GC/FT-IR Analysis of Novel 4,6,9-Triene and 2,4,6,9-Tetraene Occurring in a Female Pheromone Gland of Arctia plantaginis (Erebidae: Arctiinae)

Fifteen subspecies of the wood tiger moth, Arctia plantaginis (Lepidoptera: Erebidae: Arctiinae), have been recorded in the Northern Hemisphere. An analysis of crude pheromone extracts by GC equipped with an electroantennographic (EAG) detector showed four EAG-active components (Comps. I–IV) that were commonly involved in the pheromone glands of two subspecies inhabiting Japan and Finland. Comp. I is a major component (>75%) and the others are minor components (3% 15%). Their mass spectra, measured by GC/MS, revealed the chemical structures of C21 unsaturated hydrocarbons as follows: 3,6,9-triene for Comp. I, 4,6,9-triene for Comp. II, 1,3,6,9-tetraene for Comp. III, and 2,4,6,9-tetraene for Comp. IV. Comps. I and III are known Type II pheromone compounds, and their retention times coincide with those of the authentic standards with all Z configurations. As a next step, the extract was analyzed by GC/FT-IR to determine the configuration of Comps. II and IV. Their IR spectra showed two characteristic C-H bending absorptions around 990 and 945 cm due to the conjugated dienyl moieties; thus, Z and E configurations were assigned to the double bonds at the 2and 4-positions, respectively. Their Z double bonds at the 6and 9-positions are indicated by no absorptions around 970 cm, due to the isolated double bonds with E configurations. Finally, the structures of Comps. II and IV were confirmed by synthesis using a double Wittig reaction. The synthetic (4E,6Z,9Z)-4,6,9-triene and (2Z,4E,6Z,9Z)-2,4,6,9-tetraene showed strong EAG activity, and their How to cite this paper: Muraki, Y., Yamakawa, R., Yamamoto, M., Naka, H., Honma, A., Mappes, J., Suisto, K. and Ando, T. (2017) GC/FT-IR Analysis of Novel 4,6,9-Triene and 2,4,6,9-Tetraene Occurring in a Female Pheromone Gland of Arctia plantaginis (Erebidae: Arctiinae). American Journal of Analytical Chemistry, 8, 645-656. https://doi.org/10.4236/ajac.2017.810047 Received: September 15, 2017 Accepted: October 23, 2017 Published: October 26, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Lepidopteran sex pheromones, which have been identified from more than 670 species, are mainly sorted into two types according to their chemical structures [1] [2] [3]. Type I pheromones are composed of unsaturated fatty alcohols, acetates, and aldehydes, and are most commonly found from female moths in many families. Type II pheromones are composed of polyunsaturated hydrocarbons and their epoxides and are mostly produced by females in highly evolved groups: Erebidae (including former Arctiidae and Lymantriidae [4]) and Geometridae [5] [6]. Species-specific pheromones are created by the blending of some compounds with different structures, such as chain length, unsaturated degree, and positions of the double bond and epoxy ring. Arctiinae (former Arctiidae) is divided into three tribes: Lithosiini, Synthomini, and Arctiini. To date, sex pheromones of 23 species in the tribe Arctiini have been reported, and the pheromones of 16 of these species are composed of Type II compounds [5]. Only three species inhabiting Japan are included, while more than 100 Arctiini species have been recorded in Japan. To increase the information about the Japanese species, we started to investigate a sex pheromone of the wood tiger moth, Arctia plantaginis (former Parasemia plantaginis [7]), which belongs to Arctiini. Large distribution area of this species and great geographic variation in their phenotypes gives a unique possibility to study whether this species is its early steps of divergence. It is noteworthy that 15 subspecies of A. plantaginis have been recorded in the Northern Hemisphere, and four of these subspecies are distributed in Japan. It should be interesting to know whether differences in sex pheromones are one of the most important candidates driving pre-zygotic isolation.
Lepidopteran pheromones have usually been investigated by gas chromatography (GC) with an electroantennographic (EAG) detector (GC-EAD) and GC combined with mass spectrometry (GC/MS) [2]. GC-EAD can differentiate biologically active compounds from others in the crude extract, and then chemical structures of the pheromone candidates are identified by GC/MS [8]. The sensitivity of both instruments is so high that a pheromone gland extract of one female sometimes provides enough information to clarify the chemical cues when compared with published data of known compounds. However, the configuration of a double bond is scarcely determined by only GC/MS analysis because with Fourier transform infrared spectrometry (GC/FT-IR) to reveal configurations of Type I pheromone compounds produced by the nettle moth [9]. Our previous research provided a good opportunity to experience the high sensitivity of GC/FT-IR that was comparable to that of GC/MS. In this study, we successfully applied the same instrument to determine the structure of novel Type II compounds occurring in the pheromone glands of the wood tiger moth.

