Clay-Catalyzed Nitration of a Carbamate Fungicide Diethofencarb

Article abstract of DOI:10.1021/jf0346049

The unique nitration of the carbamate fungicide diethofencarb (Powmyl, isopropyl 3,4-diethoxycarbanilate) was examined in 14 Japanese soils and three types of clays under the aerobic conditions using the ¹⁴C-Iabeled compound. Nitration at the 6

Full text of DOI:10.1021/jf0346049

7730 J. Agric. Food Chem. 2003, 51, 7730−7737
Clay-Catalyzed Nitration of a Carbamate Fungicide
Diethofencarb
RIKA KODAKA,* TERUMI SUGANO, TOSHIYUKI KATAGI, AND
YOSHIYUKI TAKIMOTO
Sumitomo Chemical Company, Ltd., Environmental Health Science Laboratory, 2-1,
Takatsukasa 4-chome, Takarazuka 665-8555, Japan
The unique nitration of the carbamate fungicide diethofencarb (Powmyl, isopropyl 3,4-diethoxy-
carbanilate) was examined in 14 Japanese soils and three types of clays under the aerobic conditions
using the 14C-labeled compound. Nitration at the 6-position of the 3,4-diethoxyphenyl ring was a
clay-catalyzed reaction and extremely enhanced under the dry conditions. Kinetic and product analysis
on nitration of nine 14C-labeled carbamate analogues in the kaolinite thin layer showed the nitration
proceeding electrophilically. Requirement of molecular oxygen and retardation of nitration by radical
scavengers and spin-trap reagents together with semiempirical AM1 molecular orbital calculations
strongly suggested contribution of a radical mechanism, and these different speculations on the
reaction mechanism might originate from the heterogeneous reaction environment on clay.
KEYWORDS: Nitration; clay mineral; abiotic transformation
INTRODUCTION
(Clayfen) (8), which efficiently catalyzes nitration of aromatic
molecules under the mild conditions. The reaction mechanism
is still obscure, but nitrosonium ion and aromatic cation radical
were proposed to be involved.
We have investigated the effect of soil characteristics such
as clay species, soil pH, organic content, and moisture content
as dominant factors controlling nitration of diethofencarb, and
the possible involvement of microbial metabolism was also
examined together with requirement of oxygen in nitration.
Furthermore, a series of 14C-labeled diethofencarb analogues
were synthesized and subjected to nitration in the presence of
kaolinite clay and kinetic analysis was conducted to estimate a
reaction mechanism.
When applied to plants, the liquid of a pesticide formulation
intercepted by fruits, foliage, or stem exhibits its biological
activity and the other portion passes through to a ground surface.
On the soil surface, a pesticide usually undergoes various
transformations such as sunlight photodegradation, abiotic
hydrolysis, and metabolic degradation by soil microorganisms.
The contribution of each conversion highly depends on the
chemistry of a pesticide, field conditions including meteological
factors, plants, and population of microorganisms. Diethofencarb
(Powmyl, isopropyl 3,4-diethoxycarbanilate) is a carbamate
fungicide possessing preventive and curative activity against
benzimidazole resistant strains of Botrysis spp. on grapes and
vegetables (1). Because N-phenyl carbamate is known to be
resistant to hydrolysis under the natural conditions (2) and
sunlight irradiance is usually shaded by plants, the examination
of soil metabolism was considered to be of importance in
assessing an environmental fate of diethofencarb. Sakata et al.
(3) showed its rather rapid degradation under the aerobic
conditions with half-lives of 0.3-6.2 days in Japanese soils and
reported the very unique nitration reaction at the 6-position of
the 3,4-diethoxyphenyl moiety. The similar nitration of pesticide
has been recently reported for the phenyl ring of famoxadone
and pyrimethanil (4, 5). Although many reactions where
microorganisms and abiotic soil components participate are
known in the soil environment, oxidation and reduction are
predominant and nitration is only known as the clay-catalyzed
reaction (6, 7). Cornelis et al. have prepared montmorillonite
treated with transition metal nitrates such as ferric nitrate
MATERIALS AND METHODS
Chemicals. The chemical structures of carbamates used in this study
are listed in Table 1 and Figure 1. Diethofencarb (I) (isopropyl 3,4-
diethoxycarbanilate) and its analogues, II-VII, were synthesized from
the corresponding aniline and isopropyl chloroformate in our laboratory
according to the reported method (1). Compound VIII was prepared
from IV by treating with boron tribromide in dichloromethane.
Compound VII was reduced by 0.5% Pd-C to the corresponding
aniline delivative, which was further reacted with methyl iodide in the
presence of potassium carbonate to prepare IX or with acetic anhydride
in the presence of a catalytic amount of pyridine to prepare X. These
carbamates were purified by preparative silica gel thin-layer chroma-
tography (TLC) with their chemical purities determined to be >99%
by high-performance liquid chromatography (HPLC) and their chemical
1
identities were confirmed by H NMR and liquid chromatography-
mass spectrometry (LC-MS). [14C]I uniformly labeled in the phenyl
ring (8.9 MBq/mg, radiochemical purity > 99%) and [14C]II-X labeled
at the isopropyl methine carbon (8.2-10.8 MBq/mg, radiochemical
purity > 99%) were similarly prepared in our laboratory. The nitrated
* To whom correspondence should be addressed. E-mail: kodaka@
sc.sumitomo-chem.co.jp.
