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Determination of the Stereochemistry of the Aggregation
Pheromone of Harlequin Bug, Murgantia histrionica
Ashot Khrimian &Shyam Shirali &Karl E. Vermillion &
Maxime A. Siegler &Filadelfo Guzman &Kamlesh Chauhan &
Jeffrey R. Aldrich &Donald C. Weber
Received: 16 July 2014 /Revised: 17 October 2014 /Accepted: 4 November 2014
#Springer Science+Business Media New York (outside the USA) 2014
Abstract Preparation of a complete stereoisomeric library of
1,10-bisaboladien-3-ols and selected 10,11-epoxy-1-
bisabolen-3-ols was pivotal for the identification of the aggre-
gation pheromone of the brown marmorated stink bug,
Halyomorpha halys. Herein, we describe syntheses of the
remaining 10,11-epoxy-1-bisabolen-3-ols, and provide addi-
tional evidence on the assignment of relative and absolute
configurations of these compounds by single-crystal X-ray
crystallography of an intermediate, (3S,6R,7R,10S)-1-
bisabolen-3,10,11-triol. To demonstrate the utility of this ste-
reoisomeric library, we revisited the aggregation pheromone
of the harlequin bug, Murgantia histrionica, and showed that
the male-produced pheromone consists of two stereoisomers
of 10,11-epoxy-1-bisabolen-3-ol. Employment of eight cis-
10,11-epoxy-1-bisabolen-3-ol stereoisomeric standards, two
enantioselective GC columns, and NMR spectroscopy
enabled the identification of these compounds as
(3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol and
(3S,6S,7R,10R)-10,11-epoxy-1-bisabolen-3-ol, which are pro-
duced by M. histrionica males in 1.4:1 ratio.
Keywords Stink bug .Aggregation pheromone .
(1S,4S)-4-((R)-4-((S)-3,3-dimethyloxiran-2-yl)butan-2-yl)-1-
methylcyclohex-2-enol .(1S,4S)-4-((R)-4-((R)-3,3-
dimethyloxiran-2-yl)butan-2-yl)-1-methylcyclohex-2-enol .
Hemiptera .Pentatomidae
Introduction
The bisabolane skeleton is a recurring structural motif in the
semiochemistry of stink bugs (Hemiptera: Pentatomidae).
Epoxides of bisabolene were identified as male-specific pher-
omones of Nezara viridula (Aldrich et al. 1987; Baker et al.
1987)andChinavia hilaris (Aldrich et al. 1989; McBrien et al.
2001). Zingiberene, β-sesquiphellandrene and α-curcumene
constitute part of the pheromone of Thyanta pallidovirens
(McBrien et al. 2002), and β-sesquiphellandrene was identi-
fied as a pheromone component of Piezodorus hybneri (Leal
et al. 1998). 1,10-Bisaboladien-3-ols were identified as part of
the male-produced sex pheromone of the rice stalk stink bug,
Tibraca limbativentris (Borges et al. 2006), and 10,11-epoxy-
1-bisabolen-3-ol (“murgantiol”) has been reported as an ag-
gregation pheromone of the harlequin bug, Murgantia
histrionica (Zahn et al. 2008,2012). Several sesquiterpenes
were isolated from Zingiber officinale, among them a 1,10-
bisaboladien-3-ol, called zingiberenol (Terhune et al. 1974).
The stereo structures of the pheromones of both
T. lim b a t iv entris and M. histrionica, as well as zingiberenol
have not been determined. A sex pheromone of the rice stink
bug, Oebalus poecilus, also has been recently identified as
zingiberenol; more specifically, (3R,6R,7S)-1,10-
Electronic supplementary material The online version of this article
(doi:10.1007/s10886-014-0521-2) contains supplementary material,
which is available to authorized users.
A. Khrimian (*):S. Shirali :F. Guzma n :K. Chauhan :
J. R. Aldrich:D. C. Weber
US Department of Agriculture, Agricultural Research Service,
Invasive Insect Biocontrol and Behavior Laboratory, Beltsville,
MD 20705, USA
e-mail: ashot.khrimian@ars.usda.gov
K. E. Vermillion
US Department of Agriculture, Agricultural Research Service,
National Center for Agricultural Utilization Research, Peoria,
IL 61604, USA
M. A. Siegler
Department of Chemistry, Johns Hopkins University, Baltimore,
MD 21218, USA
Present Address:
J. R. Aldrich
Department of Entomology, University of California, Davis,
CA 95616, USA
JChemEcol
DOI 10.1007/s10886-014-0521-2
bisaboladien-3-ol (de Oliveira et al. 2013). The absolute con-
figuration of it has been assigned based on the correlation to
natural zingiberene and similarities of
13
C NMR spectra of a
synthetic mixture containing the pheromone and (R,R)-
quercivorol. Thus, until the recent synthesis of Khrimian
et al. (2014), no single isomer of 1,10-bisaboladien-3-ol and/
or 10,11-epoxy-1-bisabolen-3-ol had been synthesized to as-
sist identifications of stereo structures of the above natural
products. It is noteworthy that identification of the aggregation
pheromones of O. poecilus,T. limbativentris and
M. histrionica were supported by laboratory bioassays, but
field trapping experiments with identified pheromones have
not been reported. Murgantia histrionica is an important pest
of cole crops in the U.S. (Wallingford et al. 2011), and devel-
opment of an attractive pheromone as bait in monitoring traps
or in management applications would be highly desirable.
