Remarkable incorporation of the first sulfur containing indole derivative: Another piece in the biosynthetic puzzle of crucifer phytoalexins

Article abstract of DOI:10.1039/b714743k

The first sulfur labelled compound, [²H₄, ³⁴S]indolyl-3-acetothiohydroxamic acid, is incorporated into the phytoalexins cyclobrassinin and spirobrassinin and the indole glucosinolate glucobrassicin, indicating that both bi

Full text of DOI:10.1039/b714743k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Remarkable incorporation of the first sulfur containing indole derivative:
another piece in the biosynthetic puzzle of crucifer phytoalexins†,‡
M. Soledade C. Pedras* and Denis P. O. Okinyo
Received 25th September 2007, Accepted 25th October 2007
First published as an Advance Article on the web 2nd November 2007
DOI: 10.1039/b714743k
2
34
The first sulfur labelled compound, [ H
4
, S]indolyl-3-
due to their own importance, has been investigated for more than
four decades. Hence, most of the genes and intermediates of
10
acetothiohydroxamic acid, is incorporated into the phy-
toalexins cyclobrassinin and spirobrassinin and the indole
glucosinolate glucobrassicin, indicating that both biosyn-
thetic pathways are closely related.
the pathway to glucobrassicin (3) are known. On the other hand,
2
no genes of the brassinin branch have been reported. However,
camalexin (7) is produced in Arabidopsis thaliana, a wild species
where a strong effort in functional genomics is concentrated. Thus,
9
some intermediates and genes of the camalexin branch are known.
Plants have a complex range of defence responses that con-
tribute to basal and specific disease resistance to many microbial
pathogens. Phytoalexins are chemical defences produced de novo
by plants to ward off pathogens and other stresses, including
1
intense heat and UV irradiation. Because crucifers are widely
cultivated crops and include valuable oilseeds, vegetables, and
condiments, their resistance to pests and diseases is an ongoing
concern. Considering the negative impact of pesticides and
fungicides on the environment, it is crucial to devise safer strategies
to protect cropping systems. Evidently, safer and sustainable
strategies must derive from a better molecular understanding of
plant defences and their metabolic pathways. Toward this end,
the biosynthetic pathways of crucifer phytoalexins and related
2
metabolites are of great importance, albeit an ongoing challenge.
3,4
Specifically, the variety of phytoalexin structures and some
unique metabolic reactions make mapping out these pathways
5
Scheme 1 Biosynthetic precursors of (i) indolyl glucosinolates 3 and 4,
(
(
ii) phytoalexins brassinin (5), methoxybrassinin (6), and (iii) camalexin
7).
very difficult. For example, the similarity between the chemical
structures of brassinins 5 and 6 and indolyl glucosinolates 3
and 4 led to an earlier consensus that glucobrassicin (3) was a
precursor of brassinin (5). Thus, it was rather surprising to find
that glucobrassicin was not incorporated into brassinin. This
lack of incorporation suggested to us that if not glucobrassicin
Earlier work showed that the sulfur of glucobrassicin (3) was
6
derived from cysteine. Furthermore, it was suggested that the C–S
lyase that cleaves S-cysteinyl thiohydroximate is tightly coupled
to the S-donating enzyme, and that the product of the C–S
(
3), one of its precursors would be the branch point between the
10
11
brassinin and glucobrassicin pathways. Subsequently, indolyl-3-
lyase is a thiohydroximic acid. As well, Monde et al. reported
that the sulfur of the thiocarbonyl group of brassinin (5) was
derived from cysteine and the SMe from methionine. Altogether
the current information suggests to us that there should be
a sulfur containing metabolite common to both brassinin (5)
and glucobrassicin (3). Based on this reasoning, we synthesised
acetaldoxime (1) was shown to be a precursor of brassinin (5),
6
methoxybrassinin (6) and related phytoalexins.
