The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Article abstract of DOI:10.1016/j.phytochem.2009.05.015

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to "quasi-natural" products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.

Full text of DOI:10.1016/j.phytochem.2009.05.015

Phytochemistry 70 (2009) 1129–1138
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De
novo incorporation of deuterated tryptophans and quasi-natural compounds
*
M. Soledade C. Pedras , Denis P. Okinyo-Owiti, Ken Thoms, Adewale M. Adio
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoantici-
pins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain
unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated
Received 20 March 2009
Received in revised form 14 May 2009
Available online 26 June 2009
D
- and L D- and L-tryptophans and other indol-3-yl derivatives into pertinent
-10-methoxytryptophans,
phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp.
rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these
being analogues of biosynthetic intermediates that might lead to ‘‘quasi-natural” products (products
similar to natural products but not produced under natural conditions). No detectable incorporations
of deuterium labeled 10-methoxytryptophans into phytoalexins or glucobrassicin were detected.
Keywords:
Brassica napus L. ssp. rapifera
Brassicaceae
Rutabaga
Brassinin
L-tryptophan was incorporated in a higher percentage than
D-tryptophan into both phytoalexins and
Crucifer
phytoanticipins. However, in the case of the phytoalexin rapalexin A, both
D
- and -tryptophan were
L
Glucobrassicin
incorporated to the same extent. Furthermore, the transformations of both 10-methylindolyl-30-acet-
aldoxime and 10-methylindolyl-30-acetothiohydroxamic acid (quasi-natural products) into 10-methylg-
lucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic
pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy
substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime
enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A
higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively
lower structural diversity among phytoalexins than among glucosinolates.
Indolyl-3-acetaldoxime
Indolyl-3-acetothiohydroxamic acid
Indolyl glucosinolate
Rapalexin A
Rutalexin
Phytoalexin
10-Methoxytryptophan
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
pathways is of great interest. This is particularly important in cru-
cifers where the engineering of desirable metabolic pathways is
Crucifers are one of the most valuable group of cultivated
plants, representing globally consumed vegetables such as broc-
coli, cauliflower, cabbage, and rutabaga, as well as extensively cul-
tivated oilseed crops and condiments. Similar to other plant
families, crucifers (family name Brassicaceae) respond to stress
employing metabolic pathways that involve the biosyntheses of
numerous and structurally diverse secondary metabolites. Phytoal-
exins and phytoanticipins are among the cruciferous metabolites
that have crucial ecological roles in defense. Phytoalexins are bio-
synthesized de novo in response to stress, including microbial at-
tack, heavy metal salts, or UV radiation (Bailey and Mansfield,
1982; Pedras et al., 2007). By contrast, phytoanticipins are consti-
tutive plant defenses whose concentrations can increase under
stress conditions (VanEtten et al., 1994). Considering the involve-
ment of phytoalexins and phytoanticipins in plant resistance to
diseases and pests, a molecular understanding of their metabolic
becoming more common (Yan and Chen, 2007).
The phytoalexins of any given plant family derive usually from a
common primary biosynthetic precursor; however, phytoantici-
pins are biosynthesized from different precursors and thus can
have very diverse chemical structures. Coincidently, the crucifer-
ous phytoanticipins glucobrassicin (6, i.e. indolyl-3-methyl gluco-
sinolate) (Bender and Celenza, 2009) and indolyl-3-acetonitrile
(3) (Agerbirk et al., 2009) and the phytoalexins derived from brass-
inin (8) share their primary precursor, i.e. tryptophan (Pedras et al.,
2007). Specifically, the biosynthetic pathways of brassinin (8) and
those of glucobrassicin (6) and indolyl-3-acetonitrile (3) start with
the conversion of
L-tryptophan (1) to indolyl-3-acetaldoxime (2)
and appear to diverge into various branches, including (a) indo-
lyl-3-acetonitrile (3, for which additional biosynthetic pathways
are known, Grubb and Abel, 2006) and the phytoalexin camalexin
(4) (Glawischnig, 2007), (b) glucobrassicin (6), and (c) brassinin (8)
and related phytoalexins 9 and 10 (Fig. 1) (Pedras and Okinyo,
2008). While most of the genes of the glucobrassicin (6) biosyn-
thetic pathway have been cloned (Yan and Chen, 2007; Halkier
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.05.015

1130
M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
CO2H
NH3
NHOH
NCS
NOH
4'
7'
S
3'
5'
6'
1
2'
N
1'
N
H
N
H
N
2
3
4
H
5
H
7
L-tryptphan (1)
a
c
b
CN
NH
4
7
SCH3
β-D-glc
3
S
5
6
2
1
S
N OSO3K
N
H
N
H
8
N
6
H
S
N
N
SCH3
S
N
9
N
H
H
OCH3
NCS
NH
SCH3
3
d
NOH
CH3
O
N
1
S
N
H
N
N
13
O
OCH3
S
OCH3
N
12
10
11
H
Fig. 1. Biosynthetic pathways of L-tryptophan (1) and indolyl-3-acetaldoximes (2) and (11): (a) indolyl-3-acetonitrile (3, phytoanticipin, additional biosynthetic pathways are
known) and camalexin (4, phytoalexin); (b) glucobrassicin (6, phytoanticipin); (c) phytoalexins brassinin (8), cyclobrassinin (9) and rutalexin (10); structure 7 is a postulated
intermediate; (d) phytoalexin 1-methoxybrassinin (12); phytoalexin rapalexin (13).
and Gershenzon, 2006), the brassinin (8) pathway is far from such
an achievement. This status reflects the relative novelty of brassi-
nin (8) and consequently its biosynthetic pathway, but not a lack
of its importance. Indeed, the first cruciferous phytoalexins (brass-
inin (8), cyclobrassinin (9) and 1-methoxybrassinin (12)) were re-
ported in 1986 (Takasugi et al., 1986), while reports of
glucosinolates date back to the 19th century and related biosyn-
thetic studies were initiated in the 1960s (Walker and Gray,
1970; Underhill et al., 1974). Hence, current glucosinolate issues
are associated with the regulation and control of their metabolism
(Yan and Chen, 2007), whereas the understanding of cruciferous
phytoalexin metabolism poses more fundamental questions.
To this end, an important milestone was recently achieved by
establishing the incorporation of the first sulfur containing indolyl
derivative 5 (indolyl-3-acetothiohydroxamic acid) into the phyto-
alexins cyclobrassinin (9) and rutalexin (10) and into the phytoan-
ticipin glucobrassicin (6) (Fig. 1) (Pedras and Okinyo, 2008). From
this work, it can be inferred that the enzymes involved in the trans-
formation of intermediates 2 and 5 are common to both gluco-
brassicin (6) and brassinin (8) biosynthetic pathways but not to
the camalexin (4) pathway (Glawischnig, 2007). In connection with
naturally occurring 1-methoxyindol-3-yl phytoalexins, it was
shown that 10-methoxyindolyl-30-acetaldoxime (11) was incorpo-
rated into 1-methoxybrassinin (12, d, Fig. 1); however, the poten-
tial intermediacy of 10-methoxytryptophan has not been evaluated
(Pedras and Montaut, 2004). Furthermore, the biosynthetic origin
of the structurally unique phytoalexin rapalexin A (13, Fig. 1) re-
mains to be determined. In continuation of our previous studies
(Pedras and Montaut, 2004; Pedras and Okinyo, 2008), we investi-
gated de novo incorporations of several perdeuterated tryptophans
and other indol-3-yl derivatives into cruciferous metabolites (phy-
toalexins and indolyl glucosinolates) in rutabaga (Brassica napus L.
ssp. rapifera) roots. As well, by analogy to traditional precursor-di-
rected biosynthetic studies (Thiericke and Rohr, 1993), we investi-
gated the potential transformation of quasi-natural compounds,
that are derivatives of biosynthetic intermediates that might lead
to a new generation of ‘‘quasi-natural” products (products struc-
turally similar to natural products but not produced under natural
conditions). Herein we report the results of our de novo incorpora-
tions and metabolic transformations to unravel fundamental
pieces of the biosynthetic puzzle of cruciferous defense
metabolites.
2. Results
2.1. Synthesis of potential precursors
Among isotopically labeled compounds of potential use in bio-
synthetic studies, perdeuterated compounds are particularly effec-
tive since the small amounts usually incorporated by plant tissues
can be detected by mass spectrometry, e.g. ESI-HRMS or EI-HRMS
(Pedras and Okinyo, 2006). To this end, several biosynthetic pre-
cursors of cruciferous metabolites and derivatives containing dif-
ferent amounts of deuterium were synthesized (Table 1). As well,
the corresponding natural abundance compounds were synthe-
sized using the methods reported for perdeuterated compounds,
to ensure that deuterated products do not bring about artifacts in
incorporation experiments. In addition, quasi-natural compounds
(non-isotopically labeled) that would probe the specificity of the
biosynthetic enzymes and lead to quasi-natural products if
transformed (Amir-Heidari et al., 2008; Goss et al., 2006), were
prepared: 10-methylindolyl-30-acetaldoxime (26), 10-methylindol-
yl-30-acetothiohydroxamic acid (28), and 1-acetylindolyl-3-methy-
lisothiocyanate (32). 10-t-Butylindolyl-30-methylisothiocyanate
(29) was also prepared because it was expected to be substantially
more stable than 10-acetylindolyl-3-methylisothiocyanate (32).
