Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

Article abstract of DOI:10.1016/j.phytochem.2008.09.015

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.

Full text of DOI:10.1016/j.phytochem.2008.09.015

Phytochemistry 69 (2008) 2966–2971
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Structure and biological activity of maculansin A, a phytotoxin
from the phytopathogenic fungus Leptosphaeria maculans
*
M. Soledade C. Pedras , Yang Yu
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2008
Received in revised form 8 September 2008
Available online 30 October 2008
During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus
Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], the
selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic
data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual
chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans
virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant
plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, how-
ever, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition,
other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were iso-
lated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola.
Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, mac-
ulansins could assist in chemotaxonomic studies to group the diverse isolates.
Keywords:
Brassicaceae
Brassica juncea
Brassica napus
Crucifer
Indolyl-3-acetonitrile
Isonitrile
Mannitol
Phoma lingam
Sirodesmin
Ó 2008 Elsevier Ltd. All rights reserved.
Spirobrassinin
Maculansin A
1. Introduction
produced during early growth stages in MM culture as well as in
infected susceptible plants (Pedras and Biesenthal, 1998; Pedras
The plant pathogenic fungus Leptosphaeria maculans (Desm.)
Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.]
causes blackleg disease in rapeseed (Brassica napus L. and B. rapa
L.) and canola (B. napus L. and B. rapa L.) in Canada and worldwide
(Chen and Fernando, 2006; Fitt et al., 2006; Howlett et al., 2001).
Recent canola field surveys showed the prevalence of L. maculans
and an increase in more aggressive isolates in both Canada and
USA (Chen and Fernando, 2006; Kutcher et al., 2007). Consistent
with these field surveys, metabolite profiles of isolates of L. macu-
lans collected in different countries suggested that this species
comprises distinct groups (Pedras and Biesenthal, 2000; Pedras,
unpublished), one of which is highly virulent on canola. In a chem-
ically defined liquid medium (minimal medium, MM), these viru-
lent isolates produce mainly sirodesmin PL (1) (Pedras, 2001), a
non-host-selective phytotoxin recently detected in infected canola
(Elliott et al., 2007). Other sirodesmins, including deacetylsirodes-
min PL (2), and sirodesmins H (3), J (4), and K (5) and phomamide
(6) are minor metabolites co-produced with 1 in liquid MM (Ped-
ras, 2001). In addition, the host-selective toxin phomalide (7) is
et al., 1993). A rather different metabolite profile was obtained in
cultures grown in potato dextrose broth (PDB) where no phytotox-
ins appeared to be produced, but from which 2,4-dihydroxy-3,
6-dimethylbenzaldehyde (8) was isolated (Pedras et al., 2007).
Previous to our work, 8 had been isolated from Valsa ambiens
and a few other fungi (Mitova et al., 2006; Ayer et al., 1993) and
shown to have root and hypocotyl growth inhibition effect on
lettuce seedlings (Jiao et al., 1994) (see Fig. 1).
Using an elicitor-toxin bioassay to detect both elicitor and phy-
totoxic activity, we searched for bioactive metabolites in cultures
of virulent L. maculans grown in PDB, where no sirodesmins were
produced. We discovered a unique selective phytotoxin derived
from
D-mannitol and containing two 2-isocyano-3-methyl-2-bute-
noyl substituents, which was named maculansin A (9); as well,
maculansin B (10), a related metabolite was isolated. The structure
of maculansin A (9) was determined using NMR and MS spectro-
scopic data and chemical degradation. Surprisingly, this metabolite
caused larger lesions on leaves of resistant brown mustard (B. jun-
cea L.) than on susceptible canola, although it was produced by L.
maculans isolates virulent on canola. In addition, metabolite 8
was isolated and shown to display significant growth inhibitory
activity on roots of canola and mustard.
