Induction of clovamide by jasmonic acid in red clover

Article abstract of DOI:10.1016/S0031-9422(00)00098-4

The effect of jasmonic acid (JA) on the secondary metabolism of 5-day- old red clover seedlings was investigated. Induction of the formation of four compounds was found in roots after treatment with 50 μM JA for 48 h, while no induction was observed in the shoots. These compounds, whose formation was induced by JA addition, were isolated and identified as caffeoyl DOPA (clovamide), caffeoyltyrosine, p-coumaroyl DOPA and p-coumaroyltyrosine, by ion-spray MS and ¹H NMR analyses, and by chemical synthesis. Among them, clovamide was the most abundant, while the other amides represented only a minor portion. Clovamide started to increase in amount 24-36 h after treatment and reached a maximum after 96 h (2.81 nmol/mg fr. wt.). The induction of their formation was observed even with 5 μM of JA, and the amount increased with concentrations up to 100 μM. Treatment with 1 mM CuCl₂, which elicits accumulation of the phytoalexin maackiain in red clover, caused a decrease in clovamide amount. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00098-4

Phytochemistry 54 (2000) 387±392
www.elsevier.com/locate/phytochem
Induction of clovamide by jasmonic acid in red clover
a, , 1
Shin-ichi Tebayashi * , Atsushi Ishihara , Mitsuya Tsuda , Hajime Iwamura
a,1
b
a,1
a
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Division of Environmental Sciences and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b
Received 7 July 1999; received in revised form 25 January 2000
Abstract
The e€ect of jasmonic acid (JA) on the secondary metabolism of 5-day-old red clover seedlings was investigated. Induction of
the formation of four compounds was found in roots after treatment with 50 mM JA for 48 h, while no induction was observed
in the shoots. These compounds, whose formation was induced by JA addition, were isolated and identi®ed as ca€eoyl DOPA
(
clovamide), ca€eoyltyrosine, p-coumaroyl DOPA and p-coumaroyltyrosine, by ion-spray MS and 1H NMR analyses, and by
chemical synthesis. Among them, clovamide was the most abundant, while the other amides represented only a minor portion.
Clovamide started to increase in amount 24±36 h after treatment and reached a maximum after 96 h (2.81 nmol/mg fr. wt.). The
induction of their formation was observed even with 5 mM of JA, and the amount increased with concentrations up to 100 mM.
2
Treatment with 1 mM CuCl , which elicits accumulation of the phytoalexin maackiain in red clover, caused a decrease in
clovamide amount. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Trifolium pratense; Leguminosae; Red clover; Hydroxycinnamic acid amide; Clovamide; Jasmonic acid; Induction
1
. Introduction
rice phytoalexins, oryzalexins, momilactones, and
sakuranetin, in leaves and cell cultures of the plant
(Nojiri et al., 1996; Kodama, 1996). More recently, ac-
cumulation of a hydroxycinnamic acid amide, p-cou-
maroylagmatine, a precursor of antifungal hordatine
compounds, was reported in barley leaves following
MeJA treatment (Lee et al., 1997).
Jasmonic acid (JA) and its methyl ester (MeJA) are
widely distributed in the plant kingdom. It has been
suggested that JA acts as a signal molecule in response
to various environmental stimuli including wounding
and infection by pathogens, on the basis of accumu-
lation of JA under stress conditions, and activation of
stress-related genes and enzymes after exogenous JA/
MeJA treatments (Sembdner and Parthier, 1993; Creel-
man and Mullet, 1997). Treatments with JA/MeJA
also a€ect secondary metabolism in plants. Gundlach
et al. (1992) described induction of formation of sec-
ondary metabolites by treatment with MeJA in cell
suspension cultures of 36 species of mono- and dicoty-
ledonous plants. JA strongly elicits the production of
In red clover (Trifolium pratense L.), a number of
secondary metabolites have been identi®ed. Among
them, maackiain has been characterized as a pterocar-
panoid phytoalexin (Higgins and Smith, 1972), and its
glucoside and malonyl glucoside occur as constitutive
components. Iso¯avonoids such as formononetin and
biochanin A have been detected in both free and con-
jugated forms, and formononetin has been regarded as
a precursor of maackiain (Dewick, 1975, 1977; Dewick
and Ward, 1978). Another class of characteristic sec-
ondary metabolites in red clover include the hydroxy-
cinnamic acid amides conjugated to aromatic amino
acids. For example, ca€eoyl DOPA (clovamide) has
been isolated (Yoshihara et al., 1977), although no bio-
*
Corresponding author. Fax: +81-75-753-6408.
