Synthesis and characterization of 3,13- and 2,13-octadecadienyl compounds for identification of the sex pheromone secreted by a clearwing moth, Nokona pernix

Article abstract of DOI:10.1271/bbb.70.508

Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M - 18]⁺ at m/z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth, Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.

Full text of DOI:10.1271/bbb.70.508

Biosci. Biotechnol. Biochem., 70 (2), 508–516, 2006
Synthesis and Characterization of 3,13- and 2,13-Octadecadienyl
Compounds for Identification of the Sex Pheromone Secreted
by a Clearwing Moth, Nokona pernix
Hideshi NAKA,1 Tomotake NAKAZAWA,2 Mieko SUGIE,2 Masanobu YAMAMOTO,2
Yoshiteru HORIE,3 Ryohei WAKASUGI,3 Yutaka ARITA,3 Hajime SUGIE,1
y
2;
Koji TSUCHIDA,4 and Tetsu ANDO
1Entomology Group, Department of Biological Safety, National Institute for
Agro-Environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan
2Graduate School of Bio-Applications and Systems Engineering (BASE), Tokyo University of Agriculture
and Technology, Koganei, Tokyo 184-8588, Japan
3Zoological Laboratory, Faculty of Agriculture, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
4Laboratory of Entomology, Faculty of Applied Biological Sciences, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan
Received September 30, 2005; Accepted October 31, 2005
Several geometrical isomers of 3,13- and 2,13-octa-
decadien-1-ols and their acetates were synthesized
starting from 1,8-octanediol or 1,9-nonanediol utilizing
acetylene coupling reactions. In addition to commer-
cially available compounds, all geometrical isomers
of each dienyl compound were analyzed by NMR and
GC-MS to accumulate chemical data for studies of sex
pheromones secreted from clearwing moths classified
into the family Sesiidae of Lepidoptera. Although
acetoxy derivatives of the 3,13- and 2,13-dienes showed
almost the same mass spectra, the alcohols were
distinguished by comparing the relative intensities of
½M ꢀ 18ꢁþ at m=z 248, indicating direct differentiation
of the two positional isomers without derivatization.
Furthermore, each geometrical isomer eluted from a
high-polar GC column with a different retention time.
Base on these data, a pheromone gland extract of a
sesiid moth, Nokona pernix, was analyzed by GC-EAD
and GC-MS, and two EAG-active components were
identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of
3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the
synthetic compounds mixed in 9:1 ratio attracted
N. pernix males well, while a single component scarcely
attracted the males. The number of attracted males
peaked in the middle of June, and a small second peak
was observed in August.
ioral ecologists, because the behaviors affect either pre-
mating isolation via recognition mechanisms of conspe-
cies or reproductive success depending on the variation
of the behaviors.1) Sex pheromone as a chemical cue is
important in communication between sexes for species
copulating in nocturnal conditions lacking light. In
addition to the nocturnal species, whereas diurnal insects
can possibly find their mating partners by means of visual
cues, females of diurnal species in the family of Sesiidae
(Lepidoptera), which have clear wings and mimic bees,
also secret a species-specific sex pheromone to attract
males. The first identification of sesiid sex pheromones
was conducted by Tumlinson et al. from two pests of
a peach tree, Synanthedon pictipes and S. exitiosa.2)
Females of the former species secret a diunsaturated
compound, (3E,13Z)-3,13-octadecadienyl acetate (E3,
Z13-18:OAc), and those of the latter species secret its
(3Z,13Z)-isomer (Z3,Z13-18:OAc). Referring to this
identification, Nielsen et al. evaluated the synthetic
pheromones and successfully attracted males of eight
other sesiid species.3) Since then, pheromone and attract-
ants of many sesiid species composed of 3,13- and 2,13-
octadecadienyl compounds have been found in studies
mainly conducted in the United States and Europe.4–7)
In Japan, about 40 sesiid species have been record-
ed,8) and attraction of thirteen species to the lures baited
with 3,13-octadecadienyl compounds has been docu-
mented.5–10) In addition to these attractants, an effective
method has been developed to disrupt the mating
communication of two harmful pests in Sesiidae,
Key words: octadecadien-1-ol; octadecadienyl acetate;
sex attractant; Lepidoptera; Sesiidae
Studies on insect mating behaviors have been provid-
ing fascinating themes for both evolutionary and behav-
Synanthedon hector11) and S. tenuis as further agricul-
*
tural application of their lures. Identification of pher-
y
To whom correspondence should be addressed. Fax: +81-42-388-7278; E-mail: antetsu@cc.tuat.ac.jp

Pheromonal Octadecadienyl Compounds of a Clearwing Moth
509
omone components in females, however, has never been
published for any of the Japanese sesiid species because
the larvae burrowing in stems of plants are not found
readily. In order to understand the diversity of their
communication systems, it is necessary to collect the
insects and examine the components actually produced
by the females. As a first step in studies on sexual
communication systems of the diurnal species, we
started to synthesize commercially unavailable isomers
of sesiid pheromones and to accumulate analytical data
on them to support chemical studies on the Japanese
species. Utilizing these data, we tried to identify
pheromone components of a sesiid moth, Nokona
pernix, one of the most common species among the
Japanese clearwing moths. While the larvae feeding on
Paederia scandens (Rubiales: Rubiaceae), an ordinary
garden weed, were not agricultural pests, they were
rather easily found and could be subjected to future
study on the interaction of chemical and visual cues in a
mating communication system.
