Detoxification of the cruciferous phytoalexin brassinin in Sclerotinia sclerotiorum requires an inducible glucosyltransferase

Article abstract of DOI:10.1016/j.phytochem.2004.08.033

The phytoalexins, brassinin, 1-methoxybrassinin and cyclobrassinin, were metabolized by the stem rot fungus Sclerotinia sclerotiorum into their corresponding glucosyl derivatives displaying no detectable antifungal activity. Importantly, co-incubation of S. sclerotiorum with camalexins, various phytoalexin analogs, and brassinin indicated that a synthetic camalexin derivative could slow down substantially the rate of brassinin detoxification. Furthermore, inducible brassinin glucosyltransferase (BGT) activity was detected in crude cell-free extracts of S. sclerotiorum. BGT activity was induced by the phytoalexin camalexin, and the brassinin analogs methyl tryptamine dithiocarbamate and methyl 1-methyltryptamine dithiocarbamate. The overall results suggest that the fungus S. sclerotiorum in its continuous adaptation and co-evolution with brassinin producing plants, has acquired efficient glucosyltransferase(s) that can disarm some of the most active plant chemical defenses.

Full text of DOI:10.1016/j.phytochem.2004.08.033

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2685–2694
www.elsevier.com/locate/phytochem
Detoxification of the cruciferous phytoalexin brassinin in Sclerotinia
sclerotiorum requires an inducible glucosyltransferase
*
M. Soledade C. Pedras , Pearson W.K. Ahiahonu, Mohammad Hossain
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
Received 9 April 2004; received in revised form 8 June 2004
Abstract
The phytoalexins, brassinin, 1-methoxybrassinin and cyclobrassinin, were metabolized by the stem rot fungus Sclerotinia sclero-
tiorum into their corresponding glucosyl derivatives displaying no detectable antifungal activity. Importantly, co-incubation of S.
sclerotiorum with camalexins, various phytoalexin analogs, and brassinin indicated that a synthetic camalexin derivative could slow
down substantially the rate of brassinin detoxification. Furthermore, inducible brassinin glucosyltransferase (BGT) activity was
detected in crude cell-free extracts of S. sclerotiorum. BGT activity was induced by the phytoalexin camalexin, and the brassinin
analogs methyl tryptamine dithiocarbamate and methyl 1-methyltryptamine dithiocarbamate. The overall results suggest that the
fungus S. sclerotiorum in its continuous adaptation and co-evolution with brassinin producing plants, has acquired efficient gluco-
syltransferase(s) that can disarm some of the most active plant chemical defenses.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Brassicaceae; Crucifer; Brassinin; Camalexin; Cyclobrassinin; Glucosyltransferase; Methoxybrassinin; Methyl 2-naphthylmethyl dithio-
carbamate; Sclerotinia sclerotiorum
1. Introduction
pathogen would not develop or spread. This control
strategy would be environmentally advantageous if the
inhibitors had minimal effect on other organisms. To-
wards this end, we have been investigating the metabo-
lism and detoxification of cruciferous phytoalexins by
economically important phytopathogenic fungi (Pedras
et al., 2000). Crucifers (family Cruciferae or Brassica-
ceae), which comprise economically important oilseed
and condiment crops and many vegetable species, pro-
duce unique phytoalexins such as brassinins 1–3, con-
taining a dithiocarbamate group (Pedras et al., 2000,
2003). Coincidentally, synthetic dithiocarbamates have
been used extensively both as fungicides and herbicides
(Leroux, 2003; Caldas et al., 2001). The phytopathogenic
fungus Sclerotinia sclerotiorum (Lib.) de Bary causes
stem rot disease in a vast range of plant species and fam-
ilies, making it one of the most economically important
fungal pathogens (Boland and Hall, 1994; Purdy,
1979). The control of stem rot disease within Brassica
A number of plant pathogens produce detoxifying en-
zymes that can effectively disarm phytoalexins (VanEtten
et al., 2001). Such detoxification processes may be detri-
mental to the plant as phytoalexins are crucial chemical
defenses produced de novo by plants to fight pathogens
(Bailey and Mansfield, 1982; Essenberg, 2001). A better
understanding of mechanisms of phytoalexin detoxifica-
tion could lead to new approaches to control plant path-
ogens. For example, selective inhibitors of phytoalexin
detoxifying enzymes produced by phytopathogenic fungi
could be designed and exploited to protect the plant.
Such inhibitors might allow the plant to build up the nat-
urally occurring phytoalexins to a level at which the
*
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966
4730.
E-mail address: s.pedras@usask.ca (M. Soledade C. Pedras).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.08.033

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M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
species is a worldwide problem as no sources of disease
resistance are known currently (Kohli et al., 1995).
Hence, it was of interest to investigate the reactions of
S. sclerotiorum to the strongest antifungal crucifer
phytoalexins.
brassinin (1) indicated that a synthetic derivative of
camalexin (4) could slow down substantially the rate
of brassinin detoxification. Furthermore, inducible gluc-
osyltransferase activity was detected in crude cell-free
extracts of S. sclerotiorum; this activity was induced by
camalexin (4), methyl tryptamine dithiocarbamate (14),
methyl 1-methyltryptamine dithiocarbamate (24) and
the phytoalexin spirobrassinin (23).
R1
H
N
1 R=R1=H
2 R=OCH3; R1=H
3 R=H; R1=OCH3
SCH3
5
2 S
N
2. Results
R
7
2.1. Metabolism, identification of metabolites and anti-
fungal activity
An evaluation of the biological activity of diverse
phytoalexins against S. sclerotiorum indicated that
camalexin (4) and 6-methoxycamalexin (5) could inhibit
completely the growth of the pathogen (Pedras and Ahi-
ahonu, 2002). Furthermore, both camalexins 4 and 5
were detoxified via 6-hydroxycamalexin (6) to 6-O-(b-
Phytoalexins 1, 2 and 11 were synthesized as previ-
ously reported (Pedras et al., 2003); the minimum inhib-
itory concentration (5.0 · 10ꢀ4 M) of brassinin (1) and
1-methoxybrassinin (2) to S. sclerotiorum was deter-
mined in liquid cultures. Due to lower solubility of
cyclobrassinin (11) in aqueous solutions, the minimum
inhibitory conc could not be determined; a slight inhib-
itory effect was observed at 5.0 · 10ꢀ5 M. Subsequently,
brassinins 1 and 2 and cyclobrassinin (11) were sepa-
rately incubated (at 1.0 · 10ꢀ4 M) with S. sclerotiorum,
with culture samples withdrawn, extracted, and ana-
lyzed as described in the Experimental. Cultures incu-
bated with DMSO or CH3CN were used as controls.