E-mail address: tebayasi@kais.kyoto-u.ac.jp (S. Tebayashi).
CREST, Japan Science and Technology Corporation (JST),
1
Japan.
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00098-4

3
88
S. Tebayashi et al. / Phytochemistry 54 (2000) 387±392
logical function was elucidated. In this study, we inves-
tigated the e€ect of JA on secondary metabolism in
red clover, including biosynthesis of pterocarpanoids,
iso¯avonoids and hydroxycinnamic acid amides, in
order to better understand the role of JA in the defen-
sive systems of red clover.
larger amount than in the roots, although the over all
magnitude was little a€ected by treatment with JA.
Compounds 1 and 4 were isolated from 5-day-old
seedlings by three chromatographic steps, and 2 and 3
were puri®ed as a mixture in the same manner. The
structures of the compounds were identi®ed by use of
1
ion spray-MS and H NMR spectroscopy. Pseudomo-
+
lecular ions, [M+H] , in positive ion spray-MS spec-
2
. Results
tra, revealed the molecular weights of compounds 1±4
to be m/z 359, 343, 343 and 327, respectively. Com-
pounds 1 and 2 gave a diagnostic ion at m/z 163,
whereas 3 and 4 gave an ion at m/z 147, suggesting
that 1 and 2 have a ca€eoyl moiety, and that 3 and 4
have a p-coumaroyl moiety, respectively. The presence
of a ca€eoyl moiety in 1 and 2, and a p-coumaroyl
E€ects of JA on the composition of secondary
metabolites in red clover (Trifolium pratense L.) were
examined using 5-day-old seedlings. Shoots and roots
were separately extracted with MeOH containing 2%
HOAc, and the extracts were analyzed by reversed
phase HPLC (Fig. 1). In the extracts from roots trea-
ted with 50 mM JA for 48 h after wounding, increases
in the size of three peaks at Rt 3.95 min (1), Rt 5.20
1
moiety in 3 and 4, was con®rmed by H NMR spec-
1
troscopy. In the H NMR spectra of 1 and 2, the pre-
sence of a trans double bond and a 1,3,4-trisubstituted
benzene ring was assigned to the ca€eoyl moiety,
whereas, in 3 and 4, the trans double bond and a 1,4-
disubstituted benzene ring were assigned to the p-cou-
(
2+3) and Rt 7.35 (4) were observed. Neither wound-
ing alone nor JA treatment on intact roots caused any
changes. In intact shoots, these peaks were found in a
1
maroyl moiety. The rest of the signals in the H NMR
spectra of 1 and 3, including methine, methylene and
1
,3,4-trisubstituted benzene ring resonances, indicated
a 3,4-dihydroxyphenylalanine (DOPA) structure; in an
analogous manner, methine, methylene and 1,4-disub-
1
stituted benzene ring resonances in the H NMR spec-
tra of 2 and 4 revealed the presence of a tyrosine
moiety. The chemical shift of the proton connected to
the nitrogen atom established an amide linkage of the
hydroxycinnamic acids and amino acids in each case.
From these data, 1±4 were identi®ed as hydroxycin-
namic acid amides, ca€eoyl DOPA (clovamide), caf-
feoyltyrosine,
p-coumaroyl
DOPA
and
p-
coumaroyltyrosine, respectively. Finally, the identity of
each amide was established by chemical synthesis from
Fig. 1. HPLC chromatograms of the extracts from intact roots (a),
wounded roots (b), intact roots treated with 50 mM JA (c) and
wounded roots treated with 50 mM JA (d). 1: ca€eoyl DOPA (clova-
mide), 2: ca€eoyltyrosine, 3: p-coumaroyl DOPA, 4: p-coumaroyltyr-
osine, MaG: maackiain 7-O-glucoside, MaGM: maackiain 7-O-
glucoside-60-malonate, F: formononetin, FG: formononetin 7-O-glu-
coside, FGM: formononetin 7-O-glucoside-60-malonate.
