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Discovery of the Aggregation Pheromone of the Brown Marmorated Stink Bug (Halyomorpha halys) through the Creation of Stereoisomeric Libraries of 1-Bisabolen-3-ols

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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.
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Discovery of the Aggregation Pheromone of the Brown Marmorated
Stink Bug (Halyomorpha halys) through the Creation of
Stereoisomeric Libraries of 1Bisabolen-3-ols
Ashot Khrimian,*
,
Aijun Zhang,
Donald C. Weber,
Hsiao-Yung Ho,
Jerey 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
*
SSupporting 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 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 congurations. 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 identied 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 eld 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 eld crops and is also a serious nuisance
pest.
The bisabolane skeleton is a recurring structural motif in the
semiochemistry of stink bugs (Hemiptera: Pentatomidae).
Bisabolene epoxides comprise male-specic pheromones of
Nezara viridula
1,2
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 identied as a pheromone component of
Piezodorus hybneri.
6
More recently, two stereoisomeric 1,10-
bisaboladien-3-ols
7
were identied 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 congurations of the 1,10-bisaboladien-3-ols from T.
limbativentris have not been determined. Reliable assignment of
relative congurations across the cyclohexene ring of the
murgantiol structure was problematic, and 1H and 13C NMR
recordings of murgantiol failed to provide a conclusive answer.
9
Several related compounds were isolated from the oil of ginger,
Zingiber off icinale, among them a 1,10-bisaboladien-3-ol, called
zingiberenol.
11
The latter was assigned a trans-conguration
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-
isomer
11
and the absolute conguration has not been disclosed.
A sex pheromone of the rice stink bug, Oebalus poecilus, has
recently been also identied as zingiberenol and, more
specically, (1R,4R,1S)-(1,5-dimethylhex-4-enyl)-1-methyl-
cyclohex-2-en-1-ol.
13
The absolute conguration 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
Article
pubs.acs.org/jnp
© 2014 American Chemical Society and
American Society of Pharmacognosy 1708 dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 17081717
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 identications.
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, eld crops,
and ornamentals, with signicant 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 identication of a
male-produced aggregation pheromone of H. halys.
16
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
17
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
nd 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
dicult to synthesize,
18
we studied Rh-catalyzed additions of
trimethylaluminum with mixtures of these diastereomeric
ketones.
19
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 congurations: 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 dened 1,3,10-bisabolatrienes
(see further in the text), the major compound was identied as cis
and assigned structure 5; the minor product was found to have
trans relative conguration and assigned structure 6.The
stereochemical course of the trimethylaluminum addition in
Scheme 1. Syntheses of Bisaboladienol Stereoisomers 512 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.
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.
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 diculties in separation of
stereoisomers having the same relative (cis/trans) conguration
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,
17
(6R,7R)-1was highly
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)-conguration (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 ecient production of
5was due to two factors: rst, 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 sucient cis-stereoisomer 7to signicantly 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
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.
Journal of Natural Products Article
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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) Congurations of
1,10-Bisaboladien-3-ols. Assignments of the relative cong-
urations of 1,10-bisaboladien-3-ols are largely absent from the
literature. We observed that, regardless of the absolute
conguration 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 conguration for at least one stereoisomer would suce
for assigning relative congurations of all eight stereoisomers of
1,10-bisaboladien-3-ols.
We found that the Sharpless asymmetric dihydroxylation
20
of
stereoisomer 8with AD-mix αproceeded smoothly and provided
crystalline triol 13 (Scheme 2). After crystallization of 13 using a
liquidliquid diusion technique, we obtained single crystals
suitable for X-ray structure determination using Cu Kαradiation
and unequivocally proved its absolute conguration. The
displacement ellipsoid plot of crystalline 13 presented in Scheme
2 clearly shows a (3R,6S,7R,10S)-absolute conguration, 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 conguration 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-conguration.
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
corresponding trans-isomers.
21
Comparison of 13C NMR spectra of menth-2-en-1-ols and
1,10-bisaboladien-3-ols supported assignments of the relative
(and absolute) congurations. Resonances from C-1 and C-2 of
stereoisomer 5occurred at 133.9 and 133.6 ppm, respectively,
and the signals from the olenic carbons of cis-(S,S)-menthenol
and cis-(R,R)-menthenol were remarkably similar: 133.5 and
133.8 ppm
22
and 133.0 and 133.4 ppm,
23
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 olenic 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 dierences in chemical shifts of C-1 and
C-2, with trans-isomers displaying greater Δδ(3.14.6 ppm)
than cis-isomers (0.31.2 ppm) regardless of the absolute
conguration 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 congurations.
