Characterization of metabolites of fungicidal cymoxanil in a sensitive strain of Botrytis cinerea

Article abstract of DOI:10.1021/jf8010917

The metabolism of cymoxanil [1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea] by a very sensitive strain of Botrytis cinerea toward this fungicide was studied by using [2-¹⁴C]-cymoxanil. Labeled cymoxanil was added either in a culture of this strain or in its enzymatic extract. The main metabolites, detected in biological samples, were isolated and identified by mass and NMR spectrometry. Their identification allowed us to show that this strain quickly metabolized cymoxanil according to at least three enzymatic pathways: (i) cyclization leading, after hydrolysis, to ethyl parabanic acid, (ii) reduction giving demethoxylated cymoxanil, and (iii) hydrolysis and reduction followed by acetylation leading to N-acetylcyanoglycine. In a cell-free extract of the same strain, only the first and the second enzymatic reactions, quoted above, occurred. Biological tests showed that, among all the metabolites, only N-acetylcyanoglycine is fungitoxic toward this sensitive strain.

Full text of DOI:10.1021/jf8010917

8050 J. Agric. Food Chem. 2008, 56, 8050–8057
Characterization of Metabolites of Fungicidal
Cymoxanil in a Sensitive Strain of Botrytis cinerea
FREDERIQUE TELLIER,*,† RENE FRITZ,† LUCIEN KERHOAS,‡ PAUL-HENRI DUCROT,§
†
JACQUES EINHORN,‡ ABEL CARLIN-SINCLAIR,| AND PIERRE LEROUX
UMR 1290, UR 501, and UMR 206, INRA, route de Saint-Cyr, F-78026 Versailles, France, and
De´partement de Chimie, Universite´ de Versailles-Saint-Quentin en Yvelines, 45 avenue des
Etats-Unis, F-78001 Versailles, France
The metabolism of cymoxanil [1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea] by a very sensitive
strain of Botrytis cinerea toward this fungicide was studied by using [2-14C]-cymoxanil. Labeled
cymoxanil was added either in a culture of this strain or in its enzymatic extract. The main metabolites,
detected in biological samples, were isolated and identified by mass and NMR spectrometry. Their
identification allowed us to show that this strain quickly metabolized cymoxanil according to at least
three enzymatic pathways: (i) cyclization leading, after hydrolysis, to ethyl parabanic acid, (ii) reduction
giving demethoxylated cymoxanil, and (iii) hydrolysis and reduction followed by acetylation leading
to N-acetylcyanoglycine. In a cell-free extract of the same strain, only the first and the second
enzymatic reactions, quoted above, occurred. Biological tests showed that, among all the metabolites,
only N-acetylcyanoglycine is fungitoxic toward this sensitive strain.
KEYWORDS: Metabolism; fungicide; cymoxanil; mass spectrometry; Botrytis cinerea
INTRODUCTION
degradation pathways. Two pathways involve an initial cycliza-
tion to form either a six-membered ring compound, 1-ethyl-
2,4-dioxo-5-methoxyimino-6-imino(1H,3H)pyrimidine 1, or a
five-membered ring compound, 1-ethyl-5-methoxyamino-5-
cyano-2,4-dioxoimidazolidine 2, which is thereafter rapidly de-
graded into 1-ethyl-5-methoxyimino-2,4-dioxoimidazolidine
(IIa) and then 1-ethyl-2,4,5-trioxoimidazolidine (IIb). The third
degradation pathway involves a direct cleavage of the C-1 amide
bond to give 2-cyano-2-methoxyimino acetic acid 3 (Figures
1 and 2). All three pathways eventually lead to the formation
of oxalic acid. In soil, cymoxanil is degraded by both hydrolytic
and microbial actions with half-life time < 2 days 8. The
degradation in soil also follows a series of C5- and C6-
cyclization and/or hydrolysis reactions similar to those observed
in solution. Thus, during the initial phase of degradation, the
metabolites 1, 3, and 1-ethyl-5-methoxyamino-2,4-dioxo-5-
imidazolidinecarboxamide are detected as major soil degradation
products and 2-cyano-2-hydroxyiminoacetic acid 4 and IIa as
minor products. Cymoxanil metabolism in plants (grapes, tomato
fruit, and potatoes) 10–12 and in animals (rats) 13 was also
reported. In these living organisms, it is rapidly metabolized,
mainly to glycine which is reincorporated into natural compo-
nents such as sugars. In the case of rats, most of the metabolites
formed are eliminated via urinary excretion. In addition to
glycine, metabolites synthesized by the pathways previously
quoted such as the hydrolysis products, 3 and 4, and the
cyclization products, 1 and IIb, are also identified in urine.
In this article, we present our results concerning the biotrans-
formation of cymoxanil by a highly sensitive strain of B. cinerea.
