Probing crucial metabolic pathways in fungal pathogens of crucifers: Biotransformation of indole-3-acetaldoxime, 4-hydroxyphenylacetaldoxime, and their metabolites

Article abstract of DOI:10.1016/S0968-0896(03)00241-4

Indole-3-acetaldoxime is an intermediate of crucial importance in the biosynthesis of diverse plant secondary metabolites of Cruciferae. The metabolism of indole-3-acetaldoxime to indole-3-acetic acid via indole-3-acetonitrile by fungi that cause important plant diseases in crucifers, Leptosphaeria maculans (asexual stage Phoma lingam) causative agent of blackleg disease, Rhizoctonia solani causative agent of root rot disease, and Sclerotinia sclerotiorum causative agent of stem rot disease, is described. As well, the antifungal activity of indole-3-acetaldoxime and metabolites and the synthesis and biotransformation of 4-hydroxyphenylacetaldoxime by the same plant pathogens and by an insect fungal pathogen, Beauveria bassiana, are reported.

Full text of DOI:10.1016/S0968-0896(03)00241-4

Bioorganic & Medicinal Chemistry 11 (2003) 3115–3120
Probing Crucial Metabolic Pathways in Fungal Pathogens of
Crucifers: Biotransformation of Indole-3-Acetaldoxime,
4-Hydroxyphenylacetaldoxime, and Their Metabolites
M. Soledade C. Pedras* and Sabine Montaut
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, Canada S7N 5C9
Received 15 December 2002; revised 7 April 2003; accepted 8 April 2003
Abstract—Indole-3-acetaldoxime is an intermediate of crucial importance in the biosynthesis of diverse plant secondary metabolites
of Cruciferae. The metabolism of indole-3-acetaldoxime to indole-3-acetic acid via indole-3-acetonitrile by fungi that cause impor-
tant plant diseases in crucifers, Leptosphaeria maculans (asexual stage Phoma lingam) causative agent of blackleg disease, Rhi-
zoctonia solani causative agent of root rot disease, and Sclerotinia sclerotiorum causative agent of stem rot disease, is described. As
well, the antifungal activity of indole-3-acetaldoxime and metabolites and the synthesis and biotransformation of 4-hydro-
xyphenylacetaldoxime by the same plant pathogens and by an insect fungal pathogen, Beauveria bassiana, are reported.
# 2003Elsevier Science Ltd. All rights reserved.
Introduction
show selective antifungal activity and to be inducible in
brown mustard (Brassica juncea) but constitutive in
turnip (B. rapa), rapeseed (B. napus), and canola (B.
napus).2
Indole-3-acetaldoxime (1) is an intermediate of crucial
importance in the biosynthesis of diverse plant second-
ary metabolites of Cruciferae. Cruciferous chemical
defenses such as indole-3-acetonitrile (3), brassinin (5),
and brassilexin (6),1,2 as well as the indole glucosinolate
2 (glucobrassicin)3,4 and the plant hormone indole-3-
acetic acid (4)5 are derived from (S)-tryptophan via
acetaldoxime 1 (Scheme 1). Additional acetaldoxime
dependent metabolic pathways also operating in cruci-
fers lead to important aryl glucosinolates such as 8 via
4-substituted phenylacetaldoxime (7) (Scheme 2). Fur-
thermore, acetaldoximes are also important inter-
mediates in the biosynthesis of cyanogenic glucosides.4
The various acetaldoxime dependent biosynthetic path-
ways co-occur in a good number of cruciferous species
and aldoxime formation is a step common to amino
acid precursors of more than 120 glucosinolates.3,4
While the role of compounds 5 and 6 as elicited plant
defenses, that is, phytoalexins, has been established, the
ecological roles of 2, 3, 8, and 9 remain unclear. For
example, glucosinolates 2 and 8 (R=H or OH) are
constitutive in a great number of crucifers and cause a
range of biological effects,3,4 whereas nitrile 3 appears to
Despite producing multiple chemical defenses, crucifers
are susceptible to numerous pests and diseases. The
susceptibility of crucifers to infection by a wide range of
fungi appears to be related to an effective detoxification
of their chemical defenses by these phytopathogens.6,7
Because indole-3-acetaldoxime (1) has a pivotal role in
the biogenesis of cruciferous defenses, it is most impor-
tant to establish if common fungal pathogens can
metabolize it, as such a process could be highly detri-
mental to the plant.
