Metabolism of the Strobilurin Fungicide Mandestrobin in Wheat

Article abstract of DOI:10.1021/acs.jafc.8b03639

The metabolic fate of a new fungicide, mandestrobin, labeled with ¹⁴C at the phenoxy or benzyl ring was examined in wheat after a single spray application at 300 g/ha. Mandestrobin penetrated into foliage over time, with both radiolabels showing similar ¹⁴C distribution in wheat, and 2.8-3.3% of the total radioactive residue remained on the surface of straw at the final harvest. In foliage, mandestrobin primarily underwent mono-oxidation at the phenoxy ring to produce 4-hydroxy or 2-/5-hydroxymethyl derivatives, followed by their subsequent formation of malonylglucose conjugates. In grain, the cleavage of its benzyl phenyl ether bond was the major metabolic reaction, releasing the corresponding alcohol derivative, while the counterpart 2,5-dimethylphenol was not detected. The constant RS enantiomeric ratio of mandestrobin showed its enantioselective metabolism to be unlikely on/in wheat.

Full text of DOI:10.1021/acs.jafc.8b03639

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Metabolism of the Strobilurin Fungicide Mandestrobin in Wheat
Daisuke Ando,*,† Takuo Fujisawa,† and Toshiyuki Katagi‡
†Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1, Takarazuka, Hyogo 665-8555, Japan
‡Bioscience Research Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98, Kasugade-naka 3-chome, Konohana-ku, Osaka-city, Osaka
554-8558, Japan
ABSTRACT: The metabolic fate of a new fungicide, mandestrobin, labeled with 14C at the phenoxy or benzyl ring was
examined in wheat after a single spray application at 300 g/ha. Mandestrobin penetrated into foliage over time, with both
radiolabels showing similar 14C distribution in wheat, and 2.8−3.3% of the total radioactive residue remained on the surface of
straw at the final harvest. In foliage, mandestrobin primarily underwent mono-oxidation at the phenoxy ring to produce 4-
hydroxy or 2-/5-hydroxymethyl derivatives, followed by their subsequent formation of malonylglucose conjugates. In grain, the
cleavage of its benzyl phenyl ether bond was the major metabolic reaction, releasing the corresponding alcohol derivative, while
the counterpart 2,5-dimethylphenol was not detected. The constant RS enantiomeric ratio of mandestrobin showed its
enantioselective metabolism to be unlikely on/in wheat.
KEYWORDS: mandestrobin, wheat metabolism, malonylglucose conjugate, enzymatic hydrolysis
In this study, we investigated the metabolic behavior of 1 in
wheat sprayed once at the representative agricultural
application rate of 300 g/ha, using two different [14C]1
separately radiolabeled at the phenoxy and benzyl rings to
clarify the fate of individual substructures as a general approach
for the safety evaluation of pesticides. The study revealed the
distribution of 14C and metabolites in wheat at a series of
growth stages, i.e., forage, hay, straw, and grain. Due to the
presence of the chiral center, 1 potentially undergoes
isomerization or enantioselective degradation/fractionation
on and in plants either by abiotic and/or biotic reactions.5−7
To confirm such possibility, we also quantitated the RS
enantiomeric composition of 1 after foliar application on
wheat.
INTRODUCTION
■
Mandestrobin [1, (RS)-2-methoxy-N-methyl-2-[α-(2,5-xyly-
loxy)-o-tolyl]acetamide] is a new fungicide developed by
Sumitomo Chemical Co., Ltd., possessing a 2-methoxy-N-
methylacetamide substructure as a unique toxophore that is
different from other strobilurin fungicides on the market and
shows cross-resistant character with them. The fungicide was
registered in various countries (e.g., Japan, South Korea,
European Union, Canada, Australia, and United States) with
the aim to control Sclerotinia rot, fruit tree scab, and wide
varieties of crop diseases by acting on mitochondrial
respiratory chain complex III as a quinone outside inhibitor
(QoI).1 1 has an asymmetric center at the 2-carbon of the
acetamide moiety and is manufactured as a racemate and, thus,
can be categorized as a chiral pesticide. For these groups, less
or no bioactivity has often been identified on either one of the
enantiomers. In the case of 1, the R isomer possesses superior
fungicidal activity to that of the antipode.1,2
Incidentally, it is essential to examine and clarify the
metabolic fate of a synthetic pesticide in crop(s) to evaluate its
toxicological risks for consumers who intake both the pesticide
and its metabolites/degradates from agricultural commodities.
Several research articles on metabolic profiles of strobilurin
fungicides in crops have been published. For instance,
azoxystrobin, which was discovered at an early stage of the
synthetic strobilurin fungicide development history and has an
enol ether/ester toxophore, like natural strobilurin A, is known
to undergo cleavages of the ester/ether linkage and the
pyrimidine ring as major metabolic reactions combined with
sunlight-driven E/Z isomerization at the olefin bond on the
plant surface.3,4 Basically, similar profiles have been reported
for kresoxim-methyl and trifloxystrobin, which have an oxime
ether moiety as an alternative to the enol function.4 Unlike
these precedent strobilurins, 1, which does not possess enol/
oxime ether, ester, or olefin functions, may have the potential
to undergo a dissimilar metabolic pathway.
