Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide

Article abstract of DOI:10.1016/j.bmc.2016.11.017

The biotransformations of the rutabaga phytoalexins rutalexin, brassicanate A, isalexin and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite produced by A. brassicicola. These fungal transformations are detoxification reactions likely carried out by different enzymes. The discovery of rapalexin A resistance to detoxification suggests that this phytoalexin in combination with additional phytoalexins could protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.

Full text of DOI:10.1016/j.bmc.2016.11.017

Accepted Manuscript
Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassici-
cola: Unveiling the first hybrid metabolite derived from a phytoalexin and a
fungal polyketide
M. Soledade C. Pedras, Abbas Abdoli
PII:
S0968-0896(16)31026-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.017
BMC 13384
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 October 2016
8 November 2016
11 November 2016
Please cite this article as: Pedras, M.S.C., Abdoli, A., Biotransformation of rutabaga phytoalexins by the fungus
Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.017
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Pedras and Abdoli
1
Biotransformation of rutabaga phytoalexins by the fungus
Alternaria brassicicola: unveiling the first hybrid metabolite
derived from a phytoalexin and a fungal polyketide
M. Soledade C. Pedras* and Abbas Abdoli
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon, SK, S7N 5C9,
Canada
Corresponding author:
M. Soledade C. Pedras
Email: s.pedras@usask.ca;
Telephone: 1-306- 966-4772;
Fax: 1-306-966-4730

Pedras and Abdoli
2
Abstract
The biotransformations of the rutabaga phytoalexins rutalexin, brassicanate A, isalexin
and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the
biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to
fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite
named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite
produced by A. brassicicola. These fungal transformations are detoxification reactions likely
carried out by different enzymes. The discovery of rapalexin A resistance to detoxification
suggests that this phytoalexin in combination with additional phytoalexins could protect crucifers
against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to
provide the producing plants with higher disease resistance levels.
Keywords: Alternaria brassicicola; antifungal; Brassicaceae; biotransformation;
brassicanate A; crucifer; detoxification; phytoalexin; rapalexin A; rutalexin; rutapyrone.

Pedras and Abdoli
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1
.
Introduction
In a broad sense biotransformation is a chemical modification of a compound carried out
by living cells or enzymes. Biotransformations of organic compounds using microbial cells such
1
,2,3
as bacteria or fungi have been often used to obtain either products of great value, or to get rid
4
of highly detrimental materials such as organic pollutants, pesticides and explosives. In an
ecological context, microbial biotransformations are often a survival strategy of the microbe to
cope with environmental stress. For example, to colonize plant tissues, fungal pathogens produce
5
,6
enzymes that can transform antimicrobial plant defenses, namely phytoalexins, to non-toxic
7
,8
products. Conceptually these fungal transformations, highly detrimental to plants, could be
prevented if inhibitors of the enzymes mediating these transformations were available. Such
9
8,10
inhibitors, known as paldoxins, could be applied to protect crops from fungal attack. To design
paldoxins, an understanding of the metabolic detoxification pathways of phytoalexins carried out
by fungal plant pathogens is crucial. Because the intermediates and products of these pathways
are in general unique to each fungal species, it is necessary to evaluate separately the interaction
of each phytoalexin with each phytopathogenic fungal species. Toward this end, we have been
investigating the detoxification of cruciferous phytoalexins by fungal pathogens of economically
important crops (crucifer crops, e.g. canola, Brassica napus, B. rapa, B. juncea) and chemical
8
,9,10,11
strategies to inhibit these processes.
The fungal plant pathogen Alternaria brassicicola (Schwein.) Wiltshire, together with A.
brassicae (Berk.) Sacc. causes black spot disease and substantial yield losses of various crucifer
1
2,13
crops. It has been established that A. brassicicola is able to transform and detoxify several
1
4
15
16
cruciferous phytoalexins including brassinin (1) cyclobrassinin (2), and camalexin (4), but its
resistance to the phytoalexins of rutabaga (Brassica napus L. ssp. rapifera) remains unknown.

Pedras and Abdoli
4
Brassinin (1) cyclobrassinin (2) and rutalexin (3) accumulate in leaves of B. juncea infected with
1
4
8
A. brassicicola, whereas camalexin (4) is produced in wild crucifer species. Rutalexin (3),
1
7
brassicanate A (5) and isalexin (6) are phytoalexins first isolated from rutabaga, together with
1
8
rapalexin A (7), brassinin (1), cyclobrassinin (2) and others. In continuation of that work, we
have analyzed the biotransformation of phytoalexins 3, and 5–7 in cultures of A. brassicicola and
report here results of these studies. This investigation demonstrates that A. brassicicola can
efficiently metabolize rutalexin (3), brassicanate A (5), and isalexin (6), but not rapalexin A (7).
A hybrid metabolite resulting from the interaction "rutalexin – A. brassicicola" was discovered
that could alleviate oxidative stress of fungal cells exposed to this phytoalexin. Importantly,
resistance of rapalexin A (7) to A. brassicicola suggests that this phytoalexin in combination with
additional defense metabolites like camalexin (4) could effectively protect crucifers against this
pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the
producing plants with higher disease resistance levels.
Figure 1. Phytoalexins produced by crucifers: brassinin (1), cyclobrassinin (2), rutalexin (3),
camalexin (4), brassicanate A (5), isalexin (6) and rapalexin A (7).

