PHEROMONES AND SEMIOCHEMICALS IN ANIMAL COMMUNICATION OVERVIEW AND

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

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

Introduction : chemical ecology 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. Karlson et al. , Nature, 183 (1959), 55 -56

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/

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

Introduction : chirality in pheromones § 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

§ 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

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

Collection : SPME, body, flask Farine et al. , PLo. S ONE, 7 (2012), e 40396

Analysis Four specific detectors are used in GC : § GC-EAG (Electroantennogram) § GC-EAD (Electroantennographic Detector) § GC-SCR (Single Cell Recording) § GC-BB (Behavioural Bioassay) Sensibility : 0. 1 pg-100 ng

GC-EAG : Electroantennogram 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). 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-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. , 30 c (1975), 722 -725 Wyatt, Pheromones and animal behaviour (2003), 30

GC-SCR : Single Cell Recording Injection of a sample of cotton : 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. 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.

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

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.

Behavioural bioassay in wind tunnel 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. Valeur, http: //www. pheromone. ekol. lu. se/vt 2. html

Pheromones in pest management § Population monitoring : àFollow the density of a specific species with baited traps. § Mating disruption : àFlood the air with synthetic pheromones. Prevent adult males and females finding each other to mate. § Lure and kill, mass trapping : àAttract 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

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

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

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

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)

Domesticated silkmoth (Bombyx mori) sex pheromone

Domesticated silkmoth pheromone § 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, KMn. O 4), UV, IR, total synthesis and then mp Butenandt et al. , Z. Naturforsch. , 14 b (1959), 283 -284 Hecker et al. , Techniques in Pheromone Research (1985), 1 -44

Domesticated silkmoth pheromone 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/bombykolv. htm

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

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

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

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

Pine moth (Dendrolimus spectabilis) pheromone

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/

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

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

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

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

Tse-Tse fly (Glossina austeni) contact sex pheromone

Tse-Tse fly pheromone § Sleeping sickness caused by the parasite Trypanosoma brucei gambiense § 50, 000 to 70, 000 people infected § Cost 1, 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

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

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

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

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

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

Southern House mosquito (Culex quinquefasciatus) oviposition kairomone

Mosquito oviposition kairomone § 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 http: //www. ivmproject. net/about/index. cfm? fuseaction=static&label=lymphaticfilariasis

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

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

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

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

Lesser grain borer (Rhyzopertha dominica) aggregation pheromone

Lesser grain borer pheromone § 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 http: //australianmuseum. net. au/Lesser-Grain-Borer

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

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

Lesser grain borer pheromone Oxidative Kinetic Resolution (OKR) IL-bridged Salen Complex : ‘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

Gypsy moth (Lymantria dispar) sex pheromone

Gypsy moth pheromone § 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 Thorpe, Entomologia Experimentalis et Applicata, 125 (2007), 223– 229 http: //www. clintoncountypa. com/Gypsy%20 Moth%20 Website/general%20 info. htm

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

Southern pine beetle (Dendroctonus frontalis) aggregation pheromone

Southern pine beetle pheromone § Most destructive insect pest of pine in the southern US § 900, 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 Smith, USDA Forest Service Gen. Tech. Rep. , (1990), PSW-121

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

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