Discovery of the aggregation pheromone of the brown marmorated stink bug (Halyomorpha halys) through the creation of stereoisomeric libraries of 1-bisabolen-3-ols

Article abstract of DOI:10.1021/np5003753

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 addition of trimethylaluminum to diastereomeric mixtures of cyclohex-2-enones 1 and 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, (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) and (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 3 and 4 were 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 pest.

Full text of DOI:10.1021/np5003753

Article
pubs.acs.org/jnp
Discovery of the Aggregation Pheromone of the Brown Marmorated
Stink Bug (Halyomorpha halys) through the Creation of
Stereoisomeric Libraries of 1‑Bisabolen-3-ols
Ashot Khrimian,*,† Aijun Zhang,† Donald C. Weber,† Hsiao-Yung Ho,‡ Jeffrey R. Aldrich,†,§
Karl E. Vermillion,⊥ Maxime A. Siegler,∥ Shyam Shirali,† Filadelfo Guzman,† 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
S
* Supporting Information
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 1 and 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,
(3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) and
(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
3 and 4 were 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
pest.
he bisabolane skeleton is a recurring structural motif in the
semiochemistry of stink bugs (Hemiptera: Pentatomidae).
Several related compounds were isolated from the oil of ginger,
Zingiber officinale, among them a 1,10-bisaboladien-3-ol, called
“zingiberenol”.11 The latter was assigned a trans-configuration
based on similarities of its IR spectrum with that of trans-p-
menth-2-en-1-ol,12 but the structure was presented as the cis-
isomer11 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
specifically, (1R,4R,1′S)-(1′,5′-dimethylhex-4′-enyl)-1-methyl-
cyclohex-2-en-1-ol.13 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
T
Bisabolene epoxides comprise male-specific pheromones of
Nezara viridula1,2 and Chinavia (=Acrosternum) spp.3,4 The
related zingiberene, β-sesquiphellandrene, and α-curcumene
constitute part of the Thyanta pallidovirens pheromone,5 and β-
sesquiphellandrene was identified as a pheromone component of
Piezodorus hybneri.6 More recently, two stereoisomeric 1,10-
bisaboladien-3-ols7 were identified as part of the male-produced
pheromone of the rice stalk stink bug, Tibraca limbativentris,8 and
10,11-epoxy-1-bisabolen-3-ol (called “murgantiol”) has been
reported as an aggregation pheromone of the harlequin bug,
Murgantia histrionica.9,10 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
1
murgantiol structure was problematic, and H and 13C NMR
Received: May 6, 2014
recordings of murgantiol failed to provide a conclusive answer.9
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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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)
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.
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 1 and 2 were
difficult to synthesize,18 we studied Rh-catalyzed additions of
trimethylaluminum with mixtures of these diastereomeric
ketones.19
The brown marmorated stink bug, Halyomorpha halys (Stal),
̊
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
the U.S.14,15 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.16
Thus, the reaction of a ∼1:1 mixture of (6S,7R)-1 and
(6R,7R)-1 with 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
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 enones17 to
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.17 We did not
1
parameters, X-ray crystallography, H 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
B
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a
Scheme 2. Syntheses of Triols and Epoxy Alcohols Including H. halys Pheromone Components 3 and 4
a
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.
the presence of (R)-BINAP is shown in Scheme 1 (left). Of the
two diastereomers, (6S,7R)-1 is 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.17 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.