Pedras and Abdoli
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2
.
Results and discussion
2
.1 Chemical synthesis of phytoalexins
Rapalexin A (7) was synthesized and purified as previously reported, whereas the
1
9
syntheses of rutalexin (3), brassicanate A (5), and isalexin (6) were modified, as summarized
below.
Rutalexin (3) was previously prepared in modest yield (24% overall yield) by oxidation
of N-t-Boc-2-chloroindole-3-carboxaldehyde (8) to the corresponding acid, followed by
1
7
conversion into amide 9 upon treatment with thionyl chloride and methanamine. The resulting
amide 9 was allowed to react with NaHS to yield the key intermediate sulfanylamide 10.
However, during the current work it was discovered that oxidation of sulfanylamide 10 to
disulfide 13 occurred spontaneously during acidic work-up and chromatographic fractionation.
17
The dimerization of 10 to 13 could explain the lower yield of rutalexin (3) obtained previously.
20,21
Similar dimerization of thiols/thiones have been previously reported under various conditions.
The structure of sulfanylamide 10 was confirmed by methylation of the thiol group followed by
deprotection using TFA (20%) in DCM to afford 12 in 91% overall yield; the disulfide 13 was
deprotected under similar conditions to yield 14 in 90% yield (Scheme 1). In the current rutalexin
synthesis, to prevent formation of disulfide 13, methyl chloroformate was added to the crude
reaction mixture containing sulfanylamide 10, to yield compound 11 in almost quantitative yield
(
95%) after 30 min. Cyclization of compound 11 in the presence of Et N followed by
3
deprotection using TFA proceeded smoothly; after concentration of the crude reaction mixture to
dryness, the crude residue was rinsed with Et O to yield rutalexin (3) (93% yield, vi - vii; ca.
2
8
1% overall yield). Disulfide 14 was previously synthesized in three steps from 1-
phenylsulfonyl-1H-indol-3-carbonyl chloride to evaluate its ability to inhibit tyrosine kinase

Pedras and Abdoli
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2
0
activity.
Scheme 1. Synthesis of rutalexin (3). Reagents and conditions: i) NaClO KH PO4, 2-methyl-2-
2
,
2
butene, tert-butanol, H O, rt, 4 h; ii) SOCl THF/DMF, rt, 3 h; iii) CH NH THF, 0 °C, 1 h, 95%
2
2,
3
2,
(
°
i–iii); iv) NaHS, DMF/H O, 0 °C, 1 h; v) methyl chloroformate, 30 min, 95%; vi) Et N, THF, 50
2
3
C, 3 h; vii) 20% TFA-DCM, rt, 4 h, 93% (vi-vii); viii) CH I, THF, 30 min, rt, 97%; ix) 20%
3
TFA-DCM, rt, 4 h, 90%; x) 20% TFA-DCM, rt, 4 h, 90%.
Brassicanate A (5) was previously synthesized in 51% overall yield from oxidation of
1
7
aldehyde 8 to the corresponding acid, followed by methylation with diazomethane. In this work,
brassicanate A (5) was synthesized in quantitative yield from indole-2-thione (15) by methylation
with MeI followed by treatment with phosgene (COCl ) and MeOH (Scheme 2). Isalexin (6) was
2
previously prepared by directed O-lithiation of N-Boc-m-anisidine with n-BuLi in 71% overall
1
7
yield. To avoid n-BuLi, 4-methoxyindole (18) was oxidized with PCC and AlCl to yield
3
isalexin (6) in 53% yield (Scheme 3). Sriram et al. have reported the oxidation of indole (16, 100
2
2
mmol scale) to isatin (19) using PCC–SiO and AlCl in DCE in 88% yield; our attempts to
2
3
optimize the reaction conditions of a smaller scale reaction (1 mmol) yielded isatin (19) and
isalexin (6) in much lower yields (Scheme 3). Similar oxidation of 4-methoxyindole (18) using

Pedras and Abdoli
7
2
3
PCC, but replacing SiO with PANI yielded isalexin in 26% yield.
2
Scheme 2. Synthesis of brassicanate A (5). Reagents and conditions: i) CH I, Na CO , acetone, rt,
3
2
3
1
0 h; ii) COCl THF, rt, 2 h; iii) MeOH, rt, 30 min, quantitative.
2
,
Scheme 3. Synthesis of isalexin (6). Reagents and conditions: i) CH I, K CO , acetone, 35 °C, 18
3
2
3
h, 90%; ii) PCC, AlCl , DCE, 80 °C, 2 h, 53%; iii) PCC-PANI, DCE, 80 °C, 7 h, 26%; iv) PCC,
3
,
AlCl , DCE, 80 °C, 2 h, 42%; iii) PCC-PANI, DCE, 80 °C, 7 h, 25%.
3
,
2
.2 Biotransformations
To establish the biotransformation pathways of rutalexin (3), brassicanate A (5), isalexin
6) and rapalexin A (7) in cultures of A. brassicicola, the concentration was chosen based on the
solubility and inhibitory activity of compounds (0.10 mM). Mycelial cultures of A. brassicicola
48-h-old) were incubated with each phytoalexin and samples were withdrawn from cultures
(
(
immediately after addition of each compound (zero hours) and then up to 96 h. Culture samples
were either frozen or immediately subjected to neutral, acidic and basic extractions, and the
concentrated extracts were analyzed by HPLC-DAD-ESI-MS, as reported in the Experimental
Section 4.3.1. Samples of medium incubated with each phytoalexin separately (control solutions)

Pedras and Abdoli
8
were extracted and analyzed similarly to determine chemical stability and recovery. Similarly,
samples of cultures of A. brassicicola were extracted and analyzed to evaluate metabolite
production in the absence of phytoalexins (control cultures).
2
.2.1 Rutalexin (3)
The HPLC chromatograms of extracts of cultures of A. brassicicola incubated in medium
or water) containing rutalexin (3) showed a fast decrease in the area of the peak corresponding to
(t = 8.8 ± 0.2 min) and the appearance of new peaks at t = 9.6 ± 0.2 min and 10.2 ± 0.2 min
(
3
R
R
after 6 h (not detected in control cultures). The ESI-MS and the UV spectra of the peak at 9.6 min
was not available in our virtual libraries. After complete metabolism of 3, the area of the peak at
9
.6 min remained constant, whereas the area of peak at 10.2 min increased up to 12 h and
decreased there on (not detected after 48 h). No other significant peaks could be detected in the
-
chromatograms. The ESI-MS of the compound at 10.2 min displayed an ion at m/z 409 [M-1]
that suggested it to be disulfide 14, obtained during rutalexin synthesis (summarized in Scheme 1
and described in Section 4.2.5). The progress curves for transformation of rutalexin (3) by A.
brassicicola either in medium or in water indicated that it was metabolized quite fast (t = <6 h,
1
/2
Fig. 2); rutalexin (3) incubated in medium or in water was stable for the duration of the
experiments.

