Microbial Transformations of the Fungicide Cyprodinil (CGA-219417)

Article abstract of DOI:10.1021/jf970298l

A collection of 12 microbial cultures, known to contain cytochrome P-450 monooxygenase or other degradative enzymes, was screened for their ability to degrade the Novartis Crop Protection Inc. developmental fungicide cyprodinil (CGA-219417; 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine). Ten of the 12 cultures produced a monohydroxylated metabolite in yields ranging from 1.2 to 35.6%. The filamentous fungus, Beauveria bassiana ATCC 7159, produced a methoxylated glycoside of the monohydroxylated metabolite with a yield of 80%. Dihydroxylated metabolites and a molecular cleavage product, 4-cyclopropyl-6-methyl-2-pyrimidamine, were also detected in certain cultures. The overall results of the study indicated that cyprodinil was readily metabolized by a variety of microbial species. Metabolites generated by these cultures can potentially be used as analytical reference standards to support animal, plant, and soil metabolism studies.

Full text of DOI:10.1021/jf970298l

J. Agric. Food Chem. 1997, 45, 3647
−3651
3647
Micr obia l Tr a n sfor m a tion s of th e F u n gicid e Cyp r od in il
(
CGA-219417)
Mark J . Schocken,*,†,‡ J ohn Mao,†,§ and Daniel J . Schabacker|
Springborn Laboratories, Inc., Wareham, Massachusetts 02571, and Novartis Crop Protection, Inc.,
Greensboro, North Carolina 27419
A collection of 12 microbial cultures, known to contain cytochrome P-450 monooxygenase or other
degradative enzymes, was screened for their ability to degrade the Novartis Crop Protection Inc.
developmental fungicide cyprodinil (CGA-219417; 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidin-
amine). Ten of the 12 cultures produced a monohydroxylated metabolite in yields ranging from 1.2
to 35.6%. The filamentous fungus, Beauveria bassiana ATCC 7159, produced a methoxylated
glycoside of the monohydroxylated metabolite with a yield of 80%. Dihydroxylated metabolites and
a molecular cleavage product, 4-cyclopropyl-6-methyl-2-pyrimidamine, were also detected in certain
cultures. The overall results of the study indicated that cyprodinil was readily metabolized by a
variety of microbial species. Metabolites generated by these cultures can potentially be used as
analytical reference standards to support animal, plant, and soil metabolism studies.
Keyw or d s: Cyprodinil; microbial transformations; metabolite identification; reference standards
INTRODUCTION
In this study, a collection of 12 microbial cultures (9
filamentous fungi, 2 streptomycetes, and 1 bacterium)
was screened for their ability to generate metabolites
of the developmental fungicide cyprodinyl (Heye et al.,
The use of common microbes to synthesize potential
metabolites of agrochemicals in sufficient quantities can
provide an important alternative to conventional chemi-
cal synthesis or to predict environmentally relevant
degradation pathways. Examples from the published
literature include insecticides (Sariaslani et al., 1987;
Maloney et al., 1988) and herbicides (Schocken et al.,
1
994; Figure 1). The objective was to generate metabo-
lite reference standards by microbial rather than chemi-
cal means as well as to predict the environmental fate
of this agrochemical. Selection of cultures was, in
general, based on published literature demonstrating
their ability to metabolize a wide variety of xenobiotics.
1
989; Dietrich et al., 1995; Liu et al., 1996; Kulowski et
al., 1997). Most interestingly, these common microor-
ganisms can metabolize xenobiotics in a manner re-
markably similar to that shown by mammals, birds,
fish, soil, and, to some extent, plants. This similarity
in metabolite profile is largely explained by the presence
of the enzyme cytochrome P-450 monooxygenase in
these organisms. This enzyme is capable of causing
aliphatic and aromatic hydroxylations as well as N-, S-,
and O-dealkylations on a wide range of xenobiotic
substrates. Because of this similarity in metabolite
profiles to biological systems tested in the agrochemical
industry (e.g., in rat, goat, poultry, fish, plant, and soil
metabolism studies), microbial cultures can be used to
synthesize metabolites in sufficient quantities (i.e.,
milligrams) to obtain spectra for identification purposes.
Subsequently, these metabolites can be generated in
larger quantities (i.e., grams) through larger scale
transformations to serve as reference standards in
support of agrochemical metabolism and environmental
fate studies. In addition to providing an alternative to
chemical synthesis, the microbial approach can also be
used to predict metabolites in soil, mammals, birds, fish,
and plants before the bona-fide metabolism studies are
conducted.