The biosynthetic pathway of crucifer phytoalexins and that of
indolyl glucosinolates starts with the conversion of tryptophan
to indolyl-3-acetaldoxime (1) and appears to diverge into various
branches, three of which are pertinent to consider here: (i) indolyl
2
34
H and S isotopically labelled indolyl-3-acetothiohydroxamic
7
6,8
glucosinolates (e.g. 3, 4), (ii) brassinins (e.g. 5, 6)
(
and
acid (10a) and administered it to UV-irradiated rutabaga tubers
(Brassica napus L. ssp. rapifera). Remarkably, incorporation of
this labelled compound into cyclobrassinin (14), spirobrassinin
(15) and glucobrassicin (3) was established for the first time. Here
we describe details of this biosynthetic investigation as well as the
synthesis of the first doubly labelled indolyl thiohydroxamic acid.
A detailed biosynthetic pathway for brassinin (5) is proposed that
integrates all current knowledge.
Because indolyl-3-acetothiohydroxamic acid (10) had not been
characterised,§ we synthesised first non-labelled material to de-
termine its chemical stability in aqueous methanol solutions
to use in plant feeding experiments. Thus, non-labelled 10 was
9
iii) camalexin (7, Scheme 1). The biosynthesis of glucosinolates,
Department of Chemistry, University of Saskatchewan, Saskatoon, SK, S7N
5
9
†
C9, Canada. E-mail: s.pedras@usask.ca; Fax: 1 306 966 4730; Tel: 1 306
66 4772
Support for the authors work was obtained from the Natural Sciences
ꢀ
and Engineering Research Council of Canada (Discovery Research Grant
to M.S.C.P.) and the University of Saskatchewan (graduate assistantship
to D.P.O.O.). We acknowledge the technical assistance of K. Thoms and
K. Brown, Department of Chemistry, in MS and NMR determinations,
respectively.
‡
Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b714743k
This journal is © The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 51–54 | 51

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◦
Scheme 2 Synthesis of indolyl-3-acetothiohydroxamic acid (10). Reagents and conditions: (i) CH
i-PrOH, SiO , CHCl (71%); (iii) KH, (Me Si) S, THF (74%).
3 2 4 4
NO , NH OAc, 128–130 C (98%); (ii) NaBH ,
2
3
3
2
ꢀ
ꢀ
ꢀ
ꢀ
2
34
prepared from indole-3-carboxaldehyde (8) in three steps. Indole-
4
Table 1 Metabolism of [4 ,5 ,6 ,7 - H ]indolyl-3-[ S]acetothiohydroxa-
ꢀ
mic acid (10a) in elicited tubers of rutabaga (Brassica napus ssp. rapifera)
3
-carboxaldehyde (8) was transformed to 3-(2 -nitrovinyl)-indole
12
upon treatment with ammonium acetate in nitromethane. Sub-
sequent sodium borohydride reduction yielded 3-(2 -nitroethyl)-
indole (9) which upon treatment with potassium hydride, fol-
lowed by hexamethyldisilathiane yielded 10 in ca. 51% overall
yield (Scheme 2). When sodium hydride was used instead of
potassium hydride, only indolyl-3-acetonitrile (18) and indolyl-3-
acetaldoxime (1) were obtained in a 1 : 1 ratio. The C NMR
data of indolyl-3-acetothiohydroxamic acid (10) indicated that
structure 10 is the most likely tautomer present in solution, since
the chemical shift of C-1 is 189.0 ppm. This conclusion is in
Incorporation (%
ꢀ
2
34
a
Labelled product
of H and S)
13
3
4
4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
b
14
S-[4 ,5 ,6 ,7 - H ]Cyclobrassinin (14a)
7%
7%
10%
2%
4
3
2
b
S-[4 ,5 ,6 ,7 - H
4
]Spirobrassinin (15a)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
c
[
4 ,5 ,6 ,7 - H
4 ,5 ,6 ,7 - H
4
]Rutalexin (16a)
2
c
[
4
]Brassicanate A (17a)
1
3
34
ꢀ
ꢀ
ꢀ
ꢀ
2
c
S-[4 ,5 ,6 ,7 - H
4
]Glucobrassicin (3a)
2%
a
2
34
The % of H and S incorporation was established by HRMS-ESI
±
±
±
according to the following equation: % = {[M + n] /([M] +[M + n] )}
b
c
×100 (n = 4 or 6). Positive ion mode. Negative ion mode.
15
agreement with current data. In general, thiohydroximates (e.g.
