Elucidating the transformation pattern of the cereal allelochemical 6-methoxy-2-benzoxazolinone (MBOA) and the trideuteriomethoxy analogue [D 3]-MBOA in soil

Article abstract of DOI:10.1021/jf0509052

To deduce the structure of the large array of compounds arising from the transformation pathway of 6-methoxybenzoxazolin-2-one (MBOA), the combination of isotopic substitution and liquid chromatography analysis with mass spectrometry detection was used as

Full text of DOI:10.1021/jf0509052

J. Agric. Food Chem. 2006, 54, 1075−1085 1075
Elucidating the Transformation Pattern of the Cereal
Allelochemical 6-Methoxy-2-benzoxazolinone (MBOA) and the
Trideuteriomethoxy Analogue [D3]-MBOA in Soil
THOMAS ETZERODT,†,‡ SUSAN T. NIELSEN,‡ ANNE G. MORTENSEN,‡
CARSTEN CHRISTOPHERSEN,† AND INGE S. FOMSGAARD*,‡
Chemical Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark,
and Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg,
DK-4200 Slagelse, Denmark
To deduce the structure of the large array of compounds arising from the transformation pathway of
6-methoxybenzoxazolin-2-one (MBOA), the combination of isotopic substitution and liquid chroma-
tography analysis with mass spectrometry detection was used as a powerful tool. MBOA is formed
in soil when the cereal allelochemical 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) is
exuded from plant material to soil. Degradation experiments were performed in concentrations of
400 µg of benzoxazolinone/g of soil for MBOA and its isotopomer 6-trideuteriomethoxybenzoxazolin-
2-one ([D3]-MBOA). Previously identified metabolites 2-amino-7-methoxyphenoxazin-3-one (AMPO)
and 2-acetylamino-7-methoxyphenoxazin-3-one (AAMPO) were detected. Furthermore, several novel
compounds were detected and provisionally characterized. The environmental impact of these
compounds and their long-range effects are yet to be discovered. This is imperative due to the
enhanced interest in exploiting the allelopathic properties of cereals as a means of reducing the use
of synthetic pesticides.
KEYWORDS: Isotope labeling; transformation; identification; LC-MS; 6-trideuteriomethoxybenzoxazolin-
2-one; allelochemicals; soil; degradation; allelopathy
INTRODUCTION
has been determined by Kim et al. (13). The formation of APO
from BOA was shown to occur through the intermediate
2-aminophenol. The formation of APO from 2-aminophenol
occurred both as a chemical process and as a microbial process
(14). Besides APO and AMPO, it has been shown that N-(2-
hydroxyphenyl)malonamic acid (HPMA) and N-(2-hydroxy-4-
methoxyphenyl)malonamic acid (HMPMA) have been identified
in several cases from endophytic fungi (1, 15, 16). An
understanding of the total process by which these allelopathic
compounds are metabolized would give a better insight into
the involved degradation products that might be formed and
their environmental impact. Further possible metabolization of
the AMPO derivatives by different enzymes in bacteria can be
expected.
Many reversible enzymatic reactions are carried out by
microorganisms including oxidation/reduction and acetylation/
hydrolysis (16). AMPO itself is therefore a potential substrate.
The purpose of this study was to elucidate unknown degrada-
tion products from MBOA in soil using an isotope-labeled
analogue of MBOA as well as to establish whether MBOA (and
other methoxy-containing metabolites) undergoes demethylation
followed by methylation.
Secondary metabolites from wheat, rye, and corn are known
to have allelopathic activity. These metabolites, such as ben-
zoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one
(MBOA), have been investigated extensively for more than 15
years. An extensive list of the various acronyms and abbrevia-
tions used is given listed in Table 1. Major agricultural crops
such as wheat (Triticum aestiVum) and rye (Secale cereale)
produce BOA and MBOA, respectively, from their correspond-
ing hydroxamic acids 2,4-dihydroxy-1,4-(2H)-benzoxazin-3-one
(DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-
3-one (DIMBOA). The hydroxamic acids are bound as gluco-
sides in plant vacuoles and released by â-glucosidases upon
disruption of cellular integrity (1). Further degradation products
have been identified as 2-amino-3H-phenoxazin-3-one (APO)
and 2-amino-7-methoxy-3H-phenoxazin-3-one (AMPO) from
BOA and MBOA, respectively (2-6). The formation of the
further degradation products is expected to occur through the
action of several enzymes such as phenoxazine synthetase (7-
12). The overall structure of the phenoxazine synthetase enzyme
* Corresponding author (telephone +45 89993610; fax +45 89993501;
e-mail Inge.Fomsgaard@agrsci.dk).
The chemical structures of HPMA, HMPMA, DIBOA, and
DIMBOA are given by Macias et al. (17). Structures other than
these four are shown in Table 2.
† University of Copenhagen.
‡ Danish Institute of Agricultural Sciences.
10.1021/jf0509052 CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/24/2006

1076 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Table 1. Acronyms and Abbreviations Used in This Paper
Etzerodt et al.