Towards this end, we investigated and report here
results of the metabolism of 1 by fungi that cause
important plant diseases in crucifers: Leptosphaeria
maculans (Desm.) Ces. et Not. asexual stage Phoma lin-
gam (Tode ex Fr. ) Desm. causative agent of blackleg
disease, Rhizoctonia solani Kuhn causative agent of root
rot disease, and Sclerotinia sclerotiorum (Lib.) de Bary
causative agent of stem rot disease. Furthermore, con-
sidering the co-occurrence of acetaldoximes 1 and 7a in
cruciferous species, we also investigated the metabolism
of 7a by the same plant pathogens. To determine if these
transformations were common to non plant pathogenic
fungi, we examined and report the metabolism of 1 and
*Corresponding author. Tel.: +1-306-966-4772; fax: +1-306-966-
4730; e-mail:
0968-0896/03/$ - see front matter # 2003Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00241-4

3116
M. S. C. Pedras, S. Montaut / Bioorg. Med. Chem. 11 (2003) 3115–3120
The chemical synthesis of [UL-14C]-4-hydroxy-
phenylacetaldoxime from tyrosine applied enzymatic
conversion of L-[UL-14C]tyrosine to 4-hydroxy-
phenylpyruvic acid, which was allowed to react with
hydroxylamine to form 4-hydroxyphenylpyruvic acid
oxime, followed by reduction with NaBH4 and dec-
arboxylation to 4-hydroxyphenylacetaldoxime.10
A
more efficient synthesis of 7a started with oxidation of
2-(4-hydroxyphenyl)-1-ethanol with sulfur trioxide-pyr-
idine complex followed by chromatography and oxima-
tion with hydroxylamine hydrochloride.11 We devised a
very simple preparation starting from commercially
available 4-hydroxyphenylacetic acid, which was con-
secutively esterified, reduced with DIBAL to the alde-
hyde, and the aldehyde allowed to react with
hydroxylamine hydrochloride to yield, after column
chromatography, a mixture of E/Z oximes 7a in good
overall yield.
Subsequently, synthetic oximes 1 and 7a were incubated
separately with each fungal isolate (P. lingam, S. sclero-
tiorum, R. solani, B. bassiana), and culture samples were
extracted and analyzed over a five day period (all the
biotransformation studies were carried out using com-
pounds at a concentration not toxic to these fungi, 10ꢀ4
M). Because these oximes were not stable in our usual
chemically defined fungal culture medium,7 all bio-
transformation experiments were carried out in water,
as described in the experimental (in the absence of fungi
all compounds and intermediates were stable in water
for the duration of the experiments; the E/Z oximes
quickly equilibrate in water).
Scheme 1. Metabolism of indole-3-acetaldoxime (1) in cruciferous
plants.1ꢀ5
Scheme 2. Metabolism of 4-substituted-phenylacetaldoxime (7) in
cruciferous plants.3,4
HPLC analysis of the extracts of fungal cultures incu-
bated with 1 (mixture of E/Z isomers) indicated that
both isomers were metabolized by each fungal species to
indole-3-acetic acid (4), as summarized on Schemes 3and
4. The structures of all metabolites were established by
UV, NMR and MS spectroscopic data and comparison
with authentic samples (commercially available). Inter-
estingly, while the insect pathogenic species converted 1
via reduction to tryptophol (10, 2-(3-indolyl)-1-ethanol),
all plant pathogenic species converted 1 via dehydration
to indole-3-acetonitrile (3). Similarly, acetaldoxime 7a
(mixture of E/Z isomers) was converted to 4-hydroxy-
phenylacetic acid (14) via 4-hydroxyphenylacetonitrile
7a by a fungus pathogenic to insects, Beauveria bassi-
ana. As well, we describe a simple and efficient synthesis
of acetaldoxime 7a, and the antifungal activity of com-
pounds 1–3 and 10. The main objective of this work is
to establish if successful cruciferous pathogens can
metabolize oxime 1, the key precursor of tryptophan
derived phytoalexins.