Previously 6, we were able to follow the disappearance of
Cymoxanil, 1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea,
Ia is a fungicidal cyanooxime effective against plant diseases
caused by fungi belonging to the Perenosporales. In practice, it
is mainly used against downy mildew of vine (Plasmopara
Viticola) and potato blight (Phytophthora infestans) 1. Although
this compound is not effective against the gray mold, in
laboratory tests, a few strains of Botrytis cinerea Pers. Ex Fr.
appeared sensitive 2–4. In previous works 5, 6, we showed, by
monitoring the disappearance of cymoxanil in culture media of
different phenotypes of B. cinerea, that the sensitivity of strains
toward cymoxanil was correlated to its capacity to metabolize
it. In a culture of a highly sensitive strain, it was completely
and quickly metabolized (half-life time e 2 h). This correlation
suggests that cymoxanil is a profungicide. That means that it
must initially undergo a biotransformation by the fungus to be
activated into one (or several) fungitoxic metabolite(s).
The cymoxanil metabolism has been study in several condi-
tions 7, 8. Cymoxanil is rapidly degraded in neutral to alkaline
solutions, and its rate of degradation is strongly dependent on
both pH and temperature 8, 9. Morrica et al. 9 reported that the
degradation in solution takes place according to three competing
* Author to whom correspondence should be addressed. Telephone:
+33 (0)1 30 83 30 48. Fax: +33 (0)1 30 83 31 19. E-mail: ftellier@
versailles.inra.fr.
† UMR 1290, INRA.
‡ UR 501, INRA.
§ UMR 206, INRA.
| Universite´ de Versailles-Saint-Quentin en Yvelines.
10.1021/jf8010917 CCC: $40.75  2008 American Chemical Society
Published on Web 08/12/2008

Metabolism of Cymoxanil in a Strain of B. cinerea
J. Agric. Food Chem., Vol. 56, No. 17, 2008 8051
Figure 1. Structures of cymoxanil metabolites according to refs 8, 9,
13.
cymoxanil but not the appearance and evolution of the main
metabolites, in particular in the mycelium. For this reason, we
used [2-14C]-cymoxanil, which was added in a culture medium
containing the mycelium of a highly sensitive strain and in an
enzymatic extract obtained from its mycelium. The labeled
metabolites were directly monitored by thin-layer chromatog-
raphy (TLC). After these chromatographic analyses, the main
metabolites were isolated from biological samples and identified
by mass and NMR spectrometry analyses.
Figure 2. Cymoxanil metabolism in a culture of the strain “L” of B. cinerea
(* indicates 14C localization in radiolabeled cymoxanil).
acetone-EtOH (50:50, v/v). After 48 h at 4 °C, it was filtered through
a Whatman GFA filter and washed with 5 mL of acetone-EtOH (50:
50, v/v). The filtrate was evaporated in vacuo. Then, 200 µL of
acetone-EtOH (50:50, v/v) was added and 50 µL was deposited on a
plate.
MATERIALS AND METHODS
Chemicals. Cymoxanil Ia was prepared according to a method
described in the literature 13 (Figure 2). Cymoxanil with 15N into the
oxime function and deuterium into the methoxy group were respectively
obtained with sodium 15N-nitrite- and d6-dimethyl sulfate. The product
IIb 14, 15 was obtained by slowly adding oxalylchloride (0.87 mL, 10
mmol) in a solution of ethyl urea (0.88 g, 10 mmol) in 15 mL of Et2O.
The mixture was refluxed during 2 h. After evaporation, 20 mL of
H2O was added. The reaction mixture was extracted with AcOEt. The
solvent was evaporated, and the product IIb was obtained as a solid
(yield ) 80%). N-Acetylcyanoglycine IId was prepared as follows 16,
17: ethyl acetamidocyanoacetate (1.7 g, 10 mmol) was added to a
solution of KOH (0.56 g, 10 mmol) in 5 mL of H2O and 10 mL of
EtOH and stirred at room temperature for 3 days. Most of the solvent
was then removed in vacuo, and 3 mL of H2O was added to the residue.
This aqueous solution was extracted with CH2Cl2 to remove the
unreacted ester. The aqueous layer was adjusted at pH 0.5 with
concentrated HCl to precipitate IId. This one was filtered and washed
[2-14C]-Cymoxanil Metabolism in a Crude Enzyme Extract.
Mycelial pellets of B. cinerea strain “L” were prepared by seeding 150
mL of yeast extract liquid medium (about 106 conidia/mL) in a 250-
mL Erlenmeyer flask 18 and incubating at 23 °C on a rotary shaker at
150 rpm for 24 h. The mycelium was harvested by filtration through a
bolting cloth of 100 µm and resuspended in 1200 mL of yeast extract
liquid medium in a 2000-mL Erlenmeyer flask and incubated again
for 24 h as described above. The mycelium was harvested by filtration
through a bolting cloth of 100 µm, washed with H2O, and lyophilized
for 24 h. This one was kept cold until use. Cell-free extracts were
prepared by grinding the mycelium (500 mg) in a mortar with liquid
nitrogen. To this ground mycelium was added 8 mL of cold phosphate
buffer (50 mM, pH ) 6.65), containing ethylenediaminetetraacetic acid
(1 mM) and dithiothreitol (1 mM). This mixture was crushed with
Potter-Elvehjem for 2 min, and 8 mL of phosphate buffer was added.