Pedras and Abdoli
9
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
12
24
36
48
Incubation time (h)
Figure 2. Progress curves of transformation of rutalexin (3) (ꢀ), formation of metabolites 21(ꢀ)
and 14 (ꢀ) in cultures of Alternaria brassicicola, and recovery of rutalexin (3) from medium (ꢁ)
(
HPLC, method A).
Furthermore, a peak at t = 6.7 min was observed in the chromatograms of both control
R
cultures and cultures incubated with rutalexin (3). This peak was detected after 6 h in cultures
incubated with rutalexin (3), whereas in control cultures (no rutalexin (3)) it was detected only
after 24 h of incubation; production in both cultures was comparable after 48 h. Analysis of the
peak areas corresponding to this metabolite (established to be phomapyrone G (20), ESI-MS and
1
4,24
UV spectra of an authentic sample) suggested that it was produced in larger amounts in
rutalexin (3) amended cultures than in control cultures (Fig. 3).

Pedras and Abdoli
10
1800
1500
1200
900
600
300
0
0
12
24
36
48
60
72
84
Incubation time (h)
Figure 3. Progress curves of formation of metabolite at t = 6.7 min, phomapyrone G (20), in
R
cultures of Alternaria brassicicola incubated with rutalexin (3) (ꢀ) and in control cultures (no
rutalexin) (ꢁ) (HPLC, method A).
Larger scale cultures of A. brassicicola incubated with rutalexin (3) up to 72 h allowed
the isolation of the metabolite with t = 9.6 min and its spectroscopic characterization, as
R
described in Section 4.3.2. The HREI-MS spectrum (456.1719 Da, C H N O S) suggested that
2
4
28
2
5
this compound had higher MW than rutalexin (3) (232.0306 Da, C H N O S), i.e. an additional
1
1
8
2
2
1
moiety with the molecular formula C H O . The H NMR spectrum obtained in CD CN
1
3
20
3
3
displayed five indolyl protons (δ: 9.90, br s, 1H; 7.95, d, 1H; 7.30 d, 1H; 7.14 dd, 1H; 7.07 dd,
H
1
3
H), three protons at δ 7.33 (br, 1H), 6.37 (s, 1H), 6.10 (s, 1H), six methyl groups at δ 3.84 (s,
H
H
H), 2.90 (d, 3H), 2.10 (s, 3H), 1.80 (s, 3H), 1.51 (s, 3H), 1.39 (d, 3H) and one methine proton at
13
δ 3.56 (q, 1H). The C NMR spectrum displayed signals for 24 carbons, namely eight indolyl
H
carbons with chemical shifts similar to those of rutalexin (3), six methyl carbons, four carbons
above 160 ppm and six other carbons. The NMR spectroscopic data indicated the presence of an
indolyl moiety containing an N-methyl amide (δ 2.90, d, J = 5 Hz, 3H), corresponding to the
H
moiety derived from rutalexin (3). Detailed analysis of HSQC and HMBC spectroscopic data
showed correlations corresponding to the additional C H O moiety. For example, in the HMBC
1
3
20
3

Pedras and Abdoli
11
spectrum, the singlet proton at δ 6.37 correlated to carbons at δ 160.5, 76.8 and 26.8 (C6', C9' and
H
C
C12', respectively), the singlet proton at δ 6.10 correlated to carbons at δ 102.5, 160.5 and 129.7
H
C
(
C3', C6' and C7', respectively) and the methyl protons at δ 1.39 (d, J = 7.2 Hz) correlated with
H
carbon at δ 76.8 (C9) (Table 1). These signals suggested a structure resulting from condensation
C
1
4,24
of a rutalexin-derived moiety with one of the pyrone metabolites produced by A. brassicicola.
Based on this consideration and all spectroscopic data, the hybrid structure 21 derived from
rutalexin-phomapyrone G was deduced (Fig. 4). This structure is consistent with NMR and UV
spectroscopic data and the predicted chemical reactivity of phomapyrone G (20) (C H O ), a 2-
1
4
18
4
2
5
pyrone polyketide first isolated from Leptosphaeria biglobosa. In addition, NOE data showed
correlations between H5' and H13', and H13' and H8', which indicated the configuration of the
double bond as cis, consistent with the structure of phomapyrone G (20). The chemical structure
of the hybrid metabolite suggests that it results from addition of sulfanylamide 23 to the least
sterically hindered carbon of the epoxide ring of phomapyrone G (20). To the best of our
knowledge, compound 21 is the first hybrid natural product derived from a phytoalexin and a
fungal metabolite, for this reason it is named rutapyrone (21).
Figure 4. Chemical structure of rutapyrone (21) and selected HMBC and NOE correlations.

Pedras and Abdoli
12
Table 1
1
13
H (600 MHz) and C NMR (150 MHz) spectroscopic data of rutapyrone (21) in CD CN
3
δH, multiplicity
J, Hz)
δH, multiplicity
(J, Hz)
Atom #
δ
Atom #
δ
C
C
(
1
2
3
-
9.90, br s
1′
2′
3′
4′
5′
6′
-
-
130.9
115.6
127.8
111.9
124.1
-
165.2
102.5
167.1
94.0
-
-
-
3
4
5
a
-
-
7.30, d (8.4)
7.07, dd (7.8,
6.10, s
-
160.5
7
.2)
7.14, dd (7.2,
.4)
6
121.7
7′
129.7
-
8
7
7
8
9
1
121.8
137.3
166.4
-
7.95, d (7.8)
8′
9′
10′
11′
12′
136.8
76.8
57.6
17.6
26.8
13.5
9.0
6.37, s
a
-
-
-
3.56, q (7.2)
1.39, d (7.2)
1.51, s
7.33, br s
2.90, d (4.8)
0
26.4
1
3′
2.10, s
1
4′
1.80, s
OH
-
4.20, br s
3.84, s
(
O)CH3 57.3
In addition, isolation of a very small amount of the metabolite with t = 6.7 min from the
R
larger scale cultures confirmed it to be phomapyrone G (20). As expected from analysis of the
time-course experiments, disulfide 14 was not isolated from these large scale cultures because it
was not present in cultures after 48 h of incubation. It is likely that disulfide 14 results from
spontaneous oxidation of sulfanylamide 23, similar to results obtained during the chemical
synthesis of rutalexin (3). Furthermore, disulfide 14 was not stable either in culture medium or
water, oxidizing spontaneously to various undetermined products, including sulfonic acid 22.
Sulfonic acid 22 was obtained from disulfide 14 upon oxidation with m-CPBA (Scheme 4). Due

Pedras and Abdoli
13
to its low chemical stability the fate of disulfide 14 in cultures could not be determined.
Scheme 4. Synthesis of sulfonic acid 22. Reagents and conditions: i) m-CPBA, MeOH, 2.5 h, rt,
59%.
The pathway for transformation of rutalexin (3) by A. brassicicola is proposed in Scheme
. That is, in fungal cultures rutalexin (3) is enzymatically hydrolyzed and decarboxylated to
5
sulfanylamide 23, which in turn adds to the least hindered epoxide carbon C10' of phomapyrone
G (20) to produce rutapyrone (21). This addition is most likely an enzyme mediated
transformation. As well, sulfanylamide 23 can oxidize spontaneously to disulfide 14 (the major
product), which is similarly unstable in aqueous or organic solutions and oxidizes to several other
products including sulfonic acid 22.
Scheme 5. Proposed pathway of biotransformation of rutalexin (3) in cultures of Alternaria
brassicicola with formation of hybrid metabolite rutapyrone (21).

