Male-specific sesquiterpenes from phyllotreta flea beetles

Article abstract of DOI:10.1021/np100608p

Flea beetles in several genera are known to possess male-specific sesquiterpenes, at least some of which serve as aggregation pheromones that attract both sexes. In continuing research on the chemical ecology of Phyllotreta flea beetles, six new male-specific sesquiterpenes were identified, one from P. striolata (hydroxyketone 9) and five from P. pusilla (aldehydes 10-12 and 14 and alcohol 13); both species are crop pests. The minute amounts from beetles provided mass spectra and chromatographic data but were insufficient for complete structure determination. However, it was discovered that the new compounds could all be produced by applying organic reactions to previously identified flea beetle sesquiterpenes, and the resulting, larger amounts of material permitted definitive structure analysis by NMR. Molecular modeling was used in conjunction with NMR to define relative configurations of several newly created stereogenic centers. The absolute configurations of natural 9-14 were established by chiral gas chromatography/mass spectrometry. In electrophysiological tests (GC-EAD) conducted with P. striolata, compound 9 was detected with high sensitivity by the beetle antennae, which is consistent with a pheromonal function. The research opens new possibilities for using behavioral chemicals to monitor or manage these pest species.

Full text of DOI:10.1021/np100608p

ARTICLE
pubs.acs.org/jnp
Male-Specific Sesquiterpenes from Phyllotreta Flea Beetles
‡
,
§,†
†
†
†
Robert J. Bartelt,* Bruce W. Zilkowski, Allard A. Coss ꢀe , Udo Schnupf, Karl Vermillion, and
†
Frank A. Momany
†
USDA Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 North University Street,
Peoria, Illinois 61604, United States
‡
Department of Food Science, Stocking Hall, Cornell University, Ithaca, New York 14853-7201, United States
S Supporting Information
b
ABSTRACT: Flea beetles in several genera are known to possess
male-specific sesquiterpenes, at least some of which serve as
aggregation pheromones that attract both sexes. In continuing
research on the chemical ecology of Phyllotreta flea beetles, six
new male-specific sesquiterpenes were identified, one from
P. striolata (hydroxyketone 9) and five from P. pusilla
(
aldehydes 10-12 and 14 and alcohol 13); both species are
crop pests. The minute amounts from beetles provided mass
spectra and chromatographic data but were insufficient for
complete structure determination. However, it was discovered that the new compounds could all be produced by applying organic
reactions to previously identified flea beetle sesquiterpenes, and the resulting, larger amounts of material permitted definitive
structure analysis by NMR. Molecular modeling was used in conjunction with NMR to define relative configurations of several
newly created stereogenic centers. The absolute configurations of natural 9-14 were established by chiral gas chromatography/
mass spectrometry. In electrophysiological tests (GC-EAD) conducted with P. striolata, compound 9 was detected with high
sensitivity by the beetle antennae, which is consistent with a pheromonal function. The research opens new possibilities for using
behavioral chemicals to monitor or manage these pest species.
4
lea beetles (order Coleoptera, family Chrysomelidae, sub-
family Alticinae) are a large, important, and interesting group
of small insects (adults mostly <4 mm in length). The group
from a plant source, citronella oil. Blends of the racemic
9
10
F
compounds and of pure enantiomers were demonstrated to
be attractive to P. cruciferae of both sexes in the field. Thus 1-6 or
some subset of these constitutes a male-produced aggregation
pheromone in this species. We have now investigated volatile
emissions from additional Phyllotreta species, P. striolata
Fabricius) and P. pusilla Horn. P. striolata is an important
pest of oilseed rape in Canada and also attacks Brussels
sprouts, cabbage, and other cruciferous vegetables. P. pusilla
is particularly damaging to mustard, radish, and turnip crops
in the western regions of the United States, and losses can be
contains significant agricultural pests such as the crucifer flea
1
beetle, Phyllotreta cruciferae Goeze, and the eggplant flea beetle,
2
Epitrix fuscula Crotch. Adults are defoliators, while larvae live
in the soil and feed on roots. However, the group also con-
tains beneficial species. Some Aphthona spp. are specialists on
leafy spurge (Euphorbia esula L.) and have been introduced
into the U.S. as biological control agents on this invasive, noxious
(
11
2
3
weed.
Pheromones, which are typically produced by just one sex,
have become important scientific tools for monitoring and
managing economic insects, and some information now exists
about the chemistry of sex-specific compounds of flea beetles,
including P. cruciferae, several Aphthona species, and E. fuscula.
Males of P. cruciferae emit a group of six sesquiterpenes, 1-6,
2
especially serious for seedling plants in early spring.
Six new male-specific sesquiterpenes (9-14) were discov-
ered. The minute amounts from beetles made NMR of the
natural compounds impracticable, but NMR became accessible
when ad hoc methods were developed to synthesize 9-14 from
related sesquiterpenes. Relative configurations at several stereo-
genic centers were established with the aid of molecular model-
ing. Subsequently, chiral GC was used to determine absolute
configurations. Preliminary electrophysiological analysis was
conducted by coupled gas chromatography/electroantenno-
graphic detection (GC-EAD).
4
that are lacking from females, and the same compounds plus 7
4
and 8 occur in males of three Aphthona species. Sesquiterpenes
1
(
, 3, 5, 7, and 8 are also emitted by male E. fuscula, but
2E,4E,6Z)- and (2E,4E,6E)-2,4,6-nonatrienal are the major
5
components in their blend. Component ratios in the emissions
from the beetles are generally species-specific.
6
Compounds 1, 3, and 5-8 were synthesized in racemic form,
7
and 1, 3, 5, and 6 were synthesized as pure enantiomers, which
allowed the absolute configurations of the beetle compounds to
be unambiguously assigned. Sesquiterpene 4 is obtainable
Received: September 1, 2010
Published: February 22, 2011
7,8
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
ARTICLE
Figure 1. Example gas chromatograms of volatiles collected from
Phyllotreta flea beetles feeding on cabbage. Compounds 1-6 were pre-
viously known, and 9-14 were new. Peak marked * is an artifact from
rearrangement of 9 (see text). Three different sets of GC conditions
were used: DB-1 with 0.1 μm film, 10 °C/min rate (P. striolata groups of
1
0); DB-1 with 0.25 μm film, 10 °C/min rate (P. striolata single male);
DB-1 with 0.25 μm film, 15 °C/min rate (P. pusilla). See text for other
GC parameters.
the previously identified ketone 6, but there were five others
(
10-14) that were not encountered before, based on mass spec-
tra (Figure 2). Hydrocarbons 1-5 were not found in P. pusilla
samples.
