IDENTIFICATION of CHIRAL SEX PHEROMONE SECRETED by GIANT GEOMETRID MOTH, Biston Robustum Butler

Journal of Chemical Ecology, Vol. 26, No. 11, 2000

IDENTIFICATION OF CHIRAL SEX PHEROMONE SECRETED BY GIANT GEOMETRID MOTH, Biston robustum Butler

MASANOBU YAMAMOTO,1 MASAAKI KISO,2,3 HIROYUKI YAMAZAWA,1 JUN TAKEUCHI,2 and TETSU ANDO1,*

1Graduate School of Bio-Applications and Systems Engineering Tokyo University of Agriculture and Technology Koganei, Tokyo 184-8588, Japan 2Agricultural Technology Center Hachijo-jima, Tokyo 100-1401, Japan

(Received November 15, 1999; accepted July 8, 2000)

Abstract—Biston robustum Butler, a polyphagous defoliator, multiplied on Hachijo-jima Island in 1997–1998. Based on GC-MS data of authentic standards, an analysis of a pheromone gland extract of the females indicated that it included (Z,Z)-6,9-nonadecadiene (I), (Z,Z,Z)-3,6,9-nonadecatriene (II), cis-(Z)-6,7-epoxy-9-nonadecene (III), and cis-(Z,Z)-6,7-epoxy-3,9-nonadecadiene (IV) in a ratio of 13 : 2 : 70 : 15. The structure of III was confirmed by a GC-MS analysis of another extract treated with dimethyl disulfide (DMDS). This epoxymonoene was successfully converted into the corresponding DMDS adduct that showed diagnostic ions fragmented at an epoxy ring and at thiomethoxy groups reflecting the position of an original double bond. Furthermore, the 6S,7R configuration was assigned for the epoxy ring of III by chiral HPLC analysis. Field examination of synthetic lures revealed that the two epoxy compounds (III and IV) with the 6S,7R configuration were essential components and that the two unsaturated hydrocarbons (I and II) showed a synergistic effect on male attraction.

Key Words—Female sex pheromones, Lepidoptera, Geometridae, Ennomi- nae, epoxynonadecene, epoxynonadecadiene, chiral epoxide, chiral HPLC, dimethyl disulﬁde adduct.

*To whom correspondence should be addressed. 3 Present address: Tokyo Metropolitan Agricultural Experimental Station, Tachikawa, Tokyo 190- 0013, Japan; e-mail: [email protected]

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0098-0331/ 00/ 1100-2579$18.00/ 0  2000 Plenum Publishing Corporation 2580 YAMAMOTO ET AL.

INTRODUCTION

The larva of the giant geometrid, Biston robustum Butler (Geometridae: Ennomi- nae), is a common polyphagous defoliator, and the adult appears only once in the early spring. In 1997 and 1998, this univoltine species multiplied to an extraor- dinary population level on the Japanese island of Hachijo-jima, which is a busy tourist resort 280 km south of Tokyo. The southern part of the island is covered with broadleaf trees, the Japanese chinquapin, cherry, and others, which were devastated by the unusually high population of larvae. Authorities are eager to develop a technique for monitoring and further controlling the population. There- fore, we have begun to identify the sex pheromone of B. robustum. Female sex pheromones from about 40 species in the family of Geometri- dae have been chemically characterized. These pheromones are predominantly composed of (Z,Z)-6,9-dienes and (Z,Z,Z)-3,6,9-trienes with a C17–C23 straight chain and their monoepoxy derivatives, which lack a terminal functional group (Arn et al., 1992, 1997). The pheromonal epoxymonoenes and epoxydienes are expected to be synthesized from the dienes and trienes in the pheromone gland by oxidation catalyzed with a monooxygenase (Miyamoto et al., 1999). Variation in the epoxy ring position and the carbon chain length causes com- plexity of the geometrid pheromones, playing an important role in reproductive isolation. In this paper, we report the identification of C19 components in the pheromone extract of B. robustum. The determination of the epoxy ring position was achieved by a GC-MS measurement, which proposed some diagnostic fragment ions that had been reported in earlier studies (Ando et al., 1993, 1995). Furthermore, the structure of epoxymonoene was confirmed by a GC-MS analysis of a pheromone extract treated with dimethyl disulfide (DMDS), and the stereochemistry of the epoxy ring was determined by chiral HPLC. The results of the preliminary field test with a synthetic chiral pheromone are also reported.

