Induction of resistance against the leafminer, Liriomyza trifolii, by jasmonic acid in sweet pepper

Article abstract of DOI:10.1271/bbb.70033

Sweet pepper (Capsicum annuum) leaves at the mature stage have strong ovipositional deterrence against Liriomyza trifolii (Burgess) (Diptera, Agromyzidae), whereas the cotyledons are fiercely attacked by the fly. Treatment of the cotyledons with 50 μM and

Full text of DOI:10.1271/bbb.70033

Biosci. Biotechnol. Biochem., 71 (6), 1521–1526, 2007
Induction of Resistance against the Leafminer, Liriomyza trifolii,
by Jasmonic Acid in Sweet Pepper
1
;y
1
1
1
Shin-ichi TEBAYASHI, Yoh HORIBATA, Eriko MIKAGI, Takehiro KASHIWAGI,
1
1
2
1
Daniel Bisrat MEKURIA, Aman DEKEBO, Atushi ISHIHARA, and Chul-Sa KIM
1
Department of Bioresources Science, Faculty of Agriculture, Kochi University,
B200 Monobe, Nankoku 783-8502, Japan
2
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
Received January 15, 2007; Accepted February 27, 2007; Online Publication, June 7, 2007
[doi:10.1271/bbb.70033]
Sweet pepper (Capsicum annuum) leaves at the
pupate on the leaves or on the ground. Adult flies
emerge from the pupa within a week. The high fre-
quency of mines made by the larvae can reduce both the
plant growth and crop yield. Furthermore, in leafy vege-
tables and ornamental plants, some crops could lose
marketable worth even by only a single mine. To over-
come these problems, an excess application of pesticides
against the American serpentine leafminer, L. trifolii has
been used, but it has acquired high resistance to various
pesticides. As the leafminer has a short life cycle and
high resistance to chemicals, it is getting difficult to
control them by using pesticides. So, a pest management
technique without depending on chemical pesticides
would be essential. One of the answers is the utilization
of natural chemical products. With this aim, we have
already studied the resistance of the sweet pepper to this
leafminer and found that resistance developed at the
mature stage have strong ovipositional deterrence
against Liriomyza trifolii (Burgess) (Diptera, Agromyzi-
dae), whereas the cotyledons are fiercely attacked by the
fly. Treatment of the cotyledons with 50 ꢀM and 100 ꢀM
of a jasmonic acid (JA) solution caused the plant to
acquire strong oviposition deterrence against the leaf-
miner. An HPLC analysis of the JA-treated cotyledons
revealed the inducible accumulation of a compound.
Based on spectroscopic analysis and chemical methods,
the induced compound was identified to be caffeoylpu-
trescine (CP). The accumulated amounts of CP in the
cotyledons treated with 0, 10, 50 and 100 ꢀM of JA were
6
.0, 43.0, 105 and 140 ꢀg/g fr. wt., respectively. Treat-
ment of the cotyledons with CP resulted in a significant
decrease in the number of punctures made by L. trifolii,
indicating that the JA treatment enhanced the deter-
rence against the leafminer by inducing CP accumu-
lation.
3
)
mature-stage of the plant. It was elucidated that this
resistance was mainly attributable to the much higher
content of luteolin 7-O-ꢀ-D-apiofuranosyl-(1!2)-ꢀ-D-
glucopyranoside in the mature-stage plant than at its
Key words: caffeoylputrescine; jasmonic acid; Liriomy-
za trifolii; Capsicum annuum; ovipositional
deterrent
4
,5)
younger stage.
Induced resistance against pathogens has recently
6
)
been investigated in many plant species, and research
on its application to crop protection has also been con-
Liriomyza trifolii (Burgess) (Diptera, Agromyzidae),
a leafminer native to North America, causes serious
damage to a wide variety of vegetables and nursery
crops in greenhouses and open fields throughout the
world.1 This leafminer is a small fly, measuring 2 mm
in length, with yellow marking on a black body. The
female flies make many punctures on the leaf surface by
their ovipositor and lay eggs in some of those punctu-
7
)
ducted. This resistance is caused by an accumulation of
secondary metabolites, expression of pathogenesis-re-
lated genes and proteins, generation of reactive oxygen
)
8,9)
species and other unknown processes. The inducible
resistance by insect feeding has also been studied by
1
0,11)
many groups.
monic acid (JA) played an important role as a signaling
cure.
