On the Mechanism of the Oxidation of Toluenes in Artificial P450 Model Systems: Formation of Benzyl Alcohols, Benzaldehydes and Phenols

Article abstract of DOI:10.1246/bcsj.76.2353

Systems with pentafluoroiodosylbenzene (PFIB) and hemin (FeTPPCl8Cl) in dichloromethane were adopted to study the activities of the model system using toluenes as substrates for P450 enzymes. The oxidation products were mainly corresponding benzyl alcohols and benzaldehydes. Previously reported suspension systems were extended to homogeneous mixed solution systems of CH2Cl2/CH3OH/H2O to study the oxidation of benzyl alcohols to the corresponding benzaldehydes: the Hammett relation with ρ = -0.86 against ?⁺. As benzaldehydes were scarcely observed in natural P450 systems, the formation of benzaldehyde seemed characteristic only of the "open" model systems. In suspension systems, the product ratios between corresponding benzaldehydes and benzyl alcohols (ald/alc) were about 0.1-0.4, specific to the substituents and conditions applied. Curiously, the ratios (ald/alc) increased with the electronegativity of the substituents on the phenyl rings of the toluene derivatives. Time course experiments in suspension systems indicated that benzyl alcohols and benzaldehydes were formed not stepwise, but simultaneously from toluene. Separate experiments indicated that the reaction of benzyl alcohol to benzaldehyde was four-fold faster than that of toluene to benzyl alcohol. The rate was not enough to elucidate the amount of benzaldehyde. We suggest that the benzyl radical is formed by hydrogen abstraction and is attacked by the second oxidant, PFIB. Rebounding of the hydroxyl radical and the reaction with the oxidant were competitive depending on the conditions. Additionally, small amounts of phenols were formed from toluenes with electron-donating substituents. This was a minor reaction on which the aromatic hydroxylation occurred via epoxidation under the present conditions.

Full text of DOI:10.1246/bcsj.76.2353

ꢀ
2003 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 76, 2353–2360 (2003) 2353
On the Mechanism of the Oxidation of Toluenes in Artificial P450 Model
1
Systems: Formation of Benzyl Alcohols, Benzaldehydes, and Phenols
ꢀ
Hiroko Kakuda, and Yoshihiro Mori
Taku Nakano, Sally Kawabata, Tamami Sugihara, Noriko Agatsuma,
Department of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University,
2630 Sugitani, Toyama-shi, Toyama, 930-0194
Received May 16, 2003; E-mail: tnakano@ms.toyama-mpu.ac.jp
Systems with pentafluoroiodosylbenzene (PFIB) and hemin (FeTPPCl8Cl) in dichloromethane were adopted to
study the activities of the model system using toluenes as substrates for P450 enzymes. The oxidation products were
mainly corresponding benzyl alcohols and benzaldehydes. Previously reported suspension systems were extended to ho-
mogeneous mixed solution systems of CH2Cl2/CH3OH/H2O to study the oxidation of benzyl alcohols to the correspond-
þ
ing benzaldehydes: the Hammett relation with ꢀ ¼ ꢁ0:86 against ꢁ . As benzaldehydes were scarcely observed in nat-
ural P450 systems, the formation of benzaldehyde seemed characteristic only of the ‘‘open’’ model systems. In suspension
systems, the product ratios between corresponding benzaldehydes and benzyl alcohols (ald/alc) were about 0.1–0.4, spe-
cific to the substituents and conditions applied. Curiously, the ratios (ald/alc) increased with the electronegativity of the
substituents on the phenyl rings of the toluene derivatives. Time course experiments in suspension systems indicated that
benzyl alcohols and benzaldehydes were formed not stepwise, but simultaneously from toluene. Separate experiments
indicated that the reaction of benzyl alcohol to benzaldehyde was four-fold faster than that of toluene to benzyl
alcohol. The rate was not enough to elucidate the amount of benzaldehyde. We suggest that the benzyl radical is formed
by hydrogen abstraction and is attacked by the second oxidant, PFIB. Rebounding of the hydroxyl radical and the reaction
with the oxidant were competitive depending on the conditions. Additionally, small amounts of phenols were formed
from toluenes with electron-donating substituents. This was a minor reaction on which the aromatic hydroxylation oc-
curred via epoxidation under the present conditions.
Cytochrome P450 is a family of enzymes which catalyze the
oxidation of physiological substrates, including foreign
benzyl alcohols and benzaldehydes to estimate the reactivity
17
of toluenes. The reaction proceeded having a Hammett rela-
tion with ꢀ ¼ ꢁ1:5 against ꢁ. Our results supported the hy-
droxylation mechanism initiated by the hydrogen abstraction
and followed by the rebounding of the hydroxyl radical
2–4
chemicals.
model systems have successfully explained the nature of the
The mechanistic studies of P450 using artificial
5,6
7,8
heme protein enzymes. The difference between natural and
model systems still remains unknown in understanding of the
30
(ꢂOH) as shown in Fig. 1(b). The assumption was that benzyl
alcohols initially produced were further oxidized to corre-
sponding benzaldehydes (Fig. 1(c)), where k1 and k2 were rate
constants from toluene to benzyl alcohol and from benzyl alco-
hol to benzaldehyde, respectively. No benzoic acids were
found in the system, indicating no further oxidation of
benzaldehyde. The product ratios of benzaldehydes to the cor-
responding benzyl alcohols were about 0.1–0.4.
