Synthesis and antioxidant activity of [60]fuIlerene-BHT conjugates

Article abstract of DOI:10.1002/chem.200501495

Fullerene derivatives incorporating one or two 3,5-di-tert-butyl-4- hydroxyphenyl groups were synthesized by 1,3-dipolar cycloaddition of azomethine ylides to Q₆₀. The O-H bond dissociation enthalpies (BDEs) of these compounds were estimated by

Full text of DOI:10.1002/chem.200501495

DOI: 10.1002/chem.200501495
Synthesis and Antioxidant Activity of [60]Fullerene–BHT Conjugates**
Roger F. Enes,[a] Augusto C. TomØ,*[a] JosØ A. S. Cavaleiro,[a] Riccardo Amorati,[b]
Maria Grazia Fumo,[b] Gian Franco Pedulli,*[b] and Luca Valgimigli[b]
Abstract: Fullerene derivatives incor-
porating one or two 3,5-di-tert-butyl-4-
hydroxyphenyl groups were synthe-
sized by 1,3-dipolar cycloaddition of
also determined by measuring the rate
constants for their reaction with perox-
yl radicals in controlled autoxidation
experiments and compared to that re-
corded under identical experimental
settings for [60]fullerene itself and un-
linked BHT. The results indicate that
linking of the BHT structure to C60
does not substantially alter the thermo-
chemistry and kinetics of its reaction
with peroxyl radicals, but such adducts
may behave as interesting bimodal rad-
ical scavengers. The inherent rate con-
stant for trapping of peroxyl radicals
by C60 per se (kinh =3.1ꢁ1.1”
102 mꢀ1 sꢀ1) indicates that, contrary to
previous reports, [60]fullerene is an ex-
tremely weak chain-breaking antioxi-
dant.
ꢀ
azomethine ylides to C60. The O H
bond dissociation enthalpies (BDEs) of
these compounds were estimated by
studying, by means of EPR spectrosco-
py, the equilibration of each of these
phenols and 2,6-di-tert-butyl-4-methyl-
phenol (BHT) with the corresponding
phenoxyl radicals. The antioxidant ac-
tivity of the investigated phenols was
Keywords: antioxidants
·
EPR
spectroscopy · fullerenes · radical
reactions · radicals
Introduction
and their organic derivatives can trap several radicals per
molecule; these compounds may be regarded as “radical
sponges”.[9,10]
This makes [60]fullerene an interesting lead structure in
the development of novel radical-scavenging compounds
with specific functionalities. For instance, it was recently
shown that a number of water-soluble fullerene derivatives
behave as potent ROSscavengers in cell cultures and can
protect human skin keratinocytes from UV irradiation and
oxidative damage by tert-butyl hydroperoxide.[11]
Recently, some of us described the synthesis of new fuller-
ene derivatives bearing structural moieties with known anti-
oxidant activity (e.g., flavonoids).[12] Here we report the syn-
thesis and antioxidant activity of four C60 derivatives incor-
porating one or two aryl groups structurally related to
BHT,[13] a phenolic antioxidant widely used in the food in-
dustry.[14,15]
The aim of this project is to develop phenolic antioxidants
with special functionalities, such as limited diffusion in poly-
meric matrices and biomembranes, or extended reactivity
toward alkyl radicals,[16] and to investigate possible varia-
tions in the antioxidant behavior of the BHT-like structure
as a consequence of its conjugation with [60]fullerene, par-
ticularly a synergistic effect between the two type of antioxi-
dants (the [60]fullerene moiety and the phenolic antioxi-
dant).
Oxidative damage plays a significant pathological role in
human diseases such as cancer and age-related neurodege-
nerative diseases.[1,2] This oxidative damage, mainly due to
free radicals and reactive oxygen species (ROS), can be pre-
vented by antioxidants. It is generally accepted that diets
rich in food containing antioxidants help to reduce oxidative
damage in humans.[3,4] In recent years, it was shown that full-
erene derivatives can be used as protective drugs against
neurodegenerative diseases related to oxidative stress.[5–8]
This observation is directly related to the fact that fullerenes
[a] Dr. R. F. Enes, Prof. A. C. TomØ, Prof. J. A. S. Cavaleiro
Departamento de Química
Universidade de Aveiro, 3810-193 Aveiro (Portugal)
Fax : (+351)234-370-084
E-mail: actome@dq.ua.pt
[b] Dr. R. Amorati, Dr. M. G. Fumo, Prof. G. F. Pedulli,
Dr. L. Valgimigli
Dipartimento di Chimica Organica “A. Mangini”
Università di Bologna, Via S. Giacomo 11
40126 Bologna (Italy)
Fax : (+39)51-209-5688
E-mail: gianfranco.pedulli@unibo.it
[**] BHT=2,6-Di-tert-butyl-4-methylphenol.
