of ZnCytc-3 is con-
cerned, the presence of the different ions has no impact on the
chromophore deactivation.
2
, and c) 13-fold excess.
variation of the preparation method of ZnCytc or by the
complexation with 2 was found, since the protein-dominated
region of the CD spectra at 200 to 260 nm is not affected.[
Previous NMR spectroscopic studies also support the fact that
the replacement of iron by zinc in Cytc does not cause
perturbation of the protein conformation.[
18]
Contrary to what happens for 2 or 3, a complementary assay
performed with a positively charged trans-2 bis-pyrrolidini-
30]
[
29]
um
salt 13 did not lead to quenching of the ZnCytc
By interaction of Cytc with 2, different complex structures
are possible.The distinct 1:1 and 2:1 complexes (Figure 5)
were investigated by MD simulations.We used corrected
X-ray structure data[ and a semiempirically optimized
fluorescence.The reason for this different behavior relates to
31]
the repulsive forces that dominate the interactions between
the two fragments.On the nanosecond scale, evidence for the
longer-lived triplet excited state is gathered, which then–on
the lower microsecond timescale–starts to decay.The triplet
decay depends linearly on the bis-pyrrolidinium salt concen-
tration and infers an intermolecular deactivation of ZnCytc.
For the reaction of photoexcited ZnCytc with 13, an
Figure 5.Different possible structures of Cyt c-2 hybrids.
8
À1 À1
intermolecular electron-transfer rate constant of ꢀ10 m
s
model of 2.The cytochrome molecule carries a net charge of 8
at pH7.For the fullerene, almost complete deprotonation of
the carboxylic end groups of the dendrimer is assumed on the
basis of the titration experiments, which leads to a highly
negative net surface charge (Figure 6a).Both molecules were
separately preoptimized by a subsequent geometrical force-
field optimization and MD simulation over a period of 510 ps.
The conformation of the protein remains the same during
preoptimization, whereas the charges on the dendrimer
sidechains of the fullerene cause a strong electrostatic
repulsion and lead to widely extended dendrimer branches.
First, we determined whether there is a energetically favored
complexation site found for protein ± protein complexes at the
heme edge region,[ or if the strong electrostatic attraction
was determined.
To elucidate further details on the structure of the Cytc-2
complexes, circular dichroism (CD) spectra and molecular
dynamics (MD) simulations were carried out.CD spectro-
scopy demonstrated that Fe was successfully replaced by Zn
since a spectrum (Figure 4) identical to that reported in the
literature was obtained.[ The complexation of ZnCytc with 2
itself is demonstrated by the difference in the CD spectra of
parent ZnCytc and ZnCytc-2 (Figure 4) which is comparable
to the changes in the CD spectra observed by the complex-
ation of free base uroporphyrin and tetrakis(4-carboxyphe-
18]
[26]
nyl)porphyrin with cytochromec. No indication for signifi-
32]
cant changes of the protein conformation, either by the
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D.M.Guldi, A.Hirsch et al.
FULL PAPER
with 2 directed towards the preferred site for the protein ±
protein complexes[ is shown in Figure 6b.The simulations
revealed no significant difference between the different
complexation sites with respect to the observed binding
energy or the number and nature of the formed interactions.
In addition to the expected salt bridges between the
carboxylate end groups of 2 and the charged lysine residues
of Cytc, several other interactions with the protein×s sidechain
as well as with its backbone were observed (Figure 7).
Analogous MD simulations of the formation of the 2:1
complexes (Figure 6c) revealed qualitatively the same strong
complexation with an over supply in electrostatic interactions.
Interestingly, 1:1 complexes of Cytc and 3 show an even
stronger interaction than 1:1 complexes with 2.The complete
spatial adaptation of the single dendritic branch of 3 to the
surface of the Cytc seems to be facilitated compared to the
two dendritic branches of 2.The major limitation of these
calculations carried out in the gas phase is the irreversible
formation of the electrostatic interactions between Cytc and
DF.However, the gas phase conditions provide the possibility
to evaluate many different interaction patterns of such
complexes on a reasonable timescale.More detailed simu-
lations of the complexes in a periodic water box are currently
under way.
