Identification and formation pathway of laccase-mediated oxidation products formed from hydroxyphenylureas

Article abstract of DOI:10.1021/jf060351i

Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.

Full text of DOI:10.1021/jf060351i

5046 J. Agric. Food Chem. 2006, 54, 5046−5054
Identification and Formation Pathway of Laccase-Mediated
Oxidation Products Formed from Hydroxyphenylureas
‡,§
C. JOLIVALT,*,† L. NEUVILLE,† F. D. BOYER,‡ L. KERHOAS,‡ AND C. MOUGIN
Laboratoire de Synthe`se se´lective organique et produits naturels, UMR CNRS 7573, ENSCP,
11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France, and Unite´ de Phytopharmacie et
Me´diateurs Chimiques, INRA, route de Saint-Cyr, 78026 Versailles Cedex, France
Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and
biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability
of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of
five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish
that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature
of the transformation products. Structural characterization by spectroscopic methods (NMR, mass
spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to
quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as
final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining
substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation.
A reaction pathway is proposed for each of the identified products. These results demonstrate that
fungal laccases could assist the transformation of hydroxyphenylureas.
KEYWORDS: Laccase; white rot fungi; T. versicolor; transformation pathway; hydroxyphenylurea;
quinone; polyaromatic oligomers; reaction mechanism
INTRODUCTION
N′,N′-dimethyl-N-(4-chloro-2-hydroxyphenyl)urea (7). Of par-
ticular importance, direct and indirect photolysis of substituted
phenylureas exposed to sunlight leads to several photoproducts,
including those resulting from ring hydroxylation and substitu-
tion of a chlorine atom by a hydroxyl group (8-14). Function-
alization by hydroxylation of the aromatic ring considerably
extends the range of metabolic reactions able to further transform
the pollutants (5-7).
In this context, white rot fungi, such as T. Versicolor, offer
many advantages in secreting nonspecific extracellular oxidases.
Among these enzymes, laccases (EC 1.10.3.2, p-diphenol:
dioxygen oxidoreductases) have been reported to catalyze the
transformation of a wide range of xenobiotics, including phenols
and chlorinated phenols. They convert these chemicals to
radicals, which react to form stable polymers by coupling to
each other or with humic substances in soils (15-17). Such a
oxidative coupling process is a key mechanism for the formation
of bound residue in the soil and for the precipitation of insoluble
oligomers in waters. Because these polymers can be easily
removed from water by filtration, such an enzymatic approach
can be of particular interest in the decontamination of aqueous
effluents (18).
Phenylurea compounds have become extensively widespread
in the environment since their discovery in the 1950s. Most of
them are mainly used as herbicides for the selective protection
of cereal crops and for the maintenance of roads and railways.
Others enter the industrial processes as biocides and/or protective
agents against oxidation. Nevertheless, these compounds are
persistent contaminants of the environment, including soil and
both surface and ground waters (1, 2). As a consequence, there
is an increasing interest in the biotic and abiotic degradation
pathways of these chemicals (3). Biodegradation of phenylureas
in soils is governed by many environmental factors, such as
pH, temperature, and the presence of degrading micro-organisms
(4). Only one bacterial strain, Arthrobacter sp. N2 (5), was
identified as an efficient degrader of diuron [N-(3,4-dichlo-
rophenyl)-N′,N′-dimethylurea] into its corresponding aniline. In
soils, fungal strains transform phenylureas to N-demethylated
and/or hydroxylated derivatives (6). Hydroxylation of the
aromatic ring of phenylureas during their metabolization in
higher plants has also been reported for monuron [N-(4-
chlorophenyl)-N′,N′-dimethylurea], which is transformed to
* Corresponding author. E-mail: claude-jolivalt@enscp.fr. Telephone:
33 (0)1 44 27 67 54. Fax: 33 (0)1 44 27 67 01.
† UMR CNRS 7573.
Up to now, our work was mainly focused on the transforma-
tion of nonchlorinated derivatives by fungal laccases (19). In
the present study, we included chlorohydroxylated phenylurea
compounds, so that the hydroxylated phenylurea derivatives
studied in this work are representative of the range of hydroxy-
‡ INRA.
§ Present address: Unite´ Physicochimie et Ecotoxicologie de Sols
d’Agrosyste`mes Contamine´s, INRA, route de Saint-Cyr, 78026 Versailles
Cedex, France.
10.1021/jf060351i CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/15/2006

Oxidation Products Formed from Hydroxyphenylureas
J. Agric. Food Chem., Vol. 54, No. 14, 2006 5047
a UV detector. Retention times for the hydroxyphenylureas tested
ranged from 7 to 11 min. Experiments with 2HF-5Cl were duplicated.
Time Course of Hydroxyphenylurea Transformation. Hydroxyphen-
ylureas (1 mM) were shaken at 150 rpm at 30 °C for 2 h with a specified
amount of enzyme in 10 mL of 25 mM citrate/phosphate buffer at
various pH values. Incubations without enzyme served as the control.
After defined periods of incubation, the reaction assays were stopped
with 10% (v/v) concentrated HCl and filtered (Millipore 0.22 µm)
before HPLC analysis.
Scheme 1. Structure of Phenylurea Derivatives Used as Laccase
Substrates
Incubation with Phenylureas and Extraction of Oxidation Products.