Results and Discussion
Indole-3-acetaldoxime (1) (a mixture of E/Z isomers)y
and glucobrassicin (2) were synthesized as previously
reported,2 whereas a new synthesis was devised for 4-
hydroxyphenylacetaldoxime (7a) (a mixture of E/Z iso-
mers).y Although acetaldoxime 7a has been widely used
in a great number of biological studies,3,4 current
syntheses of either 7a or its corresponding aldehyde are
rather
phenylacetaldehyde was prepared from synephrine
(2-amino-1-(4-hydroxyphenyl)-1-ethanol) following
inefficient.
For
example,
4-hydroxy-
a
pinacol-pinacolone type rearrangement in 85% H3PO4
in very low yield.8 Another procedure involved con-
densation of 4-hydroxybenzaldehyde with [14C]-nitro-
methane followed by catalytic reduction using platinum
and zinc to yield [1-14C]-4-hydroxyphenylacetaldoxime.9
Scheme 3. Metabolism of indole-3-acetaldoxime (1) by fungi: (i)
Phoma lingam BJ-125; (ii) P. lingam Laird 2; (iii) P. lingam Mayfair 2;
(iv) Sclerotinia sclerotiorum clone #33; (v) Rhizoctonia solani AG 2-1;
(vi) Beauveria bassiana ATCC 7159; Rt=HPLC retention time in min
using mobile phase A (see Experimental).
yThe E/Z isomers are separable, but in solution both isomers inter-
converte to equilibrate in a solvent dependent ratio.

M. S. C. Pedras, S. Montaut / Bioorg. Med. Chem. 11 (2003) 3115–3120
3117
since no indole containing products were detected in the
cultures.
To determine if the biotransformations observed were a
detoxification process, the antifungal activity of indole-
3-acetaldoxime (1) and indole-3-acetonitrile (3) were
determined utilizing radial mycelial growth inhibition
assays as described in the experimental. The results on
the Table 1 showed that indole-3-acetaldoxime (1) was
less inhibitory to P. lingam BJ-125 (54% at 5.0ꢁ10ꢀ4
M) than indole-3-acetonitrile (70% at 5.0ꢁ10ꢀ4 M).
Similarly, oxime 1 was also less toxic to R. solani (57%
at 5.0ꢁ10ꢀ4 M) than nitrile 3 (71% at 5.0ꢁ10ꢀ4 M). By
contrast, oxime 1 was more inhibitory to S. sclerotiorum
(42% of inhibition at 5.0ꢁ10ꢀ4 M after 24 h) than the
nitrile (no effect) but showed lower inhibitory effect on
S. sclerotiorum than on P. lingam and R. solani. Indole-
3-acetic acid (4) had no substantial inhibitory effect on
any of the species at the concentrations tested. Neither
indole-3-acetaldoxime (1) nor indole-3-ethanol (10)
showed inhibitory activity against B. bassiana. Overall,
the metabolism of indole-3-acetaldoxime (1) lead to
detoxification. However, glucosinolate 2 showed no
inhibition against P. lingam, thus its metabolism did not
amount to detoxification.
Scheme 4. Metabolism of 4-hydroxyphenylacetaldoxime (7a) by fungi:
(i) Phoma lingam BJ-125; (ii) Sclerotinia sclerotiorum clone #33; (iii) Rhi-
zoctonia solani AG 2-1; (iv) Beauveria bassiana ATCC 7159; Rt=HPLC
retention time in min using mobile phase A (see Experimental).
