1. PHEROMONES AND SEMIOCHEMICALS IN ANIMAL COMMUNICATION : OVERVIEW AND CHEMISTRY Thibaut Denoel Department of Chemistry Cyclotron Research Center (CRC) Advisor : André Luxen

2. Table of contents   Introduction :   Chemical ecology   Pheromones   Chirality in pheromones   Collection :   Cold trapping volatiles   SPME, body, flask   Analysis :   GC-EAG   GC-EAD   GC-SCR   Characterization :   Structural   Stereoisomers   Behavioural bioassay in wind tunnel   Pheromones in pest management :   Population monitoring and mating disruption   Different kinds of traps   Synthetic overview

3. Introduction : chemical ecology Karlson et al., Nature, 183 (1959), 55-56 Chemical ecology study chemicals in interactions of living organisms. Semiochemicals are of four types : pheromone, allomone, kairomone, synomone.  Pheromone - from Greek word pherein (to transfert) and hormone (to excite) : semiochemical exchanged between individuals of the same species.  Induces specific reaction such as special behaviour or developmental process.  Allomone : interspecific that benefit the originator but not the receiver.  Kairomone : interspecific that benefit the receiver but not the emitter.  Synomone : interspecific that benefits both parties.

4. Introduction : pheromones  Sexual attraction, trail-following, territorial boundaries, alarm, aggregation…  Arthropods (insects, arachnides…)  Sub nanogram to a few micrograms emitted  1000 molecules/ml of air to elicit a behaviour  7000 species and 3500 semiochemicals (2012)  Different functions : hydrocarbon, alcohol, aldehyde, ketone, acetal, epoxide, ester, lactone, terpene, peptide, steroid…  Chirality is important for bioactivity Hummel, Techniques in Pheromone Research (1985), VII-VIII Kaissling, Biologically inspired signal processing, SCI, 188 (2009), 45-52 http://www.pherobase.com/

5. Introduction : chirality in pheromones  One enantiomer is bioactive, and the antipode is not inhibitor  One enantiomer is bioactive, but the antipode is inhibitor  One enantiomer is bioactive, but a diastereomer is inhibitor Mori, Bioorg. Med. Chem., 15 (2007), 7505–7523

6.  One natural enantiomer and the antipode is also active  Mixture of enantiomers and both enantiomers are separately active  Different enantiomers - different species Mori, Bioorg. Med. Chem., 15 (2007), 7505–7523 Introduction : chirality in pheromones

7.  Both enantiomers are necessary  One enantiomer is most active but a stereoisomer can enhance it  One enantiomer is active on males, the other is active on females  Only the meso-isomer is active

8. Collection : cold trapping volatiles The volatiles liberated by the beetles are carried over by an airflow and captured by three traps connected in series : two empty tubes cooled with liquid nitrogen, and a tube containing cyclohexane. Yarden et al., Journal of Chemical Ecology, 20 (1994), 2673-2685

9. Collection : SPME, body, flask Farine et al., PLoS ONE, 7 (2012), e40396

10. Analysis  GC-EAG (Electroantennogram)  GC-EAD (Electroantennographic Detector)  GC-SCR (Single Cell Recording)  GC-BB (Behavioural Bioassay) Sensibility : 0.1 pg-100 ng Four specific detectors are used in GC :

11. GC-EAG : Electroantennogram Schneider, Vergl. Physiol., 40 (1957), 8-41 (EAG) Moorhouse et al., Nature, 223 (1969), 1174-1175 (GC-EAG) Howse et al., Insect pheromones and their use in pest management (1998) GC column effluent is split between FID and a reservoir. Every 15 sec N 2 flush it toward an insect antennae coupled to an amplifier to measure an electroantennogram (EAG).