32]
Conclusion
On account of the pronounced electrostatic interaction
between the positively charged ZnCytc and the polyanionic
dendrofullerenes (DF) 2 and 3, stable protein ± fullerene
hybrids are formed.Both the dendrofullerenes and the
protein contain redox-active chromophores that can be used
as reporter moieties for the detection of hybridization and
subsequent electronic interaction within the complexes.If a
large excess of DF is used, fluorescent measurements revealed
the 1:1 hybrids ZnCytc-2 and ZnCytc-3.The association
5
À1
constants were determined to be ꢀ10 m .The hybridization
to ZnCytc-2 and ZnCytc-3 causes fluorescence quenching of
the porphyrine emission within ZnCytc.The reason for the
fluorescence quenching is a photoinduced electron transfer
between the redox protein and the fullerene chromophore.[
Our photophysical results prove unequivocally that the
connection between ZnCytc and DF (either 2 or 3) is affected
by the pH and by the ionic strength of the medium, which
leads to an acute control over the number of protonated/
deprotonated carboxylate groups of DF.The hybridization
leading to ZnCytc-2 and ZnCytc-3 was further investigated by
CD spectroscopy and molecular modeling.This new hybrid-
ization concept that uses the fullerene chromophore as a
reporter unit has a great potential for the investigation of
electronic and functional properties of a broad range of
charged proteins.Moreover, the discovery of very strong
electrostatic interactions between the protein and charged
dendrimers can be used for the self-assembly of molecular
electronic devices.The investigation of layer-by-layer assem-
blies of Cytc and the dendrofullerenes 2 and 3 leading to
photoconductive nanomaterials are currently underway.
33]
Figure 6.a) Optimized structure (PM3) of 21 in the gas phase, trans-
lucent surface net charge (red).b) Result of the MD simulation of the
hybrid Cytc-2 with the dendrofullerene 2 directed towards the preferred
site for the protein ± protein complexes.[ The fullerene, dendrimer, and
heme moieties are colored purple, red, and green, respectively.The protein
backbone is shown in blue.c) Result of the MD simulation of the hybrid
Cytc-2 (2:1 stoichiometry).
6À
25]
does not allow for a specific site selection.Therefore, we
compared the number of salt bridges as well as the total
energy of six different complexes after MD simulation (see
the Experimental Section).In the starting geometry of these
six complexes, 2 was located at six cubic sites at a distance of
3
0 ä to the protein.The resulting complex of the simulation
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Electrostatic Complexation between Fullerene Dendrimers and Cytochromec
3867±3875
Preparation of dendrofullerene 3
-Benzyloxybutyric acid (5): A solu-
tion of benzyl bromide (20 mL,
68 mmol) and g-butyrolactone
4
1
(
(
3.19 mL, 41.8 mmol) in toluene
75 mL) was treated with freshly
crushed 85% KOH (10 g, 178 mmol).
The suspension was stirred under
reflux for three days, then diluted with
Et
aqueous layer was washed with Et
2 Â 40 mL).The Et O/toluene layers
were set aside.The aqueous layer was
acidified with 3m H SO (40 mL) and
the product extracted into CH Cl
The organic layer was dried over
anhydrous MgSO , filtered, and con-
2 2
O (40 mL) and H O (75 mL).The
2
O
(
2
2
4
2
2
.
4
centrated under reduced pressure to
yield a yellowish oil (2.11 g; 26%).
Figure 7.Examples for hydrogen bonding in the Cyt c-DF complexes (average length of hydrogen bonds: 1.75 ä).
The Et
trated to ꢀ80 mL.The solution was
treated with a solution of NaOH (4 g) in H O (20 mL) and heated to reflux
overnight.After hte mixture had been cooled, the aqueous layer was
diluted with H O (50 mL) and extracted with Et
O (3 Â 20 mL).The
aqueous layer was acidified with a slurry of H SO (5 mL) in ice (20 mL)
and extracted with CH Cl
2
O/toluene layer was concen-
Experimental Section
2
2
2
General: H NMR and 13C NMR: Jeol JNM EX400 and Jeol JNMGX 400.