’
RESULTS AND DISCUSSION
Typical amounts of 9 emitted from P. striolata males in groups
of 10 were 20-50 ng per male per day, and 1-6 were less
abundant. Interestingly, amounts of 9 in collections from in-
dividuals (100-300 ng/male/day) were nearly an order of
magnitude greater on a per capita basis than from groups. We
focused primarily on collections from individuals rather than
groups because the total amount of material obtained would be
similar and there would be no ambiguity with respect to species
composition (see Experimental Section). In 41 analyzed collec-
tions, the male-specific compounds were detected from six of the
seven individual males; the seventh died during the first week.
Emission began within 2 days of setup and usually continued
until the beetles died (as long as 5 weeks).
For individual males of P. pusilla, the emission rate of 6 was as
high as 400 ng per day. Nineteen of the 24 males produced a
pattern of volatiles as in Figure 1 (6 was detectable in 127 out of
the 174 collections). Production usually began about 2 weeks
after capture in the field, but the compounds were not detected
from one male until 5 weeks. Emission continued for as long as
Emission of Male-Specific Compounds. GC-MS compar-
ison of volatiles collected from male and female P. striolata in the
presence of host material (cabbage) revealed eight compounds
in samples from males that were not present from females
(
Figure 1, peaks 1-6, 9, and that marked with *). Based on
EIMS and GC retention times relative to synthetic standards,
-6 from P. striolata were identical to compounds previously
1
encountered from P. cruciferae and three Aphthona species;
however, the mass spectrum of peak 9 was new (Figure 2). An
earlier peak in the chromatogram, marked (*) in Figure 1, had a
very similar spectrum but was subsequently found to be an
artifact, presumably a thermal rearrangement of 9 from injection
into a hot (250 °C) GC inlet. This peak was small (<10% the size
of 9) when the inlet temperature was 150 °C and was absent
altogether with injection through a cool-on-column inlet at
5
3 °C. Thus, only 9 was investigated further.
Male-specific compounds were also found consistently from
P. pusilla on cabbage (Figure 1). The most abundant of these was
5
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Journal of Natural Products
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Figure 2. Mass spectra of compounds 9-14. Unless otherwise noted, spectra are EI (70 eV ioization energy).
1
2 weeks once it began. The five beetles that did not produce
hexanes, consistent with aldehydes; 13 eluted with 25% Et O in
hexanes, indicative of an alcohol; and 9 was the most polar,
2
died soon after setup.
Mass Spectra and Polarity. None of the EI mass spectra in
Figure 2 were identifiable from the spectral library. The spectra of
elution requiring 50% Et O in hexanes.
2
Mass spectra were not sufficient to define structures, and
NMR was not feasible on the minute amounts of the compounds
obtainable from the beetles. Serendipitously, an unintentional
preparation of 9 and 14 led to a synthetic approach for solving the
unknown structures. During an earlier project, compound 1 was
1
0-14 showed prominent, likely molecular ions at m/z 218 or
2
20, but the molecular ion of 9 was not readily apparent. Single
mass chromatograms of ions m/z 218, 236, and 193 of 9 (0.4%
and 0.06% of the base peak and the base peak, respectively) all
had peaks that coincided in retention time; thus 236 was believed
the likely molecular weight of 9, with 218 representing an ion
formed by dehydration. With isobutane CI (Figure 2), m/z 219
4
aromatized to 5 prior to measurement of optical rotation, and
leftover 1 was removed by oxidation with potassium per-
12
manganate. Examination of the oxidation products by GC-
MS revealed traces of 9 and 14, which were identical to the beetle
compounds by MS and GC retention on an achiral column.
Conversion Precursors. Enantiomerically pure 1 and 6 (pre-
pared as in Scheme 1) were precursors for reactions leading to
9-14; the 1 and 6 fully matched prior authentic standards and
was the base peak (M þ 1 - H O), but m/z 237 (M þ 1, 2% of
2
base peak) and m/z 293 (M þ 57, a typical ion in isobutane CI,
0
.4%) were significant.
All of the compounds were recovered after passage through
silica gel. Compounds 10, 11, 12, and 14 eluted with 5% Et O in
2
5
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Journal of Natural Products
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a
Scheme 2. Synthesis of 9-14 from 1 and 6
Figure 3. Modeled epimers of synthetic compound 9. Numbering
corresponds to Table 1. The predicted minimum interproton distances
for the two epimers aided the interpretation of NOE results (see text).
Scheme 1. Synthesis of Compounds 1 and 6 from
a
(
R)-(þ)-Pulegone (15) by Previously Described Methods
a
Conditions, results: (a) 3-chloroperbenzoic acid (2.2 mol), CH Cl ,
2
2
RT, 1 h, yield 44%, purity 99% after chromatography; (b) (methoxy-
methyl)triphenylphosphorane (2 mol), dry THF, 0 °C to RT, 1 h, yield
a
19
þ 19
6
5%, purity 97% after chromatography; (c) conc HI in THF or hexanes,
Conditions: (a) HCl gas, 0 °C; (b) NaOH(aq), RT, then H ;
7
6
or Et O saturated with HClO , RT, ∼1 h, monitor by GC; (d) Dess-
(
c) 8 steps; (d) 3 steps. Compound 3, a synthetic byproduct, was not
2
4
Martin periodinane (1.4 mol), dry CH
1% after initial chromatography; (e) Et
RT, 12: yield 5%, purity 90% after chromatography, 14: yield 18%, purity
2
Cl
2
, RT, 1 h, yield 30%, purity
removed.
8
2
O saturated with HClO
4
, 24 h,
were the specific enantiomers of 1 and 6 found in P. cruciferae.
