A quantitative approach to the recycling of α-tocopherol by coantioxidants

Article abstract of DOI:10.1021/jo026501f

A systematic investigation is reported on the regeneration of α-tocopherol (α-TOH) in homogeneous solution by coantioxidants in order to better understand the mechanism and the factors responsible for the effectiveness of this process. The current availability of thermochemical data concerning the reactants involved in the regeneration reactions, as well as a large number of the kinetic constants for the various reactions involved, allowed us to rationalize the experimental observations collected so far. Three limiting cases have been considered. The first case is that of a coantioxidant irreversibly regenerating α-TOH, where the effectiveness of the recycling process depends on the magnitude of the rate constant k_r. The second case is that of a coantioxidant reversibly recycling α-TOH, where regeneration can only be observed if the bond dissociation enthalpy value of the coantioxidant is lower or at least close to that of the O-H bond of α-tocopherol. The third case is that of a catechol derivative (chosen as a model compound for polyphenolic antioxidants), where recycling of α-TOH is feasible even though the BDE value is significantly higher than that of vitamin E. In this case, the driving force for the recycling process is the removal of the semiquinone radical from the catechol derivative by the α-tocopheroxyl radical, which makes the regeneration of α-TOH practically irreversible.

Full text of DOI:10.1021/jo026501f

A Qu a n tita tive Ap p r oa ch to th e Recyclin g of r-Tocop h er ol by
Coa n tioxid a n ts
Riccardo Amorati, Fiammetta Ferroni, Marco Lucarini, Gian Franco Pedulli,* and
Luca Valgimigli
Dipartimento di Chimica Organica “A. Mangini”, Universita` di Bologna,
Via S. Donato 15, I-40127 Bologna, Italy
pedulli@alma.unibo.it
Received October 1, 2002
A systematic investigation is reported on the regeneration of R-tocopherol (R-TOH) in homogeneous
solution by coantioxidants in order to better understand the mechanism and the factors responsible
for the effectiveness of this process. The current availability of thermochemical data concerning
the reactants involved in the regeneration reactions, as well as a large number of the kinetic
constants for the various reactions involved, allowed us to rationalize the experimental observations
collected so far. Three limiting cases have been considered. The first case is that of a coantioxidant
irreversibly regenerating R-TOH, where the effectiveness of the recycling process depends on the
magnitude of the rate constant kr. The second case is that of a coantioxidant reversibly recycling
R-TOH, where regeneration can only be observed if the bond dissociation enthalpy value of the
coantioxidant is lower or at least close to that of the O-H bond of R-tocopherol. The third case is
that of a catechol derivative (chosen as a model compound for polyphenolic antioxidants), where
recycling of R-TOH is feasible even though the BDE value is significantly higher than that of vitamin
E. In this case, the driving force for the recycling process is the removal of the semiquinone radical
from the catechol derivative by the R-tocopheroxyl radical, which makes the regeneration of R-TOH
practically irreversible.
In tr od u ction
Despite the large number of experimental reports, the
regeneration mechanism of vitamin E by other coantioxi-
dants has not yet been fully clarified since the efficiency
of the recycling process has been found to depend not only
on the structure of the coantioxidant but also on its
relative concentration and on the microenvironment of
the reaction medium. We have therefore undertaken a
systematic investigation of the regeneration of R-toco-
pherol in homogeneous solution in order to better un-
derstand the mechanism and factors responsible for the
effectiveness of this process, following a first attempt to
rationalize the synergistic behavior of two antioxidants
reported by Mahoney some time ago.9 The current
availability of thermochemical data concerning the re-
actants involved in the regeneration reactions, as well
as the pertinent kinetic constants, allowed us to rational-
ize the experimental observations collected so far.
The observation that the antioxidant activity of vita-
min E (tocopherols) is enhanced by the presence in the
system of ascorbic acid (vitamin C) was reported for the
first time by Columbic and Mattill in 1941.1 Since then,
several investigations have demonstrated that these two
vitamins show a synergistic behavior and their mixtures
display much better antioxidant properties than expected
on the basis of the activity of the single components.
Exhaustive studies have been reported by Niki et al.2
in homogeneous solutions, by Ingold and co-workers
and by Niki et al.3 in phospholipid liposomes, and by
Barclay et al.4 in micelles. Synergism with R-tocopherol
has also been reported for ubiquinol-10 in liposomes,5 for
ubiquinol-3 in homogeneous solution and in liposomes,6
and for flavonoids such as epicatechin, epigallocatechin,
epicatechin gallate, etc. both in solution and micelles.7,8
Resu lts a n d Discu ssion
(1) Golumbic, C.; Mattill, H. A. J . Am. Chem. Soc. 1941, 63, 1279-
1280.
(2) Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. J . Biol. Chem.
1984, 259, 4177-4182.
These studies were performed by investigating the
thermally initiated autoxidation of styrene either in the
(3) Doba, T.; Burton, G. W.; Ingold, K. U. Biochim. Biophys. Acta
1985, 835, 298-303. Niki, E.; Kawakami, A.; Yamamoto, Y.; Kamiya,
Y. Bull. Chem. Soc. J pn. 1985, 58, 1971-1975.
(4) Barclay, L. C. R.; Locke, G. J .; MacNeil, J . M. Can. J . Chem.
1985, 63, 366-374.
(7) J ia, Z. S.; Zhou, B.; Yang, L.; Wu, L. M.; Liu, Z. L. J . Chem.
Soc., Perkin Trans. 2 1998, 911-915. Zhou, B.; J ia, Z. S.; Chen, Z. H.;
Yang, L.; Wu, L. M.; Liu, Z. L. J . Chem. Soc., Perkin Trans. 2 2000,
785-791. Chen, Z. H.; Zhou, B.; Yang, L.; Wu, L. M.; Liu, Z. L. J . Chem.
Soc., Perkin Trans. 2 2001, 1835-1839.
(5) Frei, B.; Kim, M. C.; Ames, B. N. Proc. Natl. Acad. Sci. 1990,
87, 4879-4883.
(8) Pedrielli, P.; Skibsted, L. H. J . Agric. Food Chem. 2002, 50,
submitted for publication.
(6) Landi, L.; Cabrini, L.; Fiorentini, D.; Stefanelli, C.; Pedulli, G.
F. Chem. Phys. Lipids 1992, 61, 121-130.
(9) Mahoney, L. R. Angew. Chem., Int. Ed. Engl. 1972, 8, 547.
10.1021/jo026501f CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/26/2002
J . Org. Chem. 2002, 67, 9295-9303
9295

Amorati et al.
F IGURE 1. Oxygen consumption observed during the AMVN
(0.046 M) initiated autoxidation at 30 °C of styrene (7.5 M) in
chlorobenzene in the presence of (a) 5 × 10-5 M R-TOH, (b)
F IGURE 2. Oxygen consumption traces observed during the
AMVN (0.023 M) initiated autoxidation at 30 °C of styrene
(7.5 M) in chlorobenzene in the presence of 2.5 × 10-5
M
1.05 × 10-4 M MPTZ, and (a + b) a mixture of 5 × 10-5
R-TOH and 1.05 × 10-4 M MPTZ.