Fig. 2. Time course of the accumulation of amide compounds in
wounded roots treated with 50 mM JA (.) and untreated (w). (a):
ca€eoyl DOPA (clovamide) (1), (b): ca€eoyltyrosine (2) plus p-cou-
maroyl DOPA (3), (c): p-coumaroyltyrosine (4). Each data point
expresses the mean of four experiments.

S. Tebayashi et al. / Phytochemistry 54 (2000) 387±392
389
the corresponding hydroxycinnamic acids and amino
acids using dicyclohexylcarbodiimide.
function of various JA concentrations (Fig. 3). The
amount of each compound was determined 48 h after
treatment. The induction of 1 was observed even with
5 mM JA, and the amount increased with JA concen-
tration up to 100 mM (1.20 nmol/mg fr. wt.). At 500
mM, the amount of 1 decreased to a level as low as
that in the control roots. The dose-response curves for
other amides were similar to that for 1, though the
amounts were small.
Various substances were ®nally tested for induction
of accumulation of clovamide and maackiain. In this
regard, JA addition resulted in the increased accumu-
lation of 1 but not maackiain, whereas hexa-N-acetyl-
chitohexaose, chitosan, laminarin, and salicylic acid
were all ine€ective on increasing the levels of both 1
and maackiain. Copper chloride, on the other hand,
caused the decrease of 1 to an undetectable level at 1
mM, although it elicited the increased production of
maackiain (0.57 nmol/mg fr. wt.).
The e€ects of JA were also investigated as regards
e€ects on the amounts of maackiain, maackiain 7-O-
glucoside, maackiain 7-O-glucoside-60-malonate, for-
mononetin, formononetin 7-O-glucoside and formono-
netin 7-O-glucoside-60-malonate (Fig. 1). Maackiain
was not detected in either JA-treated or untreated
seedlings. Although other compounds were found in
varying amounts, the changes in relative amounts were
minor as an e€ect of JA treatment.
Changes in the amounts of amides following treat-
ment with 50 mM JA were next investigated (Fig. 2).
The amount of 1 started to increase after a 24-h lag
period and reached a maximum 96 h post-treatment
(
2.81 nmol/mg fr. wt.), being ca. 10 times greater than
in control roots. Thereafter, compound 1 decreased to
0% of the maximum after 120 h. Compounds 2 and 3
8
were quanti®ed as a mixture, where their over all ac-
cumulation varied in a fashion similar to 1, although
the amount (0.54 nmol/mg fr. wt. as mixture) was ap-
proximately one-®fth that of 1 at 96 h. The level of
compound 4 also increased slightly after JA-treatment
3. Discussion
(
0.20 nmol/mg fr. wt. at 48 h), and remained at a con-
In the present study, it was demonstrated that ac-
cumulation of clovamide congeners (1±4) is increased
by JA treatment in roots of red clover. Compound 1
has been identi®ed as a constitutive component
(Yoshihara et al., 1977), but for 2±4 this is the ®rst
report of their occurrence in this plant. Hydroxycin-
namic acids linked to an amine function are common
types of hydroxycinnamic acid conjugates (Strack,
stant level thereafter up to 120 h. On the other hand,
the amounts of 1±4 in roots wounded, but not treated
with JA, did not change throughout the time period
examined.
The dependency of the induced formation of hydro-
xycinnamic acid amides was next investigated, as a
1
997), but amides with aromatic amino acids have
been found in a relatively few plants (Van Heerden et
al., 1980; Cli€ord et al., 1988). Hydroxycinnamic acid
amides have been implicated in inducible defense sys-
tems of plants. Avenanthramides, a series of substi-
tuted
characterized as oat phytoalexins (Mayama et al.,
981; Miyagawa et al., 1995). In Solanaceous plants,
N-cinnamoylanthranilates,
have
been
1
such as potato (Clarke, 1982), tobacco (Negrel and
Martin, 1984) and tomato (Pearce et al., 1998), the
production of N-hydroxycinnamoyltyramine is acti-
vated in response to various environmental stimuli.
Moreover, increased accumulation of p-coumaroyl-
and feruloylagmatines, precursors of the antifungal
compounds known as hordatines, was demonstrated
after fungal infection in barley (Peipp et al., 1997).