Hydroxyphenylureas (25-50 mg) were incubated with 5-10 U of
laccase in 25-50 mL of aerated citrate/phosphate buffer (50 mM) at
30 °C in darkness. The extent of transformation was checked by HPLC,
and the incubation stopped when the totality of the initial phenylurea
was transformed. Reaction mixtures were acidified to pH 2 by addition
of 1 M HCl. The reaction mixture (aqueous phase and precipitate) was
treated with sodium chloride (2.5 mol‚L-1 final concentration) and
extracted with dichloromethane (25 mL) three times. The extracts were
combined, dried over anhydrous sodium or magnesium sulfate, and
evaporated on a vacuum evaporator at 40 °C. The residues were
dissolved in 5 mL of ethyl acetate and separated on a silica column
using ethyl acetate as an elution solvent. Identification of pure
compounds isolated from the column was performed by mass and NMR
spectroscopy.
lated metabolites of some phenylurea herbicides found on the
fields.
The potential of fungal laccases in catalyzing the transforma-
tion reactions of these pollutants as a function of the pH was
investigated. In addition, we determined the chemical structures
of the main products formed from nonchlorinated and chlori-
nated hydroxyphenylureas during the enzymatic reactions,
allowing us to propose transformation pathways and reaction
mechanisms. Our results better define (i) the fate of phenylureas
in the environment and (ii) the potential application of laccases
in decontamination efforts.
Analytical and Spectroscopic Methods. MS and MSMS spectra
were obtained using a triple quadripole instrument Nermag R 30-10
(Quad Service, Poissy, France) in electronic impact (EI) and chemical
ionization (CI) modes. A Quattro LC (Micromass, Manchester, U.K.)
equipped with an electrospray source ionization (ESI) (Z-spray from
Micromass) was used for LC-MS determinations.
Nermag R 30-10 source conditions were set as follows: temperature,
130 °C; electron energy, 70 eV (EI) or 95 eV (CI). Samples were
introduced either by gas chromatography or with a direct insertion probe
for ionization by EI or by desorption chemical ionization (DCI). NH3
was used as reagent gas at 10-4 Torr pressure in the source housing,
and for MS-MS experiments argon was used as collision gas (4 × 10-2
Torr).
The Quattro LC electrospray source parameters were as follows:
capillary, 3.25 kV; extractor, 2 V; source block temperature, 120 °C;
desolvation gas, 500 L/h N2; temperature, 400 °C. The sampling cone
voltage was varied usually from 20 to 40 V. Data acquisition and
processing were carried out using the software MassLynx version 4.0.
The compounds were introduced by infusion (Harvard Apparatus,
Holliston. MA).
1H and 13C NMR spectra were recorded at 293 K, on Bruker AC
200 equipment or on a Varian Mercury plus 300 instrument in
deuteriochloroform (CDCl3), benzene-[D6], or DMSO-[D6]. Chemical
shifts are reported in δ ppm relative to CHCl3 (CDCl3) as internal
reference: 7.27 ppm for 1H (77.14 ppm for 13C) or measured with SiMe4
as internal reference following standard techniques. For the other cases,
residual solvent was also used as internal standards (23). Coupling
constants (J) are given in hertz (Hz). Multiplicities are recorded as s
(singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), and br
(broad).
MATERIALS AND METHODS
Chemicals. Acetonitrile (HPLC grade) was obtained from Carlo Erba
(Val de Reuil, France). Chemicals used as buffers and silica for
chromatography (Gerudan Si 60, 40-63 µm) were purchased from
VWR (Fontenay sous bois, France). Chlorzoxazone (5-chloro-2-
benzoxazolone) was from Acros (Noisy le Grand, France). All other
chemicals or reagents used for synthesis and assays, including 2,2′-
azinobis(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS), were obtained
from Sigma (Saint Quentin Fallavier, France).
N′,N′-Dimethyl-N-(4-hydroxyphenyl)urea (4HF), N′,N′-dimethyl-N-
(2-hydroxyphenyl)urea (2HF), N′,N′-dimethyl-N-(4-chloro-2-hydroxy-
phenyl)urea (2HF-4Cl), N′,N′-dimethyl-N-(5-chloro-2-hydroxyphenyl)-
urea (2HF-5Cl), and N′,N′-dimethyl-N-(3-chloro-4-hydroxyphenyl)urea
(4HF-3Cl) were prepared according to procedures adapted from the
literature (19-21) (see Supporting Information). The chemical structures
of these compounds are shown in Scheme 1.
Production of T. Wersicolor Laccase. The production and purifica-
tion of laccase have been described elsewhere (22). Briefly, the enzyme
was produced from Trametes Versicolor cultures, induced by 2,5-
xylidine in a 5-L bioreactor. The purification included two steps of
chromatography, a first one on a DEAE 52 anion exchange column,
and a second one on a Phenyl Sepharose (Pharmacia HiTrap) column.
The purification procedure led to a homogeneous sample, as checked
by electrophoresis, with a specific activity of 300 U/mg, where one
unit of enzyme activity (U) is defined as the amount of enzyme that
oxidizes 1 µmol of ABTS in 1 min.
Assays for laccase activity were performed by measuring the
enzymatic oxidation of ABTS at 420 nm (ꢀ ) 3.6 × 104 cm-1 M-1 for
the oxidation product). The reaction mixture contained 1 mM ABTS
in 0.1 M Na2HPO4/citric acid buffer (pH 3.0) in a final volume of 1
mL. The buffer solution was saturated with air by bubbling prior to
the experiment. After the enzymatic solution was added, the increase
in absorbance was followed during the first 2 min, at 30 °C.
