Journal of Chemical Ecology, Vol. 19, No. 4, 1993
HYDROCARBONS WITH A HOMOCONJUGATED POLYENE SYSTEM AND THEIR MONOEPOXY DERIVATIVES: SEX ATTRACTANTS OF GEOMETRID AND NOCTUID MOTHS DISTRIBUTED IN JAPAN
TETSU HIDEKI
ANDO, l'* HIROKAZU KISHI, 1 YUTAKA
OHSAWA,
OKAMURA,
1'3 T A D A H I R O
l and SATOSHI
UENO, 1
HASHIMOTO
2
1Department of Applied Biological Science, Faculty of Agriculture Tokyo University of Agriculture and Technology Fuchu, Tokyo 183, Japan 2Department of Zoology Natural History Museum and Institute, Chiba 955-2 Aoba-cho, Chuo-ku, Chiba 260, Japan (Received August 31, 1992; accepted December 1, 1992)
Abstract--Although several sex pheromones of the family Geometridae have been characterized, investigations on Japanese species are limited. In order to obtain more information, screening using known sex pheromones and their analogs was carried out. The (Z,Z,Z)-3,6,9-triunsaturated and (Z,Z)-6,9-diunsaturated hydrocarbons with straight C~9-C~ chains were synthesized by the Grignard coupling reaction as a key step starting from linolenic and linoleic acids, respectively. Oxidation of the homoconjugated trienes with m-chloroperoxybenzoic acid yielded a 1 : 1 : 1 mixture of three monoepoxy derivatives that could be separated by silica gel chromatography. The chemical structure of each positional isomer was confirmed using two-dimensional NMR techniques and MS measurements, which enabled characteristic fragment ions from the isomers to be identified. Field tests using lures incorporating only one of the above six hydrocarbons or nine epoxides were carried out in a forest in Tokyo. Consequently, attraction of male moths of 14 geometrid species in addition to four species in another family, the Noctuidae, was observed. It was concluded that hydrocarbons with a homoconjugated polyene system and the monoepoxy derivatives are important components of sex pheromones produced by Japanese lepidopterous insects, particularly the geometrid moths. *To whom correspondence should be addressed. 3present address: Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-33, Japan 787 0098-0331/93/0400-0787507.00/0 • 1993 Plenum Publishing Corporation
788
A N D O ET AL.
Key Words--Sex pheromone, lepidopterous attractant, field test, unsaturated hydrocarbon, epoxydiene, Geometridae, Noctuidae.
INTRODUCTION
Lepidopterous sex pheromones have been identified from females of more than 300 species since the first investigation on bombykol was carried out. Analyses of their chemical structures (Am et al., 1986; Ando, personal data base), showed that ca. 85 % of them contained linear aliphatic alcohols, aldehydes, or acetates, with a Cm-C~ 8 straight chain and one or two olefinic linkages. These types of compounds with a terminal functional group, which have been identified from 16 families of Lepidoptera, are among the most ubiquitous pheromone components. Another type of pheromone component lacking in a functional group at the terminal position was identified in the last decade, as shown in Table 1 (Ando, personal data base). To date, hydrocarbons with a homoconjugated triene or diene system and their monoepoxy derivatives have been identified from 38 species mainly in the families of Geometridae and Arctiidae. Pheromone components with the above terminal functional group, however, have not been identified from female moths in these two families. Furthermore, screening tests using these types of synthetic pheromones and the analogs revealed the attraction