3
or 4 or the sodium salt of 11) have a lower d for C-1 (ca. 162–
C
16
1
72 ppm).¶
bers incubated with non-labelled indolyl-3-acetothiohydroxamic
With indolyl-3-acetothiohydroxamic acid (10) in hand, its
acid (10).
ꢀ
ꢀ
ꢀ
ꢀ
chemical stability in aqueous solution was determined by HPLC
analysis; 10 decomposed on standing (solution of water–methanol,
Additional experiments were carried out using [4 ,5 ,6 ,7 -
2
H ]indolyl-3-acetonitrile (18a) and indolyl-3-acetonitrile (18), as
4
9
5 : 5, v/v) to indolyl-3-acetonitrile (18, ca. 50% in 7 h) with
reported above for acetothiohydroxamic acids 10a and 10. LC-
HRMS-ESI analysis of each fraction showed the same compo-
nents as above and indicated that none of the phytoalexins or
indolyl glucosinolates contained deuterium. Interestingly, indolyl-
3-acetonitrile (18) was completely metabolised within 24 h to
indolyl-3-acetic acid (19), which in turn was metabolised to indole-
3-carboxylic acid (20) and methyl indole-3-carboxylate (21).
Remarkably, as summarised in Table 1, LC-HRMS data
indicated that cyclobrassinin (14a), spirobrassinin (15a) and
extrusion of sulfur. However, since the product of indolyl-3-
acetothiohydroxamic acid (10) decomposition did not contain
sulfur, any sulfur containing products obtained from incorpo-
ꢀ
ꢀ
ꢀ
ꢀ
2
34
ration of [4 ,5 ,6 ,7 - H
4
]indolyl-3-[ S]acetothiohydroxamic acid
(
10a) would inevitably derive from 10a itself. Nonetheless, to
obtain a comprehensive map of these metabolic pathways, addi-
tional control experiments were carried out using tetradeuterated
indolyl-3-acetonitrile (18a) as well.
ꢀ
ꢀ
ꢀ
ꢀ
2
34
ꢀ
ꢀ
ꢀ
ꢀ
2
The synthesis of [4 ,5 ,6 ,7 - H
4
]indolyl-3-[ S]acetothiohydroxa-
glucobrassicin (3a) incorporated intact [4 ,5 ,6 ,7 - H ]indolyl-3-
4
2
34
±
mic acid (10a) started with [4,5,6,7- H
4
]indole-3-carboxaldehyde
[ S]acetothiohydroxamic acid (10a) since only the [M + 6] peak
was detected. By contrast, the phytoalexins rutalexin (16a) and
brassicanate A (17a) showed incorporation of only four deuteria
17
(
(
8a) and followed the steps shown in Scheme 2, but using
Me
3
4
34
18
3
Si) S (prepared from S/Na and chlorotrimethylsilane),
2
ꢀ
ꢀ
ꢀ
ꢀ
2
±
as described in the ESI.‡ The synthesis of [4 ,5 ,6 ,7 - H
4
]indolyl-
(only [M + 4] peak) and no sulfur. Because rutalexin (16) is
biosynthesised from cyclobrassinin (14), these results indicate
17
19
3
-acetonitrile was carried out as previously described.
ꢀ
ꢀ
ꢀ
ꢀ
2
34
Next, solutions of [4 ,5 ,6 ,7 - H
hydroxamic acid (10a) and indolyl-3-acetothiohydroxamic acid
10) were separately added to UV-elicited rutabaga tubers, the tis-
4
]indolyl-3-[ S]acetothio-
that the sulfur 34 of cyclobrassinin (14) was exchanged with
unlabelled sulfur. Thus, it is likely that another sulfur donor
(possibly also cysteine) was used in this conversion. A similar
explanation could be used for brassicanate A (17a), since it
(
sues were incubated, extracted, and the extracts were fractionated
to a non-polar fraction, containing phytoalexins, and a polar frac-
tion, containing indolyl glucosinolates, as described in the ESI.‡
HPLC analysis of the fractions using photodiode array and mass
detectors and comparison of the spectra of the components with
those available in our metabolite library showed the presence of
the phytoalexins cyclobrassinin (14), spirobrassinin (15), rutalexin
19
appears to be derived from brassinin (5). The fact that among
the three indolyl glucosinolates only glucobrassicin (3a) showed
incorporation of four deuteria and sulfur 34 is consistent with
8
our previous findings indicating that oxidation at position N-1 of
indole occurs immediately after oxime formation, i.e. upstream of
the step yielding thiohydroxamic acid 10.