* 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 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.09.015

M.S.C. Pedras, Y. Yu / Phytochemistry 69 (2008) 2966–2971
2967
2. Results and discussion
O
HO
RO
O
1 n=2, R = Ac
2 n=2, R=H
3 n=1, R = Ac
4 n=3, R = Ac
5 n=4, R = Ac
O
2.1. Structure determination of maculansins A (9) and B (10)
N
Sn
N
H
We have reported that cultures of L. maculans appeared to pro-
duce mainly benzaldehyde 8 in PDB (Pedras et al., 2007). However,
an accidental power failure that caused an increase in the room
temperature from 23 to 28 °C appeared to induce the production
of additional metabolites not available in our fungal metabolite
libraries (Fig. 2). To isolate these potentially new metabolites
produced in PDB cultures, their production was optimized by ana-
lyzing the relative concentration of each metabolite (determined
by peak areas of HPLC chromatograms) in function of the incuba-
tion time (four and seven days) and temperature (23–29 °C). It
was established that the production of compound 8 (Rt = 11.4 min)
was maximized at 25 °C after seven days of incubation, whereas
the production of the new metabolites with Rt = 16.4 and
18.3 min (later determined to be compound 9 and 10) was maxi-
mized around four days of incubation at 27 °C (Fig. 2).
Next, cultures of L. maculans virulent isolate BJ 125 were grown
in PDB medium and extracted as reported in the Experimental. The
EtOAc extract of the broth showed phytotoxic activity using the
elicitor-toxin bioassay. As previously reported, no sirodesmins
(1–5) were detected in the extract obtained from cultures grown
in PDB medium, as determined by HPLC analysis. Metabolite 8
(ca. 1.7 mg/l, Rt = 11.4 min) together with maculansin A (9) (ca.
1.6 mg/l, Rt = 16.4 min) were the main components isolated from
the culture extract, as described in the Experimental.
OH
O
O
O
O
O
O
O
O
HN
O
O
NH
O
HN
NH
N
H
HO
6
7
Me
CHO
OH
HO
Me
8
Fig. 1. Metabolites produced by Leptosphaeria maculans in liquid minimal medium
(1–7) and in potato dextrose medium (8).
scopic data showed that the methine proton at dH 3.89 was coupled
to both H-2 and H(O–) signals, and that the methyl resonances at
NC
OH
O
NC
OH
O
O
2
O
6
6
3
AcO
5
AcO
4
OAc
4
OAc
1
1
OH
O
2'
OH
CN
O
1'
O
O
6'
CN
5'
1'
2'
4'
3'
3'
10
9
NC
OAc
3
O
NC
O
O
OH OH
3
6
OH
O
O
NC
1
O
6
O
O
O
OH OH
11
1
OH
OAc O
CN
O
O
12
The HRMS–EI of the metabolite with Rt = 16.4 min (9) indicated the
molecular formula C22H28N2O10 (m/z 480.1749), however, the 1H
and 13C NMR spectra showed only 14 and 11 signals, respectively,
suggesting a symmetry element in the molecule. The 1H NMR spec-
trum of 9 displayed signals for two methylene protons at dH 4.64
(dd, J = 12.5, 2.5 Hz, Ha-1) and dH 4.41 (dd, J = 12.5, 4.2 Hz, Hb-1),
two methines at dH 5.13 (m, H-2) and dH 3.89 (t, J = 8.2, 8.2 Hz,
H-3), an exchangeable proton at dH 3.00 (d, J = 7.7 Hz, OH), and
three methyl singlets at dH 2.11, 2.18 and 2.31 (i.e. 14 protons, Ta-
ble 1). The 13C NMR spectrum showed 11 carbons, six of which
were sp3 hybridized, four carbons were sp2 hybridized and one
was sp hybridized (Table 1). COSY, HMQC and HMBC NMR spectro-
dH 2.31 and 2.18 correlated to sp2 carbons at dC 158.3 and 115.0.
In addition, the H-2 signal (dH 5.13) showed a correlation with an
sp2 carbon at dC 160.5, whereas a carbon at dC 168.0 did not show
detectable correlations. Altogether, these NMR correlations sug-
gested that the carbonyl at dC 160.5 was conjugated with an iso-
propenyl fragment. Since the IR spectrum contained
a
characteristic absorption at 2116 cmꢀ1 attributable to an isonitrile
group and the signal at dC 168.0 was consistent with an isonitrile
carbon (Karuso et al., 1989), a 2-isocyano-3-methyl-2-butenoyl
partial structure was assigned. The overall spectroscopic data sug-
gested that 9 was derived from a polyol containing two acetyl and
two 2-isocyano-3-methyl-2-butenoyl substituents and two free

2968
M.S.C. Pedras, Y. Yu / Phytochemistry 69 (2008) 2966–2971
two epimers at C-20 (due to the acidity of H-20) and is proposed
to be 1,6-diacetyl-2-(2-isocyano-3-methyl-2-butenoyl)-5-(2-iso-
cyano-3-methylbutanoyl)-D-mannitol and named maculansin B
(10).