M
R-TOH and 1,9-dimethylphenothiazine (MPTZ) at concentra-
tions of (a) 0 M, (b) 2.6 × 10-5 M, (c) 5.25 × 10-5 M, and (d)
7.9 × 10-5 M. Insert reports the length of the induction period
(see text) as a function of the MPTZ concentration.
absence or in the presence of the antioxidants. The
reaction was carried out in a closed system under
controlled conditions at 30 °C in air-saturated solutions
of the oxidizable substrate in chlorobenzene. The autoxi-
dations were initiated with 2,2′-azobis(2,4-dimethylvale-
ronitrile) (AMVN) or 2,2′-azobis(isobutyronitrile) (AIBN)
and followed by monitoring the oxygen consumption with
an automatic recording gas absorption apparatus using
a differential pressure transducer.10
At first we investigated the sparing effect on R-toco-
pherol (R-TOH) of 1,9-dimethylphenothiazine (MPTZ),
which is a coantioxidant characterized by a bond dis-
sociation energy (BDE) at the N-H bond (77.7 kcal/mol)11
very close to that measured at the O-H bond of TOH
(78.2 kcal/mol),12 although much less reactive than R-to-
copherol toward peroxyl radicals because of the steric
crowding about the N-H group due to the presence of
the methyl substituents in positions 1 and 9.
after which the slope of the oxygen uptake trace increases
suddenly reaching the same value observed in the
absence of any antioxidant (not shown) and indicating
that R-TOH has been completely consumed. With MPTZ,
(trace b) no clear induction period can be observed so that
the methylated phenothiazine behaves as a retardant13
rather than as an inhibitor. The mixture R-TOH/MPTZ,
in a 1:2 ratio (trace c), inhibits the autoxidation for a
period (τAH + CoAH), which is about twice as long as that
observed with only R-TOH, and then retards the oxygen
consumption for some more time.
The plots of Figure 1 can be interpreted in terms of
partial regeneration of R-tocopherol by the methylated
phenothiazine, so that the period where the autoxidation
is strongly inhibited is longer than that observed in the
presence of R-TOH alone. Since, in the absence of a
coantioxidant, each molecule of R-TOH as well as of
MPTZ11 is known to trap approximately two peroxyl
radicals and since the length of the induction period is
nearly doubled by the 1:2 mixture, about one-half of the
MPTZ molecules are capable of regenerating R-tocopherol
under the present conditions. This conclusion is further
supported by the plots of Figure 2 showing the oxygen
uptake traces observed when inhibiting the autoxidation
of styrene with R-TOH and with 1:1, 1:2, and 1:3 R-TOH/
MPTZ mixtures. The insert shows that the induction
period increases linearly with the phenothiazine concen-
tration, the slope being consistent with a regeneration
of about 40%. It is also important to point out that the
retarding effect at the end of the induction period
increases with the MPTZ concentration, this indicating
that after R-TOH is completely consumed, a remarkable
amount of the coantioxidant is still present in solution.
The rate of oxygen consumption determined during
the initiated autoxidation of styrene in PhCl in the
presence of R-TOH (5.0 × 10-5 M) and MPTZ (1.05 × 10-4
M), i.e., a concentration twice as large as that of R-TOH
and equal concentrations of both antioxidants, is plotted
in Figure 1.
It appears that the autoxidation reaction is strongly
inhibited by R-TOH (trace a) for an initial period (τΑΗ
)
These results can be expressed quantitatively by
means of eq 1, which gives the stoichiometric factor nCoAH
of the coantioxidant in the mixture with AH. The value
of nCoAH obtained for MPTZ from the data reported in
(10) Lucarini, M.; Pedulli, G. F.; Valgimigli, L.; Amorati, R.; Minisci,
F. J . Org. Chem. 2001, 66, 5456-5462.
(11) Lucarini, M.; Pedrielli, P.; Pedulli, G. F.; Valgimigli, L.; Gigmes,
D.; Tordo, P. J . Am. Chem. Soc. 1999, 121, 11546-11553.
(12) (a) Lucarini, M.; Pedrielli, P.; Pedulli, G. F.; Cabiddu, S.;
Fattuoni, C. J . Org. Chem. 1996, 61, 9259. (b) Lucarini, M.; Pedulli,
G. F.; Cipollone, M. J . Org. Chem. 1994, 59, 5063.
(13) Waters, W. A.; Wickham-J ones, C. T. J . Chem. Soc. 1951, 51,
812. Howard, J . A.; Ingold, K. U. Can. J . Chem. 1964, 42, 2324.
9296 J . Org. Chem., Vol. 67, No. 26, 2002

Recycling of R-Tocopherol by Coantioxidants
F IGURE 4. R-Tocopherol (b) and 1,9-dimethylphenothiazine
(2) consumptions observed at 60 °C during the thermal
decomposition of 5 × 10-3 M AIBN in chlorobenzene in the
presence of 5 × 10-5 M R-TOH and 1 × 10-4 M MPTZ. The
oxygen (solid line) trace has been obtained in the presence of
4.3 M styrene under otherwise identical conditions. Dotted
lines represent computer simulations (see text).
F IGURE 3. R-Tocopherol (R-TOH) consumptions observed at
60 °C during the thermal decomposition of 5 × 10-3 M AIBN
in chlorobenzene in the presence of (2) 5 × 10-5 M R-TOH
and (b) 5 × 10-5 M R-TOH and 1 × 10-4 M 1,9-dimethylphe-
nothiazine. Plot shows also the oxygen uptake traces obtained
under similar conditions except for the presence of 4.3 M
styrene added to amplify the oxygen consumption after the
end of the induction period (traces a and b refer to the
experiments carried out in the presence of only R-TOH and
the mixture R-TOH/MPTZ, respectively).
vonoids/vitamin E blends, we have investigated the
initiated autoxidation of styrene inhibited by antioxidant
mixtures containing R-tocopherol and, as a model com-
pound for flavonoids, 4-tert-butylcatechol (BC).