MATERIALS AND METHODS
■
Chemicals. Two kinds of 14C-radiolabeled 1 were prepared in our
laboratory. The compound was uniformly radiolabeled at the
dimethylphenoxy and benzyl rings, abbreviated as [Ph-14C]1 [specific
radioactivity 120 mCi/mmol (4.44 GBq/mmol)] and [Bz-14C]1 [123
mCi/mmol (4.55 GBq/mmol)], respectively. The radiochemical
purity of each 14C compound exceeded 98.9% in reversed-phase high-
performance liquid chromatography (HPLC) analysis. The RS
isomeric ratio was confirmed to be 50:50 by the chiral HPLC
analysis. Nonradiolabeled authentic standards of 1 (R:S, 50:50) and
its metabolites were prepared in our laboratory. The structures of the
following compounds are given in Figure 1: (RS)-2-[2-(2-hydrox-
ymethyl-5-methylphenoxymethyl)phenyl]-2-methoxy-N-methylaceta-
mide, 2; (RS)-2-[2-(4-hydroxy-2,5-dimethylphenoxymethyl)phenyl]-
2-methoxy-N-methylacetamide, 3; (RS)-2-[2-(5-hydroxymethyl-2-
methylphenoxymethyl)phenyl]-2-methoxy-N-methylacetamide, 4;
(RS)-3-{2-[1-methoxy-1-(N-methylcarbamoyl)methyl]benzyloxy}-4-
methylbenzoic acid, 5; (RS)-2-hydroxy-N-methyl-2-[α-(2,5-xylyloxy)-
Received: July 11, 2018
Revised: September 9, 2018
Accepted: September 12, 2018
Published: September 12, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.8b03639
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. Proposed metabolic pathway of mandestrobin (1) on/in wheat plant after the foliar application. The symbols * and † in 1 indicate the
radiolabeled positions of [Ph-14C]1 and [Bz-14C]1, respectively.
o-tolyl]acetamide, 6; (RS)-2-(2-hydroxymethylphenyl)-2-methoxy-N-
methylacetamide, 7. Compounds 6 and 7 were prepared according to
the previous methods.1,8 Other compounds were synthesized in our
laboratory as follows with structural confirmation by high-resolution
mass spectrometry (HRMS) in electron spray ionization (ESI) mode
and 1H-nuclear magnetic resonance (1H NMR) analyses. In addition,
2,5-dimethylphenol (8) was purchased from Sigma-Aldrich Co. (St
Louis, MO). The chemical purity of each standard was >98%. All the
reagents and solvents used in this experiment were of analytical grade.
The enzymes β-glucosidase (EC 3.2.1.21., from almond, 37 units/mg)
and Driselase (from Basidiomycetes sp., cellulase >100 units/g,
laminarinase >10 units/g, xylanase >3 units/g) were purchased
from Oriental Yeast Co. Ltd. (Tokyo, Japan) and Sigma-Aldrich Co.
(Tokyo, Japan), respectively. All other chemicals were of a reagent
grade and obtained from the commercial suppliers.
CH2OH), 4.65 (1H, d, J = 12.4 Hz, CH2OH), 4.97 (1H, s,
−CH(OCH3)CO−), 5.11 (1H, d, J = 11.2 Hz, −CH2O−), 5.37 (1H,
d, J = 11.2 Hz, −CH2O−), 6.76−7.47 (8H, m, CONHCH3, Ph).
Compound 3. p-Xyloquinone (5.00 g, 36.7 mmol, Tokyo Chemical
Industry, Tokyo, Japan) dissolved in 70 mL of diethyl ether was
partitioned with 35 mL of sodium hydrosulfate (Tokyo Chemical
Industry, Tokyo, Japan), washed with water, and dried using
anhydrous sodium sulfate to obtain p-xylohydroquinone (3.63 g,
yield 71.6%, pale yellow powder). To the THF solution dissolving the
hydroquinone (1.09 g, 7.90 mmol) and 7 (1.57 g, 7.50 mmol) were
added triphenylphosphine (1.97 g, 7.50 mmol) and diisopropyl
azodicarboxylate (1.52 g, 7.50 mmmol), and the mixure reacted under
nitrogen atmosphere for 18 h at room temperature with stirring. The
reaction mixture was then evaporated and purified by silica gel
column using hexane:ethyl acetate (2:1, v:v) to obtain 3 (550 mg,
yield 22.3%, white solid). HRMS (ESI+): m/z 330.1690 [M + Na]+,
Compound 2. To 7 (1.50 g, 7.17 mmol) dissolved in
tetrahydrofuran (THF, 30 mL) were added 4-methylsalicylic acid
methyl ester (1.43 g, 8.60 mmol, Tokyo Chemical Industry, Tokyo,
Japan), triphenylphosphine (2.26 g, 8.60 mmol, Tokyo Chemical
Industry, Tokyo, Japan), and diisopropyl azodicarboxylate (1.74 g,
8.60 mmol, Tokyo Chemical Industry, Tokyo, Japan), and the mixture
reacted under nitrogen atmosphere for 4 h with stirring at room
temperature. The reaction mixture was evaporated and purified by
silica gel column chromatography using toluene:acetone (2:1, v:v) to
obtain the methyl ester of 2 (2.43 g, yield 94.8%, white solid). To 1.57
g (4.40 mmol) of the methyl ester product dissolved in dichloro-
methane (CH2Cl2, 20 mL) was added diisobutylaluminum hydride (1
mol/L in CH2Cl2, 13.2 mL, 13.2 mmol, Tokyo Chemical Industry,
Tokyo, Japan) dropwise under nitrogen atmosphere at −78 °C with
stirring, and the mixture reacted for 3 h. The reaction mixture was
then poured into 2 M HCl with ice, and then extracted using CH2Cl2,
dried with anhydrous magnesium sulfate, and purified by silica gel
column using hexane/ethyl acetate (2:1, v:v) to obtain 2 (692 mg,
yield 47.7%, white solid). HRMS (ESI+): m/z 352.1511 [M + Na]+,
1
−2.952 ppm error for C19H24O4N. H NMR (400 MHz, CDCl3): δ
2.15 (3H, s, hydroquinone-CH3), 2.19 (3H, s, hydroquinone-CH3),
2.83 (3H, d, J = 4.8 Hz, CONHCH3), 3.33−3.35 (3H, m, OCH3),
3.83 (3H, s, COOCH3), 4.72 (1H, s, OH), 4.99 (1H, d, J = 11.6 Hz,
CH2OH), 5.04 (1H, s, −CH(OCH3)CO−), 5.38 (1H, d, J = 11.6 Hz,
CH2OH), 6.58 (1H, s, hydroquinone ring), 6.71 (1H, s, hydro-
quinone ring), 6.82−6.83 (1H, br, −CONHCH3), 7.26−7.52 (4H, m,
Ph).
