Discovery of the Aggregation Pheromone of the Brown Marmorated
Stink Bug (Halyomorpha halys) through the Creation of
Stereoisomeric Libraries of 1‑Bisabolen-3-ols
Donald C. Weber,
Jeﬀrey R. Aldrich,
Karl E. Vermillion,
Maxime A. Siegler,
and Tracy C. Leskey
U.S. Department of Agriculture, Agricultural Research Service, Beltsville Area, IIBBL, Maryland 20705, United States
The Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan 115
U.S. Department of Agriculture, Agricultural Research Service, NCAUR, Peoria, Illinois 61604, United States
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United States
U.S. Department of Agriculture, Agricultural Research Service, AFRL, Kearneysville, West Virginia 25430, United States
ABSTRACT: We describe a novel and straightforward route
to all stereoisomers of 1,10-bisaboladien-3-ol and 10,11-epoxy-
1-bisabolen-3-ol via the rhodium-catalyzed asymmetric addi-
tion of trimethylaluminum to diastereomeric mixtures of
cyclohex-2-enones 1and 2. The detailed stereoisomeric
structures of many natural sesquiterpenes with the bisabolane
skeleton were previously unknown because of the absence of
stereoselective syntheses of individual stereoisomers. Several of
the bisabolenols are pheromones of economically important
pentatomid bug species. Single-crystal X-ray crystallography of
underivatized triol 13 provided unequivocal proof of the
relative and absolute conﬁgurations. Two of the epoxides,
(3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (4), were identiﬁed as the main components of a male-produced aggregation
pheromone of the brown marmorated stink bug, Halyomorpha halys, using GC analyses on enantioselective columns. Both
compounds attracted female, male, and nymphal H. halys in ﬁeld trials. Moreover, mixtures of stereoisomers containing epoxides
3and 4were also attractive to H.halys, signifying that the presence of additional stereoisomers did not hinder attraction of H.
halys and relatively inexpensive mixtures can be used in monitoring, as well as control strategies. H. halys is a polyphagous
invasive species in the U.S. and Europe that causes severe injury to fruit, vegetables, and ﬁeld crops and is also a serious nuisance
The bisabolane skeleton is a recurring structural motif in the
semiochemistry of stink bugs (Hemiptera: Pentatomidae).
Bisabolene epoxides comprise male-speciﬁc pheromones of
and Chinavia (=Acrosternum) spp.
related zingiberene, β-sesquiphellandrene, and α-curcumene
constitute part of the Thyanta pallidovirens pheromone,
sesquiphellandrene was identiﬁed as a pheromone component of
More recently, two stereoisomeric 1,10-
were identiﬁed as part of the male-produced
pheromone of the rice stalk stink bug, Tibraca limbativentris,
10,11-epoxy-1-bisabolen-3-ol (called “murgantiol”) has been
reported as an aggregation pheromone of the harlequin bug,
As with murgantiol, the relative and
absolute conﬁgurations of the 1,10-bisaboladien-3-ols from T.
limbativentris have not been determined. Reliable assignment of
relative conﬁgurations across the cyclohexene ring of the
murgantiol structure was problematic, and 1H and 13C NMR
recordings of murgantiol failed to provide a conclusive answer.
Several related compounds were isolated from the oil of ginger,
Zingiber off icinale, among them a 1,10-bisaboladien-3-ol, called
The latter was assigned a trans-conﬁguration
based on similarities of its IR spectrum with that of trans-p-
but the structure was presented as the cis-
and the absolute conﬁguration has not been disclosed.
A sex pheromone of the rice stink bug, Oebalus poecilus, has
recently been also identiﬁed as zingiberenol and, more
The absolute conﬁguration has been
assigned based on the correlation to natural zingiberene and
similarities of 13C NMR spectra of a synthetic mixture containing
the pheromone and (R,R)-quercivorol. However, the pher-
omone of O. poecilus has not been synthesized in pure form and
Received: May 6, 2014
Published: June 25, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy 1708 dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 1708−1717
characterized, nor has any single stereoisomer of 1,10-
bisaboladien-3-ol and/or 10,11-epoxy-1-bisabolen-3-ol been
synthesized elsewhere to assist identiﬁcations.
The brown marmorated stink bug, Halyomorpha halys (Stål),
is an invasive pest from Asia, now well established in the mid-
Atlantic region and spread to most of the continental U.S. as well
as parts of Canada and central Europe. H. halys is a polyphagous
pest of many crops including tree fruits, vegetables, ﬁeld crops,
and ornamentals, with signiﬁcant economic damage recorded in
A monitoring tool to assess the presence,
abundance, and seasonal activity of H. halys was urgently sought
to determine the need for and timing of management actions. In
this study, we describe all eight stereoisomers of 1,10-
bisaboladien-3-ol and selected stereoisomers of 10,11-epoxy-1-
bisabolen-3-ol that provided guidance for the identiﬁcation of a
male-produced aggregation pheromone of H. halys.
■RESULTS AND DISCUSSION
Syntheses of Individual Stereoisomers of 1,10-Bisabo-
ladien-3-ol. We used a rhodium-catalyzed asymmetric 1,2-
addition of organoaluminum compounds to enones
synthesize bisaboladienol intermediates. This catalytic reaction
was highly enantioselective (>96% ee), with unsubstituted
cyclohex-2-enone providing (R)-1-methyl-2-cyclohexen-1-ol
with (S)-BINAP and (S)-1-methyl-2-cyclohexen-1-ol with (R)-
BINAP chiral ligands complexed to the rhodium.
We did not
ﬁnd any example of such a reaction with a cyclohex-2-enone
substituted at position 4; yet one might anticipate that the
diastereotopic face selectivity of this reaction would be
dependent on the size and spatial orientation of substituents.