Figure 2 is approximately 0.76, i.e., much smaller than
the stoichiometric factor of R-TOH (nAH ) 2) and that of
MPTZ alone (nAH ) 1.7) measured by studying the
inhibited autoxidation of cumene.11
Figure 5 shows the effect on the oxygen uptake traces
of the addition to the reaction medium of R-TOH (5 ×
10-5 M), BC (1 × 10-4 M), and both R-TOH (5 × 10-5 M)
and BC (5 × 10-5, 1 × 10-4, and 1.5 × 10-4 M). It appears
from the dashed trace that the antioxidant activity of BC
is not as good as that of R-TOH; actually, the inhibition
nCoAH ) Ri (τAH + CoAH - τAH) /[CoAH]
(1)
The occurrence of partial regeneration of R-tocopherol
by MPTZ is also supported by the time dependence of
the concentrations of the two antioxidants, measured by
HPLC-MS using electrospray ionization (ESI-MS), during
the thermal decomposition of AIBN at 60 °C (Figure 3),
i.e., under experimental conditions similar to those used
when carrying out the autoxidation reactions. In these
experiments, no styrene was introduced to avoid interfer-
ence with the phenol analytes in the ESI-MS analysis
due to matrix effects, and the temperature was kept
higher than 30 °C to accelerate the decomposition of the
azo initiator. Parallel autoxidation experiments per-
formed in the presence of 4.3 M styrene (see Figure 3)
show induction periods identical to those obtained from
the disappearance of R-TOH. The plots obtained in the
presence of both antioxidants indicate that the lengthen-
ing of the induction period is due to a slower net
consumption of R-tocopherol. From the data of Figures
4, which report also the disappearance of the coantioxi-
dant MPTZ, it appears that both antioxidants are con-
sumed at similar rates and that when R-TOH has reacted
completely, about 50% of MPTZ is still present, this being
the reason for the retarding effect observed in the oxygen
consumption traces after the end of the induction period.
As already pointed out in the Introduction, other
important examples of coantioxidants showing a recycling
effect toward R-tocopherol are those of flavonoids, a class
of natural compounds found in nearly every plant, whose
antioxidant properties are mostly due to the presence in
their structure of a catechol ring and which are almost
invariably accompanied by some vitamin E. With the aim
of better understanding the synergistic behavior of fla-
rate constant k′ has been measured in the present
inh
work as 5.6 × 105 M-1 s-1 from inhibited autoxidation
studies of styrene in chlorobenzene at 30 °C. This value
is six times lower than that of R-tocopherol (3.2 × 106
M-1 s-1) under the same experimental conditions. How-
ever, when using the two inhibitors together, the anti-
oxidant activity of the mixtures is much better than
expected on the basis of a purely additive effect, since
the rate of oxidation during the induction period is the
same as the inhibited rate observed when only vitamin
E was used. This behavior indicates that some vitamin
E is still present in the system until the end of the
induction period and, thus, that R-TOH is recycled by
4-tert-butylcatechol during the autoxidation reaction.
We also measured by HPLC-ESI-MS the rate of
consumption of both R-TOH and 4-tert-butylcatechol
observed during the thermally induced decomposition of
AIBN under air (in the absence of styrene, see above).
The results, reported in Figure 5, show that the first
antioxidant to be consumed is the catechol derivative
even though its reaction with peroxyl radicals is 6 times
slower than that of R-tocopherol. Only after the coan-
tioxidant has been completely consumed is the disap-
pearance of R-tocopherol observed; thus, at the end of the
induction period, no coantioxidant is present anymore in
the reaction mixture. Therefore, unlike the system
R-TOH/MPTZ, with BC, no additional retarding of the
autoxidation seems to be observed at the end of the
induction period τAH
.
+ CoAH
Mech a n ism of Regen er a tion . To explain the experi-
mental results, the mechanism of the inhibited autoxi-
J . Org. Chem, Vol. 67, No. 26, 2002 9297

Amorati et al.
F IGURE 5. (Left) Oxygen consumption during the AMVN (0.046 M) initiated autoxidation at 30 °C of styrene (7.5 M) in
chlorobenzene in the presence of 1 × 10-4 M 4-tert-butylcatechol (BC) (dashed line). Solid lines have been recorded in the presence
of 5 × 10-5 M R-TOH and BC concentrations of (a) 0 M, (b) 5 × 10-5 M, (c) 1 × 10-4 M, and (d) 1.5 × 10-4 M. (Right) Disappearance
of R-TOH (O) and BC (9) observed during the thermal (60 °C) decomposition under air of AIBN (0.002 M) in chlorobenzene
containing both antioxidants at initial concentrations of 1 × 10-4 M. Full and dashed lines represent computer simulations (see
text).
SCHEME 1
dation of an hydrocarbon, RH, should be considered. The
overall reaction scheme is given by the following series
of equations, where eqs 2 and 3 represent the initiation
and eqs 4 and 5 the propagation, and eq 6 represents
the termination steps.14
Ri
9
8 InOO•
(2)
(3)
(4)
InOO• + RH f InOOH + R•
R• + O2 f ROO•
ratio and on the relative concentrations of the two
species, so that the inhibition effect of the mixture would
be purely additive without any synergism between the
two antioxidants. If, however, hydrogen exchange takes
place at a rate larger or comparable to that of the
termination reaction (eq 10) leading to the disappearance
of the R-tocopheroxyl radicals, the latter ones will ab-
stract a hydrogen atom from the coantioxidant, thus
regenerating R-TOH. As a result, R-tocopherol will be
consumed at a lower rate, while the coantioxidant will
disappear at a rate higher than predicted on the basis of
the inhibition rate constants kinh and k′inh. At the same
time, the oxygen uptake will show an inhibition period
longer than expected on the basis of the initial R-toco-
pherol concentration, followed by a second period where
the autoxidation is retarded by the residual coantioxi-
dant. The apparent stoichiometric factor n of R-tocopherol
will appear larger than 2, its value depending on the
relative rates of reactions (reactions 8-11). The overall
behavior can be conveniently visualized on the basis of
the simplified Scheme 1.
kp
9
ROO• + RH
8 ROOH + R•
(5)
(6)
2kt
2ROO• 8 products
In the presence of a chain-breaking antioxidant, AH,
such as R-tocopherol and a coantioxidant CoAH, chain-
propagating peroxyl radicals can be trapped by either or
by both of them (eqs 7, 8) depending on the relative rate
constants kinh and k′ .
inh
kinh
ROO• + AH
8 ROOH + A•
(7)
(8)
(9)
k′
ROO• + CoAH inh8 ROOH + CoA•
A• + CoAH ykr z AH + CoA•
k-r
ROO• + A• f products
ROO• + CoA• f products
(10)
(11)
In practice, regeneration of R-tocopherol by a coan-
tioxidant can only be observed under the experimental
conditions where the rate of reaction 9 is larger or at least
comparable to that of reaction 10. If the regeneration
reaction is irreversible, i.e., k-r , kr or k11[ROO•] .
k-r[AH], this condition can be simply expressed by eq 12
or 13.
The resulting R-tocopheroxyl radical can react with a
molecule of the coantioxidant to regenerate R-tocopherol
(eq 9) with a rate constant kr or react with a second
peroxyl radical to give termination products (eq 10).
Similarly, the radical from the coantioxidant, CoA•, can
either give the hydrogen exchange reaction with R-TOH
(eq -9) or terminate with ROO• (eq 11).
k10[ROO•][Α•] e kr[CoAH][Α•]
[CoAH] g (k10/kr) [ROO•]
(12)
(13)
In the absence of the hydrogen atom exchange reaction
(eq 9), R-tocopherol and the coantioxidant would be
consumed with rates only depending on the kinh/k′
inh
(14) Denisov, E. T.; Khudyakov, I. V. Chem. Rev. 1987, 87, 1313.