9
–12
enzymatic mechanism and the chemistry of hemin.
tribution of products is the reflection of the structure and the be-
The dis-
13,14
havior of the active center. The multi-character of the in-
termediate of the P450 model was demonstrated in the case
of epoxidation of olefins.
Toluenes are reactive and useful for systematic studies and
there exist a lot of data in vivo. Products in natural systems
are benzyl alcohols and a small amount of phenols or cresols
The former group is the result of hydroxylation
at the benzylic position, and the latter group is produced by hy-
15,16
17
18–20
It had been assumed that additional benzaldehydes were
formed by the faster consecutive oxidation of the resulting ben-
zyl alcohols. However, curiosity remained in these observa-
21–23
(Fig. 1(a)).
24
17
droxylation on the aromatic ring. Benzaldehydes are scarcely
reported in natural systems,
tions as shown in Fig. 2. More benzaldehyde was formed
25,26
while benzoic acid is found in a
than previously expected, and product ratios (benzaldehyde/
benzyl alcohol) were larger with substrates having more elec-
tronegative substituents, while total yields were lower.
If these reactions occurred consecutively, two conditions
must be satisfied to elucidate the feature; 1) benzaldehyde for-
mation from benzyl alcohol derivatives with electronegative
groups is faster than those with electron-donating groups, and
2) benzyl alcohol oxidation is much faster than toluene
hydroxylation. We report here whether the two oxidations,
27
few cases. There is no report of finding bibenzyl derivatives.
On the other hand, in the model systems so far reported, prod-
ucts include benzyl alcohols and benzaldehydes, and no benzo-
ic acid is reported. Similarly, in cases of n-alkanes and cyclo-
hexane, no aldehydes, ketones, nor cyclohexanone were report-
ed in natural systems. These were, however, produced, along
28,29
with the corresponding alcohols, in models.
Previously, we examined the product formation rates for
Published on the web December 15, 2003; DOI 10.1246/bcsj.76.2353

2
354 Bull. Chem. Soc. Jpn., 76, No. 12 (2003)
Artificial P450 Model Systems
31
IC6F5. Methanol prohibited the formation of a similar adduct
with benzyl alcohol, which disturbed to react with the catalyst,
and which resulted in the formation of benzaldehyde when
heated on the GLC machine. The homogeneous solution sys-
(a)
CH3
CH2OH
CHO
CH3
C6F5IO
+
X
+
OH
32,33
FeTPPCl8Cl/CH2Cl2
tem had been introduced previously in olefin epoxidations.
X
X
X
1
a1
1a2
1a3
1a4
Experimental
Materials. Tetrakis(2,6-dichlorophenyl)porphinatoiron(c)
chloride (FeTPPCl8Cl) and pentafluoroiodosylbenzene (PFIB)
(
b)
17
were prepared as reported before.
Toluenes were purified by
H
H
H
H
C
C
O
C
O
H
passing through small SiO2 columns just before use. In case sub-
strates were solid, they were dissolved in dichloromethane and
passed through the columns. Other chemicals were reagent grade.
Apparatus. For recording spectra, a JASCO V-530 Spectrom-
eter and a Hitachi 220A Spectrometer were used. Products were
measured using a Shimadzu GC-12A Gas Chromatograph: DB-
WAX (J&W Scientific), 0.25 mm (width), 0.25 mm (i.d.) ꢃ 30 m
H
k1
Rebound
fast
H
H
H
O
+
Fe
Fe
Fe
1b3
Cage
1
b1
1b2
(c)
(
length).
Oxidation of Toluenes in Suspension Systems with PFIB.
For typical runs, 2 mg of PFIB (final concentration: 64 mmol/
CH3
CH2OH
CHO
34
k1
k2
3
C6F5IO
C6F5IO
dm ) was placed in a small glass tube (6 mm (i.d.) ꢃ 5 mm
FeTPPCl8Cl/CH2Cl2
8 2
FeTPPCl Cl/CH Cl
2
X
X
X
(length)) with a silicone rubber septum and a calculated amount
of dichloromethane was injected. After injecting an appropriate
amount of substrate, a calculated volume of hemin solution
1
c1
1c2
1c3
Fig. 1. Reaction of toluenes in P450 model system with
FeTPPCl8Cl/PFIB/CH2Cl2. (a) Product distribution. (b)
Hydroxylation mechanism. (c) Previously proposed step-
wise formation of benzaldehydes.
(
centrations of the catalyst and the substrate were arranged to be
CH2Cl2) was injected to get the reaction started. The final con-
3
3
1
.0 mmol/dm and 2.0 mol/dm , respectively. The total volume
3
of the solution was fixed at 0.1 cm . The reaction mixture was
shaken vigorously until the solution became clear at room temper-
ꢄ
ature (25 C) and then cooled in ice water to stop further reactions.
0
0
0
0
0
.4
.3
.2
.1
.0
Products were determined by GLC using the resulting pentafluoro-
iodobenzene as an internal standard. The calibration of detection
in GLC was done using authentic samples.
m-Cl
m-F
p-Cl
Oxidations of Benzyl Alcohols in Homogeneous Solution
34
Systems. For checking the oxidation of benzyl alcohol, a homo-
geneous system was needed. A solution containing methanol and
water was used as a solvent (CH2Cl2/CH3OH/H2O = 80/18/2).
Methanol formed an adduct with PFIB prior to benzyl alcohol.
