Sinalbins A and B, phytoalexins from Sinapis alba: Elicitation, isolation, and synthesis

Article abstract of DOI:10.1016/S0031-9422(00)00277-6

The chemical structure and synthesis of sinalbin A is described. This cruciferous phytoalexin is produced by white mustard (Sinapis alba) after treatment with biotic and abiotic elicitors. In addition, a related metabolite, named sinalbin B, is present in extracts from elicited plants, but not in those from non-elicited controls. Sinalbin B, which was also synthesized appears to be both a phytoalexin and a biosynthetic precursor of sinalbin A. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00277-6

Phytochemistry 55 (2000) 213±216
www.elsevier.com/locate/phytochem
Sinalbins A and B, phytoalexins from Sinapis alba:
elicitation, isolation, and synthesis
M. Soledade C. Pedras *, Irina L. Zaharia
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK, S7N 5C9, Canada
Received 22 February 2000; received in revised form 15 June 2000
Abstract
The chemical structure and synthesis of sinalbin A is described. This cruciferous phytoalexin is produced by white mustard
(Sinapis alba) after treatment with biotic and abiotic elicitors. In addition, a related metabolite, named sinalbin B, is present in
extracts from elicited plants, but not in those from non-elicited controls. Sinalbin B, which was also synthesized, appears to be both
a phytoalexin and a biosynthetic precursor of sinalbin A. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sinapis alba; Cruciferae; Crucifer; White mustard; Phytoalexin; Sinalexin; Alternaria brassicae; Alternaria blackspot; Destruxins; Phoma
lingam; Blackleg
1. Introduction
Sacc.] fungi, as well as through elicitation with destruxin
B, homodestruxin B, hydroxydestruxin B, and CuCl2.
Plant defense mechanisms may involve de novo bio-
synthesis of antimicrobial compounds, some of which
have low molecular weights and are known as phytoa-
lexins (Bailey and Mans®eld, 1982; Brooks and Watson,
1985). The phytoalexins from crucifers have unique
structures containing indole or indole related rings and
at least one sulfur atom, thus far detected only in this
plant family (Pedras et al., 2000).1 Because white mus-
tard (Sinapis alba) is resistant to economically important
fungal diseases such as Alternaria blackspot and Phoma
blackleg (Saharan, 1993; Hemingway, 1995), we have
been studying its defense mechanisms, including phy-
toalexin production. We have reported the ®rst phytoa-
lexin produced by white mustard, sinalexin (1), under
biotic and abiotic elicitation (Pedras and Smith, 1997).
In continuation of these studies, we wish to report the
elicitation, detection, and synthesis of two new metabo-
lites, named sinalbins A (5) and B (4), produced by leaf
and stem tissues of white mustard upon biotic elicitation
with the blackleg [Phoma lingam (Tode ex Fr.) Desm.,
asexual stage of Leptosphaeria maculans (Desm.) Ces. et
de Not.] and blackspot [Alternaria brassicae (Berk.)
2. Results and discussion
HPLC chromatograms of extracts of elicited leaves of
white mustard suggested that a compound with tR=
17.9 min, not present in extracts of non-elicited leaves,
was being produced over a period of several days. For
example, upon elicitation with destruxin B, CuCl2, P.
lingam, or A. brassicae the maximum production of this
compound occurred at 24 h, whereas elicitation with
homodestruxin B or hydroxydestruxin B appeared to
induce slower responses, at 48 and 96 h, respectively.
In order to obtain a reasonable amount of elicited
tissue to identify the new compound unambiguously,
white mustard plants were elicited with CuCl2, and the
leaves were extracted with EtOAc. The extract was
fractionated, the fractions were analyzed by HPLC and
further separated by preparative TLC, as described in
* Corresponding author. Tel;+1-306-966-4772; fax: +1-306-966-
4730.
E-mail address: soledade.pedras@usask.ca (M.S.C. Pedras).