TABLE 1. NUMBEROF LEPIDOPTEROUSSPECIES, SEX PHEROMONESOR ATTRACTANTS REPORTED TO BE HYDROCARBONSWITH A POLYENE SYSTEM, AND/OR THEIR MONOEPOXY DERIVATIVES
Number of species Superfamily Family Geometroidea Geometridae
Noctuoidea Arctiidae Ctenuchidae Lymantriidae Noctuidae
Total
Subfamily
Ennominae Larentiinae Oenochrominae
Catocalinae Herminiinae Hypeninae Ophiderinae
Sex pheromone
Attractant
16 4 1
20 10 1
9 2
0 0
1
0
4 0 0
3 3 3
1
1
38
41
789
POLYENE HYDROCARBONS
of 41 lepidopterous species in Canada and Europe (Millar et al., 1990b; and references therein). Although some of them are distributed in Japan, information about sexual communication utilizing the unsaturated hydrocarbons and epoxides is very limited. Therefore attempts were made to synthesize these types of compounds and to examine their field attractancy against Japanese Lepidoptera. METHODS AND MATERIALS
Synthesis of 3,6,9-Trienes and 6,9-Dienes. Three homoconjugated trienes (Z3,Z6,Z9-19 :H, Z3,Z6,Z9-20: H, and Z3,Z6,Z9-21 : H) 4 and three dienes (Z6,Z9-19:H, Z6,Z9-20: H, and Z6,Z9-21 :H) were synthesized by a modification of the method described by Conner et al. (1980) and Underhill et al. (1983). A mixture of linolenic and linoleic acids in a ratio of ca. 3:1 (25 g, 90 mmol), purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), was esterified with ethanol and reduced to alcohols with LiA1H4 in dry ether. After silica gel column chromatography, this alcohol mixture was treated with p-toluenesulfonyl chloride in pyridine to yield the tosylates (27 g, 72% yield from the acids). A portion of the tosylates (5.0 g, 12 mmol) was supplied for Grignard coupling with methylmagnesium bromide (16.7 ml, 15.9 mmol) [0.95 M tetrahydrofuran (THF) solution, Kanto Chemical Co., Inc., Tokyo, Japan] in dry THF (100 ml) under the mediation of Li2CuC14 (Fouquet et al., 1974) (1 ml) (0.1 M THF solution, Aldrich Chemical Co., Inc., Milwaukee, Wisconsin) to prepare a mixture of Z3,Z6,Z9-19:H, and Z6,Z9-19:H. The C~9 triene (1.5 g, 48% yield from the tosylates) and diene (0.6 g, 19% yield) were separated by a silica gel column impregnated with 20% AgNO3 using a n-hexanebenzene solvent system. The C20 and C~ analogs were synthesized in the same manner using ethylmagnesium bromide (0.93 M THF solution, Kanto Chemical Co.) and n-propylmagnesium bromide prepared from n-propyl bromide, respectively. Synthesis of Monoepoxy Derivatives. m-Chloroperoxybenzoic acid (Tokyo Kasei Kogyo Co., 70% pure, 490 mg, 2.0 mmol) was added to a solution of Z3,Z6,Z9-19 : H (470 mg, 1.8 mmol) in dry CH2C12 (30 ml) and stirred at 0~ for 1 hr. After further stirring at room temperature for 2 hr, the reaction mixture was washed with a saturated aqueous solution of NaHCO 3 (20 ml • 2) and dried with Na2SO4. Silica gel column chromatography gave a mixed product of the three cis-monoepoxy derivatives (240 mg, 48% yield) in a ratio of ca. 1 : 1 : 1. Since the epoxydiene mixture showed three separate spots on a silica gel TLC plate (60 F254, Merck, Darmstadt, Germany) [solvent system: n-hexane-benzene (1:1), Rf values: epo3,Z6,Z9-19:H, 0.34; Z3,epo6,Z9-19:H, 0.43; and 4Compounds were abbreviated as follows: Z3,Z6,Z9-19:H is (Z,Z,Z)-3,6,9-nonadecatriene, epo3,Z6,Z9-19: H is (Z,Z)-6,9-cis-3,4-epoxynonadecadiene.
790
A N D O ET AL.
