Effect of orientation of the peptide-bridge dipole moment on the properties of fullerene-peptide-radical systems

Article abstract of DOI:10.1021/ja303696s

We synthesized two series of compounds in which a nitroxide radical and a fullerene C₆₀ moiety were kept separated by a 3₁₀-helical peptide bridge containing two intramolecular C=O???H-N hydrogen bonds. The direction of the resulting molecular dipole moment could be reversed by switching the position of fullerene and nitroxide with respect to the peptide nitrogen and carbon termini. The resulting fullerene-peptide-radical systems were compared to the behaviors of otherwise identical peptides but lacking either C₆₀ or the free radical moiety. Electrochemical analysis and chemical nitroxide reduction experiments show that the dipole moment of the helix significantly affects the redox properties of both electroactive groups. Besides providing evidence of a folded helical conformation for the peptide bridge, IR and NMR results highlight a strong effect of peptide orientation on the spectral patterns, pointing to a specific interaction of one of the helical orientations with the C₆₀ moiety. Time-resolved EPR spectra show not only that for both systems triplet quenching by nitroxide induces spin polarization of the radical spin sublevels, but also that the coupling interaction can be either weak or strong depending on the orientation of the peptide dipole. As opposed to the concept of dyads, the molecules investigated are thus better described as fullerene-peptide-radical systems to stress the active role of the bridge as an important ingredient capable of tuning the system's physicochemical properties.

Full text of DOI:10.1021/ja303696s

Article
pubs.acs.org/JACS
Effect of Orientation of the Peptide-Bridge Dipole Moment on the
Properties of Fullerene−Peptide−Radical Systems
Luca Garbuio,† Sabrina Antonello,† Ivan Guryanov,† Yongjun Li,‡ Marco Ruzzi,† Nicholas J. Turro,*,‡
and Flavio Maran*,†
†Department of Chemistry, University of Padova, Via Marzolo 1, 35131 Padova, Italy
‡Department of Chemistry, Columbia University, New York, New York 10027, United States
S
* Supporting Information
ABSTRACT: We synthesized two series of compounds in
which a nitroxide radical and a fullerene C60 moiety were kept
separated by a 310-helical peptide bridge containing two
intramolecular CO···H−N hydrogen bonds. The direction
of the resulting molecular dipole moment could be reversed by
switching the position of fullerene and nitroxide with respect
to the peptide nitrogen and carbon termini. The resulting
fullerene−peptide−radical systems were compared to the
behaviors of otherwise identical peptides but lacking either
C60 or the free radical moiety. Electrochemical analysis and
chemical nitroxide reduction experiments show that the dipole moment of the helix significantly affects the redox properties of
both electroactive groups. Besides providing evidence of a folded helical conformation for the peptide bridge, IR and NMR
results highlight a strong effect of peptide orientation on the spectral patterns, pointing to a specific interaction of one of the
helical orientations with the C60 moiety. Time-resolved EPR spectra show not only that for both systems triplet quenching by
nitroxide induces spin polarization of the radical spin sublevels, but also that the coupling interaction can be either weak or strong
depending on the orientation of the peptide dipole. As opposed to the concept of dyads, the molecules investigated are thus
better described as fullerene−peptide−radical systems to stress the active role of the bridge as an important ingredient capable of
tuning the system’s physicochemical properties.
In this Article, we describe properties and compare the
behavior of compounds in which a free radical and a fullerene
C60 moiety are separated by a peptide. We particularly focused
on compounds that show different properties only because of
the orientation of the peptide bridge with respect to the above
moieties. Our main aim was to explore the effect of reversing
the peptide orientation and thus the direction of the dipole
moment of the bridge on the spectral, electrochemical, and
magnetic properties of the fullerene−radical dyad systems. In
this context, some interesting published results are worth
mentioning. We previously reported that an oriented peptide
dipole moment affects the redox potentials of electroactive
groups12 and even gold nanoparticles.13 Thanks to the use of
stiff helical peptide scaffolds, Galoppini and Fox observed that
the rate of photoinduced intramolecular electron transfer is
affected by the direction of the peptide dipole moment.14
Similar results were later obtained on electroactive self-
assembled monolayers on gold electrodes.15 Concerning
fullerene derivatives, that the onset of an ordered peptide
helical structure affects the way by which donor and acceptor
interact was evidenced by Polese et al. in their study about the
solvent dependence of intramolecular electron transfer in
INTRODUCTION
■
Since its discovery in 1985,1 the properties of the C60 fullerene
have been extensively investigated. Thorough understanding of
its chemical reactivity has led to the synthesis of a large number
of fullerene derivatives whose properties and applications have
been studied from many viewpoints.2 Among them, interesting
molecular systems are featured by fullerene−radical dyads
where C60 is covalently linked, through formation of full-
eropyrrolidines,3 either directly or via a molecular spacer to a
free radical function.4,5 Besides interest in synthesis and
properties of C60 and its derivatives, endohedral fullerenes6 in
which atom or small molecule is encapsulated in the C60 nano
cavity have become a field that attracts more attention due to
their potential applications in medicine and biology.7,8 In
particular, the recently successful synthesis9 of H2@C60 by the
molecular surgery method makes the material available in
quantities from tens to hundreds of milligrams. Therefore, to
probe the interactions of the motion of the endohedral
molecule with the outside environment,10 a series of H2@C60
derivatives covalently linked to a nitroxide radical has been
synthesized in which the nitroxide moiety acts as a relaxation
agent, and the effect of the nitroxide radical on proton nuclear
spin relaxation of the endohedral molecule has been
investigated.11
Received: April 17, 2012
Published: May 31, 2012
© 2012 American Chemical Society
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Chart 1
fullerene−peptide−[Ru(bpy)3]2+ systems.16 In a very recent
paper, de Miguel et al. studied the photoinduced electron
transfer between zinc(II)porphyrins and fullerenes connected
via triazole bridges and reached the conclusion that the nature
of the bridge affects charge separation and charge recombina-
tion dynamics.17
bridge could affect (i) the electrochemical behavior of the two
electroactive moieties, that is, fullerene and nitroxide, (ii) the
interaction between the nitroxide and the fullerene excited
state, and (iii) the overall behavior of the couple in terms of
nuclear magnetic resonance and electron paramagnetic
resonance behaviors.