with HBr and protection of the residual hydroxyl group
with 2,3-dihydropyran, 1,8-octanediol (1a) was con-
verted into a THP ether of bromohydorin (2a), which
was coupled with 1-hexyne to prepare a C14 acetylene
(3a). Birch reduction of 3a using Li metal yielded a
monoene compound with an (E)-9-double bond (4a),
and an iodo compound (5a) was synthesized by
deprotection of 4a and succeeding iodination of the
recovered hydroxyl group by treatment with a complex
of iodine and triphenylphosphine. Coupling of 5a with a
THP ether of 3-butyn-1-ol gave a C18 enyne (6a), which
was reduced by catalytic hydrogenation over Pd-BaSO4
poisoned with quinoline and deprotected to accomplish
the synthesis of (3Z,13E)-3,13-octadecadien-1-ol (Z3,
E13-18:OH). This alcohol was quantitatively acetylated
with acetic anhydride in pyridine to yield (3Z,13E)-3,13-
octadecadienyl acetate (Z3,E13-18:OAc). In the same
manner, the corresponding 2,13-octadecadienyl com-
pounds, Z2,E13-18:OH and Z2,E13-18:OAc, were syn-
thesized starting from 1,9-nonanediol (1b) via a cou-
pling reaction between a C15 iodo compound (5b) and
a THP ether of propargyl alcohol.
Materials and Methods
1
Instruments. H and 13C NMR spectra were recorded
Synthesis of (E,E)-isomers of 3,13- and 2,13-octade-
cadienyl compounds (Fig. 1B and C). The enyne
compound (6a) was reduced to a dienyl compound with
3E,13E configuration by the Birch reduction, and
deprotection of its THP ether produced E3,E13-18:OH.
After deprotection, the triple bond at 2-position of the
enyne compound (6b) was reduced to an (E)-double
bond by treatment with LiAlH4, and E2,E13-18:OH was
obtained. These alcohols were also acetylated with
acetic anhydride in pyridine.
with a Jeol Alpha 500 Fourier transform spectrometer at
500.2 and 125.7 MHz respectively for CDCl3 solutions
containing TMS as an internal standard. GC-MS was
conducted in the EI mode (70 eV) with an HP5973 mass
spectrometer system (Hewlett-Packard, Wilmington,
DE, USA) equipped with a DB-23 column (0.25
mm ID ꢀ 30 m, 0.25 mm, J & W Scientific, Folsom,
CA, USA) or an HP-5 column (0:25 mm ꢀ 30 m,
0.25 mm, Hewlett-Packard). The column temperature
program for the former column was 100 ꢁC for 2 min,
20 ꢁC/min to 175 ꢁC, and 6 ꢁC/min to 220 ꢁC, and that
for the latter column was 50 ꢁC for 2 min, 10 ꢁC/min to
160 ꢁC, and 4 ꢁC/min to 260 ꢁC. The carrier gas was He,
and the ion source temperature was 230 ꢁC. The electro-
antennogram (EAG) activity of natural pheromone
components was measured using a gas chromatograph
equipped with an electroantennographic detector (GC-
EAD).12) The GC involved an HP-5890 Series II gas
chromatograph (Hewlett-Packard) and the same DB-23
capillary column for GC-MS analysis. The column
temperature program was also the same as the GC-MS
with this column. The effluent from the column was split
into two lines, which were led to a flame ionization
detector (FID) and EAD at a ratio of 1:1. An antenna
was excised at the base from the male moth, and a few
distal segments were cut off. Each end of the antenna
was attached to a droplet of a saline solution on an
electrode of the EAD device such that the sensilla faced
the airflow from the GC.
Synthesis of (Z,Z)-isomer of 2,13-octadecadienyl
compounds (Fig. 1D). The THP group of a C15 acety-
lene (3b) was converted to iodine, and the produced
iodoacetylene (7) was coupled with a THP ether of
propargyl alcohol to yield diyne compound (8). Both
triple bonds were simultaneously reduced to (Z)-double
bonds by hydrogenation on a Pd catalyst poisoned with
quinoline. Removal of the THP group accomplished the
synthesis of Z2,Z13-18:OH, which was acetylated to
yield Z2,Z13-18:OAc.
Other compounds. E3,Z13-18:OAc, Z3,Z13-18:OAc,
and E2,Z13-18:OAc were supplied by Shin-Etsu Chemi-
cal Co., Ltd., (Tokyo) with purity levels exceeding 98%.
The corresponding alcohols were obtained by hydrolysis
with methanolic NaOH.