0.92 g/mL and diethyl ether a density of 0.71 g/mL, this procedure
when repeated four times left ∼90% of the NaH essentially free of
mineral oil. After the last washing, dry THF (150 mL) and p-
toluenesulfonyl chloride (49.7 g, 0.26 mol) were added, the stirring
was started, and the mixture was cooled to -5 °C. While the
temperature was held below 0 °C, trideuteriomethanol (10 g, 0.28 mol)
was added dropwise during 1 h. The reaction mixture was stirred further
for 4 days at room temperature, and diethyl ether (130 mL) added.
Water (90 mL) was added dropwise, at first followed by a vigorous
evolution of hydrogen due to unreacted sodium hydride. An additional
amount of water was added until all precipitate had dissolved, and the
aqueous phase was separated and washed three times with ethyl ether
(100 mL). The combined organic phases were dried with magnesium
sulfate, the solvent was removed in vacuo, and the residue was distilled
(bp 117 °C/2.5 mbar) to give colorless 1 (23.58 g, 48% yield): GC-
MS, m/z 189 (M•+, 48), 155 (40), 107 (15), 91 (100), 65 (38); 1H NMR
(CDCl3), δ 7.71 (d, 2H, HA, J ) 8.2 Hz), 7.29 (d, 2H, HB, J ) 8.2
Hz), 2.38 (s, 3H, CH3); 13C NMR (CDCl3), δ 144.7 (C4), 132.1 (C1),
129.7 (C3), 127.9 (C2), 21.5 (CH3); IR (cm-1) 3067 w (CH), 2926
mw (CH), 2263 w-2087 w (CD), 1598 m, 1494 m and 1451 m (arom
CdC), 1363 s and 1180 s (SdO), 1104 s (CsO), 996 s, 817 s, 741 s
(SsOsC). Found: C, 50.72; S, 16.99. Calcd for C8H7D3O3S: C, 50.77;
H, 6.92; S, 16.94.
Resorcinol-O-mono-trideuteriomethyl Ether (2). Synthesis of 2 was
carried out following the directions given by Bird et al. (19). With
magnetic stirring, sodium metal (5.72 g, 0.249 mol) was dissolved in
MeOH (240 mL, dried over molecular sieves), maintaining gentle reflux.
Resorcinol (52.9 g, 0.48 mol) was added in one portion. The solution
first turned dark orange and then light yellow, indicating changes in
equilibrium. Addition of 1 (22.72 g, 0.12 mol) followed by further reflux
for 2.5 h was followed by changes in color from violet/black to orange/
yellow. Water (70 mL) was added, followed by 4 M NaOH until a pH
of 11 was reached, where both the mono-[D3]-methyl ether and
resorcinol were converted to salts. The dimethyl ether was removed
by extraction three times with 100 mL of toluene and discarded.
Acidification to pH 1 with 3 M HCl liberated the phenols. The
monomethyl ether 2 was secured by extraction with toluene (100 mL,
9 times) (20), in which resorcinol is almost insoluble. The combined
toluene phases were dried with MgSO4 and evaporated in vacuo,
yielding 9.4 g, 62%, of crude product. Both NMR and GC-MS indicated
that resorcinol was present in the product in a 1-5% amount. The crude
product was used without further purification for the synthesis of
compound 3: MS (DI), m/z 127 (M•+, 85), 110 (100), 95 (30), 81 (34);
1H NMR (CDCl3), δ 7.14 (t, 1H, H-5), 6.50 and 6.44 (dd, each 1H, J
) 7 and 2 Hz, H-4 and H-6), 6.42 (s, 1H, H-2), 4.57 (brs, 1H, OH);
13C NMR (CDCl3), δ 101.4, 106.3, 107.7, 130.0, 156.5, 160.8; IR
(cm-1) 3387 s, br (OH), 2221 m and 2073 m (CD), 1597 s and 1490
s (arom CdC), 1293 s and 1153 s (CsO), 1017 ms, 960 m, 898 m,
838 ms, 765 ms, 685 ms.
acronym or
abbreviation
13C NMR
1H NMR
AAMPO
abs EtOH
AMPO
APO
carbon nuclear magnetic resonance
proton nuclear magnetic resonance
N-acetyl-2-amino-7-metoxy-3H-phenoxazin-3-one
99% ethanol
2-amino-7-metoxy-3H-phenoxazin-3-one
2-amino-3H-phenoxazin-3-one
ASE
accelerated solvent extractor
BOA
benzoxazolin-2-one
bp
boiling point
[D3]-
trideuterio-
DIBOA
DIMBOA
ESI
2,4-dihydroxy-1,4(2H)-benzoxazin-3-one
2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one
electrospray ionization
EtOAc
GC-MS
HMNPAA
HMNPHAA
HMNPMA
HMPAA
HMPHAA
HMPMA
HPLC
HPMA
LC-MS
m/z value
MAP
ethyl acetate
gas chromatography interfaced to mass spectroscopy
N-(2-hydroxy-4-methoxynitrophenyl)acetamide
N-(2-hydroxy-4-methoxynitrophenyl)-2-hydroxyacetamide
N-(2-hydroxy-4-methoxynitrophenyl)malonamic acid
N-(2-hydroxy-4-methoxyphenyl)acetamide
N-(2-hydroxy-4-methoxyphenyl)-2-hydroxyacetamide
N-(2-hydroxy-4-methoxyphenyl)malonamic acid
high-performance liquid chromatography
N-(2-hydroxyphenyl)malonamic acid
liquid chromatography interfaced to mass spectroscopy
mass-to-charge ratio of ions formed in the mass spectrometer
2-amino-5-methoxyphenol
MBOA
MeCN
MeOH
mp
6-methoxybenzoxazolin-2-one
acetonitrile
methanol
melting point
RP18
RT
reverse phase C18-column material
retention time
TLC
thin-layer chromotography
MATERIALS AND METHODS
Chemicals. MBOA (98% purity, purchased from Lancaster Chemi-
cals) and [D3]-MBOA (95% purity) synthesized in our own laboratory
were used for the degradation experiments. AMPO was provided by
Dr. Scott Chilton, University of North Carolina, and Dr. Francisco A.