MATERIALS AND METHODS
Ch em ica ls. Cyprodinil (4-cyclopropyl-6-methyl-N-phenyl-
2
-pyrimidinamine) was synthesized by Novartis Crop Protec-
tion, Inc., and had a purity of >99.9%, based on high-
1
4
performance liquid chromatography (HPLC). [2- C-(pyri-
midinyl)]Cyprodinil was also synthesized by Novartis Crop
Protection, Inc., and had a specific activity of 51.1 µCi/mg.
Radiochemical purity was determined to be 100% by HPLC
with radiometric detection (RAM). Solvents used for HPLC
and extractions were purchased from Burdick and J ackson and
were of HPLC grade. PIC A Low UV, an ion-pairing reagent
that minimizes background interference in the low-UV range,
was purchased from Waters Corp. Water was obtained with
a Sybron/Barnstead NANOpure II system and meets ASTM
Type IIA requirements. Soybean grits were purchased from
Archer Daniels Midland Co., Decatur, IL. All other materials
were of the highest purity commercially available.
Micr oor ga n ism s. The microbial cultures used for this
study were obtained from the American Type Culture Collec-
tion (ATCC), located in Rockville, MD (Table 1). The pure
cultures were maintained on agar slant tubes containing
appropriate growth media as recommended by the ATCC.
Microbial transformations were conducted by the two-stage
incubation protocol previously described by Betts et al. (1974).
1
4
Cyprodinil (10 mg of unlabeled cyprodinil and 3 µCi of [ C-
pyrimidinyl]cyprodinil) was dissolved in 500 µL of ethanol and
added to 24-72-h-old stage II, 50-mL cultures (200 mg/L; 0.9
mM). Cultures were harvested after 6 or 7 days. Cells were
separated from broths by centrifugation (except for Rhizopus
oryzae, for which vacuum filtration was employed). The pH
of the broths was recorded and adjusted to approximately pH
*
Author to whom correspondence should be ad-
dressed.
†
Springborn Laboratories, Inc.
Present address: Uniroyal Chemical Co., Inc., Mid-
‡
dlebury, CT 06749.
§
Present address: Vion Pharmaceuticals, Inc., New
6
and subsequently partitioned with 3 × 50 mL of ethyl
Haven, CT 06511.
acetate. Ethyl acetate portions were combined, concentrated
by rotary evaporation at 37 °C, and dried under a stream of
|
Novartis Crop Protection, Inc.
S0021-8561(97)00298-7 CCC: $14.00
© 1997 American Chemical Society

3648 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Schocken et al.
HPLC profiling was accomplished with a Waters Model 510
solvent pump equipped with a Hewlett-Packard M-1050 au-
tosampler, an Autochrom M-112-2 gradient controller, either
a Kratos 757 or a Hewlett-Packard 1049A UV detector, and a
Radiomatic A-280 radioactivity detector. Metabolites were
separated on a Metachem Nucleosil C18 analytical column (4.6
×
250 mm) by using a 20-min linear gradient starting with
1
1
8
00% of solvent A [acetonitrile/water/PIC A Low UV (20:80:
)] to 100% of solvent B [acetonitrile/water/PIC A Low UV,
0:20:1)] and then holding 100% solvent B for an additional 9
-1
min. The mobile phase flow rate was 1.0 mL min . Detection
was accomplished by UV at 230 nm and by flow-through
radiometric detection using a 500-µL liquid flow cell with a
F igu r e 1. Chemical structure of cyprodinil (CGA-219417; MW
225).
)
-
1
cocktail flow rate (Flo-Scint II) of 3.0 mL min
.
Ta ble 1. P r od u ction of th e Mon oh yd r oxyla ted
Meta bolite of Cyp r od in il (Meta bolite 1) by Micr obia l
Cu ltu r es
Thin-layer chromatography (TLC) for isolating metabolites
of cyprodinil was performed with Whatman 20 × 20 cm, 250-
µm thick, fluorescent TLC plates. Plates were developed to
1
5 cm in a solvent system comprised of methylene chloride/
methanol (9:1). After evaporation of solvent from the plates
was allowed for, parent compound and metabolites were
located by the quenching of short-wavelength UV light. Bands
corresponding to major metabolites were traced lightly with
a lead pencil and scraped from the plates using a spatula.