10.1021/jf0346049 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/12/2003

Clay-Catalyzed Nitration of Diethofencarb
J. Agric. Food Chem., Vol. 51, No. 26, 2003 7731
Table 1. Chemical Structures of Carbamates with Spectroscopic and Chromatographic Properties
a
1H NMR
R c
f
chemicals
I
R
R
phenyl (J, Hz)
LC-MS (m/e)
268 (M + H)
R (min)b
A
B
t
3,4-(OC H )
4.07 (4H, ddd)
1.29 (6H, d)
7.16 (1H, d, J ) 7.2)
6.75 (2H, dd, J ) 7.5/8.1)
7.38 (2H, d, J ) 8.4)
7.30 (2H, dd, J ) 6.9−8.1)
7.05 (1H, dddd, J ) 7.3/1.2)
7.25 (2H, d, J ) 8.1)
7.10 (2H, d, J ) 8.1)
7.28 (2H, d, J ) 8.3)
6.84 (2H, d, J ) 8.3)
7.35 (2H, d, J ) 7.0)
7.25 (2H, d, J ) 7.0)
7.60 (2H, d, J ) 7.8)
7.51 (2H, d, J ) 7.8)
8.20 (2H, d, J ) 12)
7.54 (2H, d, J ) 12)
7.15 (2H, d, J ) 8.1)
6.73 (2H, dt, J ) 8.7/3.5/2.1)
7.22 (2H, d, J ) 8.4)
6.70 (2H, dd, J ) 8.3/2.2)
7.42 (2H, d, J ) 7)
26.2
24.0
0.67
0.56
2
5 2
II
H
180 (M + H)
138
0.73
0.68
III
4-CH
2.30 (3H, s)
3.80 (3H, s)
194 (M + H)
152
210 (M + H)
168
212 (M − H)
26.1
23.2
28.0
25.0
26.0
21.0
15.7
20.2
0.77
0.76
0.66
0.55
0.67
0.32
0.68
0.25
0.70
0.64
0.63
0.53
0.66
0.34
0.03
0.20
3
IV
V
4-OCH
3
4-Cl
VI
VII
VIII
IX
X
4-CN
4-NO
203 (M − H)
223 (M − H)
194 (M − H)
223 (M + H)
2
4-OH
4-N(CH )
4.71 (1H, bs)
2.91 (6H, d, J ) 3.1)
3 2
4-NHCOCH
7.21 (NH, bs)
237 (M + H)
3
2.2 (3H, t, J ) 2)
7.32 (2H, d, J ) 6.9)
195
a In chloroform-d1, δ ppm vs TMS. Chemical shifts of aromatic protons are described. b HPLC retention time. c In solvent system of A, chloroform/methanol (9:1, v/v),
and B, toluene/ethyl acetate/acetic acid (5:7:1, v/v/v).
recovery of 14C in a sample oxidizer was >90% when 9 mL of Packard
Carb-CO2 absorber and 15 mL of Packard Permaflour oxidizer
scintillator were used. The background levels were 0.8 Bq, and more
than 1.7 Bq of radioactivity was quantitatively determined.
Chromatographies. The extracts from each sample were individu-
ally analyzed by reversed phase HPLC. A Hitachi L-6200 liquid
chromatograph equipped with a Sumipax ODS A-212 column (5 µm,
6 mm i.d. × 15 cm, Sumika Analytical Service, Ltd., Osaka) was
operated at a flow rate of 1 mL min-1. The composition of the mobile
phase was changed stepwise as follows: 0 min, %A (acetonitrile)-
%B (0.01% trifluoroacetic acid), 10:90; 0-30 min, linear, 90:10 at 30
min; 30-45 min, isocratic. The UV absorbance at 254 nm was
monitored with a Hitachi model L-4000 UV detector. The radioactivity
of column effluent was monitored with a Packard Flow-one/Beta A-100
radio detector equipped with a 500 µL liquid cell using Ultima-Flo
AP (Packard) as a scintillator. Each 14C peak was identified in HPLC
cochromatography by comparing its retention time with those of
nonradiolabeled authentic standards detected by the UV detector. The
typical retention times of I and related compounds are listed in Table
1.
Figure 1. Chemical structure of carbamates and their nitro derivatives in
clay-catalyzed nitration.
derivative of I at the 6-position of the phenyl ring (XI) was synthesized
as previously reported (3). The similar nitrated derivatives of II-X
formed via reaction on the kaolinite surface were synthesized by treating
with NO2BF4 in dichloromethane under nitrogen atmosphere in our
laboratory.
K15NO3 and Na15NO3 were purchased from ISOTEC Inc. (Miamis-
burg). 2,2,6,6-Tetramethyl-4-piperidiol (4-OH-TEMP), 5,5-dimethyl-
1-pyrroline-N-oxide (DMPO), triethylamine, N-tert-butyl-R-phenyl-
nitron, acetochlor (2-chloro-2′-ethyl-6′-methyl-N-ethoxymethylacet-
anilide), alachlor (2-chloro-2′,6′-diethyl-N-methoxymethylacetanilide),
and the other reagents were purchased from Wako Pure Chemical
Industries Ltd. (Osaka, Japan). Famoxadone (3-anilino-5-methyl-5-(4-
phenoxyphenyl)-1,3-oxazolidine-2,4-dione) was a courteous gift from
DuPont de Nemource and Company. These chemicals were used
without further purification. Kaoline, montmorillonite KSF, and mont-
morillonite K10 were purchased from Wako Pure Chemical Industries
Ltd. Activated earth (montmorillonite) was purchased from Kanto
Chemical Co., Inc. (Tokyo, Japan).
Precoated silica gel 60F254 chromato plates (20 cm × 20 cm, 0.25
mm thickness, E. Merk) were used in TLC for analytical and preparative
puposes. The Rf values of I and related compounds are also listed in
Table 1 together with the solvent systems used in development. The
nonradiolabeled reference standards were detected by ultraviolet light
at 254 nm. Autoradiograms were prepared by exposing the TLC plate
to BAS-IIIS Fuji imaging plate (Fuji Photo Film Co., Ltd.) for several
hours. The radioactivity was quantified by Bio Imaging Analyzer BAS-
1500 (Fuji Photo Film Co., Ltd.).
