Metalloporphyrins as biomimetic models for cytochrome P-450 in the oxidation of atrazine

Article abstract of DOI:10.1021/jf062462n

The aim of this work was to evaluate whether metalloporphyrin models could mimic the action of cytochrome P-450 in the oxidation of atrazine, a herbicide. The commercially available second-generation metalloporphyrins 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin metal(III) chloride [M(T-DCPP)Cl] and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin metal(III) chloride [M(TFPP)Cl] (metal = Fe or Mn) and the oxidants iodosylbenzene and metachloroperbenzoic acid were employed in this study. Results showed that the metalloporphyrins used here can oxidize atrazine. Yields as high as 32% were obtained for the Mn(TFPP)Cl/PhIO system, which shows that these catalysts can mimic both the in vivo and the in vitro action of cytochrome P-450, with formation of the metabolites DEA and DIA. The formation of five other unknown products was also detected, but only one of them could be identified, since the other four were present in very low concentrations. The compound COA, identified by mass spectrometry, was the main product in most of the oxidation reactions.

Full text of DOI:10.1021/jf062462n

J. Agric. Food Chem. 2006, 54, 10011−10018 10011
Metalloporphyrins as Biomimetic Models for Cytochrome P-450
in the Oxidation of Atrazine
MARIA C. A. F. GOTARDO, LUIZ A. B. DE MORAES, AND MARILDA D. ASSIS*
Departamento de Qu´ımica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade
de Sa˜o Paulo, Av. Bandeirantes 3900, 14040-901 Ribeira˜o Preto, SP, Brazil
The aim of this work was to evaluate whether metalloporphyrin models could mimic the action of
cytochrome P-450 in the oxidation of atrazine, a herbicide. The commercially available second-
generation metalloporphyrins 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin metal(III) chloride [M(T-
DCPP)Cl] and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin metal(III) chloride [M(TFPP)Cl] (metal
) Fe or Mn) and the oxidants iodosylbenzene and metachloroperbenzoic acid were employed in this
study. Results showed that the metalloporphyrins used here can oxidize atrazine. Yields as high as
32% were obtained for the Mn(TFPP)Cl/PhIO system, which shows that these catalysts can mimic
both the in vivo and the in vitro action of cytochrome P-450, with formation of the metabolites DEA
and DIA. The formation of five other unknown products was also detected, but only one of them
could be identified, since the other four were present in very low concentrations. The compound
COA, identified by mass spectrometry, was the main product in most of the oxidation reactions.
KEYWORDS: P450; herbicide metabolism; metalloporphyrin; atrazine
INTRODUCTION
may help scientists understand detoxification mechanisms, thus
enabling the development of novel pest inhibitors (4).
The cytochrome P-450 enzymes (P450) are one of the most
important systems responsible for pesticide detoxification in
plants (1, 2). Increased P450 activity is related to the develop-
ment of a resistance mechanism by weeds through which toxic
compounds are metabolized and thus inactivated. Therefore,
understanding the way these enzymes act, as well as gaining
knowledge about the potential development of plant resistance
to new pesticides on the part of weeds, has become of great
importance for control of herbicide tolerance in plantations and
weeds, as well as for optimization of novel active compounds
to be used in agriculture (1, 3).
The majority of the studies showing the role of P450 in the
transformation of herbicides in weeds have been carried out in
vivo. This is because isolating enough amounts of microsomal
fractions from these plants is extremely difficult. As for higher
plants, the participation of P450 in herbicide detoxification can
be investigated by using plant microsome in vitro assays (1).
In this context, synthetic metalloporphyrins have been used
as biomimetic models for more than 20 years, especially in drug
metabolism studies (5, 6). These complexes have provided a
better understanding of the metabolism of active compounds
in vivo by helping to identify which functional groups in the
new drugs are sensitive to metabolism. This information has
enabled the large-scale production of metabolites (on the scale
of milligrams) for pharmacological studies and toxicological
tests.
Despite the good results obtained with the use of synthetic
metalloporphyrins in drug oxidation, few studies have been
devoted to the application of these models in pesticide trans-
formation. Keseru¨ and co-workers are one of the few groups
working in this field, and they have shown that Fe(TFPP)Cl
can mimic the action of insect P450 in the chemical transforma-
tion of insecticides belonging to the carbamate class of
compounds (7).
As the isolation of P450 genes from plants is extremely
difficult, the first reactions employing this hemoprotein’s genes
were carried out with bacterial and mammalian P450. Only in
recent years have genes of P450 enzymes been isolated from
plants, and the first reactions confirmed that these enzymes take
an active part in herbicide detoxification (1).
Fukushima et al. have recently compared the metabolism of
the insecticide pyriproxyfen in tomatoes with results obtained
by means of a biomimetical study employing metalloporphyrin
models (8). These authors succeeded in showing that metal-
loporphyrins are able to mimic the in vivo action of P450.