(9) by the plant pathogenic fungi, whereas the insect
pathogenic fungus carried out similar transformation
via 2-(4-hydroxyphenyl)-1-ethanol (13). The inter-
mediacy of nitriles 3 and 9 and alcohols 10 and 13 in the
fungal transformations of each oxime was demonstrated
by separately feeding each of these compounds to fungal
cultures and isolating the resulting products. Avirulent
isolates of P. lingam and R. solani further bio-
transformed indole-3-acetic acid (4) to indole-3-car-
boxylic acid (11), whereas B. bassiana converted 4 into
2-oxo-indole-3-acetic acid (12).
These results are consistent with a previous observation
that avirulent isolates of P. lingam can further trans-
form indole-3-acetic acid (4) to indole-3-carboxylic
(11).12 The transformation rates of oximes 1 and 7a to
the respective nitriles 3 and 9 were similar in each spe-
cies (completed in ca. 24 h), whereas the transformation
rates of nitriles 3 and 9 to the respective carboxylic acids
4 and 14 were substantially different. While S. sclero-
tiorum converted nitriles 3 and 9 in ca. 24 h, transfor-
mations by P. lingam were achieved in ca. 48 h, but R.
solani converted only 75% of each nitrile in five days (%
determined by HPLC). Because in plants indole-3-ace-
tonitrile (3) is partly produced from glucosinolate 2,4 we
also analyzed if similar pathway occurred in P. lingam.
The transformation of 2 proceeded at relatively slower
rates than oxime 1 and lead to no detectable products
(ca. 55% of 2 remained untransformed after incubation
for five days). That is, the transformation did not
appear to proceed via oxime, nitrile, or acid since these
compounds were not detected over a five day period.
In summary, the overall results of these biotransforma-
tion studies show that all the species were able to meta-
bolize oxime 1. The plant pathogens, independently of
the species, metabolized 1 via nitrile 3, one of the inter-
mediates also formed in plants. The enzymes respon-
sible for this transformation might be specific to
crucifers and to their pathogens, specially considering
that the biotransformation of 1 by a non phytopathogen
(B. bassiana) yielded indole-3-acetic acid via a different
intermediate (10, tryptophol).
The metabolism of indole-3-acetaldoxime (1) has been
widely studied in tissues of plants representing diverse
families, including Cruciferae.3ꢀ5 It appears that the
ability to transform indole-3-acetaldoxime (1) to indole-
3-acetic acid (4)3ꢀ5 and tryptophol (10)13 is widespread.
In a previous study, we also reported that tryptophol
(10) was a metabolite of indole-3-acetaldoxime (1) in
turnip roots.1,2 Nonetheless, to the best of our knowl-
edge this is the first study demonstrating that the crucial
intermediate oxime 1 is metabolized in both crucifers
and their pathogens via similar pathways.
Contrary to our previous findings on the metabolism of
phytoalexins by phytopathogens, where transformation
pathways appeared characteristic of a particular phyto-
pathogenic species, the results described above indicate
that the metabolism of indole-3-acetaldoxime (1) is
similar in all plant pathogenic species. Since both acet-
aldoximes 1 and 7a were converted to the corresponding
nitriles by all plant pathogens, we suspected that the
metabolizing enzymes might not be substrate specific.