Compound 4. To the ethanol solution (30 mL) dissolving 3-
hydroxy-p-toluic acid (20 g, 131 mmol, Tokyo Chemical Industry,
Tokyo, Japan) was added 0.71 mL of concentrated sulfuric acid, and
the mixture refluxed for 13 h. After evaporation, 50 mL of water was
added to the reaction mixture, the pH was adjusted to 7 using sodium
carbonate, and then the mixture was extracted with tert-buthyl methyl
ether. The organic solution was then dried using anhydrous sodium
sulfate and evaporated to obtain 3-hydroxy-p-toluic acid ethyl ester
(21.8 g, yield 92.2%, pale yellow liquid). A 1.55 g portion of 3-
hydroxy-p-toluic acid ethyl ester (8.60 mmol) and 1.50 g of 7 (7.17
mmol) were dissolved in 30 mL of THF. To the solution were added
triphenylphosphine (2.26 g, 8.60 mmol) and diisopropyl azodicarbox-
ylate (1.74 g, 8.60 mmol) and, the mixture was reacted under nitrogen
1
−2.384 ppm error for C19H23O4NNa. H NMR (400 MHz, CDCl3):
δ 2.36 (3H, s, Ph−CH3), 2.78 (3H, d, J = 4.8 Hz, CONHCH3), 3.34
(3H, s, OCH3), 3.83 (3H, s, COOCH3), 4.54 (1H, d, J = 12.4 Hz,
B
DOI: 10.1021/acs.jafc.8b03639
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Journal of Agricultural and Food Chemistry
Article
atmosphere for 3 h at room temperature with stirring. The reaction
mixture was then evaporated and purified by silica gel column using
hexane:ethyl acetate (3:2, v:v) to obtain the ethyl ester of 5 (2.03 g,
yield 76.1%, white solid). To the ethyl ester of 5 (1.89 g, 5.10 mmol)
dissolved in CH2Cl2 (25 mL) was added diisobutylaluminum hydride
(1 mol/L in CH2Cl2, 15.3 mL, 15.3 mmol) dropwise at −78 °C with
stirring under a nitrogen atmosphere, and the mixture reacted for 3 h.
The reaction mixture was poured into 2 M HCl with ice and was then
extracted using CH2Cl2, dried with anhydrous magnesium sulfate, and
purified by silica gel column using hexane/ethyl acetate (2:1, v:v) to
obtain 4 (940 mg, yield 56.0%, white solid). HRMS (ESI+): m/z
Radioanalysis. Radioactivity in the plant rinsates and extracts was
determined by liquid scintillation counting (LSC) with a model
2900TR spectrometer (Packard Instrument Co.) or model LS6500
instrument (Beckman Coulter Inc.) after mixing each aliquot with
Emulsifier Scintillator Plus or Ultima Gold (PerkinElmer Co., Ltd.).
The detection limit of LSC analysis was 40 dpm (0.67 Bq). The
postextracted bound residues of plants were combusted using a model
OX-500 or OX-700 biological oxidizer with oxidizer LSC cocktail (RJ
Harvey Instrument Co.). The radioactivity therein was quantitated by
LSC. The efficiency of combustion was determined to be greater than
95.0%.
1
330.1695 [M + H]+, −1.498 ppm error for C19H24O4N. H NMR
Spectroscopy. Liquid chromatography−electrospray ionization
mass spectrometry (LC−ESI-MS) analysis was conducted to
characterize metabolites of 1 using a TQD tandem quadruple
spectrometer equipped with an Acquity UPLC (ultraperformance
liquid chromatograph) and an Acquity photodiode array detector
(Waters Co.). The following parameters controlled by MassLynx
software (version 4.1, Waters Co.) were used for the typical analysis:
source temperature, 150 °C; desolvation temperature, 450 °C;
capillary voltage, 3.2 kV; cone voltage, 10−40 V; collision energy, 5−
20 V. The HPLC column eluent was diverted in the ratio 4:1 and
introduced to the mass spectrometer and radiodetector, respectively.
For the confirmation of synthetic standards, HRMS was obtained
using a Q-Exactive Focus (Thermo Fisher Scientific Inc.) mass
spectrometer with infusion injection. The analytical parameters at the
mass module controlled by the Xcalibur software (version 2.2) are as
follows: sweep gas flow, 10; source temperature, 100 °C; desolvation
temperature, 350 °C; capillary voltage, 3.5 kV, cone voltage, 10−40
(400 MHz, CDCl3): δ 2.25 (3H, s, Ph−CH3), 2.83 (3H, d, J = 4.8 Hz,
CONHCH3), 3.35 (3H, s, OCH3), 4.61 (1H, brs, CH2OH), 4.99
(1H, s, −CH(OCH3)CO−), 5.17 (1H, d, J = 12.4 Hz, −CH2O−),
5.45 (1H, d, J = 12.4 Hz, −CH2O−), 6.82−7.53 (8H, m, CONHCH3,
Ph).