The use of chemical model systems mimicking P450 might
therefore be a very useful tool for overcoming the difficulty in
working with enzymes in vivo and in vitro, and these models
Atrazine (2-chloro 4-ethylamino-6-isopropylamino-1,3,5-tri-
azine; ATZ) is a pre- and post-emerging selective herbicide for
weed control. This compound acts as an effective inhibitor of
the Hill reaction in photosynthesis, thus reducing the amount
of CO2 fixed in the plant (9-12). This compound is extremely
important in agriculture, especially because of its tolerance by
* Corresponding author. Telephone: +55 16 36023799. Fax: +55 16
36023848. E-mail: mddassis@usp.br.
10.1021/jf062462n CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/08/2006

10012 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Gotardo et al.
Figure 2. Metallopophyrins used in this study (M
)
FeIII or MnIII).
2. MATERIALS AND METHODS
All the chemicals used in this work were HPLC-grade or P.A.,
purchased from Merck, Aldrich, Acros, Fluka, or Mallinckrodt, and
used as received. Atrazine (99.2% purity) was provided by Supelco
and Syngenta. Deethylatrazine (99.9% purity), deisopropylatrazine
(96.1% purity), deethyldeisopropylatrazine (98.3% purity), hydroxyatra-
zine (96.0% purity), deethylhydroxyatrazine (98.7% purity), and
deethyldeisopropylhydroxyatrazine (99.6% purity) were provided by
Supelco. Table 1 lists the structures, common names, chemical names,
and abbreviations of atrazine and its metabolites. Metachloroperbenzoic
acid (m-CPBA) was provided by Acros (70% purity). Iodosylbenzene
(PhIO) was prepared by hydrolysis of iodosylbenzene diacetate,
following the method of Sharefkin and Saltzman (15), and its purity
was 93%, as determined by iodometric titration. Imidazole (Im) was
provided by Acros (99% purity). The porphyrins Fe(TFPP)Cl, H2-
(TDCPP), and H2(TFPP), were purchased from Mid-Century and used
as received. Iron and manganese insertion into the free base porphyrins
H2(TDCPP)Cl and H2(TFPP)Cl was carried out using the method of
Adler et al. (16). Figure 2 shows the structures of the metalloporphyrins
used in this study.
2.1. Oxidation Reactions. The standard catalyst/oxidant/substrate
molar ratio used in the oxidation of atrazine was 1:60:120. To this
end, 2.5 × 10-7 mol of the metalloporphyrin, 1.5 × 10-5 mol of the
oxidant, and 3 × 10-5 mol of ATZ in 1500 µL of solvent were
employed (MeOH or ACN). Another catalyst/oxidant/substrate molar
ratio with excess oxidant was also used, namely 1:240:120. Imidazole
was added to some reactions, at a catalyst/ligand molar ratio of 1:10.
All reactions were carried out at room temperature under magnetic
stirring. At the end of the reaction (24 h) or at regular intervals, an
aliquot of the reaction mixture (200 mL) was withdrawn. The porphyrin
was removed from this aliquot before analysis by HPLC, by addition
of hexane (600 mL) and a mobile phase (400 mL). This mixture was
vortex-mixed and centrifuged; the lower phase (mobile phase) contain-
ing the oxidation products was then injected into the chromatographic
system.
Figure 1. Atrazine metabolism in corn (adapted form ref 11).
maize. In fact, it is one of the most widely employed herbicides
in the United States.
In this culture, the main herbicide detoxification routes consist
of glutathione conjugation catalyzed by glutathione s-transferase
and N-dealkylation, as shown in Figure 1. Although P450 is
thought to catalyze N-dealkylation, this has not been demon-
strated in vivo.
The first N-dealkylation reduces herbicide affinity for the
target receptors, thus reducing its toxicity. The second N-
dealkylation culminates with herbicide detoxification in the plant
(12).
In the environment, the main ATZ metabolites are its
N-dealkylated products. Nevertheless, this herbicide is highly
persistent and mobile, being one of the main underground water
contaminants in Europe. Degradation of this compound by
microbial catalysis in soil environments is a relatively slow
process, with a half-life of 60-100 days (13, 14).
The aim of this work is to evaluate whether metalloporphyrin
systems can mimic the action of P450 in the oxidation of
atrazine. To this end, we employed the commercially available
second-generation metalloporphyrins 5,10,15,20-tetrakis(2,6-
dichlorophenyl)porphyrin metal(III) chloride [M(TDCPP)Cl]
and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin metal(III)
chloride [M(TFPP)Cl] (metal ) Fe or Mn).
2.2. HPLC Analysis. The analytical HPLC analyses were performed
on a SHIMADZU liquid chromatograph equipped with an LC-10AS
Table 1. Chemical Structure of Atrazine and Its Metabolites
abbreviations
compounds
R1
R2
R3
ATZ
DEA
DIA
DAA
2-chloro-4-(isopropylamino)-6-(ethylamino)-s-triazine (atrazine)
2-chloro-4-(isopropylamino)-6-amino-s-triazine (deethylatrazine)
2-chloro-4-(ethylamino)-6-amino-s-triazine (deisopropylatrazine)
2-chloro-4,6-diamino-s-triazine (deethyldeisopropylatrazine)
2-hydroxy-4-(isopropylamino)-6-(ethylamino)-s-triazine (hydroxyatrazine)
2-hydroxy-4-(isopropylamino)-6-amino-s-triazine (deethylhydroxyatrazine)
2-hydroxy-4,6-diamino-s-triazine (deethyldeisopropylhydroxyatrazine)
2-chloro-4-(acetamido)-6-(isopropylamino)-s-triazine
−
Cl
−
−
−
−
−
−
−
−
CH2CH3
H
CH2CH3
H
CH2CH3
H
H
−
−
−
−
−
−
−
−
CH(CH3)2
CH(CH3)2
H
−
−
−
−
−
−
−
Cl
Cl
Cl
OH
OH
OH
Cl
H
OHA
CH(CH3)2
CH(CH3)2
H
OHDEA
OHDAA
COA
NHCOCH3
NHCH(CH3)2

Metalloporphyrins as Biomimetic Models for P-450
J. Agric. Food Chem., Vol. 54, No. 26, 2006 10013
abstraction from the substrate by the latter species, with
formation of FeIVOHP and the substrate radical. Further OH
group transfer from FeIVOHP to the substrate radical results in
hydroxylation of the carbon adjacent to the nitrogen atom,
leading to an unstable carbinolamine that decomposes into an
amine and a carbonyl derivative.