Enzymatic Reactions with Hydroxyphenylurea Derivatives. pH
ActiVity Profile of T. Versicolor Laccase. Determination of the optimum
pH was conducted in 50 mM citrate/phosphate buffer in the range of
pH 2.5-7. The chlorinated hydroxyphenylureas were solubilized at 0.1
g/L, and 0.05 U of laccase was added in a total volume of 5 mL.
Samples were incubated and shaken (150 rpm) at 30 °C for 15 min.
The reaction was then stopped by adding 0.5 mL of concentrated HCl.
Quantitative analysis of phenylurea transformation was conducted using
an HPLC system (Varian) with a reverse phase column (NovaPak C8,
Waters, 2.3 mm × 150 mm). Elution was carried out at a flow rate of
1 mL min-1 using a solvent gradient from 100% water to 100%
acetonitrile within 20 min. Detection was performed at 244 nm using
RESULTS
Influence of pH on the Transformation of Chlorinated
Hydroxyphenylureas by Laccase. pH-curves (Figure 1) exhibit
the same bell shaped profile for all the chlorinated hydroxy-
phenylureas with a maximum between pH 4 and 5. A slight
difference was observed for 2HF-5Cl, whose pH-curve was
found to be broader. In the case of 4HF-3Cl and 2HF-4Cl,
laccase showed only residual activity at pH 7, while, with 2HF-
5Cl, the enzyme retained up to 15% of its maximal activity.
Time Dependence of the Transformation of 4HF-3Cl by
Laccase. The analytical method used to follow the transforma-
tion of the chlorinated hydroxyphenylurea derivatives as a
function of the pH showed that pH influences the nature of the
reaction products. A similar behavior was observed for all the

5048 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Jolivalt et al.
constant. The formation of both products, thus, seems to proceed
via a competitive process, since product V did not seem to result
from the further transformation of product I.
Transformation of all chlorinated hydroxyphenylureas dis-
played a similar behavior to that observed with 4HF-3Cl: a
quinonic predominant product at pH 3, more polar than its parent
compound. When increasing the reaction pH, the quinonic
compound concentration decreased, favoring one or more less
polar oxidation products.
Identification of Products Formed from Hydroxyphenyl-
ureas by Laccase. Proposed structures for the transformation
1
products are shown in Tables 1-3, including their H NMR
and mass spectroscopic data.
The1H NMR spectrum of compound I showed only one
singlet (δ 6.74 ppm), suggesting a symmetric localization of
hydrogen atoms on the aromatic ring. The molecular mass was
found to be 108. Comparison (spectroscopic data, retention time)
with commercial standards led to its identification as 1,4-
benzoquinone (Table 1).
Figure 1. Influence of pH on the transformation of chlorinated hydroxy-
phenylureas by laccases:
Substrate initial concentration: 0.1 g/L in 0.1 M citrate/phosphate buffer;
30 C. Laccase concentration: 0.011 units for 2HF-5Cl; 0.05 units
([) 2HF-5Cl; (b) 4HF-3Cl; (9) 2HF-4Cl.
1
The H NMR spectrum of compound II (Table 1) showed
T
)
°
only the presence of three aromatic hydrogen atoms. According
to the observed coupling constants and the multiplicity of the
peaks, the signal pattern is consistent with the presence of three
protons in the 3, 5, and 6 positions on the ring. From mass
spectroscopy, we know that the compound retained its chlorine
atom during the transformation. Together with a molecular mass
of 142, this information is in agreement with the structure of
2-chloro-1,4-benzoquinone, as confirmed by comparison with
the spectroscopic data of the commercial standard.
for 4HF-3Cl and 2HF-4Cl. Incubation duration: 15 min.
The 1H NMR spectrum of compound III (Table 1) indicated
the presence of a N(CH3)2 group (δ 3.08 ppm). Chemical
ionization mass spectrometry indicated that the parent molecule
loses its chlorine atom during the oxidation process, leading to
a compound with a molecular mass of 194, which is in
accordance with the structure shown in Table 1.
Compound IV was the major product obtained by oxidation
of 4HF at pH 5. The NMR spectrum (Table 2) indicated the
presence of a N(CH3)2 group and of 2 × 2 aromatic protons.
The MS fragmentation pattern (EI spectrum) gave a m/z of 178
with fragments 134 and 106 representative of the successive
fragmentations of the substituted urea group on the parent
molecule. On the basis of these large similarities with the parent
molecule, together with published 1H NMR data on acetyl
p-benzoquinone imine (24), it was concluded that compound
IV was the 1,1-dimethyl-3-(4-oxocyclohexa-2,5-dienylidene)-
urea resulting from the oxidation of 4HF.
Compound V was obtained from transformation of 4HF-3Cl.
From 1H NMR spectrum analysis (Table 2), it can be assumed
that the molecule retained the dimethylated substituted phenyl-
urea motif. The aromatic hydrogen substitution pattern on the
1H NMR spectrum is very similar to that observed for II. Data
from mass spectrometry indicated a molecular mass of 212, i.e.,
loss of two mass units compared to the parent molecule and
the presence of one chlorine atom. The 13C spectrum showed
three CH (140.92, 134.04, 127.79) and two quaternary carbons
(179.02 and 161.96), whose chemical shift is very close to that
observed for a 2-chloro-1,4-quinonimine cycle, confirming the
structure of V as (3E)-3-(3-chloro-4-oxocyclohexa-2,5-dienyli-
dene)-1,1-dimethylurea. It was noticed that the 13C NMR and
1H NMR spectra contained additional peaks (around 40% of
the total intensity) assigned to the corresponding (3Z) isomeric
form of the molecule.