Compound 5. To the ethyl ester of 5 (1.30 g, 3.50 mmol)
dissolved in ethanol (50 mL) was added 10 mL of 30% NaOH, and
the mixture reacted for 1.5 h at room temperature with stirring. The
reaction solution was washed with diethyl ether, and the aqueous
solution was pH-adjusted to 2 using concentrated HCl. The aqueous
solution was extracted with ethyl acetate, and then, the organic layer
was dried using anhydrous sodium sulfate and evaporated to obtain 5
(1.17 g, yield 97.1%, white solid). HRMS (ESI−): m/z 342.1341 [M
− H]−, 1.435 ppm error for C19H20O5N. 1H NMR (400 MHz,
CDCl3) δ 2.27 (3H, s, Ph−CH3), 2.59 (3H, d, J = 4.8 Hz,
CONHCH3), 3.30 (3H, s, OCH3), 5.00 (1H, s, −CH(OCH3)CO−),
5.25 (1H, d, J = 12.2 Hz, −CH2O−), 5.45 (1H, d, J = 12.2 Hz,
−CH2O−), 6.83 (1H, br m, CONHCH3), 7.28−8.12 (1H, m,
CONHCH3, Ph).
1
eV. In addition, H NMR spectra of the standards were recorded in
CDCl3 using a Unity-400 spectrometer at 400 MHz (Varian Inc.)
with tetramethylsilane as an internal standard.
Chromatography. The reversed-phase HPLC system to analyze
the test substance and its metabolites consisted of a series 1200 LC
module (Agilent, CA) or a L-7000 module (Hitachi, Tokyo, Japan).
The column used was a 5 μm, 150 × 6 mm, SUMIPAX ODS A-212
(Sumika Chemical Analytical Service, Ltd., Osaka, Japan). The
following gradient system consisting of 0.1% formic acid (solvent A)
and acetonitrile (solvent B) was employed at a flow rate of 1 mL/min:
0−2 min, 5% B; 10 min, 30% B; 30 min, 35% B; 40−45 min, 0% B.
The typical retention times of 1 and other synthetic standards were
42.2 min (1), 33.4 min (2), 36.4 min (3), 35.8 min (4), 37.5 min (5),
39.2 min (6), 17.1 min (7), and 25.7 min (8). In addition, three
unknown metabolites with no corresponding reference standards were
detected at 22.0 min (9, 10) and 26.5 min (11); thus, another HPLC
condition was developed for the separation, quantitation, and
isolation of the coeluted peaks 9 and 10: a 4 μm, 4.6 mm i.d. × 30
cm, Synergi Polar-RP column (Phenomenex Inc.) was applied with
isocratic elution [acetonitrile with 0.1% formic acid:0.1% formic acid,
25:75 (v:v)]. The chiral analysis for the R and S isomers of 1 was
conducted with a LC-module Series 1100 (Hewlett-Packard,
Waldbronn, Germany) using a 5 μm, 150 × 4.6 mm Chiralpak AD
RH column (Daicel Chemical Industries, Tokyo, Japan) with an
isocratic eluent of acetonitrile:water, 1:1 (v:v), at a flow rate 1 mL/
min (retention times: R isomer/5.6 min, S isomer/7.1 min). The
radioactivity in the column effluent was measured using a Radiomatic
525TR or 625TR (PerkinElmer Co., Ltd.) flow scintillation analyzer
with Ultima-Flo A or M (PerkinElmer, Co., Ltd.) as the scintillator.
The detection limit of the HPLC analyses was 150 dpm (2.5 Bq).
Thin-layer chromatography (TLC) analysis was carried out for an
analytical purpose using 20 × 20 cm, 0.25 mm thickness LK5DF thin-
layer silica gel plates (Whatman). The nonradiolabeled reference
standards were detected by exposing the chromatoplates to ultraviolet
light for direct visualization. The radioactivity in each spot on the
plate was detected by a AR2000 imaging scanner (Bioscan Inc.) The
solvent systems for TLC were as follows: chloroform:methanol, 9:1
(v:v) [Rf values of 0.68 (1), 0.63 (2), 0.62 (3), 0.60 (4), 0.21 (5),
0.65 (6), 0.66 (7)]; ethyl acetate:methanol:acetic acid, 18:2:1 (v:v:v)
[Rf values of 0.65 (1), 0.58 (2), 0.65 (3), 0.50 (4), 0.20 (5), 0.56
(7)]; hexane:2-propanol, 5:2 (v:v) [Rf values 0.56 (1), 0.43 (2), 0.49
(3), 0.33 (4), 0.25 (5), 0.50 (7)].
Plant Materials and Maintenance. The wheat “Promontory”
was purchased from Granite Seed (Lehi, UT). The soil used for the
wheat cultivation was a combination of local natural loamy sand from
Rochester, MA, and a commercial potting soil (Metro Mix 360,
SunGro Horticulture Distribution, Inc.) blended at a 9:1 ratio. The
loamy sand was collected from a fallow field where no pesticide
application was recorded in the previous 3 years. The soil used in the
study had no detectable 14C-radioactivity exceeding the minimum
quantifiable level. The pH of the soil mixture was adjusted from 4.5 to
6.2 by the addition of powdered limestone. The characterization of
the soil was as follows: soil texture, sand 71%, silt 22%, clay 7%; soil
classification, sandy loam; organic carbon content, 8.3%; pH 6.2;
moisture, 29%. Wheat seeds (approximately 30 seeds per pot) were
planted in each of the 27 pots (11-in. diameter). Wheat plants were
grown in a greenhouse located at Smithers Viscient (Wareham, MA)
with the light cycle of 16 h day/8 h night. The temperature in the
greenhouse was set at 28 °C day/21 °C night, and continuously
monitored/recorded using a thermometer. The relative humidity in
the greenhouse ranged from 19−85% for the duration of the study.
The pots were watered with either well water or a dilute liquid
fertilizer as necessary until harvest. The plants were fertilized with
Peters 20−20−20 at a concentration of 200 mg/L.
