The first naturally occurring aromatic isothiocyanates, rapalexins A and B, are cruciferous phytoalexins

Article abstract of DOI:10.1039/b615424g

The discovery of the first naturally occurring aromatic isothiocyanates, indole-3-isothiocyanates, their first synthesis, antimicrobial activity and proposed biogenetic origin in canola plants are reported. The Royal Society of Chemistry.

Full text of DOI:10.1039/b615424g

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The first naturally occurring aromatic isothiocyanates, rapalexins A
and B, are cruciferous phytoalexins{
M. Soledade C. Pedras,* Qing-An Zheng and Ravi S. Gadagi
Received (in Cambridge, MA, USA) 26th October 2006, Accepted 14th November 2006
First published as an Advance Article on the web 29th November 2006
DOI: 10.1039/b615424g
The discovery of the first naturally occurring aromatic
isothiocyanates, indole-3-isothiocyanates, their first synthesis,
antimicrobial activity and proposed biogenetic origin in canola
plants are reported.
Isothiocyanates (ITCs) are a remarkable class of natural products
produced by plants of various families, including Brassicaceae
Biotrophic fungi and oomycetes are capable of colonizing plant
(syn. Cruciferae). Plant ITCs are enzymatic products resulting
tissues in ways that prevent immediate recognition by the host,11
from hydrolysis of glucosinolates by plant enzymes trivially known
which favour the invaders by avoiding the counter-chemical
as myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1,
warfare. To understand the interaction of crucifers with such
Scheme 1).1 ITCs are known to have crucial ecological roles in
‘‘disguised’’ microbial pathogens, we have analysed canola plants
protecting plants against various pests, including insects and
(Brassica rapa) infected with the white rust oomycete (Albugo
microbial systems.2 Hence, ITCs are part of a group of constitutive
candida, Pers. ex Chev., Kuntze). Gratifyingly, from infected leaf
plant chemical defences known as phytoanticipins. In addition to
tissues of canola, two unique indole-3-isothiocyanates were
phytoanticipins, plants under stress biosynthesise de novo metabo-
isolated and demonstrated to be phytoalexins. The strong
lites known as phytoalexins, which have selective antimicrobial
inhibitory activity of these isothiocyanates suggests a defensive
activity. Both phytoanticipins and phytoalexins are involved in the
role against the white rust oomycete, an economically important
chemical warfare between plants and their invaders.3
pathogen of many crucifers.
The phytoalexin arsenal of crucifers includes an unusual range
Leaves of canola were inoculated with A. candida for various
of functional groups and indolyl structures that have sulfur and
periods, as described in ESI. HPLC chromatograms of extracts of
nitrogen, in addition to the most common organic elements (C, H,
infected leaves indicated the presence of several peaks not present
O).4 The phytoalexins brassinin (1), 1-methoxybrassinin (2) and
in control leaves (non-inoculated). Several of these peaks
cyclobrassinin (3) were the first members of this unique group,5
corresponded to known phytoalexins; however, two of the peaks
whereas erucalexin (4)6 and caulilexin A (5)7 were isolated very
found no match in our libraries (DAD and MS).12 To obtain
recently. Notwithstanding the large number of crucifers surveyed
sufficient amounts of these unidentified compounds, additional
for ITC production, to date no cruciferous phytoalexins containing
plants were infected and the infected leaves were incubated,
an ITC group have been reported, nor have aromatic ITCs.2
excised, frozen in liquid nitrogen, and extracted with methanol, cf.