-
9
0
6% after chromatography; (f) LAH (4 mol H ), Et O, 0 °C to RT,
2
(
The stock of 1 contained 24% of 3 as an impurity; these can
.5 h, yield ∼100%, purity 90% after workup; (g) dimethylsulfonium
6
interconvert under acidic conditions. ) Racemic 1 and 6 were
also available and would afford racemic 9-14, needed as stan-
dards for chiral GC.
methylide (1.8 mol), dry THF, 0 °C then RT, 1.5 h, presumed inter-
mediate 19 not isolated, yield of 10, 14%, and of 11, 12%, purity after
chromatography ∼80% (10 þ 11).
13
Hydroxyketone 9. Excess 3-chloroperbenzoic acid was
1
superior to KMnO for converting 1 to 9 (Scheme 2). The
methine protons had sufficient separation in the H NMR spec-
4
product was identical to natural 9 by MS, by GC retention on an
achiral column, and by polarity on silica. Although the product of
the reaction was not fully predictable, enough 9 was obtained so
that the structure could be studied by NMR.
trum so that all of the couplings could be measured and assigned
to particular pairs of protons; simulation (not shown) supported
the assignments.
The configuration at C-3 was established through molecular
modeling and NOE experiments (the configurations at C-6 and
C-7 were expected to remain as in starting material 1). The (3R)-
9 and (3S)-9 epimers were modeled separately (Figure 3).
Observed NOESY correlations (especially that between H-6
and H-10a) assisted modeling of the flexible seven-membered
ring. The generally good agreement between observed and pre-
The NMR data for 9 (Table 1) indicated that 15 carbons and
4 hydrogens were present, and a molecular weight of 236 would
2
require two additional oxygen atoms. From the proton and
carbon spectra, there were four aliphatic methyls (three tertiary
and one secondary), one trisubstituted double bond, one carbo-
nyl group, and one tertiary hydroxy group. The HMBC spectrum
established that the carbon skeleton of 1 was conserved and that
the keto-hydroxy-olefin system was as shown, except that the
configuration at C-3 was ambiguous. The 10 methylene and two
1
13
dicted H and C NMR shifts and coupling constants (Table 1)
indicated that the modeled ring conformation was realistic.
NOESY correlations involving Me-15 protons were observed
5
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Journal of Natural Products
ARTICLE
1
13
Table 1. H (500 MHz) and C (125 MHz) NMR Data in C D for Compound 9, Assigned J Couplings, and Predicted NMR
6
6
Values for Epimers (3S)-9 and (3R)-9 (Figure 3)
Observed NMR Data and Assignments
a
b
position
δ
C
, mult.
δ
H
, (J in Hz)
HMBC
NOESY
assigned J (Hz)
1
2
3
4
5
152.3, C
125.7, CH
70.3, C
5a, 5b
-14.4
5.6
5.71, s
1,4,6,11,15
13, 15
5a, 6
OH: 2.14 v br s
5b, 6
2.5
211.0, C
6, 7
10.1
4.7
41.1, CH
2
5a: 2.61, dd, (14.4, 5.6)
1, 4, 6, 7
1, 4, 6, 7
1, 2, 4
5b, 6
7, 8a
5
b: 2.50, dd, (14.4, 2.5)
5a, 6, 14
7, 8b
8.3
6
7
8
41.8, CH
39.1, CH
37.3, CH
2.12, ddd (10.1, 5.6, 2.5)
5a, 5b, 10a, 12, 14
14, 15 (weak)
8b, 9b
7, 14
6.5
1.24, dddq (10.1, 8.3, 4.7, 6.5)
8a: 1.57, dddd (13.6, 7.6, 4.7, 2.9)
8a, 8b
8a, 9a
8a, 9b
8b, 9a
8b, 9b
9a, 9b
9a, 10a
9a, 10b
9b, 10a
9b, 10b
10a, 10b
-13.6
7.6
2
2
2
6, 7, 9, 14
8
b: 0.95, dddd (13.6, 9.4, 8.3, 3.4)
9a: 1.44, ddddd (14.2, 7.6, 7.5, 3.4, 2.5)
b: 1.18, ddddd (14.2, 10.7, 9.4, 2.9, 2.4)
10a: 1.37, ddd (14.2, 10.7, 2.5)
0b: 1.30, ddd (14.2, 7.5, 2.4)
8a, 9a, 10a
8b, 9b
2.9
9
21.9, CH
40.8, CH
3.4
9
8, 10
1, 9
8a, 9a
9.4
1
0
6, 8b, 12
12, 13
-14.2
2.5
1
1, 9
1
1
1
1
1
1
2
3
4
5
38.2, C
7.5
30.7, CH
27.1, CH
19.4, CH
24.9, CH
3
3
3
3
0.97, s
1, 10, 11, 13
1, 10, 11, 12
6, 7, 8
6, 10a, 10b
2, 10b
10.7
2.4
1.10, s
0.85, d (6.5)
1.44, s
5b, 6, 7
-14.2
2, 3, 4
2, 7 (weak)
Predicted NMR Values for (3S)-9 and (3R)-9
predicted δ
C
predicted δ
H
predicted J (Hz)
(3S)-9
position
(3S)-9
(3R)-9
(3S)-9
(3R)-9
description
(3R)-9
1
2
3
4
5
150.9
122.0
69.5
150.0
122.3
72.6
5a, 5b
5a, 6
-17.6
6.2
-14.1
6.1
5.66
5.61
OH: 1.51
OH: 2.99
5b, 6
2.5
3.1
213.4
40.6
213.0
40.0
6, 7
10.6
5.3
10.6
5.3
5a: 2.58
5a: 2.68
5b: 2.36
2.38
7, 8a
5
b: 2.36
7, 8b
11.2
6.7
11.4
6.6
6
7
8
42.3
41.0
40.7
45.3
40.1
39.6
2.32
7, 14
1.32
1.40
8a, 8b
8a, 9a
8a, 9b
8b, 9a
8b, 9b
9a, 9b
9a, 10a
9a, 10b
9b, 10a
9b, 10b
10a, 10b
-15.8
5.5
-15.8
5.5
8a: 1.65
8a: 1.63
8b: 1.05
9a: 1.30
9b: 1.31
10a: 1.37
10b: 1.46
8
b: 1.08
9a: 1.35
b: 1.28
10a: 1.45
0b: 1.47
2.1
2.2
9
24.2
40.8
22.9
40.1
2.9
3.0
9
12.9
-16.5
0.3
12.9
-16.4
0.4
1
0
1
1
1
1
1
1
1
2
3
4
5
40.5
28.5
25.1
18.4
23.7
39.7
28.1
24.1
16.9
27.0
9.6
9.5
0.95
1.02
0.69
1.17
0.93
1.02
0.75
1.11
10.8
0.3
11.0
0.3
-16.4
-16.4
a
b
HMBC correlations are from proton(s) stated to the indicated carbon. NOESY correlations are between proton(s) stated and the other indicated
protons.