Because individual stereoisomers of ketones 1and 2were
diﬃcult to synthesize,
we studied Rh-catalyzed additions of
trimethylaluminum with mixtures of these diastereomeric
Thus, the reaction of a ∼1:1 mixture of (6S,7R)-1and
(6R,7R)-1with trimethylaluminum in the presence of chloro-
(1,5-cyclooctadiene)rhodium(I) dimer and (R)-BINAP yielded
two easily separable compounds (Scheme 1, left). The major
product had a higher retention factor (Rf) by TLC analysis on
silica gel eluted with hexane/EtOAc, but a lower retention time
by GC analysis on an HP-5 column as compared to the minor
product. Including the stereocenter at C-3, 1,10-bisaboladien-3-
ols can exist in two relative conﬁgurations: cis, if the hydroxy
group at C-3 and the alkyl group at C-6 are on the same side, and
trans, if these groups are on the opposite sides of a plane formed
by C-6, C-1, C-2, and C-3. On the basis of the chromatographic
parameters, X-ray crystallography, 1H and 13C NMR data, and
dehydration to stereochemically deﬁned 1,3,10-bisabolatrienes
(see further in the text), the major compound was identiﬁed as cis
and assigned structure 5; the minor product was found to have
trans relative conﬁguration and assigned structure 6.The
stereochemical course of the trimethylaluminum addition in
Scheme 1. Syntheses of Bisaboladienol Stereoisomers 5−12 by the Reaction of ∼1:1 Diastereomeric Mixtures of (6S,7R)-1 and
(6R,7R)-1 (Left) and (6S,7S)-2 and (6R,7S)-2 (Right) with Trimethylaluminum (2 equiv) in the Presence of [Rh(cod)Cl]2(0.05
equiv) and(R)- or (S)-BINAP (0.12 equiv)
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the presence of (R)-BINAP is shown in Scheme 1 (left). Of the
two diastereomers, (6S,7R)-1is the favored isomer because the
substituent at position 6 is oriented above the plane formed by C-
6, C-1, C-2, and C-3 (si-face) and does not cause steric hindrance
to the delivery of the methyl group from the re-face as postulated
in the original report.
Thus, the reaction was highly
diastereoselective and provided (3S,6S,7R)-stereoisomer 5.In
the case of (6R,7R)-1, the re-face is shielded by the side chain and
the si-face is intrinsically restricted.
As a result, the Rh-catalyzed addition of trimethylaluminum
was disfavored and accompanied by side reactions, including
polymerization. Nevertheless, the re-face approach was still
prevalent over the si-face, leading to (3S,6R,7R)-stereoisomer 6,
albeit in low yield. Both 5and 6were isolated in greater than 95%
chemical purities. Because of diﬃculties in separation of
stereoisomers having the same relative (cis/trans) conﬁguration
and the ease of separation of cis stereoisomers from trans, alcohol
5was cross-contaminated with cis-stereoisomer 7, arising from
(6R,7R)-1, and alcohol 6contained some of trans-isomer 8
originating from (6S,7R)-1. Diastereomeric ratios of 96:4 for 5/7
and 86:14 for 6/8were found by integration of H-14 signals in
the 1H NMR spectra available in the Supporting Information.
As expected from the results above, the reaction of a ∼1:1
diastereomeric mixture of ketones 1with trimethylaluminum in
the presence of chloro(1,5-cyclooctadiene)rhodium(I) dimer
and (S)-BINAP provided stereoisomers 7and 8as major
products (Scheme 1, left). In accordance with an si-face approach
postulated in the presence of (S)-BINAP,
favored in this reaction and provided (3R,6R,7R)-alcohol 7. Due
to steric constraints, the addition of trimethylaluminum to
(6S,7R)-1was low-yielding and nondiastereoslective (si-face/re-
face, 62:38). In this milieu, cis-alcohol 7was cross-contaminated
with cis-alcohol 5and trans-alcohol 8with trans-alcohol 6with
diastereomeric ratios of 91:9 and 89:11, respectively. NMR
signals from minor stereoisomers 5and 6were easily discernible
because they were major products when (R)-BINAP was used.
The four remaining stereoisomers of 1,10-bisaboladien-3-ol
were synthesized from a diastereomeric mixture of ketones 2
with (7S)-conﬁguration (Scheme 1, right). The stereochemis-
tries of the addition of trimethylaluminum to the carbonyl group
in the presence of chloro(1,5-cyclooctadiene)rhodium(I) dimer
and (S)- and (R)-BINAP were essentially governed by the same
rules as described for the (7R)-ketones. With (R)-BINAP
enabling an re-face approach, (6S,7S)-2was a preferred
diastereomer, leading to the (3S,6S,7S) stereoisomer 9in 93:7
dr, and the sterically disfavored (6R,7S)-2provided primarily
byproducts but, nonetheless, yielded the (3S,6R,7S)-stereo-
isomer 10 in 96:4 dr. With (S)-BINAP enabling an si-face
approach, (6R,7S)-2did not sterically hinder the approaching
nucleophile and provided the (3R,6R,7S)-isomer 11 in 96:4 dr as
the main product, while (6S,7S)-2led (as a disfavored
diastereomer) to the (3R,6S,7S)-stereoisomer 12 in 93:7 dr.
Surprisingly, alcohol 5, a precursor to the main component of
the H. halys pheromone (3S,6S,7R,10S)-10,11-epoxy-1-bisabo-
len-3-ol (3), was obtained in 96:4 dr. The eﬃcient production of
5was due to two factors: ﬁrst, the highly diastereotopically
selective addition of trimethylaluminum to (6S,7R)-1and,
second, because (6R,7R)-1present in the mixture with
(6S,7R)-1primarily underwent side reactions rather than
producing suﬃcient cis-stereoisomer 7to signiﬁcantly contam-
inate isomer 5. Thus, low yields from disfavored reactions
allowed ready production of cis-stereoisomers of 1,10-
bisaboladien-3-ols. Conversely, trans-stereoisomers were isolated
from disfavored reactions because only trace amounts of trans-
isomers were produced from favored reactions. As a result, a pair
of diastereomerically enriched stereoisomers (one cis and
Scheme 2. Syntheses of Triols and Epoxy Alcohols Including H. halys Pheromone Components 3 and 4
a, AD-mix-α; b, AD-mix-β; c, (1) MsCl/Py, (2) KOH/MeOH. Diastereomeric purities were determined by GC analysis on Hydrodex-β-6TBDM*
or Chiraldex G-TA#columns. Top right: Displacement ellipsoid plot (50% probability level) of triol 13 at 110(2) K.
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another trans) could be synthesized from a single reaction of a
diastereomeric mixture of 1or 2with trimethylaluminum in
rhodium-catalyzed asymmetric 1,2-addition conditions.