Mac´ıas, University of Cadiz; AAMPO was provided by Dr. F. Mac´ıas.
Characterization of AMPO and AAMPO and the determination of their
purity were carried out by Dr. F. Mac´ıas (17). Purity was stated to be
>98%.
The chemicals used for the synthesis of 6-trideuteriomethoxyben-
zoxazolin-2-one were the following: Propionic acid (99%) was
purchased from Aldrich. Sodium hydride as a mineral oil suspension
(60%) was purchased from Riedel-de-Hae¨n. p-Toluenesulfonyl chloride
(>97%), phenyl chloroformate (97%), and Pd on charcoal (5%) were
purchased from Fluka Chemika. Metallic sodium (>99%), resorcinol
(>99%), and sodium nitrite (99%) were purchased from Merck.
Deuterated MeOH (99.8%) was purchased from Cambridge Isotopes.
All solvents used were purchased from LAB SCAN as HPLC grades.
Analytical Equipment Used for Identification of Pure Com-
pounds. GC-MS spectra were obtained on an HP GC5890-MS5972A
Hewlett-Packard series II mass spectrometer. Direct inlet mass spectra
were obtained on a JEOL JMS-HX110A tandem mass spectrometer.
The synthesis of 3 and 4 was carried out following the directions
given by Maleski (21).
2-Nitro-5-trideuteriomethoxyphenol (3). Crude 2 (7.63 g, 60 mmol)
was dissolved in propionic acid (60 mL). The solution was cooled to
approximately -5 °C, and during the following operations the
temperature was kept below 0 °C to minimize polynitration. With
stirring, another solution of NaNO2 (4.2 g, 60.9 mmol) in water (10
mL) was added dropwise, giving a deep red mixture. The resulting
slurry was stirred for a further 1 h, and HNO3 (100%, 7.58 g, 120.3
mmol) was added dropwise under a nitrogen atmosphere. The slurry
was stirred for 1 h and allowed to warm to room temperature (2 h).
Water (50 mL) was added slowly, the precipitate was filtered by suction,
and finally washing with 50% of aqueous propionic acid (60 mL) left
a curry-yellow product. Drying over KOH in vacuo yielded pure 3 (3.33
g, yield 32%): mp 91-92 °C; MS (DI), m/z 172 (M•+, 100), 156 (3),
1H NMR spectra were recorded on a Varian 300 MHz instrument. 13
C
NMR and correlated spectra (COSY, HETCOR) were recorded on a
Unity 400 MHz spectrometer. Infrared spectra were recorded as a KBr
sandwich using a Perkin-Elmer FT-IR spectrometer 1760X. Elementary
analyses were performed with a Flash EA 1112 series, CE instrument.
Synthesis of 6-Trideuteriomethoxybenzoxazolin-2-one. Trideu-
teriomethyl p-Toluenesulfonate (1). The compound was prepared
following essentially the directions given by Quast and Bieber (18).
Sodium hydride (30 g, 60%, 0.78 mol) was placed in a dry round-
bottom flask (500 mL). Diethyl ether (100 mL) was added, the
suspension was magnetically stirred, the hydride was allowed to settle
for 10 min, and the ether was decanted. Because NaH has a density of
1
142 (54), 114 (71); H NMR (DMSO-d6), δ 7.96 (d, 1H, J ) 9.1 Hz,
H-5), 6.62 (d, 1H, J ) 2.6 Hz, H-2), 6.58 (dd, 1H, J ) 2.6, 9.1 Hz,
H-4); 13C NMR (CDCl3), δ 157.97, 127.0, 109.5, 101.4; IR (cm-1
)
3195 w (OH), 2242 w, 2199 w and 2078 w (CD), 1620 s, 1591 s and
1527 m (m) (arom CdC), 1489 m and 1324 m (NdO), 1283 (CsO),
1204 m, 1094 s, 977 m, 834 m, 757 m, 676 m, 628 m. Found: C,
48.97; N, 8.08. Calcd for C7H4D3NO4: C, 48.84; H, 5.85; N, 8.14.