For the purpose of our study, peptides with well-defined
features, that is, a definite conformational preference, sufficient
stiffness, and a structure causing onset of a strong dipole
moment, are required. These properties are collectively
possessed by peptides based on the α-aminoisobutyric acid
(Aib) residue. Aib is characterized by marked steric hindrance
at the α-carbon and restricted torsional freedom.18 Because of
these features, Aib peptides adopt a 310-helical structure and
display the remarkable property that their tendency to form β-
turns and 310-helices is already evident starting from the
tripeptide.18−20 Aib peptides are known to be remarkably rigid
even when they are short,20,21 which is a general feature shared
by oligopeptides based on Cα-tetrasubstituted α-amino acids.18
Rigidity is ensured by a framework of intramolecular C
O···H−N hydrogen bonds. These H-bonds force the CO
and N−H groups to align significantly along the peptide axis,
and thus formation of the 310-helix is accompanied by the onset
of a strong molecular dipole moment that has its positive pole
on the nitrogen terminus of the peptide.22 The outcome of
theoretical calculations is that the dipole moment of 310-helical
peptides changes by 4.5 D/residue22b (although individual C
O dipole moments depend on the actual number of residues
along the chain),22c although experimental determinations of
short peptides point to the smaller but still considerable value
of ca. 2.3 D/residue.13 It is worth stressing that although the
onset of a strong molecular dipole moment is valid also for
other peptide secondary structures, such as the α-helix,22b
peptides based on coded α-amino acids form stable helices only
for rather long oligomers, and thus the dipole moment of short
oligomers is much less controlled than in Aib-oligopeptides. We
speculated that reversing the direction of a 310-helical peptide
We synthesized two series of compounds in which the
peptide scaffold has a length sufficient to form two intra-
molecular CO···H−N hydrogen bonds, based on the 310-
helical structure. The results obtained with the actual
fullerene−peptide−radical systems were compared to the
behaviors of the corresponding systems lacking either the C60
or the free radical moieties but still retaining the same number
of intramolecular hydrogen bonds. The numbers 1, 2, and 3 are
associated with the full fullerene−peptide−radical systems, the
corresponding fullerene−peptides, and the radical−peptide
systems, respectively (cf., Chart 1). The two series are defined
as plus and minus, and the compounds are labeled with “+” or
“−” depending on whether the positive or negative side of the
peptide dipole moment is directed toward or away from the C60
moiety, respectively. The free radical of choice was a nitroxide
amino acid derivative (2,2,6,6-tetramethylpiperidine-1-oxyl-4-
amino-4-carboxylic acid, TOAC), which also is a Cα-
tetrasubstituted α-amino acid and thus obeys the same general
rules of Aib.18 This free radical is characterized by a piperidinic
ring stabilized by the presence of two contiguous tetrasub-
stituted carbons.23 The properties of the two series were
studied and compared by IR absorption spectroscopy, electro-
1
chemistry, H NMR spectroscopy, and EPR spectroscopy.
Electrochemical analysis and chemical reduction experiments
(for the nitroxide-bearing molecules) show that the dipole
moment of the helix significantly affects the redox behavior of
the relevant groups. IR and NMR conformational analysis in
solution provides evidence of a folded helical conformation and
highlights a specific interaction of one of the helical orientations
with the C60 moiety. Analysis of the time-resolved EPR (TR-
EPR) spectra indicates that quenching of the C60 triplet by a
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quality of the solvent/electrolyte system were first checked in the
background solution by using CV. At the end of each experiment, the
reference electrode potential was calibrated against the ferricenium/
ferrocene (Fc+/Fc) couple. For DPV, we used a peak amplitude of 50
mV, a pulse width of 0.05 s, 2 mV increment per cycle, and a pulse
period of 0.1 s. For the CV experiments, we employed the feedback
correction to minimize the ohmic drop between the working and the
reference electrodes.
nitroxide radical occurs efficiently by a radical−triplet pair
(RTP) mechanism controlled by the peptide orientation.
Therefore, the investigated molecules are better defined as
fullerene−peptide−radical systems rather than “simple” full-
erene−radical dyads to stress the active role of the bridge not
only as a component separating the two moieties and affecting
their communication, but also as an important ingredient
capable of tuning the system’s physicochemical properties
significantly.
Electron Paramagnetic Resonance. For monitoring the
reduction of TOAC in the experiments with diphenylhydrazine in
CHCl3, continuous wave electron paramagnetic resonance (CW-EPR)
spectra were acquired using a continuous wave Bruker ER 04 XG X-
band spectrometer. EPR spectra were recorded with 100 kHz field
modulation, 10 dB of 200 mW microwave power, 0.10 mT modulation
amplitude, 10.24 ms time constant, and 81.92 ms conversion time.