Howard, J . A. In Free Radicals; Kochi, J . K., Ed.; Wiley & Sons: New
York, 1973; Vol. 2, pp 3-62.
This means that recycling of vitamin E will be more
easily observed at large coantioxidant concentrations or,
9298 J . Org. Chem., Vol. 67, No. 26, 2002

Recycling of R-Tocopherol by Coantioxidants
since [ROO•] ) Ri/(2kinh[AH] + 2k′ [CoAH]) = Ri/2kinh
[AH], at low rates of initiation and at high R-TOH
content.
R-tocopherol should be consumed 12.5 times faster than
MPTZ at the beginning of the oxidation reaction. If,
however, hydrogen exchange takes place at a rate greater
than or comparable to that of the termination reaction
10, R-tocopherol will be regenerated by MPTZ and the
mixture of the two antioxidants will show synergism
similar to that observed when vitamin C is the coantioxi-
dant. To check this condition, the values Ri ) 5.6 × 10-8
M s-1 and [ROO•] ) 1.75 × 10-10 M were extracted from
the plots of Figure 1, while kr and k-r were determined
by EPR, as described in the following section, as 1.5 ×
103 M-1 s-1 and 6.5 × 102 M-1 s-1, respectively, at 25 °C
in benzene solution. The minimum concentration of the
coantioxidant, (k10/kr)[ROO•], required to observe some
regeneration according to eq 12, is 1.2 × 10-5 M, i.e., a
value much lower than the experimental concentration
(1.0 × 10-4 M) of MPTZ employed. Therefore, although
the absolute rate constant for hydrogen transfer from 1,9-
dimethylphenothiazine to R-TO•, kr, is much smaller than
that with ascorbate, partial regeneration of R-tocopherol
can take place.
inh
r-Tocoph er ol/Vita m in C. A coantioxidant for which
the condition k-r , kr holds is vitamin C. Actually, the
pertinent rate constants measured in tert-butyl alcohol
are kinh ) 5.6 × 105 M-1 s-1 (eq 7; AH ) TOH)15 and k′
inh
) 7.5 × 104 M-1 s-1 (eq 8; CoAH ) vitamin C).2 Despite
the absence of experimental data for the rate constant
k-r of the reverse reaction (eq -9), this can be evaluated
from the free energy change for the reaction of the
R-tocopheroxyl radical with ascorbate to regenerate
vitamin E16 that has been estimated as -5.0 kcal/mol,
on the basis of the standard one-electron reduction
potentials of the involved species17 and calculated as -7.7
kcal/mol in a recent theoretical paper.18 Thus, the value
of k-r must be about 4-5 orders of magnitude lower than
kr ) 1.55 × 106 M-1 s-1 (eq 9; AH ) TO• and CoAH )
vitamin C),19 reaction 9 can be considered to be practically
irreversible, and eq 12 should hold. To check if the
condition expressed by eq 13 is also fulfilled, the rate
constant k10 for the combination of peroxyl with R-toco-
pheroxyl radicals should be known. Since most of the
reported kinetic constants for the reaction of peroxyl and
However, there is an important difference with respect
to vitamin C, since the hydrogen exchange reaction (eq
9) with MPTZ is almost thermoneutral and thus revers-
phenoxyl radicals are of the order of 108 M-1 s-1 20-22
we
,
ible (∆G° ) -0.5 kcal/mol at 298 K from the known
r
N-H11 and O-H12 BDE values in MPTZ and R-TOH). If
the hydrogen exchange reaction (eq 9) is faster than the
termination reactions 10 and 11, the concentrations of
the two antioxidants AH and CoAH and those of the
corresponding radicals are determined by the equilibrium
constant Kr (eq 14).
may assume for k10 the reasonable value of 1 × 108 M-1
s-1. From the rate of initiation reported by Niki et al.,2
Ri ) 2.58 × 10-8 M s-1 at 37 °C, the minimum concentra-
tion of the coantioxidant required to observe synergism
can be calculated as 3 × 10-8 M, a value much smaller
than the vitamin C concentration of 5.5 × 10-5 M used
in these regeneration experiments.2
[AH][CoA•]
[A•][CoAH]
Thus, on the basis of these data it is expected that,
when R-TOH and vitamin C are present in solution in
similar amounts, the peroxyl radicals will react first with
the former to give an R-tocopheroxyl radical that im-
mediately reacts with vitamin C to regenerate R-TOH.
This is in complete agreement with the behavior reported
in homogeneous solution by Niki et al.,2 who observed
complete disappearance of vitamin C before R-tocopherol
started to be consumed and 100% regeneration of the
latter antioxidant.
Kr ) kr/k-r
)
(14)
From the simultaneous equations describing the evolu-
tion of the species appearing in eqs 2-11, the rate laws
for the disappearance of R-tocopherol, coantioxidant, and
oxygen (in a closed system) can be straightforwardly
derived under the steady-state approximation for the
radical species ROO•, A•, and CoA• and are given by eqs
15-17.
r-Tocoph er ol/1,9-Dim eth ylph en oth ia zin e. In the
case of the mixture R-tocopherol/MPTZ, the pertinent
rate constants of the various reactions involved are kinh
Ri
d[AH]
dt
-
)
(15)
(16)
(17)
Krk11[CoAH]
k10[AH]
) 3.2 × 106 M-1 s-1 for R-TOH15,23 at 30 °C and k′
)
inh
2 1 +
(
)
1.4 × 105 M-1 s-1 for MPTZ at 50 °C.11 Thus, in the
absence of the hydrogen atom exchange reaction (eq 9),
Ri
d[CoAH]
since the ratio kinh/k′ = 23 and [AH]/[CoAH] ) 0.5,
inh
-
)
dt
k10[AH]
2 1 +
(15) Valgimigli, L.; Banks, J . T.; Lusztyk, J .; Ingold, K. U. J . Org.
Chem. 1999, 64, 3381-3383.
(16) Although reaction 9 could take place via an initial electron
transfer followed by proton transfer, a recent computational study
suggests the direct hydrogen transfer mechanism as a more likely
pathway.17
(
)
Krk11[CoAH]
kp[RH]
d[O2]
-
) Ri 1 +
[
]
dt
2(kinh[AH] + k′ [CoAH])
inh
(17) Buettner, G. R. Archiv. Biochem. Biophys. 1993, 300, 535-543.
(18) DiLabio, G. A.; Wright, J . S. Free Rad. Biol. Med. 2000, 29,
480-485.
From the last equation it can be inferred that the
oxygen consumption plots during the inhibition period
should display a slope slightly lower than that observed
in the presence of only R-tocopherol, which depends on
(19) Packer, J . E.; Slater, T. F.; Willson, R. L. Nature 1979, 278,
737-738.