The perdeuterated tryptophans
L
-[40,50,60,70-2H4]tryptophan
-[100,100,100,40,50,60,70-2H7]-
(1a),
D
-[40,50,60,70-2H4]tryptophan (21a),
L

M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
1131
Table 1
ment in yield (from 61% to 72%) and reaction time (reduced from
40 to 18 h).
Perdeuterated compounds used in precursor administration experiments.
The perdeuterated DL-tryptophan (Fig. 2) was prepared from
[4,5,6,7-2H4]indole (14a) and DL-serine in refluxing glacial acetic
acid. Next, the reaction mixture containing Nb-acetyl-DL-[40,50,60,
Deuterated compound (origin, reference)
Compound number
(#)
[4,5,6,7-2H4]indole (synthetic, Pedras and Okinyo, 2006)
(14a)
(1a)
(1b)
(21a)
(20b)
-[40,50,60,70-2H4]tryptophan (synthetic, this work)
-[20,40,50,60,70-2H5]tryptophan (commercial)
70-2H4]tryptophan (16a) was resolved into -[40,50,60,70-2H4]trypto-
L
L
-[40,50,60,70-2H4]tryptophan (21a), 47%
-aminoacylase respectively
L
phan (1a, 36% yield) and
yield) using -aminoacylase and
(Konda-Yamada et al., 2002; Yamada et al., 2005), as described in
Section 4. The non-deuterated -tryptophan (1) and -tryptophan
D
D
-[40,50,60,70-2H4]tryptophan (synthetic, this work)
L
D
L
-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan (synthetic,
this work)
D
-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan (synthetic,
this work)
(22b)
(20c)
(22c)
L
D
(21) were similarly prepared from indole and DL-serine, as
described in Section 4.
L
-[100,100,100-2H3]-10-methoxytryptophan (synthetic, this
work)
Perdeuterated DL-1-methoxytryptophan was prepared from
D
-[100,100,100-2H3]-10-methoxytryptophan (synthetic, this
work)
[10,10,10,4,5,6,7-2H7]1-methoxyindole (15a, Fig. 2) and DL-serine.
The
was resolved into
(20b, 30% yield) and
phan (22b, 46% yield) using
respectively, as described in Section 4 (Konda-Yamada et al.,
2002; Yamada et al., 2005).
-[100,100,100-2H3]-10-methoxytryptophan
-[100,100,100-2H3]-10-methoxytryptophan (22c), and non-la-
-10-methoxytryptophan (20) and -10-methoxytryptophan
Nb-acetyl-DL-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan
L
-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan
-[100,100,100,40,50,60,70-2H7]-10-methoxytrypto-
-aminoacylase and -aminoacylase,
D
10-methoxytryptophan (20b),
D
-[100,100,100,40,50,60,70-2H7]-10-meth-
L
D
oxytryptophan
(22b),
L
-[100,100,100-2H3]-10-methoxytryptophan
(20c), and
D
-[100,100,100-2H3]-10-methoxytryptophan (22c) were
L
prepared from [4,5,6,7-2H4]indole (14a) and DL-serine (Fig. 2)
(Konda-Yamada et al., 2002; Yamada et al., 2005). [4,5,6,7-2H4]
Indole (14a) was obtained from the readily available NMR solvent
[2,3,4,5,6-2H5]nitrobenzene (99%, 2H5) and 2-chloroacetonitrile
using a more efficient method than our previously published prep-
aration (Pedras and Okinyo, 2006). In short, addition of acetic acid
to a solution of [3,4,5,6-2H4]2-nitrophenylacetonitrile in ethyl ace-
tate decreased the required amount of catalyst by a third of that
previously reported to completely transform [3,4,5,6-2H4]2-nitro-
phenylacetonitrile into [4,5,6,7-2H4]indole (14a), with an improve-
(20c),
D
beled
L
D
(22) were similarly prepared.
Although indolyl-3-methylisothiocyanate (7) is a likely inter-
mediate in the biosynthesis of brassinin (8), due to its relatively
low stability it is not possible to prepare it (Hanley and Parsley,
1990; Hanley et al., 1990); however the 10-acetylindolyl-30-methy-
lisothiocyanate (32) and 10-t-butylindolyl-30-methylisothiocyanate
(29) were stable and prepared as previously reported (Kutschy
et al., 1997, 1998).
2.2. Biosynthesis
The biosynthetic incorporations of deuterated and quasi-natu-
ral precursors were determined by HPLC-DAD-HRMS analyses of
extracts of tissues administered compounds and extracts of control
tissues, as described below.
NHAc
3
4
6
3
4'
2
CO2H
3'
DL-serine
1
1
Rutabaga tubers were treated as reported in Section 4 and
then were incubated with aqueous solutions of perdeuterated
compounds, solutions of the corresponding non-deuterated com-
pounds, and solutions of quasi-natural compounds, respectively.
Additional control tissues were incubated similarly but with
aqueous solvent only. The aqueous solutions were collected after
72 h of incubation and extracted with ethyl acetate to yield or-
ganic extracts containing phytoalexins (established by HPLC-
DAD-MS). The following phytoalexins were consistently detected
by HPLC-DAD-MS in ethyl acetate extracts of elicited tissues, un-
less stated otherwise: cyclobrassinin (9), rutalexin (10), rapalexin
A (13), and spirobrassinin (23) (additional phytoalexins were de-
tected in different rutabaga batches). Extracts containing indolyl
glucosinolates (established by HPLC-DAD-MS) were obtained by
cutting tissues around the holes, soaking in methanol, and grind-
ing (mortar and pestle), followed by overnight extraction with
aqueous methanol. The following indolyl-3-glucosinolates were
detected by HPLC-DAD-MS in MeOH extracts of elicited and con-
trol tissues: glucobrassicin (6), 10-methoxyglucobrassicin (24),
and 4-methoxyglucobrassicin (25).
1'
N
R
Ac2O/AcOH
N
R
6'
14 R=H
16 R=H
17 R=OCH3
15 R=OCH3
L-aminoacylase
NHAc
CO2H
NH2
CO2H
+
N
R
N
R
18 R=H
19 R=OCH3
1 R=H (L-Trp)
20 R=OCH3
D-aminoacylase
NH2
CO2H
The percentage of deuterium incorporation was established from
analysis of data obtained by HPLC-HRMS-ESI (positive or nega-
N
R
tive ion modes; % of (2H) incorporation = {[M + n]+/ꢀ/([M]+/ꢀ
+
[M + n]+/ꢀ)} ꢁ 100 (n = 3, 4, 7), where
n is the number of
21 R=H (D-Trp)
22 R=OCH3
deuterium atoms, and M is the peak area of the molecular or
pseudo ion peak). The HPLC-HRMS-ESI data indicated that
the ions [M + n]+/ꢀ were not present in natural abundance
samples.
Fig. 2. Syntheses and resolution of
-10-methoxytryptophan (20, R = OCH3) and
L
-tryptophan (1, R = H),
D
-tryptophan (21, R = H),
L
D
-10-methoxytryptophan (22,
R = OCH3).

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M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
2.2.1. Precursor administration experiments using [4,5,6,7-2H4]indole
C
17H21N2O10S2 and C17H182H3N2O10S2, respectively. Based on the
(14a)
peak area of each ion, the percentage of deuterium incorporation
was found to be 11% (Table 2). The ion at m/z 480.0831 was not de-
tected in control samples incubated with non-labeled indole. The
10-methoxyglucobrassicin (24) peak at tR = 11.9 min indicated
two ions at m/z 477.0643 and 481.0894 that were consistent with
molecular formulae C17H21N2O10S2 and C17H172H4N2O10S2, respec-
tively. The percentage of deuterium incorporation was calculated
from the peak area of each ion and was found to be 11% (Table
2). The ion at m/z 481.0894 was not detected in control samples
incubated with non-labeled indole.
The incorporation of indole, a biosynthetic precursor of L-tryp-
tophan previously investigated (Pedras et al., 2004), was re-exam-
ined because new phytoalexins like rapalexin A (13), do not have a
carbon atom attached to C-3 and thus might not derive from
tryptophan.
HPLC-HRMS-ESI analyses of the ethyl acetate extract (contain-
ing phytoalexins) resulting from feeding [4,5,6,7-2H4]indole (14a)
displayed the rutalexin (10) peak at tR = 9.8 min (negative ion
mode) containing two ions at m/z 231.0233 and 235.0484 that
were consistent with the molecular formulae C11H7N2O2S and
C11H32H4N2O2S, respectively. Based on the peak area of each ion,
the percentage of deuterium incorporation into rutalexin (10)
was found to be 19% (Table 2). The ion at m/z 235.0484 was not de-
tected in control samples incubated with non-deuterated indole.
The spirobrassinin (23) peak at tR = 11.5 min (positive ion mode)
indicated two ions at m/z 251.0307 and 255.0558 that were
consistent with the molecular formulae C11H11N2OS2 and
C11H72H4N2OS2, respectively. The percentage of deuterium incor-
poration into spirobrassinin (23) was calculated based on the peak
area of each ion and was found to be 11% (Table 2). The ion at m/z
255.0558 was not detected in control samples incubated with non-
deuterated indole. The rapalexin A (13) peak at tR = 19.0 min (neg-
ative ion mode) indicated two ions at m/z 203.0284 and 206.0472
that were consistent with the molecular formulae C10H7N2OS and
C10H42H3N2OS, respectively. Based on the peak area of each ion,
the percentage of deuterium incorporation into rapalexin A (13)
was found to be 9% (Table 2). The ion at m/z 206.0472 was not de-
tected in control samples incubated with non-deuterated indole.