Metabolites were eluted from silica gel with 5-mL portions of
ethyl acetate or a 1:1 solution of ethyl acetate/methanol.
extent of
conversion (%)
microbial culture
1
C. echinulata var. elegans ATCC 36112
C. echinulata var. elegans ATCC 9245
C. echinulata var. echinulata ATCC 9244
A. pseudocylindraspora ATCC 24169
S. griseus ATCC 13273
S. rimosus ATCC 10970
Mucor circinellosides f. griseocyanus
ATCC 1207a
Bacillus megaterium ATCC 14581
R. oryzae ATCC 24563
B. bassiana ATCC 7159
Phanerochaete chrysosporium ATCC 24725
Penicillium chrysogenum ATCC 10002
1.2
2.0
Proton nuclear magnetic resonance spectra ( H-NMR) were
obtained on a Bruker 300 MHz at the University of Rhode
Island NMR Research Laboratory under the direction of Dr.
Michael A. McGregor. Samples were dissolved in deuterated
methanol.
LC/MS was accomplished with a Hewlett-Packard 5989A
MS engine with a particle beam interface, a Hewlett-Packard
25.6
35.6
24.8
28.0
5.4
1
050 gradient pump, and a Hewlett-Packard autosampler. In
most cases, chromatography included a Metachem Nucleosil
18, 10 µm) 4.6 × 250 mm column and an isocratic mobile
phase composed of acetonitrile/water (70:30) at a flow rate of
1.5
20.5
0
0
0
(C
-
1
0
.8 mL min . For analysis of the postextracted broth of C.
echinulata var. elegans (ATCC 9245), chromatography included
a Metachem Nucleosil (C18, 5 µm) 2 × 150 mm column and a
linear gradient starting at 25 mM ammonium acetate, pH 6.5
nitrogen. The residue was redissolved in several milliliters
of acetonitrile/water (1:1) and analyzed by HPLC-RAM and,
in selected cases, by liquid chromatography/mass spectrometry
(100%), and ending at 20% 25 mM ammonium acetate, pH 6.5/
8
0% acetonitrile. For mass spectrometry, ionization was by
(
LC/MS). Cells were lysed and extracted in either acetone or
electron impact at 70 eV with a source temperature of 250 °C,
an analyzer temperature of 120 °C, and a desolvation tem-
perature of 55 °C. Helium pressure was 55-60 psi. In most
cases, scan range was from 60 to 300 amu except in the
analysis of the postextracted broth of C. echinulata var. elegans
methanol using a Branson Model 450 sonifier. The cell
extracts were transferred to 50-mL centrifuge tubes and
centrifuged at 3000 rpm for 1 h. Portions of the cell extracts
were dried with a combination of rotary evaporation and a
nitrogen stream, redissolved in acetonitrile/water (1:1), and
analyzed by HPLC-RAM and, in selected cases, by LC/MS.
Because of substantial radioactivity remaining in the post-
partitioned broths of the Cunninghamella echinulata var.
elegans cultures (46-48%), postpartitioned broths were acidi-
fied with concentrated HCl to an approximate pH of 2 and
repartitioned with an equal volume of ethyl acetate in an
attempt to remove acidic metabolites from the aqueous phase.
Afterward, a 10-mL aliquot of the postpartitioned broth from
C. echinulata var. elgans (ATCC 9245) was adjusted to pH 12
with 1N NaOH and repartitioned with an equal volume of
methylene chloride to remove basic metabolites from the broth.
An aliquot of the acidic postpartitioned broth was concentrated
under a nitrogen stream and analyzed by HPLC-RAM and LC/
MS to identify and characterize metabolites. (The postparti-
tioned broths of Absidia pseudocylindraspora, Streptomyces
griseus, and Streptomyces rimosus also contained radioactivity,
although considerably less than the C. echinulata var. elegans
cultures. Therefore, detailed characterization of the postpar-
titioned broths from these cultures was not pursued.)
(ATCC 9245), for which the scan range was from 10 to 650
amu.
RESULTS AND DISCUSSION
The HPLC-RAM profile of the ethyl acetate extract
of the broth from S. rimosus indicated a major metabo-
lite (metabolite 1) eluting at 22.70 min (retention time
of cyprodinil was 28.40 min). The total ion chromato-
gram and the electron impact mass spectrum (EI-MS)
of this metabolite are provided in Figure 2, indicating
a molecular ion at m/z 241 and a prominent fragment
at m/z 240, suggesting a monohydroxylated metabolite
of the parent compound (molecular weight of cyprodinil
is 225). Subsequently, this metabolite was isolated in
a greater quantity by preparative TLC (ca. 200 µg), and
1
a
H-NMR spectrum was recorded as illustrated in
Figure 3. The symmetric and relatively simple proton
coupling pattern in the aromatic region (signals at 6.7
and 7.4 ppm) is consistent with a para-disubstituted
benzene structure (Silverstein et al., 1974), indicating
that the position of hydroxylation of cyprodinil appears
to have occurred on the phenyl ring, para to the amino
group (structure provided in the inset to Figure 3). The
same metabolite was also detected in the cell extract of
S. rimosus as well as in eight other cultures included
in the microbial screen in yields ranging from 1.2 (C.