In the second mechanism (B), known as hydrogen abstraction,
there is no electron transfer. The catalytically active species FeIV-
OP•+ directly abstracts a hydrogen atom from the substrate, and
the remaining steps are similar to those involved in mechanism
(A) (18).
In general, the occurrence of either mechanism A or B
depends on the substituents present on the nitrogen atom of the
substrate (18). The mechanism of P450-catalyzed atrazine
N-dealkylation is unknown, which makes studies employing
P450 chemical models an important tool for its investigation.
In this work, therefore, atrazine oxidation reactions were
carried out in the presence of Fe(TFPP)Cl, Fe(TDCPP)Cl, Mn-
(TFPP)Cl, and Mn(TDCPP)Cl (Figure 2), which are all
commercially available second-generation metalloporphyrins
well-established in the literature as good catalysts for hydro-
carbon and drug oxidations (5). Contrary to first-generation
metalloporphyrins, these compounds bear electron-withdrawing
substituents (-Cl or -F) in the meso-phenyl positions of the
porphyrin macrocycle, which confer steric hindrance to the
catalyst and avoid its self-oxidative destruction (5). These
substituents also activate the catalytic species, generally an oxo-
metal porphyrin cation radical [Me(IV)OP•+ or Me(V)OP],
making this species more electrophilic and reactive toward the
substrate.
PhIO, a two-electron oxidant, was initially used as an oxygen
donor in the present study because it is considered a standard
oxidant in the case of metalloporphyrin systems (19). Later,
m-CPBA, a peracid that is soluble in organic media and is very
frequently used in metalloporphyrin-catalyzed reactions, was
also used. The latter oxidant may undergo heterolytic cleavage
upon its coordination to the metalloporphyrin central metal ion,
leading to the formation of the active species oxoferryl or
oxomanganyl porphyrin π-cation radical, Me(IV)OP•+. Ho-
molytic cleavage of the O-O bond may also occur, leading to
the formation of a less reactive intermediate, MeIVP-OH, as
well as RO• radicals, thus favoring the occurrence of radicalar
mechanisms (20, 21).
ATZ oxidation reactions were carried out in MeOH and ACN,
taking the solubility of this herbicide and its metabolites in these
solvents into account. Solubility was also a determining factor
for the choice of reaction conditions, once the studied substrate
was a solid with limited solubility in the volume employed in
the reactions (1.5 mL). A catalyst/oxidant/substrate molar ratio
of 1:60:120, corresponding to 2.5 × 10-7 mol of catalyst, 1.5
× 10-5 mol of oxidant, and 3.0 × 10-5 mol of substrate (∼7
mg ATZ /1.5 mL of solvent), was the standard condition in our
studies.
Control reactions were carried out in the absence of metal-
loporphyrin, under the same conditions as the catalytic runs,
and gave no products.
Table 2 shows the total product yield (R%) obtained in the
ATZ oxidation reactions for each metalloporphyrin/oxidant
system, under different conditions. Figures 4 and 5 show the
plots of relative product distribution, in percent, in the case of
the reactions carried out in ACN.
Figure 3. Mechanism of P450-catalyzed N-dealkylations.
solvent pump, an SPD-M 10A VP spectrophotometric detector (λ )
220 nm) coupled to a CTO-10A VP column oven, and an SCL-10A
VP system controller. Separation of the herbicide and the oxidation
products was carried out in a Lichrospher 100RP-18 column, with a
particle size of 5 µm (125 mm × 4 mm), supplied by Merck. The
analytical column was protected by a LiChrospher guard column (4
mm × 4 mm). Elution was carried out with a gradient ranging from
phosphate buffer (pH 7)/methanol 90:10 (v/v) to phosphate buffer (pH
7)/methanol 50:50 (v/v), at a flow rate of 1.0 mL min-1. The run time
was 40 min. The mobile phase was purged with helium.
2.3. Mass Spectrometry Analysis. Products generated from the
metalloporphyrin-catalyzed atrazine oxidation were collected separately
by means of an HPLC collector, stored, and analyzed by mass
spectrometry (MS). Elution was carried out with a gradient of ACN/
MeOH/acetic acid 0.1% ranging from 8:12:80 v/v to 20:30:50 v/v, at
a flow rate of 1.0 mL min-1
.