Pedras and Abdoli
14
Albeit produced in small amount, formation of a hybrid metabolite like rutapyrone (21)
derived from plant (rutalexin (3)) and fungus (polyketide phomapyrone G (20)) metabolism is an
intriguing process posing a few questions. Specifically, A. brassicicola produces several 2-
pyrones such as 20, but until now no ecological functions, including phytotoxicity, have been
2
4,25
established. Based on this work we suggest that in fungal cultures rutalexin (3) induces the
biosynthesis of phomapyrone G (20) by oxidation of phomapyrone A (24), to reduce the
oxidative stress caused by the presence of rutalexin (3) (Scheme 6). Formation of phomapyrone
G (20) can quench and hence decrease the levels of reactive oxygen species (ROS), including
hydrogen peroxide, generated in mycelial cells incubated with rutalexin (3). The quick depletion
of hydrogen peroxide and/or other ROS can mitigate their deleterious effects on the pathogen. In
2
6
fact some phytoalexins are known to cause oxidative stress on fungal plant pathogens. For
example, we have shown that camalexin (4) induced production of superoxide dismutase (SOD)
1
0
in A. brassicicola. SOD is an enzyme produced by various organisms, including fungi, to
protect cells from ROS produced in cellular responses to oxidative stress. In short, oxidation of
phomapyrone A (24) (and stereo isomers) to phomapyrone G (20) can contribute to decrease
oxidative stress levels in A. brassicicola by lowering the concentration of ROS species. These
considerations suggest that enzymatic coupling of sulfanylamide 23 and phomapyrone G (20) is
likely to occur in the cell site where 20 is synthesized and rutalexin (3) is hydrolyzed, i.e. the
coupling enzyme is located in the same subcellular compartment where rutalexin is metabolized.

Pedras and Abdoli
15
Scheme 6. Proposed oxidation of phomapyrone A and potential isomers (24) to phomapyrone G
20) in cultures of Alternaria brassicicola.
(
2
.2.2 Brassicanate A (5)
The metabolism of brassicanate A (5) was investigated as reported above for rutalexin (3).
The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated with
brassicanate A (5) showed a fast decrease of the area of the peak corresponding to 5 (t = 15.8 ±
R
0
.2 min) and the appearance of a new peak at t =12.0 ± 0.2 min (Fig. 5). Brassicanate A (5) was
R
not detected in cultures after 4 h of incubation; the new peak at t = 12.0 min was not detected in
R
control cultures or medium incubated with brassicanate A (5). The UV spectrum of this
-
component was similar to that of 5, whereas the ESI-MS at m/z 205.8 [M-H] suggested the
presence of a carboxylic acid. The structure of this metabolite was confirmed as 2-
methylsulfanediylindole-3-carboxylic acid (26), after isolation from larger scale cultures of A.
brassicicola incubated with brassicanate A (5) for 4 h. The structure was further confirmed by
direct comparison with an authentic synthetic sample, prepared as summarized below.
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
1
2
3
4
5
6
Incubation time (h)

Pedras and Abdoli
16
Figure 5. Progress curves of transformation of brassicanate A (5) (ꢀ) and formation of
metabolite 26 (ꢀ) in cultures of Alternaria brassicicola, and recovery of brassicanate A from
medium (ꢁ) (HPLC method B).
2
-Methylsulfanediylindole-3-carboxylic acid (26) was prepared by treatment of 25 with
phosgene followed by aqueous work up (Scheme 7). Next, to establish the metabolic fate of 2-
methylsulfanediylindole-3-carboxylic acid (26), it was incubated in medium with A. brassicicola
and in medium only. Unexpectedly, HPLC analysis of extracts showed a slower decrease in the
concentration of 2-methylsulfanediylindole-3-carboxylic acid (26) (t = 12.0 ± 0.2 min) in fungal
R
cultures than in medium. These results suggested that the faster decrease of the concentration of
carboxylic acid 26 in medium was likely due to spontaneous transformation. It was hypothesized
that the faster transformation of acid 26 in medium was favored by the more acidic pH (ca. 6.5)
of medium than the pH of cultures of A. brassicicola (increases from 6.5–8.5 in 48 h). To verify
this hypothesis, solutions of medium only or water only at different pHs were incubated with acid
2
6 and each solution analyzed by HPLC every 2 h. Progress curves of the recovery of acid 26
over a 12 h period (Fig. 6) confirmed a much faster decrease of the concentration of acid 26 in
solutions at pH 6.0 than at pH 8.5. In addition, the concomitant appearance of a new peak at t =
R
23.2 ± 0.2 min and determination of its structure demonstrated that carboxylic acid 26 was
quickly transformed to 2-methylsulfanediylindole (25) (Scheme 7).