only to H-2 and H-7 (weak). The latter result was crucial and was
verified with the NOE difference experiment: irradiation at H-7
led to a slight (0.3%) but significant enhancement of Me-15.
From the predicted distances in Figure 3 for the (3S) epimer,
Overhauser enhancements could be expected between Me-15
and H-2 and between Me-15 and H-7 but not between Me-15
and H-5a. However for the (3R) epimer, enhancements would be
expected between Me-15 and H-2 and between Me-15 and H-5a
but not between Me-15 and H-7. Thus, we conclude that
synthetic 9 was the (3S) epimer.
Aldehyde 14. NMR analysis of 14 obtained during the syn-
thesis of 12 led to the data in Table 2. In the proton and carbon
spectra there was evidence for three aliphatic methyl groups (two
tertiary and one secondary), two trisubstituted double bonds,
5
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Journal of Natural Products
ARTICLE
1
13
Table 2. H (500 MHz) and C (125 MHz) NMR Data in C D for Compounds 13 and 14 and Assignments of Shifts to Position
6
6
Numbers in the Structures
compound 13
, (J in Hz)
compound 14
, (J in Hz)
a
a
position
δ
C
, mult.
δ
H
HMBC
C
δ , mult.
δ
H
HMBC
1
2
3
4
5
154.1, C
155.8, C
b
116.73 , CH
5.95, br s
1, 3, 4, 6, 11, 15
111.4, CH
139.8, C
6.64, s
3, 4, 6, 15
2, 5, 6, 15
137.7, C
b
116.70 , CH
5.52, br d (6.2)
141.9, CH
5.96, ddt (6.4, 3.0, 1.0)
2.29, ddd (18.0, 6.4, 1.7)
2.06, ddd (18.0, 7.0, 2.8)
1.74, br dd (9.8, 7.0)
1.40, m
27.8, CH
2
2.35, ddd (17.0, 6.2, 1.3)
1, 3, 4, 6, 7
7
28.9, CH
2
1, 3, 4, 6, 7
1, 3, 4, 6, 7
1, 2, 4, 5, 7, 8
2
.24, ddddd (17.0, 6.0, 2.5, 2.5, 2.5)
6
7
8
37.8, CH
34.4, CH
39.9, CH
1.80, m
1.74, m
1.79, m
1, 2, and/or 4
37.3, CH
34.5, CH
39.3, CH
2
2
2
2
2
2
1.64, m
6, 7, 9, 10, 14
6, 7, 9, 10, 14
1
.03, m
1.54, m
.25, m
1.51, m
.51, m
0.90, m
9
23.2, CH
40.8, CH
22.8, CH
40.6, CH
1.43, m
1
1.11, m
10, 11
1
0
1.41, m
1
1.41, m
11
12
13
14
15
38.7, C
38.9, C
32.5, CH
26.6, CH
22.3, CH
65.2, CH
3
3
3
2
1.16, s
1, 10, 11, 13
1, 10, 11, 12
8
32.2, CH
26.3, CH
22.0, CH
3
3
3
1.03, s
1, 10, 11, 13
1, 10, 11, 12
6, 7, 8
1.22, s
1.16, s
0.95, d (6.4)
4.07, d (12.3)
0.78, d (6.8)
9.42, s
189.9, CH
2, 3, 4
4
.04, d (12.3)
OH: 0.78, s
a
b
HMBC correlations are from proton(s) stated to the indicated carbon. Assignments may be reversed.
and one formyl group. The HMBC data supported that the
carbon skeleton and diene system of 1 were present, but in 14,
branch carbon 15 was a formyl group rather than a methyl. The
proton and carbon shifts for 14 were very similar to those
reported for 1, except in the region of the formyl group,
supporting that the relative configuration at C-6 and C-7 re-
mained as in starting material 1, as would be expected. Synthetic
results prompted the attempt to synthesize 10 and 11 from
ketone 6 by conversion to the enol ether (18), followed by
hydrolysis. The enol ether formed readily, but hydrolysis
15
proved difficult. By GC-MS, 18 was consumed over time
6
,7
following treatment with either HI or HClO (within several
4
hours or overnight, depending on conditions). Traces of 10 and
11 formed initially, but disappeared subsequently (presumably
due to an acid-catalyzed isomerization such as double-bond
migration). These results were encouraging with respect to the
proposed structures for 10 and 11, but the yield was too low for
NMR analysis.
1
4 matched the beetle compound exactly with respect to MS and
14
GC retention on an achiral column. Periodinane oxidation of
enol ether 18 was later found to be a more efficient means of
preparing 14 (Scheme 2).
Alcohol 13. The mass spectrum of 13 was similar to that of 14
except that many of the peaks, including the molecular ion, were
shifted two mass units higher (Figure 2). This, and the higher
polarity, suggested that 13 could be obtained by reducing
aldehyde 14 to the alcohol. Thus, treatment with LAH cleanly
afforded a product that was identical to the beetle compound in
MS and GC retention on an achiral column. The reaction was not
expected to affect the ring system of 14.