Z3,Z6,epo9-19:H, 0.40], 4 each pure sample (a racemic mixture) was obtained by preparative TLC purification. This chromatographic behavior corresponded to that of C~7 epoxydienes in a normal phase HPLC (Millar et al., 1987). In the same manner, the C20 and C2~ epoxydienes were prepared and purified. Spectroscopy. NMR spectra of each compound in CDC13 were analyzed with a JEOL GX 270 Fourier transform spectrometer (270.2 MHz for ~H and 67.9 MHz for J3C) using TMS as an internal standard. Signal assignments were made by two-dimensional techniques (1H-~H and IH-~3C COSY spectra) using ordinal pulse sequences (Ando et al., 1988). Electron-impact (EI) GC-MS was achieved using a JEOL JNM DX-300 mass spectrometer with a OV-1 capillary column (0.25 mm ID x 25 m, Gasukuro Kogyo Inc., Tokyo, Japan). Ionization voltage of every measurement was 70 eV and ion source temperature was 240~ Field Evaluation. Each chemical at 1 mg with purity >95% by GC (i.e., TIC trace from GC-MS) was applied to a white rubber septum (8 mm OD, Aldrich Chemical Co., Ltd.), which was placed in a sticky-type trap (30 • 27 cm bottom plate with a roof, Takeda Chemical Ind., Ltd., Osaka, Japan). A parallel experiment was conducted for each chemical, and the traps were set at a 1.5-m height from the ground. The screening test was carried out from August 1991 to July 1992 in a mixed forest area in the suburbs of Tokyo (Rolling Land Laboratory, Tokyo University of Agriculture and Technology, Hachiohji-shi, Tokyo), and species, sex, and number of moths caught were recorded every two weeks. Each lure was renewed every two months.
RESULTS AND DISCUSSION
Characterization of Synthetic Compounds. The ~H NMR and MS spectroscopic data of the synthetic hydrocarbons with a homoconjugated triene and diene system corresponded well to those previously reported (Comer et al., 1980; Underhill et al., 1983). The number of C = C double bonds and the carbon length were determined by integration of their olefinic 1H signals and molecular ions, respectively. The 13C NMR spectra of the unsaturated hydrocarbons were very similar to those of the parent fatty acids. Signals from C-1 to C-15 of the hydrocarbons corresponded to the signals from C-18 to C-4 of the acids. These 13C signals were assigned based on 1H-~3C COSY experiments (see Table 2 for C2~ diene and triene). Allylic carbon signals (C-5, C-8 and C-11 of dienes, and C-2 of trienes additionally) resonated in a higher field than that calculated for the geometrical isomers (Rossi and Veracini, 1982), indicating all Z configurations in the hydrocarbons and the parent fatty acids. Tables 2 and 3 indicate the 1H and 13C NMR assignments for three C2j cis-epoxydienes in addition to Cz~ diene and triene. The NMR spectra of the C19 and C2o analogs were almost the same as those of the C2~ compounds except
epo3,Z6,Z9-21 :H Z3,epo6,Z9-21 :H Z3,Z6,epo9-21 :H
Z3,Z6,Z9-21 :H
Z6,Z9-21 :H
Compound
0.89 0.98 1.06 0.99 0.97
H-1
2.08 - 1.6 2.07 2.08
H-2
1.2-1.4 -5.35 2.90 -5.5 -5.4
H-3
-5.35 2.96 -5.4 -5.35
H-4 2.05 2.81 2.22, 2 . 4 1 2.22, 2.42 2.81
H-5 -5.35 -5.35 -5.45 2.95 -5.5
H-6 -5.35 -5.35 -5.5 2.95 -5.45
H-7 2.78 2.81 2.81 2.22, 2.42 2.22, 2.40
H-8
Chemical shift (ppm)
-5.35 -5.35 -5.3 -5.4 2.95
H-9
-5.35 -5.35 -5.35 -5.5 2.93
H-10
2.05 2.08 2.04 2.07 - 1.5
H-11
1.2-1.4 1.2-1.4 1.2-1.4 1.2-1.4 1.2-1.5
H-12-H-20