diastereomeric ratios of 91:9 and 89:11, respectively. NMR
signals from minor stereoisomers 5 and 6 were 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)-2 was a preferred
diastereomer, leading to the (3S,6S,7S) stereoisomer 9 in 93:7
dr, and the sterically disfavored (6R,7S)-2 provided 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)-2 did 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)-2 led (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
5 was due to two factors: first, the highly diastereotopically
selective addition of trimethylaluminum to (6S,7R)-1 and,
second, because (6R,7R)-1 present in the mixture with
(6S,7R)-1 primarily underwent side reactions rather than
producing sufficient cis-stereoisomer 7 to 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
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 5 and 6 were 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
5 was cross-contaminated with cis-stereoisomer 7, arising from
(6R,7R)-1, and alcohol 6 contained some of trans-isomer 8
originating from (6S,7R)-1. Diastereomeric ratios of 96:4 for 5/7
and 86:14 for 6/8 were 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 1 with trimethylaluminum in
the presence of chloro(1,5-cyclooctadiene)rhodium(I) dimer
and (S)-BINAP provided stereoisomers 7 and 8 as major
products (Scheme 1, left). In accordance with an si-face approach
postulated in the presence of (S)-BINAP,17 (6R,7R)-1 was highly
favored in this reaction and provided (3R,6R,7R)-alcohol 7. Due
to steric constraints, the addition of trimethylaluminum to
(6S,7R)-1 was low-yielding and nondiastereoslective (si-face/re-
face, 62:38). In this milieu, cis-alcohol 7 was cross-contaminated
with cis-alcohol 5 and trans-alcohol 8 with trans-alcohol 6 with
C
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another trans) could be synthesized from a single reaction of a
diastereomeric mixture of 1 or 2 with trimethylaluminum in
rhodium-catalyzed asymmetric 1,2-addition conditions.
dienol stereoisomers with natural β-sesquiphellandrene and
zingiberene and their stereoisomers with established absolute
configurations. The reaction of a ∼1:1 mixture of 6 and 8 with
POCl3 provided 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
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
1,10-bisaboladien-3-ols.
We found that the Sharpless asymmetric dihydroxylation20 of
stereoisomer 8 with AD-mix α proceeded 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 Kα radiation
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, and 12.
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 SiO2 using hexane/EtOAc than the
corresponding trans-isomers.21
Figure 1. Gas chromatograms of bisabolatrienes on Hydrodex-β-
6TBDM at 110 °C; H2 2.0 mL/min: (a) (6R,7R)-1,3(15),10-
bisabolatriene (16) from alcohol 7 after removal of zingiberene and
other products; (b) from 6 + 8; (c) from alcohol 5.
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 5 occurred 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
133.8 ppm22 and 133.0 and 133.4 ppm,23 respectively. In contrast
to 5, resonances of C-1 and C-2 in bisaboladienol 6 appeared 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)
ppm.22,23 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
known configurations.22,23
identified by GC-MS with authentic samples of zingiberene25
and β-sesquiphellandrene,5 respectively. Diastereomeric
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 + 8 had the (7R)-configuration and the natural
(−)-β-sesquiphellandrene has the (6R,7S)-configuration,26 the
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 7 was 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).25 Compound
16 (Figure 1, panel a), which did not undergo a Diels−Alder
reaction,25 was isolated by chromatography in 97% chemical
purity. 1H and 13C NMR spectra of 16 were in good agreement
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 oxychloride24 to correlate bisabola-
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with those of (6S,7S)-1,3(15),10-bisabolatriene,26 but because
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 7 belonged 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 5 with 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 5 has 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 6 and 8 produced expected mixtures of hydrocarbons
(Scheme S1, left). Because 6 produced 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.
asymmetric dihydroxylation with AD-mix α.20 Thus, we assigned
the other triols originating from AD-mix α dihydroxylations (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
MeOH.28,29 Because this intramolecular cyclization proceeded
with inversion of configuration,28−30 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
The dehydration of a ∼1:1 mixture of 9 and 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).
This simplified our task of assigning their absolute
configurations because natural (−)-zingiberene is (6R,7S)-
1,3,10-bisabolatriene,24,27 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 1 and 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
intermediate mesylates.28,29 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
Figure 2. GC-MS total ion chromatograms of Halyomorpha halys male
and female aerations on a DB-5MS column, 40(1) to 300 °C at 10 °C/
min; He 1.0 mL/min. Two male-specific compounds (A and B) are
indicated.
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/z 93 suggesting a
methylcyclohexadiene fragment and the peak at m/z 220
possibly being a molecular ion. Chemical ionization mass spectra
of these two compounds with ammonia as a reagent gas
E
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(Figure 4). We found that (3S,6S,7R,10S)-stereoisomer 3
matched compound A in the H. halys male airborne collection.