HPLC analysis of the broth extracts of fungal cultures
incubated with brassinin (1) indicated it to be completely
metabolized to a single product (HPLC Rt = 7.9 min) in
ca. 24 h. To establish its structure, larger scale cultures
of S. sclerotiorum were incubated with brassinin (1),
were extracted, and the extract subjected to multiple
D
-glucopyranosylcamalexin (8), a very unusual detoxifi-
cation reaction in plant pathogenic fungi. In addition, S.
sclerotiorum converted 6-methoxycamalexin (5) via a
minor pathway to 1-b-D-glucopyranosylcamalexin (7),
as summarized in Scheme 1. In continuation of this
work, because both brassinin (1) and 1-methoxybrassi-
nin (2) showed strong antifungal activity against S. scle-
rotiorum, we investigated their metabolism in cell
cultures and compared it with that of the phytoalexin
cyclobrassinin (11) and dithiocarbamate 14.
Herein we report that both phytoalexins 1, 2 and 11
were metabolized to yield products displaying no detect-
able antifungal activity. Importantly, co-incubation of
S. sclerotiorum with various phytoalexin analogs and
chromatographic steps;
a single biotransformation
product substantially more polar than brassinin was fur-
ther separated by prep. TLC. The structure of this polar
metabolite (9) was determined by analyses of spectro-
scopic data as follows. Comparison of its 1H NMR spec-
trum, obtained in CD3CN, with that of brassinin (1)
indicated the presence of an intact indole moiety (dH
7.64, d J = 8 Hz, 1H, 7.44, d J = 8 Hz, 1H; dH 7.24, s,
1H; dH 7.24, ddd J = 8, 8, 1, 1H, 7.14, ddd J = 8, 8, 1
Hz, 1H), as well as the intact methylene protons of the
side-chain (dH 5.06) and the SCH3 singlet (dH 2.59) of
the dithiocarbamate group. Additional signals at dH
5.43 (d, J = 9 Hz, 1H) and several multiplets at dH
3.42–3.87 suggested the presence of a carbohydrate
moiety.
As well, the molecular formula of C17H22N2O5S2 ob-
tained by HRMS-FAB and the 13C NMR spectral data
corroborated the presence of a carbohydrate residue.
The identity of the monosaccharide unit was established
through homonuclear 1H–1H decoupling experiments
(upon addition of D2O). The coupling constants
(J = 7–9 Hz) indicated axial–axial proton couplings in
4'
5'
N
N
S
S
4
3a
i
3
5
2
4 or 5
N
N
R
HO
H
H
7
6
4 R=H
5 R=OCH
3
i
ii
5
N
N
S
S
1
N
R
N
RO
H CO
3
H
8
7
HO
O
R=
HO
HO
OH
Scheme 1. Detoxification of camalexin (4) and 6-methoxycamalexin
(5) by the plant pathogenic fungus S. sclerotiorum: (i) major pathway;
(ii) minor pathway (Pedras and Ahiahonu, 2002).

M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
2687
a pyranose ring, thus allowing the assignment of a b-
glucopyranose substituent. HMBC spectral data
confirmed that the b-glucopyranose unit was located at
N-1 (correlations of the anomeric proton H-1 with C-2
and C-7a of indole) and thus the structure of this meta-
CH3-(O)) and HMBC (correlation between H-10 and
C-7 at dC 144.0) spectral data confirmed that the b-
glucopyranose unit was located at C-7 and not at C-4.
Thus, the structure of this metabolic product was as-
signed as 7-(O-b-
(10) (Scheme 2).
D-glucopyranosyl)-1-methoxybrassinin
bolic product of brassinin (1) was assigned as 1-b-D-glu-
copyranosylbrassinin (9) (Scheme 2). Furthermore,
cultures of S. sclerotiorum incubated with 9 indicated
that this glucoside was further transformed but no bio-
transformation products were detected or isolated.
Similarly, HPLC analysis of the broth extract of fun-
gal cultures incubated with 1-methoxybrassinin (2) indi-
cated that 2 was completely metabolized a major
product (HPLC Rt = 9.2 min) in ca. 12 h. To establish
its structure, larger scale cultures of S. sclerotiorum were
incubated with 1-methoxybrassinin (2) as above steps.
The structure of the polar metabolite so obtained was
determined by analyses of spectroscopic data, as de-
scribed above for brassinin metabolite 9. Additional sig-
nals suggested the presence of a carbohydrate moiety,
which was corroborated by the 13C NMR spectral data
and HRMS-ESI. The identity of the monosaccharide
unit was established through homonuclear 1H–1H
decoupling experiments (upon addition of D2O). In
addition, as summarized in Fig. 1, NOE difference
experiments (enhancement of the H-6 signal at dH 7.02
upon irradiation of H-10 at dH 5.14 and vice-versa;
enhancement of both CH2 and H-5 at dH 5.00 and
7.06, respectively, upon irradiation of H-4 at dH 7.33;
enhancement of H-2 at dH 7.41 upon irradiation of
Similar to the metabolism of brassinins 1 and 2,
HPLC analysis of the broth extract of fungal cultures
incubated with cyclobrassinin (11) indicated it to be
completely metabolized to a major product (HPLC
Rt = min 8.6) in ca. 12 h. As described in the above
examples, to establish the structure of this metabolic
product, larger scale cultures of S. sclerotiorum incu-
bated with cyclobrassinin (11) for 8 h, were filtered, ex-
tracted, and the broth extract fractionated by column
chromatography followed by prep. TLC to yield a major
metabolite (12) and also a minor metabolite (13). Spect-
roscopic analysis as described above for metabolites 9
and 10 indicated that cyclobrassinin was metabolized
to the N-glucosyl derivative 12 (Scheme 3). The struc-
ture of the minor product was established to be brassica-
nal A (13) by comparison with a synthetic sample
(Pedras and Khan, 1996).