TABLE 2. ~H N M R ASSIGNMENTS FOR HENEICOSADIENE ( Z 6 , Z 9 - 2 1 : H ) , HENEICOSATRIENE (Z3,Z6,Z9-21 :H), AND EPOXYHENEICOSADIENE ( e p o 3 , Z 6 , Z 9 - 2 1 : H , Z 3 , e p o 6 , Z 9 - 2 1 : H , and Z 3 , Z 6 , e p o 9 - 2 1 :H)
0.88 0.88 0.88 0.88 0.88
H-21
z
O
>
m ,
HYDROCARBONS WITH A HOMOCONJUGATED POLYENE SYSTEM AND THEIR MONOEPOXY DERIVATIVES: SEX ATTRACTANTS OF GEOMETRID AND NOCTUID MOTHS DISTRIBUTED IN JAPAN
TETSU HIDEKI
ANDO, l'* HIROKAZU KISHI, 1 YUTAKA
OHSAWA,
OKAMURA,
1'3 T A D A H I R O
l and SATOSHI
UENO, 1
HASHIMOTO
2
1Department of Applied Biological Science, Faculty of Agriculture Tokyo University of Agriculture and Technology Fuchu, Tokyo 183, Japan 2Department of Zoology Natural History Museum and Institute, Chiba 955-2 Aoba-cho, Chuo-ku, Chiba 260, Japan (Received August 31, 1992; accepted December 1, 1992)
Abstract--Although several sex pheromones of the family Geometridae have been characterized, investigations on Japanese species are limited. In order to obtain more information, screening using known sex pheromones and their analogs was carried out. The (Z,Z,Z)-3,6,9-triunsaturated and (Z,Z)-6,9-diunsaturated hydrocarbons with straight C~9-C~ chains were synthesized by the Grignard coupling reaction as a key step starting from linolenic and linoleic acids, respectively. Oxidation of the homoconjugated trienes with m-chloroperoxybenzoic acid yielded a 1 : 1 : 1 mixture of three monoepoxy derivatives that could be separated by silica gel chromatography. The chemical structure of each positional isomer was confirmed using two-dimensional NMR techniques and MS measurements, which enabled characteristic fragment ions from the isomers to be identified. Field tests using lures incorporating only one of the above six hydrocarbons or nine epoxides were carried out in a forest in Tokyo. Consequently, attraction of male moths of 14 geometrid species in addition to four species in another family, the Noctuidae, was observed. It was concluded that hydrocarbons with a homoconjugated polyene system and the monoepoxy derivatives are important components of sex pheromones produced by Japanese lepidopterous insects, particularly the geometrid moths. *To whom correspondence should be addressed. 3present address: Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-33, Japan 787 0098-0331/93/0400-0787507.00/0 • 1993 Plenum Publishing Corporation
788
A N D O ET AL.
Key Words--Sex pheromone, lepidopterous attractant, field test, unsaturated hydrocarbon, epoxydiene, Geometridae, Noctuidae.
INTRODUCTION
Lepidopterous sex pheromones have been identified from females of more than 300 species since the first investigation on bombykol was carried out. Analyses of their chemical structures (Am et al., 1986; Ando, personal data base), showed that ca. 85 % of them contained linear aliphatic alcohols, aldehydes, or acetates, with a Cm-C~ 8 straight chain and one or two olefinic linkages. These types of compounds with a terminal functional group, which have been identified from 16 families of Lepidoptera, are among the most ubiquitous pheromone components. Another type of pheromone component lacking in a functional group at the terminal position was identified in the last decade, as shown in Table 1 (Ando, personal data base). To date, hydrocarbons with a homoconjugated triene or diene system and their monoepoxy derivatives have been identified from 38 species mainly in the families of Geometridae and Arctiidae. Pheromone components with the above terminal functional group, however, have not been identified from female moths in these two families. Furthermore, screening tests using these types of synthetic pheromones and the analogs revealed the attraction