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1077
Table 2. MBOA, [D3]-MBOA, and Their Metabolites and Parameters for the Various Compounds Discussed in This Paper
6-[D3]-Methoxy-2(3H)-benzoxazolone (4). Water (∼5 mL) was
transferred into a 250-mL round-bottom flask. Formic acid (1.72 mL,
0.044 mol) was added, followed by KOH (2.51 g, 0.045 mol) dissolved
in abs EtOH (25 mL). Magnetic stirring and a N2 atmosphere were
applied during the whole procedure. Pd on charcoal (5%, 0.0574 g)
was transferred to the reaction mixture along with 3 (2.25 g, 0.013
mol). The resulting slurry was heated for 3 h at 70 °C while the color
changed from green to black. The reaction was completed after 2.5 h
as indicated by TLC (40% EtOAc in n-heptane, alumina plates coated
with silica gel 60 F254 obtained from Merck). After the contents of the
flask had cooled to room temperature, degassed water (7.5 mL) was
added. Phenyl chloroformate (PCF, 2.324 g, 0.014 mol) was transferred
via syringe and cannula to the reaction mixture, keeping the temperature
at 25-30 °C during the addition. The mixture was cooled to 25 °C,
and the resulting cherry red slurry was stirred for a further 0.5 h. A
solution of NaOH (0.5934 g, 0.015 mol) in degassed water (11 mL)
was added, followed by heating until a temperature of 45-50 °C was
reached. The mixture was allowed to react for 1 h at this temperature,
filtered, and washed with 50% of EtOH. Aqueous HCl (10%) was added
to the combined filtrates, resulting in precipitation of product 4.
The crude product was separated using a glass column equipped
with RP18 (15 µm) material (h, 3.8 cm; L, 2.2 cm) (obtained from
Novo Nordisk A/S). A gradient of MeCN in water was used, starting
with 30% of MeCN and increasing by 5% of MeCN until the first
fraction of [D3]-MBOA was observed. The chromotographing was then
continued isocratically. Recrystallization from toluene yielded pure light
reddish 4 (223.1 mg, yield 55%): mp 151.5-153 °C; MS (DI), m/z
Experimental Design of Degradation Studies. Two series of
degradation experiments for the benzoxazolinone compounds were
performed at 400 µg/g of soil. The experiments were performed with
MBOA and with the trideuterated analogue 6-deuteriomethoxybenzox-
azolin-2-one ([D3]-MBOA). A blank soil incubation series was included
as well, sampling and analyzing at the same time intervals as the
benzoxazolinone incubations. The same soil was used for all of the
experiments. A total of 59 soil samples, including control, stability,
MBOA and [D3]-MBOA samples, were incubated in the dark at 17
°C. At preselected time intervals (see Samples) the incubation of
selected samples was interrupted and the samples were freeze-dried
and extracted for analysis.
Soil Data. Sandy loam soil (Typic Agrudalf, classified according
to USDA Soil Taxonomy) (22) was collected in September 2003 from
the upper 0-10 cm at Nørregård, a cultivated field near the Research
Center Flakkebjerg, Denmark. The field had previously been cultivated
with spring-sown barley in 2001, 2002, and 2003. The upper 0-10
cm of soil was passed through a fine 2.0-mm sieve to separate larger
objects such as stones and plant parts, leaving only finer particles of
soil. It was stored at -17 °C until experimental usage. The character-
istics of the soil were as follows: fine sand (0.063-0.2 mm), 31.2%;
coarse sand (0.2-2.0 mm), 24.2%; silt (2-20 µm), 15.0%; coarse silt
(20-63 µm), 8.8%; clay (<2 µm), 18.0%; humus, 2.8%; potassium,
5.9%; phosphorus, 1.7%; magnesium, 6.2%; pH 6.8.
Degradation Experiment. Five grams of chemically and microbially
inert Ottawa sand (particle size, 20-30 mesh; Fisher Chemicals) was
added to a 100 mL Erlenmeyer flask. Methanolic solutions of MBOA
or [D3]-MBOA were added to obtain a final concentration of 400 µg/g
of soil for each incubation sample. Addition of the compounds in
methanolic solution to Ottawa sand and subsequent evaporation of
methanol was used to improve the distribution of MBOA and [D3]-
MBOA in soil added as follows: 11.48 g of moist Nørregård soil
corresponding to 10 g of dry soil was added to the 100 mL flask and
mixed; 0.52 mL of water was added to obtain a water content
corresponding to a measured field capacity of 17.4% water content.
1
168 (M•+, 100), 150 (35), 139 (5), 122 (8), 112 (10), 106 (18); H
NMR (CDCl3), δ 8.42 (br s, 1H, NH), 6.95 (d, 1H, J ) 8.8 Hz, H-4),
6.83 (d, 1H, J ) 2.3 Hz, H-7), 6.71 (dd, 1H, J ) 8.8 Hz, 2.34 Hz,
H-5); 13C NMR (CDCl3), δ 156.1 (C3), 155.9 (CdO), 144.5 (C1), 122.5
(C6), 110.0 (C5), 109.6 (C4), 97.5 (C2), 29.6 (CD3); IR (cm-1) 3166
s (NH), 3113 s and 3020 m (CH), 2260 w and 2074 w (CD), 1785 vs
(CdO), 1635 mw and 1460 mw (arom CdC), 1496 vs (NH), 1319 s
(CsN), 1208 ms-1097ms (CsO), 881 w, 845 m. Found: C, 56.88;
N, 8.15. Calcd for C8H4D3NO3: C, 57.14; H, 5.99; N, 8.33.

1078 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Etzerodt et al.
The flask was closed with a rubber stopper with cotton allowing air
circulation. All samples were incubated in the dark at 17 °C.
Samples. Incubations were terminated after 0, 2, 4, 7, 9, 14, 18, 22,
26, 33, 46, 60, 75, 90, and 105 days for control, MBOA, and [D3]-
MBOA. Each sample was stored at -17 °C until freeze-drying followed
by extraction by accelerated solvent extraction (ASE). Before ASE,
the freeze-dried samples were crushed homogeneously.