Figure 2. Time course of 4HF-3Cl and product concentrations during
the transformation of 4HF-3Cl by T. versicolor laccase (0.03 units/mL) at
pH 3 (A) and 5 (B): (b) 4HF-3Cl; (9) product I; (2) product V.
tested compounds. Consequently, transformation of 4HF-3Cl
was chosen here as a typical example. The time course of 4HF-
3Cl transformation was carried out in the presence of laccase
at two pH values: 3 and 5 (Figure 2). At pH 3 (Figure 2A),
the enzymatic transformation of 4HF-3Cl led to the formation
of one predominant product, I, whose retention time (tR) 3.4
min) was smaller than that of 4HF-3Cl (tR ) 7 min), indicating
a higher polarity of the product. At pH 5 (Figure 2B), the
transformation of 4HF-3Cl was more rapid than that at pH 3
and led to the formation of two products, I and V (tR ) 7.4
min). After 45 min, 97% of the 4HF-3Cl was transformed
whereas the concentration of each of the two products reached
a maximum at this time. The amount of compound V slightly
decreased thereafter, whereas that of product I remained
The oxidation products formed during the transformation of
ortho-hydroxylated phenylureas 2HF and 2HF-4Cl at pH 5 were

Oxidation Products Formed from Hydroxyphenylureas
J. Agric. Food Chem., Vol. 54, No. 14, 2006 5049
Table 1. 1H NMR and Mass Spectroscopic Data for Hydroxyphenylurea Transformation Products at pH 3
Table 2. 1H NMR and Mass Spectroscopic Data for Hydroxyphenylurea Transformation Products at pH 5
identified as dimeric species resulting from a carbon-carbon
coupling of the parent molecules (Table 2). Mass spectrometry
analysis (positive and negative modes) of the 2HF transforma-
tion extract indicated a molecular mass of 356 and exhibited
fragments at (M - 45) and (M - 2 × 45) in the negative mode,
which is representative of two successive losses of the dimeth-
of the major isomer, VI, was representative of a symmetrical
dimer. The signal assignment is consistent with the presence
of a dimethylated phenylurea fragment and of three aromatic
protons in the 3, 4, and 6 positions. 13C NMR confirmed the
presence of the urea carbonyl (154.71 ppm) and indicated the
presence of one aromatic quinone (180.81 ppm), together with
two quaternary carbons (138.62, 135.17) and three CH (134.12,
128.16, 111.26) on each aromatic cycle. These results are in
agreement with the structures of VI and VIbis proposed in
1
ylated amine fragment. H NMR spectrum resulted from the
superposition of the signals of two regioisomers, compound VI
(more than 80%) and compound VIbis. The 1H NMR spectrum

5050 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Jolivalt et al.
Table 3. 1H NMR and Mass Spectroscopic Data for 2HF-5Cl Transformation Products at pH 6.5
Table 2, with the E conformation being assigned to the major
product (compound VI), which is sterically favored.
showing that each aromatic cycle kept its urea moiety. The 1H
NMR signal at δ 8.11 ppm disappeared in the presence of D2O
and was thus assigned to NH protons. On the basis of the
assignment of the two 1H NMR singlet protons in 2-acetamido-
5-ethoxy-1,4-benzoquinone (25), it was hypothesized that VIII
could contain two hydrogens in the para position on a
disubstituted 1,4-benzoquinone (δ 8.01 and 6.02 ppm in C6D6).
The 1H NMR multiplets and coupling constants of the aromatic
protons of VIII are representative of a 1, 2, 5 aromatic
substitution pattern. A NOE experiment showed correlation
between H3 and H3′ protons, which is consistent with assigning
H3, H4, H6 on one hand and H3′, H6′ on the other hand to two
different aromatic cycles.
Because of the poor chromatographic separation of the crude
extract after 2HF-4Cl transformation at pH 5, identification was
based on the mass spectrometry analysis of fractions collected
from HPLC. The purest fraction contained, together with
unreacted parent 2HF-4Cl, one major product compound (VII).
Mass spectra indicated a molecular mass of 426 for VII,
including two chlorine atoms. Two successive losses of a
dimethylated amine in the fragmentation pattern could be
1
attributed to the presence of two urea groups on VII. The H
NMR spectrum contained four singlets (Table 2). The signal
at δ 7.89 ppm disappeared in the presence of D2O and so can
be assigned to NH, by analogy with the chemical shift of the
parent molecule 2HF-4Cl (δ 7.79 ppm). Two singlets were
assigned to aromatic hydrogen atoms in the para position. To
account for the molecular mass, compound VII was thus
identified as the dimeric hydroxyl compound, derived from the
coupling of the parent phenylurea in the para position, with
reference to the hydroxyl group.