(20) Griva, A. P.; Denisov, E. T. Int. J . Chem. Kinet. 1973, 5, 869.
(21) Mahoney, L. R.; DaRooge, M. A. J . Am. Chem. Soc. 1975, 97,
4722.
the value of k′ and on the concentration of CoAH.
inh
(22) Bowry, V. W.; Ingold, K. U. J . Org. Chem. 1995, 60, 5456-
5467.
(23) Burton, G. W.; Doba, T.; Gabe, E. J .; Hughes, L.; Lee, F. L.;
Prasad, L.; Ingold, K. U. J . Am. Chem. Soc. 1985, 107, 7053-7065.
Equation 15 can be used to obtain the induction time, τ,
for the AH and CoAH mixture, which is the time at which
a sudden change of slope will be observed in the oxygen
J . Org. Chem, Vol. 67, No. 26, 2002 9299

Amorati et al.
consumption plots. Since this will occur when R-toco-
pherol is completely consumed, τ ) τAH + τCoAH will be
given by eq 18.
stituent effect of this group -1.75 kcal/mol,25 the BDE
value for BC can be calculated as 81.1 kcal/mol; thus,
the free energy change for reaction 9 (with CoAH ) BC)
and its equilibrium constant Kr at 303 K can be estimated
as 2.9 kcal/mol and 8.1 × 10-3, respectively. Assuming
that the rate constants for the termination with peroxyl
radicals of R-TO• and of the semiquinone radical from the
catechol derivative are the same (k10 = k11), the regenera-
tion factor R should be equal to Kr. Since the calculated
value of Kr is too small to justify the observed complete
recycling of R-TOH by 4-tert-butylcatechol (R ) 1), some
other reaction not considered so far must be taken into
account in the system R-TOH/BC. This conclusion is
supported by the observation that no regeneration at all
was found27 when investigating the antioxidant activity
of a 1:1 mixture of R-TOH and a 2,4,6-tri-tert-butylphenol
having nearly the same BDE (81.2 kcal/mol)26 and Kr
values of BC.
Krk11[CoAH]
[AH]
2[AH]
τ )
)
1 +
(18)
(
)
Ri
-d[AH]/dt
k10[AH]
Therefore, under equilibrium conditions for the hydro-
gen exchange reaction 9, the plot of the measured
induction period as a function of the concentration ratio
of the two antioxidants [CoAH]/[AH] should be linear
with a slope R equal to Krk11/k10. On the basis of the
definition of the stoichiometric coefficient nCoAH of the
coantioxidant in the mixture, given in eq 1, it can be
easily shown that for coantioxidants capable of scaveng-
ing two peroxyl radicals such as phenols and aromatic
amines, eq 19 holds.
The complete recycling of R-TOH by catechol deriva-
tives can be explained in terms of the two-step mecha-
nism, exemplified in eqs 20 and 21, similar to that
previously suggested in the case of the reduced form of
ubiquinone Q3, which is also able to recycle vitamin E.5
In the first step (the analogue of reaction 9), reversible
hydrogen transfer takes place between BC and R-TO• to
give a semiquinone radical, while in the second step (eq
21) the resulting semiquinone reacts with another R-to-
copheroxyl radical to give R-TOH and 4-tert-butyl-ortho-
quinone.28
nCoAH ) 2 R
(19)
In the case of a coantioxidant such as vitamin C, which
can scavenge only one peroxyl radical, eq 19 becomes
nCoAH ) R, where R represents in both cases a measure
of the effectiveness of the regeneration process.
Good linearity, with slope R ) 0.38, has actually been
observed (see the insert of Figure 2) when plotting the
induction times measured during the initiated autoxi-
dation of styrene against the ratio [1,9-dimethylphe-
nothiazine]/[R-tocopherol]. Since the equilibrium constant
for reaction 9 is 2.3 for the couple AH ) R-TOH and
CoAH ) MPTZ, the plot provides also the ratio k11/k10 as
0.17. Thus, the reaction of peroxyl with R-tocopheroxyl
radicals is ca. 6 times faster than that with dimethylphe-
nothiazinyl radicals. To check whether the k11 value
obtained by R is reasonable, we tried to fit the oxygen
and antioxidants consumption traces obtained in the
experiments carried out in the presence of the MPTZ/R-
TOH mixtures by using a method of chemical reaction
kinetics simulation (see experimental). These simulations
are shown in Figure 4 as dotted lines for the traces of
the calculated time dependence of the concentrations of
R-tocopherol, 1,9-dimethylphenothiazine, and oxygen,
superimposed to the experimental measurements. The
good agreement with the experimental results indicates
that the assumed kinetic model is satisfactory.
On the basis of the known heat of formation ∆H° of
f
catechol (-63.9 kcal/mol)30 and that calculated for ortho-
quinone (-30.0 kcal/mol) by using the Benson additivity
rules31 and the estimated O-H BDE value in catechol
(83.0 kcal/mol),24 the heats of reactions 20 and 21, with
(25) Lucarini, M.; Pedrielli, P.; Pedulli, G. F. In Free Radicals in
Chemistry, Biology and Environment; Minisci, F., Ed.; Kluwer Aca-
demic Publishers: Norwell, MA, 1997; pp 169-179.
r-Tocoph er ol/4-ter t-Bu tylca tech ol (BC). The mix-
ture R-TOH/BC provides another example of regeneration
of vitamin E through a mechanism characterized by some
important differences with respect to the previous ones.
On the basis of the known values of the rate constants
(26) Mahoney, L. R.; Ferris, F. C.; DaRooge, M. A. J . Am. Chem.
Soc. 1969, 91, 3883-3889.
(27) Amorati, R.; Calabrese, V.; Lucarini, M.; Pedulli, G. F., Val-
gimigli, L. Results to be published.
kinh ) 3.2 × 106 M-1 s-1 for R-TOH and k′ ) 5.6 × 105
(28) Evidence for the formation of 4-tert-butylquinone (4-BQ) as the
reaction product of the regeneration of R-tocopherol by 4-tert-butyl-
catchol was obtained by analyzing aliquots of a typical autoxidation
mixture (inhibited by 1 × 10-4 M R-TOH and 1 × 10-4 M BC) by a
specific spectrophotometric assay recently developed by some of us29
based on the reaction of 4-BQ with ADA (4-diethylaminoaniline) to
yield an intensely blue adduct (λmax ) 625 nm, ꢀ ) 11 120 M-1cm-1 in
CH2Cl2). By this assay, an amount of 4-BQ of about 10% the value
expected from complete conversion of BC was observed toward the end
of the inhibited period. The lack of quantitative recovery of 4-BQ will
be a matter of further work.
inh
M-1 s-1 for BC (vide infra) and of the estimated value of
the equilibrium constant Kr for the hydrogen transfer
reaction between the substituted catechol and R-toco-
pheroxyl radicals, the regeneration factor R should be
negligibly small, as it is shown below, while experimen-
tally it is found to be close to 1 (i.e., 100% regeneration).