GLC checking resulted in the negligible formation of benzalde-
hyde without the catalyst. The other reaction conditions were sim-
ilar to the suspension systems. The partially formed possible ad-
duct was killed by adding sodium dithionite before GLC detection.
Oxidation of Toluenes in Homogeneous Solution Systems
with PFIB. To compare the reactivity of toluenes and benzyl al-
cohols, the reaction was necessarily carried out in mixed solution
systems (CH2Cl2/CH3OH/H2O = 80/18/2). After shaking (or
p-F
H
p-Xy
m-Xy
-0.2
-0.1
0.0
0.1
σ value
0.2
0.3
0.4
Fig. 2. Plotting of the product ratio (ald/alc) against ꢁ value
in suspension systems. The final concentrations of catalyst
and oxidant were arranged to be 1.0 and 64 mmol/dm .
There is no direct thermodynamic relation in the figure.
stirring) the reaction mixture for 3 min, 2 mg of sodium dithionite
and 0.2 cm3 of water were added and the mixture was shaken
3
again. The reaction mixture was centrifuged and cooled in ice
water. Products in the organic layer were measured by GLC.
Competitive Reactions in Homogeneous Solution Systems.
The competitive reactions using two kinds of benzyl alcohol were
performed under the similar conditions in order to obtain the ratio,
kX=kH, where kX and kH are rate constants, with X and H standing
for substituted and unsubstituted benzyl alcohols. Absolute values
of ks were not estimated at this stage, but ratios of them were
obtained. The total concentration of the substrates was set at 2.0
the formation of benzyl alcohol and benzaldehyde, occurs si-
multaneously or in steps, and propose a reaction mechanism
in ‘‘open’’ (without surroundings but solvent) model systems.
It should be noted that the oxidation by PFIB was considered
to occur quickly close to the surface of a solid before each ox-
idant species migrated away. The possible dependence of the
rate on the surface structure of solidified PFIB could not be
ruled out at this stage.
3
mol/dm . The substrates were chosen to avoid overlapping of re-
In the case of the benzyl alcohol oxidation, we needed to em-
ploy homogeneous solution systems containing methanol and
water which dissolved PFIB in the form of adduct, MeO(HO)-
tention times. The competitive reactions between toluenes and
benzyl alcohols were also performed in homogeneous systems.
Time Course of the Oxidation in Suspension Systems. Four

T. Nakano et al.
Bull. Chem. Soc. Jpn., 76, No. 12 (2003) 2355
mg of PFIB was placed at the bottom of a glass tube (6 mm (i.d.) ꢃ
p-CH3O
0
.8
y = - 0.86x + 0.004
4
5 mm (length)) with a silicon rubber stopper. Into this tube a cal-
R = 0.921
culated amount of dichloromethane and toluene derivative were
injected. Then, a catalyst solution (final concentration: 1.0
0.4
p-F
3
mmol/dm ) was injected, and the mixture was shaken vigorously.
3
p-CH
3
H
The total volume was 0.2 cm . At the time chosen, 4 mg of sodium
3
0
.0
p-Br
p-Cl
m-F
dithionite was added after taking off the stopper. Then, 0.1 cm of
m-CH
3
m-Br
water was injected, and the stopper was replaced immediately. The
tube was shaken vigorously for 10–20 s, and centrifuged for 30 s.
The reaction mixture was then ice-cooled to prevent further
reaction. Products in the organic layer were determined by
GLC. The reactions were repeated for each reaction time of 15,
m-CH O
3
-
-
0.4
0.8
m-Cl
m-CF
3
-
1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8
σ+ value
3
0, 45, 60, 90, and 120 s.
Product Ratios (Ald/Alc) in Suspension Systems at Various
Fig. 3. Relative reaction rates in competitive reactions of
þ
Concentrations of Catalyst and Oxidant. Product ratios of
benzaldehydes to corresponding benzyl alcohols were similarly
measured in suspension systems at various amounts of the catalyst
and the oxidant. Substrates typically selected were toluene, p-xy-
benzyl alcohols vs ꢁ values. Oxidations were performed
competitively in the homogeneous mixed solution systems
(
CH2Cl2/CH3OH/H2O). kH and kX stood for the rate con-
stants for benzyl alcohol and substituted (X) benzyl
3
lene, and p-fluorotoluene. Concentrations were 2.0 mol/dm . Ox-
idations depending on catalyst with experimentally possible con-
alcohols. kX=kH were obtained based on product yields.
þ
Plotting against ꢁ resulted in better fitting than for ꢁ.
3
centrations (0.25–1.0 mmol/dm ) were performed at a constant
3
If plotted for ꢁ, ꢀ ¼ ꢁ1:1 and R ¼ 0:88. PFIB did not re-
act directly with benzyl alcohol to form benzaldehyde at
room temperature. Possibly formed adducts of PFIB were
decomposed by adding reductant before GLC measure-
ments.
concentration of the oxidant, PFIB (64 mmol/dm ). The effects
3
of PFIB (0.003–0.1 mol/dm ) were examined with a constant con-
3
centration of catalyst, FeTPPCl8Cl (1.0 mmol/dm ).
Results
Reactions in Homogeneous Solution Systems. In Table 1,
products in homogeneous solution systems with toluenes are
listed. Benzyl alcohols and benzaldehydes were produced
but with lower yields than in suspension systems. Phenols were
not detected in homogeneous systems. The ratios of these prod-
ucts (ald/alc) depended on the ꢁ values. The similarity of the
results allowed us to consider the reaction mechanism to be
treated totally in the suspension and the homogeneous systems.
