Stability and reactivity of aryloxyl radicals derived from a novel antioxidant BO-653 and related compounds. Effects of substituent and side chain in solution and membranes

Article abstract of DOI:10.1021/ja9942080

2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) is a novel antioxidant designed as a drug for inhibition of lipid peroxidation in vivo. To understand the dynamics of action of BO-653 as an antioxidant, the effects of substituents and side chains on the stability and reactivity of the BO-653 derived radical were studied and compared with the aryloxyl radicals derived from related compounds including α-tocopherol. The rate constants for the reactions of the aryloxyl radicals with themselves, lipid, hydroperoxide, and ascorbate were measured with a stopped-flow electron spin resonance (ESR) spectroscope equipped with a rapid mixing device. The ortho substituents exerted profound effects on the rate of bimolecular decay reactions and the reaction with ascorbic acid, while the effects on the reactions with methyl linoleate and tert-butyl hydroperoxide were much less significant. The side chain at the 2-position did not exert any effect in organic solution but the two pentyl side chains, when compared with two methyl side chains, diminished the apparent reactivities in the liposomal membranes.

Full text of DOI:10.1021/ja9942080

5438
J. Am. Chem. Soc. 2000, 122, 5438-5442
Stability and Reactivity of Aryloxyl Radicals Derived from a Novel
Antioxidant BO-653 and Related Compounds. Effects of Substituent
and Side Chain in Solution and Membranes
Akira Watanabe,† Noriko Noguchi,† Akio Fujisawa,† Tatsuhiko Kodama,† Kunio Tamura,§
Osamu Cynshi,§ and Etsuo Niki*,†
Contribution from the Research Center for AdVanced Science and Technology, UniVersity of Tokyo,
4-6-1 Komaba, Tokyo 153-8904, Japan, and Fujigotemba Research Laboratories,
Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
ReceiVed December 2, 1999
Abstract: 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) is a novel antioxidant
designed as a drug for inhibition of lipid peroxidation in vivo. To understand the dynamics of action of BO-
653 as an antioxidant, the effects of substituents and side chains on the stability and reactivity of the BO-653
derived radical were studied and compared with the aryloxyl radicals derived from related compounds including
R-tocopherol. The rate constants for the reactions of the aryloxyl radicals with themselves, lipid, hydroperoxide,
and ascorbate were measured with a stopped-flow electron spin resonance (ESR) spectroscope equipped with
a rapid mixing device. The ortho substituents exerted profound effects on the rate of bimolecular decay reactions
and the reaction with ascorbic acid, while the effects on the reactions with methyl linoleate and tert-butyl
hydroperoxide were much less significant. The side chain at the 2-position did not exert any effect in organic
solution but the two pentyl side chains, when compared with two methyl side chains, diminished the apparent
reactivities in the liposomal membranes.
Introduction
that the antioxidant suppresses atherosclerosis4,5 and the epi-
demiological studies suggest that high intake of vitamin E
reduces the risk of coronary heart disease.6 Probucol, 4,4′-
(isopropylidenedithio)bis(2,6-di-tert-butylphenol), is a synthetic
antiatherogenic drug used commercially, but it is known to have
the side effect of decreasing high-density lipoprotein in plasma.