Furthermore, the significant ITC contents of cruciferous
ESI. The organic residue was separated by multiple chromato-
vegetables appear to make a remarkable contribution to their
graphy to yield the two unidentified compounds, A (0.5 mg) and B
highly desirable anti-carcinogenic activity.8 In fact, mechanisms
(1.4 mg). The HREI-MS of compound A indicated the molecular
through which ITCs and indoles may protect mammals against
formula C10H8N2OS; the NMR spectroscopic data{ indicated a
disubstituted indole at C-3 and C-4 or at C-3 and C-7, an
diverse cancers have been identified (e.g. induction of phase II
enzymes such as glutathione transferases, NAD(P)H:quinone
reductase, epoxide hydrolase).9,10
exchangeable proton attributable to N-1, and
a methoxy
substituent, accounting for C9H8NO. On biogenetic grounds4
and HMBC spectroscopic data,{ the methoxy substituent was
placed at C-4. The three remaining atoms (CNS) required to fulfil
the molecular formula were located at C-3; two arrangements
could be suggested for the substituent at C-3, isothiocyanate
(–NLCLS) or thiocyanate (–S–CMN). The very strong absorption
at ca. 2100 cm21 in the FTIR spectrum suggested an isothiocya-
nate group. Based on this evidence, compound A was deduced to
be 4-methoxyindole-3-isothiocyanate (9).
Scheme 1 Enzymatic formation of isothiocyanates (ITCs) from gluco-
sinolates.1
The HREI-MS spectroscopic data of compound B indicated the
molecular formula C10H8N2O2S, i.e. an additional oxygen relative
to compound A (9). The NMR spectra{ were consistent with the
molecular formula and, relative to compound A, displayed an
additional exchangeable proton (dH 6.53) and an identical number
Department of Chemistry, University of Saskatchewan, 110 Science
Place, Saskatoon, SK, S7N 5C9, Canada. E-mail: s.pedras@usask.ca;
Fax: 1-306-9664730; Tel: 1-306-966-4772
{ Electronic supplementary information (ESI) available: Experimental
details, synthesis and spectroscopic data of all new compounds 7–10 and
12–18. See DOI: 10.1039/b615424g
368 | Chem. Commun., 2007, 368–370
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of carbons (with a lower field carbon at dC 143.2). The strong
FTIR absorption characteristic of isothiocyanates (2139 cm21
(lower H2 pressures did not reduce the N–Bn bond). As in the case
of isothiocyanate 9, reaction of amine 17 with thiophosgene
yielded 5-hydroxy-4-methoxyindole-3-isothiocyanate (18). The
spectroscopic and chromatographic properties of synthetic
5-hydroxy-4-methoxyindole-3-isothiocyanate (18) were identical
in all respects to those of compound B isolated from canola. To
prepare thiocyanate 14, reduction of dinitrostyrene 12 with Pd/C in
methanol and AcOH afforded indole 13 in 29% yield (Scheme 3).19
Treatment of 5-hydroxy-4-methoxyindole (13) with ammonium
thiocyanate afforded 5-hydroxy-4-methoxyindole-3-thiocyanate
(14) in 49% yield.
)
was observed as well. Thus, compound B was deduced to be
5-hydroxy-4-methoxyindole-3-isothiocyanate (18). The structures
of compounds A and B were confirmed by synthesis to be
isothiocyanates 9 and 18, respectively, as described below. The
corresponding thiocyanates 10 and 14 were synthesised as well, to
compare the antimicrobial activity of both functional groups.
Facile syntheses of aromatic isothiocyanates employ treatment
of the corresponding aromatic amines with thiophosgene.13
However, because the required amines (4-methoxyindole-3-amine
(8) and 5-hydroxy-4-methoxyindole-3-amine (17)) were unknown,
procedures for their preparation were developed. The preparation
of indole-3-amines by reduction of 3-nitroindoles14 appeared an
attractive strategy, although such nitrations do not appear to have
been reported for 4-methoxyindoles. Thus, 4-methoxyindole15 was
nitrated using silver nitrate and benzoyl chloride in 30% yield,
followed by hydrogenation to yield 4-methoxyindole-3-amine (8)
in modest yield. In attempts to improve the yield of this
conversion, the indole nitrogen was protected with benzyl and
tosyl groups.16 Whereas the nitration of 1-benzyl-4-methoxyindole
proceeded in yields similar to those of non-protected indole 6,
nitration of 4-methoxy-1-tosylindole yielded the 7-nitro derivative
almost exclusively. In addition, changing the nitration reagent to
HNO3–Ac2O yielded 3-nitroindole 7 in substantially lower yield
(11%). Finally, reaction of amine 8 with thiophosgene13 in
CH2Cl2–water yielded 4-methoxyindole-3-isothiocyanate (9)
(Scheme 2). In addition, reaction of 4-methoxyindole with
iodine–ammonium thiocyanate yielded 4-methoxyindole-3-thio-
cyanate (10, Scheme 2).17 The spectroscopic and chromatographic
properties of synthetic 4-methoxyindole-3-isothiocyanate (9) were
identical in all respects to those of compound A isolated from
canola leaves.