In additional experiments, dithiocarbamates 14, 16
and 17 were synthesized and administered to cultures
of S. sclerotiorum to probe the specificity of the enzymes
involved in the metabolism of brassinins 1 and 2. Cul-
tures were analyzed with metabolic products detected
and isolated as described for brassinin (1). Similar to
the brassinins, 14 was completely metabolized to a
glucoside whose structure was deduced from spectro-
scopic data to be metabolite 15, as described above for
glucosides 9 and 10. Furthermore, incubation of 16
and 17 with S. sclerotiorum showed that both com-
pounds were metabolized in 48 h; however, the 1-naph-
thyl derivative 16 was transformed to several
undetermined products, whereas the 2-naphthyl deriva-
tive 17 yielded one major product (18, HPLC Rt = 9.4
min). The HRMS-FAB mass measurement of 18 gave
a molecular formula of C19H23NO6S2. The FTIR spec-
tral data had a broad absorption band in the 3300
cmꢀ1 region indicative of the presence of hydroxyl
H
N
H
N
4
SCH3
SCH3
5
2 S
2 S
1
6
N
7a
N
R
R
7
R1
9 R=β-D-glc; R1=H
10 R=OCH3; R1=O-β-D-glc
1 R=H
2 R=OCH3
HO
HO
O
β-D-glc =
HO
1'
HO
H
1
groups. The H NMR spectrum showed a broad singlet
Scheme 2. Transformation of brassinin (1) and 1-methoxybrassinin (2)
by the phytopathogen S. sclerotiorum.
at d 8.54 (D2O exchangeable), and resonances for a
naphthalene ring system with substitutions at C-2 and
C-5 or C-8 (d 8.32, d, J = 9 Hz, 1H, 7.76, s, 1H, 7.53,
d, J = 7.5 Hz, 1H, 7.46, dd, J = 9, 1 Hz, 1H, 7.42, dd,
J = 7.5, 7.5 Hz, 1H, 7.15, d, 7 = 7.5 Hz, 1H), signals
for an intact side chain (d 5.05, CH2, 2.59, SCH3), and
H
H
H
H
N
SCH
3
H
H
4
S
6
H
resonances for a b-D-glucopyranose unit. As in the pre-
7a
N
1
vious examples, homonuclear H–1H decoupling exper-
iments established the identity of the glucopyranosyl
moiety. NOE difference experiments were used to deter-
mine the position of the glucosyl moiety either at C-5 or
C-8, as shown in Fig. 2 (enhancement of H-6 at dH 7.15
OCH
3
O
H
1'
O
10
Fig. 1. Selected NOE (dashed lines) and HMBC (full line) correlations
for 7-(O-b- -glucopyranosyl)-1-methoxybrassinin (10).
D
upon irradiation of H-10 at dH 5.09 of the O-b-
D-

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M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
N
N
CHO
SCH
N
H
SCH
SCH
3
3
S
S
+
3
1
N
H
1
1
N
R
11
12 R=β-D-glc
HO
HO
13
O
β-D-glc =
HO
1'
HO
H
Scheme 3. Transformation of cyclobrassinin (11) by the phytopathogen S. sclerotiorum.
dial mycelial growth inhibition assay (Table 2). After
six days of incubation, the mycelium of control plates
incubated with S. sclerotiorum covered the plates al-
most completely, while plates containing 1, 2 and 4
(5 · 10ꢀ4 M) showed no mycelial growth. By contrast
glucosides 9, 10, 12, 15 and 18 under similar condi-
tions showed no inhibitory activity against S.
sclerotiorum.
H
H
1
S
8
N
H
SCH
3
H
H
6
O
O
18
Fig. 2. Selected NOE (dashed lines) correlations for 5-(O-b-
glucopyranosyl)-2-naphthylmethyldithiocarbamate (18).
D-
2.2. Co-metabolism of brassinin, camalexins and related
indole derivatives
Previous work (Pedras and Ahiahonu, 2002) showed
that camalexin (4) and related synthetic analogs were
inhibitory against S. sclerotiorum. Subsequently, to eval-
uate a potential interaction between brassinins and cam-
alexins, brassinin (1, at 5.0 · 10ꢀ5 M) and camalexin (4,
at 1.0 · 10ꢀ4 and 5.0 · 10ꢀ5 M) were co-incubated with
S. sclerotiorum; brassinin (1) was administered to each
culture only after camalexin (4) was incubated for 10
min. Control cultures of S. sclerotiorum containing only
brassinin (1) or camalexin (4) were incubated separately.
The cultures were incubated for different periods and
analyzed by HPLC. Additional co-incubation of brassi-
nin (1) with either 6-fluorocamalexin (19), 6-fluoro-1-
methylcamalexin (20), 6-fluoroindole-3-carboxaldehyde
(21), 6-fluoro-l-methylindole-3-carboxaldehyde (22), or
dithiocarbamates 14, 16 and 17 were carried out. HPLC
analysis of extracts of each culture over a 7-day period
showed that only 6-fluorocamalexin (19) affected sub-
stantially the rate of metabolism of brassinin (1) (Table
3). For example, when brassinin (1) was co-incubated
with 6-fluorocamalexin at 5.0 · 10ꢀ5 M, it was metabo-
lized in ca. 96 h, whereas in cultures co-incubated with
6-fluorocamalexin at 1.0 · 10ꢀ4, brassinin (1) was de-
tected (2–5%) even after seven days.
Scheme 4. Transformation of methyl 2-naphthylmethyl dithiocarba-
mate (17) by the phytopathogen S. sclerotiorum.
glucopyranose unit, and vice-versa, and enhancement of
H-8 at dH 7.53 upon irradiation of H-10 at dH 7.76 and
vice-versa). As expected, the 13C NMR spectral data of
metabolite 18 displayed 19 carbon signals, one of which
indicated a deshielded sp2 carbon atom at dC 154.0.
Thus, the structure of this metabolic product was as-
signed as methyl 5-(O-b-D-glucopyranosyl)-2-naphthyl-
methyl dithiocarbamate (18) (Scheme 4).
The results of the biotransformations of metabolism
of brassinin (1), 1-methoxybrassinin (2), cyclobrassinin
(11) and analogs are summarized in Table 1; the percent-
ages of the products obtained do not account for the to-
tal amount of compounds used in the incubation
experiments since the respective glucosyl products are
further transformed to unidentified substances.
CHO
N
S
N
F
R
1
N
R
R
1
21 R=H
22 R=CH
19 R=H; R =F
The antifungal activity of 1, 2, 11, 14, 16 and 17
against S. sclerotiorum was determined utilizing a ra-
1
3
20 R=CH ; R =F
3

M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
2689
Table 1
Products of metabolism of brassinin (1), 1-methoxybrassinin (2), cyclobrassinin (11) and analogs 14, 16 and 17 by S. sclerotiorum
Compound (1.0 · 10ꢀ4 M) added to fungal culturesa
Products of metabolism (% relative to compound added)
Brassinin (1)
1-Methoxybrassinin (2)
Cyclobrassinin (11)
Complete biotransformation to 9 (75%)b in 24 h
95% Biotransformation to 10 (15%)b in 12 h
90% Biotransformation to 12 (36%)b and 13 (6%) in 12 h
Complete biotransformation to 15 (73%)b in 48 h
Complete biotransformation to undetermined products in 48 h
Methyl tryptamine dithiocarbamate (14)
Methyl 1-naphthylmethyl dithiocarbamate (16)
Methyl 2-naphthylmethyl dithiocarbamate (17)
a
Biotransformation to 18 (41%)c in 48 h
Compounds 1, 14, 16 and 17 were dissolved in DMSO, and 2 and 11 were dissolved in CH3CN, added to six-day-old cultures and incubated at
24 1 ꢁC.
b
Percentage yields of products determined by HPLC analyses using calibration curves; glucosyl derivatives 10, 12 ,15 and 18 were further
transformed to undetermined products.
c
Percentage yields of products represent isolated yields.
ence of compounds related to brassinin (1) such as
camalexin (4), methyl tryptamine dithiocarbamate
(14), methyl 1-methyltryptamine dithiocarbamate
(24), or spirobrassinin (23), BGT activity was detected
consistently. As shown in Table 4, camalexin (4),
methyl tryptamine dithiocarbamate (14), and methyl
1-methyl tryptamine dithiocarbamate (24) were better
inducers of BGT activity than spirobrassinin (23) (spe-
cific activity 0.07 vs. 0.02 nmol/min/mg of protein).