Insects and pheromone extraction. About 40 over-
wintering N. pernix larvae were collected in Toyota
City, Aichi Prefecture, Japan, in December 2003 and
vernalized under winter conditions (8 ꢂ 1 ꢁC, dark
condition) for one month. After vernalization, they were
placed in a plastic cage under laboratory conditions
(23 ꢂ 1 ꢁC, 13L:11D) until the emergence of the adults.
Synthesis of (Z,E)-isomers of 3,13- and 2,13-octade-
cadienyl compounds (Fig. 1A). By half bromination
*
Mochizuki, personal communication.

510
H. NAKA et al.
Fig. 1. Synthetic Routes to (A) (3Z,13E)-3,13- and (2Z,13E)-2,13-Octadecadien-1-ols and Their Acetates, (B) (3E,13E)-3,13-Octadecadien-1-ol and
Its Acetate, (C) (2E,13E)-2,13-Octadecadien-1-ol and Its Acetate, and (D) (2Z,13Z)-2,13-Octadecadien-1-ol and Its Acetate.
(a) HBr (47%)/toluene/18 h, refluxing; (b) 2,3-dihydropyran/3 h, < 45 ꢁC; (c) n-C4H9CꢃCLi (from n-C4H9CꢃCH and n-C4H9Li)/THF-
HMPA/1 h, room temp.; (d) Li/EtNH2/1 h, ꢄ5 ꢁC; (e) p-TsOH/EtOH/3 h, refluxing; (f) I2-PPh3-imidazole/Et2O-MeCN/1 h, 0 ꢁC; (g)
LiCꢃC(CH2)n-OTHP (from HCꢃC(CH2)n-OTHP and n-C4H9Li)/THF-HMPA/1 h, room temp.; (h) H2/Pd (5%)-BaSO4-quinoline/1 h, room
temp.; (i) Ac2O/pyridine/0.5 h, room temp.; (j) LiAlH4/THF/3 h, refluxing.
Their sexes were determined at the adult stage based on
their hair tuft.8) The females showed calling-position
when they were placed in direct sunlight; hence the
abdominal ends of one- or two-day-old virgin females
were excised after irradiation with direct sunlight and
separately immersed in hexane (250 ml/female) for
5 min. The crude extracts of two female equivalents
(FE) were combined and purified on a micro column
packed with Florisilꢀ (100–200 mesh, Floridin Co.,
200 mg). Substances were eluted successively with
1 ml each of hexane and 5%, 10%, 20%, and 50%
diethyl ether in hexane.
Takeda Chemical Ind. Ltd., Osaka, Japan), which was
set 1.5 m above the ground. The positions of the traps
were rotated after counting the captured moths to
eliminate any positional effect (Latin-square design),13)
and pheromone in the dispensers was replaced at the end
of June. The replicated data on numbers of catches
(Tables 4 and 5) were analyzed by means of one-way
ANOVA; means were log-transformed to ensure the
normality and homoscedasticity of the data. The means
that had significant differences were separated using the
Tukey–Kramer test. The level of significance in all tests
was 5%.
Field tests. The synthetic pheromone was examined
in three neighboring fields: Kasugai City, Japan (the
Experimental Farm of Meijo University) from May 25
to September 5, 2004 and August 15 to September 19,
2005; Nagoya (the Shiogama-guchi campus of Meijo
University) from May 25 to July 30, 2004; and Seto City
(a private coppice) from May 25 to July 30, 2004. A
rubber septum (white rubber, 8 mm OD, Sigma-Aldrich
Co., St. Louis, MO, USA) was used as a dispenser of the
synthetic pheromone and placed in the center of a sticky
board trap (30 cm ꢀ 27 cm bottom plate with a roof,
Results
Synthesis of 3,13- and 2,13-octadecadieny compounds
There are several reports on the synthesis of sesiid
pheromones.2,14,15) We synthesized several geometrical
isomers modifying the method published by Tumlinson
et al.2) Namely, the C18 3,13-dienyl skeleton (3,13-18)
was constructed with C8, C6, and C4 synthons and the
C18 2,13-dienyl skeleton (2,13-18) with C9, C6, and C3
synthons using acetylene coupling reactions, as shown in
Fig. 1. Triple bonds were stereoselectively converted