Extraction. The extraction was performed on an accelerated solvent
extractor (from Dionex) for each sample. The freeze-dried samples were
transferred to an extraction cell (30 mL) followed by extraction with
acidified methanolic solution (0.5%). The program for the extraction
procedure was as follows: preheat, 5 min; heat, 50 min; static, 5 min;
flush, 60%; purge, 60 s; cycles, 2; pressure, 1500 psi; temperature, 80
°C. Subsequently the samples were evaporated to a volume of 20 mL
with a N2 flush in a TurboVap LV evaporator from Zymark. Finally,
all extracts were stored at -17 °C until analysis by LC-MS.
Analysis. The samples were analyzed with LC-MS on a Sciex API
2000 model from Applied Biosystems. ESI was used as ionization
method, obtaining spectra in both positive and negative modes according
to the difference in sensitivity of the analytes for the two modes. For
the chromatographic part a RP18 BDS Hypersil column, 250 × 2.1
mm, particle size ) 5 µm and pore size ) 130 Å, was used. Two
mobile phases (A, 10% MeOH in water; and B, MeOH, both containing
20 µM glacial acetic acid) were used in a gradient system as follows
with a flow rate of 200 µL/min: step 0, 0-1 min, 90% A + 10% B;
step 1, 1-8 min, 30% A + 70% B; step 2, 8-15 min, 30% A + 70%
B; step 3, 15-16 min, 90% A + 10% B; step 4, 16-23 min, 90% A
+ 10% B. Standard solutions of MBOA, AMPO, and AAMPO were
prepared in methanol and used for verification of the signals of these
compounds. Recovery experiments for validation of the extraction
method have been performed earlier. The recoveries were 89% for
MBOA, 35% for AMPO, and 65% for AAMPO (23).
Figure 1. Aminophenoxazine basic structure with possible sites for
oxidation indicated by R1−R9 and O10. R′ ) H, Ac.
Table 2. Novel theoretical metabolites yet to be verified are
presented in Table 3.
We verified new masses using both MBOA and its trideu-
terated analogue 6-trideuteriomethoxybenzoxazolin-2-one to
establish whether unknown transformation products still con-
tained the 6-methyl group or not. By using LC-MS we could
control this by investigating an array of m/z values from ESI+
and ESI- mass spectrometry. Compounds exhibiting the same
transformation pattern as well as the same retention time (RT)
for the same m/z value for both transformation series (MBOA
and D3-MBOA) no longer contained the methyl group from
the original 6-position in MBOA and [D3]-MBOA. If the m/z
values differed by 3 according to the mass difference of the
MeO and D3CO groups, all other factors being equal the
transformation products still contained the original methyl group.
The detection of m/z values higher than the corresponding mass
of MBOA and [D3]-MBOA appearing at the same RT as MBOA
and [D3]-MBOA and with uniform transformation patterns was
caused by the formation of adducts of MBOA and [D3]-MBOA,
respectively. This was controlled using external standards for
MBOA and [D3]-MBOA. The results of all samples were
compared with results from blank soil, incubated for the same
time interval and analyzed with the same procedure. The
measurements from the blank samples were subtracted from the
measurements of the benzoxazolinone samples.
RESULTS AND DISCUSSION
Several types of microorganisms are responsible for the
enzymatic transformation of secondary metabolites. This trans-
formation includes hydroxylation by hydroxylases such as regio-
and chemoselective non-heme iron-monoxidases (24-28), re-
gioselective flaVoprotein monoxygenases (28-30), and cyto-
chrome P450 enzymes (31). The latter is neither regio- nor
chemoselective, giving rise to several possible hydroxylated
derivatives. Furthermore, N-oxidation is possible by flaVopro-
teins and cytochrome P450 enzymes (32-36). Additional
metabolization is also possible via methyl transferases (upon
previous introduction of OH groups) (37) and N-acetyl trans-
ferases.
Fomsgaard et al. (38) published a review on microbial
transformation products of BOA, MBOA, and 2-hydroxy-1,4-
benzoxazin-3-one (HBOA). The compounds that were listed in
the review, formed from BOA, MBOA, or HBOA by fungi in
liquid media, were N-(2-hydroxyphenyl)acetamide and further
hydroxy- and nitro-substituted derivatives thereof, N-(2-hydroxy-
phenyl) malonamic acid, N-(2-hydroxy-4-methoxyphenyl)ma-
lonamic acid, APO, various hydroxylated and acetylated sub-
stitutes of APO, and AMPO (1, 15, 16, 39, 40). APO, AMPO,
and in some cases the acetylated derivative have been reported
as soil transformation products of BOA and MBOA (2-6, 14,
23). A 4-acetyl derivative of BOA has also been observed (41).
Several m/z values corresponding to the different possible
hydroxy and methoxy derivatives of the APO structure were
investigated. A large number of metabolites from BOA and
MBOA are supposed to be identical or analogously formed.
Thus, a reasonable hypothesis regarding m/z values to search
for could be established.
For transformation products discovered in this experiment,
consult Schemes 1 and 2, respectively. In the schemes are also
given the possible relationship between the respective metabo-
lites.