After centrifugation at 9000 rpm at 4 °C for 30 min, the supernatant
obtained was collected under vacuo through a 0.22-µm membrane
(PVDF) in an Erlenmeyer flask and kept in ice, giving a filtrate to be
used in all enzyme assays. In a tube of 2 mL were added 500 µL of
crude enzyme extract and then 20 µL of 2 × 10-2 M ethanolic solution
of cymoxanil. The mixture was incubated at 38 °C, and after 0, 10, 20,
and 30 min, the enzymatic reactions were stopped by addition of 500
µL of acetone. The samples were centrifuged at 5000 rpm during 10
min. The supernatant (1 mL) was filtered through 0.2-µm membranes
(regenerated cellulose) before deposit of an aliquot of 50 µL.
TLC Procedure. The disappearance of [2-14C]-cymoxanil, correlated
with the appearance of labeled metabolites in the cultures (medium and
mycelium) and in the cell-free extracts of B. cinerea, was followed
according to time, by TLC. This technique has the advantage to avoid the
samples preliminary purification and the problematic extraction of the polar
metabolites. Chromatographies were performed both on silica gel (60 F254
plates, 0.2-mm thick, 10 × 10 cm, Merck KGaA Germany, cat no. 1.05628)
and reverse-phase (RP 18WF254s plates, 0.2-mm thick, 10 × 10 cm, Merck
KGaA Germany, cat no. 1.13124). The presence of the most polar products
was investigated by ion-pairing TLC. This technique consists in pretreating
the reverse-phase plates with quaternary ammonium salts before the deposit
of samples to separate the products from the medium during elution and
also to slow down their migration. Before samples were laid down, the
reverse-phase plates were impregnated by vertical dipping for 5 min in a
methanolic solution of tetrabutylammonium bromide at the concentration
of 70 mM and then dried at 80 °C for 5 min. Fifty microliters of each
sample was deposited onto HPTLC plates which were eluted in a horizontal
development chamber. The silica gel plates were eluted with a solution of
hexane-AcOEt-AcOH (70:30:1, v/v/v) up to 70 mm and the reverse-
phase plates with a solution of phosphate buffer (0.01 M, pH )
6.0)-MeOH (55:45, v/v) up to 50 mm. The plates were scanned for
radioactivity using a Berthold (LB 2832) automatic TLC-linear analyzer
and then were subjected to autoradiography to locate the radioactive
bands.
1
with ice water and then Et2O to give 0.6 g of IId (yield ) 42%). H
NMR 300 MHz (DMSO-d6) δ (ppm) 1.95 (s, 3, CH3), 5.6 (d, 1, J )
6 Hz, CHNH), 9.1 (d, 1, J ) 6 Hz, CHNH), 13.8 (s, 1, OH); 13C NMR
75.5 MHz (DMSO-d6) δ (ppm) 22.82 (CH3), 44.96 (CH), 117.15
(Ct N), 166.47 (CdO), 170.74 (CdO).
[2-14C]-Cymoxanil provided by Du Pont de Nemours had a
radiochemical purity of about 95% and a specific activity of 2.79 MBq/
mg. Radiolabeled cymoxanil ethanolic solution used in our studies was
prepared by dissolving unlabeled cymoxanil (1.75 mg, 8.75 µmol) and
[2-14C]-cymoxanil (0.25 mg, 1.25 µmol, 0.67 MBq/mg) in 500 µL of
EtOH.
[2-14C]-Cymoxanil Metabolism in a B. cinerea Culture. In our
study, we used the strain “L” of B. cinerea collected near a Bordeaux
vineyard that is very sensitive to cymoxanil 5. It was maintained on a
malt agar medium containing yeast extract. After 15 days under light,
mycelial pellets of B. cinerea were prepared by seeding about 106
conidia/mL in a yeast extract liquid medium 18 and incubating at 23
°C on a rotary shaker at 150 rpm for 24 h. Then, 3 mL of this culture
was taken to inoculate 150 mL of yeast extract medium in 250-mL
Erlenmeyer flasks and incubated again for 24 h as described above to
obtain the mycelium used in the cultures. The mycelium was harvested
by filtration through a bolting cloth of 100 µm, washed with 50 mL of
H2O, and resuspended in 150 mL of H2O. The [2-14C]-cymoxanil
metabolism studies were carried out on 5 mL of these suspensions
dropped in 25-mL Erlenmeyer flasks. After 25 µL of 2 × 10-2
M
ethanolic solution of cymoxanil was added, the treated cultures were
incubated as described above. After 0, 15, 30, 60, 120, 240, and 360
min, each mycelium was separated by filtration from its culture medium.
The mycelium was washed with 2.5 mL of H2O. Both the culture
medium and the mycelium were analyzed by TLC deposit. The filtrate
was lyophilized and dissolved in 1.25 mL of a mixture of H2O-EtOH
(75:25, v/v), and then an aliquot of 50 µL of this solution was deposited
on a plate. The mycelium was extracted with 5 mL of a mixture

8052 J. Agric. Food Chem., Vol. 56, No. 17, 2008
Tellier et al.