Mass spectra were obtained on a Quattro LC mass spectrometer
(Micromass) equipped with an ionization electrospray source in the
positive mode (ESI+).
2.4. Nash Test. The Nash reagent (5 mL; 37.5 g of ammonium
acetate, 0.75 mL of acetic acid, and 0.5 mL of pentane-1,4-dione in
250 mL of distilled water) (17) was added to an aliquot of the reaction
mixture (500 µL) in a 10-mL volumetric flask, and the volume was
completed with distilled water. This solution was incubated at 40 °C
for 40 min, and the solution absorbance was recorded at 412 nm.
2.5. Catalytic Intermediate Studies through UV-Vis Spectra.
UV-vis spectra were obtained on a Hewlett-Packard 8452, diode array
spectrometer. A quantity of 500 µL of PhIO solution (0.02 mol L-1
)
was added to a quartz cell (10-mm de path length) containing 1000 µL
of Fe(TFPP) and ATZ solution in MeOH or ACN (2 × 10-6 and 1.8
× 10-5 mol L-1, respectively). Consecutive spectra were recorded.
2.6. Catalytic Intermediate Studies through EPR Spectra. The
EPR spectra were recorded on a Varian E-4 spectrometer operating at
the X band frequency (9 GHz) with a gain of 103, 200 mW microwave
power, and 10 G amplitude modulation at 4 K. Quantities of 100 µL
of PhIO solution (0.02 mol L-1) and 100 µL of ATZ (0.04 mol L-1
were added to an EPR tube containing 100 µL of Fe(TFPP)Cl solution
in ACN (3.3 × 10-4 mol L-1). The mixture was stirred manually.
Consecutive spectra were recorded.
)
3. RESULTS AND DISCUSSION
P450-catalyzed ATZ metabolism occurs via N-dealkylation
of the ethyl and propyl side chains of the secondary amines in
various biological systems (1, 2). According to reports in the
literature, P450-catalyzed N-dealkylations occur via initial iron-
(III)porphyrin oxidation, which gives the high-valent catalytic
species oxoferrylporphyrin π-cation radical, Fe(IV)OP•+. There-
after, there are two proposed mechanisms for the reaction, as
shown in Figure 3.
The first mechanism (A) is known as the electron-transfer
mechanism, where there is a one-electron transfer from the
substrate to the catalytically active species FeIVOP•+, rendering
the ferryl species FeIVOP. This is followed by hydrogen atom
From Table 2, it can be seen that the metalloporphyrins
studied here are poor catalysts for atrazine oxidation in MeOH,
leading to product yields of only around 1%. The catalytic

10014 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Gotardo et al.
Figure 4. Product distribution (in percentage) obtained in the metalloporphyrin-catalyzed oxidation of ATZ by PhIO in ACN in the standard conditions.
Figure 5. Product distribution (in percentage) obtained in the metalloporphyrin-catalyzed oxidation of ATZ by m-CPBA in ACN in the standard conditions.
P I, P II, P III, P IV, and P V are unknown products.
that the low yields obtained in the ATZ oxidation reactions
carried out in MeOH are because this solvent is preferentially
oxidized in these conditions, thus consuming 75% of the oxidant
present in the reaction medium.
Table 2. Total Yield of the Metalloporphyrin-Catalyzed ATZ Oxidation
Reactions, under Different Reaction Conditions
catalyst
oxidant
MeOH (R%)
ACN (R%)
Fe(TFPP)Cl
1
2
3
4
5
6
7
8
PhIO
m-CPBA
PhIO
m-CPBA
PhIO
m-CPBA
PhIO
∼
1
2
1
1
1
1
1
1
10
10
8
14
32
11
3
In ACN, a solvent that does not compete with ATZ for the
catalytically active species, the metalloporphyrins were able to
oxidize the herbicide with total oxidation product yields as high
as 32%, in the case of the Mn(TFPP)Cl/PhIO system (Table 2,
entry 5). This result demonstrates that these metalloporphyrin
complexes are good catalysts, especially if we consider that ATZ
is a herbicide that persists in the environment and that its
degradation is difficult even in the presence of advanced
oxidation processes (13, 14).
Fe(TDCPP)Cl
Mn(TFPP)Cl
Mn(TDCPP)Cl
∼
∼
∼
∼
∼
∼
m-CPBA
10
activity remained low even upon changes in the porphyrin ligand
(TFPP2- and TDCPP2-; Figure 2), metal (Fe3+ and Mn3+), and
oxidant (PhIO and m-CPBA). Therefore, these three variables
are not the main variables responsible for the low yields. A
possible explanation for the inefficiency of these systems could
be the fact that the solvent MeOH could be acting as substrate,
thus competing with ATZ for the catalytic species. To inves-
tigate whether this was the case, formaldehyde, the oxidation
product generated from MeOH, was quantified by means of the
Nash test (21, 22) in the case of the Fe(TFPP)Cl/PhIO and Mn-
(TFPP)Cl/PhIO systems. The Nash test consists of the spectro-
photometric determination of formaldehyde, which, in the
presence of a diketone and an ammonium salt, forms diacetyl-
hydrolutidine (DDL), a compound that absorbs at 412 nm.
