GC-FT-IR Analyses of Sex Pheromones Secreted by Nettle Moths

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Naka 2 1 Graduate School of BASE, Tokyo University of Agriculture and Technology,

1 28 th ISCE Annual Meeting Vilnius, Lithuania (July 24, 212) GC-FT-IR Analyses of Sex Pheromones Secreted by Nettle Moths T. Ando, 1 * H. Shibasaki, 1 R. Yamakawa, 1 and H. Naka 2 1 Graduate School of BASE, Tokyo University of Agriculture and Technology, Tokyo , Japan * antetsu@cc.tuat.ac.jp 2 Tottori University (cm 1 )

2 Lepidopteran sex pheromones Identified from females of more than 63 species General procedures of the identification 1. GC-EAD to find an active component 2. GC-MS to determine the chemical structure Functional group Carbon chain length 16 Double bond Number 2 Position 1, 12 Configuration E, Z

Lepidopteran sex pheromones Identified from females of more than 63 species General procedures of the identification O-H 1. GC-EAD to find an active component 2.

3 Lepidopteran sex pheromones Identified from females of more than 63 species General procedures of the identification O-H 1. GC-EAD to find an active component 2. GC-MS to determine the chemical structure trans cis Functional group Carbon chain length 16 Double bond Number 2 Position 1, 12 Configuration E, Z Determined by the IR spectrum, if a large amount of the pure pheromone is available. IR analysis is not utilized, because the species-specific pheromones are composed of multi components, which are produced around ng level.

4 GC-FT-IR Capillary GC High separation FT-IR (solid phase) High sensitivity Familiarized spectrum Lq. N 2 Discover IR <Spectra Analysis, Inc.> GC capillary IR beam Detector ZnSe disk cooled at -3ºC

Identification of terminal conjugated dienes [Limacodidae] Nettle moths Parasa lepida in Japan Oil palm defoliators (Azharul et al., 29. B. B. B., 73: 1156-1162) 54 67 Setothosea asigna (Sasaerila et al.

5 Identification of terminal conjugated dienes [Limacodidae] Nettle moths Parasa lepida in Japan Oil palm defoliators (Azharul et al., 29. B. B. B., 73: ) Setothosea asigna (Sasaerila et al., 1997) O in Indonesia E9,11-12:Ald H Darna trima (Sasaerila et al., 2) in Malaysia O O O O E7,9-1:Acyl derivatives Z7,9-1: Terminal conjugated structure is easily determined by fragment ions at m/z 54 and 67. How to know the configuration? By GC?

Z:E = 1:1 Z:E = 1:1 Natural pheromone 1. 15. t R (min) 2. 25.

6 Separation of terminal conjugated dienes Synthetic mixture (1) GC E7,9-1: Column: DB-23 (.25 mm X 3 m) Z7,9-1: Z:E = 1:4 (2) HPLC Column: ODS Eluent: 45 % H 2 O in Me Z7,9-1: E7,9-1: Z:E = 1:4 Z:E = 1:1 Z:E = 1:1 Natural pheromone t R (min) t R (min) The females exclusively produce the (Z)-isomer. Reversed-phase HPLC is better than GC. (Azharul et al., 29) Other detector of capillary GC? FT-IR

Sex pheromone of the oriental moth Monema flavescens

I Eclosion in July (Univoltine) [M-18] + m/z 166 (1) GC-EAD

II M + m/z 182 Comp. I DMDS derivative 8-monoene 1. 15.

7 Sex pheromone of the oriental moth Monema flavescens (Limacodidae) Wintering in cocoon (2) GC-MS Comp. I Eclosion in July (Univoltine) [M-18] + m/z 166 (1) GC-EAD Comp. I Comp. II Column: DB-23 (.25 mm X 3 m) Comp. II M + m/z 182 Comp. I DMDS derivative 8-monoene t R (min) (3) GC-FT-IR Comp. II Mass spectrum 7,9-diene Configuration of the double bonds??