Figure 3. TLC monitoring of [2-14C]-cymoxanil metabolism in the culture
medium: Autoradiography of a Si chromatoplate. Migration with hexane-
AcOEt-AcOH (70:30:1, v/v/v) up to 70 mm.
Figure 4. TLC monitoring of [2-14C]-cymoxanil metabolism in the mycelium:
Autoradiography of a Si chromatoplate. Migration with hexane-AcOEt-
AcOH (70:30:1, v/v/v) up to 70 mm.
LC-MS and NMR Analyses. LC-MS and LC-MS/MS studies
were carried out with a Waters (Alliance 2695) HPLC system coupled
to a Quattro LC instrument (Micromass) equipped with an electrospray
ionization (ESI) or an atmospheric pressure chemical ionization (APCI).
Data acquisition and processing were performed by the MassLynx NT
4.0 system. The ESI and APCI source potentials were as follows:
capillary voltage, 3.0 kV (positive mode) or 2.75 kV (negative mode);
corona voltage 2.5 kV (in APCI mode); extractor voltage, 2 V. The
sampling cone voltage was varied usually from 8 to 20 V for mass
spectra, and the specific value of the cone voltage for each collision-
induced dissociation (CID) experiment was set to optimize the parent
ion. The source block and desolvation gas were respectively heated at
120 and 400 °C. Argon as collision gas was set at 3.5 × 10-3 mbar
for the CID experiments. Separation was achieved on a reverse-phase
column (Uptisphere C18 5 µm, 150 × 2 mm i.d., Interchrom) equipped
with a precolumn (Interchrom). Isocratic elutions were performed using
a mixture H2O-CH3CN with 0.5% acetic acid [for IIb: (75:25, v/v);
for IIc: (50:50, v/v); for IId: (95:5, v/v)] (the flow rate was 0.2 mL/
min and column temperature was constant at 24 °C). For IIc, UV
detection was set at 255 nm. 1H NMR and 13C NMR spectra were
recorded on Varian VXR 300 (1H: 300 MHz and 13C: 75.5 MHz) and
Bruker Avence 600 (1H: 600 MHz and 13C: 150 MHz) spec-
trometers.
1
136 (15 eV), m/z 121, 109, 42, 26. H NMR 300 MHz (DMSO-d6) δ
(ppm) 1.16 (t, 3, J ) 7.2 Hz, CH3CH2), 1.74 (s, 6, 2 CH3), 3.32 (qd,
2, J ) 7.2 Hz, J ) 5.7 Hz, CH3CH2NH), 8.0 (t, 1, J ) 5.7 Hz, CH2NH);
13C NMR 75.5 MHz (DMSO-d6) δ (ppm) 15.16 (CH3), 24.01 (CH3),
34.67 (CH2), 90.36 (C quat.), 111.80 (Ct N), 144.92 (CdN), 149.77
(CdO), 159.28 (CdO).
Metabolite IId. Metabolite IId was studied from the mycelium
extract obtained after 6 h. IId undergoes three successive steps of
purification. First, this extract was purified by solid-phase extraction
on a C18 cartridge (Varian). The extract was dissolved in H2O and
deposited on a column and eluted with H2O. The presence of IId was
detected on the TLC plate by staining with iodine vapors. Then, the
solution of IId was concentrated by freeze-drying before purification
by preparative TLC. An aqueous solution was deposited onto the silica
gel plate, which was eluted with a mixture of CH2Cl2-MeOH (60:40,
v/v) up to 80 mm. Then, silica was scraped from 30 to 45 mm and
extracted with MeOH. Methanolic solution was evaporated and then
H2O was added. The aqueous solution was acidified with 0.1 N HCl to
pH 2 and extracted with AcOEt. IId was in the organic layer which
was analyzed by mass spectrometry. The spectral data of the metabolite
IId obtained from the mycelium extract were identical to those of the
synthetic product. HRMS (ESI-): m/z ) 141.0298 [M - H]- (calcd
m/z ) 141.0300 for C5H6N2O3); LC-MS (ESI-): peak at 3.8 min, m/z
283, 141 [M - H]-, 97; MS/MS CID 141 (10 eV), m/z 97, 70, 42, 41,
26; MS/MS CID 97 (15 eV), m/z 70, 42, 41, 26. MS/MS CID 70 (10
eV), m/z 42, 41; MS (ESI+): m/z 181, 165, 143 [M + H]+; MS/MS
CID 143 (10 eV), m/z 125, 115, 73, 69, 43. IId obtained from [15N]-
cymoxanil: MS (ESI-): m/z 142 [M - H]-; MS/MS CID 142 (10 eV),
m/z 98, 71, 26; MS/MS CID 98 (15 eV), m/z 71, 43, 41, 26; MS/MS
CID 71(10 eV), m/z 43, 41; MS (ESI+): m/z 144 [M + H]+; MS/MS
CID 144 (10 eV), m/z 126, 116, 74, 70, 43. Derivatization of IId with
diazomethane: MS (ESI-): m/z 155 [M - H]-; MS/MS CID 155
(15 eV), m/z 123, 95, 79, 64, 53, 26; MS/MS CID 123 (10 eV), m/z
79, 64; MS/MS CID 79 (10 eV), m/z 64, 52, 26; MS/MS CID 64
(15-25 eV), m/z 26; MS (ESI+): m/z 195, 179, 157 [M + H]+.