By means of a calibration curve previously built from standard
solutions, the concentration of the formaldehyde generated in
the reaction could be calculated, and the percentage of oxidized
methanol could thus be estimated. Both in the presence and in
the absence of ATZ, the yield of formaldehyde was 75% in the
case of both Fe(TFPP)Cl and Mn(TFPP)Cl. This result shows
By analyzing the product distribution obtained in the metal-
loporphyrin-catalyzed oxidation of ATZ in ACN (Figures 4 and
5), it can be seen that the metabolite DEA was one of the main
products in all the reactions. In some reactions, formation of
the metabolite DIA was also detected. These two compounds
correspond to the main metabolites obtained in the ATZ
metabolism by cytochrome P450 in vivo (10-12), which
indicates that the metalloporphyrins used here can be considered
biomimetic models of this enzyme.
Apart from the metabolites DEA and DIA, we also detected
the formation of five other products during ATZ oxidation,
which were designated P I, P II, P III, P IV, and P V, according
to the elution order. Compound P V was the most abundant
product for all the metalloporphyrin/oxidant systems, except for
Mn(TFPP)Cl/m-CPBA, which led to DEA as the main product
(Figure 5). Compound P V was isolated and analyzed by mass
spectrometry (ESI/MS) and ESI-MS/MS in the positive mode.
This compound gave rise to a signal at m/z 230, which

Metalloporphyrins as Biomimetic Models for P-450
J. Agric. Food Chem., Vol. 54, No. 26, 2006 10015
IV is probably formed via a radicalar mechanism, once its
formation coincides with the use of the peracid as oxidant, which
may undergo homolytic cleavage and generate radicals.
Although the P450 system is capable of N-dealkylating two
alkyl side chains of the herbicide in vivo, which leads to the
formation of the metabolites DEA, DIA, and DAA, the
metalloporphyrin systems preferentially attach the ethyl chain
in ATZ, generating the compound COA and the metabolite DEA
as the main products, as well as traces of the metabolite DIA.
This preference was confirmed when the metabolites DEA and
DIA were used as substrates for the Fe(TFPP)Cl/PhIO/ACN
system in the standard conditions established in this work. When
the metabolite DIA was used as substrate, formation of the
metabolite DAA was detected in 10% yield, as expected.
However, when the metabolite DEA was employed as substrate,
there was no formation of DAA. In other words, attack of the
catalytically active species to the propyl chain is not favored,
and only traces of the compound OHDEA (yield ∼1%) were
obtained. The propyl chain probably confers higher steric
hindrance to the approach of the high-valent oxo-metal species
generated from the metalloporphyrins used in this study, which
all contain bulky substituents (-Cl and -F) in the phenyl
substituents on the porphyrin ring.
Table 3. Main Fragments of the Products DEA and P V Generated
during the Oxidation of ATZ as Determined by ESI-MS and
ESI-MS/MS
+
compound
[M
+
H ]
ESI-MS/MS (m/z)
DEA
PDV
188
230
188, 146, 110, 104, 79, 68
230, 188, 146, 110, 104, 79, 68
corresponds to the molecular ion [M + H+], and a fragmentation
MS/MS pattern similar to DEA, as shown in Table 3.
Comparison of these results with literature data for compounds
generated from atrazine degradation (13, 14) leads to the
identification of P V as a compound containing an amide in
the ethyl chain of ATZ (COA; Figure 6). Studies reporting ATZ
The different results obtained with the metalloporphyrin
models when compared with those obtained with the enzyme
demonstrate the importance of the protein matrix involving the
iron porphyrin in P450. The matrix is responsible for directing
the substrate approach, through the interaction with specific
amino acids, thus controlling the oxidation site. Biomimetic
models cannot reproduce such control.
As an attempt to understand the mechanism of metallopor-
phyrin-catalyzed ATZ oxidation and the formation of the various
products, some variables such as reaction time, excess oxidant,
Figure 6. Structure of COA, as determined by MS and MS/MS
spectrometry.
degradation via advanced oxidation processes have described
COA as an intermediate compound in the formation of the
metabolite DEA (13, 14, 23, 24).
Identification of the remaining unknown products (P II-IV)
was not possible because they were formed at very low
concentrations, even though P IV was the second most abundant
product in the case of the ironporphyrin/m-CPBA systems. P
Figure 7. Number of mol of COA and DEA as a function of time obtained in AZT oxidation by the Fe(TFPP)Cl/PhIO/ACN system.
Figure 8. Product distribution obtained in the MnP-catalyzed ATZ oxidation by m-CPBA in ACN, in the standard conditions and in the presence of
imidazole. P I, P II, P III, P IV, and P V are unknown products.

10016 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Gotardo et al.
Figure 9. Product distribution in percentage, obtained in the Fe(TFPP)Cl-catalyzed ATZ oxidation by PhIO in ACN, in the standard conditions and in
excess oxidant. P I, P II, P III, P IV, and P V are unknown products.
Figure 10. Product distribution, in percentage, obtained in the FeP-catalyzed AZT oxidation by m-CPBA in ACN in the standard conditions and in the
presence of imidazole. P I, P II, P III, P IV, and P V are unknown products.
presence of imidazole, and absence of O2 (argon atmosphere)
were evaluated.