MS: Varian MAT311A (EI), Micromass ZabSpec (FAB).UV/VIS:
Shimadzu UV3102 PC.TLC: Merck, silica gel 60F 254.Reagents used were
commercially available reagent grade.Solvents were distilled and dried
according to standard procedures.Products were isolated where possible by
flash column chromatography (silica gel 60, particle size 0.04 ± 0.063 nm,
1
2
4
2
2
(3 Â 30 mL).The organic layer was dried over
anhydrous MgSO
4
, and filtered.After removal of the solvent, 5 .1 9 g (64%)
1
of product were obtained.The overall yield was 73. 0 g (90%). H NMR
(
400 MHz, CDCl
3
, RT, TMS): d ˆ 1.97 (m, 2H; CH
2
CH
2
CH
2
), 2.50 (t,
),
3
3
J(H,H) ˆ 7.15 Hz, 2H; CH
2
COOH), 3.55 (t, J(H,H) ˆ 6.05, 2H; OCH
2
ICN).Compounds
procedures.
5 and 7 were prepared by modified literature
4
.53 (s, 2H; PhCH
2
), 7.36 (m, 5H, Ph), 8.90 ppm (brs, 1H; COOH);
C NMR (100.5 MHz, CDCl CH CH ), 30.72
, RT, TMS): d ˆ 24.59 (CH
(CH COOH), 68.82 (OCH ), 72.66 (PhCH ), 127.4, 128.0,128.2, 138.0 (Ph),
179.0 ppm (COOH).
tert-Butyl 4-benzyloxybutyrate (6): A solution of 4-benzyloxybutyric acid
5, 6.96 g, 35.8 mmol) in dry CH Cl (30 mL) was treated with condensed
isobutene (30 mL at À408C) and H SO (1 mL).The resultant solution was
stirred for three days at room temperature, then neutralized with a solution
of K CO (5 g) in H O (100 mL).The organic layer was washed with K CO
(saturated solution), citric acid (10 wt% in H O), and H O.After the
, filtered, and concentrated, a
[
34, 35]
13
3
2
2
2
Preparation of ZnCytc: This procedure represents a modification of the
standard given in the literature and several later alterations.[
step preparation of ZnCytc includes the demetalation of the commercially
available horse-heart FeCytc (Equus caballus) and the insertion of zinc into
the heme group of Cytc.Horse-heart cytochrome c was purchased from
Fluka.
2
2
2
16, 17]
The two-
(
2
2
2
4
Metal-free porphyrin Cytc: FeCytc (103 mg) was dissolved in Py(HF)
pyridinium poly(hydrogen fluoride, 6 mL) in a plastic vessel (e.g. PE)
inside a strong fumehood at room temperature.The color of the solution
changed almost immediately to dark purple.After the mixture had been
x
2
3
2
2
3
(
2
2
mixture was dried over anhydrous MgSO
4
1
yellow oil was obtained (7.31 g; 81.4%). H NMR (400 MHz, CDCl
3
, RT,
3
stirred with a Teflon stirring bar for 10 min at ambient temperature,
NH OAc buffer (3 mL, 50 mm) was added and the solvent was then
removed by a stream of nitrogen.The remaining solid was redissolved in
NH OAc buffer (6 mL, 50 mm) and purified by gel permeation chroma-
tography with a Sephadex (G-50 ™fine∫) column with the same buffer as the
mobile solvent.The main fraction comprising the free-base porphyrin Cyt c
was eluted first (like normal FeCytc, which has the similar molecular size).
After the first sharp fraction, a small amount remained in the tailing of this
band; this was discarded.Finally the sample fraction was concentrated to a
small volume (ꢀ2 mL) and dialyzed with aid of dialysis tubes against
phosphate buffer (pH 7.2) for 1 h. The molecular weight cut-off of the
TMS): d ˆ 1.42 (s, 9H; tBu), 1.89 (m, 2H; CH
2
CH
2
CH
2
), 2.32 (t, J(H,H) ˆ
3
7.15 Hz, 2H; CH
2
CO
2
tBu), 3.48 (t, J(H,H) ˆ 6.05 Hz, 2H; OCH
2
), 4.48 (s,
, RT,
tBu), 69.14
3 3
) ), 127.41, 127.48, 128.23, 138.35
4
1
3
2H; PhCH
TMS): d ˆ 25.12 (CH
(OCH ), 72.73 (PhCH
(Ph), 172.7 ppm (CO
1730, 1496, 1455, 1392, 1366, 1317, 1254, 1154, 1107, 1029, 959, 908, 847, 736,
698, 612 cm
2
), 7.31 ppm (m, 5H; Ph); C NMR (100.5 MHz, CDCl
CH CH ), 27.97 (CH ), 32.17 (CH CO
), 79.94 (C(CH
3
4
2
2
2
3
2
2
2
2
2
tBu); IR (NaCl): nÄ ˆ 3065, 3031, 2976, 2863, 1778,
À1
.