As for the oxidation of benzyl alcohols, the relationship be-
used as a reference substrate. The results estimated from the
product ratios indicated that the benzyl alcohol oxidation was
only fourfold faster than the hydroxylation of toluene. Howev-
er, if benzaldehyde was formed stepwise, as in Fig. 1(c), with
k1=k2 ¼ 4:0, the amount of formed benzyl alcohol at the initial
stage was not enough to compete with the excess amount of tol-
uene substrate to approve the stepwise process.
Time Course of Oxidation and Product Ratios. The benz-
aldehyde formation at the initial stage was confirmed through
the time course reactions in suspension systems. Some of the
results are shown in Figs. 5 and 6. There were some induction
periods at early stages observed that were not elucidated at this
stage. Nevertheless, in all cases, the formation of benzaldehyde
was observed at the very beginning of the reaction, and the
product ratios (ald/alc) did not change much from the begin-
ning, as plotted in Fig. 7. The slight changes in the ratios might
be caused by the consumption of oxidant, except in the case of
p-xylene. It was indicated that the amount of benzaldehyde
formed secondarily from the resulting benzyl alcohol was small
when the amount of substrate was in excess. Again, stepwise
reactions were not plausible.
tween the log of the relative reaction rate, logðkX=kHÞ, and the
þ
ꢁ
ꢀ
value is shown in Fig. 3. The linear Hammett plotting gave
¼ ꢁ0:86.
The Reaction from Benzyl Alcohol to Benzaldehyde Was
Fourfold Faster Than That from Toluene to Benzyl Alcohol.
The ratio of the reaction rates of toluene hydroxylation and ben-
zyl alcohol oxidation was obtained from competitive reactions
between p-xylene and toluene, and between p-xylene and ben-
zyl alcohol in homogeneous solution systems. p-Xylene was
Table 1. Hydroxylation of Toluenes in Homogeneous
Mixed Solution Systemsa)
Substrate
Toluene
p-Xylene
Alcohol yield/%
Aldehyde yield/%
Product Ratio (Ald/Alc) Depends on the Concentration
of Oxidant, but not on That of the Catalyst. As the stepwise
reactions were ruled out, the oxidant for the second oxidation
must be assigned. The examinations with various concentra-
tions of catalyst did not change the ratios (ald/alc) when the
amount of oxidant was in excess (data not shown). On the other
1.94
11.26
4.38
9.67
1.57
8.67
1.67
0.43
2.24
1.02
1.56
0.46
2.03
0.52
m-Xylene
p-Fluorotoluene
m-Fluorotoluene
p-Chlorotoluene
m-Chlorotoluene
3
hand, the effects of PFIB (0.003–0.1 mol/dm ) were clearly ob-
served at the constant concentration of catalyst (1.0 mmol/
3
dm ), as shown in Fig. 8. As the amount of oxidant increased,
a) Mixed solution: CH2Cl2/CH3OH/H2O(80/18/2). Concen-
3
tration of oxidant, PFIB, was 64 mmol/dm and that of cata-
3
the ratios (ald/alc) increased. The concentrations of oxidant
were about 3–100 fold in excess to that of the catalyst. The
conditions with a lower amount of oxidant were technically im-
possible at this stage. The dependence on PFIB was not simple.
lyst, FeTPPCl8Cl, was 1.0 mmol/dm . Yields were calculated
on the added PFIB. Dithionite did not reduce the formed prod-
ucts under the present conditions.

2
356 Bull. Chem. Soc. Jpn., 76, No. 12 (2003)
Artificial P450 Model Systems
X
50
X
X
H
O
C
H
H
O
H
40
30
20
10
0
H
C
H
C
p-Chlorotoluene
H
H
O
H
O
O
O
Fe
Fe
p-Chlorobenzyl alcohol
p-Chlorobenzaldehyde
Fe
4
a
4b
4c
40
X
m-Xylene
X
H
30
H
C
C
H
O
O
O
20
10
0
H
m-Methylbenzyl aocohol
m-Methylbenzaldehyde
H2O
+
Fe
4
e
4d
4
3
0
0
Fig. 4. Proposed mechanism for oxidation of benzyl alco-
hols in the homogeneous mixed-solution systems. The in-
termediate (Fe =O) was supposed to be same as in suspen-
sion systems. The step from 4c to 4d is the rebound of hy-
droxyl radical.
m-Chlorotoluene
þ
20
m-Chlorobenzyl alcohol
m-Chlorobenzaldehyde
1
0
0
0
20
40
60
80
100
120
5
4
0
0
Time/s
p-Xylene
p-Methylbenzyl alcohol
Fig. 6. Time courses of the oxidations of toluenes in suspen-
sion reaction system-2. Substrates were p-chlorotoluene,
m-xylene, and m-chlorotoluene. Additions of reducing
reagent, dithionite, at the time stopped further oxidations.
The final concentrations of catalyst and oxidant were ar-
3
2
0
0
0
.1
p-Methylbenzaldehyde
3
0
0
ranged to be 1.0 and 64 mmol/dm . Products were corre-
4
3
sponding benzyl alcohols and benzaldehyde. There
seemed some induction periods in the reactions, though
the reason was not clear at this stage.