Figure 8 shows the product distribution for the MnP/m-CPBA
systems in both the presence and absence of imidazole. It can
be seen that imidazole favors formation of the metabolite DEA,
with concomitant decrease in the formation of the compound
COA, or even its absence. This result shows that these two
products are formed via distinct and competitive mechanisms,
which involve different catalytically active species. The me-
tabolite DEA is probably formed via the MnVOP species, which
is stabilized by imidazole (25), whereas COA is formed via the
MnIVOP intermediate, which can be formed by reduction of
MnVOP in the absence of the nitrogen ligand (25).
Because the compound COA is an intermediate in the
formation of the metabolite DEA in ATZ degradation studies
using advanced oxidation mechanisms (13, 14, 23, 24), the
reaction using the Fe(TFPP)Cl/PhIO/ACN system under the
standard conditions was monitored as a function of time (10
min, 20 min, 1 h, 2 h, 3 h, and 5 h). This was done to investigate
whether formation of the metabolite DEA occurred via COA
consumption. Figure 7 shows a plot of the number of mol of
each of the main reaction products (COA and DEA) versus the
reaction time. It was observed that the compound COA and the
metabolite DEA are formed independently, contrary to what is
reported in the literature for other catalytic systems (13, 14,
23, 24).
Analogously, in the case of iron porphyrins, the metabolite
DEA would be formed via the catalytic species FeIVOP•+ (or
FeVOP), mimicking the P450 metabolism in vivo, whereas COA
would be formed via the FeIVOP species.
Results obtained in the reactions carried out in the presence
of imidazole also confirm our findings. The effect of imidazole
was evaluated for the metalloporphyrins Mn(TFPP)Cl, Mn-
(TDCPP)Cl, and Fe(TFPP)Cl, using m-CPBA as oxidant.
Nitrogen ligands, such as imidazole, act as cocatalysts in systems
involving manganese porphyrins, leading to increased reaction
rates, catalytic yields, and selectivity (25, 26). This is because
these ligands can coordinate to the manganese porphyrin in the
position trans to the metal-oxo bond, thus stabilizing the
intermediate catalytic species MnVOP. This intermediate is
responsible for efficient stereoselective oxidations. The presence
of imidazole also prevents reduction of the MnVOP species to
MnIVOP, which is responsible for nonselective and little efficient
radicalar reactions (25, 26). Imidazole also acts as an acid-
base catalyst, favoring heterolytic cleavage of the peroxide bond,
with formation of the high-valent oxo-metal intermediate, thus
preventing radical formation (26).
Involvement of the FeIVOP species in these reactions in the
absence of the imidazole was confirmed by the bright orange
color acquired by the reaction solution after the addition of the
oxidant, which is typical of this species (27). Preliminary studies
of the reaction intermediates by UV-vis and EPR at low
temperature also confirmed the presence of this species, which
is characterized by absorption bands at 545 and 410 nm (Soret
band) in the UV-vis spectrum (20, 28). This species also led
to the disappearance of the high-spin Fe(III) signal (S ) 5/2, g
) 6.0) in the EPR spectrum because of the formation of an
EPR silent FeIV species (29).
An increase in the yield of metabolite DEA, with concomitant
decrease in the formation of compound COA, could also be
seen in reactions carried out with excess oxidant in the case of
the Fe(TFPP)Cl/ACN/PhIO system (catalyst/oxidant/substrate
molar ratio of 1:240:120; Figure 9). In these conditions, it is
probable that COA undergoes further oxidation as a result of

Metalloporphyrins as Biomimetic Models for P-450
J. Agric. Food Chem., Vol. 54, No. 26, 2006 10017
Figure 11. Product distribution, in percentage, obtained in the FeP-catalyzed AZT oxidation by m-CPBA in ACN in the standard conditions and in the
absence of oxygen. P I, P II, P III, P IV, and P V are unknown products.
Figure 12. Scheme for MeP-catalyzed AZT oxidation.
the excess oxidant. This should lead to the formation of DEA,
as shown by the increase in the concentration of this product in
Figure 9.
Taking the product distribution under the different conditions
studied here and the above-discussed hypotheses into account,
it is possible to propose a scheme for metalloporphyrin-catalyzed
ATZ oxidation as shown in Figure 12.
No significant alterations in the DEA/COA ratio were
observed for the Fe(TFPP)Cl/m-CPBA/ACN system in the
presence of imidazole (Figure 10). Contrary to manganese
porphyrins, the cocatalytic effect of the nitrogen ligand is not
expected in this case. This is because imidazole bis-coordinates
to the axial positions of the iron porphyrin, thus blocking the
catalytic site and the formation of the catalytic species (30).
Therefore, imidazole was used in this case aiming at favoring
heterolytic cleavage of the peracid O-O bond via acid catalysis,
which should lead to formation of the oxo-ferryl porphyrin
π-cation radical, as well as hinder formation of RO• radicals
via peroxide homolytic cleavage. Figure 10 shows that the
presence of imidazole avoids formation of P IV and increases
production of COA. Bearing in mind that P IV is produced only
in reactions employing the peracid as oxidant, the absence of
this product in a system using m-CPBA in the presence of
imidazole confirms that P IV really results from the homolytic
cleavage of the peracid O-O bond.
Scheme A in Figure 12 considers the use of PhIO as oxidant.