It has been demonstrated that jas-
2)
12–15)
16)
res. First instar larvae, after hatching from the eggs
within 2 days, feed on mesophyll leaf tissue and form a
mine. After passing through three instar stages in a
week, the last instar larvae emerge from the leaves and
Constable and Ryan have revealed that the
polyphenol oxidases, which are considered to be part of
the anti-herbivore defense mechanism, were induced by
an exogenous treatment with methyl jasmonate (MeJA)
y
To whom correspondence should be addressed. Tel: +81-88-864-5203; Fax: +81-88-864-5186; E-mail: tebayasi@kochi-u.ac.jp
Abbreviations: JA, jasmonic acid; MeJA, methyl jasmonate; CP, caffeoylputrescine; pCP, p-coumaroylputrescine; FP, feruloylputrescine

1522
S. TEBAYASHI et al.
5
4
3
2
1
0
0
0
0
0
0
200
150
caffeoylputrescine
O
Punctures
Concentration
NH2
N
H
*
*
HO
OH
1
00
b
a
*
*
5
0
*
0
0
10
50
100
Concentration of JA (µM)
0
10
20 Rt (min)
Fig. 1. Effects of the Concentration of JA on the Resistance of
Cotyledons of the Sweet Pepper against Oviposition of the Leaf-
miner (L. trifolii) and Accumulation of CP.
Fig. 2. HPLC Chromatograms of Extracts from the Untreated
Cotyledon (a) and 100 mM JA-Treated Cotyledon (b).
The numbers of ovipositional punctures are represented by the
unshaded bar as the mean ꢀ S. E. (n ¼ 6) and the concentration of
CP is represented by the shaded bar as the mean ꢀ S. E. (n ¼ 4).
Data were analyzed by the Mann-Whitney U-test (P < 0:05).
dons treated with the 10 mM JA solution than with the
water-treated (control) leaves, although the difference
was not statistically significant.
The cotyledons were extracted with 90% methanol-
water, and each extract was analyzed by reversed-phase
HPLC in order to compare the amounts of secondary
metabolites (Fig. 2). In the cotyledon extract treated
with the 100 mM JA solution, a peak (compound 1) at
Rt 4.70 min was enhanced. However the induction of
luteolin 7-O-ꢀ-D-apiofuranosyl-(1!2)-ꢀ-D-glucopyra-
noside, which is an oviposition deterrent in mature
leaves, was not observed. The peak was analyzed by
LC–MS and UV without isolation to elucidate the
structure of compound 1. Compound 1 showed three
characteristic absorbances at 234, 293 and 318 in its UV
in various plants including tomato (Lycopersicon escu-
lentum), tobacco (Nicotiana tabacum), soybean (Glycine
max) and alfalfa (Medicago sativa). Especially in the
tomato plant, the resistance induced with JA or its
methyl ester (MeJA) has been well investigated. The
induced resistance factors against the noctuid caterpillar,
Spodoptera exigua, have been related to the increased of
enzyme activities including polyphenol oxidases, lypo-
1
7,18)
xygenase and peroxidase,
1
the enhanced protease
and the recruiting parasitoid
7)
inhibitor activities
wasp.1 However a chemical cure for inducible resist-
ance caused by the exogenous JA/MeJA treatment
against insects has been rarely proved successful in any
plant species. We report here the induced resistance of
sweet pepper against the leafminer by a jasmonic acid
treatment.
9)
þ
spectrum, and gave an ½M þ Hꢁ ion at m=z ¼ 251 and a
characteristic fragment ion at m=z ¼ 163 which indicat-
ed the presence of a caffeoyl moiety. Its high polarity
and spectroscopic data suggested that the induced
compound was caffeoylputrescine (CP). To confirm
this, CP was synthesized by a condensation reaction
between caffeic acid and putrescine by using dicyclo-
hexylcarbodiimide as a condensing agent. The spectro-
scopic data for 1 were identical with those of the
synthesized compound. Additionally, the identity of 1
was confirmed by co-chromatography with the synthetic
compound by reversed-phase HPLC.