Pedras and Abdoli
17
Scheme 7. Synthesis and spontaneous decomposition of 2-methylsulfanediylindole-3-carboxylic
acid (26). Reagents and conditions: (i) COCl , THF, 2 h, rt; (ii) H O, 30 min, rt, 60% (i–ii); (iii)
2
2
spontaneous decarboxylation in H O or medium.
2
A
7000
6000
5000
4000
3000
2000
1000
0
0
2
4
6
8
10
12
14
Incubation time (h)
B
0000
1
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
0
2
4
6
8
10
12
14
Incubation time (h)
Figure 6. Progress curves of decarboxylation of 2-methylsulfanediylindole-3-carboxylic acid
26) (A) and formation of 2-methylsulfanediylindole (25) (B) in medium (blue) and H O (black-
(
2
dashed) at different pHs: 8.5 (ꢀ), 7.1 (ꢁ) and 6 (ꢀ) (HPLC method C).
Although not detected in cultures of A. brassicicola incubated with brassicanate A (5),
brassicanate A sulfoxide (27) was thought to be a likely metabolite caused by ROS. To prove this

Pedras and Abdoli
18
hypothesis, brassicanate A sulfoxide (27) was prepared by oxidation of brassicanate A (5)
Scheme 8) and its biotransformation in culture and in medium was investigated. The HPLC-
(
DAD-ESI-MS of neutral extracts revealed that brassicanate A sulfoxide (27) remained in cultures
for the duration of the incubation (48 h), that is, A. brassicicola did not transform brassicanate A
sulfoxide (27). In addition, brassicanate A sulfoxide (27) was stable in medium for 48 h (Fig. 7).
Scheme 8. Synthesis of brassicanate A sulfoxide (27). Reagents and conditions: i) m-CPBA,
MeOH, 0 °C, 30 min, 79%.
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
6
12
18
24
30
36
42
48
54
Incubation time (h)
Figure 7. Progress curves of recovery of brassicanate A sulfoxide (27) from cultures of
Alternaria brassicicola (ꢀ) and medium (ꢀ) (HPLC method B).
Based on the results described above, the transformation of brassicanate A (5) by A.
brassicicola is proposed as shown in Scheme 9. In fungal cultures brassicanate A (5) is
enzymatically hydrolyzed to 2-methylsulfanediylindole-3-carboxylic acid (26), which
spontaneously decarboxylates in aqueous solutions of pH<7.

Pedras and Abdoli
19
Scheme 9. Proposed pathway of biotransformation of brassicanate A (5) in cultures of Alternaria
brassicicola with spontaneous decarboxylation (I, proposed intermediate).
2.2.3 Isalexin (6)
The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated
with isalexin (6) showed a decrease in the area of the peak corresponding to 6 (t = 9.3 ± 0.2 min)
R
and the appearance of new peak at t = 8.6 ± 0.2 min. Isalexin concentration decreased rapidly
R
(
0.12 to ca. 0.03 mM) in 6 h, but remained detectable up to 72 h of incubation, whereas in control
medium its concentration remained constant (Fig. 8). The recovery of isalexin (6) from neutral
extracts was ca. 75%, small amounts remained in both acidic and basic extracts; hence data
points of the progress curve in Fig. 8 represent the amount of 6 detected in the neutral, acidic and
basic extracts (obtained by combining the extracts). The peak at 8.6 min was not detected in
control cultures or medium incubated with isalexin (6). The ESI-MS (positive mode) spectrum of
+
the peak at 8.6 min displayed an ion at m/z 202 [M+Na] , which suggested this metabolite to be
1,3-dihydro-3-hydroxy-4-methoxyindol-2-one (28). This structure was confirmed by direct
comparison with an authentic synthetic sample prepared by NaBH reduction of isalexin (6).
4
Further incubation of cultures with 1,3-dihydro-3-hydroxy-4-methoxyindol-2-one (28) and HPLC
analysis indicated that this compound spontaneously oxidized to isalexin (6) and other
undetermined polar compounds.

Pedras and Abdoli
20
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
12
24
36
48
60
72
Incubation time (h)
Figure 8. Progress curves of transformation of isalexin (6) (ꢀ) and formation of metabolite 28
(
(
ꢀ) in cultures of Alternaria brassicicola, and recovery of isalexin from control medium (ꢁ)
HPLC method B).
Scheme 10. Proposed biotransformation of isalexin (6) in cultures of Alternaria brassicicola.
2
.2.4 Rapalexin A (7)
The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated
with rapalexin A (7) showed a consistent area for the peak corresponding to 7 (t = 12.1 ± 0.2
R
min) for the duration of the experiments (96 h). That is, under standard experimental conditions,
rapalexin A (7) was not metabolized by A. brassicicola.
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
12
24
36
48
60
72
84
96
Incubation time (h)

Pedras and Abdoli
21
Figure 9. Progress curves of recovery of rapalexin A (7) (ꢀ) in cultures of Alternaria
brassicicola and recovery from medium (ꢁ) (HPLC method A).
2
.3 Antifungal activity of phytoalexins and biotransformation products against Alternaria
brassicicola
The antifungal activities of synthetic rutalexin (3), brassicanate A (5), isalexin (6),
rapalexin A (7) and compounds 12, 14, 21, 22, 25, 26 and 28 against A. brassicicola were
evaluated as described in Section 4.4. Due to the low solubility of rutalexin (3) and some
metabolites, all phytoalexins and metabolites were tested in liquid medium.
Results of antifungal bioassays against A. brassicicola are summarized in Table 2. Among
the four phytoalexins, rapalexin A (7) displayed the highest antifungal activity, causing complete
growth inhibition of mycelia at 0.50 mM and 89% at 0.20 mM, followed by brassicanate A (5)
displaying 100% inhibition at 0.50 mM and 47% at 0.20 mM (Table 2). Due to its low solubility,
rutalexin (3) could only be tested at concentrations • 0.10 mM, whereas its metabolites were
tested at higher concentration. Metabolite 14 at 0.10 mM caused slightly higher growth inhibition
(
25% vs. 20%) than rutalexin (3), whereas 21 caused substantially lower growth inhibition (6%
vs. 20%, at 0.10 mM). The latter result suggests that transformation of rutalexin (3) to rutapyrone
21) is a detoxification reaction. Compound 12 was tested to compare its inhibitory activity with
(
that of metabolite 21, which was shown to be significantly less inhibitory than 12 at 0.10 mM. In
addition, sulfonic acid 22 was virtually devoid of antifungal activity.
Interestingly, the antifungal activity of metabolite 26 obtained from biotransformation of
brassicanate A (5) by A. brassicicola was similar to that of the parent compound (5). Because
compound 25 resulted from spontaneous decarboxylation of metabolite 26, the growth inhibitory
activity of 25 was evaluated to compare with that of 5 and 26. Compound 25 is significantly less
inhibitory than 5 or 26 at 0.50 mM, thus the apparent biotransformation of 26 to 25 is a