Interestingly, their increased accumulation was repro-
duced by treatments with JA (Lee et al., 1997),
suggesting the involvement of MeJA in their inducible
defense systems. This situation is very similar to our
®ndings of the induced levels of clovamide by JA-
treatment, regarding the chemical structure of the
induced compounds and the activity of JA. It is thus
presumed that the induction of clovamide accumu-
lation is a part of the root defense system of red clo-
Fig. 3. E€ects of the concentration of JA on the accumulation of
amides in red clover roots. Seedlings were treated with JA at various
concentrations for 48 h. Each data point expresses the mean of ®ve
experiments.

3
90
S. Tebayashi et al. / Phytochemistry 54 (2000) 387±392
ver, in a manner analogous to hydroxycinnamic acid
amides in other plants.
Diverse stimuli are known to induce formation of
hydroxycinnamic acid amides in various plants;
these include wounding (Negrel et al., 1993), chitoo-
ligosaccharides (Bordin et al., 1991), laminarin
(
(
Miyagawa et al., 1998), and heavy metal ions
Fink et al., 1990). In red clover, wounding alone
did not increase the levels of 1, although wounding
seemed to be necessary for induced formation of 1
by JA. Furthermore, the levels of 1 were not
increased by the above elicitors, indicating that the
stimulus which triggers the production of amide
compounds is species-dependent. It is of particular
4
. Experimental
interest that CuCl , which elicited accumulation of
2
4
.1. General
maackiain, caused a decrease in amounts of 1, and no
increased levels of maackiain occurred following JA
treatment. A signaling role for JA in phytoalexin in-
duction has been proposed in opium poppy, lettuce
and rice (Gundlach et al., 1992; Nojiri et al., 1996;
Kodama, 1996). However, our ®ndings suggest that
the stimulation of amide production by JA is indepen-
dent from the induction of formation of the pterocar-
panoid phytoalexins in red clover. The mechanism
1H NMR spectra were recorded on a Bruker AC-
00 spectrometer in DMSO-d solution using TMS as
3
6
an internal standard. Positive ion-spray ionization
mass spectra were obtained using Perkin-Elmer-Sciex
API-165 instrument (ion-spray voltage: 5 kV, ori®ce
voltage: 30 V, nebulizer gas: air, curtain gas: nitrogen)
combined with
equipped with an ODS column (Wakosil-II 5C18 HG,
150 mm  4.6 mm). The following HPLC conditions
were used: linear gradient: 0±65% B/A for 35 min (sol-
vent A: H O±TFA (999:1, v/v), solvent B: MeCN);
2
¯
a Shimadzu 10A HPLC system
leading to the decrease of 1 after CuCl treatment is
2
unclear. The oxidative polymerization of 1 seems to be
a possible explanation, because 1 could be in contact
with oxidative enzymes, which were localized to di€er-
ent compartments, under conditions where the integ-
rity of the membranes was lost by the toxic e€ects of
ow rate: 0.8 ml/min.
4.2. Plant material and analysis of secondary
metabolites
CuCl (Doncheva, 1998).
2
Some of the hydroxycinnamic acid amides, such as
avenanthramides (Mayama et al., 1981) and p-coumar-
oylagmatine (Stoessl and Unwin, 1970), display toxic
e€ects against pathogens. However, in our preliminary
experiments, 1±4 exhibited little antifungal activity
against Curvularia trifolii, Biopolaris oryzae, and Colle-
totrichum lagenarium up to 0.5 mM (data not shown).
Accordingly, compounds 1±4 may serve as substrates
for reinforcement of cell walls via formation of pheno-
lic polymers. Except for the minor component 4, they
have catechol groups that are easily oxidized by either
enzymatic or chemical reactions. Peroxidases are puta-
tive enzymes responsible for the incorporation of
amides into cell walls, whose activities are reportedly
induced by JA treatment (Moons et al., 1997; Curtis et
al., 1997). Incorporation of hydroxycinnamic acid
amides into cell walls has also been reported in
tobacco (Negrel and Lherminier, 1987), potato (Keller
et al., 1996) and opium poppy (Facchini, 1998). In
light of these facts, the incorporation of clovamide and
related compounds into cell wall components is under
investigation, as well as the spatial and temporal corre-
lation between oxidative enzyme activity and amide
production.