Similarly, inspection of the 1H NMR spectroscopic data of com-
ponent with Rt = 14.7 min showed a spectrum more complex than
that of 10, containing several signals for each proton and thus sug-
gesting that this sample contained a mixture of structural isomers
likely due to the presence of two 2-isocyano-3-methylbutanoyl
moieties and absence of an acetyl substituent. As in the case of
metabolite 10, the mannitol backbone appeared to remain intact;
however, due to the complexity of the spectra, structural assign-
ments could not be made unambiguously and thus structures are
not proposed. Therefore, the main components of PDB cultures of
virulent L. maculans are maculansins A (9), B (10) and a currently
inseparable mixture of related metabolites not fully characterized.
The 2-isocyano-3-methyl-2-butenoyl fragment is rare in natu-
ral products, to the best of our knowledge only antibiotic
A32390-A (11) was reported to contain it (Marconi et al., 1978).
This antibiotic, isolated from the culture broth of a Pyrenochaeta
species, is an inhibitor of dopamine-b-hydroxylase and is antimi-
crobial against fungi and gram-positive bacteria. Similarly, brassi-
cicolin A (12), isolated from cultures of Alternaria brassicicola, a
fungal pathogen of Brassica species, was the first natural product
reported to contain 2-isocyano-3-methylbutanoyl fragments and
also shown to have potent in vitro activity against the gram-posi-
tive bacteria (Gloer et al., 1988). Maculansins A (9) and B (10) to-
gether with A32390-A (11) and brassicicolin A (12) constitute a
Fig. 2. HPLC chromatograms of extracts of cultures of Leptosphaeria maculans
grown in potato dextrose medium (PDB); (A) EtOAc extract of culture incubated at
23 °C for four days; (B) EtOAc extract of culture incubated at 27 °C for four days;
numbered peaks identified as: 2,4-dihydroxy-3,6-dimethylbenzaldehyde (8), mac-
ulansin
A (9), maculansin B (10), peak at 14.7 min identified as mixture of
metabolites related to 9. Inserts; UV/Vis spectra of metabolites 8 and 9.
Table 1
13C NMR (125 MHz) and 1H NMR (500 MHz) chemical shifts (ppm) and multiplicities
(J in Hz) of maculansin A (9) (in CDCl3)
C/H #
1, 6
Maculansin A (9)
dC
dH
62.3
4.64, 2H, dd, (12.5, 2.5)
4.41, 2H, dd, (12.5, 4.2)
2, 5
3, 4
10
72.8
67.4
160.5
115.0
158.3
21.4
25.1
168.0
171.6
21.1
–
5.13, 2H, m
3.89, 2H, t, (8.2)
–
–
very unique group of fungal metabolites derived from
that display strong and diverse biological activities.
D-mannitol
20
30
–
2.2. Bioactivity
40/50
50/40
60
2.31, 6H, s
2.18, 6H, s
–
–
2.11, 6H, s
3.00, 2H, d, (7.7)
2.2.1. Maculansin A (9)
100
200
Elicitor activity results were considered positive when droplets
of a metabolite solution applied to leaves of 2-week-old plants
(4-leaf stage) elicited production of phytoalexins (detected by
HPLC analysis of leaf extracts). Toxicity was scored by measure-
ment of leaf lesions (necrosis and/or chlorosis, rated according to
the damage index number, where the number is directly propor-
tional to the size of the lesion, as described in the Experimental).
The bioactivity of maculansin A (9) was compared with that of
the toxin sirodesmin PL (1, isolated as previously reported by
Pedras et al., 1990). Maculansin A (9) did not elicit the production
of phytoalexins in B. juncea cv. Cutlass or B. napus cv. Westar
(Figs. 3 and 4). However, the damage caused by 9 on the leaves
(O)H
hydroxyl groups. The polyol backbone of metabolite 9 was deter-
mined to be -mannitol after hydrolysis, acetylation and identifica-
D
tion of one of the reaction products as hexaacetyl D-mannitol by
comparison of the 1H NMR spectroscopic data and specific optical
rotation with that of an authentic sample. Therefore, the structure
of the new metabolite 9 was elucidated as 1,6-diacetyl-2,5-bis(2-
isocyano-3-methyl-2-butenoyl)-D-mannitol.