Pheromonal Octadecadienyl Compounds of a Clearwing Moth
511
Table 1. 1H NMR Assignments for the Characteristic Signals of
3,13- and 2,13-Octadecadienyl Compounds
Table 2. 13C NMR Assignments for the Characteristic Signals of
3,13- and 2,13-Octadecadienyl Compounds
Chemical shift (ppm), [coupling constant (Hz)]
Position
(Z,Z)-Isomer (E,Z)-Isomer (Z,E)-Isomer (E,E)-Isomer
Chemical shift (ppm)
Position
(Z,Z)-Isomera (E,Z)-Isomera (Z,E)-Isomerb (E,E)-Isomerb
3,13-Octadecadien-1-ol (3,13-18:OH)
1 (2H, t)
3,13-Octadecadien-1-ol (3,13-18:OH)
3.64[6.3]a 3.62[6.3]a 3.63[6.9]a
3.62[6.9]a
1
62.3
30.8
62.0
36.0
62.3
30.8
62.0
36.0
2 (2H, dt)
3 (1H, dt)
4 (1H, dt)
5 (2H, m)
2.32[7.3]b 2.26[6.6]b 2.32[7.4]b 2.25[6.6]b
5.36[10.6]c 5.37[15.2]c 5.35[10.9]c 5.37[15.2]c
2
3
124.9
133.6
27.4
125.7
134.3
32.7
125.0
133.5
27.4
125.7
134.4
32.7
5.56
2.06
5.55
ꢆ2:02
ꢆ2:02
5.35
5.56
2.06
5.55
ꢆ2:03
ꢆ1:97
5.38
4
5
6–11
12c
12, 15 (4H, m)ꢆ2:02
ꢆ1:96
5.38
0.89
29.3–29.8
26.9
29.2–29.8
26.9
29.2–29.7
32.3
29.2–29.7
32.3
13, 14 (2H, m) 5.35
0.90
18 (3H, t)
0.90
0.89
13d
14d
15c
129.9
129.9
27.2
129.86
129.88
27.2
130.3
130.3
32.6
130.32
130.34
32.6
3,13-Octadecadienyl acetate (3,13-18:OAc)d
1 (2H, t)
4.06[7.0]a 4.06[7.0]a 4.06[6.8]a
4.06[7.0]a
2 (2H, dt)
3 (1H, dt)
4 (1H, dt)
2.37[7.3]b 2.31[6.6]b 2.37[7.5]b 2.31[6.6]b
5.34[11.2]c 5.36[15.2]c 5.32[10.9]c 5.36[15.2]c
3,13-Octadecadienyl acetate (3,13-18:OAc)e
1
2
3
4
64.0
26.8
64.2
32.0
64.0
26.8
64.2
32.0
5.51
5.51
5.50
5.50
2,13-Octadecadien-1-ol (2,13-18:OH)
1 (2H, t)
124.2
133.0
125.0
133.6
124.2
133.0
125.0
133.6
4.18[5.9]a 4.08[4.8]a 4.18[5.9]a
4.07[4.7]a
2 (1H, dt)
3 (1H, dt)
4 (2H, dt)
5.53[11.0]b 5.63[15.4]b 5.54[11.2]b 5.65[15.4]b
2,13-Octadecadien-1-ol (2,13-18:OH)
5.60[6.3]c 5.70[5.8]c 5.59[6.2]c
5.70[5.9]c
1
2
3
4
58.5
128.3
133.2
27.5
63.8
128.8
133.6
32.2
58.5
128.3
133.3
27.5
63.8
128.9
133.5
32.3
ꢆ2:05 ꢆ2:02 ꢆ2:05
ꢆ2:04
2,13-Octadecadienyl acetate (2,13-18:OAc)e
1 (2H, t)
2 (1H, dt)
3 (1H, dt)
4.61[6.6]a 4.50[6.4]a 4.62[6.8]a
4.50[6.4]a
5.52[11.0]b 5.55[15.2]b 5.52[11.0]b 5.56[15.4]b
2,13-Octadecadienyl acetate (2,13-18:OAc)f
5.64[7.3]c 5.77[6.5]c 5.65[7.3]c
5.76[6.7]c
1
2
3
4
60.4
123.3
135.5
27.6
65.4
123.7
136.7
32.3
60.4
123.2
135.5
27.5
65.3
123.7
136.7
32.3
aJ1{2 bJ2{3 cJ3{4 dCO2CH3, 2.04 ppm. eCO2CH3, 2.05–2.06 ppm.
. . .
aC-16, 32.0 ppm; C-17, 22.4 ppm; C-18, 14.0 ppm.
bC-16, 31.9 ppm; C-17, 22.2 ppm; C-18, 14.0 ppm.
c,dChemical shift values might be reversed.
eCO2CH3; 171.1 and 21.0 ppm.
into (Z)-double bonds by hydrogenation over a Pd
catalyst poisoned with quinoline, and (E)-double bonds
by Birch reduction or by reduction with LiAlH4.
f CO2CH3; 170.9 and 21.0 ppm.
1
The H and 13C NMR data for each synthetic isomer
are shown in Tables 1 and 2 respectively, in addition to
those of commercially available compounds. In these
tables, signals from the 5-position to the 18-position of
C18 3,13-dienyl acetate (3,13-18:OAc), 2,13-dien-1-ol
(2,13-18:OH), and 2,13-dienyl acetate (2,13-18:OAc)
are not listed because their chemical shifts were almost
same as those of 3,13-dien-1-ol (3,13-18:OH). Not
only the coupling constants of olefinic protons and the
chemical shifts of allylic protons in Table 1, but also the
chemical shifts of olefinic and allylic carbons in Table 2
confirm the configurations of the double bonds. These
1H NMR data coincided well with those of previous
papers,14) but there are some conflicts with published
13C NMR assignments.14)
It is noteworthy that the elution order of alcohols
is different from that of acetates, viz., E,E!E,Z!