Recently, we developed syntheses of all eight stereoiso-
mers of 1,10-bisaboladien-3-ol and six stereoisomeric
10,11-epoxy-1-bisabolen-3-ols and established their rela-
tive and absolute configurations via single-crystal X-ray
crystallography and chemical correlations (Khrimian
et al. 2014). Utilizing enantioselective gas-chromatogra-
phy, we identified two aggregation pheromone compo-
nents of the brown marmorated stink bug, Halyomorpha
halys,as(3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol
and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol
(Khrimian et al. 2014). In the current paper, we describe
preparations of the remaining stereoisomers of 10,11-
epoxy-1-bisabolen-3-ol, and the application of individual
stereoisomers and enantioselective gas-chromatography
to determine the stereochemistry of the pheromone of
the harlequin bug.
Methods and Materials
General Methods Routine GC analyses were performed on an
Agilent Technologies 6890N instrument equipped with a
flame ionization detector and a DB-5 capillary column
(30 m×0.32 mm i.d.×0.25 μm film thickness). Hydrogen
was used as carrier gas at 1 ml/min. Column temperature
was maintained at 50 °C for 3 min, and then raised to
270 °C at 10 °C/min. Enantioselective GC analyses were
performed on a Hydrodex β-6TBDM capillary column
(25 m×0.25 mm ID; Macherey-Nagel GmbH & Co. KG,
Düren, Germany), column #1, and an Astec Chiraldex G-TA
column (30 m×0.25 mm i.d.×0.12 μm film; Sigma-Aldrich/
Supelco, Bellefonte, PA, USA), column #2. Electron impact
ionization (EI) mass spectra were obtained at 70 eV with an
Agilent Technologies 5973 mass selective detector interfaced
with 6890N GC system equipped with either an HP-5MS
(30 m× 0.25 mm i.d.×0.25 μm film) column, or one of the
chiral columns described above. 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.
TLC analyses were conducted on Whatman AL SIL G/UV
plates using 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, Fair Lawn, NJ, USA).
NMR spectra of compounds 17,18,and20 were collected
on a Bruker Avance 500 spectrometer running Topspin 1.4 pl8
using a 5 mm BBO probe. Spectra were recorded inCD
2
Cl
2
at
500 MHz for
1
H and 125 MHz for
13
CNMR.Chemicalshifts
are reported as parts per million from tetramethylsilane based
on the lock solvent. COSY,
13
C-DEPT 135, HMBC, and
HSQC spectra also were recorded to assign protons and
carbons in the synthetic molecules.
1
H NMR spectra of other
compounds were obtained at 600 MHz and
13
Cspectraat
151 MHz on a Bruker AVIII-600 MHz spectrometer.
Chemical shifts are reported in δunits and referenced to the
residual CD
2
Cl
2
solvent signal.
Optical rotations were obtained using a Perkin-Elmer 241
polarimeter with a 1.0 ml cell. 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.
All reagents and solvents were purchased from Aldrich
Chemical Co., unless otherwise specified. All eight stereoiso-
meric 1,10-bisaboladien-3-ols (Scheme 1,1–8), seven inter-
mediate triols, 9,10,13,14,15,16,21, and six 10,11-epoxy-
1-bisabolen-3-ols, 25,26,29,30,31,37, have been described
previously (Khrimian et al. 2014). Syntheses of these and
remaining stereoisomeric triols and epoxybisabolenols are
presented below and in Scheme 1.
Preparation of 1-Bisabolen-3,10,11-Triols Via Asymmetric
Dihydroxylation of 1,10-Bisaboladien-3-ols Solutions of al-
cohols (2,5,6,7,and8, 1 mmol) in tert-butanol (4.7 ml) were
added to a mixture of AD-mix-α(1.38 g), and
methanesulfonamide (91 mg) in water (4.7 ml) at 0 °C.
Mixtures were stirred at 0–2 °C for 24 h, then 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 methylene chloride (4×30 ml), the combined organic
extracts were washed with 2 N KOH, brine, and dried with
Na
2
SO
4
. After evaporation of the solvent, residues were
chromatographed on SiO
2
with ethyl acetate to yield 10Striols
12 (90 % yield; M.p. 95–97 °C, ethyl acetate/heptane, 1:2), 18
(90 %), 20 (76 %), 22 (82 %), and 24 (80 %; M.p. 97–99 °C ,
ethyl acetate/hexane, 1:2), respectively. Analogously, alcohols
2,5,6,and8were dihydroxylated with AD-mix-βto yield
10Rtriols 11 (78 %; M.p. 96–98 °C, ethyl acetate), 17 (96 %),
19 (86 %; M.p. 123–124 °C, tert-butyl methyl ether), and 23
(87 %; M.p. 94–95 °C, tert-butyl methyl ether), respectively.
GC-MS (e.g., 17,m/z, %): 238 (1), 223 (3), 220 (2), 205 (6),
JChemEcol
180 (18), 162 (19), 147 (20), 134 (58), 132 (47), 121 (83), 105
(42), 94 (100), 93 (66), 79 (47), 71 (54), 59 (97), 43 (89), 41
(27). Two other cis-1-bisabolen-3,10,11-triols, 18 and 22,
have mass spectra similar to that of 17, which were consistent
with those published (Khrimian et al. 2014). GC/MS (12,m/z,
%): 238 (2), 223 (3), 220 (4), 205 (5), 180 (11), 159 (15), 145
(21), 134 (70), 132 (84), 121 (87), 105 (42), 94 (42), 93 (91),
79 (35), 71 (57), 59 (100), 43 (84), 41 (27). Other trans-1-
bisabolen-3,10,11-triols 11,19,23,20,and24 display frag-
mentations similar to 12 that were consistent with those pub-
lished (Khrimian et al. 2014). Specific rotations and
1
Hand
13
C NMR data of synthesized triols were presented in Table 1.
