Multiple active intermediates in oxidation reaction catalyzed by synthetic heme-thiolate complex relevant to cytochrome P450

Article abstract of DOI:10.1021/ja0115013

We have studied oxidation reactions using a synthetic heme-thiolate (SR complex) in order to ascertain the contributions of multiple intermediates derived from heme-thiolate to the oxygen atom transfer reaction to substrate. First, degradation of peroxyphenylacetic acid (PPAA) was examined in the presence of various substrates. The O-O bond cleavage mode of PPAA was clearly dependent on the reactivity of the substrate, and an easily oxidizable substrate enhanced heterolytic O-O bond cleavage. Second, competitive oxidations of cyclooctane and cyclooctene were carried out with various peroxybenzoic acids containing a series of substituents at the para-position as an oxygen source. The ratios of alkane hydroxylation rate/alkene epoxidation rate were dependent on the nature of the para-substituent of the oxidant. We conclude that substrate and oxidant interact with each other during the oxygen atom transfer reaction, that is, oxidation reaction occurs before O-O bond cleavage, even in the reaction catalyzed by heme-thiolate, which is considered to promote O-O bond cleavage. The results of an ¹⁸O-incorporation study that is frequently performed to determine the active intermediates derived from iron porphyrins were consistent with this conclusion.

Full text of DOI:10.1021/ja0115013

Published on Web 07/23/2002
Multiple Active Intermediates in Oxidation Reaction Catalyzed
by Synthetic Heme-Thiolate Complex Relevant to
Cytochrome P450
Noriyuki Suzuki,† Tsunehiko Higuchi,*,‡ and Tetsuo Nagano*,†
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Faculty of Pharmaceutical Sciences,
Nagoya City UniVersity, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received June 19, 2001
Abstract: We have studied oxidation reactions using a synthetic heme-thiolate (SR complex) in order to
ascertain the contributions of multiple intermediates derived from heme-thiolate to the oxygen atom transfer
reaction to substrate. First, degradation of peroxyphenylacetic acid (PPAA) was examined in the presence
of various substrates. The O-O bond cleavage mode of PPAA was clearly dependent on the reactivity of
the substrate, and an easily oxidizable substrate enhanced heterolytic O-O bond cleavage. Second,
competitive oxidations of cyclooctane and cyclooctene were carried out with various peroxybenzoic acids
containing a series of substituents at the para-position as an oxygen source. The ratios of alkane
hydroxylation rate/alkene epoxidation rate were dependent on the nature of the para-substituent of the
oxidant. We conclude that substrate and oxidant interact with each other during the oxygen atom transfer
reaction, that is, oxidation reaction occurs before O-O bond cleavage, even in the reaction catalyzed by
heme-thiolate, which is considered to promote O-O bond cleavage. The results of an 18O-incorporation
study that is frequently performed to determine the active intermediates derived from iron porphyrins were
consistent with this conclusion.
Scheme 1
Introduction
Cytochrome P450 is a group of heme-containing monooxy-
genases that play important roles in the metabolism of many
physiological substrates and xenobiotics.1 Because of their
extremely strong oxidizing ability, much interest has been
focused on P450 chemistry, and identification of the reactive
intermediate responsible for oxygen atom transfer to substrate
in catalytic oxidation by cytochrome P450 has been one of the
major goals in bioinorganic chemistry. A high-valent oxo-iron
porphyrin π-cation radical (called “compound I”, 2 in Scheme
1), formally two-electron-oxidized from the ferric state, has been
widely proposed as the active species.1-3 Indeed, compound I
is observable by spectroscopic techniques in the reactions of
oxidant and many synthetic iron porphyrins ligated by Cl-,
AcO-, MeOH, or other nonthiolate ligands4 and in the reactions
of enzymes such as peroxidase and catalase, although they also
have a nonthiolate axial ligand.5 Furthermore, Sligar et al.
recently obtained the structure of an intermediate that would
be consistent with an oxyferryl species resulting from X-ray
irradiation of ferrous-dioxygen P450cam by the use of time-
lapse X-ray crystallography.6 However, the fact that a certain
intermediate is generated by stoichiometric reaction in the
absence of substrate does not necessarily imply that the
* Author to whom correspondence may be sent. E-mail: nsuzuki@
mol.f.u-tokyo.ac.jp.
† The University of Tokyo.
‡ Nagoya City University.
(4) (a) Groves, J. T.; Watanabe, Y. J. Am. Chem. Soc. 1988, 110, 8443. (b)
Groves, J. T.; Haushalter, R. C.; Nakamura, M.; Nemo, T. E.; Evans, B. J.