22,23
Absolute Congurations of 1,10-Bisaboladien-3-ols.
The absolute congurations of stereoisomers 512 were
established on the basis of knowledge of their relative
congurations and chemical correlations. We used dehydration
reactions with phosphorus oxychloride
24
to correlate bisabola-
dienol stereoisomers with natural β-sesquiphellandrene and
zingiberene and their stereoisomers with established absolute
congurations. 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
identied by GC-MS with authentic samples of zingiberene
25
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+8had the (7R)-conguration and the natural
()-β-sesquiphellandrene has the (6R,7S)-conguration,
26
the
latter could not be used for the identication of the compounds
associated with these GC peaks. Hence, we synthesized an
individual 1,3(15),10-bisabolatriene with the (7R)-conguration
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 DielsAlder adduct 17 (Scheme S1, left).
25
Compound
16 (Figure 1, panel a), which did not undergo a DielsAlder
reaction,
25
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,
26
but because
16 was dextrorotatory, this compound was assigned the (6R,7R)-
conguration, 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 conguration. With the
absolute conguration of compound 16 established, we assigned
the faster-eluting 1,3(15),10-bisabolatriene the (6R,7R)-cong-
uration and the slower-eluting diastereomer the (6S,7R)-
conguration (Figure 1, panel b). Determinations of the absolute
congurations of the three other 1,10-bisaboladien-3-ols with the
(7R)-conguration 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)-conguration.
Because alcohol 5has the cis relative conguration (higher Rf/
shorter retention time), its absolute conguration 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)-congurations, respectively.
The dehydration of a 1:1 mixture of 9and 11 with (7S)-
congurations 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 simpliedourtaskofassigningtheirabsolute
congurations 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 identied 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
conguration 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 conguration, its absolute
conguration 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)-congurations, 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
carboncarbon 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 1823 (Scheme
2). The absolute conguration of triol 13, determined by single-
crystal X-ray crystallography, conrmed that C-10 has the S-
conguration (Scheme 2), as expected from Sharpless
asymmetric dihydroxylation with AD-mix α.
20
Thus, we assigned
the other triols originating from AD-mix αdihydroxylations (18,
20) (10S)-congurations, and triols obtained from AD-mix β
dihydroxylations (19,21,22, and 23) (10R)-congurations.
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 conguration,
2830
carbon atoms at position 10
in the epoxybisabolenols 24 and 25 were assigned the R-
conguration, and those in compounds 3,4,26, and 27 the S-
congurations. Thus, the Sharpless asymmetric dihydroxylation
of 1,10-bisaboladien-3-ols followed by the epoxide ring closure of
the intermediate triols oered 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 identication; details for
the remaining stereoisomers will be reported elsewhere.
Identication of Male-Specic 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-specic 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-specic compounds (A and B) are
indicated.
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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
histrionica.
9,10
Thus, using the authentic samples described
above, we identied 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 congurations 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)-
conguration on a Chiraldex G-TA column and identied
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)-cong-
urations (Figure S4), with the second peak, identied 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-specic 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)-conguration 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 identied 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)-conguration 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 eld 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 diered for both adult and nymphal captures (Table
1; ANOVAs for treatment eects [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 (Tukeys HSD test)
32
indicated
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-specic compounds A and B are
presented.
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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signicantly more than lures not containing the major
component.
Furthermore, eld tests of a 3:1 mixture of cis- and trans-10,11-
epoxy-1-bisabolen-3-ols with the (7R)-conguration (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 eect (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, eld
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-
noate
33
identies 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 identied the aggregation pheromone of the
brown marmorated stink bug, H. halys. Rhodium/BINAP-
catalyzed addition of trimethylaluminum to diastereomeric
mixtures cyclohex-2-enones 1and 2aorded 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 identication
of the H. halys main pheromone components, the creation of
these stereoisomeric libraries will be useful in identifying the
relative and absolute congurations of other natural products,
including the pheromones of at least two other pentatomid bugs,
Murgantia histrionica and Tibraca limbativentris.