Derivatization of IId obtained from [15N]-cymoxanil with diaz-
omethane: MS (ESI-): m/z 156 [M - H]-; MS/MS CID 156 (20
eV), m/z 124, 96, 80, 65, 54, 26.
Metabolite IIb. Metabolite IIb was studied from the culture medium
obtained after 6 h. To this end, the culture medium was concentrated
by freeze-drying and then dissolved in H2O. The aqueous solution was
extracted with AcOEt. IIb, present in the organic layer, was analyzed
by mass spectrometry. The spectral data of the metabolite IIb obtained
from the culture medium extract were identical to those of the synthetic
product.
LC-MS (ESI-): peak at 5.4 min, m/z 141 [M - H]-; MS/MS CID
141 (15 eV), m/z 113, 42.
Metabolite IIc. Metabolite IIc was isolated from the cell-free extract
obtained after 20 min. It was purified by solid-phase extraction on a
C18 cartridge (Varian); the lyophilized extract was dissolved in
H2O-CH3CN (95:5, v/v) and deposited on a column that was first
washed with H2O and then eluted with the mixture H2O-CH3CN (25:
75, v/v). It was detected in the fractions on a TLC plate by viewing
under UV light. After being freeze-dried, IIc was obtained pure as a
white solid. This solid being unstable, it was dissolved in CH3CN until
it was studied. LC-MS (APCI-): peak at 4.3 min, m/z 208 M•-, 193,
136, and 122; MS/MS CID 208 (5 eV), m/z 193, 122; MS/MS CID
Bioassays. In vitro effects toward mycelial growth and germ-tube
elongation of B. cinerea were studied. To study the mycelial growth, a
conidia suspension (106 conidia mL-1) was mixed with a molten agar

Metabolism of Cymoxanil in a Strain of B. cinerea
J. Agric. Food Chem., Vol. 56, No. 17, 2008 8053
Figure 5. TLC monitoring of [2-14C]-cymoxanil metabolism in the mycelium: Autoradiography of a C18 chromatoplate impregnated with 70 mM TBAB.
Migration with phosphate buffer-MeOH (55:45, v/v) up to 50 mm.
Figure 6. (APCI-)-MS spectrum of IIc. APCI--CID spectrum of ions m/z 208 and m/z 136.
medium (20 g of malt, 5 g of yeast extract, and 10 g of agar per liter).
Five-millimeter diameter paper filters, on which were deposited the product
(0.02 µmol) to be tested, were put on this medium. After being incubated
for 48 h at 19 °C in the dark, we checked if the mycelial growth was
inhibited or not. To test germ-tube elongation, a conidia suspension (106
conidia mL-1) was spread on the surface of an agar medium containing
10 g of glucose, 2 g of K2HPO4, 2 g of KH2PO4, and 10 g of agar per
liter. The medium was amended with the product to be tested dissolved in
ethanol (with a range of concentrations from 0.025 to 25 mg L-1). After
being incubated for 48 h at 19 °C in the dark, the lengths of germ tubes
were estimated under a microscope, using a micrometer. For each
compound, the effective concentration reducing germ-tube elongation by
half (EC 50) was determined in milligrams per liter.
RESULTS AND DISCUSSION
Labeled Cymoxanil Metabolism in a Culture. Cymoxanil
metabolism was studied by monitoring [2-14C]-cymoxanil, added

8054 J. Agric. Food Chem., Vol. 56, No. 17, 2008
Tellier et al.
enzymatic extract. The labeled cymoxanil was added to cell-
free extracts prepared from mycelium, and after 0, 10, 20, and
30 min, the enzymatic reactions were stopped by addition of
acetone. The different extracts obtained were analyzed on a silica
gel plate. We observed that cymoxanil quickly disappeared (its
half-life time is less than 10 min). After 20 min, it was
completely metabolized into three main metabolites (IIa, IIb)
and IIc, which represented, respectively, 25 and 55% of the
starting radioactivity. The extracts were also deposited on a
reverse-phase plate, but the metabolite IId was never detected.
This fact means that the enzymatic systems leading to IId
missed or were inefficient in the crude enzyme extracts.