Figure 4. Segments of GC-MS total ion chromatograms on a Chiraldex
G-TA column, He 1.0 mL/min, 50(3) to 140 °C at 10 °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.
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).31 Thus, in a pursuit of behaviorally active
compounds that could constitute an aggregation pheromone of
BMSB we focused on the field bioassay of stereoisomers 3 and 4.
Field Bioassay. Comparison of lures containing each of the
synthetic pheromone components 3 and 4 separately 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]:
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 °C at 10
°C/min. Mass spectra of main male-specific compounds A and B are
presented.
contained an ion at m/z 256 (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/z 220 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
histrionica.9,10 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
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)32 indicated
that for adult captures the major component 3 was 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 3 or 4 alone. For nymphal
captures, lures containing the major component 3 caught
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noate33 identifies a season-long attractive tool for detection,
monitoring, and potential control of this polyphagous invasive
pest of North America and Europe.
CONCLUSION
■
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 1 and 2 afforded 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.
EXPERIMENTAL SECTION
■
Figure 5. Segments of GC-MS total ion chromatograms on a Hydrodex-
β-6TBDM column, He 2.0 mL/min, 140 °C isothermal: (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-
len-3-ols.
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 CD2Cl2 at 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 CDCl3 solvent 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 μm film 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 °C at 10 °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 μm film). 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 °C at 10 °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 μm film 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).
Table 1. Captures of H. halys in Pyramid Traps Baited with
Pheromone Components
a
a
lure treatment
adults per trap
nymphs per trap
b
b
4 mg 3 + 1.1 mg 4
4 mg 3
34.6
22.6
8.2
a
54.0
32.4
20.4
4.4
a
a
b
4 mg 4
c
b
b
blank
0.8
d
a
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
b
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.
significantly more than lures not containing the major
component.
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,9 showed
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-
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 1 and 2
were synthesized following Hagiwara et al.19
Synthesis of Stereoisomers of 1,10-Bisaboladien-3-ol: Gen-
eral Procedure. Chloro(1,5-cyclooctadiene)rhodium(I) dimer ([Rh-
(cod)Cl]2, 0.05 equiv) and (R)-(−)- or (S)-(+)-2,2′-bis-
(diphenylphosphino)-1,1′-binaphthalene ((R)- or (S)-BINAP, 0.12
equiv) were placed under N2 in 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
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to 0 °C. A solution of ketone 1 or 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
h at 0 °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 SiO2 with
hexane/EtOAc, 6:1 to 3:1, to provide two main fractions. The less polar
fractions were further purified on SiO2 with 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 Rf 0.45
(SiO2, hexane/EtOAc, 3:1). The more polar fractions were further
purified on SiO2 with 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; Rf 0.32 (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 6 and 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).
dried with Na2SO4. After evaporation of the solvent, residues were
chromatographed on SiO2 with 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 μL of CH2Cl2 was placed in an NMR tube; then 110 μL of
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 Kα radiation
(mirror optics, λ = 1.5418 Å) under the program CrysAlisPro (version
1.171.36.24, 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 F2 with SHELXL-
97.34 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,10S was
established by anomalous dispersion effects in diffraction measurements
on the crystal (Scheme 2). The Flack35 and Hooft36 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−3.37
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, and 24−27 (Table S4). 1H and 13C NMR spectra of
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 × 2 cm
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
(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 CH2Cl2 and 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 N2 and
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]20 −54.2 (c 0.58, CHCl3). The specific rotation of (6S,7S)-
D
(+)-1,3(15),10-bisabolatriene was reported as +39.6 (c 0.43, CHCl3).26
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),
1
79 (21), 77 (38), 69 (100), 55 (22), 41 (47); H 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-
triene.26 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
second fraction.
(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-α or 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
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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.9,38 Therefore,
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).39 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.40 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 3 and 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.32 The
effect of sex was dropped from all models because it was not a significant
factor.