The cyclobrassinin (9) peak at tR = 24.8 min (positive ion mode)
indicated two ions at m/z 235.0358 and 239.0609 that were consis-
tent with the molecular formulae C11H11N2S2 and C11H72H4N2S2,
respectively. The percentage of deuterium incorporation into
cyclobrassinin (9) was calculated based on the peak area of each
ion and was found to be 6% (Table 2). The ion at m/z 239.0609
was not detected in control samples incubated with non-deuter-
ated indole.
2.2.2. Precursor administration experiments using L-
[20,40,50,60,70-2H5]tryptophan (1b)
L
-[20,40,50,60,70-2H5]Tryptophan (1b) 98% deuterated was pur-
chased from a chemical company. HPLC-HRMS-ESI analyses of
the phytoalexin ethyl acetate extract, resulting from administering
L
-[20,40,50,60,70-2H5]tryptophan (1b), displayed the rutalexin peak at
tR = 9.8 min (negative ion mode) containing two ions at m/z
231.0233 and 235.0484 that were consistent with the molecular
formulae C11H7N2O2S and C11H32H4N2O2S, respectively. Based on
the peak area of each ion, the percentage of deuterium incorpora-
tion was found to be 32% (Table 3). The rapalexin A peak at
tR = 19.0 min (negative ion mode) indicated two ions at m/z
203.0284 and 207.0472 that were consistent with the molecular
formulae C10H7N2OS and C10H32H4N2OS, respectively. Based on
the peak area of each ion, the percentage of deuterium incorpora-
tion was found to be 12% (Table 3). The spirobrassinin peak at
tR = 11.5 min (positive ion mode) indicated two molecular ions at
m/z 251.0307 and 255.0558 that were consistent with the molecu-
lar formulae C11H11N2OS2 and C11H72H4N2OS2, respectively. The
percentage of deuterium incorporation was calculated based on
the peak area of each ion and was found to be 17% (Table 3).
HPLC-HRMS-ESI analyses (negative ion mode) of the glucosino-
late fraction, resulting from incorporation of
L
-[20,40,50,
60,70-2H5]tryptophan (1b), displayed the glucobrassicin peak at
tR = 4.9 min containing two ions at m/z 447.0537 and 452.0788 that
were consistent with the molecular formulae C16H19N2O9S2 and
C16H142H5N2O9S2, respectively. The ion at m/z 452.0788 was not
HPLC-HRMS-ESI analyses (negative ion mode) of the glucosino-
late extract resulting from feeding of [4,5,6,7-2H4]indole (14a) dis-
played the glucobrassicin (6) peak at tR = 4.9 min containing two
ions at m/z 447.0537 and 451.0788 that were consistent with the
molecular formulae C16H19N2O9S2 and C16H152H4N2O9S2, respec-
tively. The ion at m/z 451.0788 was not detected in control samples
incubated with non-labeled indole. The percentage of deuterium
incorporation was calculated based on the peak area of each ion
and was found to be 19% (Table 2). The 4-methoxyglucobrassicin
(25) peak at tR = 10.5 min indicated two ions at m/z 477.0643 and
480.0831 that were consistent with molecular formulae
detected in control samples incubated with non-labeled L-trypto-
phan (1). The percentage of deuterium incorporation was calcu-
lated based on the peak area of each ion and was found to be
17% (Table 3). The 4-methoxyglucobrassicin peak at tR = 10.5 min
indicated two ions at m/z 477.0643 and 481.0831 that were consis-
tent with the molecular formulae C17H21N2O10S2 and
C17H172H4N2O10S2, respectively. Based on the peak area of each
ion, the percentage of deuterium incorporation was found to be
7% (Table 3). The 10-methoxyglucobrassicin peak at tR = 11.9 min
indicated two ions at m/z 477.0643 and 482.0894 that were
Table 2
Table 3
Incorporation of [4,5,6,7-2H4]indole (14a) into rutabaga phytoalexins 9a, 10a, 13a and
23a and glucosinolates 6a, 24a and 25a.
Incorporation of L-[20,40,50,60,70-2H5]tryptophan (1b) into rutabaga phytoalexins 9a,
10a, 13a and 23a and glucosinolates 6a, 24a and 25a.
Labeled products isolated (#)a
% Incorporationb
Labeled products isolated (#)a
% Incorporationb
[4,5,6,7-2H4]cyclobrassinin (9a)
6c
[4,5,6,7-2H4]cyclobrassinin (9a)
7c
[4,5,6,7-2H4]rutalexin (10a)
19d
9d
[4,5,6,7-2H4]rutalexin (10a)
32d
12d
17c
17d
12d
7d
[2,5,6,7-2H4]rapalexin A (13a)
[2,5,6,7-2H4]rapalexin A (13a)
[4,5,6,7-2H4]spirobrassinin (23a)
[40,50,60,70-2H4]glucobrassicin (6a)
[40,50,60,70-2H4]10-methoxyglucobrassicin (24a)
[50,60,70-2H3]4-methoxyglucobrassicin (25a)
11c
19d
11d
11d
[4,5,6,7-2H4]spirobrassinin (23a)
[20,40,50,60,70-2H5]glucobrassicin (6a)
[20,40,50,60,70-2H5]10-Methoxyglucobrassicin (24a)
[20,50,60,70-2H4]4-Methoxyglucobrassicin (25a)
a
a
Deuterated compounds are referred to by a number followed by the letter a.
Deuterated compounds are referred to by a number followed by the letter a.
b
b
Incorporations calculated from HPLC-HRMS-ESI (Q-TOF); % of 2H incorpora-
Incorporations calculated from HPLC-HRMS-ESI (Q-TOF); % of 2H incorpora-
tion = {[M + n]+/ꢀ/([M]+/ꢀ + [M + n]+/ꢀ)} ꢁ 100 (n = 4).
tion = {[M + n]+/ꢀ/([M]+/ꢀ + [M + n]+/ꢀ)} ꢁ 100 (n = 4 or 5).
c
c
Positive ion mode.
Negative ion mode.
Positive ion mode.
Negative ion mode.
d
d

M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
1133
Table 4
consistent with the molecular formulae C17H21N2O10S2 and
C17H162H5N2O10S2, respectively. The percentage of deuterium
incorporation was calculated from the peak area of each ion and
was found to be 12% (Table 3). The overall results of these experi-
ments are summarized in Fig. 3.
Incorporation of L-[40,50,60,70-2H4]tryptophan (1a) and D-[40,50,60,70-2H4]tryptophan
(21a) into rutabaga phytoalexins 10a, 13a and 23a and glucosinolates 6a, 24a and
25a.
Labeled products isolated (#)a
% Incorporationb
From
L
- (1a)
From D- (21a)
[4,5,6,7-2H4]rutalexin (10a)
14d
6d
3d
2.2.3. Precursor administration experiments using L-
[5,6,7-2H3]rapalexin A (13a)
6d
[40,50,60,70-2H4]tryptophan (1a) and
(21a)
D
-[40,50,60,70-2H4]tryptophan
[4,5,6,7-2H4]spirobrassinin (23a)
[40,50,60,70-2H4]glucobrassicin (6a)
[40,50,60,70-2H4]10-methoxyglucobrassicin (24a)
[50,60,70-2H3]4-methoxyglucobrassicin (25a)
9c
2c
12d
9d
61d
61d
61d
HPLC-HRMS-ESI analyses of the phytoalexin extract, resulting
5d
from administering
L
-[40,50,60,70-2H4]tryptophan (1a) and
D-
[40,50,60,70-2H4]tryptophan (21a), displayed the rutalexin peak at
tR = 9.8 min (negative ion mode) containing two ions at m/z
231.0233 and 235.0484 that were consistent with the molecular
formulae C11H7N2O2S and C11H32H4N2O2S, respectively. Based on
the peak area of each ion, the percentage of deuterium incorpora-
tion was found to be 14% (from 1a), and 3% (from 21a) (Table 4).
The rapalexin A (13) peak at tR = 19.0 min (negative ion mode)
indicated two ions at m/z 203.0284 and 206.0472 that were consis-
tent with the molecular formulae C10H7N2OS and C10H42H3N2OS,
respectively. Based on the peak area of each ion, the percentage
of deuterium incorporation was found to be 6% (from both 1a
and 21a) (Table 4). The spirobrassinin (23) peak at tR = 11.5 min
(positive ion mode) indicated two molecular ions at m/z
251.0307 and 255.0558 that were consistent with the molecular
formulae C11H11N2OS2 and C11H72H4N2OS2, respectively. The per-
centage of deuterium incorporation was calculated based on the
a
Deuterated compounds are referred to by a number followed by the letter a.
b
Incorporations calculated from HPLC-HRMS-ESI (Q-TOF); % of 2H incorpora-
tion = {[M + n]+/ꢀ/([M]+/ꢀ + [M + n]+/ꢀ)} ꢁ 100 (n = 3 or 4).
c
Positive ion mode.
Negative ion mode.
d
peak area of each ion and was found to be 9% (from 1a), and 2%
(from 21a) (Table 4).
