Detoxification of cruciferous phytoalexins in Botrytis cinerea: Spontaneous dimerization of a camalexin metabolite

Article abstract of DOI:10.1016/j.phytochem.2010.11.018

Phytopathogenic fungi are able to overcome plant chemical defenses through detoxification reactions that are enzyme mediated. As a result of such detoxifications, the plant is quickly depleted of its most important antifungal metabolites and can succumb to pathogen attack. Understanding and predicting such detoxification pathways utilized by phytopathogenic fungi could lead to approaches to control plant pathogens. Towards this end, the inhibitory activities and metabolism of the cruciferous phytoalexins camalexin, brassinin, cyclobrassinin, and brassilexin by the phytopathogenic fungus Botrytis cinerea Pers. (teleomorph: Botryotinia fuckeliana) was investigated. Brassilexin was the most antifungal of the phytoalexins, followed by camalexin, cyclobrassinin and brassinin. Although B. cinerea is a species phylogenetically related to the phytopathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary, contrary to S. sclerotiorum, detoxification of strongly antifungal phytoalexins occurred via either oxidative degradation or hydrolysis but not through glucosylation, suggesting that glucosyl transferases are not involved. A strongly antifungal bisindolylthiadiazole that B. cinerea could not detoxify was discovered, which resulted from spontaneous oxidative dimerization of 3-indolethiocarboxamide, a camalexin detoxification product.

Full text of DOI:10.1016/j.phytochem.2010.11.018

Phytochemistry 72 (2011) 199–206
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Detoxification of cruciferous phytoalexins in Botrytis cinerea: Spontaneous
dimerization of a camalexin metabolite
⇑
M. Soledade C. Pedras , Sajjad Hossain, Ryan B. Snitynsky
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytopathogenic fungi are able to overcome plant chemical defenses through detoxification reactions
that are enzyme mediated. As a result of such detoxifications, the plant is quickly depleted of its most
important antifungal metabolites and can succumb to pathogen attack. Understanding and predicting
such detoxification pathways utilized by phytopathogenic fungi could lead to approaches to control plant
pathogens. Towards this end, the inhibitory activities and metabolism of the cruciferous phytoalexins
camalexin, brassinin, cyclobrassinin, and brassilexin by the phytopathogenic fungus Botrytis cinerea Pers.
(teleomorph: Botryotinia fuckeliana) was investigated. Brassilexin was the most antifungal of the phyto-
alexins, followed by camalexin, cyclobrassinin and brassinin. Although B. cinerea is a species phylogenet-
ically related to the phytopathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary, contrary to
S. sclerotiorum, detoxification of strongly antifungal phytoalexins occurred via either oxidative degrada-
tion or hydrolysis but not through glucosylation, suggesting that glucosyl transferases are not involved. A
strongly antifungal bisindolylthiadiazole that B. cinerea could not detoxify was discovered, which
resulted from spontaneous oxidative dimerization of 3-indolethiocarboxamide, a camalexin detoxifica-
tion product.
Received 6 September 2010
Received in revised form 3 November 2010
Available online 20 December 2010
Keywords:
Brassicaceae
Antifungal
Botrytis cinerea
Botryotinia fuckeliana
Brassilexin
Brassinin
Camalexin
Crucifer
Cyclobrassinin
Detoxification
Phytoalexin
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
metabolize scopoletin (El Oirdi et al., 2010). Additional evidence
suggests that the pathogen elicits the host to induce programmed
The phytopathogenic fungus Botrytis cinerea Pers. Fr. (teleo-
morph Botryotinia fuckeliana (de Bary) Whetzel) causes grey mold
disease and great yield losses in a very large number of crops,
including grapevines (Vitis vinifera), tomatoes (Solanum lycopersicum
L.), cabbage (Brassica oleraceae L. cv. Capitata), broccoli (B. oleraceae
L. cv. Italica), tobacco (Nicotiana tabacum L.) and strawberries
(Fragaria ꢀ ananassa). Losses are caused in both field-grown and
in greenhouse-grown horticultural crops prior to and after harvest.
B. cinerea is difficult to control due to the numerous strategies it
uses to infect diverse plant genera (Williamson et al., 2007). For
example, B. cinerea produces a variety of phytotoxic metabolites
that include oxalic acid, polyketide lactones and sesquiterpenoids,
and cell-wall-degrading enzymes, together with other pathogenesis
related proteins. In addition, pathogenic isolates of B. cinerea
detoxify the phytoalexin resveratrol from grapevines and produce
an ABC transporter that increases tolerance of the pathogen
towards the phytoalexin camalexin (1) in Arabidopsis thaliana
(Choquer et al., 2007). Furthermore, the resistance of tobacco to
B. cinerea was correlated with the accumulation of the phytoalexin
scopoletin and the ability of the mycelium, not the spores, to
cell death as an infection strategy (Williamson et al., 2007).