BO-653, 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-
benzofran, is a novel antioxidant that has been designed,
synthesized, and found to exert a potent antioxidant activity
against lipid peroxidation7 and LDL oxidative modification in
vitro8,9 and also antiatherogenic activity in three different animal
models.5 Special attention has been paid in designing BO-653
to choose appropriate substituents and side chains which
determine the localization, retainment, and mobility within LDL
particle. The potency as an antioxidant is determined by many
factors as well as the chemical reactivity toward radicals.10 For
example, probucol is chemically much less reactive toward
The free radical-mediated autoxidation of organic compounds
by molecular oxygen is a double-edged sword and has both
positive and negative aspects. This is one of the key processes
in the present industrial chemistry and is applied for the
production of chemicals such as terephthalic acid and cyclo-
hexanol, important raw materials for synthetic polymers. On
the other hand, such oxidation also induces oxidative degradation
of plastics and rubbers and deterioration of foods and oils, and
therefore various antioxidants have been developed to inhibit
such oxidation. From the biological viewpoints, molecular
oxygen is essential to us for energy production, synthesis of
biologically active compounds, phagocytosis, and signal trans-
duction in vivo. At the same time, there is now increasing
experimental and clinical evidence which shows the involvement
of free radical-induced oxidative damage of lipids, proteins, and
DNA in a variety of pathological events, cancer, and aging.1
As a result, the role of antioxidants has received renewed
attention and both natural and synthetic antioxidants have been
extensively explored.2
(3) Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J. C.; Wiztum,
J. L. N. Engl. J. Med. 1989, 320, 915-924.
(4) (a) Kietchevsky, D.; Kirn, H. K.; Tepper, S. A. Proc. Soc. Exp. Biol.
Med. 1971, 136, 1216-1221. (b) Kita, T.; Nagano, Y.; Yokode, M.; Ishii,
K.; Kume, N.; Ooshima, A.; Yoshida, H.; Kawai, C. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 5928-5831. (c) Carew, T. E.; Schwenke, D. C.; Steinberg,
D. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7725-7729.
It is now generally accepted that oxidative modification of
low density lipoprotein (LDL) is a key initial event in the
progression of atherosclerosis which eventually causes coronary
heart disease and cerebral hemorrhage.3 The animal studies show
(5) Cynshi, O.; Kawabe, Y.; Suzuki, T.; Takashima, Y.; Kaise, H.;
Nakamura, M.; Ohba, Y.; Kato, Y.; Tamura, K.; Hayasaka, A.; Higashida,
A.; Sakaguchi, H.; Takeya, M.; Takahashi, K.; Inoue, K.; Noguchi, N.;
Niki, E.; Kodama T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10123-10128.
(6) Gey, K. F. BioFactors 1998, 7, 113-174.
† University of Tokyo.
§ Chugai Pharmaceutical Co., Ltd.
(1) (a) Sies, H., Ed. OxidatiVe Stress: Oxidants and Antioxidants;
Academic Press: London, 1991. (b) Halliwell, B.; Gutteridge, J. M. C.,
Eds. Free Radicals in Biology and Medicine, 3rd ed.; Oxford University
Press: Oxford, 1999.
(2) (a) Frei, B., Ed. Natural Antioxidants in Human Health and Disease;
Academic Press: San Diego, 1994, (b) Packer, L.; Cadenas, E., Eds.
Handbook of Synthetic Antioxidants; Marcel Dekker: New York, 1997.
(7) Noguchi, N.; Iwaki, Y.; Takahashi, M.; Komuro, E.; Kato, Y.;
Tamura, K.; Cynshi, O.; Kodama, T.; Niki, E. Arch. Biochem. Biophys.
1997, 342, 236-243.
(8) Noguchi, N.; Okimoto, Y.; Tsuchiya, J.; Cynshi, O.; Kodama, T.;
Niki, E. Arch. Biochem. Biophys. 1997, 347, 141-147.
(9) Mu¨ller, K.; Carpenter, K. L. H.; Freeman, M. A.; Mitchinson, M. J.
Free Rad. Res. 1999, 30, 59-71.