Results
The effect of JA on the resistance of cotyledons of
sweet pepper (Capsicum annuum L. var. grossum
Stendt) against the leafminer (L. trifolii) was investi-
gated by using 3-week-old seedlings. Cotyledons were
excised by a shape razor, and floated in solutions of JA
at various concentrations for 48 hours. The cotyledons
were then exposed to female flies in a 20 ml-glass vial
The effect of JA concentration on the induction of CP
was also investigated (Fig. 2). The cotyledons were
treated with JA for 48 h. The accumulation of CP was
enhanced with the increasing concentration of JA: the
amounts of CP in the cotyledons treated with 0, 10, 50
and 100 mM were 6.0 (0.13), 43 (0.90), 105 (2.19) and
for 24 hours. Many feeding and oviposition punctures
2
(
34:8 ꢀ 5:5 marks/cm ) made by the leafminer were
observed on the surface of cotyledons treated with water
Fig. 1). The numbers of punctures were fewer on the
2
(
140 (2.91) mg/g fr. wt. (mg/cm ), respectively (Fig. 1).
cotyledons treated with the JA solution. The numbers of
punctures on the cotyledons treated with the JA solu-
To address the significance of the induction of CP, we
treated cotyledons with CP, and analyzed its effect on
puncturing by the flies. The numbers of ovipositional
punctures on the cotyledons treated with 0 and 0.25 mM
tions of 50 mM and 100 mM were 13:4 ꢀ 3:9 and 6:6 ꢀ
2
1
:1 marks/cm , respectively. The flies made fewer
2
2
punctures (25:1 ꢀ 6:4 marks/cm ) even on the cotyle-
were 30.9 and 28.8 marks/cm , respectively, (Fig. 3)

Jasmonate-Induced Resistance against Leafminer
1523
4
3
2
1
0
0
0
0
0
directly compared, it is thought that another oviposi-
tional deterrent(s) might have been induced by the JA
treatment.
The resistance of the sweet pepper at the mature stage
against L. trifolii has been attributed to the presence of
luteolin 7-O-ꢀ-D-apiofuranosyl-(1!2)-ꢀ-D-glucopyra-
*
4
)
noside 4-aminobutanoic acid, (2S,4R)-4-hydroxy-1-
methyl-2-pyrrolidine carboxylic acid, 4-amino-1-ꢀ-D-
*
*
5)
3)
*
ribofuranosyl-2(1H)-pyrimidinone and phytol. The
plant has consistently produced defensive chemicals
against L. trifolii at the mature stage, but at the cot-
yledon stage, the plant could not produce a sufficient
amount of the chemical required to provide resistance
to attack by this species. Furthermore, based on our
preliminary experiments, the content of CP in the lower
leaves of the sweet pepper at the mature stage was
0
0.25
0.5
CP
1.0
2.0
2.0
2.0
FP
pCP
Dipping concentration of hydroxycinnnamic acids (mM)
Fig. 3. Effects of the Dipping Concentration of Hydroxycinnamic
Acids (CP, pCP and FP) on the Resistance of Cotyledons of the
Sweet Pepper against Oviposition of the Leafminer Based on the
Dipping Method.
36.0 mg/g fr. wt., although the plant had various contents
of CP depending on the growth stage or region. Gerhard
20)
and Nikolaus have also shown such variation in the
Each value is expressed as the mean ꢀ S. E. (n ¼ 6 for the control
and n ¼ 5 for a treated sample). Data were analyzed by the Mann-
Whitney U-test (P < 0:05).
contents of CP in banana pepper consistent with our
results. As a defensive compound, CP was induced by
the JA treatment at the cotyledon stage, so it is thought
the sweet pepper has multiple defense systems.
JA was induced by feeding the herbivore caterpillar,
tobacco hornworm (Manduca sexta L.) in tobacco
and thus treating with CP at 0.25 mM was not sufficient
to induce ovipositional deterrence. At higher concen-
trations, the numbers of punctures were reduced with
increasing CP concentration. The numbers of punctures
on the cotyledons treated with CP at 0.5, 1.0 and 2.0 mM
2
1)
plant, the beet armyworm (Spodoptera exigua (H u¨ b-
2
2)
ner)) in corn seedling and the cabbage butterfly (Pieris
2
3)
rapae (L.)) in Arabidopsis. Salicylate and JA were
also accumulated by attacking spider mites, Tetranychus
2
24)
were 20.1, 7.1 and 6.9 marks/cm , respectively.