The number of sequenced genomes from both saprophytic and
pathogenic fungal species has been increasing rapidly over the last
five years, providing new tools to investigate virulence factors in
fungi. The species B. cinerea and Sclerotinia sclerotiorum (Lib) de
Bary are among the first necrotrophic fungi to have their genomes
sequenced (the Broad Institute, http://www.broad.mit.edu/annota-
tion/fgi/; the Genoscope, http://www.genoscope.cns.fr/) (Fillinger
et al., 2007). Using this knowledge, the cloning, purification and
characterization of brassinin glucosyl transferase (SsBGT1), the en-
zyme from S. sclerotiorum involved in brassinin (2) detoxification,
were reported recently (Sexton et al., 2009). Alignment of the ami-
no acid sequence of SsBGT1 with glucosyltransferases from other
fungal species showed that the closest match was found in B. cine-
rea, which displayed 57% amino acid identity. This sequence simi-
larity was not surprising, particularly considering the close
phylogenetic relationship between these species, both from the
family Sclerotiniaceae (Hirschhaeuser and Froehlich, 2007).
Phytopathogenic fungi are able to overcome plant chemical de-
fenses (inducible and constitutive) through metabolism and detox-
ification, utilizing a variety of detoxifying enzymes (Pedras and
Ahiahonu, 2005). As a result of such detoxification reactions, the
plant is depleted of important antifungal metabolites and in many
⇑
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
E-mail address: s.pedras@usask.ca (M.S.C. Pedras).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.11.018

200
M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
reported (Pedras et al., 2007b). Satisfactory spectroscopic data
were obtained for all synthetic compounds.
The antifungal activity of each phytoalexin against B. cinerea
was determined employing the mycelial radial growth assay de-
scribed in the Experimental, and using CH3CN or DMSO to dissolve
each compound. The results of these assays are shown in Table 1.
Brassilexin (4) was more inhibitory to B. cinerea than camalexin
(1), brassinin (2), or cyclobrassinin (3). At 0.10 mM brassilexin
(4) inhibited completely the growth of B. cinerea, while at
0.050 mM the inhibitory effect was slightly lower (89% for UAMH
1784 and 98% for UAMH 1809). Camalexin (1) caused complete
growth inhibition at 0.20 mM, whereas brassinin (2) and cyclo-
brassinin (3) caused complete inhibition at 0.50 mM. At 0.10 mM,
the inhibitory effect of camalexin (1) was lower than brassilexin
(4), causing 57% and 54% inhibition for UAMH 1784 and UAMH
1809, respectively. Interestingly, the solvent used to dissolve each
phytoalexin affected the activity of each phytoalexin somewhat
differently (Table 1). Namely, it was noted that the mycelial
growth of the fungus was a regular circle in the presence of DMSO,
but the circle was rather irregular in the presence of CH3CN. We
attribute these differences partly to the volatility of CH3CN.
Previously, brassilexin (4) was shown to inhibit completely the
growth of S. sclerotiorum at 0.050 mM (Pedras and Hossain, 2006),
whereas camalexin (1) caused ca. 81% inhibition at similar concen-
tration and brassinin (2) was inhibitory only at 0.30 mM (Pedras
et al., 2004; Pedras and Hossain, 2007).
Fig. 1. Detoxification pathway of the cruciferous phytoalexins camalexin (1),
brassinin (2), cyclobrassinin (3), and brassilexin (4) by the phytopathogenic fungus
Sclerotinia sclerotiorum.
cases succumbs to pathogen attack. Understanding the detoxifica-
tion pathways utilized by phytopathogenic fungi could lead to new
approaches to control plant pathogens. Towards this end, we
investigated the metabolism of cruciferous phytoalexins by diverse
phytopathogenic fungi, including S. sclerotiorum (Pedras and
Ahiahonu, 2002; Pedras, 2008). It was established that detoxifica-
tion of the strongly antifungal phytoalexins camalexin (1), brassinin
(2), cyclobrassinin (3), and brassilexin (4) involved glucosylation
reactions, as summarized in Fig. 1 (Pedras and Ahiahonu, 2002;
Pedras et al., 2004; Pedras and Hossain, 2006). In addition, contrary
to other phytopathogenic fungi, S. sclerotiorum was able to metabo-
lize the strongest antifungal phytoalexin analogues (Pedras and
Hossain, 2007).
2.2. Biotransformation of phytoalexins in cultures of B. cinerea
Cultures of B. cinerea isolates UAMH 1784 and UAMH 1809 were
incubated with each phytoalexin and their transformations were
monitored by HPLC (photodiode array and ESI detection). Samples
were withdrawn from cultures immediately after addition of each
compound and then as described for each case, up to six days. The
samples were subjected to neutral, acidic and basic extractions,
and the extracts were analyzed by HPLC, as reported in the Exper-
imental. Media incubated with each phytoalexin or metabolites
(control solutions) were analyzed similarly to determine the chem-
ical stability of each compound during the incubation experiments.
The economic significance of plant diseases caused by B. cinerea
and the difficulties in controlling this pathogen using available
methodologies (Choquer et al., 2007; Williamson et al., 2007)
prompted this investigation. Although it is clear that an ABC trans-
porter is a virulence factor that increases tolerance of the pathogen
towards the phytoalexin camalexin (1) (A. thaliana) (Stefanato
et al., 2009), the pathogen produces additional macromolecules
likely to contribute to virulence. In this context, it is of great inter-
est to determine if B. cinerea is also able to detoxify camalexin or
other cruciferous phytoalexins, similar to S. sclerotiorum (Pedras
and Ahiahonu, 2002). A methodology based on this knowledge to
protect plants against B. cinerea could be devised (Pedras, 2008).