10.1021/ja9942080 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/23/2000

Stability and ReactiVity of Aryloxyl Radicals
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5439
interactions of aryloxyl radicals, time, and the concentrations
of aryloxyl radical at time t and start, respectively. The plot of
1/[I•] as a function of time gave a straight line and the rate
constnts were obtained from the slope of the plots. They are
summarized in Table 1. It shows that (i) the radicals derived
from BO-653 and BOB with tert-butyl substituents at both ortho-
positions were persistent and decayed much slower than BOM
with two o-methyl substituents, (ii) the radical from BOM with
the five-membered heterocyclic ring decayed slower than the
PMC radical with the six-membered ring, and (iii) the side chain
exerted little effect. When BOM was added to a solution
containing BOB radical, the ESR spectrum did not change, but
when BOB was reacted with BOM radical, the ESR signal of
the BOM radical was rapidly replaced by that of the BOB radical
(Figure 2). It was observed previously that when BO-653 was
added to a solution of R-tocopheroxyl radical, the ESR signal
of the R-tocopheroxyl radical disappeared rapidly and that of
the BO-653 radical appeared and the addition of galvinoxyl to
a mixture of BO-653 and R-tocopherol gave the ESR spectrum
of only the BO-653 aryloxyl radical.7
To understand the fate of the antioxidant-derived radical, the
rates of reaction of the aryloxyl radicals with lipid, hydroper-
oxide, and ascorbic acid were also measured in ethanol from
the decay of the ESR signal of the aryloxyl radical. Methyl
linoleate and tert-butyl hydroperoxide were chosen as a
representative model of polyunsaturated lipids and lipid hydro-
peroxide, respectively. The interaction with ascorbic acid was
also studied. The antioxidant was first reacted with galvinoxyl
and then mixed with the substrate. The concentrations of the
antioxidant and galvinoxyl were chosen so that galvinoxyl was
depleted by the reaction with an excess of antioxidant before
introducing a substrate.
Figure 1. Antioxidants used in this study and their abbreviations.
radicals than R-tocopherol, the most abundant and active form
of vitamin E in vivo, but probucol inhibits lipid peroxidation
in LDL more efficiently than R-tocopherol in vitro.11 When
antioxidant scavenges radical, the antioxidant-derived radical
is formed and the fate of this radical is also one of the important
factors which determine antioxidant potency. To understand the
basic chemistry of the mechanism and dynamics of the anti-
oxidant action of BO-653, this study was carried out to in-
vestigate the stability and reactivity of the aryloxyl radicals
derived from BO-653, its related compounds BOB and BOM,
R-tocopherol, and its analogue PMC (Figure 1), aiming specif-
ically at elucidating the effect of substituents and side chain, in
solution and membranes.
Results
Under these conditions, the reactions proceed as follows:
First, the reactions of the antioxidants shown in Figure 1 and
stable phenoxyl radical galvinoxyl were studied with ESR
spectroscopy. When excess antioxidant was reacted with galvi-
noxyl at 37 °C in ethanol under nitrogen, the ESR signal of
galvinoxyl disappeared and a new ESR signal appeared. Typical
examples are shown in Figure 2. As reported previously,12the
time elapsed between the mixing of reactants and the beginning
of recording operations (∼1 s) corresponded to full consumption
of galvinoxyl and the ESR signal intensity of the new aryloxyl
radical was proportional to the initial concentration of galvinoxyl
(data not shown). BO-653 and BOB gave an identical ESR
signal, while R-tocopherol and PMC gave the same ESR signal
(data not shown). The ESR signal intensities of the aryloxyl
radicals decayed at different rates. The decay was followed with
a rapid-mixing stopped-flow ESR at constant magnetic field
corresponding to the main peak in the ESR spectrum and found
to be second order with respect to the aryloxyl radical
concentrations (Figure 3).
G• + IH f GH + I•
(3)
(4)
(5)
k2
I• + I• 98 I-I
ks
9
I• + SH
8 IH + S•
where G•, IH, I-I, and SH are galvinoxyl, antioxidant, anti-
oxidant-dimer, and substrate, respectively. The rate of decay
of antioxidant radical I• is given by eq 6, where k2 and kS are
-d[I•]/dt ) k2[I•]2 + kS[I•][SH]
(6)
the rate constant for reactions 4 and 5, respectively. The
concentration of I• was calculated from eq 7.12
kS[SH][I•]0
exp(-ks[SH]t)
k2[I•]0 + ks[SH]
The decay rate of aryloxyl radicals, I•, is given by eq 1,
[I•] )
(7)
k2[I•]0
k2[I•]0 + ks[SH]
-d[I•]/dt ) k2[I•]2
(1)
(2)
1 -
exp(-ks[SH]t)
and hence
The rate constant kS was obtained from the best fit to the
experimental decay curve. The rate constants thus obtained for
the reactions of aryloxyl radicals from the antioxidants and
methyl linoleate, tert-butyl hydroperoxide, and ascorbic acid
are included in Table 1. In ethanol solution, the effect of side
chain was minimal. The rate constants of hydrogen atom
abstraction from the three substrates by the antioxidant-derived
radicals decreased in the order of R-tocopherol ∼ PMC > BOM
> BO-653 ∼ BOB, but the difference in the reactivities varied
1/[I•] ) k2t + [I•]0
-1
where k2, t, [I•], and [I•]0 are the rate constant for bimolecular
(10) Niki, E.; Noguchi, N.; Tsuchihashi, H.; Gotoh, N. Am. J. Clin. Nutri.