tert-Butyl 4-hydroxybutyrate (7): A solution of 6 (7.31 g, 29.2 mmol) in dry
ethanol (70 mL) was treated with 10% palladium on carbon (1.22 g) and H2
at room temperature.After 15 h, the suspension was filtered through Celite
and concentrated under reduced pressure to give an oily product in
dialysis membranes was 5000.UV/Vis (H
2
O) of free-base porphyrin Cytc:
max (e) ˆ 274 (36600), 403 (81400), 504 (18700), 539 (16200), 569 (14400),
22 nm (12000).
l
quantitative yield.
1
H NMR (400 MHz, CDCl , RT, TMS): d ˆ 1.36 (s, 9H;
3
6
tBu), 1.75 (m, 2H; CH CH CH ), 2.25 (t,
3
J(H,H) ˆ 7.15 Hz, 2H;
2
2
2
3
CH
HOCH
2
CO
2
tBu), 2.79 (brs, 1H; OH), 3.56 ppm (t, J(H,H) ˆ 6.05 Hz, 2H,
Insertion of zinc: The metal-free porphyrin Cytc (90 mg) was dissolved in a
minimum volume of phosphate buffer and the pH value of the solution was
lowered to 2.5 with glacial acetic acid. ZnCl or ZnAc was added in an
2 2
approximately 10-fold molar excess, and the mixture was stirred for 30 min
in a water bath at 508C to complete the reaction.The reconstitution can be
monitored by the characteristic bands in the visible absorption spectrum.
13
2
);
CH CH
C(CH ), 173.36 ppm (CO
3; elemental analysis calcd (%) for C
found: C 59.90, H 10.21.
C
NMR (100.5 MHz, CDCl
3
,
RT, TMS): d ˆ 27.75
(
CH
2
2
2
), 27.92 (CH
3
), 32.21 (CH
2
CO
2
tBu), 61.75 (HOCH
tBu); MS (EI): m/z: 160 [M] , 130, 105, 87, 57,
2
), 80.32
‡
(
3
)
3
2
4
8 16 3
H O (160.2): C 59.97, H 10.07;
After the pH was adjusted to 6 with a saturated solution of Na
ZnCytc was purified by gel filtration.The first and the last portions of the
sharp band eluted almost at the front were discarded.Dialysis followed by
lyophilization yielded a purple solid, which can be stored at À308C in the
dark for several months.Since both metal-free porphyrin Cyt c and ZnCytc
seem to be light-sensitive and less stable then FeCytc, all procedures should
2
HPO
4
,
Malonate 8: A solution of methyl malonyl chloride (0.67 mL, 6.3 mmol) in
dry dichloromethane (5 mL) was added dropwise to a cooled solution
(08C) of 7 (1.0 g, 6.3 mmol) and triethylamine (0.87 mL, 6.3 mmol) in dry
dichloromethane (50 mL) under a nitrogen atmosphere.The mixture was
stirred for 2 h at room temperature and then concentrated, and the product
was isolated by flash chromatography (silica gel, hexane/ethyl acetate 3:2)
1
be performed under exclusion of light.UV/Vis (H
67 (80300), 356sh (42800), 421 (228000), 547 (17000), 581 nm (11700).
The total yield of the two steps after lyophilization is 54%.
2
O) of ZnCytc: lmax (e) ˆ
and dried in a vacuum. Yield: 1.3 g (5.0 mmol) 80%; H NMR (400 MHz,
2
CDCl
3
, RT, TMS): d ˆ 1.46 (s, 9H; tBu), 1.96 (m, 2H; CH
2
), 2.32 (t,
),
3
J(H,H) ˆ 7.33 Hz, 2H; CH
2
COO), 3.40 (s, 2H; CH ), 3.77 (s, 3H; OCH
2
3
Chem. Eur. J. 2003, 9, 3867 ± 3875
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
3873

D.M.Guldi, A.Hirsch et al.