All triols displayed correct molecular weights in HRESIMS
analyses corresponding to 279.1936 calculated for
C
15
H
28
O
3
Na.tgroup
Preparation of 10,11-Epoxy-1-bisabolen-3-
ols Methanesulfonyl chloride (77 μl, 1.14 mmol) was added
to a stirred solution of a triol (11,12,16–20,22–24, 1.0 mmol)
in dry pyridine (1.5 ml) at 0–5 °C. The mixture was allowed to
warm to room temperature and stirred for 1 h. Then, it was
poured into ice-water (4 ml) and extracted with CH
2
Cl
2
(3×
10 ml). Combined organic extracts were washed with ice-
water, dried with Na
2
SO
4
, and concentrated to yield a crude
mesylate. This was taken into methanol (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 precipita-
tion of inorganic salts. The reaction mixture was warmed to
room temperature, stirred for 0.5 h, and concentrated to re-
move most of MeOH. The residue was combined with NH
4
Cl
solution (pH 7–8), and extracted with ether (3×10 ml).
Combined organic extracts were washed with ice-water and
brine, dried with Na
2
SO
4
, and concentrated. Flash chroma-
tography (hexane/ethyl acetate, 3:2) yielded
epoxybisabolenols 27 (56 % yield, 95 % dr, column #1), 28
(36 %, 98 % dr, column #1), 32 (61 %, 98 % dr, column #1),
33 (61 %, 94 % dr, column #1), 34 (39 %, 92 % dr, column
#1), 35 (71 %, 91 % dr, column #2), 36 (56 %, 87 % dr,
column #2), 38 (50 %, 98 % dr, column #1), 39 (20 %, 92 %
dr, column #2), 40 (36 %, 78 % dr, column #2). (Scheme 1,
Table 2). GC-MS (34,m/z, %): 220 (3), 205 (4), 187 (4), 165
(15), 147 (16), 134 (43), 132 (38), 123 (23), 121 (34), 119
(42), 109 (32), 105 (29), 93 (69), 91 (50), 79 (38), 71 (50), 55
(29), 43 (100), 41 (42). Other cis-10,11-epoxy-1-bisabolen-3-
ols (33,38) had mass-spectra similar to 34 mass-spectra
corresponding to those reported (Khrimian et al. 2014; Zahn
et al. 2008). GC-MS (28,m/z, %): 220 (7), 205 (7), 187 (5),
165 (26), 147 (21), 145 (20), 134 (57), 132 (81), 123 (28), 121
(50), 119 (83), 109 (43), 105 (42), 93(96), 91 (73), 79 (34), 71
(49), 55 (34), 43 (100), 41 (46). Other trans-10,11-epoxy-1-
bisabolen-3-ols (27,32,35,36,39,and40) had mass spectra
similar to that of 28. Optical rotations and NMR data of 10,11-
epoxy-1-bisabolen-3-ols are presented in Table 2. All synthe-
sized 10,11-epoxy-1-bisabolen-3-ols displayed correct molec-
ular weights in HRESIMS analyses corresponding to
261.1830 calculated for C
15
H
26
O
2
Na.tgroup
X-ray Structure Determination of Triol 12 Triol 12 was crys-
tallized from ethyl acetate/heptane, 1:2, then re-crystallized as
follows. Triol 12 (2.8 mg) was placed in an NMR tube and
dissolved in dichloromethane (150 μl). Then, toluene (100 μl)
was added. Lath-like crystals slowly precipitated and were
analyzed for X-ray structure determination. All reflection
intensities were measured at 110(2) K using a SuperNova
diffractometer (equipped with Atlas detector) with Cu Kα
radiation (mirror optics, λ=1.5418 Å) under the program
CrysAlisPro (Version 1.171.36.24 Agilent Technologies
2012). The program CrysAlisPro (Version 1.171.36.24
Agilent Technologies 2012) was used to refine the cell dimen-
sions. Data reduction was done using the program
CrysAlisPro (Version 1.171.36.24 Agilent Technologies
2012). The structure was solved with the program SHELXS-
97 (Sheldrick 2008), and was refined on F
2
with SHELXL-97
(Sheldrick 2008). Analytical numeric absorption corrections
based on a multifaceted crystal model were applied using
CrysAlisPro (Version 1.171.36.24 Agilent Technologies
Scheme 1 Synthesesof10,11-
epoxy-1-bisabolene-3-ols (25–
40) from 1,10-bisaboladien-3-ols
(1–8) via 1-bisabolen-3,10,11-
triols (9–24). Compounds marked
with asterisks were described in
Khrimian et al. 2014
JChemEcol
2012). The temperature of the data collection was controlled
using the system Cryojet (manufactured by 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. Their atomic coordinates (the O
−H distances were restrained to be 0.84(3) Å using the DFIX
instruction), and isotropic temperature factors were refined
freely. The structure is ordered. The absolute configuration
has been established by anomalous dispersion effects in
Tabl e 1 Specific rotations and NMR data of stereoisomeric triols
No. [α]
D20
(c, CH
2
Cl
2
)
1
HNMR
δ
a
,ppm,J,Hz
13
CNMR
δ
b
,ppm
11 −24.4 (1.0) 0.86 (d, J6.6, 3H), 1.12, 1.17, 1.24 (all s, 3H), 1.30–1.35 (m, 1H),
1.36–1.48 (m, 5H), 1.61 (m, 1H), 1.62 (td, J13.0, 3.0, 1H),
1.71 (m, 1H), 1.85 (dm, J12.5, 1H), 1.93 (m, 1H), 2.10 (m, 1H),
2.14 (d, J4.2, 1H), 3.30 (brdd, J9.8, 4.2, 1H), 5.56 (ddd, J10.3,
2.3, 1.2, 1H), 5.59 (ddd, J10.3, 2.5, 1.4, 1H)
16.6, 23.4, 24.6, 26.7, 28.7, 29.6, 31.0, 37.1,
38.8, 41.3, 69.9, 73.3, 79.0, 130.9, 135.6.