Spray Application and Sampling. The spray application was
conducted with 25% suspension concentrate (SC) formulation at the
application rate of 300 g/ha. A 4.6 mg portion of each radiolabeled
compound 1 was individually mixed with 13.7 mg of the
nonradiolabeled one for isotopic dilution (specific radioactivity of
200 kdpm/mg, 3.33 kBq/mg). Then, the blank formulation was
added to the mixture, along with water for concentration adjustment,
to prepare 25% SC spray solution. The final concentration of 1 in the
spray solution was 0.3 mg/mL. The formulation was sprayed onto the
wheat at the stem elongation stage (grown 37 days after seeding)
using a hand pump sprayer from the top of plants. After the
application, the radioactivity remaining in the bottle was recovered by
rinsing with acetonitrile and the rinsate was assayed by LSC. After 7,
14, and 104 d, forage, hay, and straw/grain were harvested by cutting
them individually at approximately 2−4 cm above the soil surface
using pruning scissors. Plant samples were removed from any
adhering soil by gentle shaking or brushing, and the total weight of
C
DOI: 10.1021/acs.jafc.8b03639
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Journal of Agricultural and Food Chemistry
Article
Table 1. 14C Distribution and Metabolites on/in Foliage
a
percent of the total radioactive residue (% TRR)
forage
hay
straw
[Ph-14C]
[Bz-14C]
[Ph-14C]
[Bz-14C]
[Ph-14C]
[Bz-14C]
surface rinses
1 (mandestrobin)
41.0
38.5
ND
ND
ND
ND
0.3
33.9
30.3
ND
ND
ND
ND
0.3
23.3
21.0
0.3
ND
ND
ND
0.5
19.1
16.4
ND
ND
ND
ND
0.2
3.9
0.3
1.1
ND
0.4
0.2
0.1
NA
2.8
0.5
0.4
0.1
0.2
0.2
<0.1
0.5
ND
2
3
4
5
6
7
9
NA
0.3
0.7
0.2
NA
0.2
ND
0.3
c
0.11
10
11
others
0.1
ND
1.8
0.2
0.2
2.0
0.2
ND
1.1
0.1
ND
2.1
ND
1.7
<0.1
0.9
b
extractables
53.2
12.5
ND
ND
ND
ND
ND
NA
10.3
3.3
60.6
29.6
0.2
ND
ND
ND
2.5
2.5
5.3
5.2
65.8
5.3
1.0
ND
ND
ND
0.2
72.8
6.3
0.9
ND
ND
ND
0.6
1.5
12.3
5.4
58.5
1.1
8.4
1.3
1.7
2.7
0.6
NA
64.7
1.5
6.0
1.4
2.7
1 (mandestrobin)
2
3
4
5
6
7
9
4.4
0.4
NA
11.0
12.9
6.3
11.3
c
c
3.9
2.8
10
11
others
6.1
4.1
11.2
6.9
38.9
1.2
37.6
ND
34.2
d
e
f
g
h
I
21.0
29.1
j
j
j
j
unextractables
5.8
5.5
10.9
8.1
37.6
32.5
total
100.0
100.0
100.0
100.0
100.0
100.0
a
b
c
ND: not detected. NA: not applicable. The sum of acetone/water and acetone/HCl extracts. Compounds 9 and 10 were not separated/
d
e
quantitated using the secondary HPLC system. Consists of more than seven components, each below 3.0% TRR. Consists of more than five
components, each below 3.3% TRR. Consists of more than eight components, each below 2.0% TRR. Consists of more than eight components,
each below 4.4% TRR. Consists of more than eight components, each below 7.3% TRR. Consists of more than seven components, each below
4.0% TRR. Further fractionated by harsh extractions.
f
g
h
I
j
each plant sample was recorded. The surface of each foliage was
individually rinsed using acetonitrile, pulverized with dry ice, and then
stored frozen until further extraction.
Metabolite Identification and Characterization. Identification
of each metabolite was basically conducted by HPLC and TLC
cochromatographies with nonradiolabeled reference standards. To
verify whether unknown metabolites 9−11 are conjugate or not,
enzymatic and alkaline hydrolysis was representatively conducted
using the [Ph-14C] hay extract. For the confirmation of glucose
conjugate, approximately 200 kdpm (3.33 kBq) of the hay extract or
50 kdpm (833 Bq) of each HPLC-isolated unknown fraction was
evaporated to dryness and dissolved in 1 mL of 10 mM acetate buffer
at pH 5.0. To the mixture, 3 mg of β-glucosidase was added, mixed
well, and incubated in a BR-180LF BioShaker (TAITEC Co. Ltd.) at
37 °C and 100 rpm. Aliquots of the mixture were sequentially
sampled and analyzed by HPLC. For malonylglucose conjugates, the
dried extract or each isolate was separately subjected to alkaline
hydrolysis by adding 1 mL of 0.03 M NaOH as pretreatment prior to
the enzymatic reaction. The mixture was reacted at room temperature
for 0.5 h and neutralized using 1 M HCl, and the reaction mixture was
subjected to HPLC and LC−MS analyses. Successively, 1 mL of 10
mM acetate buffer and 3 mg of β-glucosidase were added to the
neutralized mixture, and the reaction was periodically monitored by
HPLC.