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FULL PAPER
To evaluate the antioxidant activity of these compounds
we measured two physicochemical parameters which are be-
lieved to be crucial in determining the ease with which phe-
nolic antioxidants reduce the rate of oxidation of oxidizable
substrates, that is, the bond dissociation enthalpy (BDE) of
[19]
ꢀ
the O H bond broken during the inhibition reaction and
the rate constant kinh for the reaction of the antioxidant with
peroxyl radicals.[20,21]
Results and Discussion
Synthesis: [60]Fullerene derivatives 1–4 were obtained in
low to moderate yields (16–58%) by 1,3-dipolar cycloaddi-
tion reactions of C60 with azomethine ylides generated in
situ in refluxing toluene, as already described for similar sys-
tems.[12] Products were purified by flash chromatography on
silica with toluene/cyclohexane.
Fullerene derivatives 1 and 2 were obtained from the re-
action of the commercially available aldehydes 5 and 6, re-
spectively, with N-methylglycine and C60 (Scheme 1). While
compound 1 was obtained in 58% yield, its isomer 2 was
isolated in only 16% yield. This difference reflects the lower
reactivity of 2-hydroxybenzaldehyde derivative 6, presuma-
bly due to intramolecular hydrogen bonding.
The mass spectra of compounds 1 and 2 show the
[M+H]+ ion (m/z 982) and the NMR spectra confirm the
expected structures. The main difference in the 1H NMR
spectra of the two isomers is the position of the OH proton
signal, which appears at d=4.85 ppm in 1, but at d=
11.34 ppm in 2 due to intramolecular hydrogen bonding
with the nitrogen atom of the pyrrolidine ring. This intramo-
conditions from aldehyde 5 with La(OTf)3 as catalyst. In the
G
absence of the catalyst the reaction does not occur. Al-
though formation of two adducts 4 (with cis or trans config-
uration) could be expected, only one diastereoisomer was
detected. The absence of correlation between pyrrolidine
protons H-2 and H-5 in the NOESY spectrum of 4 presuma-
bly indicates that these protons are in a trans configuration.
In both 3 and 4, the NCH2 protons are diastereotopic and
ꢀ
lecular hydrogen bonding hampers rotation around the C2
Ar bond at room temperature.[22] This is confirmed by the
well-resolved signals corresponding to H-4 and H-6 of the
aryl group, which appear as an AB spin system at d=7.11–
7.13 ppm with J=2.4 Hz.
1
1
In the H NMR spectrum of 1 the signal corresponding to
geminal coupling is observed in the H NMR spectra. While
the ortho protons of the aryl group is a broadened singlet,
indicative of restricted rotation of this aryl group. This
effect has already been ob-
the resonances of these protons appear as a AB system (J=
13.5 Hz) in 3, in 4 they appear as two doublets (J=13.9 Hz).
served
in
similar
com-
pounds.[23,24] Synthesis of 3 and
4 required use of glycine deriv-
ative 8, which was prepared in
two steps by reductive amina-
tion of aldehyde 5 with glycine
methyl ester in 90% overall
yield (Scheme 2). The struc-
tures of 7 and 8 were confirmed
by 1H and 13C NMR and MS.
Compound 3 was obtained in
reasonable yield by reaction of
glycine derivative 8, parafor-
maldehyde and C60 in refluxing
toluene (Scheme 3). Compound
4 was obtained under similar Scheme 1.
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4647

G. F. Pedulli, A. C. TomØ et al.
solutions of 2 were photolyzed
the only signal observed was a
broad singlet centered at g=
2.0024 with a peak-to-peak line
width of 1.9 G. On the basis of
the small g factor and the line
width, we attribute this singlet
to a persistent radical adduct to
the [60]fullerene moiety. Simi-
lar spectra were observed by
Krusic et al.[25] when photolyz-
ing toluene or benzene solu-
Scheme 2.
tions of C60 in the presence of a
variety of radical precursors
and attributed to C60–radical
adducts. The same singlet, superimposed on the spectrum of
the phenoxyl radical, was also observed on prolonged pho-
tolysis of solutions of 3. The reason why no phenoxyl radical
from 2 could be detected is likely a combination of two fac-
tors, that is, its low persistency due to the absence of a
second tert-butyl substituent ortho to the radical oxygen
atom, and the very limited reactivity of the OH group
toward hydrogen abstraction due to intramolecular hydro-
gen bonding with the pyrrolidine nitrogen atom.
Scheme 3.
The signals of the ortho protons of the substituted benzyl
group in compounds 3 and 4 appear as sharp singlets at d=
7.42 and 7.52 ppm, respectively. In contrast, and as observed
1
for 1 and 2, in the H NMR spectrum of compound 4 the
resonances of the ortho protons of the aryl group at the 5-
position of the pyrrolidine ring appear as a broad singlet
due to restricted rotation of the aryl group. In the 13C NMR
spectra of compounds 3 and 4 the signal corresponding to
the ester carbonyl group appears at d=170.5 and 172.1 ppm,
respectively.
Photolysis of 4 in the presence of the peroxide gave rise
to an EPR pattern consisting of the superimposition of spec-
tra due to two different species arising from the H abstrac-
tion from the OH group on the substituted benzyl group
(4’a) and from the OH group on aryl ring directly bonded to
the pyrrolidine ring (4’b). The observed EPR spectrum and
its computer simulation are shown in Figure 1.