At pH 5, oxidation of 2HF-5Cl resulted in the formation of
2,5-benzoquinone II as a final product. Increasing the pH to
pH 6.5 allowed shifting to the formation of three different
products, identified as chlorzoxazone and compounds VIII and
IX (Table 3). They were recovered from three successive
fractions collected from the silica column. In addition to a
distinctive Rf, each compound was characterized by a different
color. The fraction with the higher Rf (0.8, ethyl acetate),
chlozoxazone, was yellow, the intermediate fraction (Rf 0.61,
ethyl acetate, product VIII) was orange, and the most retained
fraction (Rf 0.32, ethyl acetate, product IX) was pink. Chlor-
zoxazone was identified by comparison with an authentic sample
(spectroscopic data and retention time). From MS data, the
molecular mass of product VIII was 406, which corresponds
to a dimeric oligomer derived from 2HF-5Cl, and the molecule
contains one chlorine atom. 13C NMR indicated the presence
of 12 aromatic carbons in compound VIII, which confirmed
the oligomerization extent. 13C NMR showed typical signals
for quinones in the range of 180 ppm. Compound VIII contained
two carbonyls from a urea group in the range of 150 ppm,
From MS data, IX has a mass of 584, corresponding to a
trimer of its parent molecule. It contains only one chlorine atom.
Two successive losses of 45 units followed by loss of fragments
of 44 units indicate that each of the aromatic cycles of IX bears
the urea group of the parent molecule 2HF-5Cl, which is
confirmed by three 13C NMR signals in the range of urea
carbonyl (155.85, 154.09, 153.25 ppm) as well as the three
singlets (8.38, 8.19, 8.02 ppm), corresponding to NH in the 1H
NMR spectrum (Table 3). The 13C NMR spectrum also showed
the presence of 18 aromatic carbons, including two quinonic
1
carbons (182.47, 181.08 ppm). Both H NMR singlets (6.94
and 5.97 ppm in CDCl3) are supposed to be representative of
two aromatic protons in the para position on a disubstituted
1,4-benzoquinone, as encountered in compound VIII. The six
remaining 1H NMR signals can be attributed to the three protons
of each of the two aromatic cycles in the 3, 4, 6 positions on
each cycle. The highest chemical shifts were assigned to the
chlorinated ring, in accordance with related aromatic values (35).
All results led to the identification of IX as drawn in Table 3.
DISCUSSION
Study of the oxidative transformation of hydroxyphenylurea
derivatives by laccase from T. Versicolor clearly demonstrated
the influence of the pH of the incubation mixture. First, the
extent of degradation of the substrates greatly depended on the
pH. The activity curve exhibited a bell shaped profile with a

Oxidation Products Formed from Hydroxyphenylureas
J. Agric. Food Chem., Vol. 54, No. 14, 2006 5051
pathways with a common intermediate species, i.e. the carbo-
cation, is consistent with the experimental evidence that at a
given pH the concomitant formation of the imine and the p-ben-
zoquinone is observed.
Scheme 2. Possible Reaction Mechanism in the Laccase-Catalyzed
Oxidation of 4HF and 4HF-3Cl
At higher pH, 2HF and 2HF-4Cl, which are not substituted
at the para position, do not lead to the formation of an imine
but to dimeric compounds. As is the case in the previous
mechanism, two successive laccase-mediated oxidations are
leading to a cationic intermediate (Scheme 3). In a third step,
the nucleophilic addition of the parent molecule at the carbo-
cation leads to the formation of a carbon-carbon coupled dimer.
Once the cation is generated, coupling is completed without
further involvement of the enzyme. Preferential coupling at the
C4 position might be due to less sterical hindrance of the
corresponding cation, which is stabilized due to the presence
of an urea group in the meta position.
An alternative mechanism involving a laccase-catalyzed one-
electron oxidation of the substrate (2HF or 2HF-4Cl) to form
a phenoxy radical and its subsequent dimerization cannot be
ruled out (Scheme 3, radical dimerization route). In that case,
assuming that the preferred entry of a new substituent is at the
position ortho or para to the hydroxyl group, with the meta
position being unreactive, three isomers of dimers with C-C
bonds are expected to be formed from 2HF and 2HF-4Cl, as
derived from the reaction between two radicals. However, only
one of these, the 5,5-di(2-hydroxyphenylurea) dimer could be
identified, which is consistent with the cation formation route
rather than the radical dimerization one.
maximum between pH 4 and 5 for all three chlorinated
hydroxyphenylureas tested. Such behavior is representative of
phenolic substrates and was previously observed, among numer-
ous examples, with syringic and vanillic acids (27), sinapic acid
(28), and 2,4,6-trichlorophenol (29). Xu (30) postulated that this
bell shape profile is a consequence of two opposite effects: the
ascending part of the curve is generated by the redox poten-
tial difference between the reducing substrate and the type 1
copper of laccase, which increases with the pH, whereas the
descending part results from the binding of an hydroxide anion
to the type 2/type 3 coppers of laccase, which inhibits the
activity at higher pH.
For all the tested phenylureas, the para-quinonic derivative
was obtained as the predominant product at pH 3. At pH 5, the
amount of 2-chlorobenzoquinone in the incubation mixture was
only 20% of that at pH 3 (Figure 2). An earlier report of our
group in regard to the transformation products of 4HF as a result
of T. Versicolor oxidation (19) led to a similar conclusion: at
pH 3, the only products observed during the transformation of
HF derivatives were p-benzoquinones. The formation of quino-
nes in laccase-catalyzed reactions has been observed before at
low pH for the enzymatic oxidation of vanillic and syringic acids
(27) or chlorophenolic compounds (29). Recently, Niedermeyer
(26) reported that amination of alkylated p-hydroquinones with
aromatic amines using laccases proceeded through the inter-
mediary of the formation of the corresponding quinone.