J. Am. Chem. Soc. 1981, 103, 2884. (c) Yamaguchi, K.; Watanabe, Y.;
Morishima, I. J. Chem. Soc., Chem. Commun. 1992, 1721. (d) Gross, Z.;
Nimri, S. Inorg. Chem. 1994, 33, 1731. (e) Bill, E.; Ding, X.-Q.; Bominar,
E. L.; Trautwein, A. X.; Winkler, H.; Mandon, D.; Weiss, R.; Gold, A.;
Jayaraj, K.; Hatfierd, W. E.; Kirk, M. L. Eur. J. Biochem. 1990, 188, 665.
(5) (a) Baek, H. K.; Van Wart, H. E. Biochemistry 1989, 28, 5714. (b) Neptuno,
J.; Rodriguez-Lopez, J. N.; Smith, A. T.; Thornsley, R. N. T. J. Biol. Chem.
1996, 271, 4023.
(1) (a) Ortiz de Montellano, P., Ed.; Cytochrome P-450: Structure, Mechanism,
and Biochemistry, 2nd ed.; Plenum Press: New York, 1995. (b) Sono, M.;
Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. ReV. 1996, 96, 2841.
(2) (a) Meunier, B. Chem. ReV. 1992, 92, 1411. (b) Gunter, M.; Turner, P.
Coord. Chem. ReV. 1991, 108, 115. (c) Groves, J. T. J. Chem. Educ. 1985,
62, 928.
(3) (a) Gold, A.; Jayaraj, K.; Doppelt, P.; Weiss, R.; Chottard, G.; Bill, E.;
Ding, X.; Trautwein, A. X. J. Am. Chem. Soc. 1988, 110, 5756. (b) Balch,
A. L.; Latosgrazynski, L.; Renner, M. W. J. Am. Chem. Soc. 1985, 107,
2983. (c) Sugimoto, H.; Tung, H. C.; Sawyer, D. T. J. Am. Chem. Soc.
1988, 110, 2465. (d) Fujii, H.; Ichikawa, K. Inorg. Chem. 1992, 31, 1110.
(e) Fujii, H. Chem. Lett. 1994, 1491.
(6) Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson,
D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. Science 2000,
287, 1615.
9
9622
J. AM. CHEM. SOC. 2002, 124, 9622-9628
10.1021/ja0115013 CCC: $22.00 © 2002 American Chemical Society

Intermediates in Oxidation Catalyzed by Heme−Thiolate
A R T I C L E S
intermediate is responsible for the oxidation reaction even in
catalytic reactions. The catalytic reaction in the presence of
substrates could be quite different. In contrast to the conventional
simple scheme, in which compound I is the sole reactive
intermediate responsible for the oxidation reaction based on
direct and indirect evidence,7 recent studies suggest that the
actual reaction mechanism is a more complex one, as shown in
Scheme 1.8,9 Machii et al. demonstrated that the active species
responsible for the epoxidation is not always compound I in
catalytic epoxidation by synthetic iron porphyrins and also
showed that competitive epoxidation is a good indicator to
discriminate the active species.8a Nam et al. studied the
competitive epoxidation by synthetic iron porphyrins with
hydroperoxides or peracids and demonstrated that two inter-
mediates, 1 and 2 in Scheme 1, were both able to oxidize easily
oxidizable substrates such as alkenes and that the nature of the
reactive intermediates depended upon the rate of the O-O bond
cleavage of 1.8b Further, Nam et al. demonstrated that 1 could
oxidize even nonactivated alkanes to give alcohol products.9a
Collman et al. also provided evidence that the active intermedi-
ates in the oxidation reactions by iron porphyrins with iodos-
ylbenzene derivatives as oxygen donors are iron porphyrin-
oxidant complexes.9b In the case of P450 enzymes, site-specific
mutagenesis studies performed by Vaz et al. and Newcomb et
al. indicated that iron-hydroperoxide (1), iron-hydrogen
peroxide (protonated 1), and iron-oxo complex (2) all act as
electrophilic oxidants in alkene epoxidation and alkane hy-
droxylation.10
Figure 1. Structure of the SR Complex.