From another fraction, a mixture of several compounds having
structural characteristics similar to those of 9 was obtained and
further separated by prep TLC, as described in the Experimental,
to afford components with Rt = 18.3 min (10) and 14.7 min
(Fig. 2). Although HPLC analysis suggested a chromatographically
homogeneous material, preliminary inspection of the 1H NMR
spectroscopic data of the component with Rt = 18.3 min (10) indi-
cated that this sample contained a mixture of at least two com-
pounds (complex spectrum showing a polyol backbone with two
sets of signals for some protons). In addition, this spectrum showed
two doublets at dH 1.14 and 1.04 coupled to a methine proton at dH
2.38 as well as two methyl groups at dH 2.3 and 2.2, similar to those
observed in 9. That is, one of the 2-isocyano-3-methyl-2-butenoyl
fragments attached to the polyol was replaced with a 2-isocyano-
3-methylbutanoyl moiety, whereas the acetyl groups and the back-
bone remained intact. This conclusion was consistent with the
[M + 1]+ m/z 483 obtained for this sample (LC–MS–ESI). As well,
Fig. 3. HPLC chromatograms of extracts of leaves of brown mustard (Brassica juncea
cv. Cutlass, resistant to blackleg) treated with: (A) maculansin A (9) and (B)
sirodesmin PL (1); numbered peaks identified as: indole-3-acetonitrile (13), 1-
methoxy-indole-3-acetonitrile (14), 1-methoxy-indole-3-methanol (15), spiro-
brassinin (16), cyclobrassinin (17).
the polyol backbone of 10 was determined to be D-mannitol after
hydrolysis and acetylation of a mixture of 9 and 10, as reported
for metabolite 9. Therefore, the inseparable mixture 10 contained

M.S.C. Pedras, Y. Yu / Phytochemistry 69 (2008) 2966–2971
2969
Control
Treated
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Canola
Brown mustard
Fig. 6. Root length of seedlings of canola (Brassica napus cv. Westar, susceptible)
and brown mustard (B. juncea cv. Cutlass, resistant) treated with metabolite 8 at
0.5 mM.
Fig. 4. HPLC chromatograms of extracts of leaves of canola (Brassica napus cv.
Westar, susceptible to blackleg) treated with: (A) maculansin (9) and (B)
sirodesmin PL (1); numbered peaks identified as: indole-3-acetonitrile (13), 1-
methoxy-indole-3-acetonitrile (14), 1-methoxy-indole-3-methanol (15), spiro-
brassinin (16).
A
growth of susceptible and resistant host plants. It is particularly
intriguing that the inhibitory effect on the resistant plant is much
higher than that caused in susceptible plants.
of different crucifer plants appeared to depend on the plant spe-
cies. For example, 9 at 0.1 mM caused damage (2.2) on resistant
brown mustard (B. juncea cv. Cutlass), but no damage on either
susceptible canola (B. napus cv. Westar) or resistant white mustard
(Sinapis alba cv. Ochre); at higher concentration (1 mM), similar
damage on the three plant species was observed (Fig. 5). Contrary
to maculansin A (9), sirodesmin PL (1, 0.2 M) caused similar leaf
lesions in all plant species and induced the production of phytoan-
ticipins (indole-3-acetonitrile (13), 1-methoxy-indole-3-acetoni-
trile (14), 1-methoxy-indole-3-methanol (15)) and phytoalexins
(spirobrassinin (16) and cyclobrassinin (17)) in leaves of brown
mustard (Fig. 3) and canola (Fig. 4), as determined by HPLC analysis
and direct comparison with authentic samples.
The somewhat selective phytotoxicity of 9 is inconsistent with
the pathogenicity of L. maculans, because 9 caused more damage
on resistant plants (B. juncea cv. Cutlass) than on susceptible plants
(B. napus cv. Westar). Moreover, the damage caused by 9 suggested
that it is able to diffuse in the leaf tissue, causing irregular lesions,
similar to those caused by the pathogen and the toxin depsilairdin
(Pedras et al., 2004). On the other hand, the damage caused by
sirodesmin PL (1) is a defined circular lesion, indicating immediate
lyses of leaf cells.