Useful amounts of 10 and 11 were obtained by treating ketone
6 with dimethylsulfonium methylide. The expected product was
16
epoxide 19, which could give the aldehydes after subsequent
hydrolysis, dehydration, and tautomerization. In fact, the epoxide
was not isolated, and 10 and 11 were the major reaction products
upon workup. These products matched the corresponding beetle
compounds exactly by MS and GC retention on an achiral
column. Passage through an open column of silica gel removed
impurities from 10 and 11 but did not separate them from each
other. HPLC on silica separated them with difficulty, but each
pass through the column led to unacceptable losses of these labile
compounds. Thus, NMR analysis was based on a 55:45 mixture
of 10 and 11 from column chromatography, where the more
abundant compound, 10, corresponded to the earlier GC peak
from the beetles.
The NMR data for 13 (Table 2) supported the structure. Main
features were three aliphatic methyl groups (two tertiary and one
secondary), one primary alcohol group, and two trisubstituted
double bonds. Except near the alcohol group, the carbon and
proton shifts were similar to those observed previously for
6
,7
compounds 1 and 14, and HMBC results supported the ring
system as presented.
13
Aldehydes 10 and 11. The polarity of beetle compounds 10
Thirty resonances were observed in the C NMR spectrum
(Table 3). The spectrum appeared as 15 pairs of peaks, where the
members of each pair had the expected peak-height ratio of ca.
55:45, allowing assignment of signals to the proper compound.
The proton spectrum confirmed that both compounds were
and 11 (elution from silica with 5% Et O in hexanes) and mass
2
spectra with intense M - 29 fragments (at m/z 191) were con-
sistent with aldehydes. The mass spectra of 10 and 11 were
similar, suggesting the compounds could be epimers. These
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aldehydes (δ 9.40 and 9.44 for H-15, Table 3). The NMR data
were consistent with a Δ trisubstituted double bond in each. A
ring system as in 6 was supported, including the three methyl
groups on the seven-membered ring. There was no reason to
expect that the rings or the relative configurations at C-6 and C-7
would be affected by the reaction.
(Figure 4). The H-3 resonances were unobstructed for both
compounds, and these established the spatial relationship be-
tween H-3 and the C-4 methylene group. In compound 10, H-3
1
gave a multiplet at δ 2.81, with J2,3 = 3.4 Hz, J3,4a = 7.7 Hz, J3,4b
=
11.5 Hz, and J3,15 = 2.1 Hz. A simulation using these J values
(Figure 4) agreed closely with the actual signal. The relatively
large values of both J3,4a and J3,4b indicate that H-3 must be anti to
one of the C-4 methylene protons and syn to the other (Figure 4).
Given the chair conformation of the ring, H-3 must be R-
oriented and the formyl group, β-oriented, as drawn in Figure 4.
Thus, synthetic 10 must have the (3S) configuration.
Determination of the configuration at C-3 relative to C-6
1
relied on both molecular modeling and the H NMR spectrum.
The resonances for H-4 to H-6 were too complex to establish the
conformation of the six-membered ring. However, modeling
conclusions, based on the low-energy structures calculated for 10
and 11, were that H-6 is gauche to the C-5 methylene group
and that the six-membered ring has a nearly chair conformation
1
In the H NMR spectrum of compound 11, H-3 appeared as a
doublet of doublets at δ 2.58 (J2,3 = 4.1 Hz and J3,4b = 7.7 Hz);
the lack of further splitting indicated that J3,4a and J3,15 were <1
Hz. The relatively small J3,4a and J3,4b values indicated a gauche
relationship between H-3 and the C-4 methylene protons. Thus,
H-3 must be β-oriented and the formyl group, R-oriented
(
Figure 4), and synthetic 11 has the (3R) configuration.
1
Figure 4. H NMR and modeling results for compounds 10 and 11.
Conformation of the six-membered ring was established by modeling for
1
both compounds. The observed H resonances for H-3 are shown, along
Figure 5. Gas chromatograms of compound 12 on a BDEX-325 chiral
column. Mass spectra for all peaks as for 12 in Figure 2. Oven program:
50 °C for 1 min, followed by 30 °C/min increase to the final temperature
of 120 °C.
with simulations based on the observed coupling constants. The J values
were consistent between the models and the observed NMR data. The
results established that the earlier-eluting beetle compound is structure 10.
Table 4. GC Retention Times of Synthetic and Beetle-Derived Compounds on Chiral Columns
GC retention time (min)
synthetic racemic
second peak
a
compound
column
final temp (°C)
first peak
synthetic enantiomer
beetle
b
9
GDEX-225
BDEX-325
BDEX-325
BDEX-325
BDEX-325
BDEX-325
BDEX-325
150
140
140
120
140
140
140
45.3
46.8
42.4
46.7
41.7
46.3
116.0
62.2
58.8
67.9
46.7
41.7
46.3
116.1
62.2
58.7
68.0
b
10
11
12
13
14
6
41.6
b
46.2
48.0
b
116.0
117.3
b
62.1
62. 7
59.4
68.5
b
58.7
b
68.0
a
b
GC temperature program: 50 °C for 1 min, then 30 °C/min to final temperature. Peak that matched synthetic enantiomer and beetle-produced
compound.
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Figure 6. Simultaneously recorded gas chromatogram of collected volatiles from male P. striolata, containing 1 and 9 (A), and antennal activity of female
P. striolata (B). Peak marked * is a GC artifact due to injection of 9 into a hot inlet.
Aldehyde 12. This compound unexpectedly resulted from
the treatment of enol ether 18 with perchloric acid and had the
the chiral products exactly by GC on an achiral column and by
MS. As shown in Table 4, GC conditions were found so that the
enantiomers of 9-14 could be separated. Only one enantiomer
of each was detected from the beetles, and this matched the
enantiomer from the chiral synthesis in each case. The example in
Figure 5 shows the GC traces for 12. Since the chiral precursors
to 9-14 (synthetic 1 and 6) were identical to natural 1 and 6
from P. cruciferae and since the synthetic enantiomers of 9-14
were identical to the natural compounds from P. striolata and
P. pusilla, the absolute configurations at C-6 and C-7 are
conserved in the compounds from these beetle species, despite
other structural differences.
polarity of an aldehyde, eluting from silica gel with 5% Et O in
2
hexanes. The apparent molecular weight was 218 (Figure 2).