Spectroscopy. Fourier transfer (FT) NMR spectra were measured
in dilute solutions of chloroform-d1 using tetramethylsilane (TMS) as
an internal standard (δ ) 0.00 ppm) with a Varian unity 300 NMR
spectrometer at 300 MHz. API in a positive ion mode and single ion
mass (SIM) spectra were obtained with a Hitachi M-1000 mass
spectrometer. FTIR spectra were recorded with a FTIR Perkin-Elmer
Spectrum One over the range of 4000-650 cm-1 using a universal
ATR sampling accessory. Samples were mounted on the accessory and
Formation of XI in Soils and Clays. The 14 Japanese soils collected
from the top 10 cm of each ground were passed through a 2 mm sieve
prior to use to remove stones and plant debris. Their physical and
chemical properties are listed in Table 2. Ten grams of each soil on a
dry weight basis was taken into a glass beaker (3.5 cm diameter) and
preincubated in darkness at 20 ( 1 °C for 14 days by using a M-1200ES
incubator (Advantec Toyo Co., Ltd.). After preincubation, each soil
sample was dropwise fortified with 50 µL of acetonitrile solution of
[14C]I at a rate of 1 ppm by using a microsyringe. The moisture content
was adjusted to 50% of the maximum water holding capacity by
appropriately adding distilled water. The clays were suspended using
a minimal volume of water and air-dried without adjustment of moisture
scanned with an optical resolution of 2 cm-1
.
Radioassay. The radioactivity in extracts of soils and clays was
individually determined in duplicate by mixing each aliquot with 10
mL of Packard Emulsifier-Scintillator Plus in a low potassium glass
vial followed by liquid scintillating counting (LSC) with a Packard
model 2000CA liquid scintillation analyzer. The radioactivity bound
to soil or clay was quantified by combusting each aliquot (100 mg)
with a Packard 307 sample oxidizer prior to LSC measurement. The

7732 J. Agric. Food Chem., Vol. 51, No. 26, 2003
Kodaka et al.
Table 2. Soil and Clay Characteristics with Amounts of XI Formeda
physicochemical properties of the test soils
texture (%)
no.
soil name
Okayama
Noichi
Fukui
Tondabayashi
Fukushima
Sapporo
Ushiku
Takarazuka
Miyazaki
Kumamoto
Hyogo
Kasai
Tokushima
Niigata
Kaolinite
activated earth
(montmolinite)
Montmorillonite KSF
sand
silt
clay
main clay mineralb
pH (H O)
organic content (%)
XIc
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
60
53
42
64
45
43
39
86
98.2
45
90
66
55
49
0
16
14
22
32
19
23
25
24
7
1.5
21
5
17
19
27
100
100
K3+, A+, C+, I+
K3+, C2+, I+
A2+, AV+, K+
A2+, K2+, M+, Al−Vt+
A2+
A3+, I2+
A4+
A3+
A3+
A2+, K+, M+, I+
Al−Vt2+, K2+, A+, I+
A2+, K2+, Vt+, I+, M+
C2+, I2+, K2+
M3+, A+
6.7
6.5
5.5
5.3
6.3
5.3
7.4
6.5
6.2
6.6
5.8
6.6
6.1
5.0
NA
NA
1.3
2.6
2.7
2.2
2.8
12.0
6.2
1.6
0.3
10.7
<0.1
2.9
2.4
5.3
NA
NA
11.5
6.3
4.1
2.2
0.8
0.7
ND
ND
ND
ND
ND
ND
ND
ND
24.7
18.4
24
26
17
32
32
37
7
0.3
34
5
17
26
23
0
K
0
0
M
17
0
0
100
M
NA
NA
3.4
a NA, not analyzed; ND, not detected. b K, kaolinite; A, allophane; C, chlorite; I, illite; M, montmorillonite; Vt, vermiculite; Al−Vt, Al−vermiculite; +, clay mineral % in the
clay fraction (3+, 50−80%; 2+, 30−50%; +, 10−30%). c Amount of XI formed after incubation at 20 °C for 7 days.
content. Each vessel was placed in the incubator and kept at 20 ( 1
°C in darkness.
uniformly treated with [14C]I (8.9kBq, 1 µg) in acetonitrile, covered
with Parafilm, and then incubated at 20 ( 1 °C in darkness for 7 days
followed by analysis.
To examine the effect of soil characteristics on formation of XI, the
7 day incubation was first conducted for all soils and three clays. After
7 days, the soil sample was transferred to a centrifugal bottle and
extracted with 20 mL of methanol/0.2 M HCl (5/1, v/v) by mechanical
shaking for 10 min. After the samples were shaken, centrifugation was
conducted for 10 min at 5000 rpm and 4 °C, using a high-speed
refrigerated centrifuge (CX-250, Tomy). The extraction was repeated
twice in the same manner as above. The supernatant was combined
and concentrated under the reduced pressure to approximately 10 mL
and used for HPLC analysis. The observed peaks were identified by
HPLC cochromatography with the nonradiolabeled reference standards.
Because more formation of XI was detected in three clays after a 7
day incubation, kaolinite was conveniently used to examine factors
controlling nitration of I. First of all, the formation profiles of XI were
examined up to 32 days. Kaolinite equivalent to 1 g on a dry weight
basis was taken into the beaker and suspended with 2 mL of pure water
(PURIC-MX II, Organo) to prepare a thin layer. After the equivalent
was air-dried, it was treated with [14C]I in acetonitrile and incubated
similarly to the 7 day study. At appropriate intervals, the clay thin layer
was extracted and analyzed by HPLC. After extraction, the radioactivity
bound to clay was further quantified by combustion analysis to examine
the possible formation of bound 14C during incubation.