Thus, to further probe the specificity of the fungal
enzyme(s) involved in the conversion of oxime 1, indole-
3-carboxaldehyde oxime (15) was incubated with P. lin-
gam and cultures analyzed by HPLC. Results of these
analyses indicated that 15 was transformed (ca. 48–56 h)
but this transformation did not appear to occur via the
potential intermediates aldehyde, nitrile, or alcohol,
Due to the central biogenetic role played by inter-
mediate 1, it would be of great interest to establish if
during fungal infection of plants, 1 is metabolized and if
so, could such a process be inhibited without interfering
with the normal plant development. In addition, inves-
tigation of the fungal enzymes involved in these trans-
formations, and comparison with related and currently
known plant enzymes, should provide a better under-
standing of their evolution. Furthermore, considering

3118
M. S. C. Pedras, S. Montaut / Bioorg. Med. Chem. 11 (2003) 3115–3120
Table 1. Antifungal activity of indole-3-acetaldehyde oxime (1), glucobrassicin (2), indole-3-acetonitrile (3) and indole-3-ethanol (10)
Compd
Concentration
(M)
P. lingama
(% inhibition)b
R. solanic
(% inhibition)b
S. sclerotiorumd
(% inhibition)b
B. bassianae
(% inhibition)b
Indole-3-acetaldoxime (1)
5.0ꢁ10ꢀ4
2.5ꢁ10ꢀ4
10ꢀ4
54ꢃ11%
27ꢃ11%
6ꢃ4%
70ꢃ7%
22ꢃ6%
N.I.f
57ꢃ4%
N.I.f
N.I.f
71ꢃ9%
N.I.f
N.I.f
—
—
—
—
—
42ꢃ5%
N.I.f
N.I.f
N.I.f
N.I.f
N.I.f
—
—
—
—
—
N.I.f
N.I.f
N.I.f
-
–
–
N.I.f
N.I.f
N.I.f
–
–
–
Indole-3-acetonitrile (3)
Indole-3-ethanol (10)
Glucobrassicin (2)
5.0ꢁ10ꢀ4
2.5ꢁ10ꢀ4
10ꢀ4
5.0ꢁ10ꢀ4
2.5ꢁ10ꢀ4
10ꢀ4
5.0ꢁ10ꢀ4
2.5ꢁ10ꢀ4
10ꢀ4
—
—
—
N.I.f
N.I.f
N.I.f
—
—
aBJ-125, 90 h incubation.
bThe % of inhibition was calculated using the formula: 100ꢀ[(growth on treated/growth in control)ꢁ100]+standard deviation; results are the mean
of at least three independent experiments.
cAG2-1, 72 h incubation.
dClone # 33, 24 h incubation.
eATCC 7159, 120 h incubation.
fN.I.: No inhibition (growth on control medium and on medium containing compound is similar).
that a number of secondary metabolic steps12,14 co-
occur in cruciferous pathogens and their hosts, these
systems provide an ideal opportunity to investigate the
co-evolution of secondary metabolism in plants and
their pathogens.
mobile phase B),17,18 and oxindole-3-acetic acid (12)12
were prepared as previously reported.
Synthesis of 4-hydroxyphenylacetaldoxime (7a). A solu-
tion of 4-hydroxyphenylacetic acid (0.50 g, 3.3 mmol) in
MeOH (41 mL) and concd H2SO4 (1 mL) was refluxed
for 2 h, cooled to room temperature, neutralized with aq
NaHCO3 (satd), concentrated, and extracted with
EtOAc. The organic phase was dried over Na2SO4, fil-
tered, and concentrated under reduced pressure to give
the methyl ester (0.54 g, 3.25 mmol, 99%) as an yellow
oil. DIBAL-H (1.5 M in toluene, 2 mL) cooled to
ꢀ78 ꢂC was added dropwise to a stirred solution of
methyl 4-hydroxyphenylacetate (100 mg, 0.60 mmol) in
toluene (6 mL, cooled to ꢀ78 ꢂC), under Ar atmo-
sphere.19 The rate of addition was adjusted to keep the
internal temperature at ꢀ78 ꢂC, and the reaction mix-
ture was stirred for 5 h at ꢀ78 ꢂC. The reaction was
quenched by slowly adding cold MeOH (100 mL). To
the resulting white emulsion, ice-cold 1 N HCl (5 mL)
was added slowly with swirling, the emulsion was
allowed to warm up to 0 ꢂC, acidified with ice-cold 1 N
HCl (7 mL) and stirred for 2 h. The aqueous mixture
was then extracted with EtOAc, the combined organic
layers were washed with a saturated aq NaCl solution
(75 mL), dried over Na2SO4, filtered and concentrated
to yield 91.6 mg of a colorless oil. The residue was sub-
jected to flash column chromatography on silica gel
(CH2Cl2–MeOH, 99:1) to yield 61.8 mg (0.45 mmol,
75% yield from ester) of 4-hydroxyphenylacetaldehyde
and 7.6 mg (0.06 mmol, 10% from ester) of 2-(4-
hydroxyphenyl)-1-ethanol (13). Satisfactory spectro-
scopic data was obtained for both compounds.