12. GC-EAD : Electroantennographic Detector GC column effluent is split between FID and the EAD. The EAD response indicate a pheromone peak with great sensibility. Arn et al., Z. Naturforsch., 30c (1975), 722-725 Wyatt, Pheromones and animal behaviour (2003), 30

13. GC-SCR : Single Cell Recording SEM of a part of the antenna of a female cabbage moth. The sensilla hairs containing the olfactory receptor neurons are densely distributed over the antenna. Injection of a sample of cotton : GC-SCR below shows receptor neurons responding to compound E,E-TMTT (trace amount) and to E,E-α-farnesene. Ulland, Chem. Senses, 33 (2008), 509-522.

14. Structural characterization  HPLC with or without derivatization (pg – ng)  GC-FTIR (ng)  GC-MS (ng)  Microdegradation (H 2, O 3, MeSSMe…) (μg)  Chiral columns (GC, HPLC…)  NMR 1 H (< 1 μg) and 13 C (10-100 μg)

15. Stereoisomeric characterization  All possibles stereoisomers are synthesized.  They are tested on chiral-GC with bioassay techniques to determine the stereochemistry of the natural compound.  The blend of actives compounds is tested in wind tunnel against living insects in behavioural bioassay.

16. Behavioural bioassay in wind tunnel Valeur, http://www.pheromone.ekol.lu.se/vt2.html A male turnip moth is approaching a rubber septum with an applied mixture of female pheromones. The male lands and tries to mate with the pheromone emitter.

17. Pheromones in pest management  Population monitoring :  Follow the density of a specific species with baited traps.  Mating disruption :  Flood the air with synthetic pheromones. Prevent adult males and females finding each other to mate.  Lure and kill, mass trapping :  Attract with pheromones and kill responding individuals with insecticides. Advantages : not toxic, less amount of pesticides used, specific to the targeted species, low amount of chemical compound needed (g/ha vs kg/ha pesticide), no resistance. Wyatt, Pheromones and animal behaviour (2003), 255-264

18. Population monitoring and mating disruption Population monitoring of the light brown apple moth in New Zealand before and after mating disruption with pheromones (P). Suckling et al., New Zealand Journal of Crop and Horticultural Science, 18 (1990), 89–98

19. Different kinds of traps No pheromone Pheromone Pheromone content, size, color and situation are important.

20. Philip McCabe's bee beard 200 000 bees (27 kg) are attracted by swarm lures and queen bee pheromone on Philip’s skin!

21. Synthetic overview  Domesticated silkmoth sex pheromone (1962 & 2012)  Pine moth sex pheromone (2012)  Tse-tse fly contact sex pheromone (2001)  Mosquito oviposition kairomone (2012)  Lesser grain borer aggregation pheromone (2006)  Gypsy moth sex pheromone (2012)  Southern pine beetle aggregation pheromone (2011)

22. Domesticated silkmoth (Bombyx mori) sex pheromone

23.  Bombykol : first pheromone isolated (1939-1959) (Butenandt)  500 000 female scent glands of B. mori extracted  Male ‘flutter dance’ used as the detector  12 mg of bombykol obtained  Structure elucidation by chromatographic properties, derivatization, microdegradation (H 2, KMnO 4 ), UV, IR, total synthesis and then mp Butenandt et al., Z. Naturforsch., 14b (1959), 283-284 Hecker et al., Techniques in Pheromone Research (1985), 1-44 Domesticated silkmoth pheromone

24. Flutter dance : male silkmoth (right) gets excited by the female (left). She is releasing bombykol from her gland. http://www.chm.bris.ac.uk/motm/bombykol/bombykolv.htm

25.  After chromatography and derivatization 12 mg of Bombykol-NABA Structure elucidation : Hecker et al., Techniques in Pheromone Research (1985), 1-44 Domesticated silkmoth pheromone

26. Butenandt et al., Liebigs Ann. Chem., 658 (1962), 39-64 Bombykol 1962 synthesis

27. Domesticated silkmoth pheromone Several recrystallisations of the complex to get pure (E)-isomer Bombykol 1962 synthesis Butenandt et al., Liebigs Ann. Chem., 658 (1962), 39-64