Examination of Nitrogen Source. As a nitrogen source, NO3- and
+
NH4 are considered most probable in soil. Therefore, their possible
-
+
incorporation into I was examined by using NO3 and NH4 free
kaolinite. The <2 µm fractions of kaolinite were obtained by
sedimentation, and the corresponding homoionic clay was prepared by
successive treatment with 1 M CaCl2 in the usual manner. The samples
were centrifuged, washed repeatedly with deionized water, and dried
at room temperature. The clay thin layer was then prepared from a
suspension of 1 g of kaolinite in 2 mL of pure water containing either
K15NO3 or Na15NO3 at 0.2% concentration. After the water had dried
up, the clay thin layer was uniformly treated with [14C]I (53.4kBq, 6
µg), incubated for 7 days at 20 °C, and [14C]XI extracted from clay
was subjected to MS analysis to examine the incorporation of 15N into
XI.
In addition, the effect of metal cation on clay surface was examined
for montmorillonite KSF by using Ba2+, NH4+, and Na+ homoionic
clays being prepared with the corresponding 1 M metal chloride
solutions. The excess metal chlorides together with Cl- were removed
by repeated washing of the clay with water. The clay thin layer was
similarly prepared from these homoionic clays and incubated to examine
the degradation of I.
Effect of Molecular Oxygen on Formation of XI. The effect of
oxygen was investigated by the following two ways. Kaolinite is a
fine powder, and the diffusion of molecular oxygen would be reduced
when the thicker clay layer was used. Therefore, the appropriate amount
of kaolinite was added to the glass beaker, suspended in water, and
air-dried finally to realize the thickness of the clay layer to become 1,
2-3, and 10 mm. To each layer, [14C]I in acetonitrile was dropwise
applied using a microsyringe. Second, the atmosphere in incubation
was changed to the anaerobic one. The clay thin layer was prepared
from a suspension of 1 g of kaolinite in 2 mL of pure water in the
bottom of a glass beaker (3.5 cm i.d.). After the water had dried up, it
was uniformly treated with [14C]I (53.4kBq, 6 µg) in acetonitrile and
incubated under anaerobic conditions of N2:H2:CO2 ) 8:1:1 (v/v/v)
for 7 days in an anaerobic chamber (Air Tec, Japan).
Effect of Moisture Content on Formation of XI. Kaolinite
equivalent to 1 g on a dry weight basis was taken into a glass vessel
(3.5 cm diameter). The moisture content was adjusted to 0.7, 10, 50,
and 100% by adding an appropriate amount of pure water. The dry
clay was prepared by air-drying for 3 days and/or heating at 120 °C
for 2 h. Each clay thin layer was with a different moisture content
Investigation of Reaction Mechanism. Nitration is known to
proceed via cationic or radical intermediate with a different regio-
selectivity. To examine the possible involvement of a radical intermedi-
ate, some radical quenchers were utilized in the reaction of I in kaolinite.
The clay thin layer was prepared from a suspension of 1 g of kaolinite
in 2 mL of pure water. After the water had dried up, the clay thin layer
was uniformly treated 0.5-1 mL of chloroform containing 0-10 mg
of either 4-OH-TEMP, DMPO, triethylamine, or N-tert-buthyl-R-
phenylnitron, as spin-trap or radical scavengers. Molar ratios of I to
these chemicals were calculated to be 25-4400. After the solvent had
dried up (for 1 h at room temperature), the clay thin layer was uniformly
treated with [14C]I (53.4 kBq, 6 µg) and similarly incubated, and the
amount of XI was examined by HPLC analysis of extracts.
Second, the reaction mechanism was investigated by kinetic analysis
of nitration by using the structural analogues of I. The kaolinite clay
film was similarly prepared in the bottom of a glass beaker (3.5 cm
i.d.), and after it was air-dried, it was uniformly treated with [14C]II-
X (8.2-10.8 kBq, 1 µg) in acetonitrile followed by incubation at 20 (
1 °C in darkness. At an appropriate interval, samples were similarly
extracted and analyzed by HPLC cochromatography with authentic

Clay-Catalyzed Nitration of Diethofencarb
J. Agric. Food Chem., Vol. 51, No. 26, 2003 7733
nitrated derivaties. For these analogues, the rate of nitration was
calculated assuming first-order kinetics. In the case of VII-X, the
reactions in kaolinite clay layer were also conducted at 30, 50, and 80
( 1 °C.
Furthermore, the semiempirical AM1 molecular orbital (MO)
calculations (9) were applied to I to estimate possible position of
nitration, using the WINMOPAC program (version 2.0, Fujitsu Ltd.).
The initial molecular geometry of I was derived from the standard
values of bond lengths and angles together with appropriate torsional
angles. All of the geometrical parameters were fully optimized by using
an EF routine, and the energy levels of the highest occupied MO
(HOMO) and the lowest unoccupied MO (LUMO) were estimated
together with their distribution at the phenyl ring. The similar
calculations were also conducted for the cation radical of I and the
several nitrogen oxide species possibly to be involved.
Furthermore, to investigate the possible nitration for other pesticides,
acetochlor, alachlor, and famoxadone, with the aniline moiety not
substituted with an electon-withdrawing group such as Br and Cl, were
subjected to the clay reaction at 5 ppm. After 20-30 days of incubation
at 20 °C, they were similarly extracted and analyzed by HPLC and the
incorporation of nitro group was examined by SIM mass spectra.
To investigate the mode of interaction of I with the clay surface, IR
spectra of I adsorbed on kaolinite and montmorilonite were measured
as follows. After clays were sieved at 50 µm, a 50 mg aliquot was
individually mixed with 2 mL of 2% chloroform solution of I in 3 mL
screw-capped glass vials. After 3 days, the slurries were centrifuged at
3000g for 10 min, washed with pure water several times, and air-dried.