HPLC-HRMS-ESI analyses (negative ion mode) of the glucosino-
late fraction resulting from incorporation of
L
-[40,50,60,70-2H4]tryp-
tophan (1a) or
D
-[40,50,60,70-2H4]tryptophan (21a) displayed the
glucobrassicin peak at tR = 4.9 min containing two ions at m/z
447.0537 and 451.0788 that were consistent with the molecular
formulae C16H19N2O9S2 and C16H152H4N2O9S2, respectively. The
ion at m/z 451.0788 was not detected in control samples incubated
with non-labeled L-tryptophan (1) or D-tryptophan (21). The per-
centage of deuterium incorporation was calculated based on the
peak area of each ion and was found to be 12% (from 1a), and
61% (from 21a) (Table 4). The 4-methoxyglucobrassicin (25) peak
at tR = 10.5 min indicated two ions at m/z 477.0643 and 480.0831
that were consistent with the molecular formulae C17H21N2O10S2
and C17H182H3N2O10S2, respectively. Based on the peak area of each
ion, the percentage of deuterium incorporation was found to be 5%
(from 1a), and 61% (from 21a (Table 4). The 10-methoxygluco-
brassicin (24) peak at tR = 11.9 min indicated two ions at m/z
477.0643 and 481.0894 that were consistent with the molecular
formulae C17H21N2O10S2 and C17H172H4N2O10S2, respectively. The
percentage of deuterium incorporation was calculated from the
peak area of each ion and was found to be 9% (from 1a), and 6
1% (from 21a) (Table 4). The overall results of these experiments
are summarized in Fig. 3.
no
incorporation
R=OCH3
or R=CH3
CO2H
NH3
N
R
L- tryptophan, R=H (1)
2.2.4. Precursor administration experiments using
[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan (20b),
10-methoxytryptophan (20c), -[100,100,100,40,50,60,70-2H7]-10-meth
oxytryptophan (22b),
-[100,100,100-2H3]-10-methoxytryptophan (22c),
and
-10-methyltryptophan (35)
L-
L
-[100,100,100-2H3]-
R=H
D
N
SCH3
D
S
L
O
8 + 9 + 10 + 13 +
The biosynthetic pathways of 1-substituted rutabaga metabo-
N
H
lites 1-methoxybrassinin (11) and 10-methoxyglucobrassicin (24)
L
-[100,100,100,40,50,60,70-2H7]-10-methoxytry-
23
were investigated using
ptophan (20b),
-[100,100,100-2H3]-10-methoxytryptophan (20c),
-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan (22b), -[100,100,
100-2H3]-10-methoxytryptophan (22c), and -10-methyltryptophan
L
β-D-glc
β-D-glc
S
S
D
D
OCH3
L
N
OSO3K
N OSO3K
(35). After separately incubating with each compound, the aqueous
phase was collected and extracted to yield the phytoalexins cyclo-
brassinin (9), rutalexin (10), rapalexin A (13), and spirobrassinin
(23) (established by HPLC-DAD and HPLC-MS-ESI). Tissues around
the holes were extracted to yield a mixture of glucobrassicin (6),
10-methoxyglucobrassicin (24), and 40-methoxyglucobrassicin
(25) (established by HPLC-DAD-MS). HPLC-MS-ESI analyses did
not detect 1-methyl containing phytoalexins or indole glucosino-
lates. In addition, no deuterated products from administering
perdeuterated 1-methoxytryptophans were detected, neither were
+
+
6
N
N
H
OCH3
24
25
Fig. 3. Transformation of L-tryptophan (1) into phytoalexins brassinin (8), cyclo-
brassinin (9), rutalexin (10), rapalexin A (13), and spirobrassinin (23) and indolyl
glucosinolates 6, 24 and 25 in rutabaga roots; no methylated or methoxylated
phytoalexins or indolyl glucosinolates were detected from feeding 1-methoxytry-
ptophans (20 or 22) or 1-methyltryptophan (35).

1134
M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
perdeuterated 1-methoxytryptophans recovered from polar
extracts. The overall results of these experiments are summarized
in Fig. 3.
NCS
NCS
NH
S
SCH3
N
N
t-bu
2.2.5. Precursor administration experiments using 10-methylindolyl-30-
acetaldoxime (26) and 10-methylindolyl-30-acetothiohydroxamic acid
(28)
t-bu
O
O
O
O
S
29
30
The biosynthesis of rutabaga metabolites was further investi-
gated using the quasi-natural 10-methylindolyl-30-acetaldoxime
(26) and 10-methylindolyl-30-acetothiohydroxamic acid (28). Com-
pounds 26 and 28 were administered to UV-irradiated rutabaga root
slices followed by incubation and extractions. HPLC-DAD-MS-ESI
analyses of phytoalexin extracts indicated the absence of 1-
methyl-containing metabolites; however, chromatograms of MeOH
extracts established a new peak that was absent in those of control
fractions. HPLC-HRMS-ESI analyses (negative ion mode) of the new
metabolite peak at tR = 11.1 min indicated an ion at m/z 461.0656
that was consistent with the molecular formula C17H21N2O9S2 due
to the presence of 10-methylglucobrassicin (27). The overall results
of these experiments are summarized in Fig. 4.
N
SCH3
N
O
O
32
N
t-bu
O
O
31
NH
S
SCH3
N
SCH3
S
N
O
O
N
33
2.2.6. Precursor administration experiments using 1-t-butylindolyl-3-
methylisothiocyanate (29) and 1-acetylindolyl-3-
O
34
methylisothiocyanate (32)
1-t-Butylindolyl-3-methylisothiocyanate (29) was adminis-
tered to UV-irradiated rutabaga root slices followed by incubation
and extractions. The HPLC-DAD-MS-ESI analyses of phytoalexin
extracts demonstrated the presence of the phytoalexins cyclo-
brassinin (9), rutalexin (10), rapalexin A (13), and spirobrassinin
(23). In addition, two new peaks at tR = 30.1 and 34.1 min, that
were absent in chromatograms of control extracts, were detected.
HPLC-MS-ESI analyses (positive ion mode, ion trap) of the metab-
olite with peak at tR = 34.1 min showed ions at m/z 337, 230, 174
and 130. The metabolite with a peak at tR = 30.1 min contained
ions at m/z 351 and 251. The retention times, UV spectra, and frag-
mentation patterns of the metabolites at tR = 30.1 and tR = 34.1 min
were identical to those of synthetic standards, 10-(t-butoxycar-
bonyl)spirobrassinin (31) and 10-(t-butoxycarbonyl)brassinin
(30), respectively, prepared as previously reported (Kutschy et al.,
1997, 1998, 2002). These results were consistent with the involve-
ment of indolyl-3-methylisothiocyanate in the biosynthetic path-
way of indole phytoalexins. Additional experiments were
conducted by incubating rutabaga root slices with 1-(t-butoxycar-
bonyl)brassinin (30). After 72-h incubation, the aqueous phase was
collected, was extracted and subjected to preliminary clean up to
Fig. 5. Transformation of 10-t-butylindolyl-30-methylisothiocyanate (29) and quasi-
natural 1-acetylindolyl-3-methylisothiocyanate (32) in rutabaga roots.
yield a fraction that contained phytoalexins; control experiments
were carried out similarly by incubating slices with control solu-
tion. HPLC-DAD analysis of the phytoalexin fraction displayed a
new peak at tR = 30.1 min that was absent in chromatograms of
control extracts. The new metabolite was identified as 1-(t-butox-
ycarbonyl)spirobrassinin (31) by comparison of retention time, UV
spectra, and fragmentation pattern (established by HPLC-DAD and
HPLC-MS-ESI) with those of a synthetic sample (Kutschy et al.,
2002).
Because t-Boc is a relatively large group and is not known to oc-
cur in natural products, whereas the acetyl group is smaller and
known to occur in natural products, 1-acetylindolyl-3-methylis-
othiocyanate (32) was used. Compound 32 was administered to
UV-irradiated rutabaga root slices followed by incubation and
extractions. The HPLC-DAD-MS-ESI analyses of phytoalexin ex-
tracts revealed the phytoalexins cyclobrassinin (9), rutalexin (10),
rapalexin A (13), and spirobrassinin (23). In addition, a new peak
was detected at tR = 22.2 min that was absent in the chromato-
grams of control extracts. HPLC-MS-ESI analyses (positive ion
mode) of the peak at tR = 22.2 min showed ions at m/z 172 and
130. The new metabolite was identified as 1-acetylbrassinin (33)
by comparison of retention time, UV spectra, and fragmentation
pattern with those of an authentic sample. The phytoalexin ex-
tracts however, did not yield 1-acetylspirobrassinin (34) or other
1-acetyl containing phytoalexins. Additional incubation experi-
ments using rutabaga roots separately with 1-acetylbrassinin
(33) did not yield detectable 1-acetyl-containing metabolites. The
overall results of these experiments are summarized in Fig. 5.
β-D-glc
S
N OSO3K
NOH
N
CH3
N
CH3
27
26
NHOH
3. Discussion and conclusion
S
N
CH
The metabolic pathways of phytoalexins and phytoanticipins
are of great importance in plant defense and resistance to micro-
bial diseases and pests. Specifically in crucifers, where the engi-
neering of metabolic pathways is becoming more of a routine, a
number of fundamental questions need to be answered before
such pathways can be manipulated. In this work, using rutabaga
3
28
Fig. 4. Transformation of quasi-natural 10-methylindolyl-30-acetaldoxime (26) and
10-methylindolyl-30-acetothiohydroxamic acid (28) to 10-methylglucobrassicin (27)
in rutabaga roots.