Institute of Food and Agriculture, Specialty Crop Research
Initiative grant no. 2011-51181-30937.
REFERENCES
■
(1) Aldrich, J. R.; Oliver, J. E.; Lusby, W. R.; Kochansky, J. P.;
Lockwood, J. A. J. Exp. Zool. 1987, 244, 171−175.
(2) Baker, R.; Borges, M.; Cooke, N. G.; Herbert, R. H. J. Chem. Soc.,
Chem. Commun. 1987, 414−416.
(3) McBrien, H. L.; Millar, J. G.; Gottlieb, L.; Chen, X.; Rice, R. E. J.
Chem. Ecol. 2001, 27, 1821−1839.
(4) Aldrich, J. R.; Lusby, W. R.; Marron, B. E.; Nicolaou, K. C.;
Hoffmann, M. P.; Wilson, L. T. Naturwissenschaften 1989, 76, 173−175.
(5) McBrien, H. L.; Millar, J. G.; Rice, R. E.; McElfresh, J. S.; Cullen, E.;
Zalom, F. G. J. Chem. Ecol. 2002, 28, 1797−1817.
(6) Leal, W. S.; Kuwahara, S.; Shi, X. A.; Higuchi, H.; Marino, C. E. B.;
Ono, M.; Meinwald, J. J. Chem. Ecol. 1998, 24, 1817−1829.
(7) For terpene nomenclature see: Connolly, J. D.; Hill, R. A.
Dictionary of Terpenoids; Chapman and Hall: London, 1991; Vol. 1, pp
180−182.
(8) Borges, M.; Birkett, M.; Aldrich, J. R.; Oliver, J. E.; Chiba, M.;
Murata, Y.; Laumann, R. A.; Barrigossi, J. A.; Pickett, J. A.; Moraes, M. C.
B. J. Chem. Ecol. 2006, 32, 2749−2764.
(9) Zahn, D. K.; Moreira, J. A.; Millar, J. G. J. Chem. Ecol. 2008, 34,
238−251.
(10) Zahn, D. K.; Moreira, J. A.; Millar, J. G. J. Chem. Ecol. 2012, 38,
126.
(11) Terhune, S. J.; Hogg, J. W.; Bromstein, A. C.; Lawrence, B. M.
Can. J. Chem. 1974, 53, 3287−3293.
(12) Mitzner, B. M.; Mancini, V. J.; Lemberg, S.; Theimer, E. T. Appl.
Spectrosc. 1968, 22, 34−53.
(13) de Oliveira, M. W. M.; Borges, M.; Andrade, C. K. Z.; Laumann, R.
A.; Barrigossi, J. A. F.; Blassioli-Moraes, M. C. J. Agric. Food. Chem. 2013,
61, 7777−7785. [The pheromone is incorrectly named (1S,4R,1′S)-
(1′,5′-dimethylhex-4′-enyl)-1-methylcyclohex-2-en-1-ol in the title. The
correct name is (1R,4R,1′S)-(1′,5′-dimethylhex-4′-enyl)-1-methylcy-
clohex-2-en-1-ol.]
(14) Leskey, T. C.; Hamilton, G. C.; Nielsen, A. L.; Polk, D. F.;
Rodriguez-Saona, C.; Bergh, J. C.; Herbert, D. A.; Kuhar, T. P.; Pfeiffer,
D.; Dively, G.; Hooks, C. R. R.; Raupp, M. J.; Shrewsbury, P. M.;
Krawczyk, G.; Shearer, P. W.; Whalen, J.; Koplinka-Loehr, C.; Myers, E.;
Inkley, D.; Hoelmer, K. A.; Lee, D.-Y.; Wright, S. E. Outlooks Pest Manag.
2012, 23, 218.
(15) Leskey, T. C.; Short, B. D.; Butler, B. B.; Wright, S. E. Psyche 2012,
Article ID 535062.