8 GC-FT-IR analysis Column: HP-5 (.25 mm X 3 m) Peak chromatogram Comp. I Comp. II Crude pheromone extract (2FE) Disk temperature: -3ºC Disk speed: 3 mm/min (min) 2. Comp. I C-H bending vibrations Di-substituted monoene trans cm -1 cis cm -1 gem Comp II cm -1 Conjugated diene Similar absorption Discover IR-GC Recording range: 4-7 cm -1 E8-1: 966 cm (cm -1 ) E7,9-1: cm -1 93cm -1 (cm -1 ) (E) -isomer (Z)-Isomers are not directly identified.

9 Band chromatograms Column: HP-5 (.25 mm X 3 m) Peak chromatogram Comp. I Comp. II Crude pheromone extract (2FE) Disk temperature: -3ºC Disk speed: 3 mm/min (min) cm -1 (O-H) 966 cm -1 (E)-monoene 951 cm -1 (E)-diene These band chromatograms made clear the two different components with the same configuration.

10 IR spectra of (E)- and (Z)-isomers 966 E8-1: E8-1: Z8-1: Z8-1: (cm -1 ) E7,9-1: (cm -1 ) (cm -1 ) Z7,9-1: (cm -1 ) Configurations are confirmed by band chromatograms at 966 and 951 cm -1.

11 GC-FT-IR analysis of natural pheromones Oriental moth Peak chromatogram 7,9-diene Column: HP-5 (.25 mm X 3 m) 2 FE Monema flavescens Band chromatogram 951 cm Rt (min) (E) -isomer 1 15 Rt (min) Green nettle moth Peak chromatogram 7,9-diene 1 FE Parasa lepida Band chromatogram 951 cm Rt (min) Rt (min) (Z)-Configuration is confirmed by absence of absorption at 951 cm -1. (Z) -isomer

12 Field evaluation of synthetic pheromones Oriental moth Monema flavescens Lure contents (mg/septum) E8-1: E7,9-1: June 29 to July 16, 212 Captured males / trap 4.5 ±.6 a.8 ±.3 b New components of Type I pheromones Green nettle moth Parasa lepida Lure contents (mg/septum) Z7,9-1: E7,9-1: Aug. 21 to Sept. 3, 27 Captured males / trap 9.5 ±.3 a 4.5 ±.8 b 1. ±.3 c 1. ±.3 c

Further studies Identification of sex pheromones secreted by Limacodidae species About 3 species of the nettle moths inhabit Japan Conservation of the terminal conjugated dienyl structure?

13 Further studies Identification of sex pheromones secreted by Limacodidae species About 3 species of the nettle moths inhabit Japan Conservation of the terminal conjugated dienyl structure?? Structural diversity of the female pheromones?? Mechanism of reproductive isolation?? (geographical factor, moving area, northern limit of inhabitancy) Biosynthesis of the terminal conjugated dienenes Applications of GC-FT-IR Isolated dienes Conjugate dienes GC capillary IR beam Detector ZnSe disk cooled at -3ºC

3283 2921 2852 1472 328 38 292 2852 1472 3364 328

14 GC-FT-IR analysis of synthetic 3,13-dienes Z3,Z13-18: E3,Z13-18: Z3,E13-18: E3,E13-18:

15 Hiroshi SHIBASAKI Thank you for your attention!! Dr. Hideshi NAKA Chemical Ecology Laboratory of TUAT Dr. M. Yamamoto Dr. R. Yamakawa Y. Qi R. Maruyama Y. Murakami A. Kanegae Y. Sakamoto T. Fujii Y. Muraki

GC-MS and LC-MS analyses for unraveling the diversity of lepidopteran communication systems

23 rd ISCE Annual Meeting Symposium on Insect Semiochemicals I Analysis, Structures, Synthesis, GC-MS and LC-MS analyses for unraveling the diversity of lepidopteran communication systems Tetsu AND Graduate