Pedras and Abdoli
22
detoxification reaction. Hence the overall transformation of brassicanate A (5) by A. brassicicola
is a detoxification. The biotransformation of isalexin (6) to 28 yielded a metabolite displaying
significantly lower inhibitory activity, hence this biotransformation is a detoxification reaction as
well.
Table 2
a
Antifungal activity of rutalexin (3), brassicanate A (5), isalexin (6), rapalexin A (7) and
compounds 12, 14, 21, 22, 25, 26 and 28 against Alternaria brassicicola (in liquid medium).
Inhibition ± SD (%)
Compound (#)
0
.50 mM
0.20 mM
n.d.
0.10 mM
d,e,f
Rutalexin (3)
n.d.
20 ± 4
b
c
c,d
Brassicanate A (5)
Isalexin (6)
100 ± 0
47 ± 8
26 ± 4
d
d,e
e,f
40 ± 4
25 ± 3
18 ± 2
b
b
b
Rapalexin A (7)
100 ± 0
89 ± 2
66 ± 3
N-Methyl 2-methylsulfanediyl-1H-indole-3-
carboxamide (12)
e
e
f
2
9 ± 3
20 ± 3
15 ± 4
2
,2'-Disulfanediylbis(N
b
-methyl-1H-indole-3-
d
c
n. d.
32 ± 0
25 ± 3
carboxamide) (14)
e
c
e
g
Rutapyrone (21)
34 ± 3
21 ± 3
6 ± 3
3-(N-Methylcarbamoyl)-1H-indole-2-sulfonic acid
f
g
h
5
± 2
0
0
(22)
c
c,d
2
-Methylsulfanediyl-1H-indole (25)
75 ± 3
47 ± 3
23 ± 3
b
d
c,d,e
2
1
-Methylsulfanediylindole-3-carboxylic acid (26)
100 ± 0
32 ± 4
22 ± 3
,3-Dihydro-3-hydroxy-4-methoxy-indole-2-one
e
f
h
3
1 ± 2
11 ± 4
0
(28)
a
The percentage of inhibition was calculated using the formula: % inhibition = 100 – [(growth on amended/growth
in control) × 100]; values are averages of six independent experiments conducted in triplicate ± standard deviations;
n.d. =not determined due to low solubility.
For statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted αset at 0.05;
b–h
n = 6; different letters in the same column ( ) indicate significant differences (P < 0.05).
3
.
Conclusion
In conclusion, here we have developed a more efficient and simple method for the
preparation of the cruciferous phytoalexin rutalexin (3) in 81% yield starting from a simple

Pedras and Abdoli
23
1
7, 27
starting material (8), shorter than any of the currently published methods.
Similarly,
brassicanate A (5) was synthesized quantitatively from indoline-2-thione (15), a substantial
1
7
improvement over a previous synthesis (51%).
This work is the first report of the formation of a hybrid metabolite between a phytoalexin
detoxification metabolite (23) and a fungal metabolite (20), i.e. rutapyrone (21). Nonetheless,
further work that would include the isolation of the enzyme catalyzing the coupling of
hypothetical metabolite 23 and phomapyrone G (20) needs to be carried out to understand the
significance and implications of this discovery.
Previous results of the transformation of the phytoalexins brassinin (1), cyclobrassinin
1
4
(
2), and camalexin (4) indicated that A. brassicicola could detoxify 1 (ca. 12 h at 0.10 mM) and
2
8
2
(ca. 8 h at 0.10 mM) quickly, whereas 4 was detoxified relatively slower (>120 h at 0.05
1
6
mM). In this work, the biotransformation of brassicanate A (5) was the fastest in ca. 4 h (at 0.10
mM), followed by rutalexin (3) (ca. 24 h). By contrast, the transformation of isalexin (6) was
much slower (ca. 72 h), yielding several products, including product 28 that re-oxidized
spontaneously to the parent compound. These transformations are detoxification reactions likely
carried out by different enzymes. Importantly, rapalexin A (7) was not metabolized by A.
brassicicola, suggesting that higher concentrations of rapalexin A could protect the producing
plant species from infection by this pathogen. In conclusion, it is suggested that since rapalexin A
(7) is resistant to A. brassicicola and camalexin (4) is transformed very slowly, engineering the
biosynthetic pathway of 4 and increasing production of 7 in cultivated Brassica species could
have a great positive impact in obtaining resistance to A. brassicicola.

Pedras and Abdoli
24
4
.
Experimental
4
.1 General
Chemicals were purchased from Alpha Aesar, Ward Hill, MA or Sigma-Aldrich Canada
Ltd., Oakville, ON; solvents were HPLC grade and used as such. Flash column chromatography
FCC) was carried out using silica gel grade 60, mesh size 230 – 400 Å or WP C prepscale bulk
(
1
8
packing 275 Å (J.T. Baker, NJ, USA). Organic extracts were dried over Na SO and concentrated
2
4
using a rotary evaporator.
NMR spectra were recorded on Bruker 500 or 600 MHz Avance spectrometers, for H,
1
1
3
5
00.3 MHz or 600.2 and for C, 125.8 or 150.9 MHz; chemical shifts (δ) 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. FTIR data were recorded on a Bio-Rad FTS-40
spectrometer and spectra were measured by the diffuse reflectance method on samples dispersed
in KBr. HREI-MS were obtained on a VG 70 SE mass spectrometer employing a solids probe or
on a Jeol AccuToF GCv 4G mass spectrometer [field desorption (FD)] by direct insertion.
HPLC analysis was carried out with Agilent high performance liquid chromatographs
equipped with quaternary pump, automatic injector, and photodiode array detector (DAD,
wavelength range 190 - 600 nm), degasser, and a column Eclipse XDB-C18 (5 μm particle size
silica, 4.6 i.d. ×150 mm), having an in-line filter, using methods: A, mobile phase 50% H O -
2
5
0% CH OH to 100% CH OH for 25.0 min, linear gradient, at a flow rate of 0.75 mL/min; B,
3
3
mobile phase 90% H
2
O – 10% MeOH to 100% MeOH for 45.0 min, linear gradient, at a flow
rate 0.75 mL/ min; C, mobile phase 100% H O – TFA (0.10%) to 100% MeOH – TFA (0.10%)
2
for 40.0 min, linear gradient, at a flow rate 1.0 mL/min.