Z,E!Z,Z of 3,13-18:OH and E,E!Z,E!E,Z!Z,Z of
3,13-18:OAc. In the case of 2,13-dienyl compounds, this
order is quite different, viz., E,E!Z,E!E,Z!Z,Z of
2,13-18:OH and Z,E!E,E and Z,Z!E,Z of 2,13-
18:OAc. Dienyl compounds with (E)-double bond(s) at
the 3 and/or 13-positions eluted faster than those with
(Z)-double bond(s), but an opposite effect was observed
for the configuration at the 2-position of 2,13-18:OAc,
as reported for monoenyl compounds.16) When the (E)-
13-double bond changed the configuration, KI values
measured on the DB-23 column were universally
increased by about 20. Conversion of the configuration
at the 2- or 3-position caused different effects on the KI
values depending on a functional group at the terminal
position.
Geometrical isomers represented very similar mass
spectra. Furthermore, it is difficult to differentiate the
mass spectra of 3,13-18:OAc and 2,13-18:OAc. But
3,13-18:OH were easily distinguished from 2,13-18:OH
by examining their mass spectra, particularly the relative
intensity of ½M ꢄ H2Oꢅþ at m=z 248, viz., 1% of E3,Z13-
18:OH and 7% of E2,Z13-18:OH, as shown in Fig. 2.
GC-MS analysis of the synthetic standards
Table 3 shows retention times (tR) and Kovats
retention indices (KI) of the 3,13- and 2,13-dienlyl
compounds on DB-23 and HP-5 capillary columns. Each
3,13-diene eluted faster than the corresponding 2,13-
diene with the same functional group and configuration.
This result coincides with the behaviors of monoenyl
compounds, viz., 3-dodecenyl acetate eluted faster than
2-dodecenyl acetate.16) On the former polar column, four
geometrical isomers of each compound were separable
except for the (Z,Z)- and (E,E)-isomers of 2,13-18:OAc.

512
H. NAKA et al.
Table 3. Retention Times (tR) and Kovats Retention Indices (KI) of Synthetic 3,13- and 2,13-Octadecadienyl Compounds
DB-23 (0.25 mm ID ꢀ 30 m, 0.25 mm)a
HP-5 (0.25 mm ID ꢀ 30 m, 0.25 mm)b
Alcohol
Acetate
Alcohol
Acetate
tR (min)
KI
tR (min)
KI
tR (min)
tR (min)
3,13-Diene
(Z,Z)-Isomer
(E,Z)-Isomer
(Z,E)-Isomer
(E,E)-Isomer
(Z,Z)-Isomer
(E,Z)-Isomer
(Z,E)-Isomer
(E,E)-Isomer
11.39
11.09
11.20
10.91
11.67
11.49
11.46
11.30
2684
2648
2661
2627
2718
2696
2693
2673
11.01
10.91
10.84
10.74
11.02
11.17
10.85
10.99
2639
2627
2618
2606
2640
2658
2619
2636
27.13c
26.98
27.13
26.98
27.40c
27.39
27.39
27.36
29.80c
29.86
29.80
29.85
29.94c
30.21
29.92
30.19
2,13-Diene
aColumn temperature: 100 ꢁC for 2 min, 20 ꢁC/min to 175 ꢁC, and 6 ꢁC/min to 220 ꢁC.
bColumn temperature: 50 ꢁC for 2 min, 10 ꢁC/min to 160 ꢁC, and 4 ꢁC/min to 260 ꢁC.
cKI values of (Z,Z)-isomer are as follows: Z3,Z13-18:OH 2060, Z3,Z13-18:OAc 2182, Z2,Z13-18:OH 2073, Z2,Z13-18:OAc 2197.
100 -
(%)
100 -
(%)
67
55
81
(A) E3,Z13-18:OH
(B) E3,Z13-18:OAc
55
81
95
67
M+
266
194
95
50 -
50 -
222
109
121
208
248
109
205
M+
308
219
248
250
191
121
×10
-
-
0
0
100
150
200
250
248
100
150
200
300
m/z
m/z
100-
(%)
100-
(%)
(C) E2,Z13-18:OH
(D) E2,Z13-18:OAc
55
55
67
81
95
81
67
95
50 -
205
50 -
M+
266
219
109
121
191
200
109
121
M+
308
×10
205
219
248
191
200
-
-
0
0
100
150
250
100
150
250
300
m/z
m/z
Fig. 2. Mass Spectra of (A) E3,Z13-18:OH, (B) E3,Z13-18:OAc, (C) E2,Z13-18:OH, and (D) E2,Z13-18:OAc.