3. Conclusions
Altogether our past (Pedras et al., 2007) and current results
show that culture conditions have a great impact on the produc-
tion of secondary metabolites by virulent L. maculans. The diversity
of metabolite structures determined by medium composition is
particularly intriguing. One wonders which of these metabolites,
if any, affect the interaction of L. maculans with its host plants.
Our multiple attempts to identify fungal metabolites in infected
plant tissues have yet to produce consistent and convincing results.
Nonetheless, considering that L. maculans seems to be expanding
its host range to infect brown mustard as well (isolates Laird 2
and Mayfair 2 are virulent on brown mustard, Pedras et al.,
2007), maculansins could be useful in chemotaxonomic studies
for grouping the various isolates. In this context, a question arises
whether maculansin type metabolites are also produced by iso-
lates Laird 2 and Mayfair 2, as this may explain the higher phyto-
toxicity of maculansin A (9) on brown mustard and warrants
further investigations. Finally, it is also worthy to note that macu-
lansin A (9) causes obvious damage on leaf tissues but does not eli-
cit phytoalexins, an outcome very useful for the pathogen.
2.2.2. 2,4-Dihydroxy-3,6-dimethylbenzaldehyde (8)
Although 2,4-dihydroxy-3,6-dimethylbenzaldehyde (8) was
shown to have root and hypocotyl growth inhibition effects on
lettuce seedlings (Jiao et al., 1994), it was important to determine
if it affected root growth of the host plants of L. maculans. Thus, the
effect of 8 on seeds of brown mustard (resistant) and canola
(susceptible) incubated in media amended with 8 (0.5 mM) was
determined. A strong inhibitory effect was observed for both
species; while the root length of treated canola seeds was
0.3 0.1 cm, control roots measure 1.9 0.7 cm (Fig. 6). Similarly,
roots of treated brown mustard measure only 0.2 0.1 cm, while
control roots measured 4.9 1.2 cm (seeds were incubated in
darkness for 7 days). That is, 8 inhibited 6-fold and 25-fold the root
growth of canola and brown mustard, respectively. This is the first
time that a metabolite of L. maculans is reported to inhibit root
7
Brown mustard
6
5
4
3
2
1
0
Canola
4. Experimental
White mustard
4.1. General
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 in a rotary evaporator. Flash column chromatogra-
phy (FCC) was carried out using silica gel grade 60, mesh size 230–
400 Å. Prep TLC was carried out on silica gel plates, Kieselgel 60
F254 (20 ꢁ 20 cm ꢁ 0.25 mm); compounds were visualized under
1.0
0.2
0.1
Concentration (mM)
Fig. 5. Phytotoxicity of maculansin A (9) on brown mustard (Brassica juncea cv.
Cutlass, resistant), canola (B. napus cv. Westar, susceptible) and white mustard (S.
alba cv. Ochre, resistant).
UV light. Specific rotations [
perature on a polarimeter using a 1 ml, 10 cm path length cell; the
a] were determined at ambient tem-
D

2970
M.S.C. Pedras, Y. Yu / Phytochemistry 69 (2008) 2966–2971
units are 10ꢀ1 deg cm2 gꢀ1 and the concentrations are reported in
g/100 ml.
1.04 (d, J = 6.8 Hz, 3 H). 13C NMR (125 MHz, CDCl3): d 171.5 (2C),
158.8, 116.6, 115.0, 74.0, 72.5, 67.6 (2C), 62.8, 61.9 (2C), 24.8,
21.3 (2C), 20.8 (2C), 19.5, 16.6, (C-10, -60, and -100 were not de-
tected). 13C NMR spectral data were obtained from analysis of
HMQC and HMBC correlations.