The experiments were initiated by adding MBOA and [D3]-
MBOA to soil in order to identify the outcoming transformation
products. Therefore, the transformation patterns for these
compounds were determined initially. For ESI+ [MBOA + H]+,
m/z 166, and for [[D3]-MBOA + H]+, m/z 169 (RT ) 14.7
min) the observed transformation was plotted as the area of the
signals as a function of time (see Figure 2). This unique
transformation pattern was repeatedly observed for several
adduct ions besides the [MBOA + H]+ ion, including the Na+
adduct. It was notable that the transformation was very rapid.
The same unique pattern was observable in ESI- ([MBOA -
H]-, m/z 164) for which only the course of MBOA is plotted
(Figure 3) as it is representative of both compounds according
to the ESI+ mode.
Verification of m/z 243 (for MBOA incubation samples) and
246 (for [D3]-MBOA incubation samples) in ESI+ was certainly
observed as signals appeared at the same RT (17.5 min)
illustrated in Figure 4. These m/z values correspond to [AMPO
+ H]+ and [[D3]-AMPO + H]+.
In studies by Gents et al. (2) a chromatographic method very
similar to ours was used. In that study BOA appeared at RT )
14.0 min, APO at RT ) 17.0 min, and AAPO at RT ) 17.5
min. In the present studies MBOA appeared at 14.7 min, and
by analogy with this AMPO/[D3]-AMPO should appear at ∼17.7
For an overview of the parent APO structure with possible
positions for oxidation, see Figure 1. Metabolites identified in
previous studies and methoxylated analogues are shown in

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1079
Table 3. Parameters for the Various Possible Compounds Discussed in This Paper According to Our Hypothesesa
a For all isomers N-oxidation is included as an isomeric compound as well. For each compound two masses are shown: the first for the nonlabeled compound and the
second for the labeled compound.
min and AAMPO/[D3]-AAMPO at ∼18.2 min (for these
acetylated derivatives, see below).
added/g of soil APO reached the maximum concentration at
day 50, after which a decrease in concentration of APO was
observed.
This was consistent with the observed RT for these m/z
values, which were determined to be AMPO and [D3]-AMPO.
The identity of AMPO was confirmed by comparison with the
retention time and mass spectra of a pure AMPO standard.
Both AMPO and its isotopomer were present in only small
amounts on day 0, ascending to constant significant levels from
day 20 through day 105 (see Figure 4). If further transformation
products should be formed, the velocity of transformation of
AMPO/[D3]-AMPO must be almost equal to the velocity of
formation because no net change in area and thereby concentra-
tion is observed.
Studies concerning MBOA and its transformation into AMPO
at a concentration of 0.4 mg of MBOA added/g of soil (6)
showed AMPO to have only a slight decrease in concentration
after day 20. That was the same concentration of MBOA as
studied in this paper. In this paper levels of AMPO appeared
to be constant. The difference could be due to the fact that
different kinds of soil were used. This might cause a difference
in the pattern of formation and disappearance of AMPO due to
possible differences in the microbial environment in different
kinds of soil.
Fomsgaard et al. (6) showed that AAMPO was formed from
AMPO by microorganisms in soil. This could be confirmed by
the results presented in this paper. Figure 5 shows the time-
dependent course of formation of AAMPO and [D3]-AAMPO.
In ESI+ with RT of 18.2 min m/z 285 and 288 corresponding
to [AAMPO + H]+ and [[D3]-AAMPO + H]+ were observed
with a transformation pattern similar to that observed for AMPO
and [D3]-AMPO. Only the levels at which the signals were
In contrast to earlier studies of transformation of MBOA (4)
the concentration of formed AMPO remained constant, whereas
Kumar et al. (4) showed that the concentration of AMPO
decreased after day 20. The transformation rate is concentration
dependent (6). In the studies by Understrup et al. (3) APO
formed from BOA reached a maximum concentration at day 7
and was completely transformed after 20 days at a concentration
of 0.4 mg of BOA added/g of soil, whereas at 4 mg of BOA

1080 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Etzerodt et al.
Scheme 1. Metabolites Formed from MBOA in Biotransformation by Microorganisms in Soila
a Degradation products are illustrated along with their acronyms and mass values.
detected relative to AMPO were significantly smaller as also
shown by Kumar et al. (4) and Fomsgaard et al. (6).
As stated previously, a comparison with Gents et al. (2) was
possible regarding RT, and certainly a RT of 18.2 min was
observed, confirming the identity of these m/z values. The
identity of AAMPO was additionally confirmed by comparison
of retention time and mass spectrum with the retention time
and mass spectrum of a pure standard of AAMPO dissolved in
methanol and injected into the chromatographic system.
Figures 4 and 5 show that AMPO and [D3]-AMPO are
formed first, followed by the acetylation to AAMPO and [D3]-
AAMPO.
For measurements using ESI-, m/z 209 and 212 correspond-
ing to masses 210 and 213, respectively, were observed at RT
) 13.9 min. The signal appeared with a maximum at day 0
and decreased rapidly until day 10, when it reached a minimum,
which remained constant as shown in Figure 6. It was observed
for both the MBOA and the [D3]-MBOA incubation samples.
The transformation product (masses 210 and 213) has provi-
sionally been characterized to be nitro-MBOA.