Hence, to compare detoxification pathways of plant defenses in
B. cinerea and S. sclerotiorum, the cruciferous phytoalexins cama-
lexin (1), brassinin (2), cyclobrassinin (3), and brassilexin (4) were
investigated herein. It was established that the phytoalexins cam-
alexin (1) and brassilexin (4) displayed the strongest inhibitory
activity against B. cinerea, but their detoxification reactions were
rather different from those occurring in S. sclerotiorum.
Table 1
Antifungal activity of cruciferous phytoalexins (dissolved in DMSO or CH3CN) against
Botrytis cinerea isolates UAMH 1784 and UAMH 1809 (27–32 h incubation on agar
plates) under continuous light.
Phytoalexins (mM)
% Inhibitiona standard deviation
UAMH 1784 UAMH 1809
DMSO
CH3CN
DMSO
CH3CN
100
Camalexin (1)
0.20
0.10
0.050
Brassinin (2)
0.50
100
57
28
100
38
32
100
54
29
1
2
3
3
2
2
38
28
3
3
100
60
34
100
51
22
100
59
30
100
60
31
0.20
0.10
1
1
3
2
1
2
1
4
Cyclobrassinin (3)
0.50
0.20
0.10
100
70
24
Not soluble
100
66 10
41 13
Not soluble
70 10
7
5
74
36
7
4
39
8
Brassilexin (4)
0.10
0.050
100
89
54
100
88
66
100
98
83 11
100
99
80
2. Results and discussion
2
2
2
5
4
3
9
0.025
2.1. Syntheses and antifungal activity of phytoalexins
a
Percentage of growth inhibition calculated using the formula:
%
inhibi-
tion = 100 ꢁ [(growth on amended/growth in control) ꢀ 100]; values represent the
mean and standard deviation of at least two independent experiments conducted in
triplicate.
The phytoalexins camalexin (1), brassinin (2), cyclobrassinin
(3), and brassilexin (4) were synthesized and purified as previously

M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
201
2.2.1. Camalexin (1)
of medium containing thiocarboxamide 9 indicated that it was
not stable, as small amounts of the nitrile 10 and bisindolylthi-
adiazole 12 were detected in medium extracts after 6 h. Nonethe-
less, the transformation of 3-indolethiocarboxamide (9) in fungal
cultures (T1/2 = 10 h and 18 h, Fig. 4) was much faster than in con-
trol medium solutions (T1/2 = 36 h, Fig. 4), indicating that this
transformation was enzyme mediated. Formation of the bisindolyl-
thiadiazole 12 did not occur in aqueous solutions or in aqueous
acetonitrile, thus it is likely that medium components caused the
oxidative dimerization of thiocarboxamide 9 to 12.
A literature search indicated that 3,5-bisphenyl-1,2,4-thiadiaz-
oles could be synthesized from IBX (o-iodoxybenzoic acid) medi-
ated oxidative dimerization of thioamides (Patil et al., 2009).
Hence, application of this methodology to 3-indolethiocarboxa-
mide (9) yielded 12 in reasonable yield, as described in the Exper-
imental and summarized in Fig. 5. 3,5-(30,300-Bisindolyl)-1,2,
4-thiadiazole (12) was administered to cultures of isolates UAMH
1784 and UAMH 1809 and cultures analyzed over a four-day per-
iod. HPLC chromatograms indicated that thiadiazole 12 was not
metabolized by B. cinerea.
The HPLC chromatograms of neutral extracts of cultures of iso-
lates UAMH 1784 and UAMH 1809 incubated with camalexin (1)
showed that it was almost completely metabolized within 12 h
(Fig. 2). The chromatograms of neutral extracts showed the pres-
ence of camalexin (1) and additional peaks that were due to
3-indolecarboxynitrile (10, tR = 7.8 0.2 min) and 3-indolecarboxylic
acid (11, tR = 3.6 0.1 min), by comparison with authentic sam-
ples; additional peaks at tR = 4.6 and tR = 20.5 min could not be
readily identified. Isolation of these metabolites from larger scale
cultures, as described in the Experimental, and analyses of spectro-
scopic data allowed the identification of the peak with tR = 4.6 min
as 3-indolethiocarboxamide (9) (Fig. 3). Due to the small amounts
obtained, the structure of the metabolite with tR = 20.5 min was
only tentatively assigned as the bisindolylthiadiazole 12, a hitherto
unknown compound. None of these metabolites were detected
either in control cultures or in media incubated with camalexin
(1). No other metabolites were detected in either the acidic or basic
extracts of either culture. Furthermore, the rate of camalexin (1)
transformation was not light dependent.
To establish the sequence of steps of camalexin (1) transforma-
tion, the thiocarboxamide 9 was prepared as described in the
Experimental and administered separately to cultures of isolates
UAMH 1784 and UAMH 1809. The cultures were incubated, ex-
tracted and analyzed as described in the Experimental. As ex-
pected, indole-3-thiocarboxamide (9) was transformed to the
nitrile 10, which in turn was slowly (more than four days) trans-
formed to 3-indolecarboxylic acid (11) (Fig. 3). Control solutions
The antifungal activity of the metabolites of camalexin (1) to
isolates UAMH 1784 and UAMH 1809 of B. cinerea were deter-
mined employing the mycelial radial growth assay described in
Section 2 and using DMSO to dissolve each compound. The results
of these assays are shown in Table 2. Metabolites 9–11 were sub-
stantially less inhibitory to isolates UAMH 1784 and UAMH 1809
than camalexin (1) itself, indicating that both isolates detoxify
camalexin efficiently. 3,5-(30,300-Bisindolyl)-1,2,4-thiadiazole (12)
Fig. 2. Progress curves for transformation of the phytoalexin camalexin (1,
0.10 mM) by Botrytis cinerea isolates UAMH 1784 and UAMH 1809. Concentrations
were determined using calibration curves; each point is the average of at least three
independent experiments standard deviation.