Blank samples treated only with pure solvent were also prepared.
Difference spectra were obtained for clay treated with I by subtracting
the spectra of the corresponding blank clay.
Figure 2. Three-dimensional plot of percent XI vs organic matter content
(OMC %) and clay content (clay %).
RESULTS
Formation of XI on Soils and Clays. The amounts of XI
after a 7 day incubation under the aerobic conditions with the
moisture controlled are summarized in Table 2 for the 14 soils
tested. In the six soils (nos. 1-6), XI amounted to 0.7-11.5%
of the applied 14C, while no formation of XI was detected in
other soils. Among the soil characteristics possibly controlling
the nitration of I-XI, the soil pH seemed unlikely to be of
importance. The five soils (nos. 2, 5, 8, 10, and 12) showed
similar pH values of 6.3-6.6; only Noichi soil (no. 2) gave the
significant formation of XI (6.3%), and the insignificant nitration
proceeded in the other soils (e0.8%). The percentage of XI
formation after 7 days was plotted against organic matter content
(%) and percentage of clay content as shown in Figure 2. The
lesser the organic matter content and the greater the clay content,
the more formation of XI was clearly observed. These results
implied that nitration of I would most likely proceed on the
clay surface, which was clearly demonstrated by the incubation
of I in three types of clays. Kaolinite and montmorilonites
accelerated formation of XI, amounting to 3.4-24.7% of the
applied 14C.
Figure 3. Nitration rate of I on kaolinite. Total 14C ([), I (0), XI (2),
others (×), and bound 14C (4).
To examine the formation profiles of XI from I, the reaction
in the kaolinite treated with [14C]I was periodically monitored
for up to 32 days, as shown in Figure 3. Compound I was
degraded with a half-life of 5.9 days (r2 ) 0.823, first order) to
XI with the insignificant formation of other degradates (<6.7%)
and bound 14C (<2.4%). The good material balance (96.0-
98.1%) showed the insignificant formation of volatile 14C.
Because kaolinite used in this study was most likely prepared
by rigorous degradation of organic matters and repeated washing
with pure water, the nitration was most likely to proceed
abiotically on the clay surface.
Figure 4. Effect of water content on formation of XI in the kaolinite thin
layer.
content. After air dryness, the water content of kaolinite was
gravimetrically determined to be 0.3%. After 6 days, XI
amounted to 65.7% in the oven-dried kaolinite, but its formation
was retarded with an increase of a water content. Because the
water adsorbed onto the clay surface cannot be completely
removed under the tested conditions (10), the minimum coverage
of water molecules was important for the clay-catalyzed nitration
of I.
To get more information on the reaction profiles, the effect
of moisture content was examined on kaolinite at 1 ppm of I,
as shown in Figure 4. Dryness by heating in the oven at 120
°C for 2 h was conveniently considered to give the 0% water
Because the previous soil metabolism study of [14C]I using
Japanese soils showed no formation of XI under the anaerobic
conditions by purging with N2/CO2/H2 (8:1:1, v/v/v), the effect

7734 J. Agric. Food Chem., Vol. 51, No. 26, 2003
Table 3. Controlling Factions of Formation of XI in Clay
Kodaka et al.
our laboratory, and their structure was confirmed by LC-MS
and H NMR spectra, as summarized in Table 4. Isopropyl
1
carbanilate (II) labeled with 14C at the isopropyl moiety was
degraded with a half-life of 81 days, leading to formation of
nitrated derivatives with insignificant bound residues (0.6% after
30 days). The amounts of 2- and 4-NO2 derivatives were 6.0
and 4.2% of the applied 14C, respectively, with almost the
constant o/p (2-/4-) ratio of 1.5∼2:1 at any sampling time. The
substitution of electron-donating groups such as methyl (III)
and methoxy (IV) at 4-position of the phenyl ring enhanced
the nitration, and the 2-NO2 derivatives were only produced.
In contrast, introduction of electron-withdrawing groups such
as Cl (V), CN (VI), and NO2 (VII) at the 4-position inhibited
nitration even when the reaction was conducted at higher
temperatures (50 and 80 °C). When the OH (VIII), N(CH3)2
(IX), or NHCOCH3 (X) group was introduced at the 4-position,
these carbamates underwent nitration at the 3-position of the
phenyl ring. Similarly, the formation of the nitrated derivatives
of acetochlor and famoxadone was confirmed through the
incubation in kaolinite. After incubation at 20 °C for 20-30
days, SIM-MS of the kaolinite extracts exhibited ion peaks
corresponding to each pesticide (acetochlor, m/e 269; famoxa-
done, m/e 374) together with those possessing the mass greater
than the parent by m/e of 45 () NO2). In the case of
famoxadone, the two peaks observed in SIM-MS were consid-
ered to correspond to the o- and p-NO2 derivatives. Any new
MS peak corresponding to the nitrated derivative of alachlor
could not be detected through incubation even for up to 30 days.
To investigate the nitration process more in detail, the
formation rate (k) of the nitrated derivative of each analogue
was calculated assuming the pseudo-first-order kinetics based
on HPLC analysis of extracts, as listed in Table 4. Because the
change of reaction rate with a regioselectivity of nitration
seemed to imply involvement of an electrophilic substitution
mechanism, the relative reaction rates to that of (II) (k/k0) were
plotted against the Hammett σ+ values, as shown in Figure 5.
The linear regression analysis gave the following relationship.