The presence of fueruoylputrescine (FP) and p-
coumaroylputrescine (pCP) has been reported, in other
urticae, in lima bean leaves,
and these signaling
compounds accumulated by the action of an attacking
herbivore could induce resistance against insects or
pathogens. It is not known whether or not oviposition of
the fly would induce JA in a plant as far as we know, and
the cotyledon of sweet pepper did not show any re-
sistance to L. trifolii attack, so the induced amount of JA
would have been negligable or nothing. Further studies
concerning the diversity of endogenous JA and related
compounds in the sweet pepper are needed.
2
0)
cultivars of the sweet pepper so, we also examined
their oviposition deterrence. The numbers of punctures
made by the leafminer on cotyledons treated with a 2.0
mM solution of synthetic FP and pCP were 14.9 and 9.9
2
marks/cm , respectively. A significant difference in the
number of punctures was only detected with the pCP
treatment.
The presence of CP was first reported from a callus
5)
2
Discussion
tissue culture of N. tabacum, and later from the apical
2
6)
leaves, anthers and ovaries of N. tabacum. CP has also
been found in several Solanceous species including
Treatment of cotyledons of the sweet pepper with JA
at concentrations higher than 50 mM reduced the number
of punctures made by female flies as traces of ovi-
position or feeding. HPLC analysis of the cotyledons
revealed that the accumulated concentration of CP in the
JA-treated cotyledons was 20-fold higher than that in
the water-treated cotyledons. The treatment by CP of the
cotyledons effectively prevented the leafminer from
making punctures, and we thus conclude that the JA
treatment induced the resistance to L. trifolii in the cot-
yledons of sweet pepper by accumulating CP. Deter-
rence was observed at a dose of more than 0.81 mg/cm
on the surface of a cotyledon dipped into a 0.5 mM CP
solution in a dipping test and 2.2 mg/cm with a cot-
yledon treated with a 50 mM JA solution in an induction
test. As the effective doses in both tests could not be
2
0)
C. annuum, although its function has not yet been
clear. On the basis of its chemical structure, CP is
classified as a hydroxycinnnamic acid amide. This
class of compound has been identified in many plant
species, mainly Poaceae and Solanaceae. Some of them,
2
7)
namely p-coumaroylagmatine in barely and avenan-
2
8)
thramide in oats, have been shown to be toxic against
pathogenic fungi. Furthermore, hydroxycinnnamic acid
amides have been demonstrated to be induced by
various exogenous stimuli. For example, avenanthra-
2
29)
mides were induced by inoculation with the fungus, p-
coumaroyloctopamine was induced by ꢀ-1,3-glucooli-
2
30)
gosaccharide in potato tuber disks, clovamide and its
3
1)
derivatives were induced by JA in red clover and
3
2)
caffeoylputrescine was induced by MeJA in tobacco.

1524
S. TEBAYASHI et al.
Based on their toxicity and inducibility by various
exogenous stimuli, hydroxycinnnamic acid amides have
generally been established as compounds involved in the
inducible defense system against pathogens. In this
study, we have revealed that caffeoylputrescine acted as
an oviposition deterrent against L. trifolii, and this is the
first report on the biological activity of a hydroxycinn-
namic acid amide against insects. Henceforth, we should
pay more attention to the possibility of the action of
hydroxycinnnamic acid amides as defensive substances
against insects and not only against pathogens. Hydrox-
ycinnnamic acid amides should therefore be studied for
their oviposition deterrent activity, insecticidal activity,
feeding deterrent activity, repellent activity and growth
regulator activity against various insect pests.
from the seedlings with a sharp razor blade, were floated
on 10 ml of various concentrations of an aqueous
jasmonic acid solution in a Petri dish (9 i.d. ꢂ 1 cm).
ꢃ
Each container was maintained at 25 ꢀ 2 C with a 14 L-
10 D photoperiod in a growth chamber for 24 h. After
cleaning the surface of each cotyledon with water, the
treated cotyledon was used for the bioassay or extrac-
tion.