Fig. 4. Progress curves for transformation of 3-indolethiocarboxamide (9, 0.10 mM)
by Botrytis cinerea isolates UAMH 1784 and UAMH 1809, and in control medium
(CM). Concentrations were determined using calibration curves; each point is the
average of at least three independent experiments standard deviation.
Fig. 5. Synthesis of 3-indolethiocarboxamide (9) and 3,5-(30,300-bisindolyl)-1,2,4-
thiadiazole (12). Reagents and conditions: (i) CH3CSNH2, HCl/DMF, reflux; (ii) IBX
(o-iodoxybenzoic acid), CH3CN, r.t.
Fig. 3. Transformation pathway of the phytoalexin camalexin (1) by Botrytis cinerea
isolates UAMH 1784 and UAMH 1809.

202
M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
Table 2
light did not affect the biotransformation rates of camalexin (1)
in cultures of B. cinerea. S. sclerotiorum detoxified camalexin (1)
through oxidation to the corresponding 6-hydroxy derivative fol-
Antifungal activity of camalexin metabolites 9–11 and 3,5-(30,300-bisindolyl)-1,2,4-
thiadiazole (12) (dissolved in DMSO) against Botrytis cinerea isolates UAMH 1784 and
UAMH 1809 (27–32 h incubation on agar plates) under continuous light.
lowed by glucosylation to yield 6-(O-b-D-glucopyranosyl)camalex-
Compound (mM)
% Inhibitiona standard deviation
UAMH 1784 UAMH 1809
in (5) (Pedras and Ahiahonu, 2002). On the other hand, some plant
pathogenic fungi and bacteria did not transform camalexin (1),
including many isolate types of Leptosphaeria maculans and
Alternaria brassicae (Pedras et al., 1998).
3-Indolethiocarboxamide (9)
0.50
0.20
0.10
64
48
32
2
2
2
68
45
21
2
2
2
2.2.2. Brassinin (2)
3-Indolecarboxynitrile (10)
Analyses of HPLC chromatograms of neutral extracts of cultures
of isolates UAMH 1784 and UAMH 1809 incubated with brassinin
(2) showed that it was almost completely metabolized within 24 h
(Fig. 7). Furthermore, the chromatograms of neutral extracts
showed the presence of additional peaks that were due to N0-acet-
yl-3-indolylmethanamine (17, tR = 4.4 0.2 min) and 3-indolecarb-
oxylic acid (11) by comparison with authentic samples (Fig. 8). The
acidic extracts showed traces of brassinin (2) plus 11 and the basic
extracts indicated the presence of 3-indolylmethanamine (16).
None of these metabolites were detected either in control cultures
or in medium incubated with brassinin (2). HPLC detection and
quantification of amine 16% in basic extracts was carried out using
HPLC method B. Further analysis of the HPLC chromatograms of the
transformation of brassinin (2) showed that both amine 16 and
acetyl amine 17 remained in cultures for more than four days.
To establish the sequence of steps of brassinin (2) transforma-
tion, amine 16 was administered to cultures of isolates UAMH
1784 and UAMH 1809, and cultures were incubated and analyzed
as described above. The HPLC chromatograms of culture extracts
indicated that amine 16 was slowly transformed to acetyl amine
17 (more than four days), 3-indolecarboxaldehyde (18) and 3-
indolecarboxylic acid (11). Antifungal bioassays showed that the
metabolites of brassinin (2) were substantially less inhibitory to
isolates UAMH 1784 and UAMH 1809 than brassinin itself (Table 3),
indicating that both isolates detoxify brassinin (2).
0.50
0.20
0.10
65
34
24
2
2
2
60
37
11
2
2
1
3-Indolecarboxylic acid (11)
0.50
0.20
0.10
31
14
6
2
2
2
27
14
7
2
2
1
3,5-(30,300-Bisindolyl)-1,2,4-thiadiazole (12)
0.50
0.20
0.10
0.05
Not soluble
Not soluble
76
68
49
2
2
2
75
71
38
1
2
2
a
Percentage of growth inhibition calculated using the formula:
% inhibi-
tion = 100 ꢁ [(growth on amended/growth in control) ꢀ 100]; values represent the
mean and standard deviation of two independent experiments conducted in
triplicate.
was more inhibitory to isolates UAMH 1784 and UAMH 1809 than
any of the camalexin metabolites, but less inhibitory than camalex-
in (1) (ca. 76% for 12 vs. 100% for 1).