Interestingly, in the spectra of both radicals 3’ and 4’a, the
central line of the expected 1:2:1 triplet due to the benzyl
protons was not observed, while the separation between the
two outer lines (ca. 22–24 G) was twice as large as the hy-
perfine splitting constant of the methyl protons in the radi-
cal from BHT (11.2 G).[19] This means that central triplet
line is strongly broadened by spin-relaxation effects. A simi-
lar behavior was previously observed in the EPR spectra of
the phenoxyl radicals obtained from 2,6-di-tert-butylphenols
substituted at the 4-position with unsymmetrical N,N’-di-
alkylaminomethyl groups CH2N(R)R’ and attributed to the
magnetic inequivalence of the benzyl protons, which are dia-
stereotopic due to slow inversion at nitrogen on the EPR
timescale.[26] Since the same explanation can also be given in
the present case, we tried to accelerate the rate of intercon-
version between the benzyl proton splittings to reach the
EPR spectra of the free radicals obtained from 1–4: Radi-
cals were produced at room temperature inside the cavity of
an EPR spectrometer by reaction of 1–4 with alkoxyl radi-
cals generated photolytically from di-tert-butyl peroxide in
deoxygenated benzene solution [Eq. (1)]. In the case of 1, 3,
and 4 highly persistent radicals were obtained whose EPR
spectra showed good signal-to-noise ratio and g factors of
2.0046–2.0047 (see Table 1 for spectral parameters). When
Table 1. EPR spectral parameters for the phenoxyl radicals obtained by
abstraction of the OH hydrogen atom from 1, 3, and 4.
Phenol
Radical
a
G
a(N) [G]
a
N
g factor
1
3
1’
3’
1.85
1.80
0.80
1.80
2.0046
2.0047
11.20 (2H)[b,c]
12.20 (2H)[b,c]
7.60 (H-2)[a]
1.74 (H-5)[a]
4’a
4’b
1.84
1.76
1.53
1.40
2.0046
2.0046
[a] Hyperfine splitting (hfs) constants at the pyrrolidine ring protons.
[b] Average value of the hfs constants at the two diastereotopic benzyl
protons. [c] Only the two outer multiplets were observed due to selective
line-broadening effects.
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[60]Fullerene–BHT Conjugates
FULL PAPER
under the assumption that the entropic term can be neglect-
ed,[27] by means of Equation (3) from Ke and the BDE of
BHT.
BDEðArOꢀHÞ ꢂ BDEðAr0OꢀHÞꢀRT lnðKeÞ
ð3Þ
Measurements were repeated under different light intensi-
ty to check the constancy of Ke. The BDEs obtained in ben-
zene solution (Table 2) show that all the C60-containing phe-
ꢀ
Table 2. O H BDEs for 1–4 measured at room temperature in benzene
containing 10% di-tert-butyl peroxide, rate constants kinh for their reac-
tion with peroxyl radicals in cumene at 308C, and number of radicals n
trapped by each antioxidant molecule.
Compound
BDE
kinh
n
[kcalmolꢀ1
]
[103 mꢀ1 sꢀ1
]
1
2
3
4
80.1ꢁ0.1
–
7.3ꢁ1.1
0.40ꢁ0.10
8.2ꢁ1.1
6.7ꢁ1.3
2
n.d.
2
Figure 1. Room-temperature experimental (top) and simulated (bottom)
EPR spectrum observed under continuous irradiation of a deoxygenated
benzene solution of 4 containing di-tert-butyl peroxide The outer multip-
lets are due to radical 4’a (see text for additional details), and the central
part of the spectrum is due to 4’b.
80.3ꢁ0.2
80.4ꢁ0.3[a]
80.3ꢁ0.3[b]
79.9ꢁ0.1[c]
–
4
BHT
[60]fullerene
10.7ꢁ1.0
2
n.d.
0.31ꢁ0.11
[a] For the OH group linked to the substituted benzyl group (radical 4’a).
[b] For the OH group linked to the aryl ring directly bonded to the pyr-
rolidine ring (radical 4’b). [c] Recalculated from the data of ref. [15] on
fast-exchange region where the selective line-broadening
effect might disappear.[27] Thus, the sample temperature was
increased to the boiling point of benzene, but no substantial
changes in the EPR spectrum were detected.
ꢀ
the basis of the revision of the O H BDE of reference phenol
[30]
tBu3C6H2OH from 81.2 to 80.1 kcalmolꢀ1
.
The remarkable difference between the hyperfine split-
tings at the pyrrolidine nuclei (both nitrogen and protons)
in the similar phenoxyl radicals 1’ and 4’b is also noteworthy.
This is likely due to different geometry of the two radicals
arising from the very strong steric crowding in 4’.
ꢀ
nols are characterized by O H bond strengths very close to
that of their precursor BHT. This suggests that the
[60]fullerene moiety is not substantially involved in the hy-
drogen transfer reaction [Eq. (2)]. Indeed, closer inspection
of the data in Table 2 reveals that the C60-containing sub-
ꢀ
O H bond dissociation enthalpies (BDE): To measure the
ꢀ
ꢀ
O H BDEs of the title compounds we used the EPR radical
stituent slightly increases the O H BDE, perhaps due to the
weak electron-withdrawing behavior expected for [60]fuller-
ene.
equilibration technique, which, among the various experi-
mental methods for the determination of bond strengths,
seems to guarantee the best accuracy at present.[19,28] For
this purpose we measured the equilibrium constant Ke for
hydrogen-atom transfer between BHT as reference phenol
Ar’OH (revised BDE value 79.9 kcalmolꢀ1[29]), one of the
phenols 1, 3, and 4 (ArOH), and the corresponding phenox-
yl radicals [Eq. (2)] generated under continuous photolysis
in deoxygenated benzene at room temperature (258C).
