The first isocyanide of plant origin expands functional group diversity in cruciferous phytoalexins: Synthesis, structure and bioactivity of isocyalexin A

Article abstract of DOI:10.1039/c2ob25492a

Although isocyanides are not rare amongst terrestrial microbes and marine organisms, despite tens of thousands of natural products isolated from plants, isocyanides are still missing. Isocyalexin A is the first isocyanide of plant origin. Isocyalexin A was isolated from UV-irradiated rutabaga roots and shown to be a new cruciferous phytoalexin. Its chemical structure was proven by analysis of NMR spectroscopic data and chemical synthesis.

Full text of DOI:10.1039/c2ob25492a

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COMMUNICATION
The first isocyanide of plant origin expands functional group diversity
in cruciferous phytoalexins: synthesis, structure and bioactivity of
isocyalexin A†
M. Soledade C. Pedras* and Estifanos E. Yaya
Received 6th March 2012, Accepted 15th March 2012
DOI: 10.1039/c2ob25492a
methionine, valine, leucine, phenylalanine and tyrosine.5,9 By
Although isocyanides are not rare amongst terrestrial
contrast, it is relevant to note that the molecular diversity of cru-
ciferous phytoalexins, most of which are derived from (S)-trypto-
phan (Trp), is imparted by different functional groups, including
isothiocyanate (1, 1a), dithiocarbamate (2, 2a, 2b) and aldehyde
(3, 3a, 3b).7
microbes and marine organisms, despite tens of thousands of
natural products isolated from plants, isocyanides are still
missing. Isocyalexin A is the first isocyanide of plant origin.
Isocyalexin A was isolated from UV-irradiated rutabaga
roots and shown to be a new cruciferous phytoalexin. Its
chemical structure was proven by analysis of NMR spectro-
scopic data and chemical synthesis.
Naturally occurring isocyanides (ICs) comprise a diverse range
of metabolites produced by terrestrial microorganisms and
marine organisms.1–3 Remarkably, to date no metabolites con-
taining an IC (or isonitrile) group appear to have been reported
from plant sources. Unlike marine organisms such as sponges,
where both ICs and isothiocyanides (ITCs) are known to co-
occur (Fig. 1),2 ICs from the plant kingdom are unknown,‡ but
ITCs are common.4,5 On the other hand, cyanide (or nitrile, CN)
containing metabolites are common in plants and other terrestrial
organisms, as well as marine organisms.6 Natural ICs,2 ITCs4
and CNs6 are known to have biological roles related with
defence of the producing organisms.
The IC group has ambident character, as implied by its reson-
ance structures shown in Fig. 1, a versatility widely appreciated
in synthetic chemistry.10,11 Due to their unique reactivity, ICs are
very popular components of a huge number of organic reactions,
particularly since the Passerini and Ugi reactions were
discovered.12,13
The first naturally occurring aromatic ITC, rapalexin A (1),
was isolated from canola plants (Brassica rapa L.)4 and later on
shown to be produced by other cruciferous species subjected to
stress, including UV-irradiation and pathogen attack.7 These
plant defence compounds, known as phytoalexins, are essential
to protect plants, but are only biosynthesized in response to
stress. To date, there is no reasonable explanation for the absence
of ICs from plants, particularly considering that they are derived
from the metabolism of protein amino acids in terrestrial micro-
organisms such as fungi and bacteria.3,8 Metabolism of amino
acids in plants is known to afford numerous secondary metab-
olites with defensive roles.4,6,7 Specifically, ITCs from crucifer-
ous species are derived from various amino acids, including
During our previous work related with metabolic pathways of
phytoalexins in rutabaga roots,14,15 inconsistent production of an
indolyl-containing metabolite X was detected by HPLC using
diode array (DAD) and mass (MS) detectors. The UV spectrum
obtained for this component was not available in our UV spectral
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 9664772
†Electronic supplementary information (ESI) available: General exper-
imental and detailed experimental procedures for: antifungal bioassays
and syntheses and spectroscopic characterization of new compounds 4,
7, 9 and 13. See DOI: 10.1039/c2ob25492a
Fig. 1 Resonance structures of isocyanides (ICs), isothiocyanides
(ITCs) and cyanides (CNs).
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libraries, suggesting it was a new metabolite. Subsequently,
work to optimize elicitation conditions of rutabaga root slices
led to consistent production of this compound (Fig. 2).