EXPERIMENTAL SECTION
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 μmlm 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-ight 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 Scientic Instruments,
Inc.) GC equipped with a ame 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 μmlm). 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 μmlm 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
230400 mesh silica gel (Fisher Scientic).
All reagents and solvents were purchased from Aldrich Chemical Co.,
unless otherwise specied. (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.
19
Synthesis of Stereoisomers of 1,10-Bisaboladien-3-ol: Gen-
eral Procedure. Chloro(1,5-cyclooctadiene)rhodium(I) dimer ([Rh-
(cod)Cl]2,0.05equiv)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 ask. 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-
len-3-ols.
Table 1. Captures of H. halys in Pyramid Traps Baited with
Pheromone Components
lure treatment adults per trap
a
nymphs per trap
a
4mg3+ 1.1 mg 434.6 a
b
54.0 a
b
4mg322.6 b 32.4 a
4mg48.2 c 20.4 b
blank 0.8 d 4.4 b
a
Total per trap for 6 wk, June 21 through July 30, 2013, Beltsville,
Maryland, with traps adjacent to woody borders of agricultural elds, 5
randomized blocks collected and rerandomized 2×per wk and with
lures replaced every 2 wk.
b
Within each life stage (column), total trap
captures followed by a common letter do not dier by Tukeys HSD
test, p< 0.05. See text for ANOVA. Means shown are original
untransformed capture totals per trap.
Journal of Natural Products Article
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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 02°C for 20 h, then
was poured into NH4Cl solution, acidied with 10% HCl to pH 34,
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 ash chromatographed on SiO2with
hexane/EtOAc, 6:1 to 3:1, to provide two main fractions. The less polar
fractions were further puried 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
puried 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 icewater (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 puried 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
unidentied 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 rst fraction (Scheme S1, left, and Figure 1):
[α]20D54.2 (c0.58, CHCl3). The specic rotation of (6S,7S)-
(+)-1,3(15),10-bisabolatriene was reported as +39.6 (c0.43, 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),
79 (21), 77 (38), 69 (100), 55 (22), 41 (47); 1H NMR (600 MHz,
CDCl3,δ) 0.87 (d, J= 6.5 Hz, 3H), 1.141.22 (m, 1H), 1.361.46 (m,
2H), 1.471.53 (m, 1H), 1.59 (s, 3H), 1.68 (br s, 3H), 1.691.75 (m,
1H), 1.891.95 (m, 1H), 1.992.05 (m, 1H), 2.162.23 (m, 1H),
2.252.32 (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 DielsAlder 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-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 02°C for 24 h and treated with sodium sulte
(1.47 g), and the temperature was allowed to rise to 2025 °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 reection intensities were measured at 110(2) K using a SuperNova
diractometer (equipped with an Atlas detector) with Cu Kαradiation
(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 rened on F2with 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 specied) 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 dierence Fourier maps, and the OH
distances were restrained to be 0.84(3) Å using the DFIX instructions.
The structure is ordered. The absolute conguration 3R,6S,7R,10Swas
established by anomalous dispersion eects in diraction measurements
on the crystal (Scheme 2). The Flack
35
and Hooft
36
parameters rene 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 cm3,μ=
0.611 mm1, abs corr range 0.8240.963. A total of 8786 reections
were measured up to a resolution of (sin θ/λ)max = 0.62 Å1, of which
2921 were unique (Rint = 0.0163) and 2848 were observed [I>2σ(I)]. A
total of 180 parameters were rened using four restraints. R1/wR2[I>
2σ(I)]: 0.0253/0.0647. R1/wR2[all rens]: 0.0261/0.0655. S= 1.062.
Residual electron density was found between 0.13 and 0.20 e Å3.
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 05°C; then
the mixture was allowed to warm to rt and stirred for 1 h. The reaction
mixture was poured into icewater (4 mL) and extracted with CH2Cl2
(3 ×10 mL). Combined organic extracts were washed with icewater,
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 78 and
extracted with ether (3 ×10 mL). Combined organic extracts were
washed with icewater and brine, dried with Na2SO4, and concentrated.
Flash chromatography (hexane/EtOAc, 3:2) yielded epoxybisabolenols
3,4,and2427 (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 eld-
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 sunower 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 dierent containers. Insects were
Journal of Natural Products Article
dx.doi.org/10.1021/np5003753 |J. Nat. Prod. 2014, 77, 170817171715
maintained in Thermo Forma chambers (Thermo Fisher Scientic) at
25 °C and 72% relative humidity, under a 16L:8D photoperiod.