Finally, with compounds A (9) and B (18) available in sufficient
amounts, their effect on the white rust pathogen was determined.
A bioassay using impregnated cellulose membranes20 established
that compound A (9) displayed higher inhibitory effect on
zoospore germination than compound B (18), and thiocyanates
10 and 14. Compound A (9) caused complete germination
inhibition at 5.0 6 1026 M, whereas 10 and 18 caused complete
inhibition at ten times higher concentration (5.0 6 1025 M),
and 14 caused only 75% inhibition at similar concentration (5.0 6
1025 M).
Compounds A (9) and B (18) are plant metabolites biosynthe-
sised in response to biotic (A. candida) and abiotic stress (UV),
display antimicrobial activity, and are not detectable in non-
stressed plants. Consequently, these metabolites are new phytoa-
lexins for which we propose the names rapalexin A (9) and
rapalexin B (18).
As shown in Table 1, rapalexin A (9), the most potent of both
phytoalexins, was detected only eight days after inoculation
whereas rapalexin B (18) was detected five days after inoculation
in substantially larger amounts.§ Because rapalexins A (9) and B
(18) are the first crucifer phytoalexins reported to display strong
activity against a biotroph, particularly rapalexin A, their
biogenetic origin and pathway are of great importance.
Interestingly, the direct attachment of the isothiocyanate functional
group to C-3 of indole has not been observed in naturally
To prepare 5-hydroxy-4-methoxyindole-3-amine (17), first the
corresponding indole (5-benzyloxy-4-methoxyindole) was prepared
from 3-benzyloxy-2-methoxy-6-nitrobenzaldehyde (11) by conden-
sation with nitromethane to yield dinitrostyrene 12, followed by
reduction with Fe and silica gel in acetic acid, in 60% yield over
three steps.18 Nitration of 5-benzyloxy-4-methoxyindole using
benzoyl nitrate to produce 5-benzyloxy-4-methoxy-3-nitroindole
occurred in very poor yield (5%). However, protection of N-1 with
a benzyl group yielded the desirable 3-nitroindole 16 in a more
acceptable yield (20%). Reduction of 3-nitroindole 16 (H2, 40 psi)
using Pd/C, AcOH, yielded the corresponding indole-3-amine 17
Scheme 3 Synthesis of rapalexin B (18) and thiocyanate 14. Reagents
and conditions: i, CH3NO2, 18-crown-6, KF, 4-methylmorpholine, r.t.; ii,
NaOAc, Ac2O, 60 uC, 88% over two steps; iii, Fe, SiO2, toluene–AcOH,
90 uC, 75%; iv, NaH, THF, PhCH2Br, 93%; v, AgNO3, benzoyl chloride,
CH3CN, 20%; vi, Pd/C, AcOH, H2/40 psi; vii, CH2Cl2, CaCO3, CSCl2,
30% over two steps; viii, Pd/C, H2, MeOH, AcOH, 29%; ix, NH4SCN, I2,
MeOH, 0 uC, 49%.
Scheme 2 Synthesis of rapalexin A (9) and thiocyanate 10. Reagents and
conditions: i, AgNO3, benzoyl chloride, CH3CN, 30%; ii, Pd/C, AcOH, H2;
iii, CH2Cl2, CaCO3, CSCl2, 20% over two steps; iv, NH4SCN, I2, MeOH,
r.t., 75%.