Dehydroxylation, which creates this fragment ion, was
probably accelerated by migration of the double bond at
the 2-position. In addition to the different intensity of
½M ꢄ H2Oꢅþ, fragment ions at m=z 194, 208, and 222
are characteristic of 3,13-18:OH, and those at m=z 191,
205, and 219 are characteristic of 2,13-18:OH.
2684 (tR 11.84 min). These values are almost the same
as those for synthetic E3,Z13-18:OH and Z3,Z13-18:OH
respectively. Both of the EAG-active components were
eluted with 10% diethyl ether in hexane from a Florisil
column indicating their alcoholic functionality. The
authentic alcohols were recovered in a 10% diethyl ether
fraction, while the acetate derivatives were eluted with
5% diethyl ether in hexane, a less polar solvent. Their
chemical structures were further confirmed by GC-MS
analysis of the crude pheromone extract. Their spectra
coincided more closely with that of 3,13-18:OH, and the
values of tR revealed their double-bond configuration
again. The other eight GC-MS injections (1 FE each)
showed their average titers in the pheromone glands
Identification of pheromone components from Nokona
pernix
Figure 3 shows the result of GC-EAD analysis of the
crude extract (0.5 FE) on a DB-23 capillary column. The
male antennae of N. pernix reproducibly responded to
two components (I and II) with the following KI values:
component I, 2649 (tR 11.55 min), and component II,

Pheromonal Octadecadienyl Compounds of a Clearwing Moth
513
Table 4. Attraction of N. pernix Males by Traps Baited with Two
Synthetic Pheromone Components, (3E,13Z)- and (3Z,13Z)-Isomers of
3,13-Octadecadien-1-ola
Comp. I
Comp. II
Captured males/trapb
Lure (mg/septum)
(B) EAD
(mean ꢂ SD)
E3,Z13-18:OH
Z3,Z13-18:OH
Test I (May 29 to June 15, 2004)
1.00
0.90
0.50
0.10
0
0
0:5 ꢂ 0:5
49:7 ꢂ 41:9
2:7 ꢂ 1:6
0:0 ꢂ 0:0
0:0 ꢂ 0:0
0:0 ꢂ 0:0
c
a
b
c
c
c
0.10
0.50
0.90
1.00
0
0
Test II (June 15 to July 12, 2004)
1.00
0.99
0.95
0.90
0.70
0.50
0
0
0:0 ꢂ 0:0
0:0 ꢂ 0:0
8:7 ꢂ 6:5
20:7 ꢂ 13:4
7:7 ꢂ 3:8
1:3 ꢂ 1:5
0:0 ꢂ 0:0
b
b
a
a
a
b
b
0.01
0.05
0.10
0.30
0.50
0
(A) FID
5.0
0.0
10.0
tR (min)
aTested at the Experimental Farm of Meijo University (Kasugai City, Japan).
bMean number of six traps (Test I) and three traps (Test II). Values followed
by a different letter are significantly different at the 5% level by the Tukey–
Kramer test.
Fig. 3. GC Analysis (DB-23 Column) of a Crude Pheromone Extract
of N. pernix (0.5 FE) with (A) a Flame Ionization Detector (FID)
and (B) an Electroantennographic Detector (EAD).
The male antennae responded to two components, I (E3,Z13-
18:OH, KI 2649, tR 11.55 min), and II (Z3,Z13-18:OH, KI 2684, tR
11.84 min).
component caught males, no males were attracted to the
lures including the minor natural-type component
(Z3,Z13-18:OH) and another unnatural-type isomer as
a major component, as shown in Table 5.
to be 12:3 ꢂ 10:8 ng/female for E3,Z13-18:OH and
1:4 ꢂ 1:3 ng/female for Z3,Z13-18:OH. While large
individual variation was observed for the total amounts
of the two components, their mixing ratios were
approximately uniformed at 9:1. In these pheromone
extracts, no acetate components were detected.
Discussion
Since pheromone content in a female moth is very
low, synthetic standards have played an important role
in the analysis of natural components, particularly for
the determination of the positions and configurations of
double bonds.17) This study confirms that full sets of the
standards, including some commercialized compounds
and others synthesized stereospecifically by the method
shown in Fig. 1, are also meaningful for the identifica-
tion of sex pheromones in sesiid moths. Four geo-
metrical isomers of each C18 3,13- or 2,13-dienyl
compound (acetate and alcohol), whose structures were
confirmed by NMR measurements (Tables 1 and 2),
were separable on a polar GC column such as DB-23
(Table 3). Co-chromatography with the standards can
estimate configurations of the double bonds in pher-
omone components, if the unsaturated positions are
determined. The unsaturated position in monoene com-
pounds can easily be determined by characteristic
fragment ions derived from an adduct with dimethyl
disulfide (DMDS),18) and furthermore it has been
reported that mass spectra of DMDS adducts of dieneyl
compounds also inform the positions of two double
bonds.19) 3,13-18:OAc and 2,13-18:OAc, however,
dominantly produced a mono-DMDS adduct at the
common 13-position, and our trial did not succeed in
recording the spectrum of a di-DMDS adduct with
characteristic ions reflecting the double bond at the 3- or
Field attraction of Nokona pernix males
The attraction of the N. pernix males to various
mixtures of synthetic E3,Z13-18:OH and Z3,Z13-18:OH
was tested in Aichi Prefecture, Japan, where 695 males
in total were captured with pheromone lures in 2004.