Red clover seeds (Trifolium pratense L. cv. Kenland,
Yukijirushi Shubyo, Japan) were sown on moistened
germination paper and maintained at 258 with a 12-h
period of illumination from ¯uorescent lamps (60 W
� 2
m
) in growth chambers. After 120-h incubation, the
roots of red clover seedlings were wounded using car-
borundum. The seedlings were immediately placed on
a 24-well tissue culture plate (10 mm  16 mm) con-
taining 0.5 ml of aqueous JA (Wako, Japan) solution
per well. After incubation at 258 for 48 h, the seedlings
were separated into roots and shoots, and extracted
with 0.5 ml of HOAc±McOH (2:98, v/v) under ultraso-
nication. The extracts were subjected to reversed phase
HPLC analysis (column: Wakosil-II 5C18 HG, 150
mm  4.6 mm, Wako, Japan). The following HPLC
conditions were used: linear gradient: 20±65% B/A for
35 min (solvent A: HOAc±H O (3:97, v/v), solvent B:
2
MeCN); ¯ow rate: 0.8 ml/min; detection: 280 nm.
4.3. Puri®cation of hydroxycinnamic acid amides
Red clover seedlings (83.6 g, 5-day-old) were
extracted three times with 300 ml of HOAc±MeOH

S. Tebayashi et al. / Phytochemistry 54 (2000) 387±392
391
(
2:98, v/v) under ultrasonication. The combined
mm  20 mm) using H O±MeCN (77:23, v/v) as sol-
2
extract, after concentration to ca. 100 ml, was washed
with hexane and fractionated with an ODS column
(
Japan) by eluting with H O and MeOH (500 ml each).
The MeOH fraction which contained hydroxycinnamic
acid amides was fractionated on a polyamide column
vent (yield 33.4%).
Ion-spray MS, m/z (rel. int.): 360 [M+H] (52), 163
+
1
Cosmosil 75C -OPN, 50 g, 81 mm  40 mm, Nacalai,
(100); H NMR spectral data (300 MHz, DMSO-d ): d
1
8
6
2.72 (1H, dd, J = 13.9, 9.2 Hz), 2.90 (1H, dd, J =
13.9, 5.0 Hz), 4.44 (1H, ddd, J = 9.2, 8.0, 5.0 Hz),
6.41 (1H, d, J = 15.7 Hz), 6.48 (1H, dd, J = 8.0, 1.9
Hz), 6.61 (1H, d, J = 8.2), 6.62(1H, s), 6.74 (1H, d, J
= 8.1 Hz), 6.83 (1H, dd, J = 8.2, 1.8 Hz), 6.94 (1H,
d, J = 1.8 Hz), 7.20 (1H, d, J = 15.7 Hz), 8.18 (1H,
d, J = 8.0 Hz), 8.66 (1H, br s), 8.71 (1H, br s), 9.11
(1H, br s), 9.35 (1H, br s ).
2
(Polyamide C-200, 30 g, 91 mm  40 mm, Wako,
Japan) by eluting with 500 ml each of H O, H O±
2
2
MeOH (1:1, v/v), MeOH and HOAc±MeOH (3:17), re-
spectively. Finally, amides were puri®ed from the
HOAc±MeOH (3:17) fraction by reversed phase pre-
parative HPLC (column: Wakosil-II 5C18 HG, 250
mm  20 mm). The following HPLC conditions were
used: linear gradient: 0±40% B/A for 15 min (solvent
Ca€eoyltyrosine (2), p-coumaroyl DOPA (3) and p-
coumaroyltyrosine (4) were synthesized using the
above protocol. Ca€eoyltyrosine (2): ion-spray MS,
+
1
A: HOAc±H O (3:97, v/v), solvent B: MeCN); ¯ow
2
m/z (rel. int.): 344 [M+H] (50), 163 (100); H NMR
spectral data (300 MHz, DMSO-d ): d 2.72 (1H, dd, J
rate: 10 ml/min; detection: 280 nm. Because com-
pounds 2 and 3 were eluted together, they were ana-
lyzed as a mixture.
6
= 13.8, 9.3 Hz), 2.98 (1H, dd, J = 13.9, 4.8 Hz), 4.44
(1H, ddd, J = 9.3, 8.2, 4.8 Hz), 6.41 (1H, d, J = 15.7
Hz), 6.65 (2H, d, J = 8.2 Hz), 6.74 (1H, d, J = 8.2
Hz), 6.83 (1H, d, J = 8.3 Hz), 6.94 (1H, s ), 7.03 (2H,
d, J = 8.2 Hz), 7.19 (1H, d, J = 15.7 Hz), 8.21 (1H,
d, J = 8.2 Hz), 9.06 (1H, br s), 9.19 (1H, br s), 9.36
(1H, br s).