FULL PAPER
3
); 13C NMR (100.5 MHz, CDCl
), 27.93 (tBu), 31.58 (CH COO), 41.13 (CH
); 13C NMR (100.5 MHz, D
4
.20 ppm (t, J(H,H) ˆ 6.41 Hz, 2H; OCH
2
3
,
),
4.60 ppm(m, 2H; OCH
RT): d ˆ 25.45 (CH ), 27.26, 27.73, 28.02, 28.125, 29.01 (CH
spacer), 30.52 (CH CON), 46.30 (C60CCOO), 55.22 (OCH ), 55.60 (C(CH
), 58.41 (OCH ), 66.27 (sp -C60), 71.61 (sp -C60), 138.37, 139.46 140.26,
140.36, 140.49, 141.05, 141.13, 141.30 142.09, 142.54, 143.30, 143.56, 158.82
2
2
O ‡ K
2
CO
dendron and
3
(pH 7),
RT, TMS): d ˆ 23.84 (CH
2
2
2
2
2
5
(
2.35 (OCH ), 64.43 (OCH
3
2
), 80.36 (C(CH
3
)
3
), 166.30 (CˆO), 166.80
2
3
2
-
‡
3
3
CˆO), 171.87 (CˆO); MS (FAB, NBA): m/z: 260 [M] ; elemental analysis
)
3
2
calcd (%) for C12
20 6
H O (260.3): C 55.37, H 7.74; found: C 55.10, H 7.73.
(
1
2
CˆO), 159.30 (CˆO), 172.55 (CˆONH dendron), 172.96 (CˆONH spacer),
Spacer monoadduct 9: DBU (0.2 mL, 1.33 mmol) was added dropwise to a
solution of C60 (1.0 g, 1.38 mmol), malonate 8 (0.3 g, 1.20 mmol), and
tetrabromomethane (0.4 g, 1.20 mmol) in dry toluene (1 L) under a
nitrogen atmosphere.The reaction mixture was stirred at room temper-
ature for 20 h, and the progress of the reaction was monitored by TLC.The
product was isolated by flash chromatography (silica gel, toluene) and
80.31 ppm (COOH); UV/Vis (H
2
O, pH 7.2 phosphate buffer): lmax (e) ˆ
54 (88000), 322 (29000), 424 (2500), 466 nm (1700); MS (FAB, NBA):
‡
m/z: 1840 [M] .The purity ( >98%) was determined by NMR spectro-
scopy.
Photophysics: Fluorescence lifetimes were measured with a Laser Strobe
Fluorescence Lifetime Spectrometer (Photon Technology International)
with 337 nm laser pulses from a nitrogen laser fiber-coupled to a lens-based
T-formal sample compartment equipped with a stroboscopic detector.
Details of the Laser Strobe systems are described on the manufacturer×s
web site, http://www.pti-nj.com. Emission spectra were recorded with a
SLM8100 Spectrofluorometer.The experiments were performed at room
temperature.Each spectrum represents an average of at least five
individual scans, and appropriate corrections were applied whenever
necessary.Nanosecond to millisecond laser flash photolysis experiments
were performed with laser pulses from a Quanta-Ray CDR Nd: YAG
system (532 nm, 6 ns pulse width) in a front-face excitation geometry.A Xe
lamp was triggered synchronously with the laser.A monochromator
(SPEX) in combination with either a Hamamatsu R5108 photomultiplier
or a fast InGaAs diode was employed to monitor transient absorption
spectra.