Table 4 shows the results of Test I (May 29 to June 15)
and Test II (June 15 to July 12) in Kasugai City. In both
tests, 9:1 was the optimum ratio among the examined
combinations of E3,Z13-18:OH and Z3,Z13-18:OH.
Single E3,Z13-18:OH proved slightly attractive, where-
as single Z3,Z13-18:OH attracted no males. The season-
al trend of male attraction is shown in Fig. 4. In that
year, male flights started on May 29, and the number of
captured males increased up to the middle of June. The
numbers peaked on June 9 at Nagoya (16.0 males/day),
June 14 in Kasugai City (51.0 males/day), and June 17
in Seto City (8.0 males/day). The first flight season
ended in early July. In Kasugai City, a small second
peak was observed in the middle of August. The activity
of the geometrical isomers was examined in Kasugai
City (August 15 to September 19, 2005). While the lures
including the main natural-type component (E3,Z13-
18:OH) and another unnatural-type isomer as a minor

514
H. NAKA et al.
60
40
20
0
(A) Kasugai City
1
10
20
1
10
20
1
10
20
1
June
June
July
July
August
20
(B) Nagoya
0
1
1
10
10
20
20
1
1
10
10
20
30
30
20
(C) Seto City
0
20
June
July
Fig. 4. Numbers of N. pernix Males Captured by Pheromone Traps per Day in Three Fields.
Tests were conducted at three different fields in Aichi Prefecture in 2004.
Table 5. Attraction of N. pernix Males by Traps Baited with a Synthetic Lure Including Two Geometrical Isomers of 3,13-Octadecadien-1-ola
Captured males/trapb
Lure (mg/septum)
(mean ꢂ SD)
E3,Z13-18:OH
Z3,Z13-18:OH
E3,E13-18:OH
Z3,E13-18:OH
0.90
0.90
0.90
0
0.10
0
0
0
4:7 ꢂ 1:5
2:0 ꢂ 2:6
1:3 ꢂ 1:2
0:0 ꢂ 0:0
0:0 ꢂ 0:0
0:0 ꢂ 0:0
a
0.10
0
0
ab
ab
b
0
0.10
0
0.10
0.10
0
0.90
0
0
0.90
0
b
0
0
b
aTested at the Experimental Farm of Meijo University in Kasugai City, Japan from August 15 to September 19, 2005. E3,Z13-18:OH and Z3,Z13-18:OH are natural-
type isomers and E3,E13-18:OH and Z3,E13-18:OH are unnatural-type isomers.
bMean number of three traps followed by a different letter are significantly different at the 5% level by the Tukey–Kramer test.
2-position. Fortunately, 3,13-18:OH and 2,13-18:OH
showed different mass spectra without any derivatiza-
tion; thus ½M ꢄ H2Oꢅþ at m=z 248 was detected
abundantly in that of the latter alcohol (Fig. 2). This
result means that differentiation of their acetates can be
accomplished by GC-MS measurement after hydrolysis.
Based on these results, pheromone components of a
clearwing moth, Nokona pernix, were successfully
clarified. On GC-EAD analysis, two distinct EAG-active
components (I and II) were found in a crude extract of
the pheromone glands (Fig. 3). Their chemical struc-
tures and a 9:1 mixing ratio in the pheromone extract
were revealed by GC-MS measurements, which showed
identical data to those of authentic E3,Z13-18:OH
(Component I) and Z3,Z13-18:OH (Component II).
Filed evaluations showed that the male moths were
optimally attracted to these synthetic compounds when
mixed in the same ratio as detected in the pheromone
extracts (Tables 4 and 5). E3,Z13-18:OH and Z3,Z13-
18:OH have been identified from sesiid females of two
and four other species respectively. Furthermore, these
alcohols were composed of attractants of 36 sesiid
species, indicating their ordinariness in the mating
communication systems of this insect group. But it is
noteworthy that N. pernix is the first species secreting
these two alcohols as pheromone components without
any acetates.5–7) Taxonomists have demonstrated a close
relationship between the genera Nokona and Para-
nthrene in the family of Sesiidae.20–22) These alcohols
have been identified from some species in Paranthrene,
and the pheromone of N. pernix supports the taxonom-
ical results.

Pheromonal Octadecadienyl Compounds of a Clearwing Moth
515
extracts, and synthetic sex attractants. Environ. Ento-
mol., 4, 451–454 (1975).
4) Schwarz, M., Klun, J. A., Leonhardt, B. A., and Johnson,
D. T., (E,Z)-2,13-Octadecadien-1-ol acetate: a new
pheromone structure for sesiid moths. Tetrahedron Lett.,
24, 1007–1010 (1983).