M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
1135
tissues we set out to examine de novo metabolic incorporations of
several deuterated (containing three or more deuteria) primary
cation. Flash column chromatography (FCC): silica gel, grade 60,
230–400 m. Organic extracts were dried over Na2SO4 and the sol-
l
precursors: indole,
To this end, the first syntheses and resolution of several perdeuter-
ated - (21) and -tryptophans (1) and - (22) and
-10-methoxytry-
D
- (21) and
L
-tryptophan (1) and derivatives.
vents were removed using a rotary evaporator.
NMR spectra were recorded on Bruker Avance 500 MHz spec-
trometers. For 1H NMR (500 MHz) and 13C NMR (125.8 MHz) spec-
tra, the chemical shifts (d) are reported in parts per million (ppm)
relative to TMS. Fourier transform infrared (FT-IR) data were ac-
quired on a spectrometer and spectra were measured by the dif-
fuse reflectance method on samples dispersed in KBr. MS [high
resolution (HR), electron impact (EI)] were obtained on a VG 70
SE mass spectrometer employing a solids probe.
HPLC analysis was carried out with either Agilent 1100 series or
Hewlett Packard HPLC systems equipped with quaternary pump,
autosampler, diode array detector (DAD, wavelength range 190–
600 nm), degasser, and a Hypersil octadecylsilane (ODS) column
D
L
D
L
ptophans (20) were accomplished in reasonable yields using
modifications of literature procedures. Previous to this work, the
synthesis of L
-10-methoxytryptophan (20) was carried out by con-
densation of a-acetamidoacrylic acid with 1-methoxyindole fol-
lowed by enantioselective enzymatic hydrolysis of the N-acetate
using an acylase (Boger et al., 1999). In this work, we used conden-
sation of DL-serine with 1-methoxyindole (15) in Ac2O/AcOH, fol-
lowed
by
a
stepwise
resolution
of
DL-N-acetyl-10-
methoxytryptophan using two acylases to obtain for the first time
both enantiomers of 10-methoxytryptophan in reasonable overall
yields (58% for
D
(22) and 38% for
L
(20)).
(5
l
m particle size silica, 200 ꢁ 4.6 mm ID), equipped with an in-
HPLC-DAD-HRMS-ESI analysis was used to establish unambigu-
ously the incorporation of deuterated compounds or the biotrans-
formation of quasi-natural compounds by tissues of rutabaga.
[4,5,6,7-2H4]-Indole (14), a more distant precursor than trypto-
phan, was incorporated into all detectable phytoalexins and glu-
line filter. The mobile phase for HPLC-DAD consisted of either:
Method A (phytoalexins and non-polar metabolites), linear gradi-
ent of H2O–CH3CN (3:1 to 1:3 in 35 min, to 0:100 in 5 min) and a
flow rate of 1 mL/min; or Method B (indolyl glucosinolates and
other polar metabolites), linear gradient of H2O-CH3CN (100:0 to
1:1 in 25 min, to 100:0 in 5 min) and a flow rate of 1 mL/min. Sam-
ples were dissolved in CH3CN for Method A, and in H2O–CH3OH
(1:1) for Method B. HPLC-DAD-MS-ESI analysis was carried out
with an Agilent 1100 series HPLC system equipped with an auto-
sampler, binary pump, degasser, and a diode array detector con-
nected directly to a mass detector (Agilent G2440A MSD-Trap-
XCT ion trap mass spectrometer) with an electrospray ionization
(ESI) source. Chromatographic separations were carried out at
cobrassicins, and
percentage than
phytoalexin rapalexin A (13), both
were incorporated to the same extent. These results indicate that
-tryptophan (1) is not a direct precursor of rapalexin A (13), but
is a direct precursor of all other metabolites. The low incorpora-
tions of -tryptophan (21) into several metabolites suggested that
it was enzymatically isomerized to -tryptophan (1) and then
L
-tryptophan (1) was incorporated in larger
-tryptophan (21). However, in the case of the
- (21) and -tryptophan (1)
D
D
L
L
D
L
incorporated into all detectable metabolites. The immediate pre-
cursor of rapalexin A (13) may result from degradation of trypto-
phan (1), but further work needs to be carried out before a
detailed pathway can be proposed.
The absence of detectable incorporation of deuterium labeled
10-methoxytryptophans suggested that the 1-methoxy group of
phytoalexins and indolyl glucosinolate 24 would be introduced
room temperature using an Eclipse XSB C-18 column (5 lm parti-
cle size silica, 150 ꢁ 4.6 mm I.D.). The mobile phase consisted of a
linear gradient of: Method A, 0.2% HCO2H in H2O and 0.2% HCO2H
in CH3CN (3:1 to 1:3 in 35 min, to 0:100 in 5 min) and a flow rate of
1 mL/min; Method B, 0.2% HCO2H in H2O and 0.2% HCO2H in
CH3CN (9:1–1:1 in 25 min, to 90:10 in 5 min) and a flow rate of
1 mL/min. Data acquisition was carried out in positive and nega-
tive polarity modes in a single LC run, data processing using Agilent
Chemstation Software. Samples were dissolved in acetonitrile for
Method A, and in methanol-water (50:50) for Method B. HPLC-
HRMS-ESI was performed on an Agilent HPLC 1100 series directly
connected to a QSTAR XL Systems Mass Spectrometer (Hybrid
Quadrupole-TOF LC/MS/MS) with turbo spray ESI source. Chro-
matographic separation was carried out at room temperature using
downstream of L-tryptophan (1). This result is consistent with the
incorporation of both indolyl-30-acetaldoxime (2) and 10-methoxy-
indolyl-30-acetaldoxime (11) into 1-methoxybrassinin (12) (Pedras
and Montaut, 2004). The lack of transformation of L
-10-methyltry-
ptophan (35) and the transformation of both 10-methylindolyl-30-
acetaldoxime (26) and 10-methylindolyl-30-acetothiohydroxamic
acid (28) into 10-methylglucobrassicin (27), but not into phytoalex-
ins suggested that the post-aldoxime enzymes in the biosynthetic
pathway of indolyl glucosinolates are not substrate-specific. This
conclusion is consistent with previous conclusions that the
post-aldoxime enzymes involved in the biosynthetic pathway of
glucosinolates recognize the functional groups but not the side
chain (aliphatic, phenyl, indolyl) (Halkier and Gershenzon, 2006).
Hence, it would appear that the post-aldoxime enzymes of the gluc-
osinolate pathway are different from the enzymes of the phyto-
alexin pathway. A higher substrate specificity of some enzymes of
the phytoalexin pathway might explain the relatively lower struc-
tural diversity among phytoalexins than among glucosinolates. In
fact, while cruciferous phytoalexins are derived from tryptophan,
glucosinolates are derived from a variety of amino acids, including
aliphatic and aromatic amino acids as well as tryptophan.
a
Hypersil ODS C-18 column (5
l
m
particle size silica,
m particle
200 ꢁ 2.1 mm I.D.) or a Hypersil ODS C-18 column (5
l
size silica, 100 ꢁ 2.1 mm I.D.). The mobile phase consisted of a lin-
ear gradient of: Method A, 0.1% HCO2H in H2O and 0.1% HCO2H in
CH3CN (3:1–1:3 in 35 min, to 0:100 in 5 min) and a flow rate of
0.25 mL/min; Method B, 0.1% HCO2H in H2O and 0.1% HCO2H in
CH3CN (97:3–50:50 in 25 min, to 97:3 in 10 min) and a flow rate
of 0.25 mL/min. Data acquisition was carried out in either positive
or negative polarity mode per LC runs. Data processing was carried
out by Analyst QS Software. Samples were dissolved in CH3CN for
Method A and in CH3OH–H2O (1:1) for Method B.
Rutabaga roots were purchased from local markets according to
freshness and availability.