We synthesized the first synthetic heme-thiolate (SR
complex,12a Figure 1) that retains thiolate coordination during
catalytic oxidation and have found several remarkable thiolate
axial ligand effects.12a-c,13 In previous studies using SR, we
measured the rates of substrate oxidation by SR with several
alkyl hydroperoxides and found that the sixth thiolate ligand of
SR induces a 50-250-fold increase in the oxidation rates
compared with chloride ligand.12a We also examined the
degradation of terminal oxidant mediated by SR and found that
the thiolate ligand enhances the formation of the two-electron-
oxidized intermediate in high yield without the assistance of
acid or base.13 To assess the effect of thiolate ligand on the
oxidation reactivity, we examined the kinetic isotope effect and
18O incorporation from 18O-enriched oxidants in the O-de-
methylation reaction and examined the alkane oxidation rate/
alkene oxidation rate ratio in the competitive oxidations of
alkane and alkene catalyzed by SR, other synthetic iron-
porphyrins, and cytochrome P450s. From these studies, we have
established that the catalytic features of SR and P450s are very
similar and that the thiolate ligand has a marked influence on
the reactivity of the intermediate, although the electronic
structure of the reactive intermediate derived from SR remains
uncertain.12b,c
However, these previous studies using synthetic hemes were
carried out by use of nonthiolate synthetic hemes. Considering
that axial thiolate coordination is a key feature of cytochrome
P450 and that the axial ligand greatly affects both the reactivity
of heme enzyme11 and that of synthetic heme,12 thiolate-ligated
iron porphyrin would be clearly preferable as a model for
reaction mechanism analysis relevant to P450. While Vaz et
al. and Newcomb et al. obtained interesting results by using
P450 enzymes and their mutants, synthetic model studies with
thiolate-ligated iron porphyrin would be of value because one
can chose various oxidants, substrates, and reaction conditions
such as temperature, polarity of solvent, and so on, as required
for analysis of each reaction.
Now in the present study, we have carried out reaction
analysis by use of SR with various oxidants and substrates in
order to throw light on the nature of the reactive intermediate
derived from SR that is truly responsible for the oxygen atom
transfer reaction in catalytic oxidation.
Experimental Section
Materials. CH2Cl2 was distilled from CaH2 before use. Peroxy-
phenylacetic acid and other substituted peroxybenzoic acids were
prepared by a literature method14 and purified by washing with 200
mM sodium phosphate buffer (pH ) 6.5) and recrystallization from
hexane. SR complex and Fe(TMP)Cl (5,10,15,20-tetramesitylpor-
phyrinate iron(III) chloride) were prepared by a method reported
previously.12,15,16 Fe(TPFPP)Cl [5,10,15,20-tetrakis(pentafluorophen-
yl)porphyrinate iron(III) chloride] was purchased from Aldrich Chemi-
cal Co. and used without further purification. TBPH (2,4,6-tri-tert-
butylphenol) was purchased from Tokyo Kasei and purified by silica
gel column chromatography and recrystallization from EtOH-H2O.
Cyclooctane and cyclooctene were purchased from Tokyo Kasei and
distilled from CaH2 before use.
(7) (a) Nam, W.; Goh, Y. M.; Lee, Y. J.; Lim, M. H.; Kim, C. Inorg. Chem.
1999, 38, 3238. (b) Bernadou, J.; Meunier, B. Chem. Commun. 1998, 2167.
(c) Lee, K. A.; Nam, W. J. Am. Chem. Soc. 1997, 119, 1916. (d) Bernadou,
J.; Fabiano, A. S.; Robert, A.; Meunier, B. J. Am. Chem. Soc. 1994, 116,
9375.
(8) (a) Machii, K.; Watanabe, Y.; Morishima, I. J. Am. Chem. Soc. 1995, 117,
6691. (b) Nam, W.; Lim, M. H.; Lee, H. J.; Kim, C. J. Am. Chem. Soc.
2000, 122, 6641. (c) Kamaraj, K.; Bandyopadhyay, D. J. Am. Chem. Soc.
1997, 119, 8099.
(9) (a) Nam, W.; Lim, M. H.; Moon, S. K.; Kim, C. J. Am. Chem. Soc. 2000,
122, 10805. (b) Collman, J. P.; Chien, A. S.; Eberspacher, T. A.; Brauman,
J. I. J. Am. Chem. Soc. 2000, 122, 11098.
(10) (a) Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 3555. (b) Vaz, A. D. N.; Pernecky, S. J.; Raner, G. M.;
Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4644. (c) Newcomb,
M.; Shen, R.; Choi, S.-Y.; Toy, P. H.; Hollenberg, P. F.; Vaz, A. D. N.;
Coon, M. J. J. Am. Chem. Soc. 2000, 122, 2677. (d) Toy, P. H.; Newcomb,
M.; Coon, M. J.; Vaz, A. D. N. J. Am. Chem. Soc. 1998, 120, 9718.
(11) (a) Sigman, J. A.; Pond, A. E.; Dawson, J. H.; Lu, Y. Biochemistry 1999,
38, 11122. (b) Raphael, A. L.; Gray, H. B.; J. Am. Chem. Soc. 1991, 113,
1038. (c) Matsui, T.; Nagano, S.; Ishimori, K.; Watanabe, Y.; Morishima,
I. Biochemistry 1996, 35, 13118.
(12) (a) Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem. Soc. 1990, 112, 7051.