dried in a vacuum.Yield: 270 mg (0 2. 76 mmol) 24%, red brownish solid;
1
H NMR (400 MHz, CDCl
3
, RT, TMS): d ˆ 1.49 (s, 9H; tBu), 2.16 (m, 2H;
3
CH
4
2
), 2.46 (t, J(H,H) ˆ 7.36 Hz, 2H; CH
2
COO), 4.13 (s, 3H; OCH ),
3
3
13
.56 ppm (t, J(H,H) ˆ 6.34 Hz, 2H; OCH
2 3
); C NMR (100.5 MHz, CDCl ,
RT, TMS): d ˆ 24.11 (CH
60CCOO), 54.02 (OCH
0.77 (C(CH
2
), 28.14 (tBu), 31.69 (CH
2
COO), 51.96
3
3
(
C
3
), 66.34 (OCH
2
), 71.44 (sp -C60), 77.20 (sp -C60),
8
1
1
1
3
)
3
), 138.87, 139.19, 140.96, 140.99, 141.91, 141.93, 142.21,
42.97, 143.02, 143.08, 143.87, 143.89, 144.65, 144.69, 144.91, 145.07, 145.09,
45.16, 145.19, 145.21, 145.24, 145.28, 163.48 (CˆO), 164.05 (CˆO),
71.82 ppm (CˆO); UV/Vis (CH
Cl
2
):
l
max (e) ˆ 257 (107000), 325
2
‡
(
33500), 425 (2300), 476 nm (1500); MS (FAB, NBA): m/z: 978 [M] .
The purity (99%) was determined by HPLC (on NucleosilMN, 5 mm,
toluene).
Deprotected spacer-monoadduct 10: Trifluoroacetic acid (3.0 mL,
3
9.0 mmol) was added to a solution of monoadduct 9 (270 mg, 0.276 mmol)
in dry toluene (30 mL) under a nitrogen atmosphere.The reaction mixture
was stirred for 20 h at room temperature, and the progress of the reaction
was monitored by TLC.The reaction mixture was concentrated and dried
Computational methods: To examine the electrostatic charge distribution
on the protein surface, the program Grasp[
36]
was used.All other
[
37]
calculations were performed with the program package HyperChem.
The employed X-ray structure (PDB code: 1HRC)[ was corrected at the
iron that coordinates His(18) and Met(80) as well as at the two thioether
bonds that attach the heme group to the peptide at Cys(17) and Cys(14).
The total charge of the protein was kept at ‡8.0. The Amber89 force-field
parameters implemented in HyperChem were used because of the included
parameters for the heme groups.Following geometrical optimization, a
MD calculation at 300 K for 510 ps was performed to ensure that the
protein conformation remains the same.The 16-fold deprotonated
dendrimer-containing fullerene was semiempirically optimized (PM3)
and also preoptimized by MD calculation for 510 ps.The MD simulations
of the complexes were started at a distance between the protein and
fullerene of 30 ä, but at different orientations.After heating for 10 ps (1 fs
step size) without force-field cut-offs to allow the molecules to attract each
other, and to find the first salt bridge contacts, the complexes were kept at
31]
in vacuum to afford the product.Yield: 240 mg (0 2. 60 mmol) 95%, red
1
brownish solid; H NMR (400 MHz, CDCl
2
CDCl
2
, RT): d ˆ 2.19 (m, 2H;
),
2
); C NMR (100.5 MHz,
3
CH
4
2
), 2.60 (t, J(H,H) ˆ 7.08 Hz, 2H; CH
2
COO), 4.12 (s, 3H; OCH
3
3
13
.58 ppm (t, J(H,H) ˆ 6.12 Hz, 2H; OCH
CDCl
2
CDCl
2
,
RT): d ˆ 23.59 (CH
2
), 29.85 (CH
2
COOH), 51.67
3
3
(
C
60CCOO), 54.15 (OCH
3
), 65.97 (OCH
2
), 71.13 (sp -C60), 74.29 (sp -C60),
1
1
1
38.32, 138.84, 140.50, 140.56, 141.45, 141.74, 142.51, 142.59, 142.65, 143.41,
43.44, 144.12, 144.16, 144.18, 144.20, 144.25 144.47, 144.50, 144.59, 144.69,
44.74, 144.76, 144.82, 163.07 (CˆO), 163.62 (CˆO), 176.27 ppm(CˆO);
UV/Vis(CH
2
Cl
2
): lmax (e) ˆ 257 (93000), 325 (29000), 425 (2100), 475 nm
‡
(
1700); MS (FAB, NBA): m/z: 922 [M] .
Protected dendrofullerene precursor 12: A solution of dicyclohexylcarbo-
diimide (0.56 g, 0.270 mmol) and 1-hydroxybenzotriazole hydrate (0.36 mg,
0
.270 mmol) was added successively to a cooled solution (08C) of
monoadduct 10 (240 mg, 0.260 mmol) and dendron H N-G2 11 (0.39 g,
.270 mmol) in dry THF (25 mL) under a nitrogen atmosphere. The
3
00 K for another 120 ps with force-field cut-offs.Cooling the system to 0 K
2
in 20 ps (without cut-offs) enabled the determination of minimum energies
for different complexation possibilities.