Scheme 2 suggests a possible mechanism for the formation
of a p-benzoquinone from 4HF and 4HF-3Cl. First, a laccase-
catalyzed one-electron oxidation generates a phenoxy radical
by removal of hydrogen and one electron from the phenoxy
group of the parent hydroxyphenyl. A second enzymatic oxi-
dation is then assumed to proceed, leading to the formation of
a carbocation intermediate at the 4 position, favored due to a
possible stabilization by resonance via the imine form of the
molecule. At higher pH, the carbocation can lose a proton to
form an imine which becomes the major final product of the
reaction. In slightly acidic conditions, hydrolysis of the cationic
group in the para position proceeds via a nucleophilic addition
of water, leading to the formation of the corresponding
p-benzoquinone. Such a mechanism involving two different
There are only a few examples of laccase-catalyzed formation
of a C-C bond in the literature since the pioneering work of
Bollag et al. (32-33). However, similar preferential coupling
in the para position to the hydroxyl group was reported for
sinapic acid (28), although 21 dimeric products could be ex-
pected if a random coupling took place. Recently, Ciecholewski
et al. (34) performed the oxidative dimerization of salicylic esters
with laccase in an attempt to provide a new route for the
synthesis of functionalized biaryls. Selective carbon-carbon
bond formation was observed at the ortho position to the
hydroxyl group of the salycilate, likely because it was the only
unsubstituted position on the ring.
Dimers formed by coupling via C-C bond formation
remained phenolic compounds, thus likely to be potential
substrates for T. Versicolor laccase, which is known for its weak
specificity. Tridimensional structure studies (22) have shown
that the enzyme possesses a large cavity (around 10 Å) at the
reducing substrate active site. In the case of 2HF, it can thus
be suggested that a second biocatalytic oxidation takes place
following the coupling step, leading to the corresponding
quinones VI and VIbis. However, no quinonic compound was
found from 2HF-4Cl oxidation. The reason that the 2HF
phenolic dimer is more readily oxidable than the corresponding
2HF-4Cl one is most likely because of the electron-withdrawing
property of the chlorine, reducing the electron density at the
phenoxy group, thus making 2HF-4Cl more difficult to be
further oxidized. It should be noted that a dimeric quinonic
compound was previously found as the final predominant
product of enzymatic oxidation of sinapic acid (28).
Finally, it was found that the oligomers VIII and IX resulting
from the coupling of aromatic rings via an ether bond were
obtained from the laccase-catalyzed oxidation of 2HF-5Cl at
pH 6.5. The proposed pathway (Scheme 4) includes the laccase-
catalyzed formation of the corresponding p-quinone as the first
step, followed by a subsequent nonenzymatic addition at the
para position to the urea substituent via the hydroxyl group of

5052 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Jolivalt et al.
Scheme 3. Possible Reaction Mechanism in the Laccase-Catalyzed Oxidation of 2HF and 2HF-4Cl at pH 5
Scheme 4. Possible Reaction Mechanism in the Laccase-Catalyzed Oxidation of 2HF-5Cl at pH 6.5
the parent 2HF-5Cl. The presence of the p-quinone in the
reaction medium is of course a prerequisite in this pathway and
is supported by the experimental observation that the quinone
formation is the major transformation route of 2HF-5Cl, even
at pH 5. Although limited at similar pH for the other hydroxy-
phenylureas such as 4HF or 2HF, the presence of p-quinone in
the reaction medium can be explained by the more electrophilic
character of the carbocation formed after the two-electron

Oxidation Products Formed from Hydroxyphenylureas
J. Agric. Food Chem., Vol. 54, No. 14, 2006 5053
oxidation of 2HF-5Cl, due to the presence of chlorine, favoring
the subsequent addition of water. A second argument is that,
thanks to the hydrogen bond with the hydrogen of the urea
amine, the delocalization of the nonbinding doublet of its
nitrogen atom is favored, enhancing the leaving character of
the chlorine atom (Scheme 4).
compounds 2HF, 4HF, 4HF-3Cl, 2HF-4Cl, and 2HF-5Cl and
compounds I-IX. This material is available free of charge via
the Internet at http://pubs.acs.org.
LITERATURE CITED
However, quinone formation is only the first step of the 2HF-
5Cl transformation pathway. As it is known that at pH 6.5 the
transformation of the substrate is very low, the quinone is in
the presence of a rather high concentration of unreacted substrate
in solution, which is likely to undergo a nucleophilic addition
on the quinone. A similar mechanism of addition on para-
quinone was recently described for the synthesis of fungal
laccase-catalyzed aminoquinones, resulting from the amination
of p-hydroquinones with primary aromatic amines (26) at an
ortho position to the quinoic carbonyl, as observed in the present
work.
Compound IX resulted from a nucleophilic substitution on
the aromatic ring taking place para to the first C-O coupling
site. One interesting point to note is the release of chlorine ions
as a result of the coupling reaction. Chlorine atoms were released
if they happened to be attached to carbon atoms engaged in a
coupling or nucleophilic addition reaction. A similar observation
was reported for laccase-catalyzed transformation of chlorophe-
nols (15).
Conclusion. The hydroxyphenylurea derivatives studied in
this work are metabolites or photochemical degradation products
formed from phenylurea compounds widespread in the environ-
ment, including soils and waters. Functionalization of their
aromatic ring through hydroxylation makes phenylureas poten-
tial substrates for oxidative enzymes and thus for a further
degradation. This paper offers the first data on both the
transformation mechanisms and the structure of the metabolites
formed by transformation with laccase from T. Versicolor, an
oxidase produced in many soil fungi.