%of the
applied 14
C
a
thickness of
layer (mm)
atmosphere
additives
kaolinite
I
XI
aerobic
aerobic
aerobic
anaerobicc
aerobic
aerobic
aerobic
10
2−3
1
1
1
b
b
b
b
80.3
55.4
30.5
96.0
37.9
28.4
0.9
13.4
37.3
60.9
ND
KNO
54.5
65.0
90.5
3
15
1
1
K NO
3
Na15NO
3
montmorillonite KSF
aerobic
aerobic
aerobic
aerobic
aerobic
1
1
1
1
1
b
CaCl2
BaCl2
NH Cl
NaCl
80.3
62.3
39.1
23.7
39.7
9.3
25.5
42.6
32.7
49.4
4
a Incubation at 20 °C after 7 days. b Not treated. c In the atmosphere of N /
2
CO /H , 8:1:1 (v/v/v).
2
2
of molecular oxygen was first examined for the kaolinite treated
with [14C]I at 1 ppm. By changing the thickness of the kaolinite
layer from 10 to 1 mm, the amount of XI formed after 7 days
increased from 13.4 to 60.9% as summarized in Table 3.
Assuming the homogeneous distribution of I in the kaolinite
layer, the thicker layer would reduce the diffusion of molecular
oxygen, which may result in less reaction with O2. When the
kaolinite treated with [14C]I was incubated under the anaerobic
atmosphere (N2/CO2/H2, 8:1:1), no formation of XI was
observed. These results showed the importance of molecular
oxygen for the nitration of I on the clay surface.
Finally, the source of nitrogen was examined. The nitrate ion
was added to the kaolinite layer prior to incubation, since it is
one of the most abundant inorganic ions in the soil environment.
The excess amount of potassium nitrate scarcely affected the
reaction as shown in Table 3, and moreover, MS analysis around
m/z ) 312 could not demonstrate any 15N incorporation into
XI. In the presence of Na15NO3, the nitration was greatly
enhanced but without incorporation of 15N into XI, as also
evidenced by MS analysis. Although the direct evidence could
not be obtained, these results were likely to show that some
nitrogen source was firmly attached to the clay surface and
participated in nitration.
By the way, the countercation on the clay surface is known
to affect the surface environment such as surface acidity; the
several homoionic clays were examined as thin layers (at a
thickness of 1 mm). In all homoionic clays of montmorillonite
KSF, the extent of nitration increased and XI amounted to 25.5-
49.4% of the applied 14C after 7 days at 20 °C (Table 3). There
was no clear relationship between the amount of XI and cation
characterization such as ion radius (Na+, 1.16 Å; Ba2+, 1.49
Å; Ca2+, 1.14 Å) when six H2O molecules are coordinated (11).
These results showed that nitration of I in soil abiotically
proceeded on the clay surface under the dry condition via
reaction with a nitrogen source tightly bound to the clay surface
in the aid of molecular oxygen.
Nitration of Carbamate Analogues on Kaolinite. Nitration
of I and the related analogues (II-X) in the air-dried kaolinite
thin layer (1 mm thickness) at 20 °C under the aerobic conditions
were determined. After 7 days, the HPLC analysis of clay
extracts showed the appearance of the new peaks as with
incubation. The corresponding nitrated derivatives were identi-
fied by HPLC cochromatography with authentic standards. The
authentic standards of nitrated derivatives were synthesized in
log(k/k0) ) -7.746 σ+ + 0.7211 (r2 ) 0.86, n ) 5)
The large negative slope (F ) -7.746) strongly suggested the
electrophilic substitution mechanism being known for the usual
nitration by nitronium ion.
By the way, one electron oxidation of an organic molecule
has been known for various kinds of clays (12). Therefore, the
possible involvement of radical mechanism was examined. The
radical scavengers or spin-trap reagents were added to kaolinite
at three levels of concentration prior to incubation with [14C]I.
Either triethylamine as a radical scavenger or 4-OH-TEMP as
a quencher of singlet oxygen was only effective at a molar ratio
to I of 2800-4400 (addition of 10 mg), while DMPO and
N-tert-butyl-R-phenylnitron as quenchers of hydroxyl and/or
superoxide anion radicals retarded the nitration of I at a much
lower molar ratio of 25-39, as shown in Table 5. Although
the retardation of nitration in the presence of the extremely more
amounts of these reagents might simply be caused by their
coverage of the clay surface inhibiting the adsorption of I to
active sites, more effectiveness of DMPO and N-tert-butyl-R-
phenylnitron was likely to show the involvement of hydroxyl
and/or superoxide anion radicals in nitration.