CW-EPR spectra were recorded at different times after addition of
diphenylhydrazine. The concentration of the nitroxide was calculated
by digital double integration of the CW-EPR spectrum, and the ratio
between the integral value before and after addition of diphenylhy-
drazine was calculated to obtain the % reduced nitroxide value.
For TR-EPR measurements, 0.5 mM 1+ or 1− in CHCl3 was placed
in a 5 mm o.d. quartz tube, carefully degassed by several freeze−
pump−thaw cycles, and sealed under vacuum conditions (10−3 Torr).
A pulsed laser beam from a Nd:YAG laser (Quantel Brilliant, pulse
length 5 ns, pulse energy 5 mJ, pulse repetition rate 20 Hz) was used
for the optical excitation of the samples at 532 nm, where C60
absorption is significantly larger than for nitroxides. Measurements
were carried out on a Bruker ER200D (X-band) spectrometer with an
extended detection bandwidth (6 MHz), disabling magnetic field
modulation and working in direct detection mode. The temperature of
the sample inside the EPR cylindrical cavity (8 mm optical access) was
controlled by a variable-temperature nitrogen flow system. The time-
dependent EPR signals were digitized using a digital oscilloscope
(LeCroy model LT344) with a maximum acquisition rate of 500
Megasamples/s synchronized with the laser pulse. The time resolution
of the instrument was ca. 150 ns. Data collection was performed with a
personal computer and software that allowed controlling the magnetic
field and setting the digital oscilloscope. Typically, 300 transient
signals were averaged at on-resonance conditions and subtracted from
those accumulated off-resonance to eliminate the background signal
induced by the laser pulse. A complete two-dimensional (2D) data set
that shows the EPR signal as a function of both time and magnetic
field consists typically of a set of transient signals, containing 500
points each, recorded at 128 different magnetic field positions. The
400 × 128 matrix gave a two-dimensional timefield data set from
which transient spectra were extracted. CW-EPR spectra have been
simulated with the Easyspin package using the “garlic” module for
isotropic and fast-motional CW-EPR spectra of radicals in solution.27
Simulation of transient EPR spectra was carried out using a homemade
program compatible with the Easyspin package and based on the RTP
mechanism theory.
EXPERIMENTAL SECTION
■
Chemicals. Unless stated otherwise, all commercially available
chemicals were used as received. The syntheses of precursors and
peptides 1+, 2+, 3+, 1−, 2−, 3−, and 4 are described in the
Supporting Information. The following acronyms will be used: ACN,
acetonitrile; Ala, alanine; Boc, tert-butyloxycarbonyl; DCE, 1,2-
dichloroethane; DEA, diethylamine; DIEA, N,N-diisopropylethyl-
amine; EDC·HCl, N-ethyl-N′(3-dimethylaminopropyl)carbodiimide
hydrochloride; Fp, 3,4-fulleropyrrolidine; Fmoc, N-9-fluorenylmethox-
ycarbonyloxy; HOAt, 7-aza-1-hydroxybenzotriazole; Piv, pivaloyl (tert-
butylcarbonyl); TFA, trifluoroacetic acid; Z, benzyloxycarbonyl; trityl,
triphenylmethyl.
Infrared Absorption Spectroscopy. Both the solid-state (KBr
disk technique) and the solution (CHCl3, cells with 1 mm optical path
length, CaF2 windows) IR absorption spectra were recorded with a
Perkin-Elmer model 1720X FT-IR spectrophotometer, nitrogen-
flushed, equipped with a sample-shuttle device, at 2 cm−1 nominal
resolution, and averaging 100 scans.
1H Nuclear Magnetic Resonance Spectroscopy. 1H NMR
spectra were obtained with a Bruker model Ascend 500 spectrometer,
operating at 500 MHz, or with a Bruker model AC 200 spectrometer,
operating at 200 MHz. Deuteriochloroform (99.8%, Aldrich),
acetonitrile-d3 (99.8%, Aldrich), methanol-d4 (99.8%, Euriso-Top),
dimethyl sulfoxide-d6 (99.8%, Euriso-Top), and water-d2 (99.8%,
Fluka) were used as the solvents. Chemical shifts (δ) are given as parts
per million (ppm) downfield from tetramethylsilane, which was added
to each solvent as the internal standard. Well-resolved 1H NMR
spectra of compounds containing the TOAC moiety were obtained by
24
reduction of the nitroxide with Na2S2O4 or, for the fullerene
derivatives, with diphenylhydrazine.25 Splitting patterns are abbre-
viated as follows: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m)
multiplet. The proton assignments were carried out by standard
chemical shift correlations as well as by 2D rotating frame nuclear
Overhauser effect spectroscopy (ROESY) experiments in deuterio-
chloroform (for the compounds without TOAC).