METHODS AND MATERIALS

GC-MS Analysis. An electron impact GC-MS was achieved by using a Jeol JMS-AM II 20 mass spectrometer equipped with a DB-23 capillary column (0.25 mm ID × 30 m; J&W Scientific) to analyze a pheromone extract, or a DB- 1 capillary column (0.25 mm ID × 30 m; J&W Scientific) to analyze DMDS adducts derived from epoxymonoenes. The oven temperature program for the former column was 808C for 2 min, 108C/ min to 1608C, and finally 48C/ min to 2208C. The program of the latter column was 1208C for 2 min and 68C/ min to 2708C. The ionization voltage was 70 eV, and the ion-source temperature was 2408C. Chromatography. The HPLC involved a Jasco PU-980 liquid chromato- CHIRAL SEX PHEROMONE OF GEOMETRID FEMALES 2581 graph equipped with an integrator (System Instrument Chromatocorder 21J), an RI detector (Labo System RI-98SCOPE), and a Chiralpak AD column (0.46 cm ID × 25 cm; Daicel Chemical Industry Co.) or an ODS column (0.46 cm ID × 25 cm; Senshukagaku). As a mobile phase, 0.1% 2-propanol in n-hexane was used for the former normal-phase chiral column at a flow rate of 0.45 ml/ min, and 5% water in methanol for the latter reversed-phase achiral column at a flow rate of 1.0 ml/ min. GC analysis was achieved by using an HP GC System 6890 series with a flame ionization detector and a DB-23 capillary column (0.25 mm ID × 30 m, J&W Scientific). The temperature program was 508C for 2 min, 108C/ min to 1608C, and finally 48C/ min to 2208C. Insect and Pheromone Extraction. Adult females of B. robustum drawn towards a street lamp were collected at Hachijo-jima Island in the early spring of 1998 and 1999. Two or three days after the collection, pheromone glands were removed from the moths during scotophase and soaked in n-hexane for 30 min to extract the sex pheromone. The GC-MS analysis of the extract was carried out without any purification, but the extract was partially purified by HPLC with an ODS column to examine the stereochemistry of the natural pheromone by chiral HPLC. Chemicals. Synthesis, purification, and chemical characterization of all unsaturated hydrocarbons and their monoepoxy derivatives used in this report have been reported in detail previously (Ando et al., 1993, 1995). Opti- cally active epoxy compounds were prepared by resolution of a racemic mixture that used chiral HPLC (Qin et al., 1997; Yamamoto et al., 1999). In this text, chemicals are abbreviated as follows: cis-(Z)-6,7-epoxy-9-nonadecene c epo6,Z9–19 : H, cis-(Z,Z)-6,7-epoxy-3,9-nonadecadiene c Z3,epo6,Z9–19 : H, and so on. Derivatization with DMDS. Each synthetic epoxymonoene (500 ng) was dissolved in a mixture of DMDS (10 ml) and diethyl ether (30 ml) including iodine (1.8 mg). The reaction mixture was kept at 408C for 4 hr and diluted with diethyl ether after cooling. Iodine was removed by shaking with a 10% aqueous Na2S2O3 solution, and crude products were analyzed by GC-MS. The pheromone extract [10 female extract (FE)] was also treated with DMDS in the same manner. Field Tests. Field attraction of the synthetic lures was examined in 1999 by using white rubber septa (8 mm OD, Aldrich), which were totally immersed with 1 mg of epoxy compounds as follows: (a) racemic epo6,Z9–19 : H mixed with racemic Z3,epo6,Z9–19 : H in a ratio of 10 : 0, 9 : 1, 5 : 5, 1 : 9,or0 : 10, (b) optically active epo6,Z9–19 : H (6S,7R and 6R,7S isomers with 100 or 80% ee) mixed with Z3,epo6,Z9–19 : H possessing the same stereochemistry in a ratio of 9 : 1, and (c) optically active epo6,Z9–19 : H mixed with Z3,epo6,Z9–19 : H possessing the opposite stereochemistry in a ratio of 9 : 1.In2000, effect of unsaturated hydrocarbons on the attractive activity of these epoxy compounds was exam- 2582 YAMAMOTO ET AL. ined. The tested lures were baited with 0.5 mg of Z6,Z9–19 : H, Z3,Z6,Z9–19 : H, epo6,Z9–19 : H (6S,7R isomer), and Z3,epo6,Z9–19 : H (6S,7R isomer) in ratios of 9 : 1 : 81 : 9, 10 : 0 : 81 : 9,or0 : 0 : 90 : 10. Each lure was placed in a trap (30 × 27 cm bottom plate with roof; Takeda Chemical Ind.) that was set at a height of 1.5 m along a forest road in Hachijo-jima Island from February to March.