4.2. Synthesis and characterization of compounds
4. Experimental
4.2.1. Synthesis and resolution of
4.2.1.1. Synthesis. Perdeuterated compounds
tophan (1a) and
-[40,50,60,70-2H4]tryptophan (21a),
D
- and
L
-tryptophans
-[40,50,60,70-2H4]tryp-
-[100,100,100,
-[100,100,100,40,
L
4.1. General experimental procedures and plant material
D
L
40,50,60,70-2H7]-10-methoxytryptophan (20b) and
D
50,60,70-2H7]-10-methoxytryptophan (22b) were prepared from
[4,5,6,7-2H4]indole (14a) (Fig. 2). [4,5,6,7-2H4]Indole (14a) was pre-
pared from the readily available NMR solvent [2,3,4,5,6-2H5]nitro-
All solvents were HPLC grade and used as such, except for THF
(dried over sodium). Unless otherwise noted, materials were ob-
tained from commercial suppliers and used without further purifi-

1136
M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
benzene (99%, 2H5) and 2-chloroacetonitrile using an improved
method, as follows. 10% Pd on carbon (66.7 mg) was added to a solu-
tion of [3,4,5,6-2H4]-2- and [3,4,5,6-2H4]-4-nitrophenylacetonitrile
4.2.2. Synthesis and resolution of
-10-methoxytryptophan (22b)
4.2.2.1. [100,100,100,40,50,60,70-2H7]-10-Methoxytryptophan (20b). DL-Ser-
L
-10-methoxytryptophan (20b) and
D
(ca. 1:10, 200 mg, 1.20 mmol) in EtOAc (20 ml) and AcOH (322
l
l)
ine (102 mg, 0.972 mmol) was added to
a
solution of
and the reaction mixture was stirred under a H2 atmosphere (bal-
loon pressure) at r.t. for 18 h. The catalyst was removed by filtration,
with the filtrate concentrated under reduced pressure. The residue
was dissolved in CH2Cl2 (20 mL), washed with HCl (10 mL ꢁ 2,
1 M), with the organic phase dried (Na2SO4) and concentrated under
reduced pressure to yield [4,5,6,7-2H4]indole (14a, 104 mg, 72%
yield). DL-Serine (173.6 mg, 1.65 mmol) was added to a solution of
[4,5,6,7-2H4]indole (14a, 100 mg, 0.826 mmol) in glacial AcOH
[10,10,10,4,5,6,7-2H7]-10-methoxyindole (14b, 74.8 mg, 0.486 mmol)
(Pedras and Okinyo, 2008) in glacial AcOH (1.4 mL) and Ac2O
(200
lL). The reaction mixture was treated as reported above for
D-
or
L
-tryptophan 21/1 to yield N-acetyl-DL-[100,100,100,40,50,
60,70-2H7]-10-methoxytryptophan (16b, 86.1 mg, 63% yield). Deu-
terated DL-10-methoxytryptophan 22/20 was resolved as reported
above to yield
(20b, 22 mg, 30% yield) and
tryptophan (22b, 18.3 mg, 46% yield). Natural abundance
and
-10-methoxytryptophans (22) to use in control experiments
were synthesized and resolved similarly.
L
-[100,100,100,40,50,60,70-2H7]-10-methoxytryptophan
D
-[100,100,100,40,50,60,70-2H7]-10-methoxy-
- (20)
(2.4 ml) and Ac2O (340
ll) (Konda-Yamada et al., 2002; Yamada
L
et al., 2005). The reaction mixture was heated until reflux began at
78–80 °C, this being maintained for 2 h under Ar atmosphere. The
reaction mixture was allowed to cool to r.t., diluted with toluene
and concentrated. The residue obtained was subjected to FCC
D
4.2.2.2.
L
-[100,100,100, 40,50,60,70-2H7]10-Methoxytryptophan (20b). HPLC-
(CHCl3–CH3OH–AcOH, 100:8:1, v/v) to yield Nb-acetyl-DL
-
DAD Method B, tR = 14.2 min; [
a
]
D = ꢀ35 (c 0.24, CH3OH). 1H NMR
[40,50,60,70-2H4]tryptophan (16a, 170.1 mg, 99% yield).
(CD3OD) d 3.78 (dd, J = 5, 9 Hz, 1H), 3.40 (dd, J = 5, 15 Hz, 1H), 3.07
(dd, J = 9, 15 Hz, 1H). HR-EIMS m/z measured: 241.1447 (241.1444
calcd. for C12H72H7N2O3). EIMS m/z (% relative abundance); 241
[M]+ (6), 167 (100), 149 (16), 134 (22), 121 (9).
4.2.1.2. Resolution. A solution of CoCl2.6H2O (1 mL, 0.2 mg) in
phosphate buffer was added to solution of Nb-acetyl-DL
[40,50,60,70-2H4]tryptophan (16a, 85.0 mg, 0.340 mmol) in potas-
sium phosphate buffer (15.0 ml, pH 7.4). -aminoacylase (0.72 U/
a
-
L
4.2.2.3.
D
-[100,100,100,40,50,60,70-2H7]10-Methoxytryptophan (22b). HPLC-
D = +26 (c 0.24, CH3OH). 1H NMR
mg, 236 mg) was added to the reaction mixture under stirring at
36–37 °C (Konda-Yamada et al., 2002; Yamada et al., 2005). Two
parallel reactions were carried out simultaneously. After 24 h un-
der stirring, the reaction mixture was adjusted to pH 5 with 10%
HCl, and was extracted with EtOAc (20 ml ꢁ 3) and the extracts
of both reactions were combined. The organic layer was dried
(Na2SO4), and concentrated under reduced pressure to yield Nb-
DAD Method B, tR = 14.1 min; [a]
(CD3OD) d 3.79 (dd, J = 5, 9 Hz, 1H), 3.41 (dd, J = 5, 15 Hz, 1H),
3.08 (dd, J = 9, 15 Hz, 1H). HR-EIMS m/z measured: 241.1447
(241.1444 calcd. for C12H72H7N2O3). EIMS m/z (% relative abun-
dance); 241 [M]+ (6), 167 (100), 149 (14), 134 (28), 121 (8).
4.2.2.4.
[100,100,100-2H3]-10-Methoxytryptophan
(20c). DL-Serine
acetyl-
D
-[40,50,60,70-2H4]tryptophan (18a, 105 mg, 62% yield). The
(156.6 mg, 1.49 mmol) was added to a solution of [10,10,10-2H3]-1-
methoxyindole (112 mg, 0.747 mmol) in glacial AcOH (2.2 ml)
aqueous layer was concentrated to dryness and the solid residue
was extracted with CH3OH (8 ml ꢁ 3), with the extract filtered,
and concentrated to dryness. The residue obtained was subjected
and Ac2O (308
ll). The reaction mixture was treated as reported
above for - or L-tryptophan to yield to yield N-acetyl-DL-
D
to RP-FCC (H2O-CH3OH, 95:5, v/v) to yield
L
-[40,50,60,70-2H4]trypto-
[100,100,100-2H3]-10-methoxytryptophan (16c, 156.3 mg, 75% yield).
phan (1a, 50.1 mg, 36% yield) as a white solid. A solution of
Deuterated DL-10-methoxytryptophan 16c was resolved as reported
CoCl2.6H2O (0.5 mL, 0.1 mg) in phosphate buffer was added to a
above to yield
L
-[100,100,100-2H3]-10-methoxytryptophan (20c,
solution of Nb-acetyl-
D
-[40,50,60,70-2H4]tryptophan (18a, 105 mg,
15.3 mg, 30% yield) and D
-[100,100,100-2H3]-10-methoxytryptophan
0.420 mmol) in potassium phosphate buffer (19 ml, pH 7.4).
D
-
(22c, 35.1 mg, 48% yield).
Aminoacylase (8.7 mU/mg, 4 mg) was added to the reaction mix-
ture under stirring at 36 - 37 °C. After 18 h under stirring, the reac-
tion mixture was adjusted to pH 5 with 10% HCl, and was extracted
with EtOAc (15 mL ꢁ 2). The aqueous layer was concentrated to
dryness, the residue was extracted with CH3OH (5 mL ꢁ 4), was fil-
tered and was concentrated to dryness. The residue obtained was
4.2.2.5.
L
-[100,100,100-2H3]10-Methoxytryptophan (20c). HPLC-DAD
Method B, tR = 14.2 min; [
a]
D = ꢀ35 (c 0.21, CH3OH). 1H NMR
(CD3OD) d 7.67 (d, J = 8 Hz, 1H), 7.37 (d, J = 8 Hz, 1H), 7.31 (s,
1H), 7.18 (dd, J = 7.5, 7.5 Hz, 1H), 7.05 (dd, J = 7.5, 7.5 Hz, 1H),
3.80 (dd, J = 5, 9 Hz, 1H), 3.42 (dd, J = 5, 15 Hz, 1H), 3.09 (dd,
J = 9, 15 Hz, 1H). HR-EIMS m/z measured: 237.1189 (237.1192
calcd. for C12H112H3N2O3). EIMS m/z (% relative abundance); 237
[M]+ (7), 163 (100), 145 (15), 130 (24), 117 (8).
subjected to RP-FCC (H2O–CH3OH, 95:5, v/v) to yield
D-
[40,50,60,70-2H4]tryptophan (21a, 41.4 mg, 47% yield).
Non-labeled
D- and L-tryptophans to use in control experiments
were synthesized and resolved similarly.
4.2.2.6.
D
-[100,100,100-2H3]10-Methoxytryptophan (22c). HPLC-DAD
D = +24 (c 0.21, CH3OH). 1H NMR
4.2.1.3.
L
-Tryptophan (1). HPLC-DAD Method B, tR = 11.8 min;
Method B, tR = 14.3 min; [a]
[a
]
D = ꢀ33 (c 0.24, H2O). 1H NMR (D2O) d 7.59 (d, J = 8 Hz, 1H),
(CD3OD) d 7.68 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 7.32 (s,
1H), 7.19 (dd, J = 7.5, 7.5 Hz, 1H), 7.06 (dd, J = 7.5, 7.5 Hz, 1H),
3.81 (dd, J = 5, 9 Hz, 1H), 3.43 (dd, J = 5, 15 Hz, 1H), 3.10 (dd,
J = 9, 15 Hz, 1H). HR-EIMS m/z measured: 237.1193 (237.1193
calcd. for C12H112H3N2O3). EIMS m/z (% relative abundance); 237
[M]+ (7), 163 (100), 145 (15), 130 (25), 117 (9).
7.40 (d, J = 8 Hz, 1H), 7.16 (s, 1H), 7.14 (dd, J = 7.5, 8 Hz, 1H), 7.06
(dd, J = 7.5, 7.5 Hz, 1H), 3.89 (dd, J = 5, 8 Hz, 1H), 3.34 (dd, J = 5,
15 Hz, 1H), 3.15 (dd, J = 8, 15 Hz, 1H). HR-EIMS m/z measured:
204.0896 (204.0898 calcd. for C11H12N2O2). EIMS m/z (% relative
abundance); 204 [M]+ (6), 130 (100).