Because this metabolite appeared in the [D3]-MBOA incuba-
tion samples 3 m/z values higher relative to the MBOA
incubation samples, the integrity of the trideuteriomethoxy group
was conserved. The formation of this metabolite from MBOA
and [D3]-MBOA was very rapid, because the maximum signal
of the metabolite was observed at day 0. In ESI+ m/z 241 and
247 could be correlated, having the same RT ) 20.5 min
(Figure 7), the first for the MBOA samples and the latter for
the [D3]-MBOA samples corresponding to masses 240 and 246,
respectively. The signals were detected with a similar area. The
patterns of these curves were comparable to those for AMPO
and [D3]-AMPO. Both kinds of metabolites were formed to a
maximum around day 20, after which a constant level was seen.
This was believed to be N-(2,4-dimethoxyphenyl)malonamic
amide. The existence of methyl-accepting chemotaxis proteins
(MCPs) (42) indicated the possibility of transformation products

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1081
Scheme 2. Possible Structure of Metabolites Provisionally Identified from MBOA in Biotransformation by Microorganisms in Soila
a Degradation products are illustrated along with their acronyms and mass values.
from MBOA being methylated. The enzymes involved in the
studies by Albert et al. (42) used S-adenosylmethionine as the
methyl-transferring agent. For a certain amount of substrate
present, equilibrium between different degrees of methylation
occurs. Therefore, it is plausible to assume the same to be
-
Figure 2. Transformation of MBOA (m/z 166) and [D3]-MBOA (m/z 169)
Figure 3. Transformation of MBOA (m/z 164) measured using ESI (RT
)
+
measured using ESI (RT
)
14.7 min). Area is measured as a function
14.7 min). Area is measured as a function of time. Degradation
experiment was performed in Flakkebjerg soil with an initial concentration
of 400 g/g of MBOA.
of time. Degradation experiment was performed in Flakkebjerg soil with
initial concentrations of 400 g/g of MBOA and [D3]-MBOA.
µ
µ

1082 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Etzerodt et al.
Figure 7. Formation and disappearance of mass 240 (m/z 241) and 246
(m/z 247) from MBOA and [D3]-MBOA, respectively, measured using ESI
Figure 4. Formation and disappearance of AMPO (m/z 213) and [D3]-
+
AMPO (m/z 216) from MBOA and [D3]-MBOA, respectively, measured
+
(RT
)
20.5 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
using ESI (RT
) 17.5 min). Area is measured as a function of time.
concentrations of MBOA and [D3]-MBOA of 400
µ
Degradation experiment was performed in Flakkebjerg soil with initial
concentrations of 400 g/g of MBOA and [D3]-MBOA.
µ
Figure 8. Formation and disappearance of mass 257 (m/z 258) and 260
+
(m/z 261) from MBOA and [D3]-MBOA, respectively, measured using ESI
Figure 5. Formation and disappearance of AAMPO (m/z 255) and [D3]-
(RT
)
17.2 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
+
AAMPO (m/z 258) measured using ESI (RT
) 18.2 min). Measured
concentrations of MBOA and [D3]-MBOA of 400
µ
area is a function of time. Degradation experiment was performed in
Flakkebjerg soil with initial concentrations of 400
MBOA.
µg/g of MBOA and [D3]-
as fungi, which also supports the theory that this compound
could be N-(2,4-dimethoxyphenyl)malonamic amide. See Table
3 for the structural formula. For the MBOA and [D3]-MBOA
incubation samples, masses 257 and 260 were observed as m/z
258 and 261, respectively, in ESI+ (see Figure 8). The signals
were detected with a RT of 17.2 min. Both signals existed from
day 0 until the end of the experiment. The levels of both
increased until maximum around day 10. The rest of the
experiment showed a slight decrease with signals yet detectable
at day 105. The slight decrease could be due to simultaneous
formation and disappearance.
The compound was provisionally characterized to be a
dihydroxy derivative of HMPMA. Although malonamic acids
are known to have small RTs (16), possible N-oxidation and
intramolecular H-bonding can increase the RT.
Because the nonlabeled compound exhibited an odd mass, it
contained an odd number of nitrogen atoms. Compared to the
mass of HMPMA of 225, the mass difference was 32, corre-
sponding to the introduction of two additional hydroxy groups,
either in the aromatic part of the molecule or as an N-oxidation.
A different kind of metabolite observed in this experiment
was masses 287 and 290 detected as m/z 288 and 291 at RT )
17.3 min in ESI+ for MBOA and [D3]-MBOA incubation
samples, respectively (see Figure 9). The metabolite was present
in a significant amount from day 0, increasing to its maximum
around day 4. The concentration decreased rapidly from here
on until around day 14, followed by a slight decrease to the
end of the experiment.
Figure 6. Formation and disappearance of mass 210 (m/z 209) and 213
(m/z 212) from MBOA and [D3]-MBOA, respectively, measured using ESI
-
(RT
)
13.9 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
concentrations of MBOA and [D3]-MBOA of 400
µ
possible for the microorganisms in soil responsible for the
transformation of MBOA and its degradation products.