Electrochemistry. A CHI 660c electrochemical workstation was
used for both differential pulse voltammetry (DPV) and cyclic
voltammetry (CV) experiments. The solvent/electrolyte system was
DCE containing 0.1 M tetra-n-butylammonium hexafluorophosphate
(TBAH). All electrochemical experiments were conducted under an
argon atmosphere in an all-glass microcell that was thermostatted at 25
°C. The electrochemical experiments were carried out with a
homemade glassy carbon (GC) electrode prepared by sharpening a
3 mm diameter Tokai GC-20 rod to obtain a disk with a radius of 0.56
mm. The electrode was prepared as already described.26 Before
experiments, the electrode was polished with 0.015 μm alumina,
ultrasonically rinsed with ethanol for 5 min, washed with acetone, and
carefully dried with a cold air stream. The electrode was electro-
chemically activated in the background solution by means of several
voltammetric cycles at 0.5 V s−1 between the anodic and cathodic
potential limits of concern. The electrode area, 9.8 × 10−3 cm2, was
determined by measuring the voltammetric current for the oxidation of
ferrocene in N,N-dimethylformamide/0.1 M tetra-n-butylammonium
perchlorate, in which ferrocene has a diffusion coefficient of 1.13 ×
10−5 cm2 s−1. A Pt wire was the counter electrode, and an Ag wire was
used as a quasi-reference electrode. The Ag wire was kept in a tube
filled with the same electrolyte solution but separated from the main
working vessel by a Vycor frit. The behavior of the electrode and the
RESULTS AND DISCUSSION
■
Synthesis of the Peptides. The fullerene−peptides were
synthesized by solution-phase step-by-step procedures, as
schematized in Charts 2 and 3. Fp-Ala-(aminoacid)3-NHtBu
1+ and 2+, and Piv-(Aib)2-(TOAC)-NHtBu 3+ were prepared
by coupling the appropriate Nα-deprotected peptides H-
(aminoacid)3-NHtBu with freshly prepared Fp-Ala-OH and
Piv-Cl, respectively. For compounds 1−, 2−, and 3−, the
corresponding Piv-peptides with free C-terminal carboxylic
group were obtained by removal of OMe protective group by
NaOH. Amide coupling between Piv-(aminoacid)3-OH with
freshly prepared H-Ala-Fp or tBuNH2 yielded Piv-(amino-
acid)3-Ala-Fp and Piv-TOAC-(Aib)2-NHtBu, respectively.
The functionalization of C60 for obtaining compounds 1+
and 2+, and 1− and 2− was performed as illustrated in Chart 4.
Whereas for the minus series we followed a published
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of the IR absorption spectra of the amide A region, pertaining
to the stretch of the N−H groups, and the amide 1 region,
mostly related to CO stretching.18,31 The amide A region of
peptides 1+, 2+, and 3+ (Figure 1) and peptides 1−, 2−, and
Chart 2. Reaction Scheme for the Synthesis of Fullerene−
a
Peptides 1+ and 2+
a
(a) Activated ester formation with EDC/HOAt followed by
treatment with excess of tBuNH2. (b) Coupling with EDC/HOAt.
(c) Removal of Fmoc with DEA/ACN followed by coupling with
EDC/HOAt. (d) C60, paraformaldehyde, and HOOC−CH2−NH−
CHCH3−COOtBu in refluxing toluene. (e) Removal of tBuO with
TFA/CHCl3 and removal of Fmoc with DEA/ACN.
Chart 3. Reaction Scheme for the Synthesis of Fullerene−
a
Figure 1. FT-IR absorption spectra for the amide A region of the plus
series in CHCl3 at 23 °C: 1+ (red), 2+ (blue), and 3+ (black). The
spectra were background subtracted and then normalized with respect
to the main N−H band.
Peptides 1− and 2−
a
(a) Removal of Z with H2/(Pd) in MeOH followed by coupling with
EDC/HOAt. (b) Removal of Fmoc with DEA/ACN followed by
treatment with Piv-Cl. (c) Removal of OMe with 6 N NaOH/H2O/
MeOH and removal of Boc with TFA/CHCl3. (d) Coupling with
EDC/HOAt.
procedure,16 for the plus series we allowed the appropriate N-
substituted glycine to react with formaldehyde to yield an
azomethine ylide that then undergoes a [3 + 2] cycloaddition
onto the double bonds of two adjacent C60 hexagons.28 For the
synthesis of the TOAC containing peptides, the strategy based
on the base-labile Fmoc protecting group was used due to the
lability of the nitroxide group under the conditions required for
the removal of other protecting groups.29 On the other hand,
only acid-labile protecting groups were used for handling the
fullerene derivatives, which are sensitive to basic and reducing
conditions.30
Figure 2. FT-IR absorption spectra for the amide A region of the
minus series in CHCl3 at 23 °C: 1− (red), 2− (blue), and 3− (black).
The spectra were background subtracted and then normalized with
respect to the main N−H band.
IR Absorption Spectroscopy. The secondary structure of
Aib-containing peptides is conveniently studied by inspection
3− (Figure 2) shows a strong absorption band with its
maximum in the range 3368 ÷ 3329 cm−1 and weaker bands in
Chart 4. Reaction Schemes for the Synthesis of the Precursors for the Plus and Minus Series.
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the range 3470 ÷ 3434 cm−1. These values are typical IR
features of folded Aib-peptides: whereas the stronger band is
due to intramolecularly H-bonded N−H groups, the weak
bands pertain to free N−H groups (in solvents showing no
propensity to behave as hydrogen-bond acceptors such as
CHCl3, used in our experiments). The spectra of the peptides
of the plus series (Figure 1) show a very similar band for the
intramolecularly bounded NH groups. On the other hand, a
marked difference is evident for the free NH band of peptide−
reference 3+, which is significantly more intense as compared
to that of fullerene−peptides 1+ and 2+. This difference, which
is not evident for the peptides of the minus series (cf., Figure
2), points to a different number of free NH groups, and is
conceivably related to the presence of the fullerene moiety in
1+ and 2+. Concerning the minus series, the strong band of 3−
appears at a wavenumber (3367 cm−1) similar to that of the
plus series (∼3360 cm−1), in line with the wavenumber
expected for two intramolecular H-bonds in 310-helical
peptides.20,31,32 A red shift of such band is observed for 1−
(3344 cm−1) and 2− (3329 cm−1). The absence of such a shift
in the FT-IR spectra of the fullerene peptides of the plus series
may indicate a higher sensitivity of the peptide structure of the
minus series to the presence of the fullerene moiety.