Enzyme assays carried out with cell-free extracts of in-
duced cultures of S. sclerotiorum in the absence of
UDPG did not show BGT activity. Furthermore, the
substrate affinity of BGT and/or related glucosyl-
transferase(s) produced by S. sclerotiorum was exam-
ined using the compounds shown in Table 5. These
assays revealed that both brassinin (1) and methyl
tryptamine dithiocarbamate (14) were glucosylated to
a similar extent in the same amount of time. On the
other hand, neither camalexin (4) nor 6-fluorocama-
lexin (19) were transformed in cell-free extracts con-
taining BGT activity, but 6-hydroxycamalexin (6)
was glucosylated to the expected glucoside 8, though
not as efficiently as brassinin (1). Subsequently, cell-
free extracts were co-incubated with brassinin (1)
and 6-fluorocamalexin (19), under standard assay con-
ditions, and the product(s) of the enzymatic reac-
tion(s) were analyzed by HPLC. These HPLC
Table 2
Percentage of inhibition of S. sclerotiorum incubated with phytoalexins
1, 2, 4, 11, and analogs 14, (2-day incubation, constant light), 16, 17
and 19 (4 day incubation, constant light)
Compound
Concentration (M) % Inhibitiona SD
Brassinin (1)
5 · 10ꢀ4
3 · 10ꢀ4
1 · 10ꢀ4
5 · 10ꢀ4
3 · 10ꢀ4
1 · 10ꢀ4
5 · 10ꢀ4
3 · 10ꢀ4
5 · 10ꢀ5
100
100
37
8
6
6
1-Methoxybrassinin (2)
Camalexin (4)
100
100
56
100
100
81
Cyclobrassinin (11)
5 · 10ꢀ4
3 · 10ꢀ4
5 · 10ꢀ5
Not soluble
Not soluble
<10
Methyl tryptamine
dithiocarbamate (14)
5 · 10ꢀ4
3 · 10ꢀ4
1 · 10ꢀ4
100
100
75
6
1
Methyl 1-naphthylmethyl 5 · 10ꢀ4
dithiocarbamate (16)
80
0
5 · 10ꢀ5
Methyl 2-naphthylmethyl 5 · 10ꢀ4
dithiocarbamate (17)
80 1 0
0
5 · 10ꢀ5
6-Fluorocamalexin (19)
5 · 10ꢀ4
70
0
1
5 · 10ꢀ5
a
%
Inhibition = 100 ꢀ [(growth in treated/growth in con-
trol) · 100]; results are the mean of three independent experiments
conducted in triplicate.
analyses indicated clearly that 1-(O-b-D-glucopyrano-
syl)brassinin (9) was the only product formed and that
its rate of formation was affected substantially by the
presence of 6-fluorocamalexin (19) (Table 4).
2.3. Transformation of brassinin (1) in crude cell-free
extracts: evidence for brassinin glucosyltransferase
activity
N
SCH
NH
S
3
Glucosylation of brassinin (1) was obtained in
crude cell-free extracts prepared from mycelia of S.
sclerotiorum. Initially, no brassinin glucosyltransferase
(BGT) activity was detected in crude cell-free extracts;
however, when S. sclerotiorum was grown in the pres-
SCH
3
S
O
N
N
H
CH
3
24
23

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M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
Table 3
Co-metabolism of brassinin (1) and compounds 4, and 19–22 by S. sclerotiorum
Compound added to fungal culturesa
Complete transformation of brassinin
(1) to glucoside 9
Brassinin (1) (1.0 · 10ꢀ4 M)
24 h
24 h
Brassinin (1) (5.0 · 10ꢀ5 M) + camalexin (4) (5.0 · 105 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + camalexin (4) (1.0 · 10ꢀ4 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluorocamalexin (19) (5.0 · 10ꢀ5 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluorocamalexin (19) (1.0 · 10ꢀ4 M)
24 h
96 h
Traces of brassinin (<5%)b remaining
untransformed after 7 d
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoro-1-methylcamalexin (20) (5.0 · 10ꢀ5 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoro-1-methylcamalexin (20) (1.0 · 10ꢀ4 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoroindole-3-carboxaldehyde (21) (5.0 · 10ꢀ5 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoroindole-3-carboxaldehyde (21) (1.0 · 10ꢀ4 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoro-1-methylindole-3-carboxaldehyde (22) (5.0 · 10ꢀ5 M)
Brassinin (1) (5.0 · 10ꢀ5 M) + 6-fluoro-1-methylindole-3-carboxaldehyde (22) (1.0 · 10ꢀ4 M)
a
24 h
24 h
24 h
24 h
24 h
24 h
Compounds were dissolved in DMSO, added to six-day-old cultures and brassinin was added 10 min after addition of each compound;
incubation at 24 1 ꢁC.
Percentage yields determined by HPLC analyses using calibration curves.
b
Table 4
Brassinin (1) glucosyltransferase (BGT) activity of cell-free extracts of
alexins 1, 2 and 11 and dithiocarbamates 14 and 17 to
glucosylated products having no detectable antifungal
S. sclerotiorum obtained from mycelia of liquid cultures incubated for
activity. While brassinin (1), cyclobrassinin (11) and
2 h with different inducers
analog 14 (no 1-N-substituents) were detoxified through
Chemical inducer
(5.0 · 10ꢀ5 M)
Specific activitya
(nmol/min/mg of protein) SD
direct N-glucosylation, 1-methoxybrassinin (2) and
methyl 1-methyltryptamine dithiocarbamate (24) (Ahia-
honu, 2003) were regioselectively oxidized at C-7 and
then O-glucosylated. Thus, it appears that glucosylation
occurs at N-1 of brassinin-like molecules when there is
no N-substituent, otherwise glucosylation will occur
after regioselective hydroxylation at C-7. Replacement
of the indolyl moiety of brassinins with a naphthyl ring
did not prevent the enzymatic detoxification of naph-
thylmethyl dithiocarbamates 16 and 17 (Table 1). This
is not surprising as it was shown that replacing the nat-
urally occurring plant hormone 3-indolylacetic acid with
2-naphthylacetic acid caused no substantial changes on
the binding affinity to auxin binding sites of maize
(Ray, 1977). Nonetheless, 2-naphthylmethyl dith-
iocarbamate 17 appears to mimic 1-substituted indoles
better than 1-naphthylmethyl dithiocarbamate 16, as
17 was detoxified mainly to the glucoside 18 whereas
No inducer
Camalexin (4)
No activity
0.07 0.04
0.07 0.01b
Methyl tryptamine
dithiocarbamate (14)
Methyl 1-methyltryptamine
dithiocarbamate (24)
Spirobrassinin (23)
a
0.07 0.01
0.02 0.01
Results are from three independent experiments carried out in
triplicate; brassinin at 2.0 · 10ꢀ5 M was used in enzyme assays.