1
the experimental. The H and 13C NMR spectra of the
1
For a recent review see Pedras et al., 2000.
compound with tR=17.9 min displayed only aromatic
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00277-6

214
M.S.C. Pedras, I.L. Zaharia / Phytochemistry 55 (2000) 213±216
signals assignable to an indole moiety, except for three
resonances due to CH2 (ꢀH 5.42 and ꢀC 50.7), CH3O (ꢀH
4.06 and ꢀC 66.5) and CH3S (ꢀH 2.85 and dC 42.0)
groups. The assignment of structure 5 to this plant
metabolite was secured from additional spectroscopic
data (FTIR, UV, and HRMS) and corroboration by
synthesis, as shown in Scheme 1.
underway to better understand the role of these com-
pounds in the resistance of S. alba to P. lingam, and A.
brassicae, two of the most important pathogens of
canola. To the best of our knowledge, compounds 4 and
5, named sinalbin A (5) and sinalbin B (4), are new
phytoalexins from white mustard, i.e. produced by eli-
cited plant tissues, not detectable in non-elicited tissues,
and having antifungal activity against P. lingam. Con-
sidering that the closely related phytoalexin cyclo-
The synthesis of 5 was accomplished (Scheme 1) in six
steps from 1-methoxy indole, via oxime 2 and methox-
ybrassinin (3). The key step, which represents a sig-
ni®cant improvement over a previously published
synthesis of 3 (Somei et al., 1992; Yamada et al., 1993), is
the reduction of oxime 2 to the corresponding amine and
subsequent formation of methoxybrassinin (3) in 60%
overall yield. Because the synthetic intermediate 4
appeared as a likely biogenetic precursor of 5, we investi-
gated whether this compound was also produced by eli-
cited plant tissues. Interestingly, compound 4 (tR=32.2
min) was detected in extracts of white mustard stems eli-
cited by P. lingam, but not in elicited leaf tissues; how-
ever, due to the relatively smaller amount present in these
extracts, we were unable to isolate compound 4 from any
of the plant tissues. It is likely that 4 is the biosynthetic
precursor of 5, thus absence from leaf tissues suggests
its faster bioconversion in leaf tissue than in stem tissue.
Upon standing at room temperature for 13 days (in
CH3CN), compound 5 yielded mostly 1 (90%), while 4
yielded undetermined products (80%). However, 1
appears to be a naturally occurring compound since its
accumulation in elicited foliar tissue of S. alba depends
on the elicitor, plant age, and plant cultivar (e.g. cv.
Pennant accumulates sinalexin faster than cv. Ochre).
Finally, antifungal bioassays established that meta-
bolites 4 and 5 were active against P. lingam. While 5
brassinin (6) was shown to be
a
biosynthetic
intermediate of the phytoalexin brassilexin (7) in B.
juncea (Scheme 2) (Pedras et al., 1998), sinalbin B (4)
may be biosynthetic precursor of sinalexin (1) in S. alba.
3. Experimental
3.1. General
All chromatographic conditions, including HPLC
analyses, and spectroscopic techniques, were used as
previously reported (Pedras and Okanga, 1999).
3.2. Elicitation of phytoalexins
Leaves from 21-day-old plants of S. alba cv. Ochre (in
growth chamber, 16 h day/8 h night, 20ꢀC constant)
were punctured (5 leaves, 4 punctures/leaf, hypodermic
needle, 19 gauge) and each puncture was inoculated
with a 10 ml drop of spore suspension of A. brassicae
(1.5Â105 spores/ml). After 24 h, the leaf tissue (0.5 g)
was frozen in liquid N2, crushed with a glass rod and
extracted with EtOAc (50 ml, shaking for 16 h). The
EtOAc extract was separated by ®ltration, dried (Na2SO4)
and concentrated under reduced pressure. The residue
(6.4 mg) was dissolved in CH3CN and analyzed by
HPLC. Alternatively, leaves were cut, and the petioles
partly immersed in solutions of destruxin B, homo-
4
(5Â10 M) caused complete inhibition of spore germi-
nation (ED50 2Â10 4 M at 48 h) for the duration of the
assay (7 days), 4 showed moderate activity (ED50
4
4
7Â10 M at 48 h) at similar concentration (5Â10
5
M), causing about 30% germination inhibition relative
to controls, after 48 h. Further bioactivity studies are
destruxin B, or hydroxydestruxin B (2Â10 M in 2%
aq. CH3CN) for solution uptake and incubated for dif-
ferent time periods (Pedras et al., 1999). Similar elicita-
tion experiments were carried out with stems cut from
21-day-old plants and incubated with a spore suspen-
sion (5.5Â107 spores/mL) of P. lingam, or the stems
3
partly immersed in CuCl2 (2Â10
M) for solution
uptake. After incubation for di€erent times, the stems
were extracted as described for leaves.