Because the difference in mass between the transformation
product from the nonlabeled and the labeled MBOA was 6, two
of the original methyl groups appeared to be present. This was
most likely due to the formation of a methylating pool. This
pool could convert an appropriate methoxyphenol to the
corresponding dimethoxy derivative by methylation of the
phenolic OH group. Yue et al. (1) showed that aminophenols
are transformed into malonamic acids by microorganisms such
The mass of the transformation product was consistent with
an isomer of nitro-AMPO. The metabolite had an odd mass for

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1083
Figure 11. Formation and disappearance of mass 316 (m/z 315) and
Figure 9. Formation and disappearance of mass 287 (m/z 288) and 290
(m/z 291) from MBOA and [D3]-MBOA, respectively, measured using ESI
+
319 (m/z 318) from MBOA and [D3]-MBOA, respectively, measured using
-
ESI (RT
)
16.5 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
(RT
)
17.3 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
concentrations of MBOA and [D3]-MBOA of 400
µ
concentrations of MBOA and [D3]-MBOA of 400
µ
for biotransformation (16). The metabolite still contained the
original methyl group as established by the appearance in both
kinds of incubation samples at m/z values differing by 3.
Masses 316 and 319 were observed in ESI- as m/z 315 and
318 from MBOA and [D3]-MBOA incubation samples, respec-
tively. Consequently, the integrity of the original methyl group
was conserved. Both illustrations are included in Figure 11,
showing a maximum at day 9. The concentration decreased
rapidly until day 20, followed by a slight decrease throughout
the rest of the experiment. The signals were observed with RT
) 16.5 min. N-(Acetyl)-dihydroxy-AMPO, N-(hydroxyacetyl)-
hydroxy-AMPO, or N-oxidized isomers are the most likely
candidates, reflecting the lowered RT relative to AMPO. The
even mass for the nonlabeled compound corresponded to an
even number of nitrogen atoms present in the molecule. The
difference in mass relative to AMPO was 74 amu. The fact that
OH groups lower the RT implies an AMPO oxidation product.
The presence of two such groups rendered 42 amu most likely
to be an acetyl group conforming to the mentioned provisionally
characterized compounds. The time course of the formation of
the metabolites in Figures 5, 9, 10, and 11 indicates that the
addition of hydroxy and nitro groups in the AMPO molecule is
a faster process than the addition of the acetyl group.
Figure 10. Formation and disappearance of mass 303 (m/z 304) and
306 (m/z 307) from MBOA and [D3]-MBOA, respectively, measured using
+
ESI (RT
)
16.1 min). Area is measured as a function of time with initial
g/g in Flakkebjerg soil.
concentrations of MBOA and [D3]-MBOA of 400
µ
the nonlabeled compound, implying the presence of an odd
number of nitrogen atoms. When this metabolite is compared
to AMPO, the difference regarding mass was 45 m/z values,
corresponding to a nitrated derivative. The fact that acetamides
could be nitrated via endophytic fungi (16) makes this a
plausible hypothesis.
In ESI+ masses 303 and 306 were observed at RT ) 16.1
min as m/z 304 and 307 for the MBOA and [D3]-MBOA
incubation samples, respectively. This is illustrated in Figure
10. The signal appeared at day 2, reaching maximum on day 9
and followed by a decrease until a constant minimum from day
20 throughout the time of the experiment. This was likely to
be an isomer of nitro-hydroxy-AMPO according to the RT as
well as the difference in mass relative to mass 287, which
corresponded to an isomer of nitro-AMPO. When Figure 4,
the formation of AMPO, is compared with Figure 9, the
formation of nitro-AMPO, and Figure 10, the formation of
hydroxy-nitro-AMPO, the development of the curves during
time supports this theory. AMPO is formed quickly because it
is present at day 0. Nitro-AMPO can thus be formed from day
0 as well, whereas hydro-nitro-AMPO seems to not be present
in significant amounts until day 1. It must be stressed that the
formation of a metabolite of AMPO should not be expected to
coincide with the decline of the AMPO curve. During the
increase of the curve, the formation of the compound is faster
than the disappearance () further transformation), but disap-
pearance may have occurred. Mass 303 corresponded to a
metabolite containing an odd number of nitrogen atoms for
nonlabeled compounds. Therefore, it seemed to be reasonable
to expect a nitrated derivative because this is a convenient path
These results show a new range of transformation products
not detected before. A large number of transformation products
were discovered in previous experiments, emphasizing the
importance of further investigation in this field. A total outline
of the degradation path or transformation path of biologically
active secondary metabolites from cereals is crucial if the
properties of the cereals for producing these compounds are to
be utilized as a viable alternative to synthetic pesticides. The
target effect of these compounds toward soilborne pests and
insects and toward weeds depends on the biological effects of
the transformation products. The relatively limited knowledge
of the environmental impact of the degradation products makes
further identification needed as well.
The identities of the new transformation products described
in this paper have been provisionally characterized and are under
further investigation in our laboratory.
ACKNOWLEDGMENT
We are grateful to Dr. Fransisco Mac´ıas for providing AMPO
and AAMPO and to Dr. Scott Chilton, University of North
Carolina, for providing AMPO. We are also grateful to Associate
Professor Per Halfdan Nielsen from the Department of Chem-

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Received for review April 19, 2005. Revised manuscript received
October 10, 2005. Accepted December 6, 2005. The research presented
here was performed as a part of the project “FATEALLCHEM”, “Fate
and Toxicity of Allelochemicals in Relation to Environment and
Consumer”. The project was carried out with financial support from
the Commission of the European Communities under the Work
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JF0509052