Identification of Metabolites IIa and IIb. In the monitorings
of the labeled cymoxanil, the products IIa and IIb were detected
both in the culture medium and in the cell-free extract. The
metabolite IIb was analyzed by mass spectrometry after
extraction from the culture medium and identified as ethyl
parabanic acid (Figure 2). The structure of IIb was confirmed
by comparing MS data of the product present in the beforehand
extracted biological samples, with those of the synthesized
product. LC-MS/MS spectra of ions m/z 141 ([M - H]-) from
the two compounds, performed at 5, 10, 15, and 20 eV, were
identical, exhibiting mainly two ion products in various propor-
tions, m/z 113 ([M - H]- - 28) and m/z 42, corresponding to
the loss of CO and to the formation of ion [NdCdO]-,
respectively. The metabolite IIa could not be detected in the
mass spectrometry analyses. IIa is supposed to be, according
to the literature 9, 11, 13, a cyclization product of cymoxanil,
and IIb would result from the hydrolysis of the oxime function
(Figure 2). Morrica et al. reported that cymoxanil under certain
conditions of light and pH was unstable and led to the mixture
IIa-IIb 9. In our case, IIa was not due to a chemical
degradation of cymoxanil but to the presence of an enzymatic
system. Control experiments indeed showed that Ia was stable
both in our experimental conditions (pH, temperature) and
incubated in an inactivated crude extract.
13
15
Figure 7. 1H- C HMBC and 1H- N HMBC correlations for compound
IIc.
in a culture of B. cinerea strain, highly sensitive to this cy-
anooxime. The mycelium was removed from the culture medium
immediately, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h after the
adjunction of labeled cymoxanil. The main labeled metabolites
and cymoxanil were quantified at different times both in the
culture medium and in the mycelium after extraction. With time,
the radioactivity quickly decreased in the medium and increased
in the mycelium. After 15 min, 6% of the radioactivity was
found in the mycelium and 40% after 6 h.
The culture media were analyzed by using a silica gel plate
that was eluted with hexane-AcOEt-AcOH (70:30:1, v/v/v)
(Figure 3). This plate showed that cymoxanil (Rf ) 0.35) rapidly
disappeared in less than 2 h (only 3% of Ia still remained after
1 h), whereas two main labeled metabolites, less polar than Ia,
IIa (Rf ) 0.43), and IIb (Rf ) 0.46) appeared. Products IIa
and IIb were hardly separable and quantified together. The
quantity of (IIa, IIb) increased with time reaching 40% of the
starting radioactivity after 6 h. The mycelium extracts were
initially analyzed with a silica gel plate eluted as previously
(Figure 4). We detected on this plate a product IIc, less polar
(Rf ) 0.88) than Ia. The compound IIc increased during 30
min (reaching 6% of the starting radioactivity) then decreased
and disappeared after 4 h. No trace of remaining cymoxanil
was detected in the mycelium extracts. In these TLC conditions,
the major part of the radioactivity was not eluted. To detect in
the mycelium extracts the more polar products, a reverse-phase
plate impregnated with ion-pairing reagent was necessary
(Figure 5). On this pretreated plate eluted with phosphate
buffer-MeOH (55:45, v/v), we noted, in addition to IIc, the
presence of two other main labeled metabolites IId and IIe.
The product IId increased with time and represented 90% of
the radioactivity present in the mycelium and 32% of the starting
radioactivity after 6 h. The compound IIe increased for 1 h to
reach 6% of the starting radioactivity then decreased and
represented 2% after 6 h. IIe could not be studied because of
its instability. It is interesting to note that the silica gel plate
was necessary to check the complete disappearance of Ia
because, on the pretreated reverse-phase plate, Ia and Ic were
observed at the same Rf value. Metabolites IId and IIe could
also be detected on a silica gel plate eluted with a polar mix-
ture CH2Cl2-MeOH-AcOH (80:20:1, v/v/v). Their Rf values
were, respectively, 0.15 and 0.60. The ion-pairing reverse-phase
plate, however, advantageously enabled the monitoring of the
three main metabolites at the same time.
Identification of Metabolite IIc. The product IIc was
detected both in the mycelium and in the cell-free extract. It
was purified from the cell-free extract by reverse-phase solid-
phase extraction. IIc was obtained as a pure but unstable white
solid. Its structure was assigned by combining MS and NMR
studies (Figures 6 and 7).
The (APCI-)-MS spectrum of IIc showed an ion at m/z 208
that was interpreted as a M•- radical anion containing the same
even number of nitrogen atoms as that of Ia. Fragment ions at
m/z 193, m/z 136, and less so at m/z 122 were also present
(Figure 6). The CID spectrum of ion m/z 208 showed two main
product ions, indicating successive losses of a CH3 radical and
a 71 u molecule, likely CH3CH2NdCdO, leading to m/z 122.
The CID spectrum of the in-source produced ion m/z 136 gave
ion products at m/z 121, 109, 42, and 26 corresponding,
respectively, to losses of [CH3]• or HCN and formation of ions
[NdCdO]- and [Nt C]-. Ion m/z 136, not found in the CID
spectrum of ion m/z 208, might rather originate from an
undetected [M - H]- ion at m/z 207 by elimination of
CH3CH2NdCdO. Its elemental composition was determined
as C6H6N3O by ESI-HRMS. To progress in the structure
determination of IIc, we compared these MS data with those
of the derivative obtained after PtO2/CH3OH catalytic hydro-
genation. The (ESI-)-MS spectrum of hydrogenated IIc
exhibited a [M - H]- ion at m/z 209, indicating the addition of
a single molecule of hydrogen. The CID spectrum of ion m/z
209 gave ion products at m/z 182, 138, 111, and 95, resulting,
respectively, from the loss of either HCN or CH3CH2NdCdO,
In conclusion, after 6 h, the main labeled metabolites of
cymoxanil in a culture were (IIa, IIb) in the medium and IId
in the mycelium. The study of cymoxanil metabolism by
resistant strains allowed us to show that cymoxanil needs to
penetrate the mycelium to be metabolized. In these strains, the
rate of metabolism is low and 4% cymoxanil was detected in
the mycelium. In the highly sensitive strain used in this study,
it was not detected because, once penetrated, it was meta-
bolized.