12 −59.9 (1.0) 0.87 (d, J7.0, 3H), 1.11, 1.17, 1.24 (all s, 3H), 1.10–1.22 (m, 1H),
1.39–1.48 (m, 3H), 1.54 (m, 1H), 1.58–1.73 (m, 4H),
1.85 (dm, J12.2, 1H), 1.92 (m, 1H), 2.12 (m, 1H),
2.16 (d, J4.2, 1H), 3.28 (ddd, J10.2, 4.2, 2.4, 1H),
5.56 (ddd, J10.2, 2.3, 1.2, 1H), 5.59 (ddd, 10.3, 2.5, 1.4, 1H)
16.8, 23.4, 24.8, 26.7, 28.7, 30.2, 31.5, 37.5,
38.8, 41.0, 69.9, 73.3, 79.5, 130.7, 135.7
17 +27.0 (2.0) 0.89 (d, J6.8, H-14), 1.12 (s, H-12), 1.16 (m, H-8a), 1.17 (s, H-13),
1.18 (m, H-9a)
1.23 (s, H-15), 1.51 (m, H-7), 1.52 (m, H-5a), 1.53 (m, H-4a),
1.54 (m, H-9b), 1.59
(m, H-5b), 1.67 (m, H-8b), 1.80 (m, H-4b), 2.03 (m, H-6),
3.30 (d, J9.8, H-10), 5.66 (s, H-1/H-2), 5.66 (s, H-2/H-1)
c
17.0 (C-14), 22.3 (C-5), 23.36 (C-12), 26.7 (C-13),
30.0 (C-15), 30.3 (C-9), 31.2 (C-8), 37.4 (C-7),
37.8 (C-4), 41.4 (C-6), 67.5 (C-3), 79.5 (C-10),
73.3 (C-11), 133.0 (C-1), 134.3 (C-2)
c
18 −18.3 (2.8) 0.89 (d, J6.7, H-14), 1.12 (s, H-12), 1.17 (s, H-13), 1.23 (s, H-15),
1.34 (m, H-9a),
1.40 (m, H-8a), 1.41 (m, H-9b), 1.42 (m, H-8b), 1.51 (m, H-5a),
1.52 (m, H-7), 1.52 (m, H-4a), 1.59 (m, H-5b), 1.80 (m, H-4b),
2.01 (m, H-6), 3.30 (d, J9.8, H-10), 5.66 (s, H-1/H-2), 5.66
(s, H-2/H-1)
c
16.6 (C-14), 22.3 (C-5), 23.4 (C-12), 26.7 (C-13),
30.0 (C-15), 30.0 (C-9), 30.8 (C-8), 37.1 (C-7),
37.8 (C-4), 41.7 (C-6), 67.5 (C-3), 73.2 (C-11),
79.0 (C-10), 133.1 (C-1), 134.4 (C-2)
c
19 −14.7 (0.9) 0.83 (d, J6.8, 3H), 1.12, 1.17, 1.23 (all s, 3H), 1.13–1.25 (m, 2H),
1.38 (m, 1H), 1.52 (m, 2H), 1.57–1.72 (m, 3H), 1.84 (dm, J11.8, 1H),
2.14 (m, 1H), 3.29 (dd, J9.9, 1,8, 1H), 5.50 (ddd, J10.3, 2.3, 1.2, 1H),
5.59 (ddd, J10.3,2.5,1.4,1H)
16.0, 22.5, 23.4, 26.7, 28.7, 30.1, 31.7, 37.3, 38.7,
40.3, 69.8, 73.3, 79.5, 132.1, 135.3
20 −77.1 (6.6) 0.83 (d, J6.7, H-14), 1.12 (s, H-12), 1.17 (s, H-13), 1.24 (s, H-15),
1.32 (m, H-9a),
1.39 (m, H-5a), 1.41 (m, H-8a), 1.41 (m, H-8b), 1.42 (m, H-9b),
1.51 (m, H-7), 1.61 (m, H-4a), 1.67 (m, H-5b), 1.84 (dm, J12.2, H-4b),
2.13 (m, H-6), 3.30 (d, J9.9, H-10), 5.52 (ddd, J10.2,2.1,1.0,H-1),
5.59 (ddd, 10.2, 2.4, 1.5, H-2)
c
15.8 (C-14), 22.9 (C-5), 23.4 (C-12), 26.7 (C-13),
28.6 (C-15), 29.9 (C-9), 31.4 (C-8), 37.1 (C-7),
38.6 (C-4), 40.8 (C-6), 69.8 (C-3), 73.2 (C-11),
79.0 (C-10), 131.9 (C-1), 135.3 (C-2)
c
22 −22.6 (1.0) 0.85(d, J6.6, 3H), 1.11, 1.16, 1.23 (all s, 3H), 1.33 (m, 1H),
1.39–1.58 (m, 7H), 1.66 (bs, 1H), 1.79 (m, 1H), 2.01 (m, 2H),
2.26 (m, 1H), 3.30 (d, J10.2, 1H), 5.63 (dm, J10.2, 1H),
5.64 (dm, J10.2, 1H).
