PHEROMONES AND SEMIOCHEMICALS IN ANIMAL COMMUNICATION : OVERVIEW AND CHEMISTRY

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PHEROMONES AND SEMIOCHEMICALS IN ANIMAL
COMMUNICATION OVERVIEW AND CHEMISTRY

• Thibaut Denoel
• Department of Chemistry
• Cyclotron Research Center (CRC)
• Advisor André Luxen

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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

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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
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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.pher
obase.com/
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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), 75057523
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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), 75057523
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

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

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GC-EAG Electroantennogram
GC column effluent is split between FID and a
reservoir. Every 15 sec N2 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)
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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
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GC-SCR Single Cell Recording
Injection of a sample of cotton GC-SCR below
shows receptor neurons responding to compound
E,E-TMTT (trace amount) and to E,E-a-farnesene.
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.
Ulland, Chem. Senses, 33 (2008), 509-522.
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Structural characterization

• HPLC with or without derivatization (pg ng)
• GC-FTIR (ng)
• GC-MS (ng)
• Microdegradation (H2, O3, MeSSMe) (µg)
• Chiral columns (GC, HPLC)
• NMR 1H (lt 1 µg) and 13C (10-100 µg)

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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.

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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/vt2.html
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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
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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), 8998
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Different kinds of traps
Pheromone
No pheromone
Pheromone content, size, color and situation are
important.
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Philip McCabe's bee beard
200 000 bees (27 kg) are attracted by swarm lures
and queen bee pheromone on Philips skin!
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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)

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Domesticated silkmoth (Bombyx mori) sex pheromone
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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 (H2,
  KMnO4), 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
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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/bombykol/bombykolv.
htm
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Domesticated silkmoth pheromone

• After chromatography and derivatization 12 mg of
  Bombykol-NABA Structure elucidation

Hecker et al., Techniques in Pheromone Research
(1985), 1-44
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Domesticated silkmoth pheromone
Bombykol 1962 synthesis
Butenandt et al., Liebigs Ann. Chem., 658 (1962),
39-64
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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
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Domesticated silkmoth pheromone
Bombykol 1962 synthesis
Z-alkene
(10E,12E) 1 (10Z,12Z) 10 (10Z,12E)
10-3 (10E,12Z) 10-12
Butenandt et al., Liebigs Ann. Chem., 658 (1962),
39-64
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Urea inclusion complex
Linear molecules form inclusion complex with urea
Mayo et al., Journal of Solid State Chemistry,
141 (1998), 437-451
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Domesticated silkmoth pheromone
Bombykol 2012 synthesis
E-enals only
Z/E gt 91
De Figueiredo et al., J. Org. Chem., 72 (2007),
640-642
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Domesticated silkmoth pheromone
Hantzsch ester reduction
Zhu et al., J. Org. Chem., 64 (1999), 8980
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Pine moth (Dendrolimus spectabilis) pheromone
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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/
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Pine moth pheromone
Terminal alkyne
Internal alkyne
Lin et al., CN 102613177 (2012)
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Pine moth pheromone
Lin et al., CN 102613177 (2012)
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Pine moth pheromone
Z-alkene
Lin et al., CN 102613177 (2012)
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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
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Tse-Tse fly (Glossina austeni) contact sex
pheromone
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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.cf
m?id2401
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Roche ester by biotransformation
Leuenberger et al., Pure Appl. Chem., 62
(1990), 753-768
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Tse-Tse fly pheromone
Mori, Tetrahedron, 39 (1983), 3107-3109 Thomas et
al., J. Chem. Soc., Perkin Trans. 1 (1989),
507-518
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Tse-Tse fly pheromone
Kimura et al., Eur. J. Org. Chem. (2001),
3385-3390
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Tse-Tse fly pheromone
Kimura et al., Eur. J. Org. Chem. (2001),
3385-3390
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Tse-Tse fly pheromone
Kimura et al., Eur. J. Org. Chem. (2001),
3385-3390
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Southern House mosquito (Culex quinquefasciatus)
oviposition kairomone
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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?fuseacti
onstaticlabellymphaticfilariasis
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Ovitrap
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Mosquito oviposition kairomone
Yadav et al., Tetrahedron Asymmetry, 20 (2009),
17251730 Das et al., Carbohydrate Research, 358
(2012), 711
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Mosquito oviposition kairomone
E-allyl alcohol
Chandrasekhar et al., Tetrahedron Lett., 36
(1995), 5071-5074 Das et al., Carbohydrate
Research, 358 (2012), 711
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Mosquito oviposition kairomone
Dibutyltin oxide regioselective tosylation of
primary alcohols
Das et al., Carbohydrate Research, 358 (2012),
711
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Mosquito oviposition kairomone
Das et al., Carbohydrate Research, 358 (2012),
711
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Flea beetle male pheromone
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Flea beetle male pheromone
Evans chiral auxiliary
Mori, Tetrahedron Asymmetry, 16 (2005), 685692
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Asymmetric Alkylations of Evans Oxazolidinone
Auxiliaries
Evans, J. Am. Chem. Soc. 1982, 104, 1737-1739
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Flea beetle male pheromone
Weinreb amide
Mori, Tetrahedron Asymmetry, 16 (2005), 685692
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Crystallization of 3-(4-tolyl)butanoic acid
'This therefore means that (R)-acid and ()-acid
make mixed crystals, and a mixture with about 75
ee more prone to separate.'
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Aphthona flava male pheromone
(R)-ar-himachalene is dextrorotatory in hexane,
while levorotatory in chloroform
Mori, Tetrahedron Asymmetry, 16 (2005), 685692
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Weinreb ketone synthesis
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Lesser grain borer (Rhyzopertha dominica)
aggregation pheromone
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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
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Lesser grain borer pheromone
E-acrylate
Biswanath et al., Helvetica Chimica Acta, 89
(2006), 876-883
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Lesser grain borer pheromone
Synthesis of (S)-pentan-2-ol from (R)-glutamic
acid (1981)
Williams et al., J. Chem. Ecol., 7 (1981), 759
63
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), 2731
64
Jacobsen
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Sweat bee pheromone
66
Sweat bee pheromone
Fürstner et al., J. Org. Chem., 61 (1996),
3942-3943
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Sweat bee pheromone
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
68
Gypsy moth (Lymantria dispar) sex pheromone
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Gypsy moth pheromone

• Introduced in 1869 in US and infestation is
  expanding
• In 1981, a record 52,200 km2 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), 223229 http//www.clintoncountypa.com
/Gypsy20Moth20Website/general20info.htm
70
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
71
Sharpless epoxidation
Finn et al., J. Am. Chem. Soc., 113 (1991),
113-126
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Gypsy moth pheromone
Zhigang et al., Chin. J. Chem., 30 (2012), 23-28
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Southern pine beetle (Dendroctonus frontalis)
aggregation pheromone
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Southern pine beetle pheromone

• 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

Smith, USDA Forest Service Gen. Tech. Rep.,
(1990), PSW-121
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Southern pine beetle pheromone
Protective group free
25 overall yield Rac. - Gram scale 50 activity
of (-)-frontalin
Mori, Biosci. Biotechnolo. Biochem., 75 (2011),
976-981
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Cyclization
Blackett et al., Tetrahedron, 26 (1970),
1311 Wasserman et al., JACS, 91 (1969), 3674
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Thank you ! . Any questions
?