Labeled Cymoxanil Metabolism in a Crude Enzyme Ex-
tract. After having followed the metabolism of cymoxanil in a
culture, we were interested in its biotransformation in an

Metabolism of Cymoxanil in a Strain of B. cinerea
J. Agric. Food Chem., Vol. 56, No. 17, 2008 8055
Figure 8. (ESI-)-MS spectrum of IId. (ESI-)-CID spectrum of ions m/z 141 and m/z 97.
Figure 9. (ESI-)-CID spectrum of ion m/z 155 obtained from the ester derivative of IId.
or successively the latter followed by HCN or HN)CdO
elimination. These ions and their complementary anions
[NdCdO]- and [Nt C]- also observed claimed for the
conservation of most functional groups (nitrile, external or
internal amide). This decomposition pathway was confirmed by
the CID spectrum of the in-source produced ion m/z 138 that
exhibited the same ions m/z 111, 95, 42, and 26 as the parent
deprotonated molecule. The precursor of ion m/z 138 in IIc (i.e.,
m/z 136) should thus contain the original unsaturation and be
cyclic (cf. unsaturation degree).
three resonances were observed for the nitrogen atoms, one of
which (99.4 ppm) was attributed to the ethyl amide moiety and
the two remaining resonances (-14.2 and 174.9 ppm) were
correlated to the proton singlet at 1.74 ppm. This proton res-
1
onance was thereafter easily attributed in the H-13C HMBC
experiment to a gem-dimethyl moiety also correlated to two
carbon resonances at 90.36 and 144.92 ppm.
According to the MS and NMR data, the metabolite IIc was
identified as 1-(2,2-dimethyl-5-oxo-4-cyano-3-imidazolin-1-yl)-
N-ethyl amide (Figure 2). IIc probably resulted from the
reaction of an undetected cymoxanil metabolite with acetone,
which was used either to extract the mycelium or to stop the
enzymatic reactions in the cell-free extract. The precursor
of IIc is likely the demethoxylated imine, which might be
obtained by regiospecific reduction of cymoxanil. The cell-
free extract was analyzed before addition of acetone by MS
spectrometry but the precursor of IIc could not be detected.
However, it was present and stable in the extract because if
acetone was added after analysis, the product IIc was again
obtained.
1H and 13C NMR spectra were obtained for compound IIc.
1D spectra indicated the presence of nine carbon and 12 proton
1
resonances. The H spectrum first confirmed the presence of
the ethyl amide moiety, whereas the six remaining protons of
the molecule appeared as a singlet at 1.74 ppm. Since no
resonance was observed at a chemical shift compatible with
the presence of a methoxy group, this indicated that the
o-methyloxime moiety had been transformed in the reaction.
1H-13C HSQC and HMBC as well as 15N-1H HMBC
experiments (Figure 7) were thereafter performed to complete
the structure elucidation. In the 15N-1H HMBC experiment,
Identification of Metabolite IId. The product IId was only

8056 J. Agric. Food Chem., Vol. 56, No. 17, 2008
Tellier et al.