Analytical Instruments
For an analysis with gas chromatography (GC) with an electroantennographic The effluent from the column was split into two lines, which led to a flame ionization detector (FID) and EAD at a ratio of 1:1 [9]. The oven temperature was maintained at 80˚C for 1 min and then programmed at 8˚C·min −1 to 210˚C. 1 H and 13 C NMR spectra were recorded by a Jeol Delta 2 Fourier transform spectrometer (JEOL Ltd., Tokyo, Japan) at 399.8 and 100.5 MHz, respectively, for CDCl 3 solutions containing TMS as an internal standard. 1 H-1 H COSY, HMQC, and HMBC spectra were also measured with the same spectrometer, using the usual pulse sequences and parameters. GC/MS was conducted in EI mode (70 eV) with an HP5973 mass spectrometer system (Agilent Technologies) equipped with a split-splitless or a cool on-column injector and the same DB-23 column in the analysis by GC-EAD. The column temperature program was 50˚C for 2 min, 10˚C·min −1 to 160˚C, and 4˚C·min −1 to 220˚C. The carrier gas was helium. IR spectra were recorded using FT-IR (Discover IR; Spectra Analytics, Marlborough, MA) coupled to a GC (GC 7980C; Agilent Technologies), which was equipped with an HP-5 capillary column (0.25 mm ID × 30 m, 0.25 μm film; Agilent Technologies, Santa Clara, CA). A liquid nitrogen-cooled photoconductive mercury-cadmium-telluride detector was used with a FT-IR resolution of 8 cm −1 . Compounds eluting from the capillary column were solidified on a zinc selenide disk at −30˚C and rotated at 3 mm·min -1 . The distance between the column end and disk was 5 mm. The oven temperature for all GC analyses was set initially at 50˚C for 2 min. and then programmed at 10˚C·min −1 to 160˚C and 4˚C·min −1 to 220˚C. The flow rate of the carrier gas (He) was 1.0 mL·min −1 , and the GC inlet temperature was 220˚C.

Insects and Pheromone Extraction
Adults of Japanese subspecies estimated to be A. plantaginis melanomera Butler, which were collected at the Abou Pass between Gihu prefecture and Nagano prefecture in Japan (36.194˚N, 137.585˚E) in July 2013, laid eggs in a laboratory, and the larvae were reared on a semisynthetic diet for insects [Insecta LF(S), Nippon-Nosan-Kogyo Co, Yokohama, Japan] under a 16:8 L:D photoperiod at 25˚C. The specimens of Finnish A. plantaginis used in the present analysis were obtained from the laboratory stock, which was established in 2010 from wild moths collected from central and southern Finland and kept on a diet consisting mostly of dandelion (Taraxacum sp.) leaves. The resulting pupae were sexed by their abdomen tip, and the terminal abdominal segments of 2-day-old virgin females emerged from the pupae were cut off at 14 -16 hr after the start of photophase and soaked in hexane for 30 min. This extract was subjected to instrumental analyses without any purification.

Synthesis of 4,6,9-Triene (E4,Z6,Z9-21:H)
A mixture of 1,3-dibromopropane and triphenylphosphine was heated in dry benzene under a refluxing condition. The produced phosphonium salt of the dibromide was treated with NaN(SiMe 3 ) 2 in a mixed solvent of dry THF and HMPA at −80˚C under Ar gas to make bis(ylide), which was further treated with a mixture of dodecanal and (E)-2-hexenal (RCHO in Figure 1). After warming to room temperature, the crude products were extracted with hexane and cleaned by the usual workup. While this reaction produces byproducts, such as a tetraene with a short chain and a diene with a long chain, the desired E4,Z6,Z9-21:H was obtained in 18% yield, as calculated from the phosphonium salt, after the crude products were chromatographed on a silica gel column impregnated with AgNO 3 using hexane and mixtures of hexane and benzene as eluents. 1

GC-EAD Analysis of a Pheromone Extract
A pheromone gland extract of one A. plantaginis female, which was derived from a larva collected in the fields of Japan, showed four EAG-active compo-  at m/z 79, indicates a 6,9-dienyl structure commonly involved in many Type II pheromone components [8], and that its RT (17.40 min) is longer than that of the 3,6,9-triene suggests that another double bond is conjugated to the homo-conjugated dienyl moiety. Since its mass spectrum is different from that of a 6,9,11-triene synthesized previously [11], we estimated the possibility of a  Although the base peak of Comp. IV is recorded at m/z 94, the spectral similarity of these two tetraenes suggests their common moiety, such as the 6,9-dienyl structure. The GC/MS data of Comp. IV are different from those of 3,6,9,11-tetraene synthesized previously [11]. The intensity of its M + is higher than that of 1,Z3,Z6,Z9-21:H, and its RT (19.70 min) on the strong polar column is much longer than that of the tetraene that includes only one conjugated diene system. Therefore, we conceived a 2,4,6,9-tetraene that included a conjugated triene system as one possibility for Comp. IV.

Synthesis and Analysis of 4,6,9-Triene and 2,4,6,9-Tetraene
In order to confirm the structure determination of two new unsaturated hydrocarbons based on the instrumental analyses, their authentic samples were syn-

Conclusions
Four EAG-active components (I -IV) were found in the pheromone gland of Japanese In addition to the determination of a double-bond configuration, GC/FT-IR seems to be actively used for determining the functional groups in pheromone compounds [9] [15].
We carried out field tests in Japan and Finland; however, synthetic lures could not attract any males. While the subspecies-specific communication system has not been clarified, we expect that the four components found in the pheromone gland are important pheromone candidates because of their strong EAG activities. In order to attract the male by a synthetic lure in the fields, we will conduct further field tests and reexamine additional minor components in the pheromone extract.