(b) Ohno, T.; Suzuki, N.; Dokoh, T.; Urano, Y.; Kikuchi, K.; Hirobe, M.;
Higuchi, T.; Nagano, T, J. Inorg. Biochem. 2000, 82, 123. (c) Urano, Y.;
Higuchi, T.; Hirobe, M.; Nagano, T. J. Am. Chem. Soc. 1997, 119, 12008.
(d) Nam, W.; Lim, M.; Oh, S. Y.; Lee, J. H.; Lee, H. J.; Woo, S. K.; Kim,
C.; Shin, W. Angew. Chem. 2000, 39, 3646.
Instruments. GC/SIM (gas chromatography/selected ion monitoring)
analyses were performed on a Hewlett-Packard 5890 Series II gas
(13) Higuchi, T.; Shimada, K.; Maruyama, N.; Hirobe, M. J. Am. Chem. Soc.
1993, 115, 7551.
(14) Ogata, Y.; Sawaki, Y. Tetrahedron 1967, 23, 3327.
(15) Lindsey, L. S.; Wagner, R. W. J. Org. Chem. 1989, 54, 828.
(16) Kobayashi, H.; Higuchi, T.; Kaizu, Y.; Osada, H.; Aoki, M. Bull. Chem.
Soc. Jpn. 1975, 48, 3137.
9
J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9623

A R T I C L E S
Suzuki et al.
Scheme 2
Scheme 3
a high-valent oxo-iron porphyrin complex is the sole interme-
diate responsible for oxygen transfer, such phenomena should
not occur, i.e., the O-O bond cleavage mode of oxidants should
be independent of the substrate, and the reactivity of the
intermediate should be independent of the oxidant.
chromatograph with a J&W Scientific DB-5 (30 m) capillary column
and JEOL JMS-SX 102A mass spectrometer.
Effect of the Substrate on the Mode of O-O Bond
Cleavage of PPAA. First, we investigated the mode of O-O
bond cleavage mediated by SR in the presence of several
substrates with a range of reactivity, i.e., TBPH, cyclooctene,
and cyclooctane based on the hypothesis illustrated in Scheme
2. Their reactivity is in the order of TBPH > cyclooctene >
cyclooctane. Many studies have been carried out on the mode
of O-O bond cleavage of oxidants.17 We chose to use
peroxyphenylacetic acid (PPAA), which has frequently been
used as a probe for this purpose,18 because the cleavage mode
can be easily examined by quantitative determination of
degradation products derived from the oxidant. Heterolytic
cleavage of the O-O bond affords phenylacetic acid, and
homolytic cleavage affords toluene, benzyl alcohol, and ben-
zaldehyde via the benzyl radical. The O-O bond of each
acylperoxo intermediate cleaves with each inherent hetero/homo
ratio, but direct reaction of acylperoxo intermediate and substrate
affords phenylacetic acid and apparently affects the O-O bond
cleavage mode. The reactions were performed in CH2Cl2, a
relatively inert solvent that provides a hydrophobic environment
around the complex, as is generally the case at the active sites
of P450s. In each reaction, a large excess of the substrate and
just 1 mol equiv of PPAA to SR were used, because it is
necessary to minimize the decomposition of SR and to prevent
secondary oxidation of the formed phenylacetic acid by the
reactive intermediate. We confirmed the fact that secondary
oxidation of the formed phenylacetic acid did not occur under
this condition by another study (not shown). In the other
porphyrin systems, the same amounts of oxidant and substrate
as in the SR system were used to obtain comparable results.
Analysis of the O-O Bond Cleavage Mode. A methylene chloride
solution (0.8 mL) containing an iron porphyrin (0.125 mM) and a
substrate (25 mM TBPH or cyclooctene or 250 mM cyclooctane) was
prepared. The reaction was started by adding a methylene chloride
solution of PPAA (0.2 mL, 0.5 mM) at 20 °C under Ar. The reaction
was terminated by adding a methylene chloride solution of Ph3P (0.05
mL, 4 mM) after 30 s (in the case that the iron porphyrin was SR) or
10 min [in the case that the iron porphyrin was Fe(TMP)Cl or
Fe(TPPFP)Cl]. The reaction mixture was further stirred for 5 min, then
a methylene chloride solution of diethylcatechol (0.2 mL, 0.1 mM)
was added as an internal standard, and the products were analyzed by
GC-SIM.