It is noteworthy that a few bis(indole)alkaloids from marine
organisms, such as the nortopsentins A–C, containing a 2,4-bis
(3-indolyl)imidazole skeleton, exhibit antibacterial, antiviral and
cytotoxic activities. The imidazole moiety of the nortopsentins
was replaced with a thiazole, pyrazinone or pyrazine moiety to
yield bis(indolyl) alkaloids displaying anti-tumor activity (Jiang
and Gu, 2000). In addition, the synthesis of 3,5-disubstituted-
1,2,4-oxadiazoles containing indolyl moieties was also described
(Shvekhgeimer et al., 1984), but to date no 3,5-(30,300-bisindolyl)-
1,2,4-thiadiazoles have been reported.
Previous work showed that camalexin (1) was detoxified by the
phytopathogenic fungus Rhizoctonia solani Kuhn to 5-hydroxycam-
alexin (13), which was further transformed into metabolites 14
and 15 (Fig. 6), substantially less inhibitory to the pathogen than
camalexin (1) (Pedras and Khan, 2000). Interestingly camalexin
transformation to 5-hydroxycamalexin (13) was much faster under
light conditions (ca. 24 h in light vs. 8–10 days in dark); however,
The transformation pathway of brassinin (2) by B. cinerea was
similar to the pathways previously observed for L. maculans iso-
lates Laird 2/Mayfair 2 (Pedras et al., 2007a), L. biglobosa (Pedras
Fig. 7. Progress curves for transformation of the phytoalexin brassinin (2, 0.10 mM)
by Botrytis cinerea isolates UAMH 1784 and UAMH 1809. Concentrations were
determined using calibration curves; each point is the average of at least three
independent experiments standard deviation.
Fig. 6. Transformation pathway of the phytoalexin camalexin (1) by Rhizoctonia
solani virulent isolate AG 2-1.

M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
203
Fig. 8. Transformation pathway of the phytoalexin brassinin (2) by Botrytis cinerea
isolates UAMH 1784 and UAMH 1809.
Table 3
Antifungal activity of brassinin metabolites 16 and 17 (dissolved in DMSO) against
Botrytis cinerea isolates UAMH 1784 and UAMH 1809 (27–32 h incubation on agar
plates) under continuous light.
Fig. 10. Transformation pathways of the phytoalexins cyclobrassinin (3) and
brassilexin (4) by Botrytis cinerea isolates UAMH 1784 and UAMH 1809. Dashed
arrows represent potential transformation.
Compound (mM)
% Inhibitiona standard deviation
UAMH 1784 UAMH 1809
3-Indolylmethanamine (16)
0.50
0.20
0.10
26
19
14
3
3
3
38
15
15
2
0
4
Nb-Acetyl-3-indolylmethanamine (17)
0.50
0.20
0.10
38
22
16
2
2
2
30
21
11
2
2
2
a
Percentage of growth inhibition calculated using the formula:
% inhibi-
tion = 100 ꢁ [(growth on amended/growth in control) ꢀ 100]; values represent the
mean and standard deviation of at least two independent experiments conducted in
triplicate.
and Taylor, 1993) and A. brassicicola (Pedras et al., 2009). Despite
the sequence similarity between the brassinin detoxifying glucosyl
transferase from S. sclerotiorum (SsBGT1) and the putative protein
present in B. cinerea (BC1G_07249, 57% amino acid identity), no
traces of 1-b-D-glucopyranosylbrassinin (6) were detected in cul-
tures of any of the isolates of B. cinerea incubated with brassinin
(2).
Fig. 11. Progress curves for transformation of the phytoalexin cyclobrassinin
sulfoxide (19, 0.10 mM) by Botrytis cinerea isolates UAMH 1784 and UAMH 1809
and formation of brassilexin (4). Concentrations were determined using calibration
curves; each point is the average of at least three independent experiments stan-
dard deviation.
2.2.3. Cyclobrassinin (3) and brassilexin (4)
additional metabolites were detected in the acidic or basic culture
extracts. Cultures of isolates UAMH 1784 and UAMH 1809 were
also incubated with cyclobrassinin sulfoxide (19), extracted and
analyzed by HPLC. Results of these experiments indicated that
cyclobrassinin sulfoxide (19) was transformed into brassilexin (4)
(Fig. 10). However, it is not clear whether transformation of cyclo-
brassinin occurs always via 19 or both transformations occur, i.e. 3
can also give directly 4 (Figs. 10 and 11). Ultimately, purification of
the enzyme(s) catalyzing these transformations will clarify the
situation.
Analyses of HPLC chromatograms of neutral extracts of cultures
of isolates UAMH 1784 and UAMH 1809 incubated with cyclo-
brassinin (3) showed that it was almost completely metabolized
within 12 h (Fig. 9). The chromatograms of neutral extracts showed
the presence of additional peaks that were due to cyclobrassinin
sulfoxide (19, tR = 11.8 0.1 min), and brassilexin (4, tR = 9.4
0.2 min), by direct comparison with authentic samples. No
Although cyclobrassinin (3) was previously reported to decom-
pose to undetermined compounds upon treatment with MCPBA
(Devys et al., 1990), in our hands, its oxidation using MCPBA affor-
ded sulfoxide 19 in 27% yield, as described in the Experimental. By
contrast, oxidation of cyclobrassinin (3) using periodate was re-
ported to yield brassilexin (4) (Devys and Barbier, 1992). Cyclo-
brassinin sulfoxide (19) was previously described as
a
phytoalexin of B. juncea (Devys et al., 1990), however complete
NMR spectroscopic characterization is reported here for the first
time. Cyclobrassinin sulfoxide (19) was more inhibitory to B. cine-
rea than cyclobrassinin (3).