ꢀ
ꢀ
ꢀ
ꢀ
2
(
16) and brassicanate A (17) in the non-polar fraction, and the glu-
cosinolates glucobrassicin (3), 1-methoxyglucobrassicin (4), and
-methoxyglucobrassicin (22) in the polar fraction. LC-HRMS-
As expected, [4 ,5 ,6 ,7 - H ]indolyl-3-acetonitrile (18a) was not
4
incorporated into any of the phytoalexins or glucosinolates present
in elicited tissues (Table 2). This result is of interest since
4
ESI analysis of each fraction indicated the levels of deuterium and
sulfur incorporation in each metabolite, as shown in Table 1. The
indolyl-3-acetonitrile (18) appears to be an intermediate in the
biosynthesis of camalexin (7). Nonetheless, since camalexin (7)
is not produced in rutabaga we could not confirm that result.
20
±
±
[M + 4] or [M + 6] ion peaks were not detected in fractions of tu-
5
2 | Org. Biomol. Chem., 2008, 6, 51–54
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ꢀ
ꢀ
ꢀ
ꢀ 2
Table 2 Metabolism of [4 ,5 ,6 ,7 - H
4
]indolyl-3-acetonitrile (18a) in
proposed. Firstly, as depicted in Scheme 3, oxidation of indolyl-3-
acetaldoxime (1) to the corresponding nitrile oxide 12 provides
a putative intermediate suitable for nucleophilic attack by an
alkylthiol (e.g. cysteine). The resulting intermediate could be
converted through a C–S lyase to the thiohydroxamic acid 10,
possibly the last intermediate common to both pathways. Indolyl-
elicited tubers of rutabaga (Brassica napus ssp. rapifera)
Incorporation
10
2
a
Labelled product
(% of H)
ꢀ
ꢀ
ꢀ
ꢀ
2
b
[
4 ,5 ,6 ,7 - H
4
]Indole-3-carboxylic acid (20a)
≥98%
≥98%
ꢀ
ꢀ
ꢀ
ꢀ
2
b
Methyl [4 ,5 ,6 ,7 - H
4
]indole-3-carboxylate (21a)
3
-methylisothiocyanate (13) could then be thiomethylated to yield
a
2
The % of H incorporation was established by HRMS-ESI according to
the following equation: % = {[M + 4] /([M] +[M + 4] )} ×100. Negative
brassinin (5).
±
+
±
b
ꢀ
ꢀ
ꢀ
ꢀ
2
[
4 ,5 ,6 ,7 - H ]Indolyl-3-acetonitrile (18a) appeared to be the
4
ion mode.
main precursor of indolyl-3-acetic acid (19a), indole-3-carboxylic
acid (20a) and methyl indole-3-carboxylate (21a) under the
experimental conditions described in the ESI,‡ since these three
metabolites were isolated fully tetradeuterated (about ≥98%,
Table 2, Scheme 4). In agreement with these findings compounds
19–21 were not detected in control experiments.
Based on the results described above and their integration
another intermediate (10) common to
the pathways of brassinin (5) and glucobrassicin (3) can be
2,10
with reported data,
Scheme 3 Proposed biosynthetic pathway of glucobrassicin (3) and phytoalexins brassinin (5), cyclobrassinin (14), spirobrassinin (15), rutalexin (16)
and brassicanate A (17); compounds 12 and 13 are proposed intermediates.
ꢀ
ꢀ
ꢀ
ꢀ 2
Scheme 4 Incorporation of [4 ,5 ,6 ,7 - H
indole-3-carboxylate (21a).