Results are from ten independent experiments carried out in
triplicate.
b
Table 5
Substrate affinity of glucosyltransferases in cell-free extracts of mycelia
obtained from induced cultures of S. sclerotiorum
Substrate
(3.0 · 10ꢀ5 M)
Specific activitya
(nmol/min/mg) SD (brassinin = 100)
Relative activity %
Brassinin (1)
0.24 0.03
0.24 0.03
100
100
16 yielded
a number of undetermined products.
Methyl tryptamine
dithiocarbamate (14)
Cyclobrassinin(11)
Camalexin (4)
Although S. sclerotiorum detoxifies dithiocarbamates
through N-glucosylation, blackleg fungi (Leptosphaeria
maculans, asexual stage Phoma lingam) detoxified brass-
inin (1) and related dithiocarbamates mainly via oxida-
tion of the side chain at C-3 of the indole ring (Pedras
et al., 2000; Pedras and Okanga, 2000). These different
detoxification reactions suggest that these plant patho-
genic fungi have evolved rather different but selective
detoxifying enzymes and suggest that these fungal en-
zymes may be virulence traits, as in the case of other
fungi (VanEtten et al., 2001).
0.20 0.03
No detectable
conversion
83
0
6-Fluorocamalexin (19)
No detectable
conversion
6-Hydroxycamalexin (6) 0.10 0.03
0
42
a
Results are from three independent experiments carried out in
triplicate.
3. Discussion
The potential interaction between brassinin (1) and
camalexin (4) was evaluated by co-incubation of both
compounds in cell cultures; it was concluded that the
The results described above demonstrated that S.
sclerotiorum produces enzymes able to detoxify phyto-

M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
2691
Table 6
rate of co-metabolism of either 1 or 4 was not affected,
relative to when each compound was incubated alone
(Table 3). By contrast, when brassinin (1) was co-incu-
bated with 6-fluorocamalexin (19) in cell cultures, brass-
inin (1) was metabolized at substantially slower rates
(Table 3). In addition, the transformation of 6-fluoroca-
malexin (19) in cell cultures appeared to start only after
brassinin (1) was completely transformed. This interac-
tion between (1) and 19 suggests that 6-fluorocamalexin
(19) has an inhibitory effect on the glucosylation of
brassinin (1) and that this effect is not due to the anti-
fungal activity of 19 (since 19 is less antifungal than
camalexin (4), Table 2).
Enzyme assays carried out with crude cell-free mycel-
ial extracts of non-induced cultures of S. sclerotiorum or
induced cultures in the absence of UDPG did not show
BGT activity. These results indicated that BGT is an
inducible N-glucosyltransferase that requires the co-en-
zyme UDPG or equivalent (Table 4). The specific activ-
ity of crude cell-free extracts incubated separately with
brassinin (1), camalexin (4), cyclobrassinin (11), dith-
iocarbamate 14, 6-fluorocamalexin (19) or 6-hydroxyca-
malexin (6) indicated that the putative BGT is specific
(Table 5). Furthermore, 6-fluorocamalexin (19) inhib-
ited BGT and/or related glucosyltransferase(s) activity
in cell-free extracts (Table 6). That is, 6-fluorocamalexin
(19) is an inhibitor of brassinin (1) glucosylation both in
cell cultures and in crude cell-free extracts (Tables 3 and
6, respectively). However, because 19 is N-glucosylated
in cell cultures in 48 h (Scheme 5, Pedras and Ahiahonu,
Effect of 6-fluorocamalexin (19) on brassinin glucosyltransferase
(BGT) in cell-free extracts of mycelia obtained from induced cultures
of S. sclerotiorum
Substrate/concentration
Specific activitya
(nmol/min/mg)
SD
Relative activity %
(brassinin = 100)
Brassinin (1)/3 · 10ꢀ5
Brassinin (1) +
M
0.24 0.03
0.083 0.003
100
34
6-fluorocamalexin
(19)/both at 3 · 10ꢀ5
M
Brassinin (1)/3 · 10ꢀ5 M +
6-fluorocamalexin (19)/
0.076 0.003
32
6 · 10ꢀ5
M
Brassinin (1)/2 · 10ꢀ5
Brassinin (1) +
6-fluorocamalexin
M
0.027 0.004
0.012 0.002
100
44b
(19)/both at 2 · 10ꢀ5
M
a
Results are from three independent experiments carried out in
triplicate.
b
Relative to brassinin at 2 · 10ꢀ5 M.
Scheme 5. Transformation of 6-fluorocamalexin (19) by the phyto-
pathogen S. sclerotiorum (Pedras and Ahiahonu, 2002).
2002), its lack of conversion to 6-fluoro-1-(b-D-glucopy-
ranosyl)camalexin (25) in crude cell-free extracts is not
completely understood. A possible explanation is that
in cell cultures the unnatural 6-fluorocamalexin (19)
may be transformed by non-specific glucosyltransferases
which may be unstable under our extraction conditions
and/or may require a different co-enzyme. Considering
that neither camalexin (4) nor 6-fluorocamalexin (19)
are glucosylated in cell-free extracts, it is likely that
the thiazolyl ring prevents camalexins from fitting in
the catalytic site of this putative BGT. Further conclu-
sions will have to wait for detailed studies of fractions
containing purified BGT and related enzymes.
that knowledge of the mechanisms of fungal detoxifica-
tion can lead to the design of effective inhibitors that
could prevent phytoalexin detoxification.