The amide I region of the two peptide series shows a strong
band with its maximum at 1672 ÷ 1662 cm−1 (Supporting
Information, Figure S2): these wavenumbers are in keeping
with carbonyls of a peptide backbone in helical conformation,
and a component at higher energy 1681 ÷ 1679 cm−1,
particularly evident with 3+ and 3−, is related to free peptide
carbonyls.18,31 Overall and despite the limited length of the
peptide structures investigated, analysis of the amide I region
points to relatively stable helical peptides structures with
pronounced 310-helical character.
Figure 3. Background-subtracted CV curves of 1 mM 2+ () and 2−
(− − −), in DCE/0.1 M TBAH at a scan rate of 0.1 V/s.
fulleropyrrolidine nitrogen: whereas in compounds 1− and
2− an amide bond is present, a tertiary amino group
characterizes compounds 1+ and 2+ (Chart 1). On the basis
of simple substituent-effect considerations, one would thus
expect the reduction of C60 in 1− and 2− to occur at less
negative potentials than that of 1+ and 2+. For example, a
positive potential shift of 50 mV was observed on going from
N-methyl-fulleropyrrolidine to N-acyl-fulleropyrrolidine.35 The
outcome of our CV experiments, however, highlights an
opposite effect; that is, the first reduction peaks of 1+ and 2+
are less negative by 17 and 14 mV with respect to those of 1−
and 2−, respectively. The observed potential shift is thus
consistent with the effect brought about by the peptide dipole
moment.13 The peak-potential difference fades away for the
second and third reduction peaks of fullerene, suggesting that
the dipole moment effect is negligible for charged species.
Concerning the CVs of both 1+ and 1− at negative potentials,
the only relevant difference with respect to those of 2+ and 2−
is the presence of a shoulder before the third reversible
reduction peak of C60 (Supporting Information, Figure S3; for
the corresponding DPV, see Figures 4 and S4), which is due to
the irreversible reduction of the nitroxide radical function, as
was also verified in comparison with the CV behavior of 3+ and
3−, which only carry the nitroxide group.
Electrochemistry. The electrochemical measurements were
carried out by cyclic voltammetry (CV) and differential pulse
voltammetry (DPV), with a GC electrode and in DCE/0.1 M
TBAH. Because the solvent employed has a limited cathodic
discharge potential, the analysis focused on the first three
reduction peaks of the fullerene moiety of 1+, 2+, 1−, and 2−.
For all peaks, analysis of the CV and DPV peak potentials is in
agreement with fully reversible one-electron processes,33 as
expected.34 The formal potential (E°) values (Table 1) were
On the oxidation side, a single reversible oxidation peak is
observed. This is due to the oxidation of the TOAC nitroxide
radical to the oxoammonium cation.36 The DPV curves of
Table 1. Formal Potentials of the Investigated Peptides in
a
DCE/0.1 M TBAH at 25 °C
peptide
E°1red (V)
E°2red (V)
E°3red (V)
E°ox (V)
1+
2+
3+
1−
2−
3−
4
−1.056
−1.056
−1.458
−1.458
−1.973
−1.972
0.202
0.210
0.283
−1.073
−1.070
−1.460
−1.457
−1.974
−1.972
0.300
0.262
a
Potentials are against the Fc+/Fc redox standard.
obtained as the average of the cathodic and anodic CV peak
potentials. Figure 3 compares the CV behaviors of 2+ and 2−
(a very similar comparison is valid for 1+ and 1−), indicating
that the E° values of the first peak of the two compounds are
different (cf., Table 1). Apart from the different orientation of
the peptide bridge, one distinction between the plus and the
minus series concerns the chemical bond involving the
Figure 4. DPV curves of 1+ () and 1− (− − −) in DCE/0.1 M
TBAH.
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Figure 4 show that nitroxide oxidation in 1+ is considerably
easier than that of 1− by 80 mV. Therefore, the E° shift is, once
again, consistent with the direction expected on the basis of the
dipole moment effect: the nitroxide group of 1+ is on the
negative side of the peptide dipole, which makes its oxidation
easier. To check that the E° shift is indeed independent of the
presence of the fullerene moiety and only due to the peptide
dipole-moment orientation, we carried out the corresponding
CV analysis of peptides 3+ and 3−. Whereas the oxidation
peaks of 3+ and 3− are slightly more positive than those of 1+
and 1− (by 8 and 17 mV, respectively), the potential difference
is 90 mV and in very good agreement with that between 1+ and
1−. These results confirm a considerable effect exerted by the
helix dipole moment on the energy of the TOAC’s molecular
orbital hosting the unpaired electron. It is also interesting to
compare the oxidation potentials of peptides 3+ and 3− with
that of another TOAC derivative, Boc-TOAC-NHtBu (4),
which besides lacking the fullerene moiety also does not
possess an oriented dipole moment. In keeping with our
observations and interpretation, the E° oxidation potential of 4
occurs at 0.262 V, that is, between the values observed for
nitroxide oxidation in the plus and minus series (Table 1 and
Figure 5). Overall, comparative analysis of the electrochemical
incapable of acting as H-bond acceptors: a good test of the
participation of peptide amide protons in intramolecular
hydrogen bonds is to add a good H-bond acceptor such as
Me2SO-d6 and to analyze how the 1H NMR spectrum
changes.20 Both N(1)H and N(2)H protons of peptide 2−
were found to be very sensitive to stepwise addition of Me2SO-
d6, as their resonances underwent significant downfield shifts
(Figure 6). On the other hand, the resonances of the two other
Figure 6. 1H NMR titration in CDCl3. Variation of the chemical shifts
of the four amide protons of 1 mM 2− (upper graph) and 2+ (lower
graph) as a function of the addition of Me2SO-d6.