4
]indolyl-3-acetonitrile (18a) into indolyl-3-acetic acid (19a), indole-3-carboxylic acid (20a) and methyl
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As well, our findings have implications on the pathway of
indole glucosinolates. The results described above reinforce our
previous data, which indicated that glucosinolates 4 and 22
Notes and references
§
Sodium indolyl-3-thiohydroximate (23) was previously reported not to
16
6
be stable in a buffer solution at pH 6.
derive from oxidation of indolyl-3-acetaldoxime (1) and not from
glucosinolate 3. This is likely because glucosinolates 4 and 22
did not incorporate the thiohydroxamic acid 10a. Previously, it
was proposed that these indole glucosinolates (4 and 22) resulted
21
from enzymatic methoxylation of 3. In addition, our results
suggest that brassinin (5) is perhaps only a couple of steps
from the thiohydroxamic acid 10 (side-chain rearrangement and
thiomethylation).
¶ All compounds gave satisfactory spectroscopic data; in each case
the percentage of deuterated synthetic compound was ≥98%. Labelled
compounds are numbered with an additional letter a (e.g. 10a).
1
2
Phytoalexins, ed. J. A. Bailey and J. W. Mansfield, Blackie & Son,
Glasgow, UK, 1982, pp. 1–334.
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1
76; (b) M. S. C. Pedras, M. Jha and P. W. K. Ahiahonu, Curr. Org.
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3
4
5
6
7
2
007, 5, 1167–1169.
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M. S. C. Pedras and M. Hossain, Org. Biomol. Chem., 2006, 4, 2581–
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2
Eventually, with a better understanding of the biosynthetic
intermediates of crucifer phytoalexins and their corresponding
enzymes and genes, it is expected that plants may be bred to
produce a wider variety of chemical defences and display higher
levels of resistance to stress.
2
M. S. C. Pedras, S. Montaut, Y. Xu, A. Q. Khan and A. Loukaci, Chem.
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Selected spectroscopic data
1
0 For a recent review on glucosinolates see: B. A. Halkier and J.
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1
Indolyl-3-acetothiohydroxamic acid (10). H NMR (500 MHz
11 K. Monde, M. Takasugi and T. Ohnishi, J. Am. Chem. Soc., 1994, 116,
6
650–6657.
CDCl
3
): d 8.26 (br s, 1H), 7.54 (d, J = 8 Hz, 1H), 7.44 (d, J =
Hz, 1H), 7.28 (dd, J = 7.5, 7.5 Hz, 1H), 7.17–7.24 (m, 2H), 4.26
s, 2H). C NMR (125.8 MHz CDCl ): d 189.0 (s), 136.7 (s), 126.9
s), 124.5 (d), 123.5 (d), 121.0 (d), 119.0 (d), 111.9 (d), 107.2 (s),
6.9 (t). HRMS-ESI m/z measured 207.0579 (207.0586 calculated
OS). HPLC-MS-ESI m/z (relative abundance) 207
M + H] (100), 189 (22), 174 (39), 130 (35). FTIR kmax(KBr)/cm :
410, 3307, 3056, 2904, 1552, 1455, 1419, 1353, 1339, 1227, 1131,
062, 971, 744.
1
1
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8
(
(
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3
1
1
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3
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H
11
N
2
1
3, 169–194.
+
−1
[
3
1
16 D. W. Reed, L. Davin, J. C. Jain, V. Deluca, L. Nelson and E. W.
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1
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2
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1
1
8 J.-H. So and P. Boudjouk, Synthesis, 1989, 4, 306–307.
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Andreasson and B. A. Halkier, Plant Physiol., 2003, 131, 298–308.
ꢀ
ꢀ
ꢀ
ꢀ
2
34
[
4 ,5 ,6 ,7 - H
4
]Indolyl-3-[ S]acetothiohydroxamic acid (10a).
1
H NMR (500 MHz CDCl ): d 8.26 (br s, 1H), 7.20 (s, 1H), 4.27 (s,
H). HRMS-ESI m/z measured 213.0797 (213.0795 calculated for
H
3
2
2
2
C
[
2
34
10
7
H
4
+
N
2
O S). HPLC-MS-ESI m/z (relative abundance) 213
M + H] (100), 195 (15), 180 (13), 134 (18).
5
4 | Org. Biomol. Chem., 2008, 6, 51–54
This journal is © The Royal Society of Chemistry 2008