Extraction and Analysis. Each 10−30 g of pulverized plant was
placed into a plastic bottle and extracted with acetone:water (4:1, v:v,
10 mL/g plant) using a model 133 tissue homogenizer (Bio Spec
Products Inc.) at high speed for 1 min. The sample was centrifuged at
3000 rpm for 10 min to separate the extract from the plant solids. The
supernatant was removed and the extraction was repeated twice in the
same manner, and then the supernatants were combined. The
remaining plant matrices were successively extracted with acetone:-
water:concentrated HCl (80:20:1, v:v:v, 10 mL/g plant) using the
above procedures. The radioactivity in each extract was quantitated by
LSC in duplicate and analyzed by HPLC. The extraction and LSC/
HPLC analysis of each plant sample were performed within 1 month
after the harvest. The unextractable residues were allowed to dry and
then subjected to combustion analysis. The unextractable residues of
hay were further characterized by Driselase treatment [0.5−1.0 g of
residues and 40 mg of enzyme dissolved in 10 mL of 0.1 M sodium
acetate buffer (pH 4.5) were incubated at 38 °C, overnight] and 0.1
M HCl and 0.1 M NaOH extractions (40 °C, overnight). For straw,
the unextractable residues were sequentially extracted using 0.1 M
HCl (40 °C, overnight), 6 M HCl (80 °C, 4 h), 0.1 M NaOH (40 °C,
overnight), and 6 M NaOH (80 °C, 4 h). The final 14C-bound
residues in hay and straw were determined by combustion analysis.
Estimation of Chemical Property. The n-octanol/water
partition coefficient (Kow) of 7 was predicted using the Estimation
Program Interface (EPI) Suite from the U.S. EPA (version 4.11).8
D
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3.5% TRR (0.091−0.319 ppm, Driselase), 1.4−1.5% TRR
(0.090−0.133 ppm, 0.1 M HCl), and 2.3−5.8% TRR (0.208−
0.362 ppm, 0.1 M NaOH), and finally, 0.4−1.3% TRR
(0.037−0.081 ppm) remained as the bound residue for both
radiolabels. From straw, the radioactivity recovered by acidic
solvents (0.1 and 6.0 M HCl) was less than 5.9% TRR (<0.123
ppm). Successive extractions using alkaline (0.1 and 6.0 M
NaOH) released more radioactivity (10.3−16.4% TRR,
0.210−0.304 ppm), but these fractions contained large
amounts of matrices, which were unable to be analyzed
chromatographically. The final radioactivity in bound residue
of straw accounted for 0.6−1.1% TRR (0.014−0.020 ppm).
The hydroxylated products 2−4, carboxylated 5, and
demethylated 6 were considered to be typical metabolites
generated by enzymatic function(s) at inner plant tissues of
phase I detoxification processes of cytochrome P450 and/or
other oxygenases, and the malonylglucose conjugates 9−11
were regarded as phase II conjugation products of glucosyl/
malonyl transferases.10,11 In the wheat surface rinse fractions
aimed to analyze 14C-residues on the plant surface where such
phase I/II enzymes would hardly be accessible, not only 1 but
also small amounts of 2−6 and the malonylglucose conjugates
9−11 were detected. Although epiphytic microbes present on
the plant surface may have contributed to generate 2−6,12 the
components in the rinse were regarded as the portions eluted
from inner plant tissues during the surface wash, since the
majority of these products were detected in the wheat extract,
and the same theory could be applied for the existence of
malonylglucose conjugates on the surface. This assumption is
supported by the study of Myung et al.13 They investigated the
structural change of the epicuticular wax layer after washing the
surface of wheat leaf using various organic solvents and showed
that acetonitrile somewhat deteriorates the wax composition,
although it was the most mild solvent they examined. With
respect to 7, it is known as the major photodegradate rapidly
generated via photoinduced homolytic bond cleavage at the
benzyl ether bond,2,9,14 which is the reaction that could
proceed on the wheat surface by sunlight. However, especially
in straw, the degradate dominantly distributed inside the plant.
Taking into account the hydrophilic nature of 7 (log Kow
−0.26; estimated using EPI Suite), which was generally
considered unable to penetrate the hydrophobic embedding
epicuticular wax,15,16 it was assumed to be produced mainly by
enzymatic reaction(s) in wheat. The minor contribution to the
photodegradation may be due to the sunlight screening effect
by the morphological complexity of the leaf surface and the
epicuticular wax layer, which mostly consists of aliphatic
hydrocarbons but also UV-absorptive unsaturated fatty acids,
aldehydes/ketones, flavonoids, etc., preventing photolysis from
proceeding as 1 diffused within the layer.17
RESULTS AND DISCUSSION
■
Distribution of Radioactivity in Wheat. The actual
application ratios of [14C]1, determined from the 14C that
remained in the postspraying bottle, were 303 and 306 g/ha for
phenoxy and benzyl label treatments, respectively. The
recovered radioactivities in forage, hay, straw, and grain for
Ph/Bz label were (%/%) 83.8/93.0, 90.3/96.2, 23.7/30.4, and
0.006/0.008 of the corresponding sprayed radioactivities,
respectively. Since the recovery of the applied radioactivities
largely decreased at the final harvest (straw and grain), it is
considered that 1 underwent photodegradation on the plant
surface and became mineralized/volatilized. Such a tendency
was previously confirmed in the photodegradation study of 1.9
The distribution of 14C and constituents on/in foliage (forage,
hay, and straw), summarized in Table 1, was generally similar
between the two radiolabels. The total radioactive residue
(TRR) in foliage was calculated as the sum of 14C in the
surface rinse, extractable, and unextractable fraction. For
foliage, the recovered radioactivity by the surface rinse
decreased along with the cultivation period to 33.9−41.0%
TRR (3.542−4.570 ppm) in forage and 2.8−3.9% TRR
(0.069−0.070 ppm) in straw. In the surface rinses, unaltered 1