Figure 5. Background-subtracted CV curves of 3+ (), 3− (− − −),
and 4 (red −), in DCE/0.1 M TBAH and at a scan rate of 0.1 V/s.
amide protons were essentially insensitive, which indicates that
Me2SO-d6 fails to disrupt the intramolecular H-bonds involving
them. Because in the 310-helix the formation of stable
intramolecular H-bonds starts from the N(3)H proton,18,20
from the sensitivity of the first two NH protons to Me2SO-d6
addition, together with the insensitivity of the other NH
protons, we infer that peptide 2− adopts such an ordered
secondary structure. The NMR analysis is thus in agreement
with the outcome of the IR absorption spectroscopy analysis.
As far as peptide 2+ is concerned, the behaviors of the
resonances of N(2)H, N(3)H, and N(4)H protons are
qualitatively similar to that of 2−, and thus we can infer a
similar ordered structure as supported by the outcome of the IR
analysis.
One important difference between the two peptides 2+ and
2−, however, concerns the very distinct behavior of N(1)H of
peptide 2+. As compared to its usual position in a variety of 310-
helical peptides (around 6 ppm), this resonance was observed
at significantly low fields (8.17 ppm). Furthermore, its chemical
shift does not depend on Me2SO-d6 addition. This indicates
that Me2SO-d6 does not interact significantly with this
otherwise free NH group, as it does with the N(2)H proton.
This finding implies that the N(1)H protons are already
involved in some sort of rather strong interaction. We observed
behaviors of the investigated peptides points to the important
role played by helix dipole on the redox potentials, which
reflects a different charge distribution induced by the helix
dipole and its effect on the energy of fullerene’s lowest
unoccupied molecular orbital (LUMO) and TOAC’s singly
occupied molecular orbital (SOMO).
1H NMR Spectroscopy. The presence of the free radical in
compounds 1+ and 1− makes the analysis of their NMR
spectra complicated due to the paramagnetic relaxation
enhancement.37 As a first step in this direction, we used H
1
NMR (Figures S5 and S6) and ROESY (Figures S7−S10) to
obtain useful insights into the peptide secondary structure of
their analogues 2+ and 2−, where Aib replaces TOAC. ROESY
experiments were used to detect through-space connectivities
of both the CβH(i − 1)→NH(i) (between the methyl protons
and amide protons of contiguous Aib units) and the NH(i)→
NH(i + 1) types (amide protons of contiguous Aib units). In
this way, it was possible to identify the four amide protons of
both peptides (cf., Supporting Information). In addition, the
NH(i)→NH(i + 1) cross peak signals indicate that both
compounds adopt a prevailing folded structure in keeping with
the pattern expected for the 310-helix. The experiments were
carried out in chlorinated solvents (CDCl3 or CD2Cl2)
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a very similar effect for Aib-peptide monolayer protected gold
nanoclusters, in which strong interchain H-bonds between the
two otherwise free NH protons of the 310-helix and CO
groups of neighboring peptide chains form.32 For peptide 2+, it
is conceivable that the interaction is between N(1)H and the π-
system of fullerene,38 which is possible because of the favorable
orientation of the NH groups of 2+. Consistent with this
general conclusion is the outcome of the IR absorption analysis
showing that in the amide A region the bands pertaining to free
NH groups of both 2+ and 1+ are significantly less intense than
those for 3+, which does not contain the fullerene moiety. The
free NH groups of the whole minus series do not present this
feature because the free NH groups are on the other side of the
fullerene−peptide system and oppositely oriented.
1
Nitroxide Reduction. The H NMR spectra of both 1+
and 1− reduced forms could be acquired after reduction of the
nitroxide moiety with diphenylhydrazine (PH),39 as we detailed
very recently.25 For both 1+ and 1−, the presence of the free
radical was proved by CW EPR spectra (Figures S16 and S17),
which show the typical three lines pattern of the nitroxide due
to the 14N (I = 1) nuclei hyperfine coupling.4,5 The reductive
quenching of the radical by PH was monitored by the intensity
decrease of the CW EPR signal over time (Figures S16 and
S17). We found that under the same redox conditions (23 °C,
10 equiv of PH), peptide 1+ undergoes reduction at a rate 4
times slower than that of 1− (Figure 7). Once again, this is
Figure 8. 500 MHz 1H NMR in the NH resonance region of 2 mM 1+
and 2+ in CDCl3 at room temperature. The spectra correspond (from
top to bottom) to 1+ before and after reduction of the nitroxide with
50 equiv of PH, and 2+. N(3)H overlaps the peak of residual
chloroform. The peaks at 5.58, 6.85, 7.21, 7.53, and 7.93 ppm belong
to PH, which was in excess, and products from its oxidation.39
contain the fullerene moiety. The chemical surrounding for the
N(1)H proton of 1+ and 2+ is thus the same, in which both
protons experience a deshielding effect due to their interaction
with C60.40
These effects are absent for the minus series, in which the
free NH groups are on the other side of the molecule with
respect to C60 and the orientation of the N−H groups is
opposite to that of the plus series. Most of the other features
are common to both series: the peaks corresponding to the
methylene groups of the TOAC piperidinyl ring appear as two
multiplets centered at 2.2 and 2.36 ppm for 1− (Figures S12
and S14) and at 2.28 and 2.49 ppm for 1+ (Figures S15) in
good agreement with the corresponding peaks of 3− and 3+
(Figures S14 and S15); the resonances of the four TOAC
methyl groups are at higher fields, that is, in the range 1.31−
1.68 ppm for 1− and in the range 1.22−1.60 ppm for 1+
(Figures S12, S14, and S15).