Scheme 1. (i) TiCl3/NaBH3CN/NH4Ac; (ii) CS2/Py,Et3N; Mel, 60%;
(iii) CH2Cl2/NBS, 41%; (iv) m-CPBA, 20%.
Scheme 2.

M.S.C. Pedras, I.L. Zaharia / Phytochemistry 55 (2000) 213±216
215
3.3. Antifungal activity assay
80:20) to yield 1-methoxybrassinin (3) 199 mg (60%
yield from oxime). N-bromosuccinimide (7.8 mg, 0.044
mmol) was added to a stirred solution of 1-methoxy-
brassinin (3) (11.7 mg, 0.044 mmol) in anhydrous CH2Cl2
(0.5 ml) at room temperature. The reaction mixture was
stirred for 10 min at room temperature, then Et3N (13 ml,
0.088 mmol) was added and, after further stirring for 5
min, the solvent was removed under reduced pressure.
The residue was separated by FCC (silica gel, CH2Cl2±
hexane, 80:20) to yield sinalbin B (4) 5.5 mg (47% yield).
m-CPBA (16.3 mg, 0.095 mmol) was added to a cooled
stirred solution of 4 (16.7 mg, 0.06 mmol) in anhydrous
CH2Cl2 (1 ml, 0ꢀC). After 20 min at 0ꢀC, Me2S (250 ml)
was added, the reaction mixture was allowed to warm up
to room temperature (5 min) and the solvent was evapo-
rated under reduced pressure. After standard work, the
organic phase was dried (Na2SO4) and concentrated to
dryness in vacuo, the residue (16.1 mg) was separated by
prep TLC (silica gel RP-8, CH3CN±H2O, 70:30) to yield of
sinalbin A (5) 3.5 mg (20% yield).
Bioassays to determine the e€ect of compounds 4 and
5 on the germination inhibition of spores of P. lingam
were carried out after modi®cation of a method pre-
viously reported (Pedras and Biesenthal, 1998). That is,
Nunclon 4-well multidish plates containing 1Â107
spores/mL in 500 mL solutions of compound to be tested
5
(5Â10 4, 1Â10 4, 5Â10 M in 3% Tween 80 and 2%
DMSO in minimal medium) were incubated still under
constant ¯uorescent light, at 24Æ1ꢀC. The percentage of
spore germination was determined by counting about
300 spores (in randomly selected ®elds) for each repli-
cate after incubations of 24 h, 48 h and 7 days.
3.4. Isolation of sinalbin A (5)
Leaves of 21-day-old of S. alba cv. Ochre (120 plants)
were sprayed with CuCl2 solution (1Â10 2 M); after 24 h
the leaves were cut (750 g) and the tissue was frozen in
liquid N2. The leaf tissue was extracted with EtOAc (1.5 l)
and processed as described above to yield an oily resi-
due (3.32 g). The residue was fractionated by FCC
(silica gel RP-18, CH3CN±H2O, 1:1) and fractions con-
taining an HPLC peak at tR=17.9 min were combined
(17.3 mg). Further fractionation (silica gel RP-18) of this
residue gave 7.6 mg of material containing the peak of
interest. This residue was then rinsed with CH2Cl2 (3 ml)
and the CH2Cl2 soluble portion (2.7 mg) was separated
by prep TLC (silica gel RP-8, CH3CN±H2O, 65:35,
developed two times) to give (0.2 mg) of sinalbin A (5).
3.6. Sinalbin A (5)
1
HPLC tR=17.9 min; H NMR (500 MHz, CD2Cl2) :
ꢀ 7.44 (d, J=8 Hz, 1H), 7.43 (d, J=8 Hz, 1H), 7.26 (dd,
J=8, 8 Hz, 1H), 7.16 (dd, J=8, 8 Hz, 1H), 5.42 (s, 2H),
4.06 (s, 3H), 2.85 (s, 3H); 13C NMR (125.8 MHz,
CD2Cl2): ꢀ 166.2 (s), 134.8 (s), 123.2 (d), 122.3 (s), 122.0
(s), 121.5 (d), 118.3 (d), 108.8 (d), 96.7 (s), 66.5 (q), 50.7
(t), 42.0 (q); HREIMS m/z (% relative abundance)
measured: 280.0341 (280.0340 calcd. for C12H12N2
O2S2); EIMS m/z (% relative abundance): 280 [M]+
(70), 248 (12), 217 (10), 191 (92), 160 (100), 117 (51), 101
(28); FTIR ꢁmax: 2933, 2842, 1728, 1632, 1443, 1325,
1231, 1126, 1066, 950, 921, 740 cm 1; UV (CH2Cl2) lmax
(log ꢂ) 231 (4.33), 280 (3.96) nm.