1995, 62 (Suppl.), 1322S-1326S.
(11) Gotoh, N.; Shimizu, K.; Komuro, E.; Tsuchiya, J.; Noguchi, N.;
Niki, E. Biochim. Biophys. Acta 1992, 1128, 147-154.
(12) Watanabe, A.; Noguchi, N.; Takahashi, M.; Niki, E. Chem Lett.
1999, 613-614.

5440 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Watanabe et al.
Figure 2. ESR spectra of aryloxyl radicals derived from BOB and BOM. BOB or BOM (20 µM) was reacted with galvinoxyl (10 µM) and then
BOM or BOB (20 µM) was added to the solution. The ESR spectra were taken in ethanol at 25 °C under nitrogen under the conditions described
in the Experimental Section.
constants for bimolecular interactions of the aryloxyl radicals
derived from BOM, BOB, and BO-653 in PC liposomal
membranes were obtained as 3.8 × 102, 0.52, and 0.28 M-1
s-1, respectively. These rate constants in membranes were
smaller than those in homogeneous solution by 54, 48, and 25%.
The decay of BO-653 and BOB radicals was also measured in
liposomal membranes composed of soybean phosphatidylcholine
which contained about 70% linoleic acid moiety, but the decay
rates were similar to those in dimyristoyl PC liposomal
membranes, suggesting that the interaction of these aryloxyl
radicals with polyunsaturated lipids was not rapid enough. The
reduction of BOB radical and BO-653 radical in liposomal
membranes by aqueous ascorbate was also measured (Figure
4). It shows that, as described above, BOB radical undergoes
bimolecular decay faster than BO-653 radical in the absence of
ascorbic acid and that ascorbic acid reduces BOB radical faster
Figure 3. The decay of R-tocopheroxyl radical. R-Tocopherol (500
µM) and galvinoxyl (25 µM) were mixed in ethanol and the ESR signal
intensity of R-tocopheroxyl radical was followed in ethanol at 37 °C
under air. The insert is a second-order plot.
than BO-653 radical. The rate constants for the reaction of
aryloxyl radicals from BO-653 and BOB with ascorbate were
obtained as 2 and 14 M-1 s-1, respectively, in liposomal
membranes in the presence of 0.50 mM ascorbic acid. The rate
constants obtained with 12.5 mM ascorbic acid concentration
were smaller. This may be ascribed to the autoxidation of
ascorbic acid at such high concentration.14 Table 1 shows that
the rate constants for interaction of BO-653 radical and BOB
radical with ascorbic acid in liposomal membranes were 16 and
2 times smaller than those in ethanol solution, respectively.
These results suggest that side chains with different lengths
affect the apparent reactivity of aryloxyl radicals in the
membranes.
significantly with the substrates. The rate constant for the
reaction of BOM radical with BOB was obtained as (2.27 (
0.40) × 103 M-1 s-1 in ethanol at 37 °C under nitrogen.
The lipophilic antioxidants such as R-tocopherol and BO-
653 are localized in vivo in the lipophilic domain of the
membranes and lipoproteins, where the antioxidant efficacy is
affected by physical factors such as fluidity and mobility of
the microenvironment. Therefore, the action of antioxidant-
derived aryloxyl radicals was also studied in liposomal mem-
branes. The antioxidant was incorporated into the liposomal
membranes simultaneously with dimyristoyl phosphatidylcholine
(PC) and reacted with galvinoxyl to generate the aryloxyl
radical. Since the local concentration of the aryloxyl radical
within the lipophilic compartment was quite high,13 the rates
of spontaneous decay of R-tocopheroxyl and PMC radicals were
too fast to measure the rate constant accurately. The rate
Discussion
BO-653 was designed to serve as a potent, lipophilic radical-
scavenging antioxidant in the lipoproteins and cell membranes,
(14) Wayner, D. D. M.; Burton, G. W.; Ingold, K. U. Biochim. Biophys.