4. Experimental
4.1. General procedures
In concluding, it is pertinent to point out that glu-
cosylation is less usual in microorganisms, particularly
in plant pathogens, but O-glucosylation and, to a lesser
extent, N-glucosylation are common detoxification
mechanisms among plants (Pedras and Ahiahonu,
2002; Hall et al., 2000). Interestingly, various fungi used
as model systems for drug metabolism, transformed
phenothiazine to the N-glucosylated derivative (Pars-
hikov et al., 1999). The work reported here suggests that
the fungus S. sclerotiorum in its continuous adaptation
and co-evolution with brassinin producing plants, has
acquired efficient glucosyltransferase(s) that can disarm
the plant chemical defenses. Ultimately, it is anticipated
All chemicals were purchased from Sigma–Aldrich
Canada Ltd., Oakville, ON. All solvents were HPLC
grade and used as such, except for CH2Cl2 and CHCl3
which were redistilled. Organic extracts were dried (an-
hydr. Na2SO4) and solvents removed under reduced
pressure in a rotary evaporator.
NMR spectra were recorded on Bruker Avance spec-
1
trometers; for H (500 MHz), d values were referenced
to CDCl3 (CCl3H 7.27 ppm), CD3CN (CD2HCN 1.94
ppm), or (CD3)2CO (CD3COCD2H 2.05 ppm) and for
13C (75.5 or 125.8 MHz) referenced to CDCl3 (77.23
ppm), CD3CN (1.39, 118.69 ppm), or (CD3)2CO
(29.92 ppm). Fourier transform infrared (FTIR) spectra

2692
M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
were obtained on a Bio-Rad FTS-40 spectrometer using
a diffuse reflectance cell. Mass spectra (MS) were
obtained on a VG 70 SE mass spectrometer; HRMS-
FAB was obtained at 70 eV employing a solids or con-
tinuous flow probe; HRMS-ESI was obtained on a
QStar XL TOF mass spectrometer (MDS Sciex, Toron-
to, ON). Specific rotations, [a]D, were determined at
ambient temperature on a Rudolph DigiPol DP781
polarimeter using a 1 ml, 10 cm path length cell; the
units are 10ꢀ1 deg cm2 gꢀ1 and the concentrations (c)
are reported in g/100 ml.
HPLC analyses were carried out with a high perform-
ance liquid chromatograph equipped with quaternary
pump, automatic injector, and diode array detector
(wavelength range 190–600 nm), degasser, and a Hyper-
sil ODS column (5 lm particle size silica, 4.6 i.d. · 200
mm), with an in-line filter. Mobile phase: linear gradient
H2O–CH3CN (3:1) to 100% CH3CN in a 35 min period
at a flow rate 1.0 ml/min.
by HPLC. The metabolites were isolated by preparative
TLC (silica gel, CH2Cl2–MeOH, 9:1, multiple develop-
ment) and/or reversed phase preparative TLC (RP C-
18 silica gel, H2O–CH3CN).
4.4. Preparation of crude cell-free extracts, protein and
enzyme assays
S. sclerotiorum was grown in liquid cultures as reported
above; after five days the inducer compound dissolved in
DMSO (5 · 10ꢀ5 M in culture solution) was added to cul-
tures and the cultures incubated for additional 24 h. The
fungal mycelium was removed by the filteration, washed
with H2O, with the remaining H2O squeezed out between
filter paper and the mycelial pad frozen immediately. Fro-
zen mycelia (12–20 g) was mixed with ice cold standard
buffer (10–20 ml), 50 mM Tris–HCl, pH 8.0, containing
20% glycerol, 20 mM 2-mercaptoethanol, 0.1 mM phenyl-
methylsulfonyfluoride (PMSF) and 0.01% Triton X-100)
and ground using a mortar and pestle until a homogenous
mixture was obtained, similar to a previous report (Zenk
et al., 2000). The mixture was then centrifuged (27,216g
for 15 min) to obtain the cell homogenate (12–25 ml,
26–55 mg total protein), and the pellet was discarded.
The Bradford protein assay was used to quantify pro-
teins in cell-free extracts using bovine serum albumin
standard curves.
Enzyme assays were carried out at 27 ꢁC, using brass-
inin (1) (or other compounds as reported) as substrate
and UDPG as glucose donor (Orlean, 1982). In assays
using brassinin (1) as substrate, product accumulation
was linear for at least 3 h; the substrate was always pre-
sent in large molar excess over the enzyme. The specific
activity of cell-free extracts was defined as the amount
(nmol) of glucopyranosyl product formed per min per
mg of protein. The standard assay mixture contained
50 mM Tris–HCl buffer (98 ll, pH 8.0), UDPG (15
mM, 100 ll), and cell homogenate (400 ll, 0.88 lg pro-
tein). The reaction was started by adding brassinin (1) or
potential substrate in DMSO (10 mM, final concentra-
tion in assay media 3 · 10ꢀ5 M) and incubated for 3 h
with constant shaking at 27 ꢁC. The reaction was
stopped by extracting with EtOAc, the solvent was re-
moved under reduced pressure, the extract dissolved in
MeOH and analyzed by HPLC. Quantification of prod-
ucts was carried out using standard calibration curves.
4.2. Fungal cultures and antifungal assays
S. sclerotiorum (clone #33) was grown on potato dex-
trose agar (PDA) plates at 24 1 ꢁC, in the dark. Scle-
rotia were collected over a 4-week period and stored at
room temperature in the dark. Erlenmeyer flasks (250
ml) containing 100 ml of minimal media (Pedras and
Okanga, 2000) were inoculated with sclerotia of S. scle-
rotiorum and were incubated at 25 1 ꢁC on a shaker at
120 rpm under constant light. The antifungal activity of
compounds was determined as previously reported (Ped-
ras and Ahiahonu, 2002) but using either 12-well plates,
2-day incubation (22 mm diameter, 1 ml per well) or 6-
well plates, 4-day incubation (35 mm diameter, 2 ml per
well).
4.3. Metabolism
Six-day-old cultures of S. sclerotiorum were incubated
with phytoalexins or compounds at : 25 1 ꢁC on a sha-
ker at 120 rpm under constant light. Each compound dis-
solved in DMSO or CH3CN was added to fungal cultures
(final concentration 1 · 10ꢀ4 or 2 · 10ꢀ4 M) and to unin-
oculated medium (control). Samples were taken from the
flasks up to seven days and extracted with EtOAc; the
resulting aqueous layer was acidified (to pH 2 with dil
HCl) and re-extracted with EtOAc, and finally the acidic
aqueous layer was made alkaline and re-extracted with
CHCl3. After concentration of solvent, the extracts were
analyzed by HPLC.