3.5. Synthesis of sinalbins A (5) and B (4)
1-Methoxyindole (Pedras and Sorensen, 1998) was
formylated (Smith, 1954), and the corresponding oxime
2 prepared in quantitative yield. Sodium cyanoborohy-
dride (738 mg, 12.5 mmol) and NH4OAc (1.06 g, 13.7
mmol) were added to a cooled solution of 2 (237 mg,
1.25 mmol) in MeOH. To this mixture a neutralized
solution (3.5 ml, 9.9 mmol) of TiCl3 (30% by wt. in 2 M
HCl) was added. After stirring for 15 min at 0ꢀC, the
reaction mixture was diluted with 10 ml water, neu-
tralized with NaOH and extracted with CH2Cl2 (2Â15
ml). The organic phase was dried (Na2SO4) and con-
centrated to dryness in vacuo to yield crude N-
methoxyindole-3-methanamine (188 mg). To the cooled
solution of crude amine in pyridine (0.5 ml), Et3N (164
ml, 1.18 mmol) and CS2 (64 ml, 1.1 mmol) were added.
After 1 h of stirring at 0ꢀC, MeI (67 ml, 1.1 mmol) was
added and the reaction was then left at 5ꢀC for 16 h.
H2SO4 (2 ml) was added to the reaction mixture, the
mixture was extracted with Et2O (2Â10 ml), the organic
phase was separated, dried (Na2SO4) and concentrated
to dryness in vacuo to yield residue (244 mg). The resi-
due was separated by FCC (silica gel, CH2Cl2±hexane,
3.7. Sinalbin B (4)
1
HPLC tR=32.2 min; H NMR (500 MHz, CD2Cl2) :
ꢀ 7.45 (d, J=8 Hz, 1H), 7.42 (d, J=8, Hz, 1H), 7.22 (dd,
J=8, 8 Hz, 1H), 7.14 (dd, J=8, 8 Hz, 1H), 5.07 (s, 2H),
4.01 (s, 3H), 2.55 (s, 3H); 13C NMR (125.8 MHz,
CD2Cl2): ꢀ 151.1 (s), 134.8 (s), 129.7 (s), 122.7 (d), 122.4
(s), 121.1 (d), 117.9 (d), 108.8 (d), 100.2 (s), 66.0 (q), 49.0
(t), 15.6 (q); HREIMS m/z (% relative abundance)
measured: 264.0397 (264.0391 calcd. for C12H12N2OS2);
EIMS m/z (% relative abundance): 264 [M]+ (20), 232
(100), 191 (69), 159 (56), 117 (22), 89 (9); FTIR ꢁmax
2925, 1593, 1448, 1409, 1322, 1215, 1058, 935, 740 cm
UV (CH2Cl2) lmax (log ꢂ) 231 (4.13), 275 (3.80) nm.
:
;
1
Acknowledgements
We gratefully acknowledge the ®nancial support of
the Natural Sciences and Engineering Research Council

216
M.S.C. Pedras, I.L. Zaharia / Phytochemistry 55 (2000) 213±216
of Canada and the University of Saskatchewan. We
acknowledge the technical assistance in antifungal activ-
ity determinations of Soongnyeong Lee, Department of
Chemistry, University of Saskatchewan. Isolates of
Alternaria brassicae and Phoma lingam were obtained
from G. Seguin-Swartz, Agriculture and Agri-Food
Canada, Saskatoon Research Centre, Saskatoon SK.
Pedras, M.S.C., Okanga, F.I., 1999. Strategies of cruciferous pathogenic
fungi: detoxi®cation of the phytoalexin cyclobrassinin by mimicry.
Journal of Agricultural and Food Chemistry 47, 1196±1202.
Pedras, M.S.C., Okanga, F.I., Zaharia, I.L., Khan, A.Q., 2000.
Phytoalexins from crucifers: synthesis, biosynthesis, and biotrans-
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Pedras, M.S.C., Sorensen, J.L., 1998. Phytoalexin accumulation and
production of antifungal compounds by the crucifer wasabi. Phy-
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