We have shown that laccase-mediated transformation of
hydroxyphenylureas led to quinones at pH 3. By contrast, at
higher pH values, these compounds are further transformed into
oligomers by chemical processes. The polymerization process
is of special importance for the environmental fate of HF
derivatives. First, when the oxidation coupling reaction occurs
at the chlorinated site of the substrate, the induced dehaloge-
nation contributes to the overall detoxification effect since it is
generally recognized that toxicity decreases after dechlorination.
Second, as we postulated that the mechanism of covalent bond
formation involves the addition of a nucleophile, leading to the
formation of heterodimers, the same mechanism is likely to take
place between quinones resulting from the two electron oxida-
tion of hydroxyphenylureas and humic acids present in soils.
Such a polymerization process of binding to humic acid has
been previously reported for chlorophenols (15). Potentially
toxic quinones formed from laccase-catalyzed oxidation of HF
derivatives are thus likely to be bound to the organic matter of
soils and thus to lose their bioavailability and, as a consequence,
their ecotoxicity.
(1) Garmouma, M.; Teil, M. J.; Blanchard, M.; Chevreuil, M. Spatial
and temporal variations of herbicide (triazines and phenylureas)
concentrations in the catchment bassin of the Marne river
(France). Sci. Total EnViron. 1998, 224, 93-107.
(2) Nitschke, L.; Schu¨ssler, W. Surface water pollution by herbicides
from effluents of wastewater treatment plants. Chemosphere
1998, 36, 35-41.
(3) Bolte, M. Cycle de vie d’un polluant. Cas particulier de pesticides
de la famille des phe´nylure´es. L’Actual. Chim. 2004, 277-278,
33-39.
(4) Gaillardon, P.; Sabar, M. Changes in concentration of isoproturon
and its degradation products in soil and soil solution during
incubation at two temperatures. Weed Res. 1994, 34, 243-250.
(5) Tixier, C.; Sancelme, M.; Aˆıt-Aˆıssa, S.; Widehem, P.; Bonnemoy,
F.; Cuer, A.; Triffaut, N.; Veschambre, H. Biotransformation of
phenylurea herbicides by a soil bacterial strain, Arthrobacter sp.
N2: structure, ecotoxicity and fate of diuron metabolite with
soil fungi. Chemosphere 2002, 46, 519-526.
(6) Tixier, C.; Bogaerts, P.; Sancelme, M.; Bonnemoy, F.; Twagili-
mana, L.; Cuer, A.; Bohatier, J.; Veschambre, H. Fungal
biodegradation of a phenylurea herbicide, diuron: structure and
toxicity of metabolites. Pest. Manag. Sci. 2000, 56, 455-462.
(7) Frear, D. S.; Swanson, H. R. Monuron metabolism in excised
Gossypium hirsutum leaves: aryl hydroxylation and conjugation
of 4-chlorophenylurea. Phytochemistry 1974, 13, 357-360.
(8) Tanaka, F. S.; Wien, R. G.; Zaylskie, R. G. Photolysis of 3-(4-
chlorophenyl)-1,1-dimethylurea in dilute aqueous solution J.
Agric. Food Chem. 1977, 25, 1068-1072.
(9) Aguer, J. P.; Richard, C. Transformation of fenuron induced by
photochemical excitation of humic acids. Pestic. Sci. 1996, 46,
151-155.
(10) Richard, C.; Bengana, S. PH effect in the photocatlytic trans-
formation of a phenylurea herbicide. Chemosphere 1996, 33 (4),
635-641.
(11) Jirkovsky, J.; Faure, V.; Boule, P. Photolysis of diuron. Pestic.
Sci. 1997, 50, 42-52.
(12) Boulkamh, A.; Harakat, D.; Sehili, T.; Boule, P. Phototransfor-
mation of metoxuron [3-(3-chloro-4-methoxyphenyl)-1,1-dimeth-
ylurea] in aqueous solution. Pest. Manag. Sci. 2001, 57, 1119-
1126.
(13) Galichet, F.; Mailhot, G.; Bonnemoy, F.; Bohatier, J.; Bolte, M.
Iron(III) photoinduced degradation of isoproturon: correlation
between degradation and toxicity. Pest. Manag. Sci. 2002, 58,
707-712.
(14) Richard, C.; Vialaton D.; Aguer J. P.; Andreux F. Transformation
of Monuron photosensitized by soil extracted humic sub-
stances: energy or hydrogen transfer? J. Photochem. Photobiol.,
A: Chem. 1997, 111, 265-272.
(15) Park, J.-W.; Dec, J.; Kim, J.-E.; Bollag, J.-M. Dehalogenation
as a consequence of binding to humic materials. Arch. EnViron.
Contam. Toxicol. 2000, 38, 405-410.
(16) Ullah, M. A.; Bedford, C. T.; Evans, C. S. Reactions of
pentachlorophenol with laccase from Coriolus versicolor. Appl.
Microbiol. Biotechnol. 2000, 53, 230-234.
(17) Bollag, J. M.; Chu H. L.; Rao M. A.; Gianfreda L. Enzymatic
transformation of chlorophenol compounds. J. EnViron. Qual.
2003, 32, 63-69.
(18) Jolivalt, C.; Brenon S.; Caminade E.; Mougin, C.; Pontie´, M.