(16) U.S. and International Patents have been filed. See: Khrimian, A.;
Zhang, A.; Aldrich, J. R.; Leskey, T. C., Weber, D. C. Compositions and
Methods to Attract the Brown Marmorated Stink Bug Halyomorpha
halys. Int. Pat. Appl. PCT/US2012/069721, Dec 14, 2012.
(17) Siewert, J.; Sandmann, R.; von Zezschwitz, P. Angew. Chem., Int.
Ed. 2007, 46, 7122−7124.
ASSOCIATED CONTENT
* Supporting Information
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://
pubs.acs.org.
■
S
(18) Yadav, J. S.; Thirupathaiah, B.; Srihary, P. Tetrahedron 2010, 66,
2005−2009.
(19) Hagiwara, H.; Okabe, T.; Ono, H.; Kamat, V. P.; Hoshi, T.;
Suzuki, T.; Ando, M. J. Chem. Soc., Perkin Trans. 1 2002, 895−900.
(20) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;
Hartung, J.; Jeong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.; Xu,
D.; Zhang, X.-L. J. Org. Chem. 1992, 57, 2768−2771.
(21) Mori, K. Tetrahedron: Asymmetry 2006, 17, 2133−2142.
(22) Blair, M.; Tuck, K. L. Tetrahedron: Asymmetry 2009, 20, 2149−
2153.
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-504-6138. E-mail: ashot.khrimian@ars.usda.gov.
■
Present Address
§Department of Entomology, University of California, Davis, CA
95616, United States.
(23) Kashiwagi, T.; Nakashima, T.; Tebayashi, S.-I.; Kim, C.-S. Biosci.
Biotechnol. Biochem. 2006, 70, 2544−2546.
Notes
The authors declare no competing financial interest.
(24) Bhonsle, J. B.; Deshpande, V. H.; Ravindranathan, T. Indian J.
Chem. 1994, 33B, 313−316.
ACKNOWLEDGMENTS
■
(25) Millar, J. G. J. Nat. Prod. 1998, 61, 1025−1026.
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
(26) Kreiser, W.; Korner, F. Helv. Chim. Acta 1999, 82, 1427−1433.
̈
(27) Soffer, M. D.; Burk, L. A. Tetrahedron Lett. 1985, 26, 3543−3546.
(28) Frater, G.; Muller, U. Helv. Chim. Acta 1989, 72, 653−658.
̈
I
dx.doi.org/10.1021/np5003753 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(29) Moore, C. J.; Possner, S.; Hayes, P.; Paddon-Jones, G. C.;
Kitching, W. J. Org. Chem. 1999, 64, 9742−9744.
(30) Khrimian, A.; Fay, H. A. C.; Guzman, F.; Chauhan, K.; Moore C.;
Aldrich, J. R. Psyche 2012, Article ID 536149.
(31) Individual stereoisomers of trans 10,11-epoxy-1-bisabolen-3-ol
with (7S)-configurations and a mixture thereof will be described in our
subsequent paper.
(32) SAS Institute. StatView 2nd ed.; SAS Institute: Cary, NC, 1998.
(33) Weber, D. C.; Leskey, T. C.; Cabrera Walsh, G.; Khrimian, A. J.
Econ. Entomol. 2014, 107, 1061−1068.
(34) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122.
(35) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881.
(36) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr.
2008, 41, 96−103.
(37) Crystallographic data for triol 13 reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre.
(Deposition number CCDC 959242). Copies of the data can be
obtained, free of charge, on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-
mail: deposit@ccdc.cam.ac.uk).
(38) Ho, H.-Y.; Hsu, Y.-C.; Chuang, Y.-C.; Chow, Y.-S. J. Chem. Ecol.
2005, 31, 29−37.
(39) Zhang, A.; Oliver, J. E.; Aldrich, J. R.; Wang, B.; Mastro, V. C. Z.
Naturforsch., C: Biosci. 2002, 57, 553−558.
(40) Leskey, T. C.; Wright, S. E.; Short, B. D.; Khrimian, A. J. Entomol.
Sci. 2012, 47, 76−85.
J
dx.doi.org/10.1021/np5003753 | J. Nat. Prod. XXXX, XXX, XXX−XXX