Acta 1986, 884, 119-123.
(13) The volume of lipids in total aqueous suspension was about 0.5%.

Stability and ReactiVity of Aryloxyl Radicals
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5441
Table 1. Rate Constants of Aryloxyl Radicals Derived from Antioxidants for the Spontaneous Decay Reaction and the Hydrogen Atom
Abstraction Reaction from Methyl Linoleate, tert-Butyl Hydroperoxide, and Asorcic Acid in Ethanol and Phosphatidylcholine Liposomal
Membranes at 37 °C under Nitrogen
in ethanola
in membranesb
hydrogen atom abstraction (M-1 s-1
)
H- atom abstraction
spontaneous
decay (M-1 s-1
spontaeous
from ascorbic acid
)
methyl linoleate
t-BuOOH
ascorbic acid
decay (M-1 -1
)
(500 µM) (M-1 s-1
)
R-tocopherol
PMC
BOM
BOB
BO-653
1.03 ((0.03) × 103
1.10 ((0.12) × 103
7.03 ((0.02) × 102
1.08 ((0.10)
2.7 ((0.4) × 10-2
2.3 ((0.4) × 10-2
2.4 ((0.3) × 10-1
2.5 ((0.2) × 10-3
2.8 ((0.2) × 10-3
4.1 ((0.1) × 10-1
3.7 ((0.2) × 10-1
2.6 ((0.5) × 10-1
1.1 ((0.4) × 10-1
1.4 ((0.2) × 10-1
1.1 ((0.2) × 105
9.5 ((0.0) × 104
8.3 ((0.1) × 104
29 ( 8
c
c
c
c
c
14
2
3.8 × 102
0.52
1.13 ((0.15)
33 ( 5
0.28
a Data are mean ( standard deviation of four independent determinations. b The anitoxident was incorporated into dimyristoyl phosphatidylcholine
liposomal membranes. c The aryloxyl radicals decayed too fast to be measured accurately.
of ortho substituents. The rate constants were measured in the
absence of oxygen, but it has been reported that R-tocopheroxyl
radical has low reactivity toward oxygen.17
The differences in the reactivities of aryloxyl radicals toward
methyl linoleate and tert-butyl hydroperoxide were smaller than
those in the bimolecular decay reaction. The radicals from BO-
653 and BOB reacted with methyl linoleate 10 times slower
than the BOM radical,18 while they reacted with tert-butyl
hydroperoxide at about half the rate of BOM radicals. Probably,
the hydrogen atom of tert-butyl hydroperoxide is less sterically
hindered than that of methyl linoleate. On the other hand, quite
a striking steric effect was observed for the reaction with
ascorbic acid. The rate constants for the reduction of aryloxyl
radicals of BO-653 and BOB by ascorbic acid in ethanol were
smaller than those from BOM, PMC, and R-tocopherol by more
than 3 orders of magnitude. The reaction between antioxidant-
derived aryloxyl radical and ascorbate is important since it
regenerates and spares phenolic antioxidant and also inhibits
the prooxidant action of the aryloxyl radicals. The most well-
known example is the reduction of R-tocopheroxyl radical by
ascorbate19 and the synergistic inhibition of oxidation by a
combination of vitamin E and vitamin C has been observed in
many cases.10,20 The present results show that ascorbate reduces
the BO-653 radical quite slowly, about 3000 times slower than
it reduces the R-tocopheroxyl radical. In agreement with this,
it was found previously that BO-653 was not spared by ascorbic
acid during the oxidation of phosphatidylcholine liposomal
membranes7 and LDL.8 Furthermore, BO-653 reduces the
R-tocopheroxyl radical to regenerate R-tocopherol,7,8 the rate
being obtained as 1.3 × 103 M-1 s-1 in ethanol under nitrogen
at 37 °C.