Competitive Oxidation. A methylene chloride solution (0.4 mL)
containing SR complex (1.25 mM) and substrates (25 mM cyclooctene
and 250 mM cyclooctane) was prepared. The competitive oxidation
was started by adding a methylene chloride solution of a substituted
peroxybenzoic acid (0.1 mL, 5 mM) at -15 °C under Ar. The reaction
was terminated by adding a methylene chloride solution of Ph3P (0.1
mL, 50 mM) after 1 min. The reaction mixture was further stirred for
5 min at 20 °C, then a methylene chloride solution of diethylcatechol
(0.1 mL, 0.2 mM) was added as an internal standard, and the products
were analyzed by GC-SIM.
Analysis of 18O Incorporation from Bulk Water. CH2Cl2/CH3-
OH (1:1, total volume 0.4 mL) solution or CH2Cl2/CH3CN (1:1, total
volume was 0.4 mL) solution containing iron porphyrin (0.625 mM)
and cyclooctene (12.5 mM) and H218O (10%) was prepared. The
reaction was started by adding a methylene chloride solution of mCPBA
(0.1 mL, 10.0 mM) at various temperatures under Ar. The reaction
was terminated by adding a methylene chloride solution of Ph3P (0.05
mL, 25 mM) after 1 min (in the case that the iron porphyrin was SR)
or 20 min [in the case that the iron porphyrin was Fe(TPPFP)Cl]. The
reaction mixture was further stirred for 5 min, then a methylene chloride
solution of diethylcatechol (0.1 mL, 0.2 mM) was added as an internal
standard, and the products were analyzed by GC-SIM.
In the oxidation reactions catalyzed by SR with PPAA,
phenylacetic acid was predominantly formed (80-96% based
on PPAA) as the degradation product derived from the oxidant
in every reaction, compared with the reactions catalyzed by other
synthetic iron porphyrins. This result means that the thiolate
axial ligand enhances heterolytic O-O bond cleavage in all of
the reactions with various substrates, as we previously reported.13
However, significant differences in the yields of degradation
Results and Discussion
The present experiments were designed to throw light on the
nature of the reactive intermediate. If the initially formed species
(1, shown in Scheme 1) contributes to the oxidation reaction,
the substrate and oxidant could interact with each other during
the reaction. Specifically, substrates that can be readily oxidized
by the active intermediate could apparently affect the O-O bond
cleavage mode of oxidants, as shown in Scheme 2, and electron-
donating or electron-withdrawing substituents of oxidants could
affect the reactivity of the intermediate, such as substrate
selectivity, reaction rate, and so on, as shown in Scheme 3. If
(17) (a) Nam, W.; Han, H. J.; Oh, S. Y.; Lee, Y. J.; Choi, M. W.; Han, S. Y.;
Kim, C.; Woo, S. K.; Shin, W. J. Am. Chem. Soc. 2000, 122, 8677. (b)
Nam, W.; Choi, H. J.; Han, H. J.; Cho, S. H.; Lee, H. J.; Han, S. Y. Chem.
Commun. 1999, 387.
(18) (a) White, R. E.; Sligar, S. G.; Coon, H. J. J. Biol. Chem. 1980, 255, 11108.
(b) Traylor, T. G.; Lee, W. A.; Stynes, D. V. J. Am. Chem. Soc. 1984,
106, 755. (c) Traylor, T. G.; Tsuchiya, S.; Byun, Y. S.; Kim, C. J. Am.
Chem. Soc. 1993, 115, 2775.
9
9624 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002

Intermediates in Oxidation Catalyzed by Heme−Thiolate
A R T I C L E S
Table 1. Yield of Degradation Products Derived from Peroxyphenyacetic Acid Mediated by SR, Fe(TPP)Cl, and Fe(TPFPP)Cl in the
Presence of Various Substratesa
a Details of reaction conditions are described in the Experimental Section. All reactions were carried out at least in duplicate, and the data reported
represent the average values. b Yield based on PPAA. c Not detected.
products derived from PPAA were observed, depending on the
substrate, and the ratio of heterolysis to homolysis varied from
68.6 for TBPH to 9.7 for cyclooctane, as shown in Table 1.
These results demonstrate that homolysis of the O-O bond of
PPAA certainly occurs in the SR-peroxy acid system if the
reactivity of the substrate is fairly low, even though heme-
thiolate complex was considered to greatly enhance heterolytic
O-O bond cleavage13,19 and to afford the acid quantitatively,
owing to the so-called “push effect”.20 The fact that the ratio
of heterolysis to homolysis varies with substrate implies that
there is an interaction between O-O bond cleavage and
substrate oxidation, that is, the initially formed acylperoxo
intermediate that corresponds to the hydroperoxo intermediate
in P450 catalytic cycle contributes to the oxidation reaction, at
least to some extent (although the degree of contribution of the
acylperoxo intermediate is still uncertain), as shown in Scheme
2. We consider that electron transfer from the substrate to the
acylperoxo intermediate derived from SR and PPAA, in
association with the electron transfer from olefin to in situ
generated compound I,1,21 enhances heterolysis of the O-O
bond. In the Fe(TMP)Cl or Fe(TPFPP)Cl systems, significant
differences in the yields of degradation products derived from
PPAA were also observed, depending on the substrate, and
reflect the contribution of the respective acylperoxo intermedi-
ates to the oxidation reaction. The difference between these two
systems is dependent on the electronic nature of the porphyrin
ligand.