Chemical degradation of maculansin A (9). Na2CO3 (10.0 mg)
was added to a solution of maculansin A 9 (8.0 mg, 0.017 mmol)
dissolved in MeOH (4.0 ml). The reaction mixture was stirred for
4 h at room temperature then concentrated. The residue was dis-
solved in H2O (5 ml), the pH of the solution was adjusted to 2, fol-
lowed by extraction (CH2Cl2). The aqueous layer was concentrated
Nuclear magnetic resonance (NMR) spectra (1H, 13C, HMQC-het-
eronuclear multiple quantum coherence, HMBC-heteronuclear
multiple bond coherence) were recorded on Bruker Avance 500
spectrometers. High resolution (HR) electron impact (EI) mass
spectra (MS) were obtained on a VG 70 SE mass spectrometer
employing a solids probe. LC–MS–ESI data were obtained on an
Agilent 1100 HPLC system coupled to an LC/MSD ion trap detector
with electrospray ionization. Chromatographic separation was car-
ried out at room temperature using an Eclipse XSB C-18 column
(5
l
m particle size silica, 150 mm ꢁ 4.6 mm) equipped with an on-
to dryness then pyridine (500 ll) and Ac2O (500 ll) were added to
line filter. The mobile phase consisted of a gradient of 0.2% HCO2H
in H2O (A) and 0.2% HCO2H in CH3CN (B); linear gradient elution
starting with 75:25 of A/B to 25:75 of A/B in 35 min, to 100% B
in 5 min and a flow rate 1.0 mL/min. Other conditions were as pre-
viously reported (Pedras et al., 2006).
HPLC–DAD analysis was carried out with Agilent high perfor-
mance liquid chromatographs equipped with quaternary pump,
automatic injector, and diode array detector (DAD, wavelength
the residue and the reaction mixture stirred for 5 h at room tem-
perature. After concentration to dryness, the residue was separated
(prep TLC, MeOH–CH2Cl2, 4:96) to yield a compound (6.7 mg) iden-
tical to
and specific optical rotation data ([
tic -mannitol hexaacetate [ D = 12 (c 0.26, CHCl3)).
D
-mannitol hexaacetate as determined by 1H NMR, HRMS
a]D = 13 (c 0.51, CHCl3); authen-
D
a]
4.4. Bioactivity assays
range 190–600 nm), degasser, and a Hypersil ODS column (5 lm
particle size silica, 4.6 i.d. ꢁ 200 mm), having an in-line filter. Mo-
bile phase: 75% H2O-25% CH3CN to 100% CH3CN, for 35 min, linear
gradient, and at a flow rate 1.0 ml/min.
Three activity assays were carried out as described below.
Elicitor activity. Canola (B. napus cv. Westar, susceptible to L.
maculans) and brown mustard (B. juncea cv. Cutlass, resistant to
L. maculans) were grown in a growth chamber with 16 h light (fluo-
4.2. Fungal isolates and culture conditions
rescent and incandescent, 450–530 l
mol sꢀ1 mꢀ2)/8 h dark, at
24 2 °C. After two weeks, extracts dissolved in MeOH–H2O (1:1,
v/v) solutions were applied to leaves punctured with a needle
(about 20 punctures per leaf). After two days, the leaf tissues were
frozen in liquid N2, crushed with a glass rod and extracted with
50 ml EtOAc. EtOAc extract was dried over Na2SO4 and concen-
trated in a rotary evaporator. The residue was dissolved in 1%
MeOH–CH2Cl2, applied to a mini silica gel column (Pasteur pipette)
and eluted with 1% MeOH–CH2Cl2 (5 ml); the eluate was concen-
Canadian virulent isolate of L. maculans IBCN 57 (BJ 125) was
grown in Erlenmeyer flasks (250 ml) containing PDB (100 ml),
inoculated with fungal spores (108 spores/100 ml) and incubated
on a shaker at 120 rpm, at 23, 25, 27, and 29 °C. Culture samples
(20 ml) of 4-day-old or 7-day-old cultures were filtered to separate
the broth from mycelia. The broth was extracted with EtOAc
(20 ml ꢁ 3) and the EtOAc layer was dried over Na2SO4 and con-
centrated. The residue was dissolved in CH3CN (1.0 ml) and ana-
lyzed by HPLC.
trated, the residue was dissolved in CH3CN (80 ll) and analyzed
by HPLC. Control leaves were treated similarly (MeOH–H2O, 1:1)
and analyzed by HPLC. All assays were carried out in triplicate.