Semiochemical Collection and Isolation. Initially, aeration
experiments were conducted using groups of 2030 virgin adults as
was successfully employed previously for other stink bugs. Under these
conditions no sex-specic 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-specic volatiles were collected from dierent
numbers per container of virgin males only. The males were placed
separately into two 1 L, four-necked glass containers. Humidied air was
drawn into the container through 614 mesh activated charcoal (Fisher
Scientic) 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 eld 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 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 elds, in ve randomized blocks. A mixed-isomer lure at 10.7
mg was compared with unbaited traps deployed in ve 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 eects
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 Tukeys HSD test.
32
The
eect of sex was dropped from all models because it was not a signicant
factor.
ASSOCIATED CONTENT
*
SSupporting Information
Dehydration products from compounds 512 and selected gas
chromatograms thereof, entire CIF le for triol 13, as well as
Tables S1S6 and 1H and 13C NMR spectra of all new
compounds are available free of charge via the Internet at http://
pubs.acs.org.
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.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
We thank G. Cabrera Walsh, A. DiMeglio, and M. Athanas for
pheromone trap eld 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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... Adults and nymphs of H. halys also orient to both a male-produced aggregation pheromone and also to aggregation pheromones Communicated by Jarmo K. Holopainen. of other Pentatomidae (Hoebeke and Carter 2003;Leskey et al. 2015). Research has also contributed to a continually growing toolbox for the management of H. halys, including the development of several monitoring trap designs for H. halys (Leskey et al. 2015;Acebes-Doria et al. 2019), and the identification, synthesis, and optimization of the maleproduced H. halys aggregation pheromone and its synergist used as lures for field monitoring (Khrimian et al. 2014;Weber et al. 2017). The understanding of H. halys basic biology has been used to create several management strategies for H. halys including threshold-based sprays , perimeter sprays (Blaauw et al. 2015), and "attractand-kill" through heavily baited trees or insecticide-treated netting (Kuhar et al. 2017;Morrison et al. 2019;Ibrahim et al. 2020). ...
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The invasive brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), has disrupted integrated pest management programs in agroecosystems including tree fruit in North America and several other regions worldwide. While the toolbox for managing the pest continues to grow, it has not yet been determined if feeding or oviposition by H. halys onto orchard or forest trees induces changes in volatiles emitted from trees that could be exploited by foraging egg parasitoids or other insects. The goal of this study was to investigate diferences in volatile compound emissions from branches of potted peach and tree of heaven exposed directly and systemically to H. halys feeding and oviposition. Analyses indicate that H. halys feeding and oviposition induced changes in both the blend and specifc amounts of volatile compounds emitted from both tree species. Tree of heaven branches directly exposed to H. halys oviposition and feeding emitted nerolidol at a higher rate than those exposed to feeding alone and control trees. Tree of heaven compound leaves systemically exposed to H. halys feeding alone emitted higher rates of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) than trees exposed to oviposition and feeding or control trees. Conversely, for peach, both branches exposed directly or systemically resulted in a decrease in cis-3-hexenyl acetate when exposed to H. halys oviposition. Such changes in plant volatile compounds, or blends of compounds, have the potential to be used by foraging natural enemies of H. halys.
... Utilizations of aggregation pheromones, alarm pheromones, attract-and-kill, push-pull strategies along with integrated management approaches are under development [13][14][15][16][17] . Meanwhile, a monitoring protocol for H. halys has matured since the two-component attractant was developed from Pentatomidae pheromone components 7,9,18,19 . Nevertheless, new studies are emerging to identify and improve synergism of additional chemicals with undergoing attractants, as well as to increase the cost-effectiveness of the baiting methods 20 . ...