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Table 1 Production of rapalexins A (9) and B (18) in leaves of canola
(Brassica rapa) infected with Albugo candida
Notes and references
{ NMR spectroscopic data for rapalexin A (9): 1H-NMR (500.1 MHz,
CDCl3): d 7.98 (brs, N–H), 7.18 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 2.2 Hz,
1H), 6.96 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 7.9 Hz, 1H), 4.00 (s, 3H). 13C-
NMR (125.8 MHz, CDCl3): d 154.3 (s), 136.9 (–NLCLS), 136.1 (s), 125.3
(d), 118.5 (d), 114.4 (s), 108.1 (s), 105.1 (d), 101.2 (d), 55.7 (q). For
additional data see ESI. NMR spectroscopic data for rapalexin B (18): 1H-
NMR (500.1 MHz, CD3CN): d 9.31 (brs, 1H), 7.34 (d, J = 2.9 Hz, 1H),
7.09 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 3.94 (s, 3H).
1H-NMR (500.1 MHz, CD3OD): d 7.29 (s, 1H), 7.01 (d, J = 8.7 Hz, 1H),
6.82 (d, J = 8.7 Hz, 1H), 3.97 (s, 3H). 13C-NMR (CD3OD): d 143.2 (s),
138.7 (s), 133.7 (–NLCLS), 131.1 (s), 121.7 (d), 117.8 (s), 114.8 (d), 108.3 (d),
104.5 (s), 61.2 (q). For additional data see ESI.
Rapalexin A
(9) (nmol g21
fresh leaves)
Rapalexin B
(18) (nmol g21
fresh leaves)
Days after
inoculation
5
6
7
8
9
not detected
not detected
not detected
0.4–0.6
0.7–1.1
0.7–0.9
2.3–3.3
4.1–8.1
4.3–8.3
5.3–14.7
3.9–9.7
7.5–9.1
10
a
Amounts of rapalexins A and B in leaves were determined by
HPLC using calibration curves; the correlation coefficients of
phytoalexin calibration curves were ¢ 0.9998.
§ The production of rapalexins A and B is localised and restricted to areas
of infected tissues where the concentration can be around 0.1 mM (like any
other phytoalexins).
Scheme 4 Proposed biosynthetic precursors 19 and 20 of rapalexins A
(9) and B (18), respectively.
1 For a recent review on glucosinolate decomposition see, A. M. Bones
and J. T. Rossiter, Phytochemistry, 2006, 67, 1053–1067.
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occurring isothiocyanates.2 Nevertheless, considering that all
crucifer isothiocyanates reported so far derive from the corre-
sponding glucosinolates,2 rapalexins A and B may derive from yet
to be discovered indolyl glucosinolates or from closely related
indolyl thiohydroximates such as 19 and 20, via a Lossen type
rearrangement,21 as depicted in Scheme 4. Clearly, this hypothesis
requires further work with isotopically labelled precursors. That is,
the discovery of these unique isothiocyanates 9 and 18 reveals
additional pathways that need to be integrated in the biosynthetic
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8 A. Lynn, A. Collins, Z. Fuller, K. Hillman and B. Ratcliffe, Proc. Nutr.
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isolation, purification and analysis, in contrast to indolyl-3-methyl
isothiocyanates such as 21 and 22.23 It would be of interest to
determine the potential anti-carcinogenic activity of rapalexins in
mammalian systems. Further work to improve the syntheses and
establish the biosynthetic precursors of rapalexins A and B is in
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9 A. Wiseman, Trends Food Sci. Technol., 2005, 16, 215–216.
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23 (a) Both compounds 21 and 22 remain to be isolated or prepared. See,
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Financial support from the Natural Sciences and Engineering
Research Council of Canada (Accelerator Grants for Exceptional
New Opportunities to M.S.C.P.) is gratefully acknowledged. We
thank S. R. Rimmer from Agriculture and Agri Food Canada,
Saskatoon Research Centre for providing Albugo candida. We
acknowledge the technical assistance of K. Thoms and K. Brown,
Department of Chemistry, in MS and NMR determinations,
respectively.
370 | Chem. Commun., 2007, 368–370
This journal is ß The Royal Society of Chemistry 2007