A control consisted of cyprodinil added to the soybean grit-
glucose medium in the absence of microorganisms.
An a lytica l Meth od s. Radioactivity was quantified by
liquid scintillation counting (LSC) using a Beckman Model LS
5
000 TD or a Beckman Model LS 1801 liquid scintillation
counter calibrated with factory-prepared standards. Counting
efficiencies of all experimental samples were determined using
an external standard and a factory-prepared calibration curve
(Beckman Instruments). All test samples were counted for a
maximum of 5 min or until a 2σ error of 5% was attained.

Microbial Transformations of Cyprodinil
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3649
F igu r e 2. LC/MS of the ethyl acetate extract of the broth from
S. rimosus.
F igu r e 4. 1H-NMR spectrum of the major metabolite pro-
duced by B. bassiana.
nonconjugated metabolite was misleading since the
sugar portion apparently was lost as a result of the
relatively energetic ionization method. Of note, a meth-
oxylated sugar conjugated to a metabolite of the herbi-
cide propham was also formed from B. bassiana (then
called B. sulfurescens) and reported by Vigne et al.
(
1986).
In the fungus A. pseudocylindraspora, two major
metabolites of cyprodinil were produced. The major
metabolite, accounting for a 35.6% yield, was identified
1
as metabolite 1. Its identity was based on H-NMR and
chromatographic and mass spectral comparisons to the
authentic reference standard. The other metabolite
(
metabolite 3), produced in a 12% yield and somewhat
1
more polar, had a H-NMR spectrum indicating a
pattern of a para-disubstituted phenyl ring with loss of
the proton (ca. 6.5 ppm) signal on the pyrimidine ring.
The EI-MS of this metabolite indicated a molecular ion
of m/z 257, suggesting a dihydroxylated metabolite. Both
metabolites of A. pseudocylindraspora are presented in
Figure 5, which also depicts pathways of cyprodinil
metabolism in the various microbial cultures.
F igu r e 3. 1H-NMR spectrum of the major metabolite pro-
duced by S. rimosus.
echinulata var. elegans ATCC 36112) to 35.6% (A.
pseudocylindraspora). A summary of the production of
the monohydroxylated metabolite is provided in Table
The ethyl acetate extract of the broth of both isolates
of the fungus C. echinulata var. elegans (ATCC 36112
and ATCC 9245) had comparable radiocarbon contents
1
.
Additional confirmation of the identity of this
metabolite (produced by A. pseudocylindraspora) was
accomplished by cochromatography (HPLC) of the mi-
crobial metabolite with the authentic reference stan-
dard.
(13.3 and 14.5% of the dose) and showed similar HPLC-
RAM profiles. LC/MS analysis from C. echinulata var.
elegans ATCC 36112 indicated polar metabolites with
molecular ions at m/z 241 and 257, suggesting mono-
and dihydroxylated metabolites of cyprodinil. On the
basis of the EI-MS fragmentation pattern (i.e., promi-
nent fragment ions at m/z 148 and 149, representing
an unsubstituted methylcyclopropylamino pyrimidine
moiety), the position of dihydroxylation is thought to
occur on the phenyl ring rather than on the pyrimidine
or cyclopropyl rings (Figure 5; metabolite 4). LC/MS
analysis of ATCC isolate 36112 also indicated a me-
tabolite with an apparent molecular ion of m/z 149
(Figure 5; metabolite 5). This would be consistent with
a molecular cleavage product of cyprodinil. The HPLC
retention time of the authentic reference standard
(14.88 min) was similar to that of the microbial me-
tabolite (14.80 min), providing additional confirmation.
Both fungal isolates showed significant amounts of
radiocarbon (46-48%) remaining in the broth after ethyl
acetate partitioning. Therefore, extracted broths were
adjusted to an approximate pH of 2 and repartitioned
with ethyl acetate. A small portion of radiocarbon was
A predominant metabolite (metabolite 2; produced in
0% yield and present in both the broth and cell extract)
8
from Beauveria bassiana had an HPLC retention time
of 16.6 min. Its mass spectrum had an apparent
molecular ion of m/z 241 but a shorter chromatographic
retention time on LC/MS, suggesting a monohydroxyl-
ated metabolite different from the para-substituted
metabolite described above. Approximately 2 mg of this
1
metabolite was isolated by preparative TLC and a H-
NMR spectrum obtained, which is illustrated in Figure
4
.