Assignment of Relative (cis/trans) Conﬁgurations of
1,10-Bisaboladien-3-ols. Assignments of the relative conﬁg-
urations of 1,10-bisaboladien-3-ols are largely absent from the
literature. We observed that, regardless of the absolute
conﬁguration at C-7, all eight stereoisomers of 1,10-bisabola-
dien-3-ols could be divided into two groups. Four major
stereoisomers from our syntheses, 5,7,9, and 11 (Scheme 1),
had identical retention factors by TLC analyses (SiO2; hexane/
EtOAc) that were higher than those of four minor stereoisomers,
6,8,10, and 12. GC retention times of the major stereoisomers
on an HP-5 column were almost indistinguishable from each
other but were shorter than those for the minor stereoisomers,
which also eluted as a tight group. Thus, reliably proving a
relative conﬁguration for at least one stereoisomer would suﬃce
for assigning relative conﬁgurations of all eight stereoisomers of
We found that the Sharpless asymmetric dihydroxylation
stereoisomer 8with AD-mix αproceeded smoothly and provided
crystalline triol 13 (Scheme 2). After crystallization of 13 using a
liquid−liquid diﬀusion technique, we obtained single crystals
suitable for X-ray structure determination using Cu Kαradiation
and unequivocally proved its absolute conﬁguration. The
displacement ellipsoid plot of crystalline 13 presented in Scheme
2 clearly shows a (3R,6S,7R,10S)-absolute conﬁguration, under-
lining that the hydroxy group at C-3 and alkyl group at C-6 are
trans. This provided direct evidence that the lower TLC Rf/
longer GC retention time stereoisomers of 1,10-bisaboladien-3-
ol had the trans relative conﬁguration and that this rule must
apply to the other minor stereoisomers 6,10,and12.
Conversely, higher Rf/shorter retention time stereoisomers 5,
7,9, and 11 must have the cis-conﬁguration.
In fact, monoterpene analogues of 1,10-bisaboladien-3-ols
display the same chromatographic behavior. For instance,
individual stereoisomers and mixtures of cis-p-menth-2-en-1-ols
eluted faster (hence had higher retention factors) during
chromatography on SiO2using hexane/EtOAc than the
Comparison of 13C NMR spectra of menth-2-en-1-ols and
1,10-bisaboladien-3-ols supported assignments of the relative
(and absolute) conﬁgurations. Resonances from C-1 and C-2 of
stereoisomer 5occurred at 133.9 and 133.6 ppm, respectively,
and the signals from the oleﬁnic carbons of cis-(S,S)-menthenol
and cis-(R,R)-menthenol were remarkably similar: 133.5 and
and 133.0 and 133.4 ppm,
respectively. In contrast
to 5, resonances of C-1 and C-2 in bisaboladienol 6appeared at
130.5 and 135.1 ppm, analogous to signals of oleﬁnic carbons in
trans-(S,R)-menthenol at 131.5 (131.2) and 134.8 (134.5)
Other stereoisomeric 1,10-bisaboladien-3-ols com-
plied with the observed diﬀerences in chemical shifts of C-1 and
C-2, with trans-isomers displaying greater Δδ(3.1−4.6 ppm)
than cis-isomers (0.3−1.2 ppm) regardless of the absolute
conﬁguration at C-7. In addition, resonances of C-3 and C-15 in
trans- and cis-1,10-bisaboladien-3-ols and 10,11-epoxybisabolen-
3-ols closely corresponded to those of menth-2-en-1-ols of
Absolute Conﬁgurations of 1,10-Bisaboladien-3-ols.
The absolute conﬁgurations of stereoisomers 5−12 were
established on the basis of knowledge of their relative
conﬁgurations and chemical correlations. We used dehydration
reactions with phosphorus oxychloride
to correlate bisabola-
dienol stereoisomers with natural β-sesquiphellandrene and
zingiberene and their stereoisomers with established absolute
conﬁgurations. The reaction of a ∼1:1 mixture of 6and 8with
POCl3provided the expected 1,3(15),10-bisabolatriene 14 (= β-
sesquiphellandrene) and 1,3,10-bisabolatriene 15 (= zingiber-
ene) (both as mixtures of two diastereomers), plus an unknown
sesquiterpene hydrocarbon, in a 43:52:5 ratio (Scheme S1, left,
Figure 1, panel b). Major dehydration products 14 and 15 were
identiﬁed by GC-MS with authentic samples of zingiberene
1,3(15),10-bisabolatrienes and 1,3,10-bisabolatrienes were not
separated on an HP-5MS column, but 1,3(15),10-bisabolatrienes
were almost baseline-separated on a Hydrodex-β-6TBDM
column (Figure 1, panel b). Because 1,3(15),10-bisabolatrienes
derived from 6+8had the (7R)-conﬁguration and the natural
(−)-β-sesquiphellandrene has the (6R,7S)-conﬁguration,
latter could not be used for the identiﬁcation of the compounds
associated with these GC peaks. Hence, we synthesized an
individual 1,3(15),10-bisabolatriene with the (7R)-conﬁguration
as follows. Stereoisomer 7was dehydrated with POCl3
analogously to 6+8, and the mixture of hydrocarbons (Scheme
S1, left) was subjected to reaction with 4-phenyl-1,2,4-triazoline-
3,5-dione, whereupon 1,3,10-bisabolatriene acted as a dienophile
to form Diels−Alder adduct 17 (Scheme S1, left).
16 (Figure 1, panel a), which did not undergo a Diels−Alder
was isolated by chromatography in 97% chemical
purity. 1H and 13C NMR spectra of 16 were in good agreement
Figure 1. Gas chromatograms of bisabolatrienes on Hydrodex-β-
6TBDM at 110 °C; H22.0 mL/min: (a) (6R,7R)-1,3(15),10-
bisabolatriene (16) from alcohol 7after removal of zingiberene and
other products; (b) from 6+8; (c) from alcohol 5.
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with those of (6S,7S)-1,3(15),10-bisabolatriene,
16 was dextrorotatory, this compound was assigned the (6R,7R)-
conﬁguration, which was then also assigned to 7, from which 16
originated. Finally, because alcohol 7belonged to the pool of
higher Rf/shorter retention time cis-1,10-bisaboladien-3-ols, it
was assigned the (3R,6R,7R) absolute conﬁguration. With the
absolute conﬁguration of compound 16 established, we assigned
the faster-eluting 1,3(15),10-bisabolatriene the (6R,7R)-conﬁg-
uration and the slower-eluting diastereomer the (6S,7R)-
conﬁguration (Figure 1, panel b). Determinations of the absolute
conﬁgurations of the three other 1,10-bisaboladien-3-ols with the
(7R)-conﬁguration were carried out using the developed GC
method. Thus, reaction of alcohol 5with POCl3(Scheme S1,
left) produced a mixture of sesquiterpene hydrocarbons, the GC-
FID trace of which is presented in Figure 1, panel c. 1,3(15),10-
Bisabolatriene present in that mixture matched the slower-
eluting compound and, hence, has the (6S,7R)-conﬁguration.