Phytotoxic activity. Canola, brown mustard and white mustard
(S. alba cv. Ochre, resistant to L. maculans) were grown as described
above. Solutions of either fractions or purified compounds dis-
solved in MeOH–H2O (1:1, v/v) were applied to punctured leaves
4.3. Isolation and characterization of maculansins A (9) and B (10)
PDB cultures of L. maculans (5 l) were prepared as reported
above and grown in an incubator at 27 °C to maximize production
of 9. After four days, the cultures were combined and filtered, the
broth was extracted with EtOAc, and the EtOAc layer was concen-
trated to yield 360 mg of residue. The residue was separated by
FCC (RP-18, CH3CN–H2O, 10:90 to 40:60, gradient elution) to yield
30 fractions (15 ml per fraction). Fractions 8–10 were combined
and further purified by FCC (MeOH–CH2Cl2 0:100 to 5:95, gradient
elution) to give compound 8 (10.5 mg). Fractions 15 to 18 were
combined (45.1 mg) and further purified by prep TLC (MeOH–
CH2Cl2, 5:95) to yield compound 9 (9.7 mg) and a mixture of the
component with Rt = 14.7 min (mixture of isomers structural type
similar to 9). Fractions 22–25 contained a mixture of maculansin
type metabolites that were further separated by prep TLC to yield
an epimeric mixture of maculansin B (10, 1.6 mg).
(5 ll, 6 punctures per leaf). After two days, the damaged areas
were measured using a stencil having cut out circles with different
diameters, and the measured diameters were converted to the
damage index, as shown in Table 2. All bioassays were carried
out in triplicate.
Root growth inhibition activity. Seeds of brown mustard (20)
and canola (20) were sterilized by soaking in Javex (10%) for
10 minutes, washed with H2O, air dried and incubated in PDB med-
ium (0.9 g/100 ml) amended with compound 8 (0.5 mM). The
dishes were sealed with parafilm and kept in darkness at room
temperature. After 7 days the root length of seedlings was mea-
sured with a ruler.
Maculansin A (9). HPLC Rt = 16.4 min. HRMS–EI: m/z 480.1749,
C22H28N2O10, calcd. 480.1744. MS–EI: m/z 480 (3%), 465 (22%), 115
(100%). for 1H and 13C NMR spectroscopic data see Table 1. FTIR
Table 2
Scale for conversion of damaged area to damage index
(KBr): 3460, 2960, 2923, 2116, 1740, 1732, 1222 cmꢀ1
. UV
Lesion diameter (mm)
Damage index
(MeOH): kmax (log
e) 231 (4.2) nm; [
a]
D = ꢀ71 (c 0.10, CHCl3).
Maculansin B (10). HPLC Rt = 18.3 min. HRMS–ESI: [M + Na]+
m/z 505.1807, C22H28N2O10Na, calcd. 505.1792. HRMS–ESI–MS/
MS: m/z 505 (100%), 380 (40%), 378 (70%), 253 (27%). 1H NMR
(500 MHz, CDCl3): d 5.2 (m, 1 H), 5.1 (m, 1 H), 4.6 (m, 2 H), 4.4
(m, 2 H), 4.28 (d, J = 4.2 Hz, 1 H), 3.8 (m, 2 H), 3.07 (d, J = 7.4 Hz,
1 H, OH), 2.93 (d, J = 8.3 Hz, 1 H, OH), 2.4 (m, 1 H), 2.32 (s, 3 H),
2.19 (s, 3 H), 2.10 (s, 3 H), 2.09 (s, 3 H), 1.14 (d, J = 6.8 Hz, 3 H),
<1.5
0
1
2
3
4
5
6
7
1.6–2.3
2.4–3.1
3.2–3.9
4.0–4.7
4.8–5.5
5.6–6.3
6.4–7.0

M.S.C. Pedras, Y. Yu / Phytochemistry 69 (2008) 2966–2971
2971
Karuso, P., Poiner, A., Scheuer, P.J., 1989. Isocyanoneopupukeanane, a new tricyclic
sesquiterpene from a sponge. J. Org. Chem. 54, 2095–2097.
Acknowledgements
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Pedras, M.S.C., 2001. Phytotoxins from fungi causing blackleg disease on crucifers:
isolation, structure determination, detection, and phytotoxic activity. Recent
Res. Devel. Phytochem. 5, 109–117.
Pedras, M.S.C., Biesenthal, C.J., 1998. Production of the host-selective phytotoxin
phomalide by isolates of Leptosphaeria maculans and its correlation with
sirodesmin PL production. Can. J. Microbiol. 44, 547–553.
Financial support for the authors’ work was obtained from the
Natural Sciences and Engineering Research Council of Canada (Dis-
covery 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
Y.Y.). We acknowledge the technical assistance of K. Brown
(NMR) and K. Thoms (MS), from the Department of Chemistry, Uni-
versity of Saskatchewan.
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