As shown, the proton splitting pattern in the
aromatic region indicated substitution para to the amino
group on the phenyl ring. However, the multiplex of
signals in the region of 3.0-4.0 ppm is consistent with
a methoxylated carbohydrate with the anomeric proton
1
at ca. 4.8 ppm. Thus, the H-NMR spectrum indicated
a monohydroxylated metabolite of cyprodinil (para-
disubstituted phenyl ring moiety) which was conjugated
to a sugar. The apparent molecular ion at m/z 241
(obtained by the mass spectral analysis) suggesting a

3650 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Schocken et al.
F igu r e 5. Pathways of cyprodinil metabolism in microbial cultures.
removed (5%), suggesting the presence of acidic me-
tabolites. Since substantial radiocarbon remained, the
extracted broth of C. echinulata var. elegans (ATCC
The presence of conjugates in the B. bassiana and C.
echinulata var. elegans cultures is indicative of phase
II reactions in mammalian metabolism. C. echinulata
var. elegans has previously been reported to form
conjugates with a variety of polycyclic aromatic hydro-
carbon metabolites including anthracene (sulfate con-
jugate; Cerniglia, 1982), phenanthrene (sulfate and
glucoside conjugates; Casillas et al., 1996), fluoranthene
(glucoside conjugates; Pothuluri et al., 1992), pyrene
(glucoside conjugates; Cerniglia et al., 1986), benzo[a]-
pyrene (sulfate and glucuronide conjugates; Cerniglia
and Gibson, 1979), and benzo[e]pyrene (sulfate and
glucoside conjugates; Pothuluri et al., 1996).
The recovery of radioactivity in the broth and cell
extracts of the cultures used in the screen ranged from
71 to 96%. Since the majority of radioactive residues
were extractable, no further efforts were made to release
cell-bound materials.
The control showed minimal degradation of cyprodinil
(<2%) in the soybean grit-glucose medium, indicating
that observed metabolism in the screen was microbially
mediated.
Thus, the results of this study indicate that the
fungicide cyprodinil is readily degraded by a number of
microbial species known to contain the enzyme cyto-
chrome P-450 monooxygenase. The identification of the
resulting metabolites can provide important information
relevant to the metabolism of this compound in animals,
soils, and higher plants.
Although microbial cultures have been used exten-
sively for generating as well as predicting potential
mammalian metabolites of pharmaceuticals (Smith and
9
245) was brought to a pH of 12 with 1 N sodium
hydroxide and partitioned with an equal volume of
methylene chloride. Only a small amount of radiocar-
bon was found in the methylene chloride (2%), suggest-
ing that the metabolites in the extracted broth of the
C. echinulata var. elegans cultures were quite polar in
nature. The partitioned pH 2 broth was subsequently
concentrated 10-fold under a nitrogen stream and
analyzed by HPLC-RAM and LC/MS. The HPLC-RAM
profile revealed the presence of polar metabolites. The
LC/MS total ion chromatogram was quite complex.
Mass spectra were obtained on eluting compounds,
which were base-line resolved. A number of metabolites
had apparent molecular ions at m/z 241 and 257,
suggesting mono- and dihydroxylated metabolites of
parent compound. However, since these kinds of com-
pounds should have been partitioned into ethyl acetate,
it is more likely that these metabolites represent
conjugates, either hydroxylated at a variety of positions
on the cyprodinil molecule or hydroxylated in similar
positions but conjugated to a variety of sugars or other
natural products. Since molecular ions at higher mass
corresponding to an intact conjugate were not detected,
it is likely that the electron impact mode of ionization
was not “soft” enough (compared to alternative modes
of ionization such as electrospray or thermospray) to
preserve the molecular ion of the conjugates. Further
efforts to define the nature of the conjugates were not
pursued.

Microbial Transformations of Cyprodinil
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3651
Rosazza, 1975, 1983; Griffiths et al., 1991), the approach
has been used only sparingly for agrochemicals. Nev-
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published reports suggest the important utility of the
technique.
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Received for review April 10, 1997. Revised manuscript
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received J une 30, 1997. Accepted J uly 2, 1997.
J F970298L
3
73-381.
X
Heye, U. J .; Speich, J .; Siegle, H.; Wohlhauser, R.; Hubele, A.
Abstract published in Advance ACS Abstracts, Au-
CGA-219417sA novel broad-spectrum fungicide. Brighton
gust 15, 1997.