In conclusion, three important cruciferous pathogens
and an insect pathogen metabolized indolyl-3-acet-
aldoxime (1) to the corresponding carboxylic acid 4;
however, while the plant pathogen transformed 1 via
indolyl-3-acetonitrile (3), transformation of 1 by the
insect pathogen occurred via tryptophol (10).
Experimental
General experimental procedures
All chemicals were purchased from Sigma-Aldrich
Canada Ltd., Oakville, ON, except for 4-hydroxy-
phenylacetonitrile purchased from Alfa Aeser, Ward
Hill, MA, USA. All solvents were HPLC grade and used
as such, except for CH2Cl2 and CHCl3 which were redis-
tilled. Remaining conditions as previously reported.15
HPLC analysis was carried out with a high performance
liquid chromatograph equipped with quaternary pump,
automatic injector, and diode array detector (wave-
length range 190–600 nm), degasser, and a Hypersil
ODS column (5 mm particle size silica, 4.6 i.d. ꢁ200
mm), equipped with an in-line filter. Mobile phase A:
75% H2O–25% CH3CN to 100% CH3CN, for 35 min,
linear gradient, and a flow rate 1.0 mL/min. Mobile
phase B: gradient of 100% H2O–100% MeOH for 60
min, linear gradient, at a flow rate 1.0 mL/min; both
water and methanol contained 0.15% triethylamine and
0.18% formic acid.16 Compounds were identified by
comparison with authentic samples.
.
Next, a solution of NH2OH HCl (162.6 mg, 2.34 mmol)
and NaOAc (192 mg, 2.34 mmol) in H2O (2 mL) was
added to a solution of 4-hydroxyphenylacetaldehyde
(61.8 mg, 0.45 mmol) in 95% aq EtOH (2 mL). After
stirring for 6 h at room temperature, the reaction mix-
ture was concentrated to dryness, the resulting residue
was diluted with H2O (10 mL) and extracted with
Indole-3-acetaldehyde oxime (1, Rt=8.1, 8.9 min,
mobile phase A),2 glucobrassicin (2, Rt=15.7 min,

M. S. C. Pedras, S. Montaut / Bioorg. Med. Chem. 11 (2003) 3115–3120
3119
EtOAc. The organic layer was dried over Na2SO4, fil-
tered, and concentrated under reduced pressure (95.6
mg). The residue was fractionated by flash column
chromatography on silica gel (CH2Cl2–MeOH, 99:1) to
afford 4-hydroxyphenylacetaldoxime (66.8 mg, 0.44
mmol, 98% from the aldehyde, overall 73%). The spec-
troscopic data of 4-hydroxyphenylacetaldoxime (7a)
were similar to published data10,11,20ꢀ22 (since no 13C
NMR or FTIR data have been reported to date, we
report here complete spectroscopic characterization).
Rf=0.5 (EtOAc–hexane 3:1), Rt=3.7 and 4.0 min
(mobile phase A); mp 104–107 ꢂC (lit.20 110–111 ꢂC);
UV (MeOH) lmax (log e) 200 (4.5), 224 (4.0) and 278
(3.5); FTIR nmax 3373, 3257, 3089, 3029, 2898, 1514,
1443, 1243, 924 and 830 cmꢀ1; HRMS-EI m/z (% rela-
tive abundance): measured: 151.0635 [M+] (56)
(151.0634 calcd for C8H9NO2), 133.0527 (58), 107.0493
(100), 94.0416 (13), 77.0399 (27), 57.0538 (11); 1H NMR
(500 MHz, CD3CN) ꢀ 3.37 (d, J=6.3Hz, 0.2H), 3.55 (d,
J=5.4 Hz, 2H), 6.75 (m, 3.2H), 7.03 (d, J=8.6 Hz,
0.2H), 7.06 (d, J=8.5 Hz, 2H), 7.38 (t, J=6.3Hz,
0.1H), 8.80 (br s, exchangeable) (the intensities of
methylene signals at ꢀ 3.55 and 3.37 indicated that the
ratio of Z/E=10:1); 13C NMR (125.8 MHz, CD3CN) ꢀ
31.5 (Z), 35.8 (E), 116.5, 116.7, 129.5, 129.9, 131.2,
132.2, 151.4, 151.7, 156.8, 157.0.