TR-EPR. In the primary stages of photochemical reactions,
short-lived paramagnetic species are usually generated with
polarized spin sublevels, that is, with populations of spin
sublevels that differ from those at thermal equilibrium.
Chemically induced dynamic electron polarization (CIDEP),
used for indicating this phenomenon, provides a powerful tool
for investigating elementary steps of photochemical reactions.41
Triplet quenching by stable free radicals42 can produce spin
polarization in the radical spin sublevels, and a RTP mechanism
was proposed to explain the polarization effects observed in
transient EPR spectra in solution.43 Most of the published
studies focus on spin polarization of nitroxide radicals
interacting in solution with excited state molecules.44 The
RTP mechanism was also extended to molecular systems
containing a triplet precursor and a free radical connected by
chemical bonds or other interactions.45
CW-EPR spectra of peptides 1+ and 1− in CHCl3 at T = 230
K are shown in Figure S18 together with their simulations. The
two spectra are very similar and consist of the typical 14N
hyperfine triplet of lines.4,5 The same g-factor (gR = 2.0058
0.0005) and hyperfine coupling constant (aN = 15.32 G) were
obtained from simulations (cf., caption to Figure S18). By
photoexiciting 1+ and 1−, we obtained TR-EPR spectra
Figure 7. Reduction kinetics for the couples of nitroxide containing
compounds 1+ and 3+, and 1− and 3−. The reactions were conducted
at 0.05 mM concentration in CHCl3 with 10 equiv of PH, at 23 °C.
nicely in keeping with the effect of the dipole moment: the
nitroxides of the minus series (1− and 3−) feel the positive end
of the peptide dipole, and this makes their reduction easier.
1
Figure 8 compares the H NMR spectra of compounds 1+
and 2+ and shows the effect brought about by nitroxide
reduction on the NMR behavior of 1+. Before reduction, the
1H NMR spectra of the nitroxide-bearing compounds (see also
Figures S13 and S15) only display broad and unresolved peaks
in the aliphatic region; no other resonances were observable.
After reduction, however, the peaks related to the expected
products are resolved and in good agreement with the
corresponding behavior of 2+. A very similar situation holds
for the minus series, as illustrated in Figures S11, S12, and S14.
Figure 8 shows that, similarly to what was already described for
2+, also for reduced 1+ the N(1)H proton resonance occurs at
very low field (8.18 ppm). On the other hand, this resonance
shifts upfield for peptide 3+ (Figure S15), which does not
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showing different features (Figure S19). The TR-EPR spectra
show the variation of the EPR signal intensity with respect to
magnetic field and time axes. Both transient spectra and time
evolution signals can be extracted from TR-EPR data surfaces.
Figure 9 shows sections of the TR-EPR data surfaces taken at
observable, the transient spectrum of 1− only shows the wider
triplet of lines at gR, with intensities that decrease on going from
low to high fields (multiplet effect). A similar TR-EPR
spectrum was previously interpreted on the basis of the
conventional RTP mechanism.48
The TR-EPR spectrum of 1+ (Figure 9b) shows that after
photoexcitation the triplet of lines at gR has a marked E
polarization character. The latter is retained at longer delays,
similarly to 1− (cf., Figure 9a and b, black spectra). Differences
between 1+ and 1− are evident in the spectra recorded within a
few microseconds after photoexcitation and particularly
concern several additional reinforced absorptive/emissive
polarized components (multiplet with A/E/A/E/A/E polar-
ization pattern) that replace the triplet of lines at gQ (Figure 9b,
magenta, red, and blue spectra). Moreover, no inversion of
polarization is observed. This is an unusual TR-EPR behavior
previously observed, to the best of our knowledge, only for
another nitroxide-C60 derivative.49
Both of the TR-EPR spectra recorded of 1− and 1+ are in
keeping with the RTP mechanism theory.48 According to
quantum theory, the coupling of a triplet molecule (S = 1) with
a radical (S = 1/2) gives rise to an intermediate RTP that can
exist as an excited quartet Q (Q+3/2, Q+1/2, Q−1/2, Q−3/2) or
doublet D (D+1/2, D−1/2) spin states, depending on antiparallel
or parallel coupling of the two spin systems, respectively.
According to the theory, whereas the magnitude of the
exchange coupling constant J0 between the electrons of
fullerene and the unpaired electron of nitroxide provides the
energy separation between the excited D and Q states of the
pair (in particular EQ − ED = 3J0), the sign of J0 determines the
ferromagnetic (J0 > 0) or antiferromagnetic (J0 < 0) character
of the coupling.50 Both aspects affect the TR-EPR signal. For
both peptides, the triplet of lines at gR and with separation aN is
assigned to the D ground state transition of the pair separated
into three hyperfine components by the nitroxide 14N hyperfine
interaction. The emissive polarization character of these three
hyperfine lines can be interpreted in terms of quartet-precursor
RTP mechanism with J0 < 0.43 Generally, most RTPs are
characterized by a negative J0, with the positive sign assigned
only for particular pairs’ geometries48 or for systems involving
some charge transfer contribution to the excited state.51 The
differences of the spectra of 1− and 1+ are accounted for by a
different intensity of J0.52 Two situations, corresponding to
weakly and strongly coupled triplet-doublet systems, are
possible.53 The transient spectrum of 1− (Figure 9a) is
interpreted in terms of pure Q and D transitions, that is, by
assuming strong coupling (|J0| ≫ aN = 15 G). On the other
hand, the spectra of 1+ are best interpreted according to a
weaker coupling (|J0| ≤ 15 G). Note that transitions between
the Q and D states are in principle not allowed, but for 1+ they
are efficient due to the small value of J0 and thus increased
efficient mixing of states. Successful simulation of the transient
spectrum of 1+ (Figure S20)54 provides full support to the
weak-coupling mechanism. A value of J0 = 7 G was calculated
for the exchange coupling constant of this peptide.