Analyses of HPLC chromatograms of neutral extracts of cultures
incubated with brassilexin (4) at 0.05 mM (due to higher toxicity
lower conc. was used) indicated that it was almost completely
metabolized within two days (Fig. 12). Furthermore, the chromato-
grams of neutral extracts indicated the presence of a peak identi-
fied as 3-aminomethyleneindole-2-thione (20, tR = 4.9 0.2 min).
Fig. 9. Progress curves for transformation of the phytoalexin cyclobrassinin (3,
0.10 mM) by Botrytis cinerea isolates UAMH 1784 and UAMH 1809 and formation of
cyclobrassinin sulfoxide (19). Concentrations were determined using calibration
curves; each point is the average of at least three independent experiments stan-
dard deviation.

204
M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
cinerea with A. thaliana have to consider that camalexin (1) is
detoxified to products that could be presumed as plant products
(e.g. 9), but actually result from fungal metabolism. It is surmised
that detoxification reactions of phytoalexins carried out by phyto-
pathogenic fungi do not appear to be predictable, even when there
is a substantial sequence similarity between a detoxifying enzyme
from one species and a putative protein from a phylogenetically re-
lated species. Indeed, S. sclerotiorum was previously shown to glu-
cosylate brassinin (2) using SsBGT1, but no evidence of such
transformation was found in this study with B. cinerea, which pro-
duces a putative protein showing 57% amino acid identity to
SsBGT1, (Accession No. BC1G_07249, Sexton et al., 2009). S. sclero-
tiorum was previously shown to also glucosylate the phytoalexins
camalexin (1), cyclobrassinin (3) and brassilexin (4), but B. cinerea
did not carry out similar transformations under our experimental
conditions.
Fig. 12. Progress curves for transformation of the phytoalexin brassilexin (4,
0.05 mM) by Botrytis cinerea isolates UAMH 1784 and UAMH 1809. Concentrations
were determined using calibration curves; each point is the average of at least three
independent experiments standard deviation.
4. Experimental
This metabolite was further transformed enzymatically to metabo-
lites not extractable with EtOAc, likely due to their higher solubil-
ity in aqueous media. None of these metabolite(s) were detected
either in control cultures or in medium incubated with cyclobrass-
inin (3) or brassilexin (4). Aminomethyleneindole-2-thione (20)
oxidized spontaneously to brassilexin (4) at much slower rate than
its enzymatic reduction, as previously established (Pedras and
Suchy, 2005). Due to its lower stability, solutions of 20 were ob-
tained containing some brassilexin and thus 20 was not tested
for inhibitory activity against isolates UAMH 1784 and UAMH
1809. The complete transformations of cyclobrassinin (3) and
brassilexin (4) by isolates UAMH 1784 and UAMH 1809 led to
metabolites that did not inhibit mycelial growth.
Previously, cyclobrassinin (3) was found to be converted to the
phytoalexin dioxibrassinin (21) and further to non-toxic undeter-
mined metabolites by isolates of L. maculans virulent on canola
(Pedras and Ahiahonu, 2005), whereas L. maculans isolates Laird
2/Mayfair 2 transformed cyclobrassinin (3) to 3-aminomethylen-
eindole-2-thione (20) and to other polar and non-antifungal
metabolites (Pedras and Snitynsky, 2010). Considering that 20
was not detected in cultures incubated with cyclobrassinin (3) dur-
ing the first 12 h, it is not clear whether this metabolite was
formed directly from cyclobrassinin (3) or if it resulted only from
brassilexin (4). Similarly, brassilexin (4) was converted by various
isolate types of L. maculans to 3-aminomethyleneindole-2-thione
(20) and to other polar and non-antifungal metabolites (Pedras
and Suchy, 2005). It is noteworthy that S. sclerotiorum remains un-
ique in its glucosylation of the most antifungal cruciferous phyto-
alexins (Pedras and Hossain, 2006).
4.1. Chemicals and instrumentation
All chemicals were purchased from Sigma–Aldrich Canada Ltd.,
Oakville, ON; solvents were HPLC grade and used as such. Organic
extracts were dried with Na2SO4 and solvents removed under re-
duced pressure using a rotary evaporator. Flash column chroma-
tography (FCC) was carried out using silica gel grade 60, mesh
size 230–400 Å. Preparative thin layer chromatography (prep
TLC) was carried out on silica gel plates, Kieselgel 60 F254
.
Nuclear magnetic resonance (NMR) spectra were recorded on
Bruker 500 MHz Avance spectrometers, for 1H, 500.3 MHz and for
13C, 125.8 MHz; chemical shifts (d) are reported in parts per million
(ppm) relative to TMS; spectra were calibrated using the solvent
peaks; spin coupling constants (J) are reported to the nearest
0.5 Hz. Fourier transform infrared (FT-IR) data were recorded on
a spectrometer and spectra were measured by the diffuse reflec-
tance method on samples dispersed in KBr. MS [high resolution
(HR), electron impact (EI)] were obtained on a VG 70 SE mass spec-
trometer employing a solids probe.