found in the mycelium. Being polar and hardly separable from
other constituents, it could not be obtained sufficiently pure for
NMR analyses. The structure of IId was thus investigated only
by MS approaches (Figure 8). This study was made easier by
the introduction of stable isotopes into the starting cymoxanil
like 15N into the oxime function or deuterium into the methoxy
group. Comparison of spectra of IId obtained from labeled and
nonlabeled cymoxanil showed in IId the presence of 15N but
not that of the perdeutered methyl moiety. Moreover, we showed
by dissolving IId in D2O that three atoms of hydrogen in IId
were exchangeable. The ESI--HRMS spectrum of IId exhib-
ited a [M - H]- ion signal at m/z 141.0298 corresponding to
the C5H5N2O3 formula. The low-resolution ESI spectrum of IId
showed that [M - H]- was accompanied by the [2M - H]-
ion. In the positive mode, the [M + H]+ ion at m/z 143 was
observed together with the cationized adducts [M + Na]+ and
[M + K]+, confirming the molecular mass 142. The CID
spectrum of [M - H]- ion m/z 141 (Figure 8) exhibited ion
products at m/z 97, 70, and 26, indicating successive losses of
CO2 (44 u) and HCN (27 u) and the production of the anion
[Nt C]-. The initial loss of CO2 from the deprotonated molecule
suggested the occurrence of a CO2H group in IId. The CID
spectrum of the in-source generated ion m/z 97 confirmed that
this ion was thereafter responsible for the production of ions
m/z 70 and 26 during its decomposition. Interestingly also, ion
m/z 97 was found to produce two other ions at m/z 41 and 42,
likely issuing directly from m/z 70. Comparing to spectra of
the metabolite obtained from the 15N-labeled cymoxanil, it
appeared that, in the low mass region, only ion m/z 42 (i.e.,
shifted to m/z 43) contained the labeled nitrogen atom. It was
thus interpreted as a [NdCdO]- anion, whereas ion m/z 41
might correspond to a C2HO structure. To complete identifica-
tion, IId was subjected to derivatization by diazomethane. The
ESI--MS spectrum of the obtained derivative (Figure 9)
exhibited a [M - H]- ion at m/z 155, confirming the presence
of an acid function in IId. At low energy, this ion m/z 155 could
undergo successive losses of CH3OH and CO2 to lead to the
ion product m/z 79. The latter was dissociated into ion products
m/z 64 and 26, corresponding, respectively, to the loss of [CH3]•
and the formation of the anion [Nt C]-. The use of [15N]-
cymoxanil showed that the ion at m/z 64 contained also the
second nitrogen atom. A second decomposition pathway was
observed that showed an alternative transformation of ion m/z
155 into ion m/z 53 by elimination of 60 and 42 u neutrals that
could be interpreted as H3CO-CHO and CH2CO, respectively.
From the elemental composition of the parent ion m/z 155
(deduced from the exact mass of IId), ions m/z 79 and m/z 53
were, respectively, determined as C4H3N2 and C2HN2. The
presence of the two nitrogen atoms in these ions indicated a
cyanoamino acid substructure for IId. On the basis of these
results, the metabolite IId was identified as N-acetylcyanogly-
cine. The structure of IId was confirmed by comparing the mass
spectra data of the metabolite isolated from the mycelium extract
with those of the synthesized product. LC-MS/MS spectra of
ions m/z 141 ([M - H]-) and m/z 143 ([M + H]+) from both
compounds recorded at a variety of collision energies were
found to be identical. In conclusion, it can be postulated that
cymoxanil was initially hydrolyzed, reduced, and finally acety-
lated (Figure 2). The N-acetyl group should originate from the
cellular metabolism.
according to at least three enzymatic pathways: a cyclization
leading after hydrolysis to ethyl parabanic acid, a reduction
giving the demethoxylated cymoxanil, and a hydrolysis and a
reduction, followed by an acetylation leading to N-acetylcyan-
oglycine. In a cell-free extract of the same strain, only the two
first enzymatic reactions, quoted above, occurred. Among the
identified metabolites, IIb is ubiquitous in the different cy-
moxanil metabolism quoted previously 8, 9, 13, whereas IIc
and IId are obtained by specific pathways to cymoxanil meta-
bolism by a highly sensitive strain of B. cinerea.
Fungicidal Activity. The fungicidal activity of the main
metabolites obtained, IIb, IIc, and IId, and also one of the cell-
free extract before addition of acetone containing IIa, IIb, and
the precursor of IIc, was tested in vitro toward B. cinerea strains.
Only the metabolite IId exhibited a fungitoxic activity toward
the highly sensitive strain. The inhibition of the germ-tube
elongation with this metabolite was more effective (EC 50 )
0.025 mg L-1) than one obtained with cymoxanil (EC 50 )
0.25 mg L-1). On the other hand, the comparison of cymoxanil
metabolism by a highly sensitive strain to those obtained by
less sensitive ones showed that IId was obtained in 32% yield
in a highly sensitive strain, 12% in a moderately sensitive strain,
and only 2% in a resistant one. These results suggested that the
sensitivity of B. cinerea strains could be correlated with
cymoxanil biotransformation into this metabolite. The forma-
tion of N-acetylcyanoglycine probably plays an important role
in the fungitoxicity of cymoxanil, but its presence is not
sufficient to explain the fungicidal activity because it was
ineffective on the resistant strains. It is difficult to explain
the inefficiency of N-acetylcyanoglycine on these resistant
strains of B. cinerea. One could make the assumption that
cyanoglycine would be the final active compound rather than
N-acetylcyanoglycine. Indeed, cyanoglycine is reported in
the literature as a competitive inhibitor of enzymes implied
in the biosynthesis of aminoacids. For example, R-cyanogly-
cine strongly inhibits some pyridoxal phosphate-dependent
enzymes such as glutamate decarboxylase, tryptophan syn-
thase, threonine dehydratase, and cystathionine synthase
19–22. Cyanoglycine as active principle would mean that
the acetylated metabolite must be hydrolyzed by an enzymatic
system that would be ineffective in the resistant strains. On
the other hand, the inefficiency of acetylcyanoglycine on the
resistant strains could be also due to a weaker uptake of the
metabolite when added in the nutrient medium.
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Received for review April 7, 2008. Revised manuscript received June
12, 2008. Accepted June 18, 2008.
JF8010917