Inhibition rate constants: The rate constants kinh for reaction
of 1, 3, and 4 with peroxyl radicals were determined by
studying the inhibition of the thermally initiated autoxida-
tion of cumene.[21] Cumene was chosen because of its lower
oxidizability, which magnifies the antioxidant behavior of a
given compound and allows the antioxidant activity of mod-
erately effective inhibitors to be more easily differentiated.
The reaction was followed by monitoring the oxygen con-
sumption (Figure 2) during the autoxidation with an auto-
matically recording gas-absorption apparatus, built in our
laboratory and described previously,[32] which uses as detec-
tor a commercial differential pressure transducer. The reac-
tions [Eqs. (4)–(9)], initiated by the thermal decomposition
of 2,2’-azobis(2,4-dimethylvaleronitrile) (AMVN), were car-
ried out at 308C under controlled conditions in air-saturated
solutions of cumene, both in the absence and in the pres-
ence of each antioxidant; BHT was used as reference chain
breaking inhibitor.
ArOH þ Ar0OC Ð ArOC þ Ar0OH
ð2Þ
Experiments were performed on benzene solutions with
the highest concentration of the [60]fullerene derivatives
compatible with their low solubility (ca. 10ꢀ3 m). The persis-
tence of the related phenoxyl radicals guarantees achieve-
ment of equilibrium, despite the relatively low concentration
of their precursors. In the calculation of Ke, the initial con-
centrations of ArOH and Ar’OH were used, while the rela-
tive radical concentrations were determined by means of
EPR spectroscopy. The BDEs for ArOH were calculated,
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G. F. Pedulli, A. C. TomØ et al.
The true value of 2kt for cumylperoxyl radicals was ob-
tained by an indirect procedure by measuring the ratio kinh
/
pffiffiffiffiffiffiffiffi
2 kt [Eq. (10)] during the initiated oxidation of cumene in-
hibited by BHT. Under these conditions the formation of
tetroxide from cumylperoxyl radicals is almost negligible,
since most of them terminate by reacting with the antioxi-
dant [Eqs. (8) and (9)]. Then, a very accurate determination
of the value of BHT was made by using styrene as oxidiza-
ble substrate and analyzing the data obtained at different
antioxidant concentrations with the method proposed by
Darley-Usmar et al.[34] The resulting kinh value of 1.1”
104 mꢀ1 sꢀ1 is only slightly different from that determined for
BHT by Ingold et al. (1.4”104 mꢀ1 sꢀ1).[20] By inserting the in-
pffiffiffiffiffiffiffiffi
hibition rate constant in the ratio kinh
/
2 kt experimentally
1
1
ꢀ =
determined as 50.2mꢀ =
s
, the 2kt value at 308C was ob-
2
2
Figure 2. Oxygen consumption observed during the autoxidation of
cumene initiated by AMVN (5”10ꢀ3 m) at 308C in the absence of any an-
tioxidant (not inhibited, n.i.) and in the presence of BHT, [60]fullerene,
or each of the four investigated derivatives (5.0”10ꢀ6 m).
tained as 4.6”104 mꢀ1 sꢀ1.[35]
A second determination of 2kt for cumene at 308C was
made by following, by EPR spectroscopy, the decay of the
signal due to the cumylperoxyl radical in pure cumene.
Under these conditions (cumene concentration 7.2m) the
fragmentation of cumyloxyl radicals to acetophenone and
methyl radicals (kf =1.23”106 sꢀ1)[36] is 15 times slower than
hydrogen-atom abstraction from cumene (kH =2.6”
106 mꢀ1 sꢀ1)[36] and thus the measured termination rate con-
stant is expected to be very close to the true value. This de-
termination led to a 2kt value of 4.5”104 mꢀ1 sꢀ1, which is
surprisingly similar to that obtained from autoxidation ex-
periments.
The term n represents the stoichiometric coefficient, that
is, the number of peroxyl radicals trapped by each antioxi-
dant molecule, and can be determined from Equation (11)
by measuring the length of the induction period t during
which the rate of oxygen consumption in the inhibited au-
toxidation of cumene is strongly reduced. The results of
these determinations are reported in Table 2.
Ri
Initiator
RC
ð4Þ
ð5Þ
ð6Þ
ð7Þ
ð8Þ
ð9Þ
ꢀ!
RC þ O2 ! ROOC
kp
ꢀ!
ROOC þ RH
ROOH þ RC
2 kt
ROOC þ ROOC
products
ꢀ!
ROOC þ ArOH kinh ROOH þ ArOC
ꢀ!