HPLC analysis was carried out with Agilent high performance li-
quid chromatography instruments equipped with quaternary
pump, automatic injector, and diode array detector (DAD, wave-
length range 190–600 nm), degasser, and a column, having an in-
line filter. Method A: column Eclipse XDB-C18 (5 lm particle size
silica, 4.6 i.d. ꢀ 150 mm), mobile phase H2O–MeOH (1:1, v/v) to
MeOH, for 25.0 min, linear gradient, and at a flow rate 0.75 ml/
min; method B (for amines both solvents containing 0.01% propan-
amine): Zorbax ODS (3.5
l
m particle size silica, 3.0 i.d. ꢀ 100 mm),
mobile phase H2O–MeOH (1:9, v/v) to MeOH, for 5.0 min, linear
gradient, MeOH, for 15.0 min, at a flow rate of 0.50 ml/min.
HPLC–DAD-ESI-MS analysis were carried out with an Agilent
1100 series HPLC system equipped with an autosampler, binary
pump, degasser, and a diode array detector connected directly to
a mass detector (Agilent G2440A MSD-Trap-XCT ion trap mass
spectrometer) with an electrospray ionization (ESI) source. Chro-
matographic separation was carried out at room temperature using
an Eclipse XDB C-18 column (5 lm particle size silica, 4.6
i.d. ꢀ 150 mm). The mobile phase consisted of a linear gradient
of 0.2% HCO2H in H2O and 0.2% HCO2H in CH3CN (75:25 to 25:75
in 35 min, to 0:100 in 5 min) and a flow rate of 1 ml/min. Data
acquisition was carried out in positive and negative polarity modes
in a single LC run. Data processing was carried out with Agilent
Chemstation Software. HPLC–HR-ESI-MS was performed on an Agi-
lent HPLC 1100 series directly connected to a QSTAR XL Systems
Mass Spectrometer (Hybrid Quadrupole-TOF LC/MS/MS) with tur-
bo spray ESI source. Chromatographic separation was carried out
3. Conclusion
Altogether our results suggest that phytoalexin detoxification
may contribute to the virulence of B. cinerea in crucifers. Further-
more, our results demonstrate the importance of considering
detoxification of phytoalexins in plants infected with B. cinerea, be-
fore drawing conclusions based on the amounts of phytoalexins
produced. For example, metabolic studies in the interaction of B.

M.S.C. Pedras et al. / Phytochemistry 72 (2011) 199–206
205
at room temperature using a Hypersil ODS C-18 column (5
ticle size silica, 2.1 i.d. ꢀ 200 mm) or a Hypersil ODS C-18 column
(5
consisted of a linear gradient of 0.1% HCO2H in H2O and 0.1%
HCO2H in CH3CN (75:25 to 25:75 in 35 min, to 0:100 in 5 min)
and a flow rate of 0.25 ml/min. Data processing was carried out
by Analyst QS Software.
l
m par-
3-Indolethiocarboxamide (9) (100 mg, 0.570 mmol) was added
to a stirred suspension of IBX (166 mg, 0.630 mmol) in CH3CN
(2.0 ml). After completion of the reaction, the residue was filtered
off and the solvent was evaporated under reduced pressure. The
reaction mixture residue was dissolved in EtOAc (20 ml), washed
with NaHCO3 (sat. solution) and then H2O. The organic layer was
dried and concentrated under reduced pressure, and the residue
was fractionated by FCC (silica gel, CH2Cl2–MeOH, 99:1) to yield
3,5-(30,300-bisindolyl)-1,2,4-thiadiazole (12) (40.5 mg, 45%) and 3-
indolecarboxynitrile (10) (16.1 mg, 20%).
l
m particle size silica, 2.1 i.d. ꢀ 100 mm). The mobile phase
4.2. Mycelial radial growth assays
B. cinerea isolates UAMH 1784 and UAMH 1809 isolated from
soft rot on plum and dogwood leaf, respectively, were obtained
from the University of Alberta Microfungus Collection and
Herbarium.
4.4.2. 3,5-(30,300-Bisindolyl)-1,2,4-thiadiazole (12)
HPLC tR = 20.5 min (method A). 1H NMR d (500 MHz, DMSO-d6)
11.26 (1 H, s), 10.85 (1H, s), 7.61 (1H, m), 7.55 (1H, m), 6.71–6.65
(2H, m), 6.48–6.43 (2H, m), 6.38–6.34 (2H, m). 13C NMR d
(125.8 MHz, DMSO-d6) 180.2, 169.7, 136.7, 136.6, 129.3, 128.7,
125.1, 124.3, 122.8, 122.1, 121.5, 121.1, 121.5, 120.2, 112.0,
111.2, 110.2, 107.6. HREI-MS m/z: calc. for C18H12N4S: 316.0787,
found 316.0783; m/z (%): 316.0 [M]+ (81), 200.0 (19), 174.0 (100),
142.1 (57), 115.0 (18). UV (HPLC, CH3OH–H2O) kmax (nm): 220,
Spores were spotted onto potato dextrose agar plates (PDA) and
allowed to grow for three days under constant light at 23 1 °C.
Plugs (4 mm) were cut from the edges of the mycelia and placed
inverted onto six-well plates containing phytoalexins in either
DMSO or CH3CN mixed into potato dextrose agar. The final concen-
trations of each phytoalexin in agar varied from 0.02 to 0.50 mM,
with a DMSO or CH3CN concentration of 1%. The plates were al-
lowed to grow under constant light at 23 1 °C up to 48 h; the
diameter of the mycelial mat was measured and compared to con-
trol mycelia grown on plates containing DMSO or CH3CN only.