Figure 4. Decay of aryloxyl radicals derived from BO-653 (solid mark)
and BOB (open mark) in dimyristoyl phosphatidylcholine liposomal
membranes in the absence (diamond symbol) and presence of ascorbic
acid. [Ascorbic acid] ) 0.5 (square) and 12.5 mM (triangle).
above all against oxidative modification of LDL, and the
substituents and side chains were carefully considered. Admit-
tedly, the antioxidant should have high reactivity toward radicals,
but it may be noteworthy that the chemical reactivity of the
antioxidant toward radicals may be less important in LDL
particles than in homogeneous solution. For example, R-toco-
pherol inhibits lipid peroxidation much more efficiently than
2,6-di-tert-butyl-4-methylphenol or probucol in organic solution,
but the difference in antioxidant activities against lipid peroxi-
dation is smaller in the membranes.11 It appears considering
the wealth of information from the extensive studies on
antioxidants that the localization, mobility, and fate of antioxi-
dant radical are more important factors which determine the
overall antioxidant capacity in the membranes and lipoproteins.
The ortho tert-butyl substituents hinder sterically the access of
radical to the phenolic hydrogen atom but they also hinder the
coupling reaction of aryloxyl radicals to make the radicals more
persistent and also reduce their reactivities toward substrates.
The data in Table 1 show that the rate constants for bimolecular
decay of aryloxyl radicals derived from R-tocopherol and PMC
are the same and the largest. About 30% smaller rate constant
for BOM radical may be interpreted by a higher resonance
stabilization due to the five-membered heterocylic ring than the
six-membered ring.15,16 The bimolecular decay rate constants
for BO-653 and BOB radicals were found to be more than 600
times smaller than that for BOM, suggesting a significant effect
Another interesting issue that can be seen from Table 1 is
that the side chain has little effect in organic solution on the
stability and reactivity of aryloxyl radicals: similar rate constants
were obtained for R-tocopherol and PMC and also for BO-653
and BOB. A side chain of phenolic compound is another
important factor which determines biological antioxidant capac-
ity. R-Tocopherol and PMC exert similar antioxidant activity
in homogeneous solution,21 but PMC is more potent against lipid
(17) Doba, T.; Burton, G. W.; Ingold, K. U.; Matsuo, M. J. Chem. Soc.,
Chem. Commun. 1984, 461-462.
(18) We believe that the aryloxyl radicals react with methyl linoleate by
hydrogen atom abstraction rather than addition reaction, because the reaction
yields four isomeric conjugated hydroperoxides.
(19) The pKa1 and pKa2 values for ascorbic acid are 4.25 and 11.79,
respectively, and hence it should be present as the monoanion form of
ascorbate at pH 7.4.
(20) (a) Niki, E. Ann. N. Y. Acad. Sci. 1987, 498, 186-199. (b) Halpner,
A. D.; Handelman, G. J.; Harris, J. M.; Belmont, C. A.; Blumberg, J. B.
Arch. Biochem. Biophys. 1998, 259, 305-309. (c) Bendich, A.; Machlin,
L. J.; Scandurra, O. AdV. Free Radic. Biol. Med. 1986, 2, 419-444.
(21) Niki, E.; Kawakami, A.; Saito, M.; Yamamoto, Y.; Tsuchiya, J.;
Kamiya, Y. J. Biol. Chem. 1985, 260, 2191-2196.
(15) (a) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194-
201. (b) Mukai, K.; Okabe, K.; Hosose, H. J. Org. Chem. 1989, 54, 557-
560.
(16) Barclay, L. R. C. Can. J. Chem. 1993, 71, 1-16.

5442 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Watanabe et al.
peroxidation in the membranes21,22 and LDL23,24 than R-toco-
pherol in vitro. However, PMC has little biological activity
because it is readily excreted. The side chain is important for
incorporation and retainment in the membranes and lipoproteins.