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Halyomorpha halys has been recognized as a global cross-border pest species. Along with well-established pheromone trapping approaches, there have been many attempts to utilize botanical odorant baits for field monitoring. Due to sensitivity, ecological friendliness, and cost-effectiveness for large-scale implementation, the selection of botanical volatiles as luring ingredients and/or synergists for H. halys is needed. In the current work, botanical volatiles were tested by olfactometer and electrophysiological tests. Results showed that linalool oxide was a potential candidate for application as a behavioral modifying chemical. It drove remarkable attractiveness toward H. halys adults in Y-tube assays, as well as eliciting robust electroantennographic responsiveness towards antennae. A computational pipeline was carried out to screen olfactory proteins related to the reception of linalool oxide. Simulated docking activities of four H. halys odorant receptors and two odorant binding proteins to linalool oxide and nerolidol were performed. Results showed that all tested olfactory genes were likely to be involved in plant volatile-sensing pathways, and they tuned broadly to tested components. The current work provides insights into the later development of field demonstration strategies using linalool oxide and its molecular targets.
... Information on the successful application of this strategy against stink bugs is scarce. However, a study carried out by Zhang et al. (2014) showed that several plant extracts obtained from clove, lemongrass, spearmint oil, and ylang-ylang plants acted as repellents toward H. halys and could be candidates as 'push stimuli' for this pest. The repellent stimuli could be used as extracts in dispensers or as intercropping plants to repel the pest that simultaneously could be attracted by a trap crop that serves as a 'pull stimulus'. ...
Thesis
In a context of globalization and climate change, more and more insects are becoming invasive, particularly affecting agriculture on a global scale. At the heart of this network of invasions, the United States of America is experiencing the arrival of many species, mainly from Asia. Among them, stink bugs (Pentatomidae) threaten most American agrosystems and are now mainly regulated by synthetic pesticides. Bagrada hilaris (bagrada), native to Asia and Africa, is the most recent species to invade the USA, where it has been reported since 2008 in Brassicaceae crops. To answer the needs of farmers, USDA-ARS funded a classical biological control program to select and introduce from its native range one or more natural enemies of bagrada into California. The research included in this PhD thesis is part of this program. It sought to assess in a quarantine greenhouse whether the oophagous parasitoid Gryon gonikopalense (Scelionidae), native to Pakistan, could establish in California and efficiently control bagrada. Thus, four main objectives were defined: 1) to describe the general biology of G. gonikopalense; 2) describe its host foraging behavior; 3) examine the possibility of its mass production and 4) assess its specificity towards bagrada using Euro-Mediterranean non-target stink bugs. Objective 1 allowed to show that the physiology of G. gonikopalense was similar to that of many other scelionids: depending on the temperature, it completed its development in 7 to 25 days, its lifespan ranges from 30 to 150 days and it is able to parasitize hundred eggs during its lifetime. It shares with bagrada a thermal optimum between 25 and 35 °C which could allow it to settle in California. In addition to being an effective parasitoid of bagrada, the advantage of G. gonikopalense particularly involves its foraging behavior for host's eggs, which are buried in the ground in 90% of cases. We found that G. gonikopalense was able to dig into the soil to parasitize eggs. In addition, we have shown in a tri-trophic system that the parasitoid mainly attacks the buried eggs of bagrada, while being able to reach the eggs deposited on the host plant (about 10%). For the 3rd objective, we have shown that the storage of bagrada eggs at 5 °C over three weeks made it possible to optimize the breeding of the parasitoid and facilitate mass production. Concerning the objective 4, still in progress, we showed that among the 11 pentatomids tested, at least four were suitable for the parasitoid, and two of them were mainly located around the Mediterranean basin. These results complement the tests carried out in California and open a proactive discussion on the introduction of G. gonikopalense in invasive Mediterranean populations of bagrada. Finally, during this thesis, we have described several aspects, hitherto unknown, of the biology of G. gonikopalense. Its behavioral specialization towards bagrada makes this parasitoid particularly promising for the biological control program. A petition-to-release drafted on the basis of this knowledge submitted to the health authorities of the USA remains a short-term objective. They will then assess the risks and benefits of using G. gonikopalense to control bagrada in California.
... The pheromones identified to date from species in the subfamily Pentatominae are all produced by males ( Figure 2). In Neotropical species, all of these are sex pheromones that attract only females [16], whereas in Nearctic species, such as Piezodorus hybneri and Murgantia histrionica, the pheromone produced by males attracts both sexes and nymphs, working as an aggregation pheromone [23][24][25]. Pheromones that play an important role in the mating behavior of Pentatomidae are very well described for only 32 species [16,[26][27][28]; of these, 14 produce fatty acid derivatives compounds as pheromones, 15 produce sesquiterpenes, and 3 produce a combination of fatty acid and terpene derivatives as pheromone components [16]. Some Pentatomidae pheromones present chemical structures similar to plant compounds or are produced by plants. ...