4.2.1.4.
[a]
D
-Tryptophan (21). HPLC-DAD Method B, tR = 11.9 min;
4.2.3. Synthesis of 10-methylindolyl-30-acetaldoxime (26) and 10-
methylindolyl-30-acetothiohydroxamic acid (28)
D = +29 (c 0.10, H2O). 1H NMR (D2O) d 7.59 (d, J = 8 Hz, 1H),
7.40 (d, J = 8 Hz, 1H), 7.16 (s, 1H), 7.14 (dd, J = 7.5, 8 Hz, 1H), 7.06
(dd, J = 7.5, 7.5 Hz, 1H), 3.89 (dd, J = 5, 8 Hz, 1H), 3.34 (dd, J = 5,
15 Hz, 1H), 3.15 (dd, J = 8, 15 Hz, 1H). HR-EIMS m/z measured:
204.0898 (204.0898 calcd. for C11H12N2O2). EIMS m/z (% relative
abundance); 204 [M]+ (6), 130 (100).
4.2.3.1. 10-Methylindolyl-30-acetaldoxime (26). A solution of DIBAL-
H (790 lL, 1.18 mmol) in toluene was added drop wise to a solu-
tion of 1-methylindolyl-3-acetonitrile (100 mg, 0.588 mmol) in
dry toluene (6.5 mL) cooled to ꢀ78 °C under Ar atmosphere. The
reaction mixture was allowed to stir at ꢀ78 °C for 10 min, was

M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
1137
diluted with ice-cold HCl (10 mL, 2 M) and immediately extracted
with EtOAc (30 mL ꢁ 2). The combined organic extracts were
washed with H2O (10 mL ꢁ 2), dried (Na2SO4) (Pedras and Okinyo,
2006), and was concentrated under reduced pressure to yield 1-
methylindolyl-3-acetaldehyde which was used for the next step
(120 mg, 0.594 mmol) in CH3OH (9 mL). The reaction mixture was
cooled to 0 °C and NaBH4 (144.1 mg, 6.45 mmol) was added in one
portion (Kutschy et al., 1997, 1998). After stirring for 10 min, the
black precipitate that formed was removed by filtration with
the filtrate concentrated to about a third of the original volume.
The remaining mixture was poured into H2O (20 mL) containing
aqueous NH3 (1 mL, 28%), and extracted with EtOAc (30 mL ꢁ 2).
The combined organic extracts were dried (Na2SO4) and concen-
trated, with the crude mixture subjected to FCC (CH2Cl2–CH3OH–
NH4OH 28%, 92:8:1, v/v) to yield 1-acetylindol-3-ylmethanamine
(78 mg). A solution of 1-acetylindol-3-ylmethanamine (78 mg) in
CH2Cl2 (1.5 mL) was added drop wise (ca. 8 min) to a vigorously
without purification.
A
solution of HONH2ꢂHCl (104.3 mg,
1.50 mmol) and CH3COONa (123.1 mg, 1.50 mmol) in H2O (1 mL)
was added to a cooled solution (0 °C) of 1-methylindolyl-3-acetal-
dehyde in EtOH (8.5 mL) under stirring. The reaction mixture was
allowed to stir at 1 °C for 10 min, and then at r.t. for 15 min, was
concentrated, the residue was taken in H2O (15 mL), and was ex-
tracted with EtOAc (15 mL ꢁ 4). The combined organic extract
was dried (Na2SO4) and concentrated under reduced pressure. Sep-
aration by FCC (CH2Cl2-CH3OH, 98:2, v/v) yielded 10-methylindol-
yl-30-acetaldoxime (26, 73.3 mg, 66% yield).
stirred mixture of Cl2CS (31 lL, 0.41 mmol) and CaCO3 (53 mg,
0.52 mmol) in CH2Cl2–H2O (1.5:3 mL, Kutschy et al., 1997, 1998).
After being stirred for 5 min the two layers were separated and
the aqueous solution was extracted with CH2Cl2 (10 mL ꢁ 3). The
combined organic extract was filtered over charcoal, the solvent
was removed under reduced pressure and the residue was sepa-
rated by FCC (CH2Cl2) to yield 10-acetylindolyl-30-methyl isothiocy-
anate (32, 42 mg, 31% yield from 1-acetylindole-3-carboxaldehyde
oxime).
HPLC-DAD Method A, two peaks at tR = 12 and 11.3 min. 1H
NMR (500 MHz CD3CN) d 8.83 (s, OH), 7.54 (d, J = 8 Hz, 1H), 7.35
(d, J = 8 Hz, 1H), 7.20 (dd, J = 7.5, 7.5 Hz, 1H), 7.07 (dd, J = 7.5,
7.5 Hz, 1H), 7.03 (s, 1H), 6.81 (dd, J = 5.5, 5.5 Hz, 1H), 3.75 (d,
J = 5.5 Hz, 1H), 3.74 (s, 3H), 3.58 (d, J = 6 Hz, 1H). 13C NMR
(125.8 MHz CD3CN): d 150.3, 137.1, 127.7, 127.3, 121.5, 118.7,
118.6, 109.5, 32.0, 25.3, 20.7. HR-EIMS m/z measured 188.0955
(188.0950 calcd. for C11H12N2O). EIMS m/z (% relative abundance)
188 [M]+ (56), 171 (22), 144 (100), 131 (14).
HPLC-DAD Method A, tR = 24.9 min. 1H NMR (500 MHz CDCl3) d
8.46 (d, J = 8 Hz, 1H), 7.55 (s, 1H), 7.56 (d, J = 8 Hz, 1H), 7.47 (dd,
J = 8, 8 Hz, 1H), 7.36 (dd, J = 7.5, 7.5 Hz, 1H), 4.85 (s, 2H), 2.66 (s,
3H). 13C NMR (125.8 MHz CDCl3): d 168.6, 136.4, 134.7, 128.5,
126.4, 124.4, 123.6, 118.9, 117.2, 116.6, 41.1, 24.2. HR-EIMS m/z
measured 230.0520 (230.0514 calcd. for C12H10N2OS). EIMS m/z
(% relative abundance) 230 [M]+ (18), 172 (32), 130 (100), 102 (7).
4.2.3.2. 10-Methylindolyl-30-acetothiohydroxamic acid (28). NaH
(110 mg, 2.76 mmol) and ICH3 (172 lL, 2.76 mmol) were added
to a cooled solution (0 °C) of indole-3-carboxaldehyde (200 mg,
1.38 mmol) in dry THF (20 mL) with stirring under an Ar atmo-
sphere. After 45 min of stirring at 0 °C, the reaction mixture was
poured into ice-cold H2O (30 ml), extracted with EtOAc
(30 mL ꢁ 3), the combined organic extracts washed with brine,
dried (Na2SO4) and concentrated under reduced pressure. The
crude mixture was subjected to FCC (CH2Cl2–CH3OH, 100:0-98:2,
v/v) to yield 1-methylindole-3-carboxaldehyde (209 mg, 95%). 10-
Methylindolyl-30-acetothiohydroxamic acid (28) was prepared
from 1-methylindole-3-carboxaldehyde (200 mg, 1.26 mmol) in
4.2.3.4. 1-(tert-Butoxycarbonyl)indol-3-ylmethylisothiocyanate (29).
Prepared as previously reported (Kutschy et al., 1997, 1998). HPLC
Method A, tR = 37.6 min. 1H NMR (500 MHz CDCl3) d 8.18 (d,
J = 6.5 Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 8 Hz, 1H), 7.40 (dd, J = 7.5,
7.5 Hz, 1H), 7.33 (dd, J = 7.5, 7.5 Hz, 1H), 4.82 (s, 2H), 1.69 (s, 9H).
HR-EIMS m/z measured 288.0937 (288.0933 calcd. for
C15H16N2O2S). EIMS m/z (% relative abundance) 288 [M]+ (10),
232 (22), 205 (9), 174 (21), 130 (64), 57 (100).
CH3NO2 (547 lL, 10.1 mmol) as previously reported for indolyl-3-
acetothiohydroxamic acid (5) (Pedras and Okinyo, 2008) to yield
10-methylindolyl-30-acetothiohydroxamic acid (28, 71.8 mg, 67%
yield) as brownish oil which solidified on cooling to 5 °C. HPLC-
HRMS-ESI Method A, tR = 17.2 min. 1H NMR (500 MHz CDCl3): d
7.52 (d, J = 8 Hz, 1H), 7.37 (d, J = 8 Hz, 1H), 7.31 (dd, J = 7.5, 8 Hz,
1H), 7.18 (dd, J = 7.5, 7.5 Hz, 1H), 7.03 (s, 1H), 4.24 (s, 2H), 3.82
(s, 3H). 13C NMR (125.8 MHz CDCl3): d 137.2, 128.9, 127.1, 122.6,
120.2, 118.7, 109.8, 104.8, 36.5, 32.9 (thiocarbonyl carbon at ca.
189 ppm not detected). HRMS-EI m/z measured 220.0671
(220.0670 calcd. for C11H12N2OS). EIMS m/z (% relative abundance)
220 [M]+ (8), 188 (11), 169 (90), 144 (100), 128 (12), 115 (11).