ROOC þ ArOC ! products
The inhibition rate constant kinh [Eq. (8)] of each com-
pound was determined by means of a kinetic treatment[32]
consisting of measuring the initial rates of oxidation of
cumene both in the presence (ꢀd[O2]/dt=Rox) and absence
((ꢀd[O2]/dt)0 =Rox,0) of antioxidant, ArOH, and calculating
kinh from these data by means of Equation (10).
Rit
½AHŠ
n ¼
ð11Þ
Rox,0 Rox
ꢀ
nkinh½AHŠ
pffiffiffiffiffiffiffiffiffiffiffiffi
2 ktRi
0
¼
ð10Þ
From Figure 2 it can be inferred that 1 and 3 show, during
the inhibition period, moderate antioxidant activity, compa-
rable to that of BHT. In fact, the inhibition rate constants
are only slightly lower than that of BHT, that is, the pres-
ence of the [60]fullerene group does not improve the radi-
cal-scavenging ability of BHT. Phenol 2, on the other hand,
is practically devoid of any antioxidant activity, presumably
due to formation of an intramolecular hydrogen bond be-
tween the phenolic OH group and the nitrogen atom of the
Rox Rox,0
This equation allows evaluation of kinh even when the in-
hibition and termination [Eq. (7)] rates are comparable. The
use of Equation (10) requires knowledge of the initiation
rate Ri, which was determined in preliminary experiments as
described in the Experimental Section, and the termination
constant 2kt for the self-combination of cumylperoxyl radi-
cals. Unfortunately, the value of this constant, measured by
following the decay of the cumylperoxyl radicals, shows con-
siderable variations with cumene concentration.[33] This is
due to the irreversible decomposition of the tetroxide,
formed in reaction (7), to give molecular oxygen and two
caged alkoxyl radicals, 10% of which combine to give the
corresponding peroxide, while the greater portion (ca. 90%)
escapes into the reaction medium and undergoes reactions
1
pyrrolidine ring (d(OH)=11.34 ppm in the H NMR spec-
ꢀ
trum), which leads to a large increase in O H bond
strength. Compound 4, although characterized by a kinh
value similar to those of 1 and 3, shows an antioxidant effi-
cacy lasting twice as long as that of the other phenols. This
can be easily explained in terms of the presence of two radi-
cal-scavenging units.
C
typical of the RO radical such as hydrogen-atom abstraction
Careful examination of Figure 2 shows that the slopes of
the oxygen-consumption plots for 1, 3, and 4, after the end
and b scission.[21]
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[60]Fullerene–BHT Conjugates
FULL PAPER
of the inhibition period, are not exactly parallel to that ob-
served with BHT. This might suggest that these compounds,
even when no more phenolic hydrogen atoms are present in
solution, still retain a weak scavenging ability for peroxyl
radicals, conceivably due to the C60 group per se. An investi-
gation by Hwang et al.[37] attributes to C60 an antioxidant ac-
tivity in liposomes higher than that of a-tocopherol, based
on the relative ability of a series of antioxidants (at the
same concentration) to prevent pH changes in the aqueous
phase internal to the liposomes during the generation of hy-
droxyl or superoxide radicals. The authors, who were them-
selves surprised at this result, suggest that this is due to the
multiple binding sites (30 double bonds) available on C60 to
accept several peroxyl radicals per molecule, as opposed to
a-tocopherol, which is able to quench “only” two peroxyl
units. Although the ability of [60]fullerene to add several
tert-butylpexoxyl radicals to form stable multiple peroxides
has been proven by NMR spectroscopy,[38] it should be kept
in mind that the antioxidant ability of a given compound
does not merely reflect the stoichiometry of its reaction with
peroxyl radical, but it is mainly due to the rate of such reac-
tion. For this reason we measured the antioxidant activity of
unsubstituted [60]fullerene under the same experimental
conditions employed for its derivatives 1–4. The resulting
rate constant for peroxyl radical trapping (kinh =313mꢀ1 sꢀ1)
is not significantly different from the value recorded for
compound 2 (see errors in Table 2) and indicates that, con-
trary to previous reports, [60]fullerene itself is an extremely
weak chain-breaking antioxidant. When this value is divided
by 30, that is, the number of double bonds in the C60 struc-
ture, its “intrinsic” reactivity (kꢂ10mꢀ1 sꢀ1) with peroxyl
radical is not greater than that of styrene (kꢂ41mꢀ1 sꢀ1).[21]
The reason why [60]fullerene behaves as a poor antioxidant
rather than propagating the oxidation chain like styrene is
the lack of reactivity of the resulting extensively conjugated
carbon-centered radical. In this respect, C60 shows a reactivi-
ty quite similar to that of other polyenes such as b-caro-
tene.[39,40]
synthesized and their antioxidant activities determined. The
experimental results suggest that the introduction of the
[60]fullerene moieties affects only marginally the overall
rate of reaction with peroxyl radicals with respect to the
simpler precursor BHT. The lower rates of inhibition corre-
ꢀ
late nicely with the corresponding slightly higher O H BDE
values.[41]
Although these compounds behave as moderately effi-
cient antioxidants under aerobic conditions, an additional
radical-scavenging mode, that is, efficient trapping of alkyl
radicals, is expected to be contributed by the C60 group
under hypoxic conditions, and this would make these phe-
nolic C60 derivatives interesting bimodal radical scavengers
that could find interesting applications in pharmaceuticals
and polymer chemistry (e.g., as polymerization inhibitors
and antioxidants).[42] In these areas the bulky C60 unit could
also contribute additional properties to these adducts, for
example, limited mobility or preferential location in some
biological compartment.