(6) in EtOCHO and Et3N in a sealed vial at 60 °C did not
improve formamide yields.19
Next, it was reasoned that instead of dehydration of forma-
mide, reduction of the corresponding carbamate could poten-
tially yield an isocyanide. Thus, after hydrogenation of 5 in
ethanol, the resulting amine 6 was quenched at −15 °C with
phosgene to yield, after 5 h, ethyl carbamate 8 in low yield (ca.
32%). However, in our hands reduction of carbamate 8 to the
corresponding formamide under a variety of conditions (e.g.
Et3SiH, NaBH4, NABH4–NiCl2) could not be accomplished.
Thus, direct formylation of 3-aminoindole (6) was attempted by
adding a mixture of HCO2H–Ac2O to the hydrogenated reaction
mixture; under these conditions, (N′-formyl)-3-aminoindole (7)
was obtained albeit in poor yield (ca. 25%). Subsequently,
hydrogenation–formylation of 3-nitroindole (5) was carried out
in MeOH–HCO2H–AcOCHO; satisfactory yields of formamide
7 were obtained consistently. Dehydration of 7 provided a short
route to the first ever reported 3-indoleisonitrile (9, Scheme 1).
The hydrogenation of N-protected-3-nitroindoles has been pre-
viously reported in Ac2O,17 however this is the first time that a
non-protected nitroindole has been hydrogenated and formylated
in a one-pot procedure using Pd/C–H2.
After UV-irradiation of rutabaga root slices for 90 min (vs.
60 min) and further incubation, the amounts of metabolite X
increased dramatically (5–10-fold). The consistent and increased
production facilitated the isolation and chemical structure eluci-
dation of X. Rutabaga root slices UV-irradiated for 90 min and
incubated in the dark,14 were extracted and the organic extract
1
was fractionated to yield X in sufficient amount to obtain H
NMR, HRMS-EI and FTIR spectroscopic data. The molecular
formula suggested by HRMS (C10H8N2O) was consistent with
the 1H NMR spectrum¶ of 3-substituted-4-methoxyindole
(C9H8NO); the missing carbon and nitrogen atoms detected by
HRMS suggested the presence of either a CN or an IC group at
1
C-3. Since the H NMR spectral data did not match those of 4-
methoxyindole-3-carbonitrile,k a synthetic compound available
in our library, the structure of X was assigned as 4-methoxyin-
dole-3-isonitrile (4). A literature search revealed that neither this
compound nor any other substituted indole-3-isonitrile had been
previously reported. In addition, to date no ICs from plant origin
have been reported. The chemical structure of metabolite 4 was
confirmed by synthesis, as described below.
One of the most common syntheses of ICs involves formyla-
tion of the corresponding amines followed by dehydration.16
3-Aminoindoles are unstable compounds that readily oxidise to
undetermined products upon exposure to oxygen;4,17 hence in
situ transformation of 3-aminoindoles is necessary to obtain
reasonable yields of products. Initially, a route to indole-3-isoni-
triles was investigated using 3-nitroindole (5), a substrate simpler
to make than 4-methoxy-3-aminoindole (11).18 Following hydro-
genation of 3-nitroindole (5) in ethanol,4 the resulting amine was
immediately quenched with EtOCHO, without filtering off the
catalyst. After 24 h only a very small amount of the correspond-
ing formamide 7 was isolated (ca. 4%). Heating 3-aminoindole
Subsequently, to obtain metabolite 4, it was necessary to
prepare the starting material 4-methoxy-3-nitroindole (10).
This compound was previously synthesised by nitration of
4-methoxyindole in 30% yield.4 Thallation–iodination of 3-
Fig. 2 HPLC-DAD chromatograms (C-18 reverse phase column,
H2O–MeOH elution, 50 : 50 to 0 : 100 linear gradient for 25 min, detec-
tion at 220 nm) of rutabaga root extracts: UV-irradiated for 90 min (isa =
isalexin (14), ind-CN = indolyl-3-acetonitrile, 4-MeO–CHO = 4-meth-
oxyindolecarboxaldehyde, spiro = spirobrassinin, caul C = caulilexin C,
brass = brassinin, 4-MeO–brass = 4-methoxybrassinin, ruta = rutalexin,
1-MeO–brass = 1-methoxybrassinin, rap A = rapalexin A (1), cycl =
cyclobrassinin§) and control (no irradiation).
Scheme 1 Synthesis of isocyalexin A (4) and rapalexin A (1).