Pedras and Abdoli
25
4
.2 Synthesis
4
.2.1 Rutalexin (4)
A solution of NaClO (1 g, 6.80 mmol) and KH PO (1.2 g, 8.8 mmol) in water (6 mL)
2
2
4
was added to a mixture of 1-t-Boc-2-chloroindole-3-carboxaldehyde (8, 255 mg, 0.900 mmol),
tert-butanol (6.4 mL) and 2-methylbut-2-ene (6.4 mL). The reaction mixture was stirred for 4 h at
rt. The organic phase was separated and the aqueous layer was acidified pH 3 (HCl, 0.50 M) and
extracted with EtOAc. The organic layer and extracts were combined, dried and concentrated to
dryness to afford 1-t-Boc-2-chloroindole-3-carboxylic acid (270 mg, 0.900 mmol, 100%).
Thionyl chloride (250 μL , 3.75 mmol) was added to a solution of 1-t-Boc-2-chloroindole-3-
carboxylic acid (90 mg, 0.30 mmol) in dry THF (3 mL) at 0 °C followed by catalytic amount of
DMF (5 μL ). The reaction mixture was stirred for 2 h at rt. The excess of thionyl chloride was
evaporated, the reaction mixture was cooled to 0 °C and a solution of CH NH in THF (2 M, 4
3
2
mL, 8 mmol) was added slowly (10 min). The reaction mixture was stirred for an additional 1 h
at 0 °C, was diluted with water and extracted with EtOAc. The combined extracts were dried and
concentrated to dryness and the residue was subjected to FCC (silica gel, EtOAc-hexane, 1:1) to
afford amide 9 (88 mg, 0.29 mmol, 95%) as colorless oil. A solution of sodium hydrogen sulfide
(
360 mg, 3.20 mmol) in water (100 μL ) was added to a mixture of amide 9 (50 mg, 0.25 mmol) in
DMF (1.5 mL) at 0 °C and the reaction mixture was stirred at 0 °C for 1 h. Methyl chloroformate
1.2 mL, 14 mmol) was added dropwise to the reaction mixture, the ice bath was removed and the
(
reaction mixture was stirred for an additional 30 min at rt. The reaction mixture was diluted with
water, extracted with EtOAc, and the combined extracts were dried and concentrated to dryness
to afford 11 (56 mg, 0.15 mmol, 95%) as colorless oil (spectra in Supplementary Data).
Triethylamine (50 μL ) was added to a solution of amide 11 (30 mg, 0.08 mmol, 1 eq) in THF (1

Pedras and Abdoli
26
mL) and the reaction mixture was stirred at 50 °C for 3 h. After amide 11 was consumed, the
reaction mixture was cooled to rt and a mixture of 20% TFA in DCM (2 mL) was added. The
reaction mixture was kept stirring for an additional 4 h at rt, was concentrated to dryness and the
residue was rinsed with diethyl ether (2 mL × 2) to afford rutalexin (3, 17 mg, 0.07 mmol, 90%)
1
7
as white powder. The spectroscopic data were identical to those previously reported.
4
.2.2 Brassicanate A (5)
Methyl iodide (280 mg, 123 μL , 1.97 mmol, 2.7 eq) was added to a solution of indole-2-
thione (15, 100 mg, 0.74 mmol, 1 eq) and sodium carbonate (120 mg, 1.13 mmol, 1.5 eq) in
2
9
acetone (2 mL) and the mixture was stirred for 10 h at rt. The reaction mixture was diluted with
brine (10 mL), extracted with EtOAc and the combined extracts were dried and concentrated to
dryness to afford 2-methylsulfanediylindole (25, 109 mg, 0.67 mmol) as yellow oil in
quantitative yield. Phosgene (130 μL , 0.6 mmol, 4 eq) was added to a solution of 2-
methylsulfanediylindole (25, 25 mg, 0.06 mmol, 1 eq) in THF (0.5 mL), the reaction mixture was
stirred for 2 h at rt, MeOH (1 mL) was added and the reaction mixture was stirred for an
additional 30 min at rt. The reaction mixture was diluted with water, extracted with EtOAc, the
combined extracts were dried and concentrated to afford brassicanate A (5) as brownish solid in
quantitative yield (13.5 mg, 0.06 mmol). The spectroscopic data were identical to those
1
7
previously reported.
4
.2.3 Isalexin (6)
A solution of 4-methoxyindole (18, 25 mg, 0.17 mmol, 1 eq) in 1,2-dichloroethane (DCE)
2 mL) was added to a solution of pyridinium chlorochromate (PCC) (273 mg, 1.36 mmol, 8 eq)
in DCE (2 mL) followed by aluminum chloride (7 mg, 0.05 mmol, 0.3 eq). The reaction mixture
(

Pedras and Abdoli
27
was heated at 80 °C for 2 h and was concentrated to dryness. The residue was subjected to FCC
(
EtOAc-hexane, 1:1) to afford isalexin (6, 16 mg, 0.09 mmol, 53%) as a yellow powder. The
1
7
spectroscopic data were identical to those previously reported.
4
.2.4 N-Methyl-2-(methylsulfanediyl)-1H-indole-3-carboxamide (12)
A solution of NaHS (108 mg, 1.19 mmol) in water (100 μL ) was added to a mixture of
amide 9 (30 mg, 0.15 mmol) in DMF (1 mL) at 0 °C and the reaction mixture was stirred at 0 °C
for 1 h. Methyl iodide (187 μL , 3.00 mmol) was added dropwise to the reaction mixture, the ice
bath was removed and the reaction mixture was stirred for an additional 30 min at rt. The reaction
mixture was diluted with water, extracted with EtOAc, and the combined extracts were dried and
concentrated to dryness to afford t-Boc protected 12 as a light yellow solid, which was treated
with a solution of 20% TFA in DCM (2 mL). The reaction mixture was stirred for 4 h at rt,
concentrated to dryness and the residue was subjected to FCC (silica gel, EtOAc-hexane, 1:1) to
afford carboxamide 12 (15 mg, 0.070 mmol, 94%) as yellow oil.
1
H NMR (500 MHz, CDCl ): δ8.80 (br s, 1H), 8.30 (d, J= 7.0 Hz, 1H), 7.33 (d, J= 7.0 Hz,
3
1
3
1
H), 7.25-7.21 (m, 2H), 3.08 (d, J= 3.5 Hz, 3H), 2.55 (s, 3H). C NMR (500 MHz, CDCl ): δ
3
1
66.3, 136.3, 132.2, 127.6, 123.5, 121.8, 121.6, 112.3, 111.0, 26.5, 19.3. HPLC t = 2.8 ± 0.2
R
-
1
min. UV (HPLC) λ (nm): 220, 300. FTIR (KBr) ν cm : 2926, 1725, 1618, 1543, 1413, 1313,
max
max
+
1
148, 750. HRMS-EI m/z: measured 220.0668 ([M] , calcd. 220.0670 for C H N OS) (100%).
1
1
12
2
4
.2.5 2,2'-Disulfanediylbis(N-methyl-1H-indole-3-carboxamide) (14)
A solution of NaHS (108 mg, 1.92 mmol) in water (100 μL ) was added to a solution of
amide 9 (30 mg, 0.15 mmol) in DMF (1 mL) at 0 °C. The reaction mixture was stirred at 0 °C for
h, diluted with brine (10 mL) acidified (pH 3, HCl, 0.50 M), extracted with EtOAc, and the
1