After formation of the active species, MeVOP in this case, the
reaction may follow two pathways. The first one is biomimetic
and involves reaction of the active species with ATZ, leading
to the formation of the metabolite DEA. The second pathway
corresponds to the reaction between the active species and
another catalyst molecule, leading to formation of the species
MeIVOP. The latter reacts with ATZ, producing COA. Once
ATZ is a relatively inert substrate, the second pathway is
favored, so the main reaction product is COA. Because COA
is an intermediate species to DEA, it is possible that it is
oxidized again to DEA under certain conditions, such as in the
presence of excess oxidant.
Scheme B in Figure 12 considers the use of the peracid as
oxidant. Formation of the species MeVOP depends on the
heterolytic cleavage of the metal-oxo bond, which is favored
in the presence of imidazole. Formation of this species takes
us to the first pathway in scheme A, which was discussed above.
Homolytic cleavage of the metal-oxo bond leads to the formation
of the species MeIVPOH and of radicals represented by RO•, as
well as of other radicals associated with molecular oxygen,
present in the reaction medium. These should lead to P IV and
other byproducts of radicalar reactions.
The absence of P IV and an increase in COA concentration
are also observed when the reaction using the Fe(TFPP)Cl/m-
CPBA/ACN system is carried out under argon atmosphere
(Figure 11). This also confirms that P IV is formed via the
radicalar mechanism propagated by O2 present in the reaction
medium.

10018 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Gotardo et al.
(14) Chan, K. H.; Chu, W. Atrazine removal by catalytic oxidation
processes with or without UV irradiation: Part II: an analysis
of the reaction mechanisms using LC/ESI-tandem mass spec-
trometry. Appl. Catal., B 2005, 58, 165-174.
(15) Sharefkin, J. G.; Saltzman, H. Iodosobenzene. Organic Syntheses;
Wiley & Sons: New York, 1973; Collect. Vol. 5, p 658.
(16) Adler, A. D.; Longo, F. R.; Kampas, F.; Kim, J. On the
preparation of metalloporphyrins. J. Inorg. Nucl. Chem. 1970,
32, 2443-2445.
In conclusion, this work has demonstrated the ability of
metalloporphyrins to mimic the action of P450 in the oxidation
of atrazine, a highly persistent herbicide, as well as their capacity
to mimic the in vivo action of P450, with formation of two of
the metabolites found in plants: DEA and DIA.
Our studies also showed the potential application of these
biomimetic chemical models in studies that pursue the elucida-
tion of the in vivo metabolism of herbicides, thus overcoming
the difficulty in working with enzymes in vitro.
As in the case of studies on drug oxidation, it can be
envisaged that metalloporphyrins will become an important tool
for the understanding of herbicide metabolism, and they will
help elucidate some resistance mechanisms developed by weeds,
thus assisting the development of novel compounds for pest
control in agriculture.
(17) Nash, T. Colorimetric determination of formaldehyde under mild
conditions. Nature 1952, 170, 976.
(18) Karki, S. B.; Dinnocenzo, J. P.; Jones, J. P.; Korzekwa, K. R.
Mechanism of Oxidative Amine Dealkylation of Substituted N,N-
Dimethylanilines by Cytochrome P-450: Application of Isotope
Effect Profiles. J. Am. Chem. Soc. 1995, 117, 3657-3664.
(19) Groves, J. T. High-valent iron in chemical and biological
oxidations J. Inorg. Biochem. 2006, 100, 434-447.
(20) Nam, W.; Han, H. J.; Oh, S. Y.; Lee, Y. J.; Choi, M. H.; Han,
S. Y.; Kim, C.; Woo, S. K.; Shin, W. New Insights into the
Mechanisms of O-O Bond Cleavage of Hydrogen Peroxide and
tert-Alkyl Hydroperoxides by Iron(III) Porphyrin Complexes.
J. Am. Chem. Soc. 2000, 122, 8677-8684.
ACKNOWLEDGMENT
We thank Dr. Akira Ueda, from Syngenta, for kindly supplying
atrazine. We also thank Professor Otaciro R. Nascimento for
the EPR analysis.
(21) Guedes, A. A.; Santos, A. C. M.; Assis, M. D. Some factors
influencing the selectivity of styrene oxidation by active oxygen
donors catalyzed by three generations of ironporphyrins. Kinet.
Catal. 2006, 47, 572-580.
(22) Schiavon, M. A.; Iamamoto, Y.; Nascimento, O. R.; Assis, M.
D. Catalytic activity of nitro- and carboxy-substituted iron
porphyrins in hydrocarbon oxidation: Homogeneous solution and
supported systems. J. Mol. Catal. A: Chem. 2001, 174, 213-
222.
(23) Kawahigashi, H.; Hirose, S.; Ohkawa, H.; Ohkawa, Y. Phytore-
mediation of the herbicides atrazine and metolachlor by trans-
genic rice plants expressing human CYP1A1, CYP2B6, and
CYP2C19. J. Agric. Food Chem. 2006, 54, 2985-2991.
(24) Ross, M. K.; Filipov, N. M. Determination of atrazine and its
metabolites in mouse urine and plasma by LC-MS analysis.
Anal. Biochem. 2006, 351, 161-173.