To obtain larger amounts of extract to isolate the
products of metabolism of each compound, experiments
were carried out with larger batches. The extracts were
fractionated by FCC on silica gel with gradient elution:
CH2Cl2–MeOH (100:0–85:15) or on RP C-18 silica gel,
H2O–CH3CN (80:20–50:50). Each fraction was analyzed
4.5. Syntheses and spectral data
Brassinin (1), 1-methoxybrassinin (2), camalexin (4),
cyclobrassinin (11), tryptamine dithiocarbamate (14), 6-
fluorocamalexin (19), 6-fluoroindole-3-carboxaldehyde
(21), and 6-fluoro-1-methylindole-3-carboxaldehyde
(22) were synthesized as previously reported (Pedras et
al., 2003; Pedras and Ahiahonu, 2002). 6-Hydroxycama-
lexin (6) was isolated from culture as previously reported

M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
2693
(Pedras and Ahiahonu, 2002). Satisfactory spectroscopic
data were obtained for all known compounds.
Hz, 1H), 7.44 (s, 1H), 7.24 (ddd, J = 8, 8, 1 Hz, 1H),
7.14 (ddd, J = 8, 8, 1 Hz, 1H), 5.46 (d, J = 8 Hz, 1H),
5.08 (s, 2H), 3.42–3.87 (m, 10H, 4H D2O exchangeable),
2.59 (s, 3H) and a minor signal (ca. 1/8 intensity of the
major one) due to a rotamer at 2.68 (s). 13C NMR
(125.8 M Hz, CD3CN): d 198.8 (s), 137.0 (s), 128.0 (s),
125.7 (d), 122.6 (d), 120.5 (d), 119.5 (s), 112.0 (d),
111.0 (d), 85.2 (d), 79.2 (d), 77.9 (d), 72.5 (d), 70.5 (d),
61.9 (t), 42.4 (t), 17.6 (q ). HRMS-FAB m/z: measured
398.0965 (M+, calcd. 398.0970 for C17H22N2O5S2).
MS-FAB m/z (relative intensity): 398 (M+, 5), 154
(100), 137 (67), 136 (85), 107 (35) 77 (40), 57 (43), 55
(69). FTIR (cmꢀ1): 3317, 2952, 2922, 2852, 1740, 1581,
1462, 1377, 1115, 1076, 875, 743 cmꢀ1. UV (CH3CN)
kmax (log e): 218 (4.0), 270 (3.6).
4.5.1. Methyl 1-naphthylmethyl dithiocarbamate (16)
To a solution of 1-naphthaldehyde (129 mg, 0.83
mmol) in EtOH (2 ml) a solution of NH2OH Æ HCl
(115 mg, 1.65 mmol) and Na2CO3 (88 mg, 0.83 mmol)
in EtOH–H2O (0.5/1 ml) was added at room tempera-
ture. After stirring at room temperature for 2 h, the sol-
vent was removed under reduced pressure, the residue
dissolved in EtOAc (30 ml), washed with brine and dried
(Na2SO4). Concentration under reduced pressure
yielded 1-naphthaldehyde oxime (140 mg, 99%). To a
solution of 1-naphthaldehyde oxime (50 mg, 0.29 mmol)
in EtOH (10 ml), 10% Pd/C (10 mg) and glacial AcOH
(2 ml) were added. The reaction mixture was shaken un-
der 3-4 atmosphere of hydrogen at room temperature
for 2 h. The catalyst was removed by filtration, conc.
HCl (1 ml) was added to the filtrate, with the filtrate
concentrated under reduced pressure. The residue was
dissolved in H2O (5 ml), then extracted with Et2O, and
the aqueous layer was basified with 5 M NaOH. The
resulting mixture was extracted (CH2Cl2–MeOH, 9:1,
v/v), the combined extracts washed with brine, dried
(Na2SO4), and concentrated under reduced pressure to
yield crude 1-naphthylmethylamine (36 mg, 79%). This
amine was used without further purification to prepare
the dithiocarbamate. Separation of the crude dith-
iocarbamate by FCC (EtOAc: hexanes, 1:15, v/v)
yielded methyl 1-naphthylmethyl dithiocarbamate (16)
(49 mg, 89%). FTIR (KBr) (cmꢀ1) 3342, 3239, 1500,
1351; UV (CH3CN) kmax nm (log e) 272.1 (4.57), 222.3
4.5.4. 7-(b- -glucopyranosyl)-1-methoxybrassinin (10)
D
1
HPLC Rt = 9.2 min; [a]D = ꢀ 55 (c 0.54, MeOH). H
NMR (500 MHz, CD3CN): d 8.22 (br s, 1H D2O
exchangeable), 7.41 (s, 1H), 7.33 (d, J = 7.7 Hz, 1H),
7.06 (dd, J = 7.7, 7.8 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H),
5.14 (d, J = 7.7 Hz, 1H), 5.00 (d, J = 4.4 Hz, 2H), 4.14
(s, 3H), 3.42–3.84 (m, 10H, 4H D2O exchangeable), 2.59
(s, 3H). 13C NMR (125.8 MHz, CD3CN): d 198.5 (s),
144.0 (s), 126.8 (s), 125.5 (d), 123.6 (s), 121.2 (d), 113.7
(d), 108.7 (d), 107.7 (s), 101.6 (d), 77.1 (d), 76.9 (d), 74.1
(d), 70.5 (d), 67.2 (q), 61.9 (d), 42.1 (t), 17.6 (q). HRMS-
ESI m/z: measured 445.1094 ([M + H]+, calcd. 445.1097
for C18H25N2O7S2). MS-ESI m/z (relative intensity): 445
([M + H]+, 58), 414 (72), 338 (100), 249 (25). FTIR
(cmꢀ1): 3347, 2926, 2855, 1698, 1578, 1496, 1249, 1077
cmꢀ1. UV (CH3CN) kmax (log e): 221 (4.5), 270 (4.0).
1
(5.30). H NMR (300 MHz, CDCl3) d 7.91 (m, 3H),
7.58–7.42 (m, 4H), 7.03 (s br, 1H), 5.31 (d J = 5 Hz,
2H), 2.66 (s, 3H). 13C NMR (75.5 MHz, CDCl3) d
189.9, 134.1, 131.8, 131.7, 129.6, 129.1, 127.9, 127.3,
126.5, 125.7, 123.6, 50.0, 18.5. HRMS-EI m/z calcd.
for C13H13NS2 247.0489, found 247.0486.
4.5.5. 1-(b- -glucopyranosyl)cyclobrassinin (12)
D
1
HPLC Rt = 8.6 min; [a]D = ꢀ 14 (c 0.23, MeOH). H
NMR (500 MHz, (CD3)2CO): d 7.57 (d, J = 8 Hz, 1H),
7.50 (d, J = 8 Hz, 1H), 7.09–7.16 (m, 2H), 5.48 (br, s,
1H), 5.32 (d, J = 17.7 Hz, 1H), 4.77 (d, J = 17.7 Hz,
1H), 4.66 (br, s, 1H, D2O exchangeable), 3.63–4.09 (m,
8H, 2H D2O exchangeable), 2.54 (s, 3H). 13C NMR
(125.8 MHz, (CD3)2CO): d 152.5 (s), 137.0 (s), 125.5
(s), 121.9 (s), 121.8 (d), 120.4 (d), 117.3 (d), 111.1 (d),
104.5 (s), 86.1 (d), 80.4 (d), 78.1 (d), 72.7 (d), 70.8 (d),
62.4 (t), 48.5 (t), 14.7 (q). HRMS-FAB m/z: measured
397.0881 ([M + H]+, calcd. 397.0891 for C17H21N2O5S2).