Because alcohol 5has the cis relative conﬁguration (higher Rf/
shorter retention time), its absolute conﬁguration is (3S,6S,7R).
Dehydrations of the two trans (lower Rf/longer retention time)
alcohols 6and 8produced expected mixtures of hydrocarbons
(Scheme S1, left). Because 6produced primarily (6R,7R)-
1,3(15),10-bisabolatriene (Figure S1, a) and 8(6S,7R)-1,3-
(15),10-bisabolatriene (Figure S1, c), they were assigned
(3S,6R,7R)-and (3R,6S,7R)-conﬁgurations, respectively.
The dehydration of a ∼1:1 mixture of 9and 11 with (7S)-
conﬁgurations provided the expected hydrocarbon mixtures
(Scheme S1, right). Interestingly, in this case diastereomeric
1,3,10-bisabolatrienes (= zingiberenes), but not 1,3(15),10-
bisabolatrienes, were separated on a Hydrodex-β-6TBDM
column (Figure S2, panel a).
conﬁgurations because natural (−)-zingiberene is (6R,7S)-
and this compound matched (Figure
S2, panel b) the slower-eluting of the two stereoisomeric 1,3,10-
bisabolatrienes. Hence, the faster-eluting compound in Figure
S2, panel a, was identiﬁed as (6S,7S)-1,3,10-bisabolatriene, or 6-
epi-zingiberene. Dehydration of alcohol 11 provided zingiberene
(Figure S2, panel c), and because 11 is a cis-alcohol, its absolute
conﬁguration must be (3R,6R,7S). Conversely, alcohol 12
formed 6-epi-zingiberene upon dehydration (Figure S3, panel
b), and because it has a trans relative conﬁguration, its absolute
conﬁguration has to be (3R,6S,7S). Dehydrations of cis-alcohol 9
led to 6-epi-zingiberene, and trans alcohol 10 to zingiberene
(Scheme S1, right). Hence, these compounds were assigned
(3S,6S,7S)-and (3S,6R,7S)-conﬁgurations, respectively. The
presence of minor diastereomers in the 1,3(15),10-bisabolatriene
and 1,3,10-bisabolatriene dehydration products, as determined
from GC analyses on a Hydrodex-β-6TBDM column, was
ascribed to isomeric 1,10-bisaboladien-3-ols formed along with
the main stereoisomers during the Rh-catalyzed addition of
trimethylaluminum to ketones 1and 2.
Syntheses of Stereoisomers of 10,11-Epoxy-1-bisabo-
len-3-ols. For enantioselective epoxidation of the 10,11
carbon−carbon double bond of 1,10-bisaboladien-3-ols we
used a sequence of a Sharpless asymmetric dihydroxylation and
stereoselective cyclization of intermediate diols through
Dihydroxylations of 1,10-bisabola-
dien-3-ols (5,7,8, and 11) occurred regioselectively at the
trisubstituted double bonds and provided triols 18−23 (Scheme
2). The absolute conﬁguration of triol 13, determined by single-
crystal X-ray crystallography, conﬁrmed that C-10 has the S-
conﬁguration (Scheme 2), as expected from Sharpless
asymmetric dihydroxylation with AD-mix α.
Thus, we assigned
the other triols originating from AD-mix αdihydroxylations (18,
20) (10S)-conﬁgurations, and triols obtained from AD-mix β
dihydroxylations (19,21,22, and 23) (10R)-conﬁgurations.
Triols were regioselectively converted to the corresponding
mesylates of the secondary hydroxy groups, and the mesylates
were cyclized to epoxides (Scheme 2) by treatment with KOH in
Because this intramolecular cyclization proceeded
with inversion of conﬁguration,
carbon atoms at position 10
in the epoxybisabolenols 24 and 25 were assigned the R-
conﬁguration, and those in compounds 3,4,26, and 27 the S-
conﬁgurations. Thus, the Sharpless asymmetric dihydroxylation
of 1,10-bisaboladien-3-ols followed by the epoxide ring closure of
the intermediate triols oﬀered a simple two-step route to make
individual stereoisomers of 10,11-epoxy-1-bisabolen-3-ols with
predictable stereochemistry. Herein, we describe the preparation
of the six stereoisomers of 10,11-epoxy-1-bisabolen-3-ol that
were essential for H. halys pheromone identiﬁcation; details for
the remaining stereoisomers will be reported elsewhere.
Identiﬁcation of Male-Speciﬁc Aggregation Phero-
mone Components from Halyomorpha halys.We collected
airborne extracts from separate groups of male and female H.
halys. GC-MS analyses of these aerations showed that H. halys
males produced several compounds not present in the extract of
volatiles from females (Figure 2). Electron impact ionization
mass spectra of two main male-speciﬁc compounds, A and B,
were quite similar (Figure 3) and resembled the mass spectra of
sesquiterpenoids, with the ion at m/z93 suggesting a
methylcyclohexadiene fragment and the peak at m/z220
possibly being a molecular ion. Chemical ionization mass spectra
of these two compounds with ammonia as a reagent gas
Figure 2. GC-MS total ion chromatograms of Halyomorpha halys male
and female aerations on a DB-5MS column, 40(1) to 300 °Cat10°C/
min; He 1.0 mL/min. Two male-speciﬁc compounds (A and B) are
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contained an ion at m/z256 (238 +NH4), suggesting that the
molecular weight of both A and B is 238 amu, corresponding to a
molecular formula of C15H26O2, and the ion at m/z220 is formed
apparently by the loss of 18 amu (H2O) under EIMS conditions.