16.0, 20.9, 23.4, 26.7, 29.9, 30.0, 31.4, 37.1,
37.7, 41.3, 67.5, 73.3, 79.0, 134.0, 134.1
23 +52.7 (1.3) 0.86 (d, J6.8, 3H), 1.12, 1.17, 1.24 (all s, 3H), 1.10–1.22 (m, 1H),
1.26 (m, 1H), 1.39–1.48 (m, 2H), 1.54 (m, 1H), 1.57 (m, 1H),
1.58–1.73 (m, 3H), 1.85 (dm, J12.2, 1H), 1.88 (m, 1H),
2.09–2.16 (m, 2H), 3.28 (d, J10.2, 1H), 5.56 (ddd,
J10.2, 2.3, 1.2, 1H), 5.59 (ddd, 10.3, 2.5, 1.4, 1H)
16.8, 23.4, 24.8, 26.7, 28.8, 30.2, 31.5, 37.5,
38.8, 41.0, 69.9, 73.3, 79.5, 130.7, 135.7
24 +27.1 (1.0) 0.87 (d, J6.6, 3H), 1.12, 1.17, 1.24 (all s, 3H), 1.30–1.35 (m, 1H),
1.36–1.50 (m, 5H), 1.61 (m, 1H), 1.62 (td, J13.0, 3.0, 1H),
1.71 (m, 1H), 1.84 (dm, J12.5, 1H), 1.93 (m, 1H), 2.10 (m, 1H),
2.15 (m, 1H), 3.30 (bd, J10.2, 1H), 5.56 (ddd, J10.2,2.3,1.2,1H),
5.59 (ddd, J10.3,2.5,1.4,1H)
16.6, 23.4, 24.6, 26.7, 28.7, 29.5, 31.0, 37.1,
38.8, 41.3, 69.9, 73.3, 79.0, 130.9, 135.6
a
Referenced to CD
2
Cl
2
signal at 5.32 ppm
b
Referenced to CD
2
Cl
2
signal at 53.84 ppm
c
Signals were assigned based on COSY,
13
C-DEPT 135, HMBC, and HSQC recordings
JChemEcol
diffraction measurements on the crystal. The Flack and Hooft
parameters (Flack 1983; Hooft et al. 2008)refineto0.01(14)
and −0.05(5), respectively. The model has chirality at C3 S,
C6 R, C7 R, and C10 S (Fig. 1). Triol 12, Fw=256.37, thin
colorless lath, 0.56× 0.10× 0.04 mm
3
, monoclinic, P2
1
(no. 4),
a=9.4105(3), b=6.49971(18), c=12.2958(3) Å, β=
99.261(2)°, V=742.28(4) Å
3
,Z=2, D
x
=1.147 g cm
−3
,μ=
0.614 mm
−1
, abs. corr. range: 0.807–0.977. Seven thousand
eight hundred thirty reflections were measured up to a reso-
lution of (sin θ/λ)
max
=0.62 Å
−1
. Two thousand nine hundred
reflections were unique (R
int
=0.0206), of which 2844 were
observed [I>2σ(I)]. One hundred eighty parameters were
refined using 4 restraints. R1/wR2[I>2σ(I)]: 0.0306/0.0833.
R1/wR2 [all refl.]: 0.0312/0.0842. S=1.055. Residual electron
density found between −0.16 and 0.21 e Å
−3
.