Toluene
0
Benzyl alcohol
2
0
0
1
Benzaldehyde
0.4
m-Chlorotoluene
0
0
4
3
2
1
p-Fluorotoluene
0.3
0.2
0.1
0
p-Chlorotoluene
p-Fluorotoluene
p-Fluorobenzyl alcohol
0
0
0
0
p-Fluorobenzaldehyde
Toluene
m-Xylene
p-Xylene
40
0
20
40
60
80
100
120
Time/s
0
20
60
80
100
120
140
Fig. 5. Time courses of the oxidations of toluenes in suspen-
sion reaction system-1. Substrates were p-xylene, toluene,
and p-fluorotoluene. Additions of reducing reagent, di-
thionite, at the time stopped further oxidations. The final
concentrations of catalyst and oxidant were arranged to
Time/s
Fig. 7. Time course of product ratios (ald/alc) in oxidation
of toluenes in suspension systems. Data were derived from
those in Figs. 5 and 6. Product ratios indicated that benz-
aldehydes were formed even at the initial stages of the re-
actions.
3
be 1.0 and 64 mmol/dm . Products were corresponding
benzyl alcohols and benzaldehydes. There seemed some
induction period in the reactions, though the reason was
not clear at this stage.
Discussion
In the case of p-fluorotoluene, as the oxidant concentration in-
3
It is generally accepted that an oxoiron(d) complex with a
cation radical on the peripheral porphyrin ring is the active in-
creased up to about 0.02 mol/dm , the aldehyde formation in-
35–38
creased steeply, and then slowed down. In other cases, the
amount of aldehyde increased slowly. There might be a steep
increase under the conditions of less oxidant, in which case the
ratio could be extrapolated to zero.
termediate in actual P450 enzymes.
The system with iodo-
sylbenzenes in dichloromethane could be considered to pro-
duce only a high-valent oxoiron(d) intermediate, 9a, which
simplified the situation. The formation step of the oxoiron(d)

T. Nakano et al.
Bull. Chem. Soc. Jpn., 76, No. 12 (2003) 2357
0
0
0
0
0
.4
.3
.2
.1
.0
It should be noted that both of the independent reactions of
toluenes to benzyl alcohols and of benzyl alcohols to benzalde-
hydes had Hammett relations with a negative reaction factor, ꢀ.
If this scheme actually occurred in the case of toluene oxida-
tions, the consecutive formation of benzaldehyde should have
simply indicated that the product ratios would have decreased
in cases of more negative substrates. Apparently, this was
not the case, as shown in Fig. 2. There should be an opposite
factor in the formation of benzaldehyde.
The fact that aldehydes were formed at a very early stage of
oxidation is shown in Fig. 7, derived from data in the time
course experiments (Figs. 5 and 6). If the initial hydrogen ab-
straction from toluenes was true, the second oxidation must
have occurred before the rebounding of the hydroxyl radical
and should have been competitive or faster. The intermediate,
p-Fluorotoluene
Toluene
p-Xylene
0
.00
0.02
0.04
0.06
0.08
0.10
3
[PFIB]app /mol/dm
Fig. 8. Dependence of oxidant (PFIB) amount on product
ratio (ald/alc). Catalyst was FeTPPCl8Cl (1.0 mmol/
dm ) in suspension systems. The complicated effect of
the oxidant reflected the interactions between substrate
3
9
b, in Fig. 9 has been supposed to be within the solvent cage in
which the hydroxyl radical rebounding occurred before
liberation. If the ‘‘cage’’ was tight, only benzyl alcohols would
be formed. However, if the cage was loose and/or hydroxyl
radical rebounding was slow, the intermediate would have a
chance to be attacked by the second PFIB. The partially neg-
ative-charged oxygen atom of PFIB (iodine is partially posi-
tively charged) nucleophilically attacked the benzyl radical,
as schematically shown in Fig. 9. Remember that the oxygen
atom of PFIB similarly attacked at the iron(c) ion of hemin
at the initial stage. On the other hand, it was reported that
the high-valent oxoiron intermediate electrophilically attacked
olefins. As bibenzyl was not found, the secondary attack by
PFIB was faster than the dimerization of the benzyl radical un-
der the present conditions. Thus, it was suggested that benzal-
dehydes were made by the nucleophilic attack of PFIB on the
benzyl radicals. The situation should have been reflected on
the Hammett plotting with positive ꢀ values and an increase
in the ratio (ald/alc). Though the detail of the rebounding proc-
ess is not known, if electron-donating groups on the substrate
caused the rebounding to occur faster and the electron-with-
drawing groups made it slower, such that it was attacked easier
by the second oxidant, the situation would be coincident with
and oxidant in this particular system. The effects of PFIB
3
(
centration of catalyst, FeTPPCl8Cl (1.0 mmol/dm ).
0.003–0.1 mol/dm ) were examined at the constant con-
3
Table 2. Oxidation of Benzyl Alcohols in Homogeneous
Mixed Solution Systems
Substrate
Benzyl alcohol
Corresponding benzaldehyde/%
31.8
35.2
22.8
38.5
28.2
27.4
21.4
26.2
31.3
22.0
9.7
p-Methylbenzyl alcohol
m-Methylbenzyl alcohol
p-Fluorobenzyl alcohol
m-Fluorobenzyl alcohol
p-Chlorobenzyl alcohol
m-Chlorobenzyl alcohol
p-Bromobenzyl alcohol
m-Bromobenzyl alcohol
p-Methoxybenzyl alcohol
m-Methoxybenzyl alcohol
16
intermediate has been considered to be rate-determining, espe-
cially with substrates such as 2,4,6-tri-t-butylphenol and
39,40
ABTS.