5) Ando, T., Internet database (2005), http://www.tuat.
ac.jp/~antetsu/LepiPheroList.htm
6) El-Sayed, A. M., Internet database (2005), http://www.
pherobase.com/
In spite of the large number of species in the family of
Sesiidae, the structural diversity known to date for
pheromonal compounds is limited; almost all of them
have the Z,Z or E,Z configuration in the 3,13-18 or 2,13-
18 skeleton.5–7) This identification with N. pernix pro-
posed an additional example. No compounds unsaturat-
ed at the 13-position with an E configuration have been
found from sesiid species. Owing to visual cues,
diversity of chemical cues might be not necessary for
the mating communication of diurnal insects. It would
be interesting to determine whether sesiid species have
developed an enzyme to introduce the (E)-13-double
bond, and hence we intend to utilize the synthetic
compounds with a Z,E or E,E configuration in random
screening tests to find new attractors.
Many Sesiid species inhabiting the temperate regions
are single-brooded, and adults of N. pernix usually
appear once a year in June. In a warm year, adults have
been found in September, too.8) Although it has not been
confirmed that this species is fully double-brooded,
since the numbers encountered in September were very
low, field monitoring by pheromone traps has indicated
that it might be partially double-brooded (Fig. 4). The
mean temperature from March to May in 2004 in the
Nagoya region, including our three fields, was 1.3 ꢁC
higher than in normal years. This warmer weather might
have resulted in their earlier emergence, leading in turn
to a small second brood. The pheromone traps brought
an ecological finding. Furthermore, we are examining
mating behavior with several kinds of dummies of
N. pernix impregnated with the synthetic pheromone to
analyze recognition ability of visual cues.
´
7) Witzgall, P., Lindblom, T., Bengtsson, M., and Toth, M.,
Internet database (2004), http://www-pherolist.slu.se/
cgi-bin-pherolist/pherolist.cgi
8) Arita, Y., and Ikeda, M., ‘‘Sesiidae of Japan’’ (in
Japanese), Mushi-sha, Tokyo, p. 203 (2000).
9) Yaginuma, K., Kumakura, M., Tamaki, Y., Yushima, T.,
and Tumlinson, J. H., Sex attractant for the cherry
tree borer, Synanthedon hector Butler (Lepidoptera:
Sesiidae). Appl. Entomol. Zool., 11, 266–268 (1976).
10) Tamaki, Y., Yushima, T., Oda, M., Kida, K., Kitamura,
K., Yabuki, S., and Tumlinson, J. H., Attractiveness of
3,13-octadecadienyl acetates for males of clearwing
moths. Jap. J. Appl. Entomol. Zool. (in Japanese), 21,
106–107 (1977).
11) Ogawa, K., The application of insect sex pheromones
for pest control. Kagaku To Seibutsu (in Japanese), 28,
295–303 (1980).
12) Struble, D. L., and Arn, H., Combined gas chromatog-
raphy and electroantennogram recording of insect
olfactory responses. In ‘‘Techniques in Pheromone
Research,’’ eds. Hummel, H. E., and Miller, T. A.,
Springer-Verlag, New York, pp. 161–178 (1984).
13) Perry, J. N., Wall, C., and Greenway, A. R., Latin-square
designs in field experiments involving insect sex-
attractants. Ecol. Entomol., 5, 385–396 (1980).
14) Hoskovec, M., Saman, D., and Koutek, B., A convenient
synthesis of 2,13- and 3,13-octadecadienyl acetates, sex
pheromone components of the Synanthedon species.
Collect. Czech. Chem. Commun., 55, 2270–2281 (1990).
(Further references are cited here.)
Acknowledgments
We thank Mr. D. Wakatsuki and Ms. Y. Katayama at
Meijo University and Mr. H. Yamazaki at Kagoshima
University, who kindly helped prepare cocoons of
N. pernix, and Dr. F. Mochizuki at Shin-etsu Chemical
Co., Ltd., Dr. A. Mochizuki at NIAES, Mr. K. Fukuzumi
(in Nagoya) and Mr. H. Shibata of Kyoto University,
who provided us with important information. This work
was supported in part by research funds from Heart
Co., Ltd. (Tokyo).
ˆ
15) Buda, V., Maeorg, U., Karalius, V., Rothschild, G. H. L.,
Kolonistova, S., Ivinskis, P., and Mozuraitis, R., C18
¨
ˆ
Dienes as attractants for eighteen clearwing (Sesiidae),
tineid (Tineidae), and choreutid (Choreutidae) moth
species. J. Chem. Ecol., 19, 799–813 (1993).
16) Nesbitt, B. F., Beevor, P. S., Cork, A., Hall, D. R.,
David, H., and Vandagopal, V., The female sex
pheromone of sugarcane stalk borer, Chilo auricilius:
identification of four components and field tests. J.
Chem. Ecol., 12, 1377–1388 (1986).
17) Ando, T., Inomata, S., and Yamamoto, M., Lepidopteran
sex pheromones. Topics Current Chem., 239, 51–96
(2004).
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