MS-FAB m/z (relative intensity): 397 ([M + H]+, 100),
396 (63), 395 (34), 329 (50). FTIR (cmꢀ1): 3380, 2923,
2852, 1617, 1452, 1346, 1249, 1079, 901, 734 cmꢀ1. UV
(CH3CN) kmax (log e): 231 (4.4), 286 (3.9).
4.5.2. Methyl 2-naphthylmethyl dithiocarbamate (17)
Prepared as described for methyl 1-naphthylmethyl
dithiocarbamate (16) in similar yield but using as start-
ing material 2-naphthaldehyde. FTIR (KBr) (cmꢀ1
)
3203, 3006, 1520, 1353. UV (CH3CN) kmax nm (log e)
249.9 (4.59), 220.8 (5.16). H NMR (300 MHz, CDCl3)
1
6 7.82 (m, 3H), 7.74 (s, 1H), 7.50 (m, 2H), 7.41 (d, J = 8
Hz, 1H), 7.27 (s br, 1H), 5.05 (d, J = 5 Hz, 2H), 2.66 (s,
3H). 13C NMR (75.5 MHz, CDCl3) d 199.6, 133.9,
133.5, 133.2, 129.1, 128.1, 127.4, 127.3, 126.7, 126.5,
126.2, 51.5, 18.6. HRMS-EI m/z calcd. for C13H13NS2
247.0489, found 247.0485.
4.5.6. Methyl 1-(b-
iocarbamate (15)
D
-glucopyranosyl)tryptamine dith-
1
4.5.3. 1-(b-
D
-glucopyranosyl)brassinin (9)
HPLC Rt = 8.9 min.; [a]D = ꢀ 5 (c 0.80, MeOH). H
NMR (500 MHz, CD3CN): d 8.18 (br s, 1H D2O
exchangeable), 7.68 (d, J = 8 Hz, 1H), 7.52 (d, J = 8
Hz, 1H), 7.29 (s, 1H), 7.22 (ddd, J = 8, 8, 1 Hz, 1H)
HPLC Rt = 7.9 min.; [a]D = ꢀ 7.7 (c 0.20, MeOH).
1H NMR (500 MHz, CD3CN) d 8.34 (br s, 1H D2O
exchangeable), 7.64 (d, J = 8 Hz, 1H), 7.52 (d, J = 8

2694
M. Soledade C. Pedras et al. / Phytochemistry 65 (2004) 2685–2694
7.15 (ddd, J = 8, 8, 1 Hz, 1H), 5.44 (d, J = 8 Hz, 1H),
3.93 (m, 2H), 3.86 (t, J = 8 Hz, 1H), 3.45–3.76 (m, 8H,
3H exchangeable with D2O), 3.08 (m, 2H), 2.76 (s, 1H
exchangeable with D2O), 2.53 (s, 3H), and a minor sig-
nal (ca. 1/8 intensity of the major one) due to a rotamer
at 2.61 (s). 13C NMR (125.8 MHz, CD3CN): d 198.8 (s),
137.0 (s), 128.9 (s), 123.7 (d), 122.4 (d), 119.3 (d), 119.1
(d), 113.3 (s), 110.8 (d), 85.2 (d), 79.1 (d), 77.9 (d), 72.6
(d), 70.6 (d), 62.0 (t), 47.4 (t), 23.8 (t), 17.5 (q).
HRMS-FAB m/z: measured 413.1209 ([M + H]+, calcd.
413.1205 for C18H25N2O5S2). MS-FAB m/z (relative
intensity): 413 ([M + H]+, 41), 412 (25), 329 (42), 305
(30), 261 (100), 176 (98). FTIR (cmꢀ1): 3329, 2921,
2856, 1708, 1610, 1509, 1462, 1421, 1365, 1329, 1227,
1071, 1017, 930, 744, 620 cmꢀ1. UV (CH3CN) kmax
(log e): 223 (4.2), 273 (3.7).
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4.5.7. Methyl 5-(O-b-
methyl dithiocarbamate (18)
D
-glucopyranosyl)-2-naphthyl-
1
HPLC Rt = 9.4 min.; [a]D = ꢀ 5 (c 0.04, MeOH). H
NMR (300 MHz, CH3CN) d 8.54 (br s, 1H, D2O
exchangeable), 8.32 (d, J = 9 Hz, 1H), 7.76 (s, 1H),
7.53 (d, J = 7.5 Hz, 1H), 7.46 (dd, J = 9, 1 Hz, 1H)
7.42 (dd, J = 7.5, 7.5 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H),
5.09 (d, J = 7.5 Hz, 1H), 5.05 (s, 2H), 3.36–3.81 (m,
10H, 4H D2O exchangeable), 2.59 (s, 3H). 13C NMR
(125.8 MHz, CD3CN): d 198.8 (s), 154.8 (s), 138.0 (s),
137.0 (s), 128.3 (d), 127.9 (d), 127.3 (d), 127.0 (s), 124.2
(d), 123.4 (d), 110.9 (d), 102.8 (d), 78.4 (d), 75.3 (d),
71.9 (d), 63.3 (t), 51.7 (t), 19.1 (q). HRMS-FAB m/z:
measured 426.1048 ([M + H]+, calcd. 426.1045 for
C19H24NO6S2). FTIR (cmꢀ1): 3340, 3221, 2924, 2853,
1740, 1492, 1367, 1247, 1073, 929, 788 cmꢀ1. UV
(CH3CN) kmax (log e): 220 (4.6), 274 (3.7).
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Support for the authorsꢀ work was obtained from the
Natural Sciences and Engineering Research Council of
Canada (Discovery Grant to M.S.C.P.) and the Univer-
sity of Saskatchewan (Harvey Graduate Scholarship to
M.H.). We thank M. Palcic, Department of Chemistry,
University of Alberta, Edmonton, AB, for useful advice
on separation of glucosyltransferases, C. Lefol, Agricul-
ture and Agri-Food Canada Research Station, Saska-
toon SK, for kindly providing clones of Sclerotinia
sclerotiorum, Y. Xu for synthesis of compounds 16
and 17, and K. Brown and K. Thorns, Department of
Chemistry, University of Saskatchewan, for technical
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