Application of the chemicals was carried out by the
dipping method, the cotyledons being dipped into vari-
ous chemical-methanol solutions for 5 seconds. After
the solvent had been evaporated by air drying at room
temperature, the treated cotyledons were used for the
bioassay. To estimate the dose of chemicals applied to a
leaf in the dipping test, the applied solvent volume was
measured by dipping ten cotyledons into a 0.25 mM CP
methanol solution for 5 s, the consumption of the solvent
then being measured and the area of the cotyledons
recorded by a PC equipped with a scanner. The applied
FP and pCP also decreased the number of punctures
by L. trifolii, although their activity was weaker than
that of CP. FP and pCP were not detected in the cot-
yledons treated with JA in our analysis. However, the
presence of FP and pCP has previously been reported in
2
volume of solvent per cm was then calculated and used
to estimate the dose of various hydroxycinnnamic acid
amides in the dipping test.
2
0)
different cultivars of the sweet pepper. Thus, FP and
pCP may also play an important role in the defense
system against L. trifolii in other varieties of sweet
pepper.
Analytical methods. The treated and untreated coty-
ledons of sweet pepper were extracted with methanol for
48 h, and each extract was directly analyzed by HPLC or
LC–MS. HPLC analyses was carried out with an ODS
column (Wakosil-II 5C18 HG, 150 ꢂ 4:6 mm i.d., Wako
Pure Chemical Industry, Japan) with a 10ADvp system
equipped with a diode array detector (SPD-M10Avp,
Shimadzu Co., Ltd., Japan) under the following con-
ditions: linear gradient (10% A/B for the first 5 min,
then 10–30% for 5 min, and finally 30–90% A/B for 25
The inhibition by CP against L. trifolii was almost
saturated when used above a concentration of 1.0 mM
CP, while even a 2.0 mM solution of FP or pCP was
exhibited weaker activity than that of a 1 mM CP
3
3)
solution against the flies. Tanaka et al. have similarly
reported that feruloylserotonin induced in witches’
broom diseased bamboo along with p-coumaroylseroto-
nin showed weaker antifungal activity than p-coumar-
oylserotonin. It thus, seems that substitution of the
hydroxyl group in the benzene ring of hydroxycinn-
namic acid amide might be important for this activity.
min) {solvent A, H O–AcOH (99:1, v/v); solvent B,
2
acetonitrile–AcOH (99:1, v/v)}. LC–MS data were
measured by a Perkin-Elmer-Sciex API-165 or API-
3000 instrument (ion-spray voltage, 5 kV; orifice volt-
age, 30 V; nebulizer gas, air; curtain gas, nitrogen) con-
nected to a Shimadzu 10A HPLC system equipped with
the same ODS column.
Experimental
Plant materials. Sweet pepper (Capsicum annuum L.
var. grossum Stendt cv. new Sakigake Number 2) and
kidney bean (Phaseolus vulgaris L.) seeds were pur-
chased from Maekawa-syubyo nursery (Japan). The
sweet pepper seeds were sown on a tray (20 ꢂ 15 ꢂ
NMR data were recorded with a JEOL JNM-AL400
spectrometer in DMSO-d or with a Bruker Avance400
6
spectrometer in methanol-d , using TMS as an internal
4
1
cm depth) containing nursery soil (Cell Compost,
standard. Chemical shifts are represented as ꢁ units,
and the multiplicity of signals is abbreviated as singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m).
Coupling constants (J) are given in Hz.
Mitsui-Nourin Co., Ltd., Japan) and maintained at 25 ꢀ
ꢃ
2
C with a 14 L-10 D photo period in a growth cham-
ber. After 3 weeks, the seedlings, which consisted only
of cotyledons, were used for bioassays. Three kidney
bean seeds were sown in a vinyl pot containing nursery
soil (Tsuchitaro, Mitsui-Nourin Co., Ltd., Japan) and
Insect. L. trifolii was reared on the primary leaf of the
kidney bean seedling. Nine seedlings were exposed to
15 female L. trifolii for 2 days in an acrel cage (30 ꢂ
25 ꢂ 28 cm) which was fitted with nylon mesh (0.5 mm)
at the back and two sides. After removing the flies, the
kidney bean seedlings were placed in a growth chamber
ꢃ
maintained at 25 ꢀ 5 C with a natural photoperiod in a
greenhouse. After the primary leaves had developed for
4 weeks, the seedlings were used to maintain the popul-
ation of L. trifolii.
ꢃ
maintained at 25 ꢀ 2 C with a 14 L-10 D photo period.
Elicitor treatment and chemical application. The
elicitor treatment was done by the floating method.