Compound 1 (11.2 mg): ion-spray MS, m/z (rel.
+
int.): 360 [M+H] (49), 163 (100); H NMR spectral
data (300 MHz, DMSO-d ): d 2.73 (1H, dd, J =13.9,
1
6
9
.2 Hz), 2.92 (1H, dd, J = 13.9, 4.8 Hz), 4.44 (1H,
ddd, J = 9.2, 8.0, 4.8 Hz), 6.41 (1H, d, J = 15.7 Hz),
.50 (1H, dd, J = 8.1, 1.8 Hz), 6.62 (1H, d, J = 8.20
6
p-Coumaroyl DOPA (3): ion-spray MS, m/z (rel.
+
int.): 344 [M+H] (45), 147 (100); H NMR spectral
1
Hz), 6.63(1H, s), 6.76 (1H, d, J = 8.1 Hz), 6.86 (1H,
dd, J = 8.2, 1.8 Hz), 6.96 (1H, d, J = 1.8 Hz), 7.21
data (300 MHz, DMSO-d ): d 2.73 (1H, dd, J = 13.8,
6
(
9
1H, d, J = 15.7 Hz), 8.21 (1H, d, J = 7.2 Hz), 8.7±
.4 (4H, br m ).
Compounds 2±4 were isolated and identi®ed by
9.2 Hz), 2.91 (1H, dd, J = 13.8, 4.7 Hz), 4.44 (1H,
ddd, J = 9.2, 8.0, 4.7 Hz), 6.48 (1H, d, J = 8.1 Hz),
6.49 (1H, d, J = 15.4 Hz), 6.61 (1H, d, J = 8.0 Hz),
6.62 (1H, s), 6.79 (2H, d, J = 8.1 Hz), 7.28 (1H, d, J
= 15.7 Hz), 7.38 (2H, d, J = 8.3 Hz), 8.16 (1H, d, J
= 8.0 Hz), 8.68 (1H, br s), 8.73 (1H, br s), 9.83 (1H,
br s ).
comparison with authentic standards and comparison
with literature data (Yoshihara et al., 1977; Van Heer-
den et al., 1980; Cli€ord et al., 1988).
4
.4. Preparation of synthetic 1±4
p-Coumaroyltyrosine (4): ion-spray MS, m/z (rel.
+
1
int.): 328 [M+H] (59), 147 (100); H NMR spectral
data (300 MHz, DMSO-d ): d 2.80 (1H, dd, J = 13.8,
Synthesis of ca€eoyl DOPA (1) was carried out by
6
the method of Villegas and Brodelius (1990) with a
slight modi®cation. Ca€eic acid (3.0 mmol), L-3,4-dihy-
droxyphenylalanine methyl ester hydrochloride (1.5
mmol) and dicyclohexylcarbodiimide (3.3 mmol) were
dissolved in dry pyridine (10 ml) and the mixture was
stirred at room temperature for 24 h. After evapor-
ation of pyridine in vacuo, the residue was dissolved in
MeOH (20 ml). The solution was cooled in an ice bath
after which 2 M KOH (200 ml) was added. The mix-
9.2 Hz), 2.98 (1H, dd, J = 13.8, 4.8 Hz), 4.47 (1H,
ddd, J = 9.2, 8.1, 4.8 Hz), 6.48 (1H, d, J = 15.7 Hz),
6.65 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.6 Hz),
7.03 (2H, d, J = 8.4 Hz), 7.28 (1H, d, J = 15.7 Hz),
7.38 (2H, d, J = 8.6 Hz), 8.19 (1H, d, J = 8.1 Hz),
9.19 (1H, br s ), 9.82 (1H, br s).
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2
and then neutralized with HOAc. The solvent was
evaporated in vacuo and the residue was dissolved in
MeOH (50 ml).
Dicyclohexylurea and salts were removed by ®l-
tration. After evaporation of solvent, the ®ltrate was
fractionated using a polyamide column (Polyamide C-
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ca€eoyl DOPA was puri®ed by reversed phase pre-
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