Experimental Section
1H and 13C NMR spectra were recorded on Bruker Avance 300 or 500
spectrometers at 300 or 500 MHz and 75 or 125 MHz, respectively.
CDCl3 and CDCl3/CS2 were used as solvents, and TMSwas used as inter-
nal reference. Mass spectra and HRMSwere recorded on VG AutoSpec
Q and M mass spectrometer. m-Nitrobenzyl alcohol was used as matrix
for FAB+ mass spectrometry. Melting points were determined with a
Reichert Thermovar electric instrument and are uncorrected. Flash chro-
matography was carried out with silica gel 0.032–0.063 mm.
Synthesis of C60 derivatives 1 and 2: General procedure: A solution of
C60 (70 mg, 9.7”10ꢀ5 mol), N-methylglycine (43 mg, 5 equiv), and alde-
hyde 5 or 6 (24 mg, 1 equiv) in toluene (60 mL), was heated at reflux
under N2 for 6 h. The mixture was concentrated and purified by flash
chromatography with a gradient of cyclohexane/toluene as eluent. The
first fraction was unconsumed C60, and the second the monoadduct. Com-
pounds 1 and 2 were crystallized from CS2/chloroform.
1-Methyl-2-(3,5-bis-tert-butyl-4-hydroxyphenyl)[60]fullero[c]pyrrolidine
(1): M.p. >3108C. 1H NMR (500 MHz, CDCl3/CS2): d=1.36 (s, 18H;
tBu), 2.79 (s, 3H; NCH3), 4.20 (d, J=9.3 Hz, 1H; H-5), 4.85 (s, 1H; OH),
4.89 (d, J=9.3 Hz, 1H; H-5), 5.05 (s, 1H; H-2), 7.46 ppm (brs, 2H;
Thus, the rate of reaction of [60]fullerene itself, or the
[60]fullerene moiety in the investigated derivatives, with per-
oxyl radicals does not justify further development of C60-
linked phenols. However, the C60 unit is known to be an ex-
tremely efficient trap for alkyl radicals that outperforms
BHT nearly 3000-fold and even a-tocopherol 23-fold, as can
be judged by comparison of their rate constants of reaction
at room temperature.[16] Due to the high reactivity of
carbon-centered radicals with molecular oxygen, this addi-
tional radical-scavenging ability would be relevant only at
low partial pressures of oxygen, for example, in vivo under
hypoxic conditions or in bulk polymers, where oxygen diffu-
sion is limited.
ArH); 13C NMR (125 MHz, CDCl3/CS2): d=30.2 (C
ACHTREUNG
AHCTREUNG
139.4, 139.8, 140.0, 141.3, 141.4, 141.5, 141.7, 141.8, 141.90, 141.97, 142.0,
142.1, 142.4, 142.5, 142.8, 143.0, 144.2, 144.5, 144.6, 144.9, 145.0, 145.16,
145.23, 145.3, 145.4, 145.6, 145.7, 145.88, 145.92, 146.0, 146.1, 146.4, 146.7,
147.1, 153.4, 153.6, 153.9, 154.2, 156.2 ppm; MS(FAB +): m/z: 982
[M+H]+, 720 [C60 +]; HRMS(ESI): m/z calcd for C77H28NO [M+H]+:
C
982.2165, found: 982.2174.
1-Methyl-2-(3,5-bis-tert-butyl-2-hydroxyphenyl)[60]fullero[c]pyrrolidine
1
(2): M.p. >3108C. H NMR (300 MHz, CDCl3/CS2): d=1.18 (s, 9H; tBu),
1.31 (s, 9H; tBu), 3.06 (s, 3H; NCH3), 4.28 (d, J=9.5 Hz, 1H; H-5), 5.06
(d, J=9.5 Hz, 1H; H-5), 5.07 (s, 1H; H-2), 7.12 and 7.13 (AB, J=2.4 Hz,
2H; ArH), 11.34 ppm (s, 1H; OH); 13C NMR (75 MHz, CDCl3/CS2): d=
29.0, 31.3 (C
N
N
117.9, 123.7, 124.2, 135.1, 136.23, 136.27, 136.7, 138.6, 139.3, 139.7, 139.8,
140.16, 140.23, 141.05, 141.09, 141.2, 141.4, 141.6, 141.69, 141.73, 141.76,
141.81, 141.87, 141.91, 142.15, 142.22, 142.4, 143.96, 143.97, 144.2, 144.69,
144.77, 144.84, 144.86, 145.1, 145.27, 145.28, 145.46, 145.51, 145.55, 145.68,
145.79, 145.84, 145.9, 146.1, 146.2, 146.9, 147.0, 151.4, 152.5, 152.6, 152.9,
Conclusion
154.6 ppm; MS(FAB +): m/z: 982 [M+H]+, 720 [C60 +]; HRMS(ESI): m/
Three different types of [60]fullero[c]pyrrolidines bearing
one or two 3,5-di-tert-butyl-4-hydroxyphenyl groups were
C
z calcd for C77H28NO [M+H]+: 982.2165, found: 982.2180.