Pedras and Abdoli
28
combined extracts were dried and concentrated to dryness. The residue was dissolved in 20%
TFA in DCM (1.50 mL), was stirred for 4 h at rt and was concentrated to dryness. The final
residue was rinsed with chloroform to yield 14 in quantitative yield (20 mg, 0.02 mmol) as an
off-white powder, mp 232 – 234 °C.
-
1
HPLC t = 10.2 ± 0.2 min. UV (HPLC) λ (nm): 220, 310. FTIR (KBr) ν cm : 1614,
R
max
max
+
1
535, 1438, 1409, 1209, 1026, 742. HRMS-FD m/z: measured 410.0885 ([M] , calcd. 410.0871
1
13
for C H N O S ) (100%). The H and C NMR spectroscopic data were identical to those
2
0
18
4
2
2
2
0
previously reported.
4
.2.6 3-(N-Methylcarbamoyl)-1H-indole-2-sulfonic acid (22)
A solution of m-CPBA (90 mg, 0.50 mmol, 16.60 eq) in DCM (1 mL) was added to a
solution of disulfide 14 (14 mg, 0.03 mmol, 1 eq) in MeOH (0.50 mL). The reaction mixture was
stirred for 2 h at rt followed by addition of dimethyl sulfide (120 μL , 0.12 mmol). The reaction
mixture was stirred for an additional 30 min at rt and concentrated to dryness. The residue was
fractionated using a reverse-phase column (MeOH-H O) to afford sulfonic acid 22 (10 mg, 0.04
2
mmol, 59%) as white powder, mp > 300 °C.
1
H NMR (500 MHz, DMSO-d ): δ11.56 (br s, 1H), 9.07 (d, J = 3.5 Hz, 1H), 8.22 (d, J =
6
8
1
1
.0 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 7.0, 7.5 Hz, 1H), 7.05 (dd, J = 7.5, 7.5 Hz,
1
3
H), 3.16 (s, 1H), 2.78 (d, J = 4 Hz, 3H). C NMR (125.8 MHz, DMSO-d ): δ 164.6, 141.8,
6
32.9, 127.7, 122.4, 122.4, 120.5, 112.1, 105.8, 25.7. HPLC t = 1.0 ± 0.2 min (HPLC method
R
-1
C). UV (HPLC) λ (nm): 220, 280. FTIR (KBr) ν cm : 1600, 1463, 1368, 1118, 1072, 802.
max
•••
+
HRMS-FD m/z: measured 254.0351 ([M] , calcd. 254.0361 for C H N O S) (100%).
1
0
10
2
4

Pedras and Abdoli
29
4
.2.7 2-Methylsulfanediylindole-3-carboxylic acid (26)
Phosgene (195 μL , 2.7 mmol, 9 eq) was added to a solution of 2-methylsulfanediylindole
25, 50 mg, 0.3 mmol, 1 eq) in THF (2 mL) at 0 °C. The ice bath was removed and the reaction
(
mixture was stirred for 2 h at rt. Water (5 mL) was added to the reaction mixture and the mixture
was stirred for an additional 30 min at rt. The reaction mixture was diluted with water, extracted
with EtOAc and the combined extracts concentrated to dryness. The residue was subjected to
FCC on silica gel (DCM-MeOH, 98:2) to afford acid 26 (33 mg, 0.16 mmol, 52 %) as white
solid, mp 167 - 170 °C.
1
H NMR (500 MHz, CD CN): δ9.85 (br s, 1H), 7.92-7.90 (m, 1H), 7.45-7.43 (m, 1H),
3
1
3
7
.19-7.14 (m, 2H), 2.60 (s, 3H). C NMR (500 MHz, CD CN): δ167.0, 146.4, 137.6, 128.6,
3
1
22.8, 122.7, 120.9, 111.7, 103.7, 14.7. HPLC t = 12 ± 0.2 min (HPLC method B). UV (HPLC)
R
-
1
λ (nm): 240, 270, 300. FTIR (KBr) ν cm : 1639, 1498, 1485, 1448, 1301, 1201, 1060, 748.
max
max
-
HRMS-ESI m/z: measured 206.0276 ([M-H] , calcd. 206.0281 for C H N O S ).
1
0
8
1
2
1
4
.2.8 Brassicanate A sulfoxide (27)
A solution of m-CPBA (20 mg, 0.1 mmol, 1.2 eq) in DCM (0.50 mL) was added to a
solution of brassicanate A (5, 20 mg, 0.09 mmol, 1 eq) in MeOH (1 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min, dimethyl sulfide (6.8 mg, 0.80 mL, 0.11 mmol, 1.2 eq)
was added and the mixture was stirred for an additional 30 min at rt. The reaction mixture was
concentrated to dryness and the residue was subjected to FCC on silica gel (EtOAc-hexane, 1:1)
to afford sulfoxide 27 (16.6 mg, 0.07 mmol, 79%) as white solid, mp 207 °C (decomposed).
1
H NMR (500 MHz, CDCl ): δ 11.19 (br s, 1H), 8.09 (d, J= 7.5 Hz, 1H), 7.61 (d, J= 8.0
3
1
3
Hz, 1H), 7.37-7.27 (m, 2H), 3.98 (s, 3H), 3.11 (s, 3H). C NMR (500 MHz, CDCl ): δ165.2,
3