On the other hand, in the hydroxylation of toluene
derivatives, the rates depended on the substrates to indicate that
the steps between the active intermediate and substrates were
x
x
H
C
41,42
O
Fe+
CH3C6H4X
H
Rebound
H
H
C
OH
rate-determining.
As experiments under nitrogen atmos-
H
O
phere did not affect product yields and distributions (data not
shown), the participation of dioxygen was eliminated. Similar
Fe
9a
43
9b
relations were reported in other cases in P450 models.
C6F5IO
The mechanistic similarity in the suspension and homogene-
ous systems was demonstrated in the data of the homogeneous
systems shown in Table 1, though yields were lower. The ox-
idation of benzyl alcohol was checked in the homogeneous so-
lution systems (Table 2). The results of the competitive reac-
tions selecting sets of two benzyl alcohols are shown in
Fig. 3. The trend in the reactions was quite similar to that in
suspension systems. The Hammett plotting indicated again
the hydrogen abstraction mechanism. Generally, dissociation
energies of benzylic hydrogens were smaller than those of hy-
F5
I
F5
I
C F I
6
5
δ+
δ-
O
C
O
C
H
X
C
H
H
X
O
H
H
H
H
O
2
H O
O
Fe
Fe
9c
9d
44
drogens in hydroxy groups. So, we proposed that the reaction
was initiated by the abstraction of hydrogen from the benzylic
position, being followed by the quick rebounding of the hy-
droxyl radical to give acetals that turned to benzaldehyde and
Fig. 9. Proposed mechanism of benzaldehyde formation.
Second oxidant PFIB attacked at benzyl radical before re-
bound of hydroxyl radical. When the life-time of the inter-
mediate is longer and/or the rebound process is slower, al-
dehyde formation will increase.
45
water (Fig. 4).

2
358 Bull. Chem. Soc. Jpn., 76, No. 12 (2003)
Artificial P450 Model Systems
the experimental results.
Table 3. Arene Hydroxylation of Toluenes in Suspension
Systems
While the rate-determining step is the initial hydrogen ab-
straction for the overall reaction, the steps to benzyl alcohol
and benzaldehyde occurred competitively as faster processes
after forming the intermediate, 9b. Previously reported deute-
rium isotope effects altered the overall rates, but not the product
distributions. The primary deuterium isotope effects on this hy-
droxylation were clearly observed to be 6.2 for toluene and 5.7
Substrate
Benzene
Product
Yield/%
Phenol
4-Fluorophenol
m-Cresole
0.96
0.47
0.41
2.47
1.28
2.28
3.28
4.28
5.28
6.28
Fluorobenzene
Toluene
m-Xylene
m-Fluorotoluene
p-Chlorotoluene
2,4-Dimethylphenol
4-Fluoro-2-methylphenol
2-Chloro-5-methylphenol
2,4-Diethylphenol
2,4,6-Trimethylphenol
2,4-Di-t-butylphenol
17,46
for cumene in the same suspension model systems.
On the
other hand, the product ratio (ald/alc) in deuterated toluene was
obtained to be 0.18, similar to the non-deuterated case of 0.15
in the suspension system, though the product yields of the deu-
terated substrate were only modest. These facts supported
that the deuterium transfer process was not involved in the
benzaldehyde formation step.
1,3-Diethylbenzene
Mesitylene
1
,3-Di-t-butylbenzene
17
1
,3-Dimethoxybenzene 2,6-Dimethoxyphenol
mechanism is not plausible, because it requires more energy.
It was suggested from the data in Table 3 that an electrophilic
attack on the phenyl rings formed unstable epoxides which be-
came phenols. Though yields were small, it is true that under
some restricted circumstances in actual heme proteins, phenols
can be made.
Molecular orbital calculations about the rebounding mech-
47–49
anism were reexamined recently.
followed by a rebounding mechanism in the oxidation of al-
A hydrogen-abstraction
48
kanes by permanganate was examined. Only benzaldehyde
but benzyl alcohol was formed. In the paper, the authors sug-
gested that the rate-determining step was the hydrogen abstrac-
tion, and that an interaction existed between the benzylic car-
bon and the second oxygen of permanganate in the transition
state.
Conclusions
The overall mechanism of the hydroxylation of toluenes in
the ‘‘open’’ model system is summarized in Fig. 10. The main
route is the hydroxylation at the benzylic position. The mech-
anism is a hydrogen abstraction by an oxoiron(d) intermediate,
and a rebounding of the hydroxyl radical. The sub route 1,
which is only observed in ‘‘open’’ model systems, allows the at-
tack by the second oxidant on the benzyl radical to form benz-
aldehyde before the rebounding occurs. The first hydrogen ab-
straction is rate determining and the consecutive steps are
faster. It is suggested the rebounding of the hydroxyl radical
and the attack by the second oxidant are competitive, causing
the distribution of products. The ratio of the rates depends
on the electronic effect of the substituents on the ring. Exper-
Newcomb and co-workers reported their mechanistic studies
for the hydroxylation of hydrocarbons, proposing a concerted
50–52
mechanism.