253, 318. FTIR (KBr, cmꢁ1
) mmax: 3100, 1531, 1338, 1236, 744.
4.5. Synthesis of cyclobrassinin sulfoxide (19)
m-Chloroperoxybenzoic acid (MCPBA, 30 mg, 0.17 mmol) in
MeOH (1.5 ml) was added to a solution of cyclobrassinin (3,
20 mg, 0.085 mmol) in MeOH (1 ml) at room temperature, and
the reaction mixture was stirred for 30 min. The reaction mixture
was cooled to 0 °C, dimethylsulfide was added (200 ml to destroy
MCPBA) and the reaction was stirred for another 30 min. The sol-
vent was evaporated under reduced pressure, the residue was dis-
solved in EtOAc (4 ml), the solution was washed with Na2CO3,
dried over Na2SO4, and concentrated to yield the crude product.
The purified product was obtained after FCC (silica gel, hexane–
EtOAc, 2:1) to yield cyclobrassinin sulfoxide (19, 5.7 mg, 27%
yield). HPLC tR = 10.5 min (method A). 1H NMR (CD2Cl2): d 8.75
(br s, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.19
(dd, J = 7.5, 7.5 Hz, 1H), 7.14 (dd, J = 7.5, 7.5 Hz, 1H), 5.42 (s, 2H),
2.86 (s, 3H). 13C NMR (CD2Cl2): d 166.5, 137.4, 125.0, 122.9,
121.5, 120.9, 117.9, 111.4, 100.4, 50.7, 42.0. HREI-MS m/z (%): calc.
for C11H10N2OS2: 250.0234, found 250.0237 (26), 161.0 (100). MS-
ESI m/z (%): 273 [M+Na]+ (15), 251 (3), 187 (100).
4.3. Liquid fungal cultures and biotransformation studies
Liquid cultures of were grown in 250 ml Erlenmeyer flasks con-
taining 100 ml of minimal medium inoculated fungal spores for a
final concentration of 1 ꢀ 104/100 ml, or six mycelial plugs
(6 mm diameter) per 100 ml, and cultures were incubated on a
shaker at 120 rpm. After 48–72 h at 23 1 °C (96 h for cyclobrass-
inin sulfoxide), under constant light, a solution of the phytoalexin
or compound in CH3CN (100–250 ll) was added to the cultures, for
a final concentration of 0.10 mM, except brassilexin (4) that was
0.050 mM. The flasks were returned to the shaker, and samples
(5 ml) were withdrawn at various times and either extracted
immediately with EtOAc or immediately frozen. The EtOAc residue
was dissolved in MeOH or CH3CN and analyzed by HPLC, as de-
scribed above.
4.4. Syntheses of 3-indolethiocarboxamide (9) and
3,5-(30,300-bisindolyl)-1,2,4-thiadiazole (12)
Acknowledgements
3-Indolethiocarboxamide (9) was prepared from 3-indolecarb-
oxynitrile (10) by modification of a previously published procedure
(Gu et al., 1999), as follows. A mixture of 3-indolecarboxynitrile
(100 mg, 0.700 mmol) and thioacetamide (105 mg, 1.40 mmol) in
10% HCl–DMF solution (1.5 ml) was stirred at 95 °C for 12 h. The
reaction mixture was then neutralized with NaHCO3 (sat. solution),
extracted with EtOAc, the organic layer was dried and separated by
FCC (silica gel, CH2Cl2–MeOH, 99:1) to afford indole-3-thiocarbox-
amide (9) (71.5 mg, 59%). The 13C NMR spectroscopic data of 9 is
reported here for the first time, other spectroscopic data being sim-
ilar to that previously reported (Jiang and Gu, 2000).
Financial support was obtained from the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant to
M.S.C.P.), the Canada Research Chairs program, Canada Foundation
for Innovation, the Saskatchewan Government, and the University
of Saskatchewan (graduate assistantship to S.H.). We acknowledge
the technical assistance of K. Brown (NMR), P.B. Chumala (HPLC)
and K. Thoms (MS), from the Department of Chemistry.
Appendix A. Supplementary data
4.4.1. 3-Indolethiocarboxamide (9)
Supplementary data associated with this article (NMR spectra
for new compounds 9 and 12 and cyclobrassinin sulfoxide (19))
can be found, in the online version, at doi:10.1016/
j.phytochem.2010.11.018.
HPLC tR = 4.6 min (method A). 1H NMR (DMSO-d6) d 10.91 (1H,
s), 8.08 (1H, s), 7.95 (1H, s), 7.75 (1H, d, J = 7.5 Hz), 7.22 (1H, d,
J = 3 Hz), 6.56 (1H, d, J = 8 Hz), 6.29 (1H, dd, J = 7, 7 Hz), 6.26 (1H,
dd, J = 7, 7 Hz). 13C NMR (DMSO-d6) d 193.6, 136.8, 128.1, 125.9,
122.0, 121.8, 120.7, 116.3, 112.0. HREI-MS m/z: calc. for C9H8N2S2
176.0409, found 176.0408; m/z (%):176.0 [M]+ (100), 160.0 (24),
143.1 (84), 142.1 (43), 116.0 (20). UV (HPLC, CH3OH–H2O) kmax
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