Reagents and conditions: (i) H2–Pd/C, MeOH, HCO2COCH3, HCO2H;
7, 64%, 13, 66%; (ii) POCl3, THF, Et3N; 4, 41%, 9, 54%; (iii) TFA,
Tl(OAc)3; I2, CuI, DMF, NaOMe, MeOH; 10, 64%; (iv) H2–Pd/C,
EtOH; (v) Cl2CS; 12, 51%.
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carbonylindoles is known to yield substitution only at C-4.20,21
Thus, it was surmised that a nitro group at C-3 of indole had the
potential to afford similar regiocontrol, by directing substitution
to C-4. To test this hypothesis, 4-methoxylation of 3-nitroindole
(5) was attempted using Tl(OAc)3 and CuI–I2. We were
delighted to find that this reaction worked well, and that the nitro
group could direct thallation–iodination to the C-4 of indole.
This is the first time that a nitro group at C-3 of indole has been
reported to direct 4-substitution. Next, addition of MeONa to
4-iodo-3-nitroindole afforded the desired 4-methoxy-3-nitroin-
dole (10) in reasonable yield. One-pot hydrogenation and formy-
lation of 10, followed by dehydration, as reported for isocyanide
9, led to metabolite 4 in 27% overall yield. This route from
3-nitroindole (5) provided a convenient synthesis of 4 involving
4-steps, 3-pots and two chromatographic separations (Scheme 1).
Having sufficient amounts of ICs 4 and 9 in hand facilitated
spectroscopic characterization by NMR (1H and 13C NMR,
HMQC and HMBC); interestingly, spin–spin coupling between
results indicated that 4 is a novel phytoalexin produced by
stressed rutabaga tissues, which may have a significant role in
protecting rutabaga against root pathogens. Importantly, this
phytoalexin was also induced by R. solani incubation with ruta-
baga roots. Hence, this metabolite was named isocyalexin A (4).
A puzzling question regarding the biosynthetic origin of iso-
cyalexin A (4) emerged. Since this is the first isocyanide of plant
origin, there is no precedence for analogy reasoning.24 Nonethe-
less, the close structural similarity between isocyalexin A (4) and
rapalexin A (1) hinted that 4 might derive from 1 by enzymatic
desulfurization. To verify this possibility, [2H3]rapalexin A (12)
was synthesised and administered to UV-irradiated rutabaga
roots, similar to experiments described previously.14 After incu-
bation and extraction, analysis of the extracts by HPLC-MS-ESI
indicated a substantial amount of deuterium in isalexin (14a,
60% 8) relative to the total amount (i.e. deuterated 14a plus
natural abundance 14), while no deuterium incorporation into
isocyalexin A (4) was detected (Scheme 2). The high deuterium
incorporation suggests that rapalexin A (1) is a close precursor of
isalexin (14), while the lack of deuterium incorporation into iso-
cyalexin A (4) suggests that 1 is not a precursor of 4. Nonethe-
less, this hypothesis cannot be completely ruled out. It is worth
stressing that rapalexin A (1) is the first precursor of isalexin (14)
reported to date. Transformation of 1 to 14 suggests that this bio-
synthetic step is likely to involve a single oxidase, placing isa-
lexin as a potential piece at the border of this biosynthetic
puzzle.
13C-3 (δC 103.2 in 4 and 104.3 ppm in 9) and 14N (t, JC–N
=
ca. 16 Hz) of the IC group was observed. To the best of our
knowledge, a similar coupling has only been reported for brasili-
dene A, an indolyl-conjugated alkenyl isocyanide.22
To establish the potential ecological role of metabolite 4 in
rutabaga roots and correlate functional group with antifungal
activity, mycelial growth inhibition assays23 were performed
testing compounds 4, 7, 9 and 13 against four fungal species of
economically important cruciferous pathogens (Table 1).
Among the four compounds tested, metabolites 4 and 9 dis-
played the highest antifungal activity against the root pathogen
Rhizoctonia solani and the lowest activity against Leptosphaeria
maculans. Because 9 displayed almost no inhibitory activity
against L. maculans, it is concluded that the presence of the
4-MeO substituent has a stronger effect on this pathogen. These
Alternatively, a common precursor transformed by different
enzymes to isocyalexin A (4) and rapalexin A (1) could be pro-
posed (Scheme 3). Hence, by analogy with the biosynthesis of
other ITCs,9 the as yet unknown 4-methoxyindole glucosinolate
(16) could be the precursor common to both isocyalexin A (4)
and rapalexin A (1). The formation of ITCs is mediated by thio-
glucosidases** and is followed by a spontaneous Lossen-type
rearrangement. That is, rapalexin A (1) and isocyalexin A (4)
could result from different enzymatic transformations of gluco-
sinolate 16. In this context, it is of interest to point out that the
thermal rearrangement of isocyanides to cyanides is well
known,25 but equivalent enzymatic reactions do not appear to
have been reported. Furthermore, a Beckman-type rearrange-
ment26 of the aldoxime derivative 15 to the corresponding iso-
cyanide 4 could be considered also a possibility.