TABLE 1. NUMBEROF LEPIDOPTEROUSSPECIES, SEX PHEROMONESOR ATTRACTANTS REPORTED TO BE HYDROCARBONSWITH A POLYENE SYSTEM, AND/OR THEIR MONOEPOXY DERIVATIVES
Number of species Superfamily Family Geometroidea Geometridae
Noctuoidea Arctiidae Ctenuchidae Lymantriidae Noctuidae
Total
Subfamily
Ennominae Larentiinae Oenochrominae
Catocalinae Herminiinae Hypeninae Ophiderinae
Sex pheromone
Attractant
16 4 1
20 10 1
9 2
0 0
1
0
4 0 0
3 3 3
1
1
38
41
789
POLYENE HYDROCARBONS
of 41 lepidopterous species in Canada and Europe (Millar et al., 1990b; and references therein). Although some of them are distributed in Japan, information about sexual communication utilizing the unsaturated hydrocarbons and epoxides is very limited. Therefore attempts were made to synthesize these types of compounds and to examine their field attractancy against Japanese Lepidoptera. METHODS AND MATERIALS
Synthesis of 3,6,9-Trienes and 6,9-Dienes. Three homoconjugated trienes (Z3,Z6,Z9-19 :H, Z3,Z6,Z9-20: H, and Z3,Z6,Z9-21 : H) 4 and three dienes (Z6,Z9-19:H, Z6,Z9-20: H, and Z6,Z9-21 :H) were synthesized by a modification of the method described by Conner et al. (1980) and Underhill et al. (1983). A mixture of linolenic and linoleic acids in a ratio of ca. 3:1 (25 g, 90 mmol), purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), was esterified with ethanol and reduced to alcohols with LiA1H4 in dry ether. After silica gel column chromatography, this alcohol mixture was treated with p-toluenesulfonyl chloride in pyridine to yield the tosylates (27 g, 72% yield from the acids). A portion of the tosylates (5.0 g, 12 mmol) was supplied for Grignard coupling with methylmagnesium bromide (16.7 ml, 15.9 mmol) [0.95 M tetrahydrofuran (THF) solution, Kanto Chemical Co., Inc., Tokyo, Japan] in dry THF (100 ml) under the mediation of Li2CuC14 (Fouquet et al., 1974) (1 ml) (0.1 M THF solution, Aldrich Chemical Co., Inc., Milwaukee, Wisconsin) to prepare a mixture of Z3,Z6,Z9-19:H, and Z6,Z9-19:H. The C~9 triene (1.5 g, 48% yield from the tosylates) and diene (0.6 g, 19% yield) were separated by a silica gel column impregnated with 20% AgNO3 using a n-hexanebenzene solvent system. The C20 and C~ analogs were synthesized in the same manner using ethylmagnesium bromide (0.93 M THF solution, Kanto Chemical Co.) and n-propylmagnesium bromide prepared from n-propyl bromide, respectively. Synthesis of Monoepoxy Derivatives. m-Chloroperoxybenzoic acid (Tokyo Kasei Kogyo Co., 70% pure, 490 mg, 2.0 mmol) was added to a solution of Z3,Z6,Z9-19 : H (470 mg, 1.8 mmol) in dry CH2C12 (30 ml) and stirred at 0~ for 1 hr. After further stirring at room temperature for 2 hr, the reaction mixture was washed with a saturated aqueous solution of NaHCO 3 (20 ml • 2) and dried with Na2SO4. Silica gel column chromatography gave a mixed product of the three cis-monoepoxy derivatives (240 mg, 48% yield) in a ratio of ca. 1 : 1 : 1. Since the epoxydiene mixture showed three separate spots on a silica gel TLC plate (60 F254, Merck, Darmstadt, Germany) [solvent system: n-hexane-benzene (1:1), Rf values: epo3,Z6,Z9-19:H, 0.34; Z3,epo6,Z9-19:H, 0.43; and 4Compounds were abbreviated as follows: Z3,Z6,Z9-19:H is (Z,Z,Z)-3,6,9-nonadecatriene, epo3,Z6,Z9-19: H is (Z,Z)-6,9-cis-3,4-epoxynonadecadiene.
790
A N D O ET AL.