3, 4, 7a, 9, 10, and 12–15 in DMSO (final concentrations
10ꢀ4 M; final concentration of DMSO in water 0.5% v/
v) and 2 in H2O (final concentration 10ꢀ4 M) were
added. Cultures were incubated in constant light, on a
shaker at 130 rpm, at 23ꢃ2 ꢂC. Samples (5 mL) were
withdrawn at different intervals and were either imme-
diately frozen or extracted with ethyl acetate. The ethyl
acetate extracts and the aqueous residues were evapo-
rated to dryness and analyzed by HPLC using mobile
phase A, except for experiments with glucosinolate 2 in
which HPLC analyses were carried out with both
mobile phases A and B. Control flasks containing only
H2O, DMSO and the compound, or the mycelia in H2O
and DMSO were also incubated and analyzed similarly.
Bioassays
The antifungal activity of compounds 1–3 and 10 was
investigated using the following mycelial radial growth
bioassay. A DMSO solution (final concentration 1%) of
the compound to be tested (final concentration of each
compound 5.0ꢁ10ꢀ4, 2.5ꢁ10ꢀ4 and 10ꢀ4 M, compound
2 was dissolved in H2O) was added to agar medium at
ca. 50 ꢂC (for P. lingam, S. sclerotiorum and B. bassiana)
or to PDB (for R. solani), mixed quickly and poured
onto 12-well plates (1 mL). An agar plug (4 mm dia-
meter) cut from edges of 3-day-old solid cultures of
S. sclerotiorum, 5-day-old solid cultures of R. solani and
B. bassiana and 7-day-old solid cultures of P. lingam
BJ-125 was placed upside down on the center of each
plate, the plates were sealed with parafilm, and incu-
bated at 23ꢃ2 ꢂC under constant light for 24 h for
S. sclerotiorum, 72 h for R. solani, 90 h for P. lingam
BJ-125 and 120 h for B. bassiana ATCC 7159. The dia-
meter of the mycelia (in mm) was then measured and
compared with control plates containing only DMSO.
Each assay was conducted in triplicate and repeated at
least three times.
Fungal cultures
Solid cultures of P. lingam isolates BJ-125, Mayfair 2
and Laird 2 were grown on V8 agar media as previously
reported.23 Pycnidiospores of these isolates were col-
lected after 15 days of growth on V8 agar media under
constant cool fluorescent light at 23ꢃ2 ꢂC.23 R. solani,
virulent isolate AG2-1, obtained from the AAFC col-
lection (Saskatoon, SK) was grown on potato dextrose
agar (PDA) plates at 23ꢃ2 ꢂC, under constant light for
five days. S. sclerotiorum clone #33 was grown on PDA
plates at 20ꢃ2 ꢂC, under constant darkness and scler-
otia were collected after 15 days of incubation.7
B. bassiana ATCC 7159 was obtained from American
Type Culture Collection, Manassas, VA, USA. The
fungus was grown on PDA plates at 23ꢃ2 ꢂC, under
constant light.
Acknowledgements
Support for the authors’ work was obtained from: the
Natural Sciences and Engineering Research Council of
Canada and the University of Saskatchewan. We thank
C. Lefol, Agriculture and Agri-Food Canada Research
Station, Saskatoon SK, for kindly providing the clone
of Sclerotinia sclerotiorum.