Chem. Eur. J. 2006, 12, 4646 – 4653
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4651

G. F. Pedulli, A. C. TomØ et al.
Synthesis of C60 derivatives 3 and 4: General procedure: a solution of C60
(70 mg, 9.7”10ꢀ5 mol), glycine derivative 8 (36 mg, 1.2 equiv), and para-
formaldehyde (5.9 mg, 2 equiv) or aldehyde 5 (35 mg, 1.5 equiv) in tol-
uene (60 mL) was heated at reflux under N2 for 6 h. For the synthesis of
reaction was performed in a oxygen-uptake apparatus built in our labora-
tories and based on a Validyne DP15 differential-pressure transducer,
which has been previously described in detail.[31] The entire apparatus
was immersed in a thermostatically controlled bath which ensured a con-
stant temperature within ꢁ0.18C.
compound 4, La(OTf)3 (5.7 mg, 0.1 equiv) was also added. The mixture
N
was concentrated and purified by flash chromatography with a gradient
of cyclohexane/toluene as eluent. The first fraction was unconsumed C60,
and the second the monoadduct. Compounds 3 and 4 were crystallized
from chloroform/methanol.
In a typical experiment, air-saturated cumene containing the antioxidant
was equilibrated with the reference solution containing an excess of a-to-
copherol (1”10ꢀ3 to 1”10ꢀ2 m) in the same solvent at 308C. After equili-
bration, a concentrated chlorobenzene solution of AMVN was injected
into both the reference and sample flasks, and the oxygen consumption
in the sample was measured, after calibration of the apparatus, from the
differential pressure recorded with time between the two channels. This
instrumental setting allowed the N2 production and the oxygen consump-
tion due to decomposition of the azo initiator to already be subtracted
from the measured reaction rates. The antioxidant concentration was
kept constant for all measurements (5.0”10ꢀ6 m) in order to compare
more easily their behavior. Initiation rates Ri were determined for each
condition in preliminary experiments by the inhibitor method with a-to-
Methyl 1-(3,5-bis-tert-butyl-4-hydroxyphenylmethyl)[60]fullero[c]pyrroli-
1
dine-2-carboxylate (3): M.p. 147–1498C. H NMR (300 MHz, CDCl3): d=
1.53 (s, 18H; tBu), 3.89 (s, 3H; OCH3), 4.29 and 4.49 (AB, J=13.5 Hz,
2H; NCH2Ar), 4.35 (d, J=9.4 Hz, 1H; H-5), 4.98 (d, J=9.4 Hz, 1H; H-
5), 5.06 (s, 1H), 5.29 (s, 1H), 7.42 ppm (s, 2H; ArH); 13C NMR (75 MHz,
CDCl3): d=30.4 (C
N
N
75.6, 126.0, 126.5, 130.1, 135.6, 136.0, 136.2, 136.5, 137.7, 139.7, 139.8,
140.2, 140.3, 141.8, 141.9, 141.97, 142.01, 142.08, 142.11, 142.2, 142.3,
142.62, 142.64, 142.7, 143.0, 144.4, 144.48, 144.54, 144.7, 145.26, 145.27,
145.30, 145.36, 145.40, 145.5, 145.6, 145.7, 145.8, 145.9, 146.04, 146.06,
146.2, 146.3, 146.4, 147.3, 147.4, 151.3, 153.4, 153.6, 154.7, 154.8,
copherol as reference antioxidant: Ri =2
[a-TOH]/t.[21]
A
EPR and thermochemical measurements: Deoxygenated benzene solu-
tions containing the phenols (0.01–0.001m), and di-tert-butyl peroxide
(10 vol%) were sealed under nitrogen in a suprasil quartz EPR tube. The
sample was inserted at room temperature into the cavity of an EPR spec-
trometer, and photolyzed with the unfiltered light from a 500 W high-
pressure mercury lamp. The temperature was controlled with a standard
variable-temperature accessory and was monitored before and after each
run with a copper–constantan thermocouple.
170.5 ppm (C=O); MS(FAB +): m/z: 1040 [M+H]+, 720 [C60 +]; HRMS
C
(ESI): m/z calcd for C79H30NO3 [M+H]+: 1040.2220, found: 1040.2200.
Methyl 5-(3,5-bis-tert-butyl-4-hydroxyphenyl)-1-(3,5-bis-tert-butyl-4-hy-
droxyphenylmethyl)[60]fullero[c]pyrrolidine-2-carboxylate
(4):
M.p.