Effect of the Substituent of the Oxidant on the Reactivity
of the Intermediate. Next, we carried out competitive oxidation
of cyclooctane versus cyclooctene catalyzed by SR to examine
the effect of an electron-donating or electron-withdrawing
substituent of oxidants on the reactivity of the intermediate based
on the hypothesis illustrated in Scheme 3. It has been generally
accepted that alkane hydroxylation and alkene epoxidation
involving the common active intermediate, compound I, proceed
via different mechanisms. It has been proposed that the alkane
hydroxylation proceeds by an H atom abstraction-hydroxyl
radical recombination mechanism in the case of P450,22 while
alkene epoxidation proceeds via a one-electron-transfer mech-
anism in metalloporphyrin-catalyzed oxidation.21 Therefore, it
is expected that alkene/alkane competitive oxidation can be used
as a probe for discrimination of differences in chemical
properties among active intermediates, as Mansuy et al. sug-
gested.23 This competitive oxidation has been used in our
(19) (a) Adachi, S.; Nagano, S.; Ishimori, K.; Watanabe, Y.; Morishima, I.;
Egawa, T.; Kitagawa, T.; Makino, R. Biochemistry 1993, 32, 241.
(20) Dawson, J. H. Science 1988, 240, 433.
(22) Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J. Biochem.
Biophys. Res. Commun. 1978, 81, 154.
(23) Bartoli, J. F.; Brigaud, O.; Battioni, P.; Mansuy, D. Chem. Commun. 1991,
440.
(21) Gross, Z.; Nimri, S. J. Am. Chem. Soc. 1995, 117, 8021.
9
J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9625

A R T I C L E S
Suzuki et al.
Table 2. Competitive Oxidation of Cyclooctane versus
Cyclooctene Catalyzed by SR with Various Peroxybenzoic Acids
Containing a Series of Substituents at the Para-Positiona
Figure 2. Hammett plot of the results of competitive oxidation of
cyclooctane versus cyclooctene catalyzed by SR with various peroxybenzoic
acids.
of the iron porphyrin-peroxybenzoic acid complex cleaves,
because iron-oxo species should mediate the reactions with
identical selectivity, regardless of its origin. This result is
consistent with the result of PPAA oxidation, and now we can
reasonably conclude that two distinct reactive intermediates
contribute to substrate oxidation (at least, alkene oxidation) even
in the reaction catalyzed by heme-thiolate, which has been
considered to promote O-O bond cleavage.25
a Details of reaction conditions are described in the Experimental Section.
All reactions were carried out at least in duplicate, and the data reported
represent the average values. b Yield based on oxidant.
previous studies in order to discriminate differences in chemical
properties among active intermediates derived from various P450
enzymes or synthetic hemes that have various porphyrin ligands
or axial ligands, including SR and SR derivatives, from the
viewpoint that the high-valent oxo-iron porphyrin complex is
the sole reactive intermediate.12b,24
Quantitative evaluation of these substituent effects was
performed by means of a Hammett plot, as shown in Figure 2.
There seems to be a straightforward relationship between the
ratio of alkane oxidation/alkene oxidation and σp of each
substituent, except for the methoxy group. This significant
correlation means that the change of the ratio is due to the
electronic effect of the substituent of the peroxybenzoic acids
used as terminal oxidants. If the acylperoxo intermediate of an
iron porphyrin contributes to oxidation reactions, an electron-
deficient oxidant would make the active intermediate more
electron-deficient, so an electron-deficient oxidant such as
p-nitroperoxybenzoic acid should enhances alkene epoxidation.