28. Domesticated silkmoth pheromone Bombykol 1962 synthesis Butenandt et al., Liebigs Ann. Chem., 658 (1962), 39-64 (10E,12E) : 1 (10Z,12Z) : 10 (10Z,12E) : 10 -3 (10E,12Z) : 10 -12 Z-alkene

29. Urea inclusion complex Mayo et al., Journal of Solid State Chemistry, 141 (1998), 437-451 Linear molecules form inclusion complex with urea

30. De Figueiredo et al., J. Org. Chem., 72 (2007), 640-642 Domesticated silkmoth pheromone E-enals only Z/E > 9:1 Bombykol 2012 synthesis

31. Zhu et al., J. Org. Chem., 64 (1999), 8980 Hantzsch ester reduction Domesticated silkmoth pheromone

32. Pine moth (Dendrolimus spectabilis) pheromone

33. Pine moth pheromone  Pine forests defoliating insect  100 000 ha of pine trees in Korea infested in 2007  Up to 2000 caterpillars (1 kg) / tree http://www.fao.org/forestry/49410/en/prk/

34. Pine moth pheromone Terminal alkyneInternal alkyne Lin et al., CN 102613177 (2012)

35. Pine moth pheromone Lin et al., CN 102613177 (2012)

36. Pine moth pheromone Z-alkene Lin et al., CN 102613177 (2012)

37. Pine moth pheromone A blend of 100/3/25 : OH/OAc/OPr is the best attractant. Lin et al., CN 102613177 (2012) Kong et al., Kunchong Xuebao, 46 (2003), 131-137

38. Tse-Tse fly (Glossina austeni) contact sex pheromone

39. Tse-Tse fly pheromone  Sleeping sickness caused by the parasite Trypanosoma brucei gambiense  50,000 to 70,000 people infected  Cost 1,000,000,000$/year in Africa  Tse-Tse fly eradicated on Zanzibar in 1997 with the Sterile Insect Technique  Contact sex pheromone can be used to rear competitive sterile males http://www.doctorswithoutborders.org/news/issue.cfm?id=2401

40. Roche ester by biotransformation Leuenberger et al., Pure & Appl. Chem., 62 (1990), 753-768

41. Tse-Tse fly pheromone Mori, Tetrahedron, 39 (1983), 3107-3109 Thomas et al., J. Chem. Soc., Perkin Trans. 1 (1989), 507-518

42. Tse-Tse fly pheromone Kimura et al., Eur. J. Org. Chem. (2001), 3385-3390

43. Tse-Tse fly pheromone Kimura et al., Eur. J. Org. Chem. (2001), 3385-3390

44. Tse-Tse fly pheromone Kimura et al., Eur. J. Org. Chem. (2001), 3385-3390

45. Southern House mosquito (Culex quinquefasciatus) oviposition kairomone

46.  Vector of lymphatic filariasis and West Nile virus  120 million people infected by filariasis ‘Elephantiasis’  Ovitraps baited with the oviposition pheromone attract females to lay their eggs  Water in the trap contain poison to larvae Mosquito oviposition kairomone http://www.ivmproject.net/about/index.cfm?fuseaction=static&label=lymphaticfilariasis

47. Ovitrap

48. Mosquito oviposition kairomone Yadav et al., Tetrahedron: Asymmetry, 20 (2009), 1725–1730 Das et al., Carbohydrate Research, 358 (2012), 7–11

49. Mosquito oviposition kairomone E-allyl alcohol Chandrasekhar et al., Tetrahedron Lett., 36 (1995), 5071-5074 Das et al., Carbohydrate Research, 358 (2012), 7–11

50. Mosquito oviposition kairomone Das et al., Carbohydrate Research, 358 (2012), 7–11 Dibutyltin oxide : regioselective tosylation of primary alcohols

