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

Transcription

1 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 School of BASE, Tokyo University of Agriculture and Technology, Tokyo , Japan antetsu@cc.tuat.ac.jp Jena, Germany (July 23, 2007)

2 Lepidopteran sex pheromones Produced by to attract Main factor for reproductive isolation Species specific Pheromones of 580 species have been reported. Lepidoptera: ca. 150,000 species Variety of chemical structures Blending of multiple components Small insects Low content Identification by GC-EAD GC-MS HPLC and LC-MS?

3 Type I sex pheromones i) Monoenyl compounds Diamondback moth Ac H H Pest insects in Japan Z11-16:Ac Z11-16:Ald Z11-16:H ii) Dienyl compounds Pine caterpillar H Z5,E7-12:H Cherry treeborer Ac Z3,Z13-18:Ac Ac E3,Z13-18:Ac iii) Trienyl compounds Mulberry pyralid Ac E10,E12,Z14-16:Ac

4 Double-bond positions of dienes and trienes (C 13 and C 15 chain compounds are not included.) Chain Double bond position, counting from the functional group length C10 C12 C14 C16 C18

5 Determination of double-bond positions (A) MTAD (4-methyl-1,2,4-triazoline-3,5-dione) derivative 2 1 CH 3 CH N 3 N GC-MS NN NN 2 1 (B) Diimide reduction DMDS derivative NH NH H DMDS GC-MS Derivatization is difficult for natural pheromones in a trace amount. SCH CH 3 S 2 SCH 3 CH 3 S 1 If conjugated dienes specifically show characteristic fragment ions on the direct GC-MS analysis, derivatization is not necessary.

6 Mass spectra of conjugated dienes (C 12 alcohols) 5,7-diene 5, 7-diene 110 6,8-diene 4, 6-diene ,9-diene 3, 5-diene ,10-diene 2, 4-diene ,11-diene , 3-diene In the case of the a terminal conjugated diene, identification of a natural pheromone can be accomplished without derivatization.

7 Diagnostic ions of conjugated dienes Fragment ion A Fragment ion B 5, 7-4, 2-3, 5-2, 4-1, 3- diene diene diene diene diene A + (m/z) B + (m/z) In the case of the a terminal conjugated diene, identification of a natural pheromone can be accomplished without derivatization.

8 Identification of dienyl pheromones 5, 7-diene 110 4, 6-diene Z5,E7-12:H Pine caterpillar [Lasiocampidae] 3, 5-diene E8,E10-12:Ac 2, 4-diene H Ac Ac E5,Z7-12:Ac Chrysanthemum golden plusia [Noctuidae: Plusiinae] [Tortricidae: lethreutinae] Soybean pod borer Centroxena sp. 1, 3-diene 54 67

9 Identification of 1, 3-dienes dienyl pheromones [Limacodidae] Nettle moths 5, 7-diene 110 Parasa lepida il palm defoliators Setothosea asigna (Sasaerila et al., 1997) H 4, 6-diene Indonesia Z5,E7-12:H 96 E5,Z7-12:Ac 109 Chrysanthemum golden Pine caterpillar [Lasiocampidae] H E9,11-12:Ald plusia golden [Noctuidae: plusia [Noctuidae] Plusiinae] 3, 5-diene E8,E10-12:Ac 2, 4-diene H Z7,9-10:H Ac 82 Darna 95 trima (Sasaerila et al., 2000) [Tortricidae: lethreutinae] Malaysia Soybean pod borer Ac Centroxena sp. E7,9-10:Acyl derivatives 1, 3-diene 54 67

10 Identification of 1, 3-dienes [Limacodidae] Parasa lepida (A) GC-MS analysis (TIC) DB-23 column (0.25 mm X 30 m) Z-isomer E-isomer Synthetic mixture Z7,9-10:H H (B) HPLC analysis (UV 240 nm) Column: DS 2.0 mm X 25 cm Eluent: 45 % H 2 Z-isomer in MeH (0.2 ml/min) E-isomer Synthetic mixture Extract (0.2 FE) t (min)

11 Pheromones of Sesiidae species Do any females produce E13 compounds? Z2 compounds? Z3 or E3 aldehydes? Double bonds Position Configuration Number of species H Ac Ald 3,13-Diene Z3,Z13 E3,Z13 Z3,E13 E3,E ? ,13-Diene Macroscelesia spp. Z2,Z13 E2,Z13 E2,Z13-18:Ald H M. japona M. longipes

12 GC analysis of 3,13- and 2,13-dienals Cool on-column injection, DB-23 column (0.25 mm X 30 m) (A) 3,13-18:Ald Z3,Z13 (B) 2,13-18:Ald Z2,Z13 E3,Z13 Z3,E13 Ac H E2,Z13 Z2,E13 Ac H E3,E13 E2,E t (min) E2,Z13 E2,E13 Peaks without any isomerization Isomerized to E2,Z13-18:Ald Isomerized to E2,E13-18:Ald t (min) E2,Z13 E2,E13 GC-MS is not suitable for the analysis of the dienals.