To evaluate Kr we have used the recently measured O-H
BDE value24 of 3,5-di-tert-butylcatechol (79.3 kcal/mol),
which differs from 4-tert-butylcatechol only for the pres-
ence of an additional tert-butyl group. Being the sub-
(29) Rescigno, A.; Sanjust, E.; Pedulli, G. F.; Valgimigli, L. Anal.
Lett. 1999, 32, 2007-2017. Valgimigli, L.; Pedulli, G. F.; Cabiddu, S.;
Sanjust, E.; Rescigno, A. Tetrahedron 2000, 56, 659-662.
(30) Ribeiro da Silva, M. D. M. C.; Ribeiro da Silva, M. A. V. J . Chem.
Thermodynam. 1984, 16, 1149-1155.
(24) Lucarini, M.; Mugnaini, V.; Pedulli, G. F. J . Org. Chem. 2002,
67, 928-931.
(31) Estimated on the basis of the Benson additivity rules32 assum-
ing that CO - (Cd)(CO) ) CO - (CB)(CO).
9300 J . Org. Chem., Vol. 67, No. 26, 2002

Recycling of R-Tocopherol by Coantioxidants
R ) H, are calculated to be 4.8 and -23.1 kcal/mol,
respectively. Given its high exothermicity (due to the very
low O-H bond strength in the semiquinone radical, ca.
55 kcal/mol), reaction 21 is expected to be fast enough to
favorably compete with termination reaction 11. There-
fore, the semiquinone radicals produced by reaction with
either peroxyl radicals (eq 8) or R-tocopheroxyl radicals
(eq 20) will be continuously subtracted from the reaction
medium making the regeneration of R-TOH much more
efficient than predicted on the basis of the Kr value. This
mechanism can explain the experimental results shown
in Figure 5 reporting the disappearance of R-TOH and
4-tert-butylcatechol during the thermal decomposition of
AIBN in chlorobenzene.
F IGURE 6. Time evolution of the EPR signal due to the
aminyl radical generated at 298 K by UV irradiation of a
benzene solution of 1,9-dimethylphenothiazine (0.001 M)
containing tBuOOtBu (0.1 M). Irradiation was started at time
1 and stopped at time 2; R-tocopherol was injected at time 3.
No radical decay was observed, in the absence of R-TOH,
during the time of the experiment.
To estimate the unknown rates of the reactions in-
volved we have simulated the time dependence of the
antioxidant concentrations of Figure 5. To do so, we first
determined by EPR the rate constant for the hydrogen
transfer from 4-tert-butylcatechol to R-tocopheroxyl radi-
cals in benzene at 298 K (vide infra). By assuming that
reaction 20 is the rate-limiting step of the sequence
described by eqs 20 and 21, we obtained kr ) (2.8 ( 1.2)
× 103 M-1 s-1. From the calculated equilibrium constant
Kr ) 8.1 × 10-3, the value of k-r is obtained as 3.5 × 105
substituted catechol is not complete, this should only
depend on the low value of the rate constant kr for
hydrogen atom transfer.
M-1 s-1
.
Ra te Con sta n ts for th e Regen er a tion of r-Toco-
p h er ol. To assess the feasibility of the regeneration
reaction of R-tocopherol by coantioxidants (eq 9), the
knowledge of the rate constants for hydrogen atom
transfer from the coantioxidant to R-TO• (kr) is essential.
We therefore carried out the determination of kr in
benzene solution at room temperature, by the kinetic
EPR method, for several coantioxidants.
In the case of 1,9-dimethylphenothiazine, the reverse
rate constant k-r was measured for convenience, i.e., the
rate for hydrogen atom transfer from R-tocopherol to the
aminyl radical of MPTZ; then, kr was calculated from the
equilibrium constant Kr obtained from the known ∆BDE
difference. The method consisted of monitoring the decay
of a spectral line of the very persistent 1,9-dimethylphe-
nothiazinyl radical, not superimposed onto that of R-TO•,
after the instantaneous injection of a concentrated stock
solution of R-tocopherol (final concentration of ca. 1 mM)
in the sample tube, while continuously bubbling nitrogen
(see Figure 6). Under the experimental conditions em-
ployed, the EPR signal completely decayed in approxi-
mately 5-10 s after the injection of R-tocopherol, follow-
ing good pseudo-first-order kinetics. The measured rate
constant k-r was obtained as 6.5 × 102 M-1 s-1 from
which the value kr ) 1.5 × 103 M-1 s-1 in benzene at 298
K was calculated using the difference of 0.5 kcal/mol
between the BDEs of R-TOH and MPTZ.11,12
Since the value for k-r is close to the known rate
constant for the bimolecular self-decay of R-tocopheroxyl
radicals in benzene at the same temperature,12b the
observed kinetics might be due to the occurrence of fast
hydrogen transfer between R-tocopherol and the aminyl
radical followed by the spontaneous decay of the resulting
R-tocopheroxyl radical. To rule out this possibility, the
rate constants of hydrogen transfer kH (eq 22) to the
aminyl radical from MPTZ from a series of substituted
phenols having the same steric hindrance around the
reaction center as R-TOH (i.e., two methyl groups ortho
to the OH) have been measured and correlated with the
O-H BDE values of the phenols. Experiments were
Simulations (see Figure 5) were performed using the
same method previously described by leaving unchanged
the experimentally determined rate constants as well as
k10 ) 1 × 108 M-1 s-1 as in the previous simulations. The
two remaining rate constants k11 and k21 were instead
allowed to vary on a wide range of values, i.e., 1 × 108 e
k11 e 1 × 1010 M-1 s-1 and 1 × 105 e k21 e 5 × 109 M-1
s-1
.
For any value of k21, the simulations were found totally
independent of the assumed rate constant k11 up to 1 ×
1010 M-1 s-1; we therefore chose for it the reasonable
value of 1 × 108 M-1 s-1. On the other hand, to get good
agreement with the experimental data concerning the
consumption of BC, R-TOH, and oxygen, the rate constant
k21 should be at least 5 × 107 M-1 s-1. Such a high value
seems to be quite reasonable in view of the strong
exothermicity of reaction 21 and also by comparison with
the rate constants for the similar dismutation reactions
33
of catechol (3.9 × 108 M-1 s-1
)
and of 2,6-dimethylhy-
droquinone (3.0 × 108 M-1 s-1).34 It can be therefore
concluded that reaction 21, being so fast to overwhelm
reaction -20, i.e., the hydrogen transfer from R-TOH to
the semiquinone radical, represents the driving force of
the regeneration process that makes the recycling of
R-tocopherol practically irreversible under typical au-
toxidation conditions.35 Since the regeneration of R-TOH
is likely to be essentially irreversible with any catechol
derivative, with this class of coantioxidants the regenera-
tion factor R should depend only on the ratio kr/k10; in
other words, if the regeneration of R-tocopherol by a
(32) Benson, S. W. Thermochemical Kinetics; Wiley & Sons: New
York, 1968.