13
Compound 12 had 15 C NMR resonances, including one
carbonyl and four olefinic signals (Table 3). There was NMR
1
3
1
evidence ( C and H shifts and HMBC correlations) for an
isopropenyl group (C-11, C-12, and C-13), a secondary aliphatic
methyl group (C-14), and a formyl group (C-15). These groups
accounted for five carbon atoms, thus leaving no more than 10 for
a ring system. A decalin-type ring system with the substitution
13
pattern shown was supported by the HMBC data and the C and
1
H NMR shifts. The rearrangement that converted the himach-
Preliminary GC-EAD Analysis. Female P. striolata antennae
(N = 5) readily sensed the presence of the two most abundant
compounds, 1 and 9, in volatile collections from male P. striolata
(Figure 6). The other apparent antennal responses seen in
Figure 6 were random noise signals. As noted above, the peak
marked * was probably due to thermal rearrangement of 9 in the
hot (250 °C) GC inlet port. This artifact would not occur from
P. striolata in nature; correspondingly, the beetle antennae did
not detect it as it emerged from the GC column (Figure 6).
Female P. striolata antennae responded equally well to natural 9
and to synthetic 9 (results not shown). Male antennae were not
tested. GC-EAD analyses for P. pusilla were not successful due to
the small number of individuals available for study and the rapid
deterioration of the antennal preparations in this species.
Chemistry, Chemical Ecology, and Practical Implications.
The current research revealed six new male-specific compounds that
likely play a role in Phyllotreta chemical ecology, doubling the
number of male-specific compounds known from this large genus.
Five of the new compounds have the himachalane carbon skeleton
that is prevalent in the volatiles from studied flea beetle species, but
there are also structural features not encountered previously in flea
beetle sesquiterpenes. These include formyl groups (in 10-12 and
14), an unsaturated hydroxyketone substructure (9), a primary
hydroxy group (in 13), and a decalin-based ring system with unusual
branching (12). Phyllotreta pusilla is the first studied Phyllotreta
species in which none of 1-5 have been detected.
alane ring system to the decalin presumably involved C-1, C-10,
and C-11. It was expected that the configurations at C-6 and C-7
would be unaffected, but it was not clear whether the ring
junction would be cis or trans after the rearrangement at C-1.
The H NMR spectrum and, in particular, the resonance for
H-6 (δ 1.31) gave information about the ring junction. Cou-
1
plings of 10.8, 3.5, 3.5, and 1.7 Hz were observed for H-6. By the
1
H and COSY spectra, J2,6 = 1.7 Hz (“W-coupling”). The other
couplings for H-6 were to H-5a, H-5b, and H-7, and J6,7 was
concluded to be the largest of these. The width of the multiplet
1
for H-7 was ∼49 Hz, from the H and HSQC spectra. Most
of this width (∼36 Hz) was due to the three protons of Me-14
(
J
= 6.5 Hz) to H-8b (J
7,8b
≈ 12.8 Hz, axial-axial) and to
7
,14
H-8a (J7,8a ≈ 4 Hz, typical for an axial-equatorial relationship,
not directly observable). By difference, J6 would be ∼13 Hz,
,7
which was close to only one observed value, 10.8 Hz (this
magnitude indicates that H-6 and H-7 have an axial-axial
relationship and supports that the configurations at C-6 and
C-7 are as in starting material 6). Thus, both J5a,6 and J5b,6 were
concluded to be 3.5 Hz, indicating a gauche relationship between
H-6 and the C-5 methylene protons. Molecular modeling and
structural models showed that a cis ring junction would accom-
modate the gauche conformation between H-6 and C-5 methyl-
ene protons, but this relationship was impossible with a trans ring
junction. We conclude that the ring junction must be cis.
Chiral GC Analysis. Racemic 9-14 resulted when the reac-
tions (Scheme 2) were applied to racemic 6 or 1. These matched
Preliminary electrophysiological analysis revealed that one of
the new compounds, 9, has biological activity in P. striolata that is
5
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Journal of Natural Products
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consistent with a pheromonal function, although the precise
behavioral effect of the compound has yet to be determined.
The lesser per capita laboratory emission of 9 from P. striolata
in groups than from individuals may be related to a natural
mechanism for regulating the size of beetle aggregations.
Field trapping experiments and other behavioral studies are still
needed for P. striolata and P. pusilla. Nevertheless, the com-
pounds described here open new possibilities for detecting and
managing pest species of flea beetles and for bringing a new level
of understanding to the chemical ecology of flea beetles.
to confirm identity, and voucher specimens were retained at the
Smithsonian Institution, Washington, DC. Also collected in Peoria were
P. conjuncta (Gentner) and P. zimmermanni (Crotch), both of which had
yellow and black elytral markings similar to P. striolata and required
careful examination under a microscope for certain identification.
Volatile Collections. Volatiles from male and female beetles feed-
ing on chunks of cabbage were trapped in filters containing Super-Q
4
porous polymer as described previously. For P. striolata, 87 collections
were made from groups of 5-10 males, 10 were made from groups of
females, and nine were made from cabbage only, as controls. The
collection period was usually 2-10 days. Subsequently, 41 collections
were made from individual males on cabbage. The study of P. pusilla
volatiles was based on 174 collections from 24 individual males and
’
EXPERIMENTAL SECTION
2
7 from 3 individual females, for comparison.
The volatile collections (400 μL, in hexanes) were usually concen-
General Experimental Procedures. NMR spectra of synthetic
compounds were obtained on a Bruker Avance 500 spectrometer. Samples
were dissolved in C , and spectra were acquired at 300 K. Experi-
trated about 10-fold under a gentle stream of nitrogen and submitted
to GC-MS. Sex-specific compounds that occurred consistently in collec-
tions were of particular interest. Representative samples with such
compounds were submitted to column chromatography on silica gel
to gain information about compound polarity. A typical column was 0.5
by 3 cm (in a Pasteur pipet) and was eluted with hexanes, followed by 5,
6 6
D
1
13
ments included H, C, COSY, HSQC, and HMBC. The DEPT-135
experiment was conducted for the sample containing 10 and 11, and
the NOESY and 1D NOE difference experiments were conducted for 9.