Though we could not exclude the possibility
of a concerted mechanism in some of natural enzyme systems,
the present model system clearly supports the hydrogen ab-
straction and hydroxyl radical rebounding mechanism. If the
concerted mechanism was involved, the benzaldehyde formed
in the model system could not be rationalized unless a different
mechanism coexisted.
Modest amount of phenols were observed in the suspension
systems. The mechanism should be different from the hydrox-
ylation at the benzylic positions. The hydrogen abstraction
+
Fe
C6F5IO
X
X
X
CH3C6H4X
H
H-abstraction
H
C
Rebound
O
H
H
C
C
H
H
H
H
Fe
OH
O
O
Fe
Fe
Main route
m-(CH3)2C6H4
C6F5IO
F5
H3C
H3C
CH3
I
OH
X
O
X
Dehydration
C
H
H
H3C
O
H
C
+
O
Fe
H
H
O
Fe
Sub route 2
Sub route 1
Fig. 10. Overall mechanism of toluene oxidations in ‘‘open’’ model systems. Main route is the ordinary hydroxylation. Sub route 1
is the process resulting in benzaldehyde formation. Additional sub route 2 forming phenols is competitive with other routes.

T. Nakano et al.
Bull. Chem. Soc. Jpn., 76, No. 12 (2003) 2359
imental results suggest that electron-donating groups on the
substrate cause the hydroxylation and the rebounding to pro-
ceed faster, and that electron-withdrawing groups get the situa-
tion reverse to make rebounding slower to be easily attacked by
the second oxidant. It is reasonable that in natural systems
benzaldehyde is not observed if the structure of the protein
pocket does not allow the second process during the
oxidation. Aromatic hydroxylation occurs through an epoxide,
as shown in the sub route 2. Totally, the benzylic and the aro-
matic hydroxylations are competitive depending on the
environment. Previous assumptions made to estimate the kinet-
ics to be kX=k0 = ([alc]x + [ald]x)/([alc]0 + [ald]0) is now veri-
fied under the following conditions: k ꢅ kalc and kald, where
kalc and kald are competitive. A still unsolved question in the
PFIB system is the whereabouts of ‘‘oxygen’’, without reacting
with substrates and destroying catalysts. The answer might be
dioxygen made through the reaction of the intermediate oxo-
iron compound with PFIB.
395 (1991).
20 T. Nakajima, R.-S. Wang, E. Elovaara, F. J. Gunzalez, H.
V. Gelboin, H. Raunio, O. Pelkonen, H. Vainio, and T. Aoyama,
Biochem. Pharmacol., 53, 271 (1997).
2
1
R. Toftgard, T. Haaparanta, and J. Halpert, Toxicology, 39,
25 (1986).
K.-H. J. Ling and R. P. Hanzlik, Biochem. Biophys. Res.
Commun., 160, 844 (1989).
H. Kim, R. S. Wang, E. Elovaara, H. Raunio, O. Pelkonen,
T. Aoyama, H. Vainio, and T. Nakajima, Xenobiotica, 27, 657
2
2
2
2
3
(
1997).
I. Johansson and M. Ingelman-Sundberg, Cancer Res., 48,
387 (1988).
D. E. Chapman, T. J. Moore, S. R. Michener, and G. Powis,
Drug Metab. Dispos., 18, 929 (1990).
H. Hanioka, M. Hamamura, K. Kakino, H. Ogata, H. Jinno,
A. Takahashi, T. Nishimura, and M. Ando, Xenobiotica, 25, 1207
1995).
W. Woiwode, R. Wordarz, K. Drysch, and H. Weichardt,
Arch. Toxicol., 43, 93 (1979).
L. Ji, M. Liu, A.-K. Hsieh, and T. S. A. Hor, J. Mol. Catal.,
2
4
5
2
5
2
6
(
2
7
2
8
References
7
0, 247 (1991).
29 D. Mansuy, J.-F. Bartoli, and M. Momenteau, Tetrahedron
Lett., 23, 2781 (1982).
1 Abbreviations: FeTPPCl8Cl; 5,10,15,20-tetrakis(2,6-di-
chlorophenyl)porphinatoiron(c) chloride, PFIB; pentafluoro-
0
iodosylbenzene, ABTS; 2,2 -azinobis(3-ethyl-2,3-dihydrobenzo-
thiazole-6-sulfonic acid).
30 J. T. Groves, J. Chem. Educ., 62, 928 (1985).
31 B. C. Schardt and C. L. Hill, Inorg. Chem., 22, 1563 (1983).
32 D. R. Leanord and J. R. L. Smith, J. Chem. Soc., Perkin
Trans. 2, 1991, 25.
33 T. G. Traylor, K. W. Hill, W.-P. Fann, S. Tsuchiya, and B.
E. Dunlap, J. Am. Chem. Soc., 114, 1308 (1992).
34 Suspension and homogeneous conditions: In this paper,
suspension conditions refer to the system with iodosylbenzene
which is suspended as a solid and homogeneous conditions refers
to the system with iodosylbenzene which is dissolved in alcohol
(methanol). Homogeneous conditions were adopted when they
were needed.
2
F. P. Guengerich and T. L. Macdonald, Acc. Chem. Res., 17,
(1984).
‘‘Cytochrome P-450: Structure, Mechanism, and Biochem-
9
3
istry,’’ ed by P. R. Ortiz de Montellano, 2nd ed, Plenum Press, New
York (1995).
4
1996).