13 LC and LC-MS analyses of the dienals Column: DS 2.0 mm X 25 cm Eluent: 16 % H 2 in CH 3 CN (0.2 ml/min) 3,13-18:Ald (A) LC analysis 215 nm ZZ EZ ZE EE 250 ng (B) LC-MS (APCI) analysis of DNP derivatives ([M-1] -, m/z 443) ZZ ZE EZ EE 50 ng 2,13-18:Ald 235 nm ZZ EZ ZE EE 50 ng ZE EZ EE ZZ 50 ng Pheromone extract of M. japona 5 FE 2.5 FE t (min) t (min) dienals 2 N NHNH 2 N 2

14 Type II sex pheromones (A) First Identification Hill et al., (1981) J. Chem. Ecol., 7: 655 saltmarsh caterpillar moth (Estigmene acrea: Arctiidae) Z3,Z6,epo9-21:H + Z9,Z12,Z15-18:Ald + Z9,Z12-18:Ald (27:6:1) (B) Identification in Japan Biston robustum (Geometridae) Milionia basalis (Geometridae) Penina nuda (Lymantriidae) epo3,z6,z9-19:h epo6,z9-19:h Z3,epo6,Z9-21:H Z3,epo6,Z9-19:H epo3,epo6,z9-21:h

15 Diagnostic ions of GC-MS analysis (EI) i) Monoepoxydienes M-29 M-58 M-72 -H -H 79 ii) Monoepoxymonoenes -H 99 ii) Diepoxymonoenes M-100 -H M-114 M H M-69 -H M H -H M M-111 M-69 -H M-125 M M-125 M-125 -H M- M-85 M-87 (-2H) M-69 -H M-139

16 Analysis by LC-TF MS (ESI) Column: DS (2.1 mm X 15 cm) Eluent: 20-5% H 2 in MeH Spray tip potential: +3,400 V Nozzle potential: +120 V C 19 compounds (=C 4 H 9 ) 221 M-57 + H + H [M+NH 4 ] [M+H] [M-H] M-71 M- -H ,4-epoxide (C 19 ) m/z 6,7-epoxide (C 19 ) X m/z 9,10-epoxide (C 19 ) X X m/z

17 LC-TF MS (ESI) analysis of pheromones 1 Column: DS (2.1 mm X 15 cm) Eluent: 20-5% H 2 in MeH Spray tip potential: +3,400 V Nozzle potential: +120 V C 18 compounds (=C 3 H 7 ) 207 M-57 + H + H [M+NH 4 ] [M+H] [M-H] M-71 M- -H 141 (A) Synthetic mixture 3,4-epoxide 9,10-epoxide (C 18 ) 3,4-epoxide 2076,7-epoxide m/z X ,7-epoxide (C 18 ) m/z m/z X (B) Extract of the mulberry looper m/z ,10-epoxide (C 18 ) X t m/z (min)

18 LC-TF MS (ESI) analysis of pheromones 2 Column: Chiralcel J- Eluent: 10% H 2 in MeH Spray tip potential: +3,400 V Nozzle potential: +120 V C 19 compounds (=C 4 H 9 ) 221 M-57 + H + H [M+NH 4 ] [M+H] [M-H] M-71 M- -H 155 (A) acemic mixture of 3,4-epoxide UV TIC m/z S,4 3,4S (B) Extract of the giant looper UV m/z TIC t (min)

19 Conclusion EI-Mass spectra of Type I pheromones (conjugated dienes) and Type II pheromones (polyunsaturated hydrocarbons and their epoxy derivatives) showed diagnostic fragment ions for the structure determination. LC and LC-MS are useful tools for unstable and inapplicable components on GC analysis. Stereochemistry of epoxy pheromones can be determined by LC-MS with a chiral column. The diversity of lepidopteran communication systems will be understood in detail using these elegant(?) and smart(?) techniques.

20 Acknowledgments Co-workers Drs. H. Naka, Y. Arita and K. Tsuchida Identification of the pheromones from Sesiidae species Drs. F. Komai, M. Kinjo and H. Ishitani identification of the pheromones from lethreutinae species Drs. K. htani, G.-Q. Pu and K. I. Karasawa Identification of the pheromone from Geometridae species Chemical Ecology Laboratory of TUAT Dr. X.-. Qin Dr. Witjaksono Dr. S. Inomata Dr. M. Yamamoto Dr. A. no Dr. H. Yamazawa Dr. L. V. Vang T. Nishida T. Nakazawa K. Matsuoka M. D. A. Islam N. Numakura M. Sugie