Z3,Z6,epo9-19:H, 0.40], 4 each pure sample (a racemic mixture) was obtained by preparative TLC purification. This chromatographic behavior corresponded to that of C~7 epoxydienes in a normal phase HPLC (Millar et al., 1987). In the same manner, the C20 and C2~ epoxydienes were prepared and purified. Spectroscopy. NMR spectra of each compound in CDC13 were analyzed with a JEOL GX 270 Fourier transform spectrometer (270.2 MHz for ~H and 67.9 MHz for J3C) using TMS as an internal standard. Signal assignments were made by two-dimensional techniques (1H-~H and IH-~3C COSY spectra) using ordinal pulse sequences (Ando et al., 1988). Electron-impact (EI) GC-MS was achieved using a JEOL JNM DX-300 mass spectrometer with a OV-1 capillary column (0.25 mm ID x 25 m, Gasukuro Kogyo Inc., Tokyo, Japan). Ionization voltage of every measurement was 70 eV and ion source temperature was 240~ Field Evaluation. Each chemical at 1 mg with purity >95% by GC (i.e., TIC trace from GC-MS) was applied to a white rubber septum (8 mm OD, Aldrich Chemical Co., Ltd.), which was placed in a sticky-type trap (30 • 27 cm bottom plate with a roof, Takeda Chemical Ind., Ltd., Osaka, Japan). A parallel experiment was conducted for each chemical, and the traps were set at a 1.5-m height from the ground. The screening test was carried out from August 1991 to July 1992 in a mixed forest area in the suburbs of Tokyo (Rolling Land Laboratory, Tokyo University of Agriculture and Technology, Hachiohji-shi, Tokyo), and species, sex, and number of moths caught were recorded every two weeks. Each lure was renewed every two months.
RESULTS AND DISCUSSION
Characterization of Synthetic Compounds. The ~H NMR and MS spectroscopic data of the synthetic hydrocarbons with a homoconjugated triene and diene system corresponded well to those previously reported (Comer et al., 1980; Underhill et al., 1983). The number of C = C double bonds and the carbon length were determined by integration of their olefinic 1H signals and molecular ions, respectively. The 13C NMR spectra of the unsaturated hydrocarbons were very similar to those of the parent fatty acids. Signals from C-1 to C-15 of the hydrocarbons corresponded to the signals from C-18 to C-4 of the acids. These 13C signals were assigned based on 1H-~3C COSY experiments (see Table 2 for C2~ diene and triene). Allylic carbon signals (C-5, C-8 and C-11 of dienes, and C-2 of trienes additionally) resonated in a higher field than that calculated for the geometrical isomers (Rossi and Veracini, 1982), indicating all Z configurations in the hydrocarbons and the parent fatty acids. Tables 2 and 3 indicate the 1H and 13C NMR assignments for three C2j cis-epoxydienes in addition to Cz~ diene and triene. The NMR spectra of the C19 and C2o analogs were almost the same as those of the C2~ compounds except
epo3,Z6,Z9-21 :H Z3,epo6,Z9-21 :H Z3,Z6,epo9-21 :H
Z3,Z6,Z9-21 :H
Z6,Z9-21 :H
Compound
0.89 0.98 1.06 0.99 0.97
H-1
2.08 - 1.6 2.07 2.08
H-2
1.2-1.4 -5.35 2.90 -5.5 -5.4
H-3
-5.35 2.96 -5.4 -5.35
H-4 2.05 2.81 2.22, 2 . 4 1 2.22, 2.42 2.81
H-5 -5.35 -5.35 -5.45 2.95 -5.5
H-6 -5.35 -5.35 -5.5 2.95 -5.45
H-7 2.78 2.81 2.81 2.22, 2.42 2.22, 2.40
H-8
Chemical shift (ppm)
-5.35 -5.35 -5.3 -5.4 2.95
H-9
-5.35 -5.35 -5.35 -5.5 2.93
H-10
2.05 2.08 2.04 2.07 - 1.5
H-11
1.2-1.4 1.2-1.4 1.2-1.4 1.2-1.4 1.2-1.5
H-12-H-20
TABLE 2. ~H N M R ASSIGNMENTS FOR HENEICOSADIENE ( Z 6 , Z 9 - 2 1 : H ) , HENEICOSATRIENE (Z3,Z6,Z9-21 :H), AND EPOXYHENEICOSADIENE ( e p o 3 , Z 6 , Z 9 - 2 1 : H , Z 3 , e p o 6 , Z 9 - 2 1 : H , and Z 3 , Z 6 , e p o 9 - 2 1 :H)
0.88 0.88 0.88 0.88 0.88
H-21
z
O
>
m ,