(33) Khudiakov, I. V.; Kuzmin, V. A.; Emanuel, N. M. Int. J . Chem.
Kinet. 1978, 10, 1005.
(34) Foster, T.; Elliot, A. J .; Adeleke, B. B.; Wan, J . K. S. Can. J .
Chem. 1978, 56, 869.
(35) Simulations were also done by assuming that dismutation of
the semiquinone radical from BC could take place. No effect was found
on the simulated traces by varying the rate constant in the range 0 e
kdis e 5 × 109 M-1 s-1
.
J . Org. Chem, Vol. 67, No. 26, 2002 9301

Amorati et al.
TABLE 1. Ra te Con sta n ts kH for th e Hyd r ogen Tr a n sfer
Rea ction (eq 22) betw een or th o-Dim eth yl P h en ols a n d
th e 1,9-Dim eth ylp h en oth ia zin yl Ra d ica l, in Ben zen e
Solu tion a t 298 Ka
from MPTZ, R-tocopheroxyl radicals undergo bimolecular
self-decay in the time scale of the reaction with BC, care
had to be taken to set the initial concentration of R-TO•
such that the bimolecular self-decay was negligible under
our experimental conditions (less than 5% of the total
apparent decay rate). Decay traces followed good pseudo-
first-order kinetics from which the value for 2kr was
obtained as (5.6 ( 2.4) × 103 M-1 s-1 at 298 K.
kH,
BDE(O-H),
(kcal/mol)
k-H
(M-1 s-1
)
(M-1 s-1
)
2,6-Me2-C6H3OH
2,4,6-Me3-C6H2OH
C6Me5OH
R-TOH
MPTZ
0.08
0.70
3.60
650
-
84.5b
82.7b
81.8c
78.2b
77.7d
6.4 × 103
2.8 × 103
3.2 × 103
1.5 × 103
Con clu sion s
a
O-H Bond Dissociation Enthalpies of the phenols and calcu-
lated rate constants k-H of the reverse reactions are also reported.
It has been shown that the recycling of R-tocopherol
by coantioxidants in homogeneous solutions may be
completely described by the proposed kinetic model. This
model has been applied to three limiting cases: in the
first one, where the coantioxidant is vitamin C, the
regeneration step is irreversible or, at least, is so fast
with respect to the reverse reaction that it can be
considered irreversible for practical purposes. Thus, the
effectiveness of the recycling process depends on the
magnitude of the rate constant kr that, under normal
experimental conditions, must be at least 103 M-1 s-1 in
order for regeneration of R-TOH to be observed.
b
d
From ref 12. c From the present work. From ref 11.
The second case is that of 1,3-dimethylphenothiazine
(a model for sterically hindered coantioxidants character-
ized by low rate constants of inhibition) where the
recycling is reversible and its effectiveness depends on
the ratio Krk11/k10, i.e., essentially on the equilibrium
constant for the hydrogen atom transfer reaction between
the two antioxidants and the corresponding radicals,
being k11/k10 normally close to 1. Since the magnitude of
Kr is determined by the BDE difference of the two
antioxidants, regeneration will only be observed when
the BDE value of the coantioxidant is lower or at least
close to that of R-TOH (78.2 kcal/mol).
The third case is that of 4-tert-butylcatechol (a model
compound for polyphenolic antioxidants) where recycling
of R-TOH is feasible even though the BDE value is
significantly higher than that of vitamin E. In this case,
the driving force for the recycling process is the removal
of the semiquinone radical from the catechol derivative
by the R-tocopheroxyl radical, which makes the regenera-
tion of R-TOH practically irreversible. This may explain
the strong antioxidant properties of natural products
containing flavonoids or other polyphenolic antioxidants
together with some amount of R-tocopherol.
F IGURE 7. Relation between the logarithm of the rate
constants kH for the hydrogen transfer reaction from phenols
to the 1,9-dimethylphenothiazinyl radical and the difference
between the strengths of the O-H bond of phenols and the
N-H bond of MPTZ (∆BDE).
performed on solutions of both antioxidants by using high
phenol concentrations because of the large BDE differ-
ence with MPTZ. The measured rate constants are
reported in Table 1 together with the pertinent BDE
values.
ArOH + Ar2N• ykH z ArO• + Ar2NH
(22)
k-H
The O-H bond dissociation enthalpy for pentameth-
ylphenol, previously unknown, was measured here from
equilibration experiments as 81.8 ( 0.4 kcal/mol.36
As shown in Figure 7, for all the investigated phenols
(including R-tocopherol), the correlation between log kr
and the difference between the BDE values of phenols
and that of 1,9-dimethylphenothiazine (BDEOH -
BDENH) is excellent. This guarantees that the measured
rate constants actually pertain to the same chemical
reaction, i.e., to the hydrogen atom transfer of eq 9.
The rate constant for the hydrogen transfer between
4-tert-butylcatechol and the R-tocopheroxyl radical (eq 20,
kr) was similarly measured in benzene by kinetic EPR
using identical procedures and directly monitoring the
decay of the EPR signal of the photolytically generated
R-TO• after the injection of a concentrated stock solution
of BC (0.25-1 mM). Since, unlike the aminyl radicals
Exp er im en ta l Section
Ma ter ia ls. Solvents were of the highest grade commercially
available and used as received. 2,2′-Azobis(2,4-dimethylvale-
ronitrile), AMVN, and R,R′-azobis(isobutyronitrile), AIBN,
were stored at -20 °C. RRR-R-Tocopherol was purified by
column chromatography on silica gel eluting with 92:8 hexane/
ethyl acetate as previously described.37 4-tert-Butylcatechol,
2,4,6-trimethylphenol, and 2,6-dimethylphenol were com-
mercially available and used without further purification. 1,9-
Dimethylphenothiazine was available from previous studies.11
Pentamethylphenol was prepared according to a literature
procedure38 from 2,4,6-trimethylphenol, paraformaldehyde,
and HBr in acetic acid. The obtained 3,5-bis(bromomethyl)-
(37) Valgimigli, L.; Banks, J . T.; Ingold, K. U.; Lusztyk, J . J . Am.
Chem. Soc. 1995, 117, 9966-9971.
(38) Nagel, M.; Hansen, H. J . Helv. Chim. Acta 2000, 83, 1022-
1048.
(36) Experimental data will be reported on a forthcoming paper.