At the same time, however, the side chain diminishes mobility
of the antioxidant within and between the membranes, the effects
being more significant with increasing side chain length.21,22
As shown in Table 1, the rate constants for bimolecular decay
reaction for BOM and BOB radicals in liposomal membranes
were about half as large as those in solution, but that for the
BO-653 radical with two pentyl side chains decreased to a
quarter in the membranes. The rate constants for the reduction
of aryloxyl radicals derived from BOB and BO-653 by ascorbate
were smaller in the membranes than in solution. It has been
shown that the reduction of radicals in the membranes and
lipoproteins by ascorbate becomes slower as the radicals go
deeper into the interior.25,26 The more significant effect of the
two pentyl side chains than two methyl groups within the
liposomal membranes may be ascribed to lower mobility in the
membranes, as observed for phytyl and methyl side chains of
R-tocopherol and PMC.22
tetramethyl-5-benzofuranol) was performed as follows. Claisen rear-
rangement of the allyl ether prepared by alkylation of 3,5-dimethylphe-
nol with 3-chloro-2-methyl-1-propene gave 3,5-dimethyl-2-(2-methyl-
2-propenyl)phenol. Then, the phenol derivative was converted into
BOM through the following three-step reactions: oxidation to the
benzoquinone derivative using Fremy’s salt, reduction to the hydro-
quinone with sodium hydrosulfite, and cyclization of the resultant
hydroquinone using boron trifluride diethyl etherate. Natural 2R,4′R,8′R-
R-tocopherol and PMC were kindly supplied by Eisai Co. Ltd. (Tokyo,
Japan). Methyl linoleate purchased from Sigma Chemical Co. (St. Louis,
MO) was purified before use as reported previously.11 Dimyristoyl
phosphatidylcholine, tert-butyl hydroperoxide, and ascorbic acid were
obtained from Sigma Chemical Co. and used as received. Other
chemicals were those of the highest grade available commercially.
Phosphatidylcholine liposomal membranes were prepared as reported
previously.11
ESR Spectra of Galvinoxyl and Antioxidant-Derived Aryloxyl
Radicals. The solution of galvinoxyl was placed into a quarts ESR
tube and an appropriate solution of the antioxidant was taken into the
sidearm. When required, the second antioxidant was taken into another
sidearm. The solutions were frozen and evacuated and then the
antioxidant was vacuum-transferred into the ESR tube. The solutions
were thawed, galvinoxyl and the antioxidant were mixed, and the ESR
spectra were recovered on an X-band JEOL JES-TE100 spectrometer
at room temperature under vacuum under the following conditions:
modulation frequency, 9.43 GHz; time constant, 0.30 s; scan time, 2
min; and microwave power, 1.00 mW. When required, the second
antioxidant was introduced to the solution.
Experimental Section
Materials. BO-653 was synthesized from 4-acetoxy-3,5-di-tert-
butylphenol and purified by column chromatography with 10% ethyl
acetate in hexane (v/v) as eluent.27 BOB and BOM were synthesized
and purified similarly.27 The synthesis of BOM (2,3-dihydro-2,2,4,6-
Stopped-Flow ESR Spectroscopy. Appropriate ethanol solutions
or aqueous suspensions of liposomal membranes containing antioxidant
and substrate were taken into the reservoir after removing air by
bubbling nitrogen and they were introduced into a rapid mixer (type
ES-SE2, JEOL, Tokyo) and then into an ESR flat cell by a pump.
(22) (a) Takahashi, M.; Tsuchiya, J.; Niki, E. J. Am. Chem. Soc. 1989,
111, 6350-6353. (b) Niki, E.; Komuro, E.; Takahashi, M.; Urano, S.; Ito,
E.; Terao, K. J. Biol. Chem. 1988, 263, 19809-19814. (c) Takahashi, M.;
Niki, E.; Kawakami, A.; Kumasaka, A.; Yamamoto, Y.; Kamiya, Y.;
Tanaka, K. Bull. Chem. Soc. Jpn. 1986, 59, 3179-3183.
Acknowledgment. The authors would like to acknowledge
the valuable contribution of Dr. M. Takahashi, Dr. H. Shi, Ms.
Y. Nakai, and Mr. H. Kubo. This study was supported in part
by the Research for the Future Program of the Japan Society
for the Promotion of Science.
(23) Gotoh, N.; Noguchi, N.; Tsuchiya, J.; Morita, K.; Sakai, H.;
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