5
1983).
6
S. Graham-Lorence and J. A. Peterson, FASEB J., 10, 206
J. T. Groves and T. E. Nemo, J. Am. Chem. Soc., 105, 6242
T. G. Traylor and P. S. Traylor, ‘‘Active Oxygen in Bio-
(
(
chemistry,’’ ed by J. S. Valentine, C. S. Foote, A. Greenberg,
and J. F. Liebman, Chapman & Hall, London (1995), pp. 84–187.
35 J. T. Groves, R. C. Haushalter, M. Nakamura, T. E. Nemo,
and B. J. Evans, J. Am. Chem. Soc., 103, 2884 (1981).
36 H. Fujii, J. Am. Chem. Soc., 115, 4641 (1993).
37 G. H. Loew and D. L. Harris, Chem. Rev., 100, 407 (2000).
38 M. Hata, Y. Hirano, T. Hoshino, and M. Tsuda, J. Am.
Chem. Soc., 123, 6410 (2001).
39 T. G. Traylor, W. A. Lee, and D. V. Stynes, Tetrahedron,
40, 553 (1984).
40 P. N. Balasubramanian, J. R. L. Smith, H. J. Davies, T. W.
Kaaret, and C. T. Bruice, J. Am. Chem. Soc., 111, 1477 (1989).
41 M. J. Nappa and C. A. Tolman, Inorg. Chem., 24, 4711
(1985).
42 R. P. Hanzlik and K.-H. J. Ling, J. Org. Chem., 55, 3992
(1990).
43 J. Huang and H. B. Dunford, Can. J. Chem., 68, 2159
(1990).
44 ‘‘Handbook of Chemistry and Physics,’’ 73rd ed, CRC Press
(1995), pp. 9138–9145.
45 V. P. Miller, R. A. Tschirret-Guth, and P. R. Ortiz de
Montellano, Arch. Biochem. Biophys., 319, 333 (1995).
46 Those reported in natural systems dispersed from around 2
to 7; a) J. I. Manchester, J. P. Dinnocenzo, L. A. Higgins, and P. J.
Jeffrey, J. Am. Chem. Soc., 119, 5069 (1997). b) J. T. Groves, G. A.
McClusky, R. E. White, and M. J. Coon, Biochem. Biophys. Res.
Commun., 81, 154 (1978). c) L. M. Hjelmeland, L. Aronow, and
J. R. Trudell, Biochem. Biophys. Res. Commun., 76, 541 (1977).
7
8
J. H. Dawson, Science, 240, 433 (1988).
T. L. Poulos, B. C. Finzel, I. C. Gunsalus, G. C. Wagner,
and J. Kraut, J. Biol. Chem., 260, 16122 (1985).
G. M. Keseru, I. Kolossvary, and B. Bertok, J. Am. Chem.
Soc., 119, 5126 (1997).
9
1
1995).
0
D. Harris and G. Loew, J. Am. Chem. Soc., 117, 2738
(
1
Montellano, J. Am. Chem. Soc., 119, 5489 (1997).
1
J. J. De Voss, O. Sibbesen, Z. Zhang, and P. R. Ortiz de
1
1
2
3
H.-X. Zhou, J. Am. Chem. Soc., 116, 10362 (1994).
M. Nishiki, T. Satoh, and H. Sakurai, J. Mol. Catal., 62, 79
(
1990).
1
4
J. R. Collins, D. L. Camper, and G. H. Loew, J. Am. Chem.
Soc., 113, 2736 (1991).
T. G. Traylor, Y. Iamamoto, and T. Nakano, J. Am. Chem.
Soc., 108, 3529 (1986).
T. G. Traylor, J. C. Marsters, Jr., T. Nakano, and B. E.
Dunlap, J. Am. Chem. Soc., 107, 5537 (1985).
T. Nakano, N. Agatsuma, S. Kodama, H. Kakuda, and D.
Dolphin, Bull. Chem. Soc. Jpn., 69, 3513 (1996).
R. E. White and M.-B. McCarthy, Arch. Biochem. Biophys.,
46, 19 (1986).
T. Nakajima, R.-S. Wang, E. Elovaara, S. S. Park, H. V.
Gelboin, E. Hietanen, and H. Vainio, Biochem. Pharmacol., 41,
1
5
1
6
1
7
1
8
2
1
9

2
360 Bull. Chem. Soc. Jpn., 76, No. 12 (2003)
Artificial P450 Model Systems
4
4
7
8
J. M. Mayer, Acc. Chem. Res., 31, 441 (1998).
T. Strassner and K. N. Houk, J. Am. Chem. Soc., 122, 7821
(1995).
51 M. Newcomb, R. Shen, S.-Y. Choi, P. H. Toy, P. F.
Hollenberg, A. D. N. Vaz, and M. J. Coon, J. Am. Chem. Soc.,
122, 2677 (2000).
(
2000).
4
9
F. Ogliaro, N. Harris, S. Cohen, M. Filatov, S. P. de Visser,
and S. Shaik, J. Am. Chem. Soc., 122, 8977 (2000).
M. Newcomb, M.-H. Le Tadic-Biadatti, D. L. Chestney, E.
S. Roberts, and P. F. Hollenberg, J. Am. Chem. Soc., 117, 12085
52 P. H. Toy, M. Newcomb, and P. F. Hollenberg, J. Am.
Chem. Soc., 120, 7719 (1998).
5
0