0
reaction mixture was stirred for 20 h at room temperature, and the progress
of the reaction was monitored by TLC.After the mixture was concentrated,
the product was isolated by flash chromatography (silica gel, toluene/ethyl
acetate 1:1), and dried in a vacuum.Yield: 250 mg (0 1. 07 mmol) 41%, red
1
Acknowledgement
brownish solid; H NMR (400 MHz, CDCl
3
, RT, TMS): d ˆ 1.44 (s, 81H;
), 2.03 (m, 2H; CH ), 2.19 (m,
CO), 4.15 (s, 3H;
), 6.22 (s, 3H; NH), 7.68 ppm
, RT, TMS): d ˆ 24.35 (CH ),
COO), 31.58 ([C(CH -) ), 31.68
tBu), 1.98 (m, 24H; C(CH
4H; CH
2
-)
3
‡ [C(CH
2
-)
3
]
3
2
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB583 Redoxaktive Metallkomplexe–Reaktivit‰tssteuerung durch mo-
lekulare Architekturen), by the European Union under the 5 Framework
Programme, HPRNT 1999 ± 00011 FUNCARS, HPRN - CT 2002 ± 00177
WONDERFULL and HPRN - CT 2002 ± 00171 FAMOUS
2
2
COOtBu ‡ CH
2
CON), 2.36 (m, 2H; CH
2
3
OCH
(
3
), 4.58 (t, J(H,H) ˆ 6.32 Hz, 2H; OCH
2
th
s, 1H; NH); 1 C NMR (100.5 MHz, CDCl
3
3
2
2
8.06 (tBu), 29.76 (CH
2
CON), 29.82 (CH
2
2
3 3
]
(
(
C
C(CH
[C(CH
60), 80.57 (C(CH
2
-)
3
), 32.63 (CH
2
CON), 52.04 (C60CCOO), 54.17 (OCH
3
), 57.45
3
3
2
-)
3
]
3
), 57.57 (C(CH
2
-)
3
), 66.72 (OCH
2
), 71.45 (sp -C60), 77.19 (sp -
3
)
3
), 139.17, 140.89, 140.96, 141.89, 142.17, 142.18, 142.95,
1
1
1
42.98, 143.02, 143.84, 143.87, 144.58, 144.63, 144.65, 144.67, 144.86, 145.10,
[
1] a) A .P .Maierhofer, M.Brettreich, S.Burghardt, O.Vostrowsky, A.
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Brettreich, S.Burghardt, C.Bˆttcher, T.Bayerl, S.Bayerl, A.Hirsch,
Angew. Chem. 2000, 112, 1915 ± 1918; Angew. Chem. Int. Ed. 2000, 39,
45.15, 145.18, 145.25, 145.27, 163.65 (CˆO), 164.13 (CˆO), 171.61 (CˆO),
2 3 3 2 3 2 2
72.66 (-CH [COO) ] ), 172.89 ppm(-CH (CON) ); UV/Vis (CH Cl ): lmax
(
e) ˆ 257 (106500), 323 (36500), 425 (4400), 469 nm (3900); MS (FAB,
‡
‡
NBA): m/z: 2345 [M] , 2367 [M‡Na] .The purity (99%) was determined
1
845 ± 1848.
by HPLC (on NucleosilMN, 5 mm, toluene/ethyl acetate 3:2).
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1
red brownish solid; H NMR (400 MHz, D
2
O ‡ K
), 2.16 (m, 24H; CH
), 2.47 (m, 2H; CH CO), 4.16 (s, 3H; OCH
2
CO
3
(pH 7), RT): d ˆ
COO ‡
),
1
.95 (m, 24H; C(CH
2
-)
3
‡ [C(CH
2
-)
3
]
3
2
CH CON and 2H; CH
2
2
2
3
J. Am .
3874
¹ 2003 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3867 ± 3875

Electrostatic Complexation between Fullerene Dendrimers and Cytochromec
3867±3875
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