Because these carbamates underwent nitration through bind-
ing to clay surfaces, the mode of interaction of I with clay was
studied by IR with an ATR mode. Figure 6 shows IR difference
spectra of I, I in kaolinite, and montmorillonite KSF at 1750-
1100 cm-1. The absorption due to stretching of the carbonyl

Clay-Catalyzed Nitration of Diethofencarb
J. Agric. Food Chem., Vol. 51, No. 26, 2003 7735
Table 4. Kinetic Analysis of Nitration of I and Its Analogues and Structure of Formation Products
rate of
formation
(µmol/s)
d
TLC R c
1H NMR of aromatic moiety
f
nitrated
derivative
R
t
(min)b
R
A
B
substituent
ring-H
MS (m/e)
σ+f
k/k0g
I
XI
33.1
0.80
0.71
4.20 (4H, ddd)
1.35 (6H, dd)
8.21 (1H, s)
7.68 (1H, s)
313 (M + H)
227
225 (M + H)
2.30 × 10-7
II
2-NO
28.8
0.80
0.72
8.58 (1H, d, J ) 8.4/0.9)
8.20 (1H, dd, J ) 9.0/1.2)
7.62 (1H, ddd, J ) 8.7/1.5)
7.10 (1H, ddd, J ) 7.5/1.5)
8.20 (2H, d, J ) 12)
7.54 (2H, d, J ) 12)
8.44 (1H, d, J ) 8.7)
7.99 (1H, d, J ) 1.5)
7.44 (1H, dd, J ) 8.7/1.8)
8.46 (1H, d, J ) 9.3)
7.65 (1H, d, J ) 3)
1.49 × 10-10
0
1
2
183, 139
4-NO
26.1
30.7
0.67
0.80
0.66
0.74
225 (M + H)
177
239 (M + H)
1.10 × 10-10
0.90 × 10-7
2
III
2-NO
2.37 (3H, s)
3.85 (3H, s)
−0.17
−0.27
398
598
2
197, 153
IV
2-NO
28.6
0.82
0.69
255 (M + H)
213, 169
1.33 × 10-7
2
7.23 (1H, m, J ) 12/3)
e
e
e
e
e
e
e
e
V
VI
VII
VIII
a
a
a
NA
NA
NA
NA
NA
NA
NA
NA
e
e
e
e
e
e
e
e
NA
NA
NA
NA
NA
NA
NA
NA
e
e
e
e
e
e
e
e
NA
NA
NA
NA
NA
NA
NA
NA
3-NO
27.9
29.8
26.5
0.61
0.68
0.40
0.55
0.80
0.26
6.51 (1H, bs)
2.8 (6H, s)
8.18 (1H, d, J ) 3)
7.61 (1H, dd, J ) 7/3)
7.12 (1H, d, J ) 9)
7.81 (1H, d, J ) 3)
7.47 (1H, d, J ) 9.3)
7.02 (1H, d, J ) 8.7)
8.49 (1H, d, J ) 9.3)
8.43 (1H, d, J ) 2.4)
7.72 (1H, d, J ) 9.3/2.4)
239 (M − H)
268 (M + H)
282 (M + H)
5.22 × 10-7
1.05 × 10-7
0.317 × 10-8
−0.37
−0.83
0
2310
463
14
2
IX
X
3-NO
2
3-NO
7.63 (NH, bs)
2.20 (3H, s)
2
a No reaction was observed at 20, 50, and 80 °C. b HPLC retention time. c In solvent system of A, chloroform/methanol (9:1, v/v), and B, toluene/ethyl acetate/acetic
acid (5:7:1, v/v/v). d In chloroform-d1, δ ppm vs TMS. Chemical shifts of aromatic protons are described. e Not analyzed. f Hammett σ plus. g k, formation rate of the nitrated
derivative; k0, formation rate of the nitrated derivative of II.
Figure 5. Hammett plot for the nitration of the carbamate analogues.
Figure 7. Energy levels of HOMO, SOMO, and LUMO of I and related
species estimated by the AM1 MO calculation.
Figure 6. IR spectra of I in clays.
adsorbed onto the clay surface by interaction through the
group (amine I band) was constantly observed at 1693 cm-1
,
carbamate moiety, possibly via ion-dipole interaction between
CdO group and exchangeable metal cation on clay (13); hence,
the phenyl ring would be located parallel to the clay surface.
AM1 Calculation. On the basis of the fully optimized
structure, the energy levels of HOMO and LUMO (also singly
occupied MO, SOMO, for the cation radical of I) were estimated
as shown in Figure 7. In general, MOs with a smaller energy
gap and more overlap of orbitals are considered to more
effectively interact with each other leading to formation of a
new bond. From this viewpoint, the reaction of I or its cation
and the N-H bending at 1602 cm-1 did not shift irrespective
of adsorption to clays. The broad absorption around 1530 cm-1
consisting of C-N and CdC stretching was observed in every
case but with its shift to a higher frequency by 6 and 1 cm-1
for kaolinite and montmorillonite, respectively. The plane
deformation mode of the COO and NHCO moieties was found
at ca. 1230 cm-1, and the corresponding adsorption in kaolinite
clealy shifted to a higher frequency by 4 cm-1. These results
together with the chemical structure of I may imply that I is

7736 J. Agric. Food Chem., Vol. 51, No. 26, 2003
Kodaka et al.
the dry conditions. When the thin layer of kaolinite was prepared
by air drying, the water content was determined to be about
0.3%; hence, the similar acid-catalyzed reaction would be most
likely to proceed for I on the surface. By the way, the strong
acid such as sulfuric acid is usually used for nitration of an
aromatic ring in organic synthesis (21), which might be in
agreement with the acidic condition of the clay surface. Nitration
proceeds via reaction with either nitrosonium or nitronium ion
and the o/p isomer ratio is known to be characteristic to these
reactive cations. In the former case, the p-NO2 derivative is
dominant, while nitronium ion affords the compounds with an
o/p ratio of 2:1. In the case of kaolinite-catalyzed niration of
II, this ratio was 1.5-2:1, indicating that the usual mechanism
via nitronium ion would operate in our system. For III and IV,
whose 4-positions were blocked by electron-donating substit-
uents, only 2-NO2 derivatives were formed. In the case of
VIII-X whose substituents at the 4-position have lone pair
electrons, the o-position to these substituents (3-position) was
selectively nitrated. Furthermore, the Hammett equation esti-
mated for these carbamates gave the large negative F value,
indicating that the nitration on the kaolinite surface was an
electrophilic reaction. Thus, the obtained regioselectivity and
Hammett analysis imply that nitration may proceed similarly
Table 5. Yields of XI on Kaolinite Treated with Radical Quenchers
XI, % of the applied 14C after 7 days
additives
(mg)
N-tert-butyl-R-
phenylnitron
triethylamine
4-OH-TEMP
DMPO
0
0.1
1
56.3
54.0
52.5
73.8
41.3 (44)a
31.4 (441)a
70.9 (28)a
54.6 (283)a
0.7 (2834)a
23.4 (25)a
9.2 (251)a
11.4 (39)a
7.7 (394)a
0.2 (3938)a
b
b
10
ND (4406)a
ND (2514)a
a Molecular ratio of additives to I. b ND, not detected.