1
3
For compounds 12 and 14, the sample amounts were too small for
C
13
NMR, and C resonances were read from the HSQC and HMBC
spectra. H and C NMR shifts were assigned to the proposed struc-
1
13
10, and 25% Et O in hexanes, Et O, and 10% MeOH in DCM.
2 2
4
,6,7
tures, using the previous assignments for compounds 1 and 6
as the
Synthetic Compounds. Single enantiomers 1 and 6 were pre-
pared as synthetic precursors for 9-14 and for other project7s
(Scheme 1). The synthesis of 6 was mostly by the published method,
but (R)-(þ)-pulegone (15) was used as the starting material rather than
starting point. Some data processing and simulations were performed
18
with Spinworks 3.1 software.
Most EIMS (70 eV) was done on a Hewlett-Packard (HP) 5973 mass
selective detector, interfaced to an HP 6890 gas chromatograph. GC
columns included DB-1 (30 m, 0.25 mm i.d., and 0.25 μm film thickness,
or 15 m, 0.25 mm i.d., and 0.1 μm film thickness, J&W Scientific,
Folsom, CA). Helium was the carrier gas (34 cm/s), and injection was
through the splitless inlet. The usual oven temperature program was
19
citronellal for reasons of cost and availability. By a known method, 15
was converted with HCl gas to hydrochloride 16, which was then
hydrolyzed to citronellic acid 17, an early intermediate in the previous
synthesis of 6. The yield of 6 was 5 g from 100 g of 15. A portion of
the 6 was used to produce a 3.2:1 mixture of 1 and 3 (1.4 g) via alcohols 7
6
5
0 °C for 1 min, then increasing at 10 or 15 °C/min to 250 °C. Inlet
and 8.
temperature was usually 250 °C. The Wiley mass spectral library was
installed on the data system. An HP 5971 mass selective detector,
coupled to an HP 5890 GC, was also used, primarily for EIMS of
synthetic products. CIMS (isobutane reagent gas) was done for one
compound from P. striolata on a Finnigan 4535 instrument with GC
inlet. Chiral GC-MS analysis was done on the HP 5973, using BDEX-
Synthetic 1 and 6 were subjected to a variety of small-scale reactions,
attempting to generate compounds identical to those from the beetles
and in amounts large enough for NMR analyses. The syntheses of 9-14
are summarized in Scheme 2. Details are given in the Supporting Infor-
mation. The successful reactions were repeated on racemic 1 and 6,
6
prepared by the achiral route, to provide standards for chiral GC.
3
25 or GDEX-225 columns (both 30 m, 0.25 mm i.d., 0.25 μm film
thickness, Supelco, Bellefonte, PA). The initial oven temperature was
0 °C; after a 1 min hold, the oven temperature was increased at 30 °C/min
NMR data for the synthetic compounds are presented in Tables 1-3.
EIMS for 9-14 were as in Figure 2. HREIMS: 9, 236.1759, calc for
C H O , 236.1776. 10, 220.1842; 11, 220.1833; and 13, 220.1822,
5
1
5 24 2
to one of the final values (given with results). HREIMS was done at
the Department of Chemistry, University of Minnesota, on a VG 70SE
instrument with GC inlet.
Some volatile collections were analyzed on an HP 5890 GC with flame
ionization detector (GC-FID) for quantitative purposes (external standard
method, relative to heptadecane). Injection was through a splitless or
cool-on-column inlet.
High-performance liquid chromatography (HPLC) of synthetic
products employed a Waters 515 pump and a Waters R-401 differential
refractometer detector. A Rainin Dynamax silica column (Si-80-125-C5,
15 24
calc for C H O, 220.1827. 12, 218.1657, and 14, 218.1650, calc for
C H O, 218.1671. Nomenclature: 9, (3S,9R,9aS)-3-hydroxy-3,5,5,9-
1
5 22
tetramethyl-1,3,5,6,7,8,9,9a-octahydro-2H-benzo[7]annulen-2-one; 10,
(3S,9R,9aS)-5,5,9-trimethyl-2,3,5,6,7,8,9,9a-octahydro-1H-benzo[7]-
annulene-3-carbaldehyde; 11, (3R,9R,9aS)-5,5,9-trimethyl-2,3,5,6,7,8,
9,9a-octahydro-1H-benzo[7]annulene-3-carbaldehyde; 12, (4aS,5R,8aS)-
5-methyl-8a-(prop-1- en-2-yl)-3,4,4a,5,6,7,8,8a-octahydronaphthalene-
2-carbaldehyde; 13, (9R,9aS)-5,5,9-trimethyl-5,6,7,8,9,9a-hexahydro-1H-
benzo[7]annulen-3-yl]methanol; 14, (9R,9aS)-5,5,9-trimethyl-5,6,7,
8,9,9a-hexahydro-1H-benzo[7]annulene-3-carbaldehyde.
4
.6 mm i.d. by 25 cm) was used with a flow rate of 1 mL/min.
Chiral GC-MS. Conditions (column type and temperature) were
sought that would give separation of enantiomers for synthetic racemic
9-14 and 6. Under the successful conditions, the synthetic enantiomers
of 9-14 and 6 were compared to compounds derived from P. striolata
and P. pusilla to determine whether these had the same or opposite
absolute configuration. Mass spectra were acquired to confirm that the
GC peaks corresponded to the expected compounds.
Separations were monitored on the refractometer, but collected frac-
tions were also analyzed by GC-MS. Solvents and separation details are
given in the Supporting Information.