Insect Rearing and Semiochemical Collection Harlequin bug
adults and nymphs were collected by hand from their host
Tabl e 2 Specific rotations and NMR data of stereoisomeric epoxybisabolenols
No [α]
D20
(c, CH
2
Cl
2
)
1
HNMR
δ
a
,ppm,J,Hz
13
CNMR
δ
b
,ppm
27 −37.7 (1.0) 0.87 (d, J6.7, 3H), 1.23, 1.24, 1.27 (all s, 3H), 1.22 (m, 1H),
1.38–1.59 (m, 6H), 1.62 (m, 1H), 1.71 (m, 1H), 1.84 (m, 1H),
2.10 (m, 1H), 2.66 (dd, J6.6, 4.8, 1H), 5.55 (ddd, J10.2, 2.2,
1.2, 1H), 5.60 (ddd, J10.2, 2.4, 1.5,1H)
16.6, 18.8, 24.5, 25.0, 27.5, 28.7, 30.8 37.1,
38.7, 41.2, 58.1, 64.7, 69.8, 130.7, 135.7
33 −1.3 (1.0) 0.90 (d, J6.8, 3H), 1.231,1.234, 1.27 (all s, 3H), 1.31–1.37 (m, 1H),
1.41–1.61 (m, 8H), 1.80 (m, 1H), 2.02 (m, 1H), 2.65 (t, J6.0, 1H),
5.64 (m,1H), 5.66 (m, 1H)
16.7, 18.9, 22.5, 25.1, 27.4, 30.0, 30.6, 37.0, 38.9,
41.4, 58.4, 64.6, 67.5, 132.8, 134.5
35 −39.2 (1.1) 0.83 (d, J6.6, 3H), 1.24 (br. s, 6H), 1.27 (s, 3H) ,1.30–1.50 (m, 6H),
1.51–1.57 (m, 1H), 1.58–1.69 (m, 2H), 1.85 (m, 1H), 2.14 (m, 1H),
2.66 (t, J6.0, 1H), 5.50 (ddd, J10.2, 2.4, 1.2, 1H),
5.59 (ddd, J10.2,2.8,1.2,1H)
15.9, 18.9, 22.7, 25.0, 27.3, 28.7, 31.1, 36.9, 38.7,
40.4, 58.4, 64.6, 69.8, 131.9, 135.4
39 +34.1 (1.8) 0.88 (d, J6.7, 3H), 1.23, 1.24, 1.27 (all s, 3H), 1.31 (m, 1H),
1.37–1.58 (m, 6H), 1.62 (m, 1H), 1.72 (m, 1H), 1.84 (m, 1H),
2.11 (m, 1H), 2.64 (t, J6.0, 1H), 5.54 (ddd, J10.2,2.4,1.2,1H),
5.60 (ddd, J10.2,2.4,1.8,1H).
16.7, 18.9, 24.6, 25.0, 27.3, 28.7, 30.8, 37.0, 38.7,
41.0, 58.3, 64.6, 69.8, 130.5, 135.7
28 −38.7 (1.1) 0.87 (d, J6.6, 3H), 1.23, 1.24, 1.27 (all s, 3H), 1.31 (m, 1H),
1.38–1.58 (m, 6H), 1.63 (m, 1H), 1.71 (m, 1H), 1.85 (m, 1H),
2.11 (m, 1H), 2.64 (t, J6.0, 1H), 5.54 (dm, J10.2, 1H),
5.60 (ddd, J10.1,2.5,1.4,1H)
16.7, 18.9, 24.6, 25.0, 27.3, 28.7, 30.7, 37.1, 38.7,
41.0, 58.4, 64.7, 69.8, 130.6, 135.8
32 +32.2 (1.0) 0.83 (d, J6.6, 3H), 1.24 (br. s, 6H), 1.27 ( s, 3H), 1.30–1.50 (m, 6H),
1.51–1.57 (m, 1H), 1.58-1.69 (m, 2H), 1.84 (m, 1H), 2.14 (m, 1H),
2.65 (t, J6.6, 1H), 5.50 (ddd, J10.2, 2.4, 1.2, 1H), 5.59 (ddd, J10.2,
2.8, 1.2, 1H)
15.9, 18.9, 22.6, 25.0, 27.3, 28.6, 31.1, 36.9, 38.6,
40.4, 58.3, 64.6, 69.8, 131.8, 135.4
34 +10.4 (1.0) 0.89 (d, J6.6, 3H), 1.232, 1.234, 1.27 (all s, 3H), 1.24 (m, 1H),
1.42–1.62 (m, 8H), 1.80 (m, 1H), 2.02 (m, 1H), 2.65 (m, 1H),
5.65 (m, 1H), 5.66 (m, 1H)
16.7, 18.8, 22.3, 25.1, 27.6, 30.0, 30.7, 37.1, 37.9,
41.6 ,58.2, 64.8, 67.5, 132.9, 134.5
38 +1.91 (0.9) 0.86 (d, J6.6 3H), 1.23 (s, 6H), 1.26 (s, 3H), 1.3 (m, 1H),
1.40–1.60 (m, 8H), 1.80 (m, 1H), 2.04 (m, 1H), 2.66 (t, J6.0,
1H), 5.61 (br. d, J10.2, 1H), 5.67 (br. d, J10.2, 1H)
15.9, 18.9, 20.6, 25.0, 27.5, 30.0, 31.1, 37.1,
37.7, 41.0, 58.2, 64.6, 67.4, 133.9, 134.1
40 +38.0 (4.7) 0.87 (d, J7.2, 3H), 1.23, 1.24, 1.27 (all s, 3H), 1.22 (m, 1H),
1.37–1.60 (m, 6H), 1.62 (m, 1H), 1.71 (m, 1H), 1.84 (m, 1H),
2.10 (m, 1H), 2.64 (t, J5.4, 1H), 5.57 (ddd, J10.2, 2.4, 1.2, 1H),
5.61 (ddd, J10.2,2.6,1.2,1H)
16.6, 18.8, 24.5, 25.0, 27.5, 28.7, 30.8, 37.1, 38.7,
41.2, 58.1, 64.7, 69.8, 130.7, 135.7
36 −31.9 (2.2) 0.83 (d, J6.6, 3H), 1.231, 1.234, 1.27 (all s, 3H), 1.23 (m, 1H),
1.35–1.57
(m,6H),1.58–1.70 (m, 2H), 1.84 (m, 1H), 2.12 ( m, 1H),
2.65 (t, J6.01, 1H) 5.51 (ddd, J10.2, 2.4, 1.2 1H), 5.58 (ddd,
J10.2,2.4,1.2,1H)
15.9, 18.9, 22.8, 25.1, 27.5, 28.7, 31.2, 37.1,
38.6, 40.7, 58.2, 64.7, 69.8, 131.8, 135,5
a
Referenced to CD
2
Cl
2
signal at 5.32 ppm
b
Referenced to CD
2
Cl
2
signal at 53.84 ppm
Fig. 1 Displacement ellipsoid plot (50 % probability level) of triol 12 at
110(2) K
JChemEcol
plants, primarily collards, kale, forage radish, and rape (cv.