RESULTS

GC-MS Analysis of Pheromone Gland Extract. A GC-MS analysis of the pheromone extract (1 FE) produced a total ion chromatogram (TIC) shown in Figure 1A. Mass chromatograms with M+ ions of tri- and diunsaturated hydrocarbons with a C17–C23 chain and their monoepoxides, which were characterized as pheromone components of geometrid moths or their biosynthetic precursors (Arn et al., 1992, 1997; Ando et al., 1997), indicated that the extract included the following four C19 compounds (I–IV) in a ratio of 13 : 2 : 70 : 15; i.e. diene (compound I) with a peak of m/ z 264 at 10.10 min (Figure 1C), triene

FIG. 1. GC-MS analysis of a pheromone extract of Biston robustum (1 FE); total ion chromatogram (TIC) (A) and mass chromatograms (B–E). The mass chromatograms moni- + tored M of C19 triene (B, m/ z 262), diene (C, m/ z 264), epoxydiene (D, m/ z 278), and epoxymonoene (E, m/ z 280). Compounds: I c Z6,Z9–19 : H, II c Z3,Z6,Z9–19 : H, III c epo6,Z9–19 : H, and IV c Z3,epo6,Z9–19 : H. CHIRAL SEX PHEROMONE OF GEOMETRID FEMALES 2583

FIG. 2. Chemical structures of compounds I–IV identiﬁed from a pheromone-gland extract of Biston robustum.

(compound II) with a peak of m/ z 262 at 10.55 min (Figure 1B), epoxymonoene (compound III) with a peak of m/ z 280 at 14.63 min (Figure 1E), and epoxydiene (compound IV) with a peak of m/ z 278 at 15.17 min (Figure 1D). The GC-MS data of compounds I and II coincided well with those of authentic Z6,Z9–19 : H and Z3,Z6,Z9–19 : H (Figure 2). Although separation of the two epoxymonoenes derived from Z6,Z9–19 : H was not effective even on a capillary column (DB-23) [epo6,Z9–19 : H (Rt c 14.63 min) and Z6,epo9–19 : H (Rt c 14.69 min)], the mass spectrum of compound III was almost the same as that of the authentic epo6,Z9–19 : H (Figure 2), which showed diagnostic fragment ions of 6,7-epoxymonoene at m/ z 180 (M-100), 166 (M-114), and 99 (Ando et al., 1995). Three epoxymonoenes derived from Z3,Z6,Z9–19 : H were separable [epo3,Z6,Z9–19 : H (Rt c 15.40 min), Z3,epo6,Z9–19 : H (Rt c 15.17 min), and Z3,Z6,epo9–19 : H (Rt c 15.43 min)], and each positional isomer showed char- acteristic fragment ions (Ando et al., 1993). Compound IV was revealed to be Z3,epo6,Z9–19 : H (Figure 2), since the mass spectrum represented the diagnostic ions of 6,7-epoxydiene at m/ z 209 (M-69), 195 (M-83), 111, and 97. GC-MS Analysis of DMDS Adducts Derived from Epoxymonoenes. Epoxy- monoenes were easily converted into DMDS adducts, which showed informa- tive fragment ions reﬂecting the position of their original double bonds. DMDS adducts derived from synthetic 6,7-epoxy-9-enes produced abundant ions at m/ z 187 and M-187, which were prepared by cleavage of the bond between the 9 and 10 positions. In addition, they showed two other signiﬁcant ions at m/ z 139 and 99, as shown in the mass spectra of the DMDS adduct of epo6,Z9–19 : H (Figure 3A). The former ion might be derived by further removal of MeSH from the m/ z 187 ion, and the latter ion by a split at the epoxy ring. On the other hand, DMDS adducts of synthetic 9,10-epoxy-6-enes produced abundant ions at m/ z 131, M- 131, M-179, and M-219, as shown in the mass spectra of the Z6,epo9–19 : H DMDS adduct (Figure 3B). The former two ions were prepared by cleavage of the bond between the 6 and 7 positions, and the latter two in a similar manner for the formation of m/ z 139 and 99 ions of the 6,7-epoxy-9-ene DMDS adducts. The relative intensities of the fragment ions produced from the DMDS 2584 YAMAMOTO ET AL.