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... Usually, large bug monitoring is based on beat samples of the tree canopy or trapping. There are a large variety of traps available (Morrison et al. 2015) and accompanying pheromone lures for many species (Borges et al. 1998;Tillman et al. 2010;Khrimian et al. 2014), but beat samples are considered to be the most accurate to determine pest presence in the pistachio canopy (Daane et al. 2016). Since beat samples of the tree canopy are time-intensive and can underestimate abundance of flying insects, we wanted to test if sweeping the groundcovers, the best sampling method to determine if hemipteran pests are in the orchard (Daane et al. 2016), could be an appropriate substitute. ...
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Using sown groundcovers as trap crops to protect a cash crop is a traditional pest management tool. Pistachio is a major crop in California's Central Valley, where high summer temperatures and little to no precipitation between May and November lead to summer dry-down of annual groundcover. Hemipteran pests that consist of 'small bugs' and 'large bugs' are a major contributor to nut damage, especially in organic production. In this 2-year field study, we tested the use of irrigated trap crop mixtures, sown between tree rows, to reduce those hemipteran pests' abundance or damage. Biweekly beat samples of the tree canopy and sweep samples of the sown groundcovers in trap crop plots and resident weedy vegetation in control plots were taken over two consecutive growing seasons. Arthropod richness and abundance were highest in the groundcover and tree canopy in the trap crop plots. Small and large bug pest populations were higher and lower, respectively, in the tree canopy in trap crop plots, indicating a mixed response of these hemipterans to the presence of the trap crops. Additionally, natural enemy populations were more abundant in the tree canopy in trap crop plots than in control plots. There was no difference in nut damage between plots with and without the trap crop. These findings suggest that populations of hemipteran pests and beneficials can be manipulated successfully with irrigated trap crops, but future studies will need to focus on doing so in a way that decreases hemipteran pistachio damage.
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Four commercial orchards in the mid-Atlantic region of the United States were surveyed weekly in 2010 and 2011 for the presence of brown marmorated stink bug and the injury caused to both apple and peaches. Among tested sampling techniques, pyramid traps baited with the aggregation pheromone of Plautia stali Scott, methyl-(2 E ,4 E ,6 Z )-decatrienoate, yielded the most brown marmorated stink bug adults and nymphs, followed by visual observations. Brown marmorated stink bugs began to feed on apples and peaches soon after fruit set and continued to feed on fruit throughout the growing season. Injury to apple was relatively inconsequential until after mid-June, whereas feeding on peaches resulted in immediate economic injury as the surface became distorted, dented, discolored, and the flesh beneath turned brown. Significantly more apples were injured and with greater severity in 2010 than in 2011. Likewise, percent injury on the exterior portion of each apple plot was significantly greater than injury reported from the interior in both years. Growers increased the number of insecticide applications nearly 4-fold from 2010 to 2011. In addition to the increased number of targeted insecticide applications, growers also reduced the interval between treatments in 2011. A metric was created to compare the relative intensity of each grower's commercial management program between seasons and amongst each other.
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The stereochemistry and absolute configuration of (-)-isozingi- berene dihydrochloride have been determined and the latter has been correlated with the cadalenic sesquiterpene, (-)-epizonarene.
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Bioassays using an olfactometer showed that Oebalus poecilus males produce the sexual pheromone and the chemical analysis demonstrated that this compound is zingiberenol. Two groups of isomers, each containing four diastereoisomers (1RS,4RS,1'S)- and (1RS,4RS,1'R)-zingiberenol were prepared. These diastereoisomers were not separated on a chiral GC column. Therefore, to determine the absolute configuration of the carbon 1, 4 and 1' of zingiberenol produced by males the following strategies were conducted. The extract containing males volatiles was submitted a dehydration microchemistry to produce zingiberene, which the isomers are separated on a chiral GC analysis and by comparison with the natural zingiberene from ginger oil the absolute stereochemistry of the carbon 4 and 1' was determined to be R and S, respectively, and the carbon 1 was determined as R from the 13C NMR spectra of quercivorol. Finally, the bioassays showed that O. poecilus females responded to racemic mixture and to (1RS,4RS,1'S)-zingiberenol.
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