(25) Gunter, M. J.; Turner, P. The role of the axial ligand in
mesotetraarylmetalloporphyrin models of the P-450 cytochromes.
J. Mol. Catal. 1991, 66, 121-126.
(26) Battioni, P.; Renaud, J. P.; Bartoli, J. F.; Artiles, M. R.; Fort,
M.; Mansuy, D. Monooxygenase-like Oxidation of Hydrocarbons
by Hydrogen Peroxide Catalyzed by Manganese Porphyrins and
Imidazole: Selection of the Best Catalytic System and Nature
of the Active Oxygen Species. J. Am. Chem. Soc. 1988, 110,
8462-8463.
(27) Bell, S. E. J.; Cooke, P. R.; Inchley, P.; Leanord, D. R.; Smith,
J. R. L. Oxoiron(IV) porphyrins derived from charged iron(III)
tetraarylporphyrins and chemical oxidants in aqueous and
methanolic solutions. J. Chem. Soc., Perkin Trans. 1991, 2, 549-
559.
LITERATURE CITED
(1) Reichhart, D. W.; Hehn, A.; Didieriean, L. Cytochrome P450
for engineering herbicide tolerance. Trends Plant Sci. 2000, 5,
116-123.
(2) Coleman, J.; Blake-Kalff, M.; Davies, E. Detoxification of
xenobiotics by plants: chemical modification and vacuolar
compartmentation. Trends Plant Sci. 1997, 2, 144-151.
(3) Fragoso, D. B.; Guedes, R. N. C.; Ladeira, J. A. Selection in
the evolution of resistance to organophosphates in Leucoptera
coffeella. Neotrop. Entomol. 2003, 32, 329-334.
(4) Bernadou, J.; Meunier, B. Biomimetic Chemical Catalysts in the
Oxidative Activation of Drugs. AdV. Synth. Catal. 2004, 346,
171-184.
(5) Meunier, B.; Robert, A.; Pratviel, G. Metalloporphyrins in
catalytic oxidations and oxidative DNA cleavage. In The
Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard,
R., Eds.; Academic Press: New York, 2000; pp 119-187.
(6) Santos, A. C. M. A.; Smith, J. R. L.; Assis, M. D. Chloroquine-
iron(III) tetra-arylporphyrin interactions and their effect on
chloroquine oxidations catalyzed by iron(III) tetra-arylporphyrin.
J. Porphyrins Phthalocyanines 2005, 9, 326-333.
(7) Keseru¨, G. M.; Balogh, G.; Czudor, I.; Karancsi, T.; Fehe´r, A.;
Berto´k, B. Chemical Models of Cytochrome P450 Catalyzed
Insecticide Metabolism. Application to the Oxidative Metabolism
of Carbamate Insecticides. J. Agric. Food Chem. 1999, 47, 762-
769.
(8) Fukushima, M.; Fujisawa, T.; Katagi, T. Tomato Metabolism
and Porphyrin-Catalyzed Oxidation of Pyriproxyfen. J. Agric.
Food Chem. 2005, 53, 5353-5358.
(9) Topal, A.; Adams, N.; Hodgson, E.; Kelly, S. L. In vitro
metabolism of atrazine by tulip cytochrome P450. Chemosphere
1996, 32, 1445-1451.
(10) Hanioka, N.; Jinno, H.; Kitazawa, K.; Tanaka-Kagawa, T.;
Nishimura, T.; Ando, M.; Ogawa, K. In vitro biotransformation
of atrazine by rat liver microsomal cytochrome P450 enzymes.
Chem.-Biol. Interact. 1998, 116, 181-198.
(11) Cherifi, M.; Raveton, M.; Picciocchi, A.; Ravanel, P.; Tissut,
M. Atrazine metabolism in corn seedlings. Plant Physiol.
Biochem. 2001, 39, 665-672.
(12) Ohkawa, H.; Tsujii, H.; Ohkawa, Y. The use of cytochrome P450
genes to introduce herbicide tolerance in crops: a review. Pestic.
Sci. 1999, 55, 867-874.
(28) Fujii, H. Eletronic structure and reactivity of high-valent oxo
iron porphyrin. Coord. Chem. ReV. 2002, 226, 51-60.
(29) Assis, M. D.; Serra, O. A.; Iamamoto, Y.; Nascimento, O. R.
An EPR and electronic spectroscopy study of intermediates in a
mono o-nitro substituted iron porphyrin reaction with iodosyl-
benzene. Inorg. Chim. Acta 1991, 187, 107-114.
(30) Evans, S.; Lindsay-Smith, J. R. The oxidation of ethylbenzene
and other alkylaromatics by dioxygen catalysed by iron(III)
tetrakis(pentafluorophenyl)porphyrin and related iron porphyrins.
J. Chem. Soc., Perkin Trans. 2 2000, 1541-1551.
Received for review August 28, 2006. Revised manuscript received
October 24, 2006. Accepted October 24, 2006. We thank the Brazilian
agencies FAPESP, CNPq, and CAPES for financial support.
(13) Arnold, S. M.; Hickey, W. J.; Harris, R. F. Degradation of
Atrazine by Fenton’s Reagent: Condition Optimization and
Product Quantification. EnViron. Sci. Technol. 1995, 29, 2083-
2089.
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