FIG. 3. GC-MS analyses of DMDS adducts derived from C19 epoxymonoenes; mass spectra of the adducts of synthetic epo6,Z9–19 : H (III) (A) and Z6,epo9–19 : H (B), TIC and mass chromatograms of their mixture (C), and those of a pheromone extract of Biston robustum (1 FE) treated with DMDS (D). Mass chromatograms monitored ions at m/ z 374 (M+), m/ z 187 and 139 [diagnostic fragment ions for the adduct of III, (X)], and m/ z 195 and 155 [diagnostic fragment ions for the adduct of Z6,epo9–19 : H, (Y)].

adducts with a C17–C23 chain and their Rts are listed in Table 1. 6,7-Epoxy- 9-ene DMDS adducts eluted from a DB-1 capillary column a little faster than the corresponding 9,10-epoxy-6-ene DMDS adducts. These GC-MS data indicate CHIRAL SEX PHEROMONE OF GEOMETRID FEMALES 2585 8 99 z / m ENES - 6 - POXY -E 10 , C. 9 8 270 219 187 139 131 and fragment ions at indicated M- min to ENES AND / - + 9 C - 8 6 187 POXY M- -E 7 , min and 6 2 179 C for M- 8 120 Relative intensity (%) of M ERIVED FROM 131 D DDUCTS )MM- z + / M m ( DMDS A m), temperature program: 30 a × 11 360 5 99 100 1 29 2 2 30 3 460623 34636 36052 374 403 388 412 402 3 416 3 150 430 3 0 2 029 346 2 0 142 0 057 374 5 0 010 388 0 29 017 402 5 100 28 0 100 416 4 0 430 4 27 0 1 84 4 83 26 1 80 4 0 22 80 100 100 18 0 99 100 100 100 100 0 60 1 100 0 55 100 37 100 0 2 100 0 87 23 45 5 100 0 0 43 2 100 23 41 1 43 0 22 3 36 2 37 0 18 25 0 20 2 2 32 0 20 4 31 7 25 1 31 4 2 28 10 28 4 28 2 3 5 32 38 3 41 5 7 6 ...... ATA OF ATA (min) 20 mm 18 20 21 22 23 25 26 18 21 22 23 25 26 t R 25 . 0 ( 1 GC-MS D . 1 -ene -ene 6 9 ABLE T -epoxy- : H : H : H : H : H : H : H : H : H : H : H : H : H : H -epoxy- 7 10 , , 17 18 19 20 21 22 23 17 18 19 20 21 22 23 6 9 – – – – – – – – – – – – – – 9 9 9 9 9 9 9 9 9 9 9 9 9 9 Z Z Z Z Z Z Z , , , , , , , 6 6 6 6 6 6 6 ,epo ,epo ,epo ,epo ,epo ,epo ,epo 6 6 6 6 6 6 6 epo Z epo epo epo epo epo epo Z Z Z Z Z Z Capillary GC; column: DB- DMDS of DMDS of a 2586 YAMAMOTO ET AL.

FIG. 4. Quantitative GC analyses of chiral HPLC-eluted epo6,Z9–19 : H (III), which were collected every 20 sec; a synthetic racemate, and a natural pheromone of Biston robustum. The chiral resolution was carried out by a Chiralpak AD column with 0.1% 2-propanol in n-hexane (ﬂow rate: 0.45 ml/ min) as a solvent system.