Cotyledons of the sweet pepper, which had been excised
The leaves were cut from the seedlings before the emer-
gence of maggots, and kept in an ice cream cup (12
i.d. ꢂ 7 cm) under the same conditions. Adult flies were

Jasmonate-Induced Resistance against Leafminer
1525
collected everyday to arrange their age for bioassay and
maintain the population.
(1H, d, J ¼ 8:3 Hz), 6.90 (1H, dd, J ¼ 8:3, 1.8 Hz), 7.02
13
(1H, d, J ¼ 1:8 Hz, ), 7.39 (1H, d, J ¼ 15:7 Hz). ). C-
NMR (CD OD) ꢁ: 27.9, 30.6, 40.2, 42.0, 56.2, 112.2,
3
Bioassay. The degree of ovipositional deterrence was
measured by counting the number of leaf punctures
made by the female flies. The treated cotyledon was
put into a 20 ml-glass vial which had a small sheet of
moistened filter paper inside. Five 2-day-old female flies
were released into the vial. After screwing on a lid, the
114.9, 118.9, 121.0, 129.5, 142.1, 150.2, 151.8, 169.2.
References
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)
Spencer, K. A., Agromyzidae (Diptera) of economic
importance. In ‘‘Series Entomologica’’ Vol. 9, Dr. W.
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vial was then placed in a growth chamber maintained at
ꢃ
2
5 ꢀ 2 C with a 14 L-10 D photo period for 24 h. The
2
)
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3
)
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7
39–742 (2005).
3
1)
method of Tebayashi et al. A mixture of caffeic acid
5 mmol), 1,4-butandiamine and dicyclohexylcarbodii-
4
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(
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6
h under N . After the mixture had been neutralized
2
with AcOH, the solvent was evaporated in vacuo, the
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þ
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(
1
35.3%). CP: LC–MS, m=z (rel. int.): 250½M þ Hꢁ (42),
þ
1
63[caffeoyl] (100); UV, ꢂmax nm: 234, 293, 318. H-
9
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7
:4 Hz), 3.32 (2H, t, J ¼ 7:4 Hz), 6.43 (1H, d, J ¼ 15:6
1
1
1
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Hz), 6.75 (1H, d, J ¼ 8:1 Hz), 6.89 (1H, dd, J ¼ 2:0,
8
1
4
1
.1 Hz, ), 6.99 (1H, d, J ¼ 2:0 Hz), 7.38 (1H, d, J ¼
1
3
5:6 Hz). C-NMR (DMSO-d ) ꢁ: 26.0, 27.6, 39.5,
6
0.4, 115.0, 116.5, 118.2, 122.1, 128.2, 142.4, 146.4,
48.9, 169.5.
pCP and FP were synthesized by using the foregoing
þ
protocol. pCP: LC–MS, m=z (rel. int.): 235½M þ Hꢁ
þ
þ
(
100), 218½M ꢄ NH2ꢁ (12), 147[p-coumaroyl] (24);
1
UV, ꢂmax nm: 225, 293, 308. H-NMR (CD3OD) ꢁ: 1.6–
1
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1
.8 (4H, m), 2.93 (2H, t, J ¼ 7:2 Hz), 3.33 (overlapping
with CD OD), 6.42 (1H, d, J ¼ 15:7 Hz), 6.80 (2H, d,
3
J ¼ 8:6 Hz, ), 7.40 (2H, d, J ¼ 8:6 Hz), 7.45 (1H, d, J ¼
1
3
1
5:7 Hz). C-NMR (CD3OD) ꢁ: 26.3, 27.5, 39.6, 40.4,
1
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1
16.8, 118.2, 127.5, 130.5, 141.9, 160.7, 169.4. FP: LC–
þ
MS, m=z (rel. int.): 265½M þ Hꢁ (100), 248½M ꢄ
þ
þ
NH2ꢁ (7), 177[feruloyl] (24); UV, ꢂmax nm: 220,
40 (sh), 290, 317. 1H-NMR (CD3OD) ꢁ: 1.50–1.65
4H, m), 2.69 (2H, t, J ¼ 7:0 Hz), 3.30 (overlapping with
CD OD), 3.86 (3H, s), 6.39 (1H, d, J ¼ 15:6 Hz), 6.87
1
2
(
3

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2