In our oxidation system, in which SR was employed, electron-
deficient oxidants certainly showed lower ratios of alkane
oxidation/alkene oxidation compared with relatively electron-
rich oxidants. Therefore, we concluded that the change in
product ratio demonstrates that the acylperoxo intermediate
contributes to oxygen atom transfer in oxidation reactions
catalyzed by SR. Another possibility that affects the ratios of
alkane oxidation/alkene oxidation is the contribution of com-
pound II, formally one-electron-oxidized from the ferric state,
formed by homolysis of the O-O bond. However, we could
ruled out this possibility for the following reasons: If the
oxidation reactions presented in this work proceeded via iron-
oxo intermediates and if compound II formed by homolysis
contributed to these oxidation reactions, an electron-deficient
oxidant such as nitroperoxybenzoic acid should exhibit a higher
ratio of alkane/alkene oxidation compared with a relatively
electron-rich oxidant. This is because compound II dose not
mediate alkane hydroxylation, so an electron-deficient oxidant
that enhances heterolysis of the O-O bond should enhance
generation of compound I and should enhance alkane hydroxy-
In this report, we used this competitive oxidation in order to
examine in detail the contribution of the acylperoxo intermediate
to the oxidation reaction. We used six peroxybenzoic acids with
a series of substituents at the para-position: p-nitroperoxyben-
zoic acid, p-cyanoperoxybenzoic acid, p-(tirfluoromethyl)-
peroxybenzoic acid, p-chloroperoxybenzoic acid, p-fluoroper-
oxybenzoic acid, and p-methoxyperoxybenzoic acid. The reactions
were performed in CH2Cl2 under argon and at -15 °C in order
to prevent direct epoxidation of peroxybenzoic acid, although
we confirmed that the direct epoxidation was negligible, because
the oxygen transfer reaction catalyzed by SR occurs immediately
after peroxybenzoic acid addition, compared with the gradual
direct epoxidation (not shown). Just as in PPAA oxidation, a
large excess of the substrate and just 1 mol equiv of oxidant to
SR were used. Cyclooctanol, cyclooctanone, and cyclooctene
oxide were determined as oxidation products. The allylic
hydroxylation of cyclooctene was negligibly small in all cases.
As shown in Table 2, SR-peroxy acid systems effectively
oxidized cyclooctane and yielded cyclooctanol and cyclo-
octanone in every reaction, despite the difficulty of C-H bond
activation, as we previously reported,12b and the ratio values of
oxidation products are similar to each other. However, Table 2
also shows significant differences in the ratio values (from 0.63
to 0.81) when different peroxybenzoic acids were used. These
results indicate that the substituents of the oxidants affected the
substrate selectivity of the reactive intermediate, that is, the
oxidation reaction occurs to some extent before the O-O bond
(24) (a) Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Uekusa, H.; Ohashi,
Y.; Uchida, T.; Kitagawa, T.; Nagano, T, J. Am. Chem. Soc. 1999, 121,
11571. (b) Dokoh, T.; Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.;
Nagano, T, J. Inorg. Biochem. 2000, 82, 127.
(25) Dawson, J. H.; Holm, R. H.; Trudell, J. R.; Barth, G.; Linder, R. E.;
Bunnenberg, E.; Djerassi, C. J. Am. Chem. Soc. 1976, 98, 3707.
9
9626 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002

Intermediates in Oxidation Catalyzed by Heme−Thiolate
A R T I C L E S
Scheme 4
Table 3. Percentage of 18O Incorporation from Bulk Water into the
Oxide Product in the Epoxidation of Cyclooctene Catalyzed by SR
and Fe(TPFPP)Cl with MCPBAa
%of 18
in oxide
product
O
% yield
of oxide
productb
catalyst
solvent
temp (°C)
SR
CH2Cl2/CH3OH
25
0
-40
25
0
-40
25
0
-40
25
0
-40
-
NDc
NDc
NDc
4.6
5.8
6.4
56.8
66.0
86.4
74.3
88.5
91.0
-
-
CH2Cl2/CH3CN
CH2Cl2/CH3OH
CH2Cl2/CH3CN
NDd
NDd
NDd
32.0
12.1
4.6
5.0
2.7
0.8
Fe(TPFPP)Cl
a Details of reaction conditions are described in the Experimental Section.
All reactions were carried out at least in duplicate, and the data reported
represent the average values. b Based on mCPBA. c No product peak was
lation. However, we obtained the opposite result in our work.
If this change of ratio was caused by the different reactivity of
each acylperoxo intermediate that was formed by each terminal
oxidant, we can readily rationalize our results as described
above.
d
detected by GC-SIM. 18O incorporation in excess of the natural
abundance was not detected.
evidence of the formation of iron-oxo intermediates. However,
the lack of oxygen exchange does not necessarily rule out the
formation of iron-oxo intermediates. In this study, there might
be several reasons for the lack of oxygen exchange. As shown
in Scheme 4, lack of oxygen exchange means k1 . kO-O (an
iron-oxo intermediate is not formed) or k2 . kex (although an
iron-oxo intermediate is formed, the rate of oxygen exchange
is much smaller than the rate of oxygen transfer from the iron-
oxo intermediate to the substrate). In addition, Nam et al.
reported that when the axial position to the oxo group of the
iron-oxo intermediate is occupied by a ligand such as imida-
zole, oxygen exchange of the iron-oxo intermediate with bulk
water is greatly inhibited, because the six-coordinated iron-
oxo intermediate does not have an appropriate binding site for
water.27 Therefore, the above results alone are not a critical test
of our hypothesis described in Scheme 1, but they are consistent
with the conclusion based on the other studies described here.