Table 6. Reaction Indices of I Estimated by the AM1 MO Calculation
C1
C2
C3
C4
C5
C6
fN
fR
fE
0.608
0.453
0.298
0.217
0.119
0.020
0.059
0.137
0.215
0.529
0.475
0.420
0.146
0.111
0.075
0.118
0.219
0.320
as the usual nitration including NO2 ion rather than NO+ as
+
an active species. Incidentally, Corne´lis et al. (8) have reported
the efficient catalyst “Clayfen” for nitration of organics, which
was an acidic K10 clay treated with ferric nitrate. “Clayfen”
affords the o/p isomer ratio of 0.81:1 for phenol different from
the usual nitration, and they proposed a cation radical mecha-
nism. A phenol was considered to be oxidized by clay to form
a cation radical, which reacted with NO2 being produced from
reaction of NO+ with NO3- and/or cleavage of a covalent nitrate
complex. By thermal analysis of the several metal nitrates on
montmorillonite K10, nitrogen oxides were reported to be
produced at lower temperatures than that in the absence of clays
(22).
radical with NO+ or NO2+ would be most probable. Because it
was very difficult to computationally estimate these reactions
along with reaction coordinates, the usual reaction indices fN
(nucleophilic), fR (radical), and fE (electrophilic) for I were
conveniently calculated by using the distribution of HOMO and
LUMO, as listed in Table 6. These results mean that the
nitration is most likely to proceed at the 6-position of the 3,4-
diethoxyphenyl ring according to the electrophilic reaction
mechanism, usually known for NO2+ or NO+. This estimation
was in agreement with the real reaction in soils and clays.
However, the possibility of a reaction with some radical species
resulting in nitration also at the 6-position could not be excluded.
In the case of the radical cation of I, AM1 calculation gave the
spin distribution as follows: 17 (C1), 22 (C2), 9.9 (C3), 13.8
(C4), 3.6 (C5), and 5.9% (C6). If the cation radical of I is
involved in nitration as propared for nitration with clay-
supported metal nitrates (7), it would less favorably proceed at
the 6-position, which disagrees with the experimental results.
Therefore, AM1 calculations only suggested that nitration of I
In our study, nitration of I proceeded only at the 6-position
of the 3,4-diethoxyphenyl ring. The AM1 calculations of I, its
•
cation radical, NO2+, and NO2 showed that the reaction of I
•
with NO2+ was energetically favorable but that either with NO2
•
or with the reaction of cation radical of I with NO2 could not
be excluded. However, the spin distribution at the phenyl ring
of the cation radical suggested the unfavorable reaction at the
6-position, and the fE and fR indices for I demonstrated that the
6-position was most probable in nitration. Furthermore, the
pretreatment of kaolinite with triethylamine inhibited the
reaction. This basic reagent as a radical quencher can neutralize
acidic sites on the clay surface; hence, this retardation could
not be simply used to clarify the reaction mechanism. The
inhibition of nitration on kaolinite by DMPO and N-tert-butyl-
R-phenylnitron showed the possible involvement of radical
process by hydroxyl and/or superoxide anion radical and that
the radical sites were only deactivated by coverage of excess
reagents, indicating that the radical species were not free but
bound to the clay surface. The presence of molecular oxygen
was found prerequisite for nitration as previously shown in soil
metabolism study of I (3). Larson and Hufnal (23) have
would proceed via electrophilic mechanism of NO2 or NO+
+
or via reaction with some radical species on clay surfaces.
DISCUSSION
Both the qualitative analysis on the relationship between
nitration of I with soil characteristics and the enhanced formation
of XI in pure kaolinite and montmorillonite clays strongly
suggested the abiotic clay-catalyzed process. The catalytic
behavior of clays is well-known and has been utilized for many
types of organic syntheses (7, 14). In relation to soil metabolism
of pesticides, the involvement of catalytic reactions on clay
surface has been reported for hydrolysis of organophosphorus
pesticides (15, 16) and carbamates (17, 18) and hydration of
cyano group of fenpropathrin (19), where the Bro¨nsted acidity
on the clay surface originating from the proton dissociation of
bound water around the exchangeable metal cations had a great
role in the reactions. The lesser water content of clays is known
to make a water molecule coordinated to a counter metal cation
more reactive for nucleophilic attack (20) and to extremely
increase the surface acidity nearly up to 90% H2SO4 (12) under
•
proposed the possible formation of hydroperoxy radical (HO2 )
on the clay surface via electron transfer from an incorporated
transition metal to molecular oxygen. In general, clays are
known to have either some defects in lattice where aluminum
is substituted with a transition metal such as iron or metal oxides
and hydroxides attacked thereon. Although the origin of nitrogen
could not be identified in our study, the nitrogen source was

Clay-Catalyzed Nitration of Diethofencarb
J. Agric. Food Chem., Vol. 51, No. 26, 2003 7737
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clays used in this study showed the nitration activity even after
rigorous washing of water or exchanging cations using metal
chlorides. Therefore, if NO2• is formed on the clay surface, the
abundant inorganic nitrate ion (24, 25) might be most probably
coordinated to such sites (radical mechanism). This process
would favorably proceed under the dry condition minimizing
side reactions with adsorbed water molecules, but at the same
time, the highly acidic environment would also be realized.
Therefore, nitronium ion may possibly be formed on the clay
surface from nitrate ion (cationic mechanism). The water content
dependency and o/p-regioselectivity with the Hammett analysis
supported the cationic mechanism, while the inhibition by spin
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contradictory result would most likely originate from the
heterogeneous reaction environment on the clay surface where
the reactive nitrogen species and I or the other carbamate were
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Received for review June 6, 2003. Revised manuscript received October
7, 2003. Accepted October 9, 2003.
JF0346049