Interestingly, we found a striking similarity between the mass
spectra of compounds A and B and that of the recently reported
aggregation pheromone of the harlequin bug, Murgantia
Thus, using the authentic samples described
above, we identiﬁed the faster-eluting compound A as a cis-10,11-
epoxy-1-bisabolen-3-ol and the slower-eluting compound B as a
trans-10,11-epoxy-1-bisabolen-3-ol. In order to determine the
absolute conﬁgurations of compounds A and B, we compared
GC retention times of the compounds in the extract of volatiles
from males with those of mixtures and individual stereoisomers
of 10,11-epoxy-1-bisabolen-3-ol on enantioselective columns.
We resolved all four cis-10,11-epoxy-1-bisabolen-3-ols with (7R)-
conﬁguration on a Chiraldex G-TA column and identiﬁed
individual compounds by co-injections with authentic samples
(Figure 4). We found that (3S,6S,7R,10S)-stereoisomer 3
matched compound A in the H. halys male airborne collection.
Hydrodex-β-6TBDM was the column of choice for separation of
all four cis-10,11-epoxy-1-bisabolen-3-ols with the (7S)-conﬁg-
urations (Figure S4), with the second peak, identiﬁed as
stereoisomer 27, being close but not accurately matching
compound A in the H. halys male extract. Compounds A and
27 were in fact clearly separated on the Chiraldex G-TA column
(Figure S5). Thus, out of eight cis-10,11-epoxy-1-bisabolen-3-ols,
only (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) matched
the main male-speciﬁc compound A present in the H. halys male
aeration. Next, we found that four trans-10,11-epoxy-1-
bisabolen-3-ols with the (7R)-conﬁguration baseline separated
on Hydrodex-β-6TBDM (Figure 5), and the third component of
that mixture matched compound B in the H. halys male airborne
extract. This stereoisomer was identiﬁed as (3R,6S,7R,10S)-
10,11-epoxy-1-bisabolen-3-ol (4). Finally, no trans-10,11-epoxy-
1-bisabolen-3-ols with the (7S)-conﬁguration matched com-
pound B (Figure S6).
Thus, in a pursuit of behaviorally active
compounds that could constitute an aggregation pheromone of
BMSB we focused on the ﬁeld bioassay of stereoisomers 3and 4.
Field Bioassay. Comparison of lures containing each of the
synthetic pheromone components 3and 4separately and
together in the natural ratio of 3.5:1 demonstrated that the
treatments diﬀered for both adult and nymphal captures (Table
1; ANOVAs for treatment eﬀects [using arcsin-square-root-
transformed block proportions of totals for the trapping period]:
F(3,16) = 59.6; p< 0.0001 for adults; F(3,16) = 15.1; p< 0.0001 for
nymphs). Means comparison (Tukey’s HSD test)
that for adult captures the major component 3was more
attractive than the minor component 4, which in turn was more
attractive than the blank lure, but the mixture at the natural 3.5:1
ratio was more attractive than either 3or 4alone. For nymphal
captures, lures containing the major component 3caught
Figure 3. GC-MS total ion chromatogram of aeration extract from virgin
H. halys males on an HP-5MS, He 1.0 mL/min, 50(5) to 270 °Cat10
°C/min. Mass spectra of main male-speciﬁc compounds A and B are
Figure 4. Segments of GC-MS total ion chromatograms on a Chiraldex
G-TA column, He 1.0 mL/min, 50(3) to 140 °Cat10°C/min. (a) Co-
injection of H. halys male aeration and four cis-(7R)-10,11-epoxy-1-
bisabolen-3-ols; (b) cis-(7R)-10,11-epoxy-1-bisabolen-3-ols, 25,26,24,
and 3; (c) H. halys male aeration.
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dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 1708−17171713
signiﬁcantly more than lures not containing the major
Furthermore, ﬁeld tests of a 3:1 mixture of cis- and trans-10,11-
epoxy-1-bisabolen-3-ols with the (7R)-conﬁguration (mixed-
isomer lure), which was prepared from (R)-citronellal without
stereoselective reactions as a mixture of eight isomers,
that this mixture was 9.6 times more attractive to adults than the
blank lure (15.4 adults per trap versus 1.6 for unbaited; ANOVA
for treatment eﬀect (using arcsin-square-root-transformed block
proportions of totals for the trapping period): F(1,8) = 70.1; p<
0.0001 for adults). No nymphs were captured during this trial
because of the early season time frame of the experiment. In
subsequent studies that will be summarized elsewhere, nymphs
were readily captured with the mixed-isomer lure. Thus, ﬁeld
bioassays demonstrated that both pheromone components were
important for optimal attraction, but the presence of additional
stereoisomers apparently does not hinder attraction of H. halys.
Therefore, relatively inexpensive mixtures of the stereoisomers
can be developed for trapping the brown marmorated stink bug.
Finally, the recently discovered synergy of the H. halys
aggregation pheromone with methyl (E,E,Z)-2,4,6-decatrie-
identiﬁes a season-long attractive tool for detection,
monitoring, and potential control of this polyphagous invasive
pest of North America and Europe.
We isolated and identiﬁed the aggregation pheromone of the
brown marmorated stink bug, H. halys. Rhodium/BINAP-
catalyzed addition of trimethylaluminum to diastereomeric
mixtures cyclohex-2-enones 1and 2aﬀorded two stereoisomers
from one reaction and thus provided an access to all eight
stereoisomers of 1,10-bisaboladien-3-ol and six of stereoisomers
of 10,11-epoxy-1-bisabolen-3-ol, previously unreported. In
addition to enabling the complete stereochemical identiﬁcation
of the H. halys main pheromone components, the creation of
these stereoisomeric libraries will be useful in identifying the
relative and absolute conﬁgurations of other natural products,
including the pheromones of at least two other pentatomid bugs,
Murgantia histrionica and Tibraca limbativentris.
General Experimental Procedures. Melting points were meas-
ured on a Thomas-Hoover capillary melting point apparatus. Optical
rotations were obtained using a PerkinElmer 241 polarimeter with a 1.0
mL cell. NMR spectra of all compounds but 16 were collected on a
Bruker Avance 500 spectrometer running Topspin 1.4 pl8 using a 5 mm
BBO probe. Spectra were recorded in CD2Cl2at 500 MHz for 1H and
125 MHz for 13C NMR. Chemical shifts are reported as parts per million
from tetramethylsilane based on the lock solvent. COSY, 13C-DEPT
135, HMBC, and HSQC spectra were also recorded to assign protons
and carbons in the synthetic molecules. The 1H NMR spectrum of 16
was obtained at 600 MHz and the 13C spectrum at 151 MHz on a Bruker
AVIII-600 MHz spectrometer. Chemical shifts are referenced to the
residual CDCl3solvent signal; coupling constants are reported in Hz.