51. Mosquito oviposition kairomone Das et al., Carbohydrate Research, 358 (2012), 7–11

52. Flea beetle male pheromone

53. Evans chiral auxiliary Mori, Tetrahedron: Asymmetry, 16 (2005), 685–692

54. Asymmetric Alkylations of Evans Oxazolidinone Auxiliaries Evans, J. Am. Chem. Soc. 1982, 104, 1737-1739

55. Flea beetle male pheromone Weinreb amide Mori, Tetrahedron: Asymmetry, 16 (2005), 685–692

56. 'This therefore means that (R)-acid and (±)- acid make mixed crystals, and a mixture with about 75% ee more prone to separate.' Crystallization of 3-(4- tolyl)butanoic acid

57. Aphthona flava male pheromone ‘(R)-ar-himachalene is dextrorotatory in hexane, while levorotatory in chloroform’ Mori, Tetrahedron: Asymmetry, 16 (2005), 685–692

58. Weinreb ketone synthesis

59. Lesser grain borer (Rhyzopertha dominica) aggregation pheromone

60.  The biggest pest of stored grain in Australia  Thousands of tonnes of stored grain are lost each year in Australia alone  Use of dominicalure baited trap to detect, monitor and control infection Lesser grain borer pheromone http://australianmuseum.net.au/Lesser-Grain-Borer

61. E-acrylate Biswanath et al., Helvetica Chimica Acta, 89 (2006), 876-883 Lesser grain borer pheromone

62. Williams et al., J. Chem. Ecol., 7 (1981), 759 Lesser grain borer pheromone Synthesis of (S)-pentan-2-ol from (R)-glutamic acid (1981)

63. ‘After one catalytic run, the catalysts could be easily separated from the reaction mixture. The IL-bridged salen Mn(III) complexes could be reused at least five times without significant loss of activity and enantioselectivity’ Chengyong et al., Catalysis Communications, 15 (2011), 27–31 IL-bridged Salen Complex : Oxidative Kinetic Resolution (OKR) Lesser grain borer pheromone

64. Jacobsen

65. Sweat bee pheromone

66. Fürstner et al., J. Org. Chem., 61 (1996), 3942-3943 Sweat bee pheromone

67. ‘Thus, a sequence of only three steps converts well accessible substrates into the 21-membered lactone in 66% overall yield’ Fürstner et al., J. Org. Chem., 61 (1996), 3942-3943 Sweat bee pheromone

68. Gypsy moth (Lymantria dispar) sex pheromone

69.  Introduced in 1869 in US and infestation is expanding  In 1981, a record 52,200 km 2 were defoliated  1.2 million ha treated by mating disruption  37.5 g (rac)-disparlure is used by ha Gypsy moth pheromone Thorpe, Entomologia Experimentalis et Applicata, 125 (2007), 223–229 http://www.clintoncountypa.com/Gypsy%20Moth%20Website/general%20info.htm

70. ‘Moderate enantioselectivity of Sharpless epoxidation for (Z)- allylic alcohol has been observed. Fortunately epoxy is solid and could be enriched.’ Zhigang et al., Chin. J. Chem., 30 (2012), 23-28 Gypsy moth pheromone

71. Sharpless epoxidation Finn et al., J. Am. Chem. Soc., 113 (1991), 113-126

72. Zhigang et al., Chin. J. Chem., 30 (2012), 23-28 Gypsy moth pheromone

73. Southern pine beetle (Dendroctonus frontalis) aggregation pheromone

74.  Most destructive insect pest of pine in the southern US  900,000,000$ of damage to pine forests from 1960 through 1990  Frontalin attract the pest to tree treated with insecticide  (rac)-frontalin is half as active as (-)- frontalin Southern pine beetle pheromone Smith, USDA Forest Service Gen. Tech. Rep., (1990), PSW-121

75. 25% overall yield Rac. - Gram scale 50% activity of (-)-frontalin Protective group free Mori, Biosci. Biotechnolo. Biochem., 75 (2011), 976-981 Southern pine beetle pheromone

76. Cyclization Blackett et al., Tetrahedron, 26 (1970), 1311 Wasserman et al., JACS, 91 (1969), 3674

77. Thank you ! …. Any questions ?