was consistently the main component that remained in forage
and hay at 30.3−38.5% TRR (3.163−4.289 ppm) and 16.4−
21.0% TRR (1.302−1.478 ppm), respectively, and then
became as minor as 0.3−0.5% TRR (0.005−0.012 ppm) in
straw. As minor products, 2, 3, 4, 5, 6, and 7 were detected at
their maximum of 1.1% TRR (0.020 ppm, straw), 0.1% TRR
(0.003 ppm, straw), 0.4% TRR (0.007 ppm, straw), 0.2% TRR
(0.004 ppm, straw), 0.5% TRR (0.031 ppm, hay), and 0.7%
TRR (0.072 ppm, forage), respectively. Three polar unknowns,
9−11, which were later characterized as malonylglucose
conjugates of 2−4, were also observed, each at below 0.4%
TRR. Several other 14C minor constituents also existed as
minors, each amounting to less than 2.1% TRR for both
radiolabels. With respect to the acetone/water and acetone/
HCl extracts, the radioactivities therein were 50.0−58.6 and
2.0−3.2% TRR (forage), 61.1−70.6 and 2.2−4.7% TRR (hay),
and 41.1−46.7 and 17.4−18.0% TRR (straw), respectively. In
the sum of extracts, i.e., acetone/water and acetone/HCl, 1
declined from 12.5−29.6% TRR (13.950−3.091 ppm, forage)
to 1.1−1.5% TRR (0.020−0.037 ppm, straw) along with the
plant growth. The relevant metabolites observed in the extracts
were 9, 10, and 11, which reached their maximum values in
hay as 11.0−12.3% TRR (0.688−1.108 ppm), 5.4−12.9% TRR
(0.484−0.803 ppm), and 6.3−6.9% TRR (0.390−0.619 ppm),
respectively. The maximum residual levels of 2, 3, and 4 were
6.0−8.4% TRR (0.151−0.156 ppm, straw), 1.3−1.4% TRR
(0.023−0.035 ppm, straw), and 1.7−2.7% TRR (0.032−0.069
ppm, straw), respectively. The benzyl-label-specific degradate 7
amounted to the maximum of 11.3% TRR (0.281 ppm, straw),
while the structural counterpart 8 unique to the phenyl label
was not detected in any amount. There were no other 14C
components exceeding 7.3% TRR in the foliage extracts for
both radiolabels. The unextractable residues of forage, hay, and
straw increased from 5.5−5.8% TRR (0.569−0.642 ppm,
forage) to 32.5−37.6% TRR (0.696−0.810 ppm, straw), in
parallel with the 14C decrease on the plant surface. There was a
further attempt to characterize the unextractable residues of
hay and straw by Driselase treatment and successive harsh
extractions using HCl and NaOH solutions. By these
procedures, the unextractable residues of hay released 1.5−
With respect to grain, the distribution of radioactivity and
14C constituents is summarized in Table 2. The TRR in grain
was calculated as the sum of 14C in the extractable and
unextractable fractions because grain was extracted without a
surface rinse since it did not receive any direct 14C spray due to
the application timing prior to grain setting. The TRR of
benzyl labeled specimen showed approximately 7 times higher
14C residues than that of phenoxy label (phenoxy, 0.012 ppm;
benzyl, 0.089 ppm). The radioactivities in acetone/water and
acetone/HCl extracts were combined and subjected to HPLC/
TLC analyses due to the low residual level. For the phenoxy
label, 67.0% TRR (0.008 ppm) was extractable. No radioactive
components matched with the reference standards, but
E
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Table 2. 14C Distribution and Metabolites in Grain
RS Enantiomeric Ratio of 1 in Wheat. The RS isomeric
ratio of 1 remaining in the rinsates and extracts of forage and
hay was investigated by the chiral HPLC analysis. As
representative, the chiral HPLC chromatogram of 1 in
[Ph-14C] straw is shown in Figure 2. The RS ratio ranged
a
percent of the total radioactive residue
(% TRR)
[Ph-14C]
[Bz-14C]
b
extractables
67.0
ND
ND
NA
ND
72.7
ND
3.1
60.6
1.3
1 (mandestrobin)
2
7
c
9/10
d
e
others
67.0
7.7
unextractables
33.0
27.3
total
100.00
100.00
a
b
ND: not detected. NA: not applicable. The sum of acetone/water
c
and acetone/HCl extracts. Not separated/quantitated using the
secondary HPLC system. Consists of more than three components,
each below 25.6% TRR. Consists of core than five components, each
d
e
Figure 2. Chiral HPLC chromatogram of [Ph-14C] straw extract.
below 4.3% TRR.
from R:S = 50.1:49.9 to 50.4:49.6 in the surface rinsates and
from 50.2:49.8 to 44.1:55.9 in the extracts, mostly remaining as
racemate. The results indicated that RS isomerization at the 2-
carbon of acetamide as well as enantioselective degradation
were insignificant at both surface and inner portions of wheat
plant. Although the possibility of enantiomerization cannot be
concluded unless the behavior of each isomer is clarified, the
results are important evidence to support that the fungicidal-
active R isomer would not predominantly convert to less
effective S isomer and vice versa in crops after agricultural use.
Characterization of the Metabolites 9−11. The
unknown metabolites 9−11 detected as major components
in foliage extracts were further characterized in detail using the
hay extract treated with [Ph-14C]1. When a portion of the
original extract containing the unknowns was subjected to the
enzymatic hydrolysis using β-glucosidase, 9−11 were slowly
(deglycosylation not completed by 7 days) transformed to the
corresponding aglycons, 2−4, showing their possible identities
as glucose conjugates (Figure 3A,B). By the LC−MS analysis
of each unknown, the following ions were obtained in the
negative ion mode (C28H35NO12, Mw 577): m/z 690 [M +
CF3COO]−, 576 [M − H]−, 532 [M − COOH]−. As
representative, the mass spectrum of 9 is shown in Figure 4A.
From these results, the structures of 9, 10, and 11 were
proposed as malonylglucose conjugates of 2, 3, and 4,
respectively, and these metabolites were likely generated by
malonylation, which is a well-known reaction in plants, for
example, to stabilize labile biomolecules23 and to enhance
solubility of xenobiotics to transport them into the vacuole for
detoxification.24 As another hydrolysis experiment, the alkaline
pretreatment was introduced prior to the enzymatic digestion
in an attempt to release possible endogenous malonyl moieties.