Dwarf Essex) on gardens and small farms within 80 km of
Beltsville, Maryland, and reared under conditions of 25°
±1 °C, 50± 10 % RH, and 16:8 h L:D photoperiod in a
walk-in growth chamber. Nymphs were reared on collard
plants (Brassica oleracea L. (acephala group)) (cv.
Champion or Vates) grown in 3.8-l pots. Newly-molted adults
were separated by observing sexual differences in the terminal
abdominal segments, and males were retained separately in
small (375 ml) ventilated containers on commercial
organically-grown broccoli florets until at least 7-d-old as
adults, after which volatile collections were initiated.
Five adult harlequin bug males then were placed in a glass
jar (500 ml) aeration system with a moistened cotton ball at
the bottom and commercial organic cauliflower florets as
food. The sample was aerated with 100 ml/min activated
carbon filtered air-flow for 72 h. Volatiles released from
M. histrionica males on the cauliflower florets were trapped
onto 50 mg activated charcoal (50/80 mesh; Sigma-Aldrich,
USA) in glass tubing between two plugs of glass wool.
Trapped volatiles were removed with 1 ml dichloromethane
into a 2-ml glass vial. The aeration extract was kept in freezer
(−20 °C) before GC/MS analyses. The control aeration extract
was obtained by conducting aeration without insects. Five
replicates of the aeration extract were collected.
Results and Discussion
Syntheses Recently, we developed syntheses of all eight ste-
reoisomers of 1,10-bisaboladien-3-ols (Scheme 1,1–8),which
were used to prepare selected 10,11-epoxy-1-bisabolen-3-ols
via intermediate triols (Scheme 1, compounds marked with
asterisks) (Khrimian et al. 2014). Herein, we describe the
remaining stereoisomers of 10,11-epoxy-1-bisabolen-3-ol
and 1-bisabolen-3,10,11-triol, which completes full stereoiso-
meric libraries of three classes of 1-bisabolen-3-ols. In addi-
tion to triol 16 described earlier (Khrimian et al. 2014), five
more triols turned out to be crystalline. X-ray structure deter-
mination of triol 12 (Fig. 1) clearly demonstrates trans ar-
rangement of the OH group at C-3 and alkyl group at C-6 of
the cyclohexene ring, thus providing an additional confirma-
tion of our earlier assignments of relative and absolute
Fig. 2 GC-MS total ion
chromatogram (top)ofaeration
from virgin Murgantia histrionica
males on an HP-5MS column.
Tridecane (asterisk)andcis-
murgantiol (A, mass-spectrum
shown below) were identified;
other compounds were not
studied
JChemEcol
configurations of these stereogenic centers. Also, the 10S
configuration of triol 12 provided further proof of the stereo-
chemistry of the Sharpless asymmetric dihydroxylation,
whereby AD-mix αdelivered (S)- and AD-mix β(R)-prod-
ucts (Sharpless et al. 1992). Triols were converted to the
corresponding mesylates of the secondary hydroxy groups,
and the mesylates were cyclized to epoxides with inversion of
configuration by treatment with KOH in MeOH (Frater and
Müller 1989; Moore et al. 1999). All stereoisomers of 10,11-
epoxy-1-bisabolen-3-ols and 1-bisabolen-3,10,11-triols were
characterized by NMR spectroscopy. Thus,
13
C NMR is the
method of choice to distinguish between cis (3R*,6R*) and
trans (3R*,6S*) stereoisomers (Khrimian et al. 2014), with
trans isomers displaying greater difference in chemical shifts
of C-1 and C-2 (3.2–5.2 ppm) than cis-isomers (0.1–1.7 ppm)
regardless of configurations at C-7 and C-10. Additionally, in
trans isomers, chemical shifts of C-3 (69.4–69.9 ppm) are
higher and those of C-15 (28.2–28.7 ppm) lower than corre-
sponding signals of cis isomers (67.1–67.5 and 29.6–
30.0 ppm, respectively), consistent with those previously re-
ported (Blair and Tuck 2009; Khrimian et al. 2014).
Stereoisomeric Composition of Pheromone of Harlequin
Bug Aeration of male M. histrionica fed on cauliflower with
subsequent analysis of volatiles by GC-MS revealed two
major compounds (Fig. 2). The first was identified as
tridecane, also found in earlier work (Zahn et al. 2008), and
the second one as the expected aggregation pheromone,
murgantiol (Zahn et al. 2008). The mass-spectrum and GC-
retention time on HP-5MS column of murgantiol (compound
A) matched those of cis-10,11-epoxy-1-bisabolen-3-ols. The
male aeration extract was examined further on two
enantioselective GC columns against all eight synthetic ste-
reoisomers of cis-10,11-epoxy-1-bisabolen-3-ol. The
murgantiol, eluted as a single peak on HP-5MS, split into
two peaks on Chiraldex G-TA column (Fig. 3, III), signifying
that the aggregation pheromone of M. histrionica consists of
more than one stereoisomer. Earlier, we showed that
Fig. 3 Segments of GC-MS total
ion chromatograms on Chiraldex
G-TA column: (I) mixture of four
cis-(7R)-10,11-epoxy-1-
bisabolen-3-ols; peaks were
assigned using individual stereo-
isomers; (II) co-injection of
Murgantia histrionica male aera-
tion and the mixture of cis-(7R)-
10,11-epoxy-1-bisabolen-3-ols;
(III) M. histrionica male aeration
JChemEcol
Chiraldex G-TA column separated all fourcis-10,11-epoxy-1-
bisabolen-3-ols with 7Rconfigurations (Khrimian et al. 2014).