9302 J . Org. Chem., Vol. 67, No. 26, 2002

Recycling of R-Tocopherol by Coantioxidants
2,4,6-trimethylphenol was reduced with LiAlH4 in dry THF
to afford the title compound.
superimposed onto those of the phenoxyl radical was previ-
ously chosen in a separate set of experiments. The reaction of
the aminyl radical with 2,6-dimethylphenol, 2,4,6-trimeth-
ylphenol, and pentamethylphenol was similarly obtained,
although in this case, the two antioxidants were both present
in the system during photolysis: a deoxygenated benzene
solution containing di-tert-butylperoxide (0.1 M), 1,9-dimethyl
phenothiazine (1 × 10-4-0.1 M), and one of the phenols (0.1-1
M) was sealed in a quartz tube sitting inside the cavity of the
EPR spectrometer and shortly irradiated. The time evolution
of the EPR signal from the aminyl radicals was monitored
during and after UV irradiation using the instrumental setting
described above. The actual concentrations of the two anti-
oxidants (1,9-dimethylphenothiazine and phenol) had ratios
ranging between 1:10 and 1:2000, which were chosen taking
into account the BDE difference. The aminyl radical initially
formed then decayed, following pseudo-first-order kinetics, by
reacting with the excess phenol. For each phenol investigated,
measures were repeated at 5 different phenol concentrations.
Plots of the initial pseudo-first-order rate constant of EPR
signal decay versus the concentration of the phenol gave
excellent straight lines (r > 0.9) whose slopes provided the
second-order rate constant for hydrogen transfer from the
phenol to the aminyl radical.
The second-order rate constant for the hydrogen abstraction
by R-tocopheroxyl radical from 4-tert-butylcatechol was mea-
sured at 298 K in benzene containing di-tert-butyl peroxide
(0.1 M) by kinetic EPR by monitoring the decay of the EPR
trace of photolytically generated R-TO• radical with and
without the injection of a concentrated stock solution of 4-tert-
butylcatechol in benzene (final concentration of 0.25-1 mM).
The procedure and experimental settings were otherwise
identical to those previously described for the reaction of
R-TOH with the aminyl radical from MPTZ. The initial
concentrations of the reactants and the amount of tocopheroxyl
radical produced in solution by short UV photolysis were
accurately chosen to ensure clean decay traces under pseudo-
first-order kinetics and to minimize other reactions, including
the bimolecular self-decay of the R-tocopheroxyl radical. This
last reaction was determined for each experiment and set to
account for less than 5% the total apparent decay rate,
therefore being negligible under the employed conditions. Five
measurements were performed with different amounts of
injected BC, and from the resulting pseudo-first-order rate
constants, the bimolecular rate constant kr was obtained.
Sim u la tion of Rea ction Kin etics. Simulation of the
experimental traces of the consumption of the antioxidants and
oxygen were carried out by means of a stochastic simulation
program (CKS, developed at IBM, Almaden, San J ose, CA)
that calculates concentrations of all reactants and products
in chemical systems as function of time (for example, see ref
41). This is done by representing the reaction system with a
volume containing an adequate number of particles. The
particles are distributed among reactants present at the
beginning of the simulation according to their initial concen-
trations, and then the system is allowed to evolve following
the scheme and kinetic constants defined by the user, with
time steps inversely proportional to the reaction rate. The
simulations were carried out using the higher number of
molecules available (2 × 109) and the kinetic equations and
rate constants described above.
Au toxid a tion Exp er im en ts. Autoxidation experiments
were performed in a two-channel oxygen uptake apparatus,
based on a Validyne DP 15 differential pressure transducer,
that has already been described elsewere.39 The entire ap-
paratus was immersed in a thermostated bath that ensured a
constant temperature within (0.1 °C.
In a typical experiment, an air-saturated chlorobenzene
solution of styrene containing the antioxidant mixture (from
2.5 × 10-5 to 1.5 × 10-4 M) was equilibrated with the reference
solution containing only an excess of R-tocopherol (from 1 ×
10-3 to 1 × 10-2 M) in the same solvent at 30 or 60 °C. After
equilibration, a concentrated chlorobenzene solution of AMVN
or AIBN (final concentration from 5 × 10-2 to 5 × 10-3 M)
was injected in 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 instru-
mental setting allowed us to have the N2 production and the
oxygen consumption derived from the azo-initiator decomposi-
tion already corrected from the measured reaction rates.
Initiation rates, Ri, were determined for each condition in
preliminary experiments by the inhibitor method using R-to-
copherol as areference antioxidant: Ri ) 2[R-tocopherol]/τ.
Qu a n tita tive Deter m in a tion s of An tioxid a n ts. R-Toco-
pherol ((0.5-5) × 10-4 M), the coantioxidant (1 × 10-4 M), and
the thermal initiator AIBN (2-5 mM) in chlorobenzene were
allowed to react at 60 °C under air while stirring. Aliquots of
the reaction mixtures were sampled at time intervals, cooled
to 20 °C, diluted 1:10 with methanol, and analyzed by HPLC-
MS using electrospray ionization (ESI) in a Waters 2695
separation module (equipped with an autosampler) coupled to
a Micromass ZMD ESI-MS spectrometer. The most appropri-
ate instrumental settings were determined in a preliminary
set of experiments: injection volume, 20 µL; column, C18
(Waters X-Terra-MS, 3 × 150 mm, 3.5 µm); eluent, 97:3
methanol/water; flow rate (in column), 0.5 mL/min; splitting
ratio, 5:1; ESI type, negative ions; desolvation gas (N2), 750
L/h; cone gas (skimmer), 76 L/h; desolvation temp, 380 °C;
capillary voltage, -2.8 kV; cone voltage: -30 V; hexapole
extractor, -3 V. Calibration curves were obtained for each
analyte using the same instrumental settings and authentic
samples dissolved in 1:10 chlorobenzene/methanol.
Kin etic Mea su r em en ts by EP R. The rate of hydrogen
exchange between R-tocopherol and the aminyl radical from
1,9-dimethylphenothiazine was measured at 298 K by a
previously described kinetic-EPR method.40 Briefly, an open
EPR suprasyl quartz tube containing a benzene solution of 1,9-
dimethylphenothiazine ((1-5) × 10-5 M) and di-tert-butylp-
eroxide (0.1 M) was placed in the thermostated cavity of a
Bruker ESP 300 spectrometer equipped with a Bruker ER033M
Field-Frequency Lock accessory. For each experiment, a single
time-sweep EPR scan was recorded with the following set-
tings: microwave power ) 5 mW, time constant ) 4 ms,
conversion time ) 20-164 ms. The solution was continuously
bubbled with a fine stream of nitrogen through a capillary
glass tube, which ensured rapid mixing of the solutions and
continuous removal of oxygen. The aminyl radical was gener-
ated by a short pulse of UV light from an unfiltered 500 W
high-pressure Hg lamp. Briefly after the irradiation had been
interrupted, a concentrated stock solution of R-tocopherol (final
concentration ) (1-5) × 10-3 M was rapidly (<1s) injected
and the time-dependence of the EPR signal of a spectral line
from the aminyl radical was monitored. The EPR line not
Ack n ow led gm en t. Financial support from the Uni-
versity of Bologna and MURST (Research project “Free
Radical Processes in Chemistry and Biology: Syntheses,
Mechanisms, Applications”) is gratefully acknowledged.
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