Immobilization of laccase from Trametes Versicolor on a
modified PVDF microfiltration membrane: characterisation of
the grafted support and application in removing a phenylurea
pesticide in wastewater. J. Membr. Sci. 2000, 180, 103-113.
(19) Jolivalt, C.; Raynal, A.; Caminade, E.; Kokel, B.; Le Goffic, F.;
Mougin, C. Transformation of N′,N-4-dimethyl-N-(hydroxyphen-
ACKNOWLEDGMENT
We thank Dr. Debrauwer (UMR 1089 Xe´nobiotiques, INRA-
ENVT, Toulouse) for acquisition of the APCI mass spectrum
of compound VII and Dr. David Tepfer for the careful reading
of the manuscript.
Supporting Information Available: Synthesis procedure and
spectroscopic data (1H and 13C NMR, mass spectrometry) of

5054 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Jolivalt et al.
yl)ureas by laccase from the white rot fungus Trametes Versi-
color. Appl. Microbiol. Biotechnol. 1999, 51, 676-681.
(20) Hafjieva, B.; Kalcheva, V.; Vassilev, G.; Galabov, B.; Dimcheva,
Z. 1-(2-hydroxyaryl)-3-(2-hydroxyethyl)-and 3,3-dialkyl ureas:
synthesis, herbicidal and plant growth regulating activities. C.
R. Acad. Bulg. Sci. 1988, 41, 113-116.
(21) Smith, A. Putrescine dihydrochloride. Organic Synthesis; Wiley
& Sons New York, 1963; Collect. Vol. 4, pp 819-823.
(22) Bertrand T.; Jolivalt C.; Caminade E.; Joly N.; Mougin C.;
Briozzo P. Purification and preliminary crystallographic study
of Trametes Versicolor laccase in its native form. Acta Crystal-
logr., D 2002, 58, 319-321.
(23) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts
of common laboratory solvents as trace impurities. J. Org. Chem.
1997, 62, 7512-7515.
(24) Novak, M.; Pelecanou, M.; Pollack, L. Hydrolysis of the model
carcinogen N-(pivaloyloxy)-4-methoxyacetanilide: involvement
of N-acetyl-p-benzoquinone imine. J. Am. Chem. Soc. 1986, 108,
112-120.
(25) Calder, I. C.; Crekk, M. J.; Williams, P. J.; Funder, C. C.; Green,
C. R.; Ham, K. N.; Tange, J. D. N-hydroxylation of p-aceto-
phenetidide as a factor in nephrotoxicity. J. Med. Chem. 1973,
16, 499-501.
(26) Niedermeyer, T.; Mikolasch, A.; Lalk, M. Nuclear amination
catalyzed by fungal laccases: reaction products of p-hydro-
quinones and primary aromatic amines. J. Org. Chem. 2005, 70,
2002-2008.
(27) Leonowicz A.; Edgehill, R. U.; Bollag, J-.M. The effect of pH
on the transformation of syringic and vanillic acids by the
laccases of Rhizoctonia praticola and Trametes Versicolor. Arch.
Microbiol. 1984, 137, 89-96.
(28) Lacki, K.; Duvnjak, Z. Transformation of 3,5- dimethoxy-4-
hydroxy cinnamic acid by polyphenol oxidase from the fungus
Trametes Versicolor: product elucidation studies. Biotechnol.
Bioeng. 1998, 57 (6), 694-703.
(29) Leontievsky, A. A.; Myasoedova, N. M.; Baskunov, B. P.;
Golovleva, L. A.; Bucke, C.; Evans, C. S. Transformation of
2,4,6-trichlorophenol by free and immobilized fungal laccase.
Appl. Microbiol. Biotechnol. 2001, 57, 85-91.
(30) Xu, F. Effects of redox potential and hydroxide inhibition on
the pH activity profile of fungal laccases. J. Biol. Chem. 1997,
272 (2), 924-928.
(31) Leonowicz A.; Cho, N.-S.; Luterek, J.; Wilkolazka, A.; Wojtas-
Wasilewska, M.; Matuszewska, A.; Hofrichter, M.; Wesenberg,
D.; Rogalski, J. Fungal laccase: properties and activity on lignin.
J. Basic Microbiol. 2001, 41, 185-227.
(32) Sjoblad, R. D.; Minard, R. D.; Bollag, J. M. Polymerisation
of 1-naphthol and related phenolic compounds by an extracellular
fungal enzyme. Pestic. Biochem. Physiol. 1976, 6, 457-463.
(33) Jonas, U.; Hammer, E.; Schauer, F.; Bollag, J. M. Transformation
of 2-hydroxydibenzofuran by laccases of the white rot fungi
Trametes Versicolor and Pycnoporus cinnabarinus and charac-
terization of oligomerization products. Biodegradation 1998, 8,
321-328.
(34) Ciecholewski, S.; Hammer, E.; Manda, K.; Bose, G.; Van
Nguyen, T. H.; Langer, P.; Schauer, F. Laccase-catalyzed
carbon-carbon bond formation: oxidative dimerization of
salicylic esters by air in aqueous solution. Tetrahedron 2005,
61, 4615-4619.
(35) Pretsch, E.; Bu¨hlmann, P.; Affolter, C. In Structure Determina-
tion of Organic Compounds, 3rd ed.; Spinger-Verlag: Berlin,
Heidelberg, Germany, 2000.
Received for review February 6, 2006. Revised manuscript received
May 11, 2006. Accepted May 11, 2006.
JF060351I