Figure 9. Transient EPR spectra of 1− (a) and 1+ (b) in CHCl3 at
280 K, and recorded at increasing time delays after the laser pulse (t =
0.3, 1.4, 2.7, and 10.0 μs for magenta, red, blue, and black spectra,
respectively). Both spectra were recorded under the same experimental
conditions (microwave power, Pμw = 10 mW; photoexcitation, λ = 532
nm; E/pulse = 5 mJ). The A and E arrows indicate enhanced
absorption and emission polarization, respectively. For both peptides,
the polarized signals decay to Boltzmann equilibrium within ca. 28 μs.
increasing time delays after the laser pulse. Immediately after
photoexcitation, the transient spectra of 1− (Figure 9a) consist
of the superposition of two triplets of lines with different g
factors and hyperfine separations. The first triplet is centered at
g = 2.0064 0.0005 (i.e., g = gR within the experimental error)
and is characterized by a line separation of aN = 15.3 G
(nitroxide 14N hyperfine coupling constant). The second triplet
at a lower g factor (g = 2.0033 0.0005) consists of three lines
with a separation of a′N = 4.87 G ≈ aN/3. In the following, the g
factor of this second triplet will be indicated as gQ. Because of
the different g factors, the low-field line of the triplet centered at
gQ is superimposed to the central line of the triplet at gR.46 The
two triplets of lines display different time evolutions. In the
early stages after photoexcitation (Figure 9a, magenta
spectrum), the three lines of the triplet at gR are weakly
polarized in emission (E),47 and the emissive character is
retained even at longer delays from the laser pulse (Figure 9a,
red, blue, and black spectra). On the other hand, the lines
assigned to the triplet centered at gQ show a more complex time
evolution: as time increases, the lines reverse their polarization
from enhanced absorption (A) to emission. Eventually, while
10 μs after the laser pulse the triplet of lines at gQ is no more
These results point to different intensities of the spin
exchange coupling between the excited state of fullerene and
the radical. The same number of bonds between the fullerene
and the radical moieties occurs in both cases, and the
interacting spins are at a similar distance (about 10 Å). At
such a distance, the exchange interaction is still sensitive to the
relative orientation of the fullerene and nitroxide systems. The
stronger exchange coupling in 1− as compared to 1+, with the
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same (negative) sign, can thus be assigned to different
orientations of the pairs in the two cases and thus to the
different way by which the helix is oriented. In this context, we
note that the line width of the low-field line (mi = +1) hyperfine
component in the CW-EPR spectrum of 1+ is slightly larger
than that of 1− (cf., Figure S18). Although small, this difference
is in keeping with a less stiff system for 1−, which provides
more opportunities of optimizing the RTP coupling. This result
also is in keeping with the IR and NMR results that concur to
indicate that 1+ is stiffer because of the NH/fullerene
interaction.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full details about the syntheses of the peptide and fullerene
precursors. Additional electrochemical curves and IR absorp-
tion spectroscopy, NMR spectroscopy, and EPR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
flavio.maran@unipd.it; njt3@columbia.edu
CONCLUSIONS
Notes
■
The authors declare no competing financial interest.
The design, study, and application of new materials with
improved physicochemical properties are very important
aspects of areas such as molecular electronics, solar cells
engineering, and biosensors. In this work, we connected
fullerene C60 and nitroxide radical moieties with a sufficiently
rigid 310-helical peptide that possesses a strong molecular
dipole. The direction of the latter could be reversed by
switching the position of fullerene and nitroxide with respect to
the peptide’s nitrogen and carbon termini. Our main aim was to
verify whether this variation would have affected the electro-
chemical, optical, and magnetic behavior of the fullerene and
the nitroxide.
For all of the peptides, FT-IR and NMR analyses provided
evidence of highly folded structures, in agreement with the 310-
helix. For the fullerene peptides in which the positive end of the
dipole is near C60, the results evidenced a strong interaction
between the peptide helix and C60. Analysis of the electro-
chemical reduction and oxidation of the fullerene and nitroxide
groups, respectively, nicely shows that the formal potentials are
affected by the peptide dipole in the expected direction.
Chemical reduction of the nitroxide radical by diphenylhy-
drazine was found to proceed with rates that are significantly
different for oppositely oriented peptides, once again in keeping
with the effect brought about by the peptide dipole. TR-EPR
spectra showed that the coupling interaction in triplet
quenching by the nitroxide radical can be either weak or
strong depending on the orientation of the peptide dipole.
Overall, the results point to an important effect of the
orientation of the dipole moment and also evidence that the
fullerene moiety affects the orientation and, to some extent,
rigidity of the peptide chain for one of the two series.
ACKNOWLEDGMENTS
■
This work was financially supported by the Foundation
CARIPARO (progetto di eccellenza), the University of Padova
(PRAT grant CPDA103389), and NSF (grant CHE 11-11392).
We thank Dr. Steffen Jockusch for his assistance during the
nitroxide reduction experiments and Dr. Lorenzo Franco for
helpful discussions concerning the TR-EPR experiments and
his contribution to simulations.
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(54) Theoretical details on the simulation program are described in
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