that the 6,7- and 9,10-epoxides are unquestionably distinguished after derivatization with DMDS. Figure 3C illustrates mass chromatograms of DMDS adducts derived from a 1 : 1 mixture of authentic epo6,7–19 : H and Z6,epo9–19 : H, and Figure 3D shows the same chromatographic analysis of the pheromone extract (10 FE) treated with DMDS, confirming the 6,7-epoxy ring of the main natural pheromone component (compound III). Chiral HPLC of Pheromonal Epoxynonadecene. The separation of two enantiomers of epo6,Z9–19 : H was accomplished by chiral HPLC equipped with a Chiralpak AD column (Yamamoto et al., 1999). After injection of the synthetic epo6,Z9–19 : H (500 mg) into the chiral column, the eluted materials were collected every 20 sec, and the epoxymonoene included in each fraction was quan- titatively analyzed by utilizing achiral capillary GC. This analysis confirmed that the racemic mixture produced two peaks of enantiomers (Figure 4). The 6S,7R isomer eluted from 14.0 to 15.0 min and the 6R,7S isomer from 15.0 to 16.3 min. In contrast, the injection of natural epoxymonoene, which was purified from a crude extract (10 FE) by HPLC with an ODS column, indicated only one peak with a shorter Rt (Figure 4). The purified pheromone component was prepared by collection of the eluted materials from 14.5 to 15.5 min [synthetic epo6,Z9–19 : H (Rt c 15.0 min)] on the achiral HPLC. This result indicated that B. robustum females selectively synthesized the 6S,7R isomer of epo6,Z9-19 : H as a main pheromone component. Field Attraction. Tables 2 and 3 show the total number of B. robustum males that were captured by traps baited with synthetic compounds. After the CHIRAL SEX PHEROMONE OF GEOMETRID FEMALES 2587

TABLE 2.ATTRACTION OF B. robustum MALES BY LURES BAITED WITH RACEMIC AND OPTICALLY ACTIVE EPOXY COMPOUNDS (III AND IV) IN JAPANESE CHINQUAIN TREES, HACHIJO-JIMA ISLANDa

Treatment (mg)

epo6,Z9–19 : H (III) Z3,epo6,Z9–19 : H (IV) Total 6S,7R 6R,7S Ratio 6S,7R 6R,7S Ratio males captured

(a) 500 500 5 : 500 0 450 450 5 : 550505: 50 250 250 5 : 5 250 250 5 : 50 50 50 5 : 5 450 450 5 : 50 00 5005005: 50 (b) 900 0 10 : 0100010: 09 810 90 9 : 190109: 138 90 810 1 : 910901: 90 09000: 10 0 100 0 : 10 0 (c) 900 0 10 : 001000: 10 0 810 90 9 : 110901: 90 90 810 1 : 990109: 10 09000: 10 100 0 10 : 00 00 00 0 a The field test was carried out from February 26 to March 30, 1999 using two traps for each lure. appearance of vast numbers of adults early in the spring of 1998, the extraordi- nary multiplication of this species ceased, and the attraction of the moths to a black light was reduced significantly after 1999. Although the field examination was conducted under this unfortunate condition, the specific attrac-

TABLE 3.ATTRACTION OF B. robustum MALES BY LURES BAITED WITH UNSATURATED HYDROCARBONS (I AND II) AND (6S,7R)-EPOXY COMPOUNDS (III AND IV) IN JAPANESE CHINQUAIN TREES, HACHIJO-JIMA ISLANDa

Treatment (mg) Total Z3,Z6, Epo6,Z9–19 : H Z3,epo6,Z9–19 : H males Z6,Z9–19 : H (I) Z9–19 : H (II) (6S,7R-III) (6S,7R-IV) Ratio captured

45 5 405 45 9 : 1 : 81 : 920 50 0 405 45 10 : 0 : 81 : 99 0 0 450 50 0 : 0 : 90 : 10 1 0 0 450b 50b 0 : 0 : 90 : 10 1 00 0 0 0 a The field test was carried out from March 1 to 20, 2000 with two traps for each lure. b (6S,7R)-Epoxy compounds with 80% ee. 2588 YAMAMOTO ET AL. tion was accomplished in 1999 by using a mixture of two epoxy compounds, epo6,Z9–19 : H and Z3,epo6,Z9–19 : H with a 6S,7R configuration in a ratio of 9 : 1. Males captured by the chiral epoxy compounds with 80% ee were much larger than those captured by the optically pure stereoisomers (Table 2b). No males were captured with mixtures composed of either of the two epoxy compounds with the 6R,7S configuration or racemic compounds (Table 2a). The 6S,7R isomer of epo6,Z9–19 : H mixed with the 6R,7S isomer of Z3,epo6,Z9–19 : H was inactive (Table 2c). Furthermore, a field test in 2000 indicated that unsaturated hydrocarbons played an important role for male attraction (Table 3). Males were caught by traps baited with the hydrocarbons as third and fourth components, while the mixture of only two epoxy compounds scarcely attracted the male moths.