>3108C. 1H NMR (300 MHz, CDCl3): d=1.37 (s, 18H; 5-(ArtBu)), 1.51
(s, 18H; 1-(ArtBu)), 3.89 (s, 3H; OCH3), 4.03 (d, J=13.9 Hz, 1H;
NCH2Ar), 4.62 (d, J=13.9 Hz, 1H; NCH2Ar), 5.17 (s, 1H; 5-ArOH),
5.23 (s, 1H; 1-ArOH), 5.55 (s, 1H; H-2), 6.57 (s, 1H; H-5), 7.48 (brs,
1H; 5-ArH), 7.52 (s, 2H; 1-ArH), 7.92 ppm (brs, 1H; 5-ArH); 13C NMR
The EPR spectra were recorded on a Bruker ESP 300 spectrometer
equipped with a Hewlett Packard 5350B microwave frequency counter
for the determination of the g factors, which were corrected with respect
to that of perylene radical cation in concentrated H2SO4 (g=2.00258).
(75 MHz, CDCl3): d=30.40 (5-ArC
G
G
(5-ArC(CH3)3), 34.48 (1-ArC(CH3)3), 51.3 (CH2Ar), 51.6 (OCH3), 70.3
A
ACHTREUNG
(C60-sp3), 73.2 (C-2), 76.7 (C-5 and C60-sp3), 124.9, 127.3, 128.8, 134.9,
135.9, 136.0, 136.1, 136.2, 137.5, 139.1, 139.7, 139.8, 141.46, 141.52, 141.55,
141.9, 142.97, 142.05, 142.15, 142.18, 142.3, 142.55, 142.61, 142.7, 142.9,
145.05, 145.17, 145.20, 145.23, 145.36, 145.42, 145.47, 145.50, 145.53,
145.79, 145.86, 145.90, 145.92, 146.05, 146.08, 146.14, 146.4, 146.6, 147.0,
147.3, 147.4, 151.0, 151.6, 152.4, 153.1, 153.66, 153.75, 155.15, 156.15,
For mixtures of BHT and one of the investigated phenols, the molar ratio
of the two equilibrating radicals was obtained from the EPR spectra and
used to determine the equilibrium constant K1. Spectra were recorded a
few seconds after starting irradiation to avoid significant consumption of
the phenols during the course of the experiment.
Relative radical concentrations were determined by comparison of the
digitized experimental spectra with computer-simulated ones, as previ-
ously described.[19]
172.1 ppm (C=O); MS(FAB +): m/z: 1244 [M+H]+, 720 [C60 +]; HRMS
C
(ESI): m/z calcd for C93H50NO4 [M+H]+:1244.3734, found: 1244.3745.
Synthesis of glycine derivative 8: Glycine methyl ester hydrochloride
(154 mg, 3 equiv), K2CO3 (170 mg, 3 equiv), and La(OTf)3 (24 mg,
N
0.1 equiv) were added to a solution of aldehyde 1 (100 mg, 0.41 mmol) in
anhydrous toluene (40 mL). The reaction mixture was refluxed under ni-
trogen atmosphere for 14 h. The mixture was cooled to room tempera-
ture and filtered, and the solvent evaporated to afford imine 7 as a
yellow solid (125 mg, 100% yield). M.p. 178–1808C. 1H NMR (300 MHz,
CDCl3): d=1.46 (s, 18H; tBu), 3.77 (s, 3H; OCH3), 3.37 (s, 2H; a-CH2),
5.54 (s, 1H; OH), 7.59 (s, 2H; ArH), 8.20 ppm (s, 1H; N=CHAr);
Acknowledgement
We thank the University of Aveiro and Fundażo para a CiÞncia e a Tec-
nologia (FCT, Portugal) and FEDER for funding the Organic Chemistry
Research Unit and projects POCTI/QUI/46529/2002 and POCI/QUI/
58515/2004. R.F.E. also thanks FCT for a post-doctoral grant. The au-
thors thank Prof. A. M. S. Silva (Univ. of Aveiro) for his invaluable assis-
tance in the NMR experiments. Financial support from MIUR (Rome),
contract 2004038243 (GFP, LV), is gratefully acknowledged.
13C NMR (75 MHz, CDCl3): d=30.2 (C
61.9, 125.8, 127.6, 136.1, 156.8, 166.2, 171.0 ppm; MS(EI +): m/z (%): 305
A
N
(26) [M +], 290 (27), 246 (12), 234 (36), 219 (100), 203 (7).
Imine (125 mg, 0.41 mmol) was dissolved in anhydrous methanol
C
7
(10 mL), the solution cooled to 08C, and NaBH4 (47 mg, 3 equiv) added.
The mixture was stirred for 15 min under nitrogen atmosphere at 08C.
Acetic acid (0.8 mL) was then added and the solvent was evaporated.
The resulting residue was dissolved in chloroform (60 mL) and washed
with water (2”25 mL). The organic phase was dried (Na2SO4) and the
solvent was evaporated to afford compound 8 (113 mg, 90% yield). M.p.
72–748C. 1H NMR (300 MHz, CDCl3): d=1.43 (s, 18H; tBu), 3.45 (s,
2H; a-CH2), 3.70 (s, 2H; NCH2Ar), 3.73 (s, 3H; OCH3), 5.15 (s, 1H;
OH), 7.11 ppm (s, 2H; ArH); 13C NMR (75 MHz, CDCl3): d=30.3 (C-
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4652
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 4646 – 4653

[60]Fullerene–BHT Conjugates
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.
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Received: December 1, 2005
Published online: March 14, 2006
Chem. Eur. J. 2006, 12, 4646 – 4653
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4653