Table 1 Inhibitory activity of isocyalexin A (4) and compounds 7, 9
and 13 against myceliaa of plant pathogenic fungi Alternaria
brassicicola, Leptosphaeria maculans, Rhizoctonia solani and
Sclerotinia sclerotiorum23
Compoundsb
A. brassicicola L. maculans R. solani S. sclerotiorum
Isocyalexin A (4)
5.0 × 10−4 M 47
2.0 × 10−4 M 14
1.0 × 10−4 M
0
2
0
29
9
8
2
0
2
100
4
2
65
40
31
2
2
5
These hypotheses, as depicted in Scheme 3, need substantial
experimental work before any sort of confirmation can be
obtained. Nonetheless, such work is essential to design crops
producing higher levels of phytoalexins. Increased concen-
trations of these metabolites in infected plants are expected to
provide superior protection against plant pathogens.
51
32
Indole-3-isonitrile (9)
5.0 × 10−4 M 42
2.0 × 10−4 M 27
1.0 × 10−4 M 17
4
4
4
5
0
0
0
100
4
4
60
31
17
2
2
0
51
31
4-Methoxy−in4dole-3-formamide (13)
5.0 × 10 M 21
2
4
0
27
7
0
0
2
29
12
6
4
2
2
32
25
17
2
4
0
Finally, the discovery of the first IC of plant origin attracts
speculation concerning the absence of such metabolites from the
2.0 × 10−4 M
9
1.0 × 10−4 M
2
Indole-3-formamide (7)
5.0 × 10−4 M 42
2.0 × 10−4 M 27
1.0 × 10−4 M 17
4
4
4
31
16
5
3
2
2
15
4
0
0
25
19
15
0
2
2
a Percentage of inhibition = 100 – [(growth on medium containing
compound/growth on control medium) × 100)] standard deviation.
Results are the averages
standard deviations of independent
experiments conducted in triplicate. b Compounds were dissolved in
Scheme 2 Biotransformation of [2H3]rapalexin A (12) to [2H3]isalexin
(14a) in UV-irradiated rutabaga roots.
DMSO and added to potato dextrose agar medium (see ESI†).
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Notes and references
‡A report of constituents of floral scents of Saponaria officinalis
suggested that benzylisocyanide was detected by GC-MS analysis;
however, it was not established whether this volatile is an air pollutant or
a plant metabolite.27
§Chemical structures of compounds are provided in the ESI.†
¶Isocyalexin A (4): HPLC: tR = 11.0 min. UV (CH3CN–H2O, HPLC)
λmax (nm): 220, 269. FTIR (KBr, cm−1) νmax: 3305, 3129, 2134, 1592,
1514, 1447, 1366, 1336, 1272, 1079. 1H NMR (500 MHz, CD3OD) δ
7.45 (1H, s), 7.10 (1H, dd, J = 8, 8 Hz), 6.97 (1H, d, J = 8 Hz), 6.58
(1H, d, 8 Hz), 3.91 (3H, s). 13C NMR δ (125.8 MHz, CD3OD) δ 164.1,
154.8, 137.3, 125.5, 124.3, 113.9, 106.4, 103.2, (JC-3-N = 15 Hz), 102.1,
56.0. HREI-MS m/z 172.0633 (M+), calcd for C10H8N2O 172.0632
(100%), 157.04 (69%), 129.04 (69%). HPLC-MS-ESI m/z [M − H]−,
171.1 (11%), 156.0 (100%).
k4-Methoxyindole-3-carbonitrile does not appear to have been
described previously, synthesis and spectroscopic data are provided in
ESI.†
**Thioglucosyl hydrolases (EC 3.2.1.147) are commonly known as
myrosinases.
1 M. S. Edenborough and R. B. Herbert, Nat. Prod. Rep., 1988, 5, 229.
2 M. J. Garson and J. M. Simpson, Nat. Prod. Rep., 2004, 21, 164.
3 P. J. Sheuer, Acc. Chem. Res., 1992, 25, 433.
4 M. S. C. Pedras, Q. A. Zheng and R. S. Gadagi, Chem. Commun., 2007,
368–370.