Electron impact ionization (EI) mass spectra were obtained at 70 eV
with an Agilent Technologies 5973 mass selective detector interfaced
with a 6890 N GC system equipped with either a 30 m ×0.25 mm i.d. ×
0.25 μmﬁlm HP-5MS column or one of the chiral-phase columns
described below. The HP-5MS column temperature was maintained at
50 °C for 5 min and then raised to 270 °Cat10°C/min. Helium was
used as a carrier gas at 1 mL/min. GC-HRMS analyses were performed
by time-of-ﬂight in EI or ESI modes on a Waters GCT Premier
instrument equipped with a DB5-MS column. Routine GC analyses
were performed on a Shimadzu 17A (Shimadzu Scientiﬁc Instruments,
Inc.) GC equipped with a ﬂame ionization detector, an AOC-20s
autosampler, and an AOC-20i autoinjector and with an HP-5 capillary
column (30 m ×0.25 mm ×0.25 μmﬁlm). Hydrogen was used as carrier
gas at 1 mL/min. Column temperature was maintained at 80 °C for 5
min and then raised to 280 °Cat10°C/min. Chiral GC analyses were
performed on (i) a 25 m ×0.25 mm i.d. Hydrodex β-6TBDM capillary
column (Macherey-Nagel GmbH & Co. KG) and (ii) a 30 m ×0.25 mm
×0.12 μmﬁlm Astec Chiraldex G-TA column (Sigma-Aldrich/
Supelco). TLC analyses were conducted on Whatman AL SIL G/UV
plates using a 20% ethanol solution of phosphomolybdic acid and/or
UV for visualization of spots. Flash chromatography was carried out with
230−400 mesh silica gel (Fisher Scientiﬁc).
All reagents and solvents were purchased from Aldrich Chemical Co.,
unless otherwise speciﬁed. (S)-(−)-Citronellal (97% ee) was purchased
from Sigma-Aldrich, and (R)-(+)-citronellal (98% ee) was purchased
from Takasago International. Diastereomeric cyclohexenones 1and 2
were synthesized following Hagiwara et al.
Synthesis of Stereoisomers of 1,10-Bisaboladien-3-ol: Gen-
eral Procedure. Chloro(1,5-cyclooctadiene)rhodium(I) dimer ([Rh-
(cod)Cl]2,0.05equiv)and(R)-(−)- or (S)-(+)-2,2′-bis-
(diphenylphosphino)-1,1′-binaphthalene ((R)- or (S)-BINAP, 0.12
equiv) were placed under N2in a round-bottom three-neck ﬂask. Dry
tetrahydrofuran (30 mL) was added to the mixture, and the resulting
solution was stirred at room temperature (rt) for 0.5 h and then cooled
Figure 5. Segments of GC-MS total ion chromatograms on a Hydrodex-
β-6TBDM column, He 2.0 mL/min, 140 °Cisothermal:(a)
Halyomorpha halys male aeration; (b) co-injection of H. halys male
aeration and trans-(7R)-10,11-epoxy-1-bisabolen-3-ols; (c)
(3R,6S,7R,10S)-stereoisomer 4; (d) trans-(7R)-10,11-epoxy-1-bisabo-
Table 1. Captures of H. halys in Pyramid Traps Baited with
lure treatment adults per trap
nymphs per trap
4mg3+ 1.1 mg 434.6 a
4mg322.6 b 32.4 a
4mg48.2 c 20.4 b
blank 0.8 d 4.4 b
Total per trap for 6 wk, June 21 through July 30, 2013, Beltsville,
Maryland, with traps adjacent to woody borders of agricultural ﬁelds, 5
randomized blocks collected and rerandomized 2×per wk and with
lures replaced every 2 wk.
Within each life stage (column), total trap
captures followed by a common letter do not diﬀer by Tukey’s HSD
test, p< 0.05. See text for ANOVA. Means shown are original
untransformed capture totals per trap.
Journal of Natural Products Article
dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 1708−17171714
to 0 °C. A solution of ketone 1or 2(1 equiv) in dry THF (5 mL) was
added to the mixture followed by trimethylaluminum (2 equiv, 2.0 M in
heptane) maintaining the temperature at 0 to −5°C. After stirring for 4
hat0°C, the mixture was left in a refrigerator at 0−2°C for 20 h, then
was poured into NH4Cl solution, acidiﬁed with 10% HCl to pH 3−4,
and extracted with hexane/ether, 5:1. Combined organic extracts were
washed with water and brine and dried with Na2SO4. After evaporation
of the solvent, the residue was ﬂash chromatographed on SiO2with
hexane/EtOAc, 6:1 to 3:1, to provide two main fractions. The less polar
fractions were further puriﬁed on SiO2with CH2Cl2/EtOAc, 40:1, to
provide cis-1,10-bisaboladien-3-ols 5,7,9, and 11 of >95% chemical
purity. GC retention times were ∼18.030 min (HP-5MS) and Rf0.45
(SiO2, hexane/EtOAc, 3:1). The more polar fractions were further
puriﬁed on SiO2with CH2Cl2/EtOAc, 30:1, to provide >95% pure trans-
1,10-bisaboladien-3-ols 6,8,10, and 12. GC retention times were
∼18.240 min; Rf0.32 (hexane/EtOAc, 3:1). The isolated 1,10-
bisaboladien-3-ols are characterized in Tables S1, S2, and S3.
Dehydrations of 1,10-Bisaboladien-3-ols. (a) A solution of a
∼1:1 mixture of 6and 8(70 mg, 0.32 mmol) in dry pyridine (3 mL) was
cooled to 0 °C and treated with POCl3(58 μL, 0.58 mmol). The mixture
was warmed to rt, stirred for 18 h, poured into ice−water (5 mL), and
extracted with hexane (4 ×5 mL). The combined hexane extracts were
washed with 1 M HCl and brine and dried with Na2SO4. After
evaporation of the solvent, the residue was chromatographed with
hexane to provide a mixture of hydrocarbons (55 mg) consisting of 5%
unknown sesquiterpene, 43% 1,3(15),10-bisabolatriene 14, and 52%
1,3,10-bisabolatriene 15 (Scheme S1, left, and Figure 1).