Metabolism of compounds 1–4, 7a, 9, 10, and 12–15
Spores of P. lingam isolates BJ-125 (virulent), Mayfair 2
(avirulent) and Laird 2 (avirulent) were separately
inoculated at a concentration of 108 spores per 100 mL
of liquid minimal media (15 g of glucose, plus thiamine)
in 250 mL Erlenmeyer flasks.23 Mycelia (5 mm diameter
agar plugs) cut from the edge of the solid cultures of
R. solani and B. bassiana and sclerotia of S. sclerotiorum
were separately inoculated into 100 mL of potato dex-
trose broth (PDB). Incubations (3days for Laird 2, 4
days for B. bassiana, 5 days for R. solani and S. sclero-
tiorum, 6 days for P. lingam BJ-125 and Mayfair 2),
were carried out on a shaker at 130 rpm at 23ꢃ2 ꢂC,
under constant light. After incubation, the mycelia were
filtered, washed with sterile distilled water and trans-
ferred to an other 250 mL Erlenmeyer flask with 100
mL of sterile distilled water. Solutions of compounds 1,
References and Notes
1. Pedras, M. S. C.; Montaut, S.; Xu, Y.; Khan, A. Q.; Lou-
kaci, A. Chem. Commun. (Cambridge) 2001, 1572.
2. Pedras, M. S. C.; Nycholat, C. M.; Montaut, S.; Xu, Y.;
Khan, A. Q. Phytochemistry 2002, 59, 611.
3. Wittscock, U.; Halkier, B. A. Trends Plant Sci. 2002, 7,
263.
4. Mikkelsen, M. D.; Petersen, B. L.; Olsen, C. E.; Halkier,
B. A. Amino Acids 2002, 22, 279.
5. Normanly, J.; Bartel, B. Curr. Opin. Plant Biol. 1999, 2,
207.
6. Pedras, M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. Q.
Phytochemistry 2000, 53, 161.

3120
M. S. C. Pedras, S. Montaut / Bioorg. Med. Chem. 11 (2003) 3115–3120
7. Pedras, M. S. C.; Ahiahonu, P. W. K. Bioorg. Med. Chem.
2002, 10, 3307.
17. Chevolleau, S.; Joseph, B.; Rollin, P.; Tulliez, J. J. Label-
led Compd. Radiopharm. 1993, 33, 671.
8. Robbins, J. H. Arch. Biochem. Biophys. 1966, 114, 576.
9. Schieffer, S.; Kindl, H. J. Lab. Comp. 1970, 7, 291.
10. Møller, B. L. J. Labelled Compd. Radiopharm. 1978, 14, 663 .
11. Lin, J.; Cashman, J. R. Chem. Res. Toxicol. 1997, 10, 842.
12. Okanga, F. I. Ph.D. Dissertation, University of Saskatch-
ewan, Saskatoon, SK. 1998.
18. Gardrat, C.; Quinsac, A.; Joseph, B.; Rollin, P. Hetero-
cycles 1993, 35, 1015.
19. Garner, P.; Park, J. M. Org. Synth. 1992, 70,
18.
20. Hazato, T.; Kumagai, M.; Naganawa, H.; Aoyagi, T.;
Umezawa, H. J. Antibiot. 1979, 32, 91.
13. Rajagopal, R.; Larsen, P. Planta 1972, 103, 45.
14. Pedras, M. S. C.; Okanga, F. I. J. Agric. Food Chem. 1999,
47, 1196.
15. Pedras, M. S. C.; Khan, A. Q. Phytochemistry 2000, 53, 59.
16. Zrybko, C. L.; Fukuda, E. K.; Rosen, R. T. J. Chroma-
togr. A 1997, 767, 43 .
21. Halkier, B. A.; Olsen, C. E.; Møller, B. L. J. Biol. Chem.
1989, 264, 19487.
22. Du, L.; Lykkesfeldt, J.; Olsen, C. E.; Halkier, B. A. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 12505.
23. Pedras, M. S. C.; Biesenthal, C. J. Can. J. Microbiol. 1998,
44, 547.