Insects. The beetles were collected from cabbage plots located at
NCAUR, Peoria, IL. P. striolata were collected during July-September
of 1999, 2001, and 2002, and P. pusilla, during October and November of
1
3
1
2
002, 2003, and 2009. Beetle sex was determined under a microscope by
Computational Procedure. Calculation of C and H NMR
17
examining the ventral surface of the abdomen tip. Species were
chemical shifts was performed within the GIAO (gauge-independent
17
20,21
tentatively determined prior to volatile collections, and identifications
were checked by dissection and examination of the genitalia, once
volatile collections were completed. Example specimens were submitted
to the USDA-ARS Systematic Entomology Laboratory, Beltsville, MD,
atomic orbital)
framework using the GAUSSIAN03 suite of ab initio
2
2
programs and the PQS (Parallel Quantum Systems) density functional
theory (DFT) optimization programs, implemented on PQS 64-bit 16
processor computers. Empirical potentials (AMBER)
2
3
2
4,25
were initially
5
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Journal of Natural Products
ARTICLE
used with Insight/Discover modeling software (version 2000.3 L,
Accelrys Corp.) to derive trial structures, which were then optimized
at the B3LYP/6-31þG* level of theory. Due to the flexibility of the
seven-membered ring, many different stable conformations had to be
calculated via the DFT optimization procedure. The numbers of unique
DFT-optimized conformations considered in this study were 11 for 9
(13) March, J. Advanced Organic Chemistry, 3rd ed.; John Wiley and
Sons: New York, 1985; p 735.
(14) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7
277–7287.
15) March, J. Advanced Organic Chemistry, 3rd ed.; John Wiley and
Sons: New York, 1985; p 847.
16) Corey, E. J.; Chaykovsky, M.. J. Am. Chem. Soc. 1965,
(
(
(
(
including both possible epimers), 12 for 10, 13 for 11, and 18 for 12
including the cis and trans ring junctions). Relative energies of forma-
87, 1353–1364.
1
13
(17) Smith, E. H. Fieldana (Zoology) 1983, 28, 1–168.
tion and H and C NMR shifts (relative to TMS) were calculated for all
structures at the B3LYP/6-31þG* level of theory. The predicted
chemical shifts were further adjusted with linear regression so that the
rmsd relative to observed values was minimized (notably, the predicted
(18) SpinWorks 3.1, 2009, Kirk Marat, University of Manitoba;
simulations based on NUMARIT algorithm, Martin, J. S.; Quirt, A. R.
J. Magn. Reson. 1971, 5, 318.
(
19) Overberger, C. G.; Kaye., H. J. Am. Chem. Soc. 1967, 89,
640–5645.
20) Wolinski, K.; Hilton, J. F.; Pulay, P. J. Am. Chem. Soc. 1990,
1
3
C NMR shifts were systematically lower than the observed shifts by
5
about 4 ppm). At the same level of theory, J values were calculated for all
relevant structures.
(
112, 8251–6260.
GC-EAD Analysis. Coupled gas chromatographic-electroanten-
nographic detection (GC-EAD) analyses were made by methods and
(21) Rauhut, G.; Puyear, S.; Wolinski, K.; Pulay, P. J. Phys. Chem.
1996, 100, 6310–6316.
26
equipment generally described earlier. GC-EAD connections were
made by inserting a glass-pipet silver-grounding electrode into the back
of an excised beetle head. A second glass-pipet silver-recording probe
was placed in contact with the distal end of one antenna. Both pipettes
(22) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam; J. M.; Iyengar, S. S.; Tomasi, J.;
Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson,
G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.;
Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski,
V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui,
Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith,
T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.;
Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.;
Pople, J. A. Gaussian 03, Revision C.02; Gaussian, Inc.: Wallingford, CT,
27
were filled with Beadle-Ephrussi saline.
’
ASSOCIATED CONTENT
S
Supporting Information. Synthetic details for 9-14.
b
This information is available free of charge via the Internet at
http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
*Phone: 309-699-9851. E-mail: Robert.Bartelt@sbcglobal.net.
2
004.
(23) PQS (Version 3.3), Ab Initio Program Package; Parallel Quantum
Solutions: Fayetteville, AR, USA, 2007.
24) Momany, F. A.; Willett, J. L.; Schnupf, U. Carbohydr. Polym.
009, 78, 978–986.
25) Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.;
Notes
Retired.
§
(
2
(
’
ACKNOWLEDGMENT
Dr. A. Konstantinov of the USDA-ARS-Systematic Entomology
Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J.
J. Comput. Chem. 2005, 26, 1668–1688.
(26) Coss ꢀe , A. A.; Bartelt, R. J. J. Chem. Ecol. 2000, 26, 1735–1748.
(27) Ephrussi, B.; Beadle, G. W. Am. Nat. 1936, 70, 218–225.
Laboratory, Beltsville, MD, kindly verified the identity of Phyllotreta
beetles used in this research.
’
REFERENCES
(1) Lamb, R. J. Annu. Rev. Entomol. 1989, 34, 211–229.
(2) Capinera, J. L. Handbook of Vegetable Pests; Academic Press:
San Diego, CA, 2001.
3) Gassmann, A.; Schroeder, D.; Maw, E.; Sommer, G. Biol. Control
996, 6, 105–113.
4) Bartelt, R. J.; Coss ꢀe , A. A.; Zilkowski, B. W.; Weisleder, D.;
Momany, F. A. J. Chem. Ecol. 2001, 27, 2397–2423.
5) Zilkowski, B. W.; Bartelt, R. J.; Coss ꢀe , A. A.; Petroski, R. J.
J. Chem. Ecol. 2006, 32, 2543-2558.
6) Bartelt, R. J.; Weisleder, D.; Momany, F. A. Synthesis 2003,
17–123.
(
1
(
(
(
1
1
(
7) Muto, S.; Bando, M.; Mori, K. Eur. J. Org. Chem. 2004,
946–1952.
(8) Mori, K. Tetrahedron: Asymmetry 2005, 16, 685–692.
(9) Soroka, J. J.; Bartelt, R. J.; Zilkowski, B. W.; Coss ꢀe , A. A. J. Chem.
Ecol. 2005, 31, 1829–1843.
ꢀ
(10) T ꢀo th, M.; Csonka, E.; Bartelt, R. J.; Coss ꢀe , A. A.; Zilkowski,
B. W.; Muto, S.-E.; Mori, K. J. Chem. Ecol. 2005, 31, 2705–2720.
(
(
11) Lamb, R. J.; Turnock, W. J. Can. Entomol. 1982, 114, 827–840.
12) Pandey, R. C.; Dev, S. Tetrahedron 1968, 24, 3829–3839.
5
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dx.doi.org/10.1021/np100608p |J. Nat. Prod. 2011, 74, 585–595