(b) Alcohol 7(222 mg, 1 mmol) was treated with POCl3(193 μL,
1.93 mmol) in dry pyridine (3 mL) at 0 °C; then the mixture was stirred
2 h at rt. After the workup as described above, the products were
extracted with CH2Cl2and puriﬁed by chromatography with hexane to
provide a mixture of hydrocarbons (53 mg) consisting of 56% 1,3,10-
bisabolatriene, 31% 1,3(15),10-bisabolatriene, and 13% of an
unidentiﬁed sesquiterpene. This mixture was added to a solution of 4-
phenyl-1,2,4-triazoline-3,5-dione (31 mg) in dry THF (2.5 mL). After
0.5 h, the mixture was concentrated with a gentle stream of N2and
chromatographed with pentane/methyl acetate (99:1). (6R,7R)-
(−)-1,3(15),10-Bisabolatriene (16, 9 mg) of 97% chemical purity by
GC-MS was isolated in the ﬁrst fraction (Scheme S1, left, and Figure 1):
[α]20D−54.2 (c0.58, CHCl3). The speciﬁc rotation of (6S,7S)-
(+)-1,3(15),10-bisabolatriene was reported as +39.6 (c0.43, CHCl3).
GC-MS m/z(% relative intensity, ion) 204 (30, M+), 161 (40), 133
(40), 120 (36), 119 (15), 109 (25), 105 (21), 93 (64), 92 (36), 91 (55),
79 (21), 77 (38), 69 (100), 55 (22), 41 (47); 1H NMR (600 MHz,
CDCl3,δ) 0.87 (d, J= 6.5 Hz, 3H), 1.14−1.22 (m, 1H), 1.36−1.46 (m,
2H), 1.47−1.53 (m, 1H), 1.59 (s, 3H), 1.68 (br s, 3H), 1.69−1.75 (m,
1H), 1.89−1.95 (m, 1H), 1.99−2.05 (m, 1H), 2.16−2.23 (m, 1H),
2.25−2.32 (m, 1 H), 2.42 (dt, J= 12.0, 6.0 Hz, 1H), 4.72 (br s, 1H), 4.74
(br s, 1H), 5.09 (br t, J= 7.0 Hz, 1H), 5.70 (br d, J= 11.0 Hz, 1H), 6.15
(dm, J= 11.0 Hz, 1H); 13C NMR (CDCl3, 150 MHz, δ) 16.46, 17.68,
25.73, 26.06, 26.26, 30.50, 33.90, 36.51, 41.02, 109.94, 124.80, 129.79,
131.29, 134.02, 143.80. Mass spectrometry and NMR data are in good
agreement with those reported for (6S,7S)-(+)-1,3(15),10-bisabola-
A Diels−Alder adduct of zingiberene with 4-phenyl-1,2,4-
triazoline-3,5-dione 17 (42 mg, Scheme S1) was also isolated in the
(c) In separate experiments, alcohols 5,6,8,9+11,9,10,11, and 12
(4 mg each) in pyridine (50 μL) were treated with POCl3(4 μL), and
the resulting hydrocarbon mixtures were separated as described in
experiment (a). The mixtures were analyzed by GC-MS on HP-5MS
and by GC-FID on Hydrodex- β-6TBDM columns.
Enantioselective Dihydroxylations of 1,10-Bisaboladien-3-
ols to 1-Bisabolen-3,10,11-triols. Solutions of alcohols (1 mmol) in
tert-butyl alcohol (4.7 mL) were added to a mixture of AD-mix-αor AD-
mix-β(1.38 g), depending on the stereoisomer being synthesized
(Figure 2), and methanesulfonamide (91 mg) in water (4.7 mL) at 0 °C.
Mixtures were stirred at 0−2°C for 24 h and treated with sodium sulﬁte
(1.47 g), and the temperature was allowed to rise to 20−25 °C within 0.5
h. The mixtures were extracted with CH2Cl2(4 ×30 mL), and the
combined organic extracts were washed with 2 N KOH and brine and
dried with Na2SO4. After evaporation of the solvent, residues were
chromatographed on SiO2with ethyl acetate to yield triols characterized
in Table S4. 1H and 13C NMR spectra of triols are presented in Tables S5
and S6, respectively.
X-ray Structure Determination of Triol 13. After recrystallizing
13 (mp 125 °C) from tert-butyl methyl ether, a sample for X-ray
structure determination was prepared as follows. A solution of 2 mg of
13 in 120 μLofCH
2Cl2was placed in an NMR tube; then 110 μLof
hexane was added, allowing needle-like crystals to gradually precipitate.
All reﬂection intensities were measured at 110(2) K using a SuperNova
diﬀractometer (equipped with an Atlas detector) with Cu Kαradiation
(mirror optics, λ= 1.5418 Å) under the program CrysAlisPro (version
18.104.22.168, Agilent Technologies, 2012). This program was used for
unit cell determination and data reduction. The structure was solved
with the program SHELXS-97 and was reﬁned on F2with SHELXL-
Analytical numeric absorption corrections based on a multifaceted
crystal model were applied using CrysAlisPro. The temperature of the
data collection was controlled using the system Cryojet (Oxford
Instruments). The H atoms (unless otherwise speciﬁed) were placed at
calculated positions using the instructions AFIX 13, AFIX 23, AFIX 43,
or AFIX 137 with isotropic displacement parameters having values 1.2 or
1.5 times Ueq of the attached C atoms. The H atoms attached to O1, O2,
and O3 were found from diﬀerence Fourier maps, and the O−H
distances were restrained to be 0.84(3) Å using the DFIX instructions.
The structure is ordered. The absolute conﬁguration 3R,6S,7R,10Swas
established by anomalous dispersion eﬀects in diﬀraction measurements
on the crystal (Scheme 2). The Flack
parameters reﬁne to
0.05(13) and 0.03(4), respectively. Compound 13: fw = 256.37,
colorless plate, 0.43 ×0.38 ×0.07 mm3, monoclinic, P21(no. 4), a=
9.58434(13) Å, b= 6.33143(8) Å, c= 12.29045(17) Å, β=
92.0157(12)°,V= 745.355(17) Å3,Z=2,Dx= 1.142 g cm−3,μ=
0.611 mm−1, abs corr range 0.824−0.963. A total of 8786 reﬂections
were measured up to a resolution of (sin θ/λ)max = 0.62 Å−1, of which
2921 were unique (Rint = 0.0163) and 2848 were observed [I>2σ(I)]. A
total of 180 parameters were reﬁned using four restraints. R1/wR2[I>
2σ(I)]: 0.0253/0.0647. R1/wR2[all reﬂns]: 0.0261/0.0655. S= 1.062.