4.2.4. Synthesis of 1-acetylspirobrassinin (34) and 1-tert-
butoxycarbonylspirobrassinin (31)
4.2.4.1. 1-Acetylspirobrassinin (34). Pyridine (500
lL) was added to
a stirred solution of spirobrassinin (23, 5 mg) in Ac2O (500
lL).
After stirring for 60 min, the reaction mixture was diluted with tol-
uene and concentrated under reduced pressure to yield 1-acetyl-
spirobrassinin (34, 4.8 mg, 80% yield).
HPLC-DAD Method A, tR = 16.9 min. 1H NMR (500 MHz CD3CN)
d 8.15 (d, J = 8 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.39 (dd, J = 8,
8 Hz, 1H), 7.28 (dd, J = 7.5, 7.5 Hz, 1H), 4.66 (d, J = 15.5 Hz, 1H),
4.49 (d, J = 15.5 Hz, 1H), 2.60 (s, 3H), 2.58 (s, 3H). 13C NMR
(125.8 MHz CD3CN): d 177.3, 170.9, 162.7, 139.6, 129.9, 129.3,
125.9, 123.9, 116.2, 75.7, 65.2, 25.9, 14.9. HR-EIMS m/z measured
292.0339 (292.0340 calcd. for C13H12N2O2S2). EIMS m/z (% relative
abundance) 292 [M]+ (100), 250 (70), 203 (26), 177 (100), 145 (38),
117 (34).
4.2.3.3. 1-Acetylindol-3-ylmethylisothiocyanate (32). Pyridine (4 mL)
was added to a stirred suspension of indole-3-carboxaldehyde
(200 mg, 1.38 mmol) in Ac2O (4 mL). After 90 min, the reaction
mixture was diluted with toluene and concentrated to yield 1-ace-
tylindole-3-carboxaldehyde (253 mg, 98%) which was used in the
next step without purification.
A
solution of HONH2ꢂHCl
4.2.4.2. 1-(tert-Butoxycarbonyl)spirobrassinin (31). Prepared as pre-
viously reported (Kutschy et al., 2002). HPLC-DAD Method A,
tR = 30.1 min. 1H NMR (500 MHz CD3CN) d 7.80 (d, J = 8 Hz, 1H),
7.42 (d, J = 7.5 Hz, 1H), 7.38 (dd, J = 8, 8 Hz, 1H), 7.23 (dd, J = 7.5,
7.5 Hz, 1H), 4.62 (d, J = 15.5 Hz, 1H), 4.47 (d, J = 15.5 Hz, 1H), 2.59
(s, 3H), 1.59 (s, 9H). 13C NMR (125.8 MHz CD3CN): d 174.7, 162.5,
148.8, 139.1, 129.9, 128.8, 125.2, 124.0, 115.1, 84.4, 75.6, 64.9,
27.0, 14.9. HR-EIMS m/z measured 350.0767 (350.0759 calcd. for
C16H18N2O3S2). EIMS m/z (% relative abundance) 350 [M]+ (7),
250 (61), 203 (15), 177 (31), 149 (19), 116 (11), 57 (100).
(74.4 mg, 17 mmol) and CH3COONa (87.8 mg, 17 mmol) in H2O
(2.5 mL) was added to a solution of 1-acetylindole-3-carboxalde-
hyde (200 mg, 17 mmol) in EtOH (17 mL), the resulting mixture
was heated for 10 min (47–50 °C) with stirring. The reaction mix-
ture was concentrated under reduced pressure, the residue dis-
solved in H2O (10 mL), extracted with Et2O (30 mL ꢁ 3), with the
combined organic extract was dried (Na2SO4) and concentrated
to yield 1-acetylindole-3-carboxaldehyde oxime (182.1 mg, 84%).
A solution of NiCl2ꢂH2O (141 mg, 0.594 mmol) in CH3OH (6 mL)
was added to a solution of 1-acetylindole-3-carboxaldehyde oxime

1138
M. S. C. Pedras et al. / Phytochemistry 70 (2009) 1129–1138
Bender, J., Celenza, J.L., 2009. Indolic glucosinolates at the crossroads of tryptophan
metabolism. Phytochemistry Reviews 8, 25–37.
4.3. Biosynthesis: precursor administration experiments
Boger, D.L., Keim, H., Oberhauser, B., Schreiner, E.P., Foster, C.A., 1999. Total
Synthesis of HUN-7293. Journal of the American Chemical Society 121, 6197–
6205.
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Goss, R.J.M., Lanceron, S.E., Wise, N.J., Moss, S.J., 2006. Generating rapamycin
analogues by directed biosynthesis: starter acid substrate specificity of mono-
substituted cyclohexane carboxylic acids. Organic and Biomolecular Chemistry
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Science 11, 89–100.
Halkier, B.A., Gershenzon, J., 2006. Biology and biochemistry of glucosinolates.
Annual Review Plant Biology 57, 303–333.
4.3.1. Precursor administration and extraction of rutabaga
phytoalexins
Rutabaga (B. napus ssp. rapifera) root tubers were cut horizon-
tally in 10–15 mm thick slices and cylindrical holes (16 mm in
diameter) were made on one side of the discs with a cork-borer
(Pedras et al., 2004). The slices were kept in tightly sealed plastic
boxes and incubated at 20 °C in darkness. After 24 h, the slices
were UV-irradiated on the surface with holes for 1 h, and were
incubated for further 24 h. Compounds (5 ꢁ 10ꢀ4 M) were
dissolved in H2O in the case of tryptophans 1 and 21, in H2O–
CH3OH–Tween 80 (95/5/0.05, v/v) in the case of 1-methylindolyl-
3-acetaldoxime (26), or H2O–CH3OH (90/10, v/v) for all other
compounds. A solution of each compound was pipetted into each
Hanley, A.B., Parsley, K.R., 1990. Identification of 1-methoxyindolyl-3-methyl
isothiocyanate as an indole glucosinolate breakdown product. Phytochemistry
29, 769–771.
Hanley, A.B., Parsley, K.R., Lewis, J.A., Fenwick, G.R., 1990. Chemistry of indole
glucosinolates: intermediacy of indol-3-ylmethyl isothiocyanates in the
enzymic hydrolysis of indole glucosinolates. Journal of the Chemical Society,
Perkin Transactions 1: Organic and Bio-Organic Chemistry 8, 2273–2276.
Konda-Yamada, Y., Okada, C., Yoshida, K., Umeda, Y., Arima, S., Sato, N., Kai, T.,
hole (500 ll/hole) and the discs were further incubated at 20 °C
in darkness. Following adsorption of compound solution, the holes
were filled with distilled H2O. The aqueous solution in holes of
each slice was collected after 72 h incubation and was extracted
with EtOAc. The combined organic extracts were dried (Na2SO4)
and the solvent removed under reduced pressure. The residue ob-
Takayanagi, H., Harigaya, Y., 2002. Convenient synthesis of 70 and 60 bromo-
D-
tryptophan and their derivatives by enzymatic optical resolution using D-
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Kutschy, P., Achbergerova, I., Dzurilla, M., Takasugi, M., 1997. Synthesis of indole
phytoalexins brassinin and cyclobrassinin via [1-(tert-butoxycarbonyl)indol-3-
yl]methyl isothiocyanate as the key biomimetic intermediate. Synlett 3, 289–
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tained was dissolved in CH3CN (400 ll per slice) and analyzed by
Kutschy, P., Dzurilla, M., Takasugi, M., Torok, M., Achbergerova, I., Homzova, R.,
Racova, M., 1998. New syntheses of indole phytoalexins and related
compounds. Tetrahedron 54, 3549–3566.
Kutschy, P., Suchy, M., Monde, K., Harada, N., Maruskova, R., Curillova, Z., Dzurilla,
M., Miklosova, M., Mezencev, R., Mojzis, J., 2002. Spirocyclization strategy
toward indole phytoalexins. The first synthesis of ( )-1-methoxyspirobrassinin,
( )-1-methoxyspirobrassinol, and ( )-1-methoxyspirobrassinol methyl ether.
Tetrahedron Letters 43, 9489–9492.
HPLC-DAD. The phytoalexin containing fractions were analyzed
further by HPLC-MS-ESI and HPLC-HRMS-ESI. Control experiments
were similarly carried out by incubating rutabaga root tubers with
non-labeled precursors or with solvent only.
4.3.2. Extraction of rutabaga glucosinolates
Tissue around the holes was cut (ca. 0.5 cm thickness), soaked
in MeOH, was ground and extracted with an aqueous CH3OH solu-
tion. Extraction was carried out overnight on a shaker at 170 rpm,
after which the tissue was removed by filtration and the filtrate
concentrated. The residue obtained was subjected to RP-FCC
H2O–CH3CN (100:0; 90:10, 80:20, v/v) to yield three fractions con-
taining a mixture of indole glucosinolates. The fractions were dis-
solved in H2O–CH3OH (50:50) and were analyzed by HPLC-DAD,
HPLC-MS-ESI, and HRMS-ESI.
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Support for the authors’ work was obtained from the Natural
Sciences and Engineering Research Council of Canada (Discovery
Research Grant and AGENO Grant to M.S.C.P.) and the University
of Saskatchewan (graduate assistantship to D.P.O.O.). We acknowl-
edge the technical assistance of K. Brown, Department of Chemis-
try, in NMR determinations.
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