5 J. W. Fahey, A. T. Zalcmann and P. Talalay, Phytochemistry, 2001, 56,
5–51.
6 F. F. Fleming, Nat. Prod. Rep., 1999, 16, 597–606.
7 M. S. C. Pedras, E. E. Yaya and E. Glawischnig, Nat. Prod. Rep., 2011,
28, 1381–1405.
8 S. F. Brady, L. Simmons, J. H. Kim and E. W. Schmidt, Nat. Prod. Rep.,
2009, 26, 1488–1503.
9 A. M. Bones and J. T. Rossiter, Phytochemistry, 2006, 67, 1053–1067.
10 J. Campo, M. Garcia-Valverde, S. Marcaccini, M. J. Rojo and T. Torroba,
Org. Biomol. Chem., 2006, 4, 757–765.
11 A. de Meijere and A. V. Lygin, Angew. Chem., Int. Ed., 2010, 49, 9094–
9124.
Scheme 3 Proposed biosynthetic intermediates of isocyalexin A (4)
and rapalexin A (1).
12 A. Dömling and I. Ugi, Angew. Chem., Int. Ed., 2000, 39, 3168–3210.
13 M. V. Barybin, Coord. Chem. Rev., 2010, 254, 1240–1252.
14 M. S. C. Pedras and D. P. O. Okinyo, Org. Biomol. Chem., 2008, 6, 51–54.
15 M. S. C. Pedras and D. P. O. Okinyo, Phytochemistry, 2009, 70, 1129–
1138.
16 H. M. Walborsky and G. E. Niznik, J. Org. Chem., 1972, 37, 187–190.
17 S. Roy, S. Roy and G. W. Gribble, Tetrahedron Lett., 2008, 49, 1531–
1533.
18 R. H. Bahekar, M. R. Jain, A. Goel, D. N. Patel, V. M. Prajapati, A.
A. Gupta, P. A. Jadav and P. R. Patel, Bioorg. Med. Chem., 2007, 15,
3248–3265.
19 K. P. Dhake, P. J. Tambade, R. S. Singhal and B. M. Bhanage, Green
Chem. Lett. Rev., 2011, 4, 151–157.
20 R A. Hollins, L. A. Colnago, V. M. Salim and M. C. Seidl, J. Heterocycl.
Chem., 1979, 16, 993–996.
21 M. Somei, F. Yamada, M. Kunimoto and C. Kaneko, Heterocycles, 1984,
22, 797–801.
22 J. Kobayashi, M. Tsuda, A. Nemoto, Y. Tanaka, K. Yazawa and
Y. Mikami, J. Nat. Prod., 1997, 60, 719–720
plant kingdom. To a greater extent, this is a more pertinent ques-
tion in crucifers, since the corresponding ITCs and CNs are
abundant. Toward this end, future work should include syntheses
of IC libraries, the counterparts of naturally occurring ITCs, to
establish their occurrence and function in crucifers. In addition,
it will be of interest to determine if isocyalexin A (4) is produced
in other Brassica species and its potential synergistic activity in
naturally occurring blends of cruciferous phytoalexins.
Considering the novel chemical structure and its selective anti-
fungal activity, phytoalexin 4 is a new but prominent jigsaw
piece yet to find a position in the biosynthetic puzzle of crucifer-
ous phytoalexins. Unquestionably, the importance of cruciferous
phytoalexins would warrant further chemical and biochemical
work to solve the puzzle.
Acknowledgements
.
23 M. S. C. Pedras, Q. A. Zheng and M. G. Sarwar, Org. Biomol. Chem.,
2007, 5, 1167–1169.
24 M. S. C. Pedras, E. E. Yaya and S. Hossain, Org. Biomol. Chem., 2010,
We thank Q. A. Zheng for synthesis of 4-methoxyindole-3-
nitrile, K. Thoms for HRMS-EI data and K. Brown from the
Department of Chemistry for NMR data. Financial support for
the authors’ work was obtained from the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant to
M. S. C. P.), Canada Foundation for Innovation, the Saskatche-
wan Government, and the University of Saskatchewan (scholar-
ship to E. E. Y.).
8, 5150–5158.
25 G. W. Van Dine and R. Hoffmann, J. Am. Chem. Soc., 1968, 90, 3227–
3232.
26 T. Mukaiyama, K. Tonooka and K. Inoue, J. Org. Chem., 1961, 26,
2202–2206.
27 A. Jürgens, T. Witt and G. Gottsberger, Biochem. Syst. Ecol., 2003, 31,
345–357.
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This journal is © The Royal Society of Chemistry 2012