Residual electron density was found between −0.13 and 0.20 e Å−3.
Syntheses of Stereisomeric 10,11-Epoxy-1-bisabolen-3-ols.
Methanesulfonyl chloride (77 μL, 1.14 mmol) was added to a stirred
solution of a triol (1.0 mmol) in dry pyridine (1.5 mL) at 0−5°C; then
the mixture was allowed to warm to rt and stirred for 1 h. The reaction
mixture was poured into ice−water (4 mL) and extracted with CH2Cl2
(3 ×10 mL). Combined organic extracts were washed with ice−water,
dried with Na2SO4, and concentrated to yield a crude mesylate. This was
taken into MeOH (5 mL), cooled to 0 °C, and treated with a solution of
KOH (112 mg, 2 mmol) in MeOH (1.3 mL), which resulted in an
instantaneous precipitation of inorganic salts. The reaction mixture was
warmed to rt, stirred for 0.5 h, and concentrated to remove most of
MeOH. The residue was treated with an NH4Cl solution to pH 7−8 and
extracted with ether (3 ×10 mL). Combined organic extracts were
washed with ice−water and brine, dried with Na2SO4, and concentrated.
Flash chromatography (hexane/EtOAc, 3:2) yielded epoxybisabolenols
3,4,and24−27 (Table S4). 1Hand13CNMRspectraof
epoxybisabolenols are presented in Tables S5 and S6, respectively.
Insect Rearing. The brown marmorated stink bug colony in Taiwan
was established in 2000 from adults collected in Nangang. The H. halys
colony at Beltsville was established in 2007 from adults collected in
Allentown, PA, USA, supplemented annually with ∼20 adult bugs ﬁeld-
collected in the vicinity of Beltsville, MD, USA. Rearing was
accomplished in ventilated plastic cylinders (21 cm ×21 cm o.d.) on
a diet of organic green beans and shelled sunﬂower and buckwheat seeds
(2:1, w/w), glued onto squares of brown wrapping paper with wallpaper
paste, and distilled water supplied in two cotton-stopped 7 cm ×2cm
o.d. shell vials held together with a rubber band. Eggs were collected
weekly and hatched in plastic Petri dishes with a water vial, and after
molting to second-instars, the nymphs were transferred to the larger
rearing cages as described above for the remaining four instars. Adult
males and females were separated 1 or 2 days after emergence and
subsequently maintained in diﬀerent containers. Insects were
Journal of Natural Products Article
dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 1708−17171715
maintained in Thermo Forma chambers (Thermo Fisher Scientiﬁc) at
25 °C and 72% relative humidity, under a 16L:8D photoperiod.
Semiochemical Collection and Isolation. Initially, aeration
experiments were conducted using groups of 20−30 virgin adults as
was successfully employed previously for other stink bugs. Under these
conditions no sex-speciﬁc volatiles were detected. Eventually, evidence
in the literature indicated pheromone production of some stink bugs is
inhibited when males are grouped in large numbers.
successful aerations were originally conducted in Taiwan with one
female and three males, both 14 days old, for 2 days (Figure 2).
Thereafter, at the Beltsville Research Center volatiles were collected (as
described below) with one to three laboratory-reared virgin H. halys
adults (at least 1 week old) and with one to three wild adults (Figure 3).
Subsequently, the male-speciﬁc volatiles were collected from diﬀerent
numbers per container of virgin males only. The males were placed
separately into two 1 L, four-necked glass containers. Humidiﬁed air was
drawn into the container through 6−14 mesh activated charcoal (Fisher
Scientiﬁc) and out of the container through two traps (15 cm ×1.5 cm
o.d.) containing Super Q (200 mg each; Alltech Associates, Inc.) by
vacuum (∼1 L/min).
Insects were fed with organic green beans
(replaced weekly) and water on cotton balls and aerated continuously
for 20 to 90 days at rt and a 16L:8D photoperiod. The adsorbent traps
were changed every day (some of them in 3 days for the weekend), and
the adsorbents were eluted with CH2Cl2(0.5 mL/sample). The
solutions were stored at −30 °C before analyses.
Bioassay Methods. Pyramid traps described previously were used
for both ﬁeld trials.
Hercon Vaportape II (Hercon Environmental) was
added as a killing agent to prevent escape from traps and was replaced at
four-week intervals. H. halys adults and nymphs were removed from
traps, and the lure placement within each block was rerandomized twice
weekly, recording the numbers and sexes of adults. Rubber septa used to
evaluate pheromone treatments were replaced at two-week intervals.
Single isomers 3and 4, and a combined lure in the natural ratio, as well
as an unbaited trap (Table 1) were compared from June 21 to July 30,
2013, in Beltsville, MD, USA, with traps adjacent to woody borders of
agricultural ﬁelds, in ﬁve randomized blocks. A mixed-isomer lure at 10.7
mg was compared with unbaited traps deployed in ﬁve randomized
blocks ∼5 m from the border of apple and pear orchards at the
Appalachian Fruit Research Station, Kearneysville, WV, USA, from
March 20 to April 17, 2012. ANOVA was used to evaluate overall eﬀects
for the single-isomer test as well as the mixed-isomer test, using arcsin-
square-root-transformed block proportions of totals for the trapping
period. Adjacent means were compared using Tukey’s HSD test.
eﬀect of sex was dropped from all models because it was not a signiﬁcant
Dehydration products from compounds 5−12 and selected gas
chromatograms thereof, entire CIF ﬁle for triol 13, as well as
Tables S1−S6 and 1H and 13C NMR spectra of all new
compounds are available free of charge via the Internet at http://
*Phone: 301-504-6138. E-mail: email@example.com.
Department of Entomology, University of California, Davis, CA
95616, United States.
The authors declare no competing ﬁnancial interest.
We thank G. Cabrera Walsh, A. DiMeglio, and M. Athanas for
pheromone trap ﬁeld collection and enumeration of all Maryland
samples. Support for this study was from USDA, National
Institute of Food and Agriculture, Specialty Crop Research
Initiative grant no. 2011-51181-30937.
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