18O Incorporation Study from Bulk Water to Oxide
Product in the Epoxidation Reaction Catalyzed by SR. Since
isotopically labeled water, H218O, has been frequently used as
a probe for the formation of iron-oxo intermediates in oxidation
reactions catalyzed by iron porphyrins, as shown in Scheme
4,26 we examined the SR-catalyzed epoxidation of cyclooctene
with mCPBA in the presence of H218O to determine whether
an iron-oxo intermediate is involved in the oxidation reactions
by analyzing the extent of 18O incorporation from labeled water
into the product. If the iron-oxo intermediate is generated in
the reaction and if the rate of oxygen exchange between the
iron-oxo intermediate and bulk water is comparable to the rate
of oxygen transfer from the iron-oxo intermediate to substrate,
the oxide product should contain the labeled oxygen.
First we carried out this study in methanol-containing solvent,
as this is frequently used for 18O incorporation studies. Under
this condition, 18O was incorporated into the oxide product
generated by Fe(TPFPP)Cl-catalyzed epoxidation of cyclooctene
with mCPBA in the presence of H218O, depending on the
reaction temperature (shown in Table 3), as Nam et al.
previously reported.27 However, the epoxide product was not
formed in the reaction catalyzed by SR under this condition.
This is because active intermediates derived from SR have
extremely strong oxidizing ability and readily oxidize methanol.
Therefore, we carried out this study in acetonitrile-containing
solvent. Under this condition, 18O incorporation into the epoxide
product in the reaction catalyzed by Fe(TPFPP)Cl also occurred,
as in the reaction in methanol-containing solvent, although the
18O enrichment was smaller. In contrast, as shown in Table 3,
18O was not incorporated in excess of the natural abundance
into the oxide product formed by SR-catalyzed epoxidation of
cyclooctene with mCPBA in the presence of H218O, suggesting
that the oxygen of the active intermediate derived from SR and
mCPBA did not exchange with bulk water. In this study,
incorporation of labeled oxygen can be regarded as indirect
Conclusion
Our results clearly demonstrate that the initially formed
acylperoxo-heme-thiolate complex contributes to oxygen atom
transfer in oxidation reactions catalyzed by SR in a hydrophobic
environment resembling the hydrophobic active sites of P450s,
although the respective contributions of the acylperoxo complex
and pure iron-oxo complex to the oxidation reaction are still
uncertain. In our reaction system, each terminal oxidant forms
an intermediate, affording multiple SR-acylperoxo complexes
that have distinct oxidizing abilities. This result supports the
recent proposal about the reaction mechanism of cytochrome
P450, i.e., that iron-hydroperoxide, iron-hydrogen peroxide,
and iron-oxo complex act as multiple electrophilic oxidants
in alkene epoxidation and alkane hydroxylation.10 Because the
molecular structure, hydrophobic environment of the reaction
site, and oxidation reactivity of SR and P450 are very similar,
we suggest that the oxidation reactions in the catalytic cycle of
P450 probably occur in the same manner.
(26) (a) Nam, W.; Valentine, J. S. J. Am. Chem. Soc. 1993, 115, 1772 and
references therein. (b) Groves, J. T.; Haushalter, R. C.; Nakamura, M.;
Nemo, T. E.; Evans, B. J. J. Am. Chem. Soc. 1981, 103, 2884.
Acknowledgment. This work was supported in part by
Grants-in-Aid for Scientific Research (No. 07407079, Biome-
(27) Lee, K. A.; Nam, W. J. Am. Chem. Soc. 1997, 119, 1916.
9
J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9627

A R T I C L E S
Suzuki et al.
tallics No. 10129203, and No. 11116207) from the Ministry of
Education, Science, Sports and Culture, Japan. We thank Dr.
Y. Urano (The University of Tokyo) for his kind and fruitful
discussions. N.S. gratefully acknowledges a JSPS Research
Fellowship for Young Scientists from the Japan Society for the
Promotion of Science.
Supporting Information Available: The time course of the
oxidation reaction catalyzed by SR and Fe(TPFPP)Cl at low
temperature by which we determined the reaction time for each
complex. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0115013
9
9628 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002