DISCUSSION

GC-MS analyses of pheromone-gland extract of B. robustum treated with DMDS or untreated indicated that the females secreted cis-(Z)-6,7-epoxy-9-nonadecene and cis-(Z,Z)-6,7-epoxy-3,9-nonadecadiene. In the field test of 1999, the males were specifically attracted to a 9 : 1 mixture of the epoxymonoene and epoxydiene, which possessed the 6S,7R configuration. Chiral HPLC analysis suggested exclusiveness of this configuration as the natural main component, and we concluded that the sex pheromone of this species essentially consisted of two chiral epoxy compounds. Although the field trial was not satisfactory because of the limitation placed on it by having only one limited flight period in a year, it interestingly revealed stronger attraction qualities with the mixture of the two epoxy compounds with 80% ee compared to the attraction qualities of the optically pure epoxides. In order to use the sex pheromone as a monitoring tool, it will be important in the future to conduct research that will make clear an opti- mum mixing ratio of the chiral epoxides. In the pheromone gland, there are two other components, (Z,Z)-6,9-nonadecadiene and (Z,Z,Z)-3,6,9-nonadecatriene, which seem to be biosynthetic precursors of the C19 6,7-epoxy pheromone components, considering their chemical structures. On a related geometrid species, Ascotis selenaria cretacea, which includes the same triene in the pheromone gland as a precursor of 3,4-epoxy pheromone (Miyamoto et al., 1999), we have observed a strong synergistic effect of the hydrocarbon on male attraction (Wit- jaksono et al., 1999). In this study, we also observed that these unsaturated hydrocarbons identified in a pheromone extract of B. robustum were important components to attract the males effectively. The family of Geometridae is divided into six subfamilies, and sex pheromones of 27 species in one of the largest subfamilies, Ennominae, have been identified. Besides the pheromones, sex attractants of 38 species in Ennom- CHIRAL SEX PHEROMONE OF GEOMETRID FEMALES 2589 inae have been found by random screening tests of synthetic compounds. These compounds consist mostly of C17–C23 (Z,Z)-6,9-dienes and (Z,Z,Z)- 3,6,9-trienes, biosynthesized from linoleic and linolenic acids, respectively, and their monoepoxy derivatives. The main pheromone component of B. robustum, epo6,Z9–19 : H, has been identified from Anacamptodes humaria in this subfamily (Millar et al., 1991), while the stereochemistry of the pheromones of these species is opposite. We have observed that the racemic epoxymonoene attracts another Ennominae species, Menophra senilis (Ando et al., 1995). The stereochemistry of the epoxy components is important for composing species-specific pheromones. The minor pheromone component of B. robustum, Z3,epo6,Z9–19 : H, has been identified from the following three Ennominae species: Colotois pennaria (Hansson et al., 1990; Szocs¨ et al., 1993), Erannis defoliaria (Hansson et al., 1990; Szocs¨ et al., 1993), and Eufidoia convergaria (Millar et al., 1990a). Males of the first species are attracted to the 6R,7S isomer mixed with C19 triene. Lures including Z3,epo6,Z9–19 : H have attracted six other Ennominae species: Agriopis marginaria (Hansson et al., 1990; Szocs¨ et al., 1993), Anavitrinella pampinaria (Millar et al., 1990a), Caripeta angustiorata (Millar et al., 1990b), Colotois pennaria (Ando et al., 1993), Probole alienaria (Landolt et al., 1996), and Probole amicaria (Millar et al., 1990b). These find- ings suggest that the C19 6,7-epoxy compounds are ordinary pheromone components in this subfamily. However, this is the first case in which both of these 6,7-epoxides were identified from one Ennominae species. Two epoxymonoenes derived from each (Z,Z)-6,9-diene showed only small differences even on the GC-MS analysis with a capillary column (Ando et al., 1995). When authentic standards are not available, it is hard to determine the chemical structure of natural pheromone components secreted by female moths. In the case of many pheromones composed of unsaturated primary alcohols, acetates, and aldehydes, positions of their double bonds have been successfully determined by GC-MS analysis after making DMDS adducts (Buser et al., 1983). In this study, we found that DMDS adducts of the 6,7-epoxy-9-enes and 9,10- epoxy-6-enes also showed diagnostic fragment ions for determining the original position of their double bonds. The double bonds of these epoxymonoenes were easily attacked by DMDS without any modification of the homo-conju- gated epoxy ring. DMDS derivatization is useful to confirm chemical structures of geometrid pheromones.

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