We used that feature in the current study (Fig. 3,I)and
determined that (3S,6S,7R,10S)-epoxide 25 co-eluted with
the main murgantiol peak (A) and (3S,6S,7R,10R)-epoxide
26 with the minor murgantiol peak (B) (Fig. 3, II). On a
Hydrodex-β-6TBDM, murgantiol again produced two peaks,
but the order of elution was reversed (Fig. 4. I). All four cis-
10,11-epoxy-1-bisabolen-3-ols with 7Sconfigurations were
baseline separated (Fig. 4, II) on this column (Khrimian
et al. 2014). Co-injection of this mixture with M. histrionica
male aeration extract showed that the stereoisomer 37 eluted
closely to peak A but did match it, and that (3S,6S,7S,10R)-
epoxide 34 co-eluted with peak B. Thus, analyses of
M. histrionica male aeration extract on two enantioselective
columns revealed that the major male-specific component A
matched only one out of eight cis-10,11-epoxy-1-bisabolen-3-
ols, and hence, was unequivocally identified as
(3S,6S,7R,10S)-epoxyalcohol 25. The minor component B in
the male aeration could be either epoxide 26,orepoxide34,or
a mixture thereof. The last two compounds did not separateon
Chiraldex G-TA either; therefore, we used NMR spectroscopy
to finalize the stereochemical identification of component B.
We compared
1
Hand
13
C NMR spectra of synthetic
(3S,6S,7R,10S)-epoxide 25,(3S,6S,7R,10R)-epoxide 26 and
(3R,6R,7R,10S)-epoxide 29 with those of natural murgantiol
(Zahn et al. 2008). Compound 29 (which is an enantiomer of
34 and hence has identical NMR spectra) was chosen instead
of 34 because the spectra of 25,26,and29 were obtained on
the same instrument under identical conditions (Khrimian
et al. 2014). Resonances from H-14 of epoxyalcohols 25 and
26 in
1
H NMR spectra occur in the same field, 0.89 ppm, due
to identical configurations of C-3, C-6, and C-7. On the other
hand, the signal of H-14 in 29 appeared at 0.93 ppm signifying
that if 29 (or 34) is mixed with either 25,or26, there should be
two distinctly separated doublets from H-14 methyl groups in
1
H NMR spectra. Indeed, a small amount of 29 is easily
detectable in synthetic 25 (Khrimian et al. 2014), but only
one doublet from H-14 methyl group was present in the
1
H
NMR of the M. histrionica male volatiles (Zahn et al. 2008;
also see Acknowledgement). This confirms the presence of
epoxide 25, which was earlier proven to be the main constit-
uent A, as well as epoxide 26 as the minor component B in the
male volatiles. The absence of a second (downfield) doublet
from H-14 rules out the (3S,6S,7S,10R)-epoxide 34 as a pos-
sible male-specific compound in M. histrionica. Finally, field
bioassays with different stereoisomers confirmed that
(3S,6S,7R,10S)-epoxide 25 and (3S,6S,7R,10R)-epoxide 26
were both attractive to M. histrionica, but (3S,6S,7S,10R)-
epoxide 34 was not (Weber et al. 2014).
In summary, we completed the synthetic library of stereo-
isomers of 10,11-epoxy-1-bisabolen-3-ol, which was proven
essential in determining the stereoisomeric composition of the
harlequin bug aggregation pheromone. Field studies have
established the attractiveness of both epoxyalcohols 25 and
26, and especially their mixtures, to harlequin bug males,
Fig. 4 Segments of GC-MS total
ion chromatograms on
Hydrodex-β-6TBDM column: (I)
Murgantia histrionica male aera-
tion; (II) cis-(7S)-10,11-epoxy-1-
bisabolen-3-ols; compounds were
assigned using individual stereo-
isomers; (III) co-injection of
M. histrionica male aeration and
cis-(7S)-10,11-epoxy-1-
bisabolen-3-ols
JChemEcol
females, and nymphs, in the field (Weber et al. 2014).
Murgantia histrionica and Halyomorpha halys share the same
compound, (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol
(25), as the main aggregation pheromone component, whereas
the minor pheromone components are different in these bugs.
Further studies will investigate the sensitivity of life stages of
each species to ratios of the three identified aggregation pher-
omone components as well as other attractants, in order to
target each or both bugs for monitoring and management.
Acknowledgments We thank Dr. Jocelyn G. Millar, University of
California, Riverside, for sharing
1
H NMR spectrum of the
murgantiol described in Zahn et al. 2008. We express our gratitude
to Michael M. Athanas, Anthony DiMeglio, Matthew Klein, and Meiling
Z. Webb, for collecting, rearing, and volatile collection of the insects.
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