By the pretreatment, individual malonylglucose conjugates in
the hay extract were immediately converted to the correspond-
ing new single peaks on the HPLC chromatogram, each of
which eluted at approximately 3−4 min earlier retention times
within 0.5 h (Figure 3C), and successive β-glucosidase
hydrolysis released each aglycon by 3 h (Figure 3D,E). The
postalkaline treatment products of 9−11 were also confirmed
by LC−MS analysis. The following molecular mass and
characteristic fragments were detected in the positive ion
mode, revealing the glucose conjugate structures (C25H33NO9,
Mw 491): m/z 514 [M + Na]+, 492 [M + H]+, 330 [M −
multiple constituents were detected in which the maximum
single component amounted to 25.6% TRR (0.003 ppm).
These products were not characterized further due to their
extremely low radioactivity. For the benzyl label, 72.7% TRR
(0.065 ppm) was extractable. The metabolites detected therein
were 2, 7, and 9/10, which amounted to 3.1% TRR (0.003
ppm), 60.6% TRR (0.054 ppm), and 1.3% TRR (0.001 ppm),
respectively. Similarly with foliage, 8 was not detected. Several
unknown 14C constituents were observed, but they were not
further characterized due to their low TRR concentrations,
<0.01 ppm. The unextractable residue accounted for 33.0 and
27.3% TRR, which was equivalent to 0.004 and 0.024 ppm for
phenoxy and benzyl labels, respectively.
Both in wheat foliage and grain, 8 or its particular
derivatives, as a possible counterpart of 7, was not detected
on/in wheat, which could be explained by their biotic
(enzymatic) and abiotic (photolytic) processes. For biotic
processes, the metabolism of dimethylphenols has been
extensively studied in bacteria.18−21 It was shown that the
metabolism of 8 proceeds via oxidation of the methyl group at
the 5-position and the phenyl ring at the 4-position to form 4-
methylgentisic acid, which successively undergoes benzene ring
cleavage to produce various minor organic acids. In addition,
phenolic compounds widely distributed as plant natural
constituents in various higher plants are known to be
incorporated into lignin in cell wall and anthocyanin in
vacuole under the shikimate pathway.22 For the abiotic
process, by employing electron spin resonance (ESR) and
radical trapping detection techniques, Adachi et al.9 succeeded
in detecting the benzyl radical form of 7, whereas the
counterpart phenoxy radical of 8 was undetectable. The
authors concluded that the phenoxy radical was unstable and
rapidly decomposed to multiple components and was
mineralized via successive radical chain reactions interacting
with other molecules or via self-decomposition. Thus, in
summary, 8 is considered to be rapidly degraded into multiple
degradates and incorporated into plant natural components
once generated on/in a plant. Such explanation could be
supported by the results in which foliage and grain of the
phenoxy label treatment overall showed higher rates of
multiple degradates and unextractables than the benzyl label.
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Figure 3. Hydrolytic behavior of [Ph-14C] hay extract analyzed by HPLC. (A) original whole extract, (B) 7 days after β-glucosidase treatment, (C)
0.5 h after alkaline treatment; (D) 1 h after β-glucosidase treatment following 0.5 h of alkaline hydrolysis, (E) 3 h after β-glucosidase treatment
following 0.5 h of alkaline hydrolysis.
glucose + OH + H]+, 312 [M − glucose + H]+, 192 [M −
glucose − dimethylphenol + H]+. Figure 4B is shown as the
representative of the demalonyl conjugate of 9. These results
demonstrated the elimination of the malonyl group from the
glucose by the alkaline treatment while each glycosidic linkage
remained intact. As the results, 9, 10, and 11 were fully
characterized as 2-, 3-, and 4-malonyl glucosides, respectively.
Similar to our finding, the poor reactivity of malonylglucose
conjugates against β-glucosidase is known for a wide varieties
of substrates, both natural products (e.g., isoflavones) and
xenobiotics.25−27 The β-glucosidase from almond applied in
this study is categorized in the glycoside hydrolase (GH)
family GH 1,28 and many β-glucosidases from GH 1, GH 3,
etc., possess a similar reactive domain with highly conserved
catalytic amino acid residues.29−33 The binding of the glucose
moiety at subsite −1 is considered to be strongly controlled by
hydrogen bonds formed between the hydroxyl groups of the
glucose moieties at the 2-, 3-, 4-, and 6-positions, where
malonyl moiety bounds, and amino acid residues.29,30,34−39
Such interactions are also believed to stabilize the deglycosy-
lation transition state29−37 and enable the key glutamine
residue(s), or others, to function as catalytic acid/base and
nucleophile.29,31,35,38 Overall, on the basis of the critical
importance of hydrogen bonds formed at glucose OH groups,
G
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Figure 4. Mass spectrum of (A) malonylglucoside and (B)
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deglycosylation efficiency observed for malonylglucose can be
attributed to the malonyl additive causing unfavorable
electrostatic repulsion or steric hindrance in forming proper
hydrogen bond interactions.
In conclusion, the metabolic pathway of 1 in wheat is
proposed in Figure 1. Compound 1 penetrated into wheat
tissue along with the growth period and underwent extensive
metabolism via phase I and II oxidation followed by
incorporation into plant components. The RS isomerization
or enantioselective degradation was considered unlikely in the
wheat metabolism.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-797-74-2116. Fax: +81-797-74-2132. E-mail:
andod@sc.sumitomo-chem.co.jp.
■
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ORCID
Daisuke Ando: 0000-0003-2133-2437
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Notes
The authors declare no competing financial interest.
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I
DOI: 10.1021/acs.jafc.8b03639
J. Agric. Food Chem. XXXX, XXX, XXX−XXX