Pheromones and semiochemicals in animal communication: overview and chemistry

PHEROMONES AND

PHEROMONES AND

SEMIOCHEMICALS IN ANIMAL

SEMIOCHEMICALS IN ANIMAL

COMMUNICATION : OVERVIEW

COMMUNICATION : OVERVIEW

AND CHEMISTRY

AND CHEMISTRY

Thibaut Denoel

Thibaut Denoel

Department of Chemistry

Department of Chemistry

Cyclotron Research Center (CRC)

Cyclotron Research Center (CRC)

Advisor : André Luxen

Table of contents

Table of contents



Introduction :



Collection :

 Cold trapping volatiles  SPME, body, flask



Analysis :



Characterization :



Behavioural bioassay in wind tunnel



Pheromones in pest management :

 Population monitoring and mating disruption  Different kinds of traps

Introduction : chemical

Introduction : chemical

ecology

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_Pheromone - from Greek word - from Greek word phereinpherein (to transfert) and (to transfert) and hormonehormone (to (to excite) : semiochemical exchanged between individuals of the

excite) : semiochemical exchanged between individuals of the same same species.

species.

 Induces specific reaction such as special behaviour or developmental Induces specific reaction such as special behaviour or developmental process.

process.

 AllomoneAllomone : : interspecificinterspecific that that benefitbenefit the the originatororiginator but not the receiver. but not the receiver.

 KairomoneKairomone : : interspecificinterspecific that that benefitbenefit the the receiverreceiver but not the emitter. but not the emitter.

Introduction : pheromones

Introduction : pheromones



Sexual attraction, trail-following, territorial

_{Sexual attraction, trail-following, territorial}

boundaries, alarm, aggregation

boundaries, alarm, aggregation

…

…



Arthropods (

_{Arthropods (}

insects

insects

, arachnides…)

, arachnides…)



Sub

_Sub

nanogram

nanogram

to a few micrograms

to a few micrograms

emitted

emitted



1000

₁₀₀₀

molecules/ml

molecules/ml

of air to elicit a behaviour

of air to elicit a behaviour



_{7000 species and}

_{7000 species and}

_{3500 semiochemicals}

_{3500 semiochemicals}

₍₂₀₁₂₎

₍₂₀₁₂₎



Different functions

_{Different functions}

: hydrocarbon, alcohol,

: hydrocarbon, alcohol,

aldehyde, ketone, acetal, epoxide, ester,

aldehyde, ketone, acetal, epoxide, ester,

lactone, terpene, peptide, steroid…

lactone, terpene, peptide, steroid…



Chirality is important

_{Chirality is important}

for bioactivity

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

Introduction : chirality in

pheromones

pheromones



One enantiomer

_{One enantiomer}

is bioactive, and the

is bioactive, and the

antipode is not inhibitor

antipode is not inhibitor



One

_One

enantiomer

enantiomer

is bioactive, but the

is bioactive, but the

antipode is inhibitor

antipode is inhibitor



One enantiomer

_{One enantiomer}

is bioactive, but a

is bioactive, but a

diastereomer is inhibitor

diastereomer is inhibitor

O O (4S,6S,7S)-serricornin (4S,6S,7R)-serricornin ACTIVE INHIBITOR (CH₂)₉Me O (+)-(7R,8S)-disparlure (CH₂)₉Me O (-)-(7S,8R)-disparlure ACTIVE INHIBITOR O O 1 ₅ ()-(1S,5R)-frontalin O O 5 ₁ ()-(1R,5S)-frontalin

ACTIVE NOT AN INHIBITOR



One natural enantiomer

_{One natural enantiomer}

and the

and the

antipode

antipode

is also

is also

active

active



Mixture of enantiomers

Mixture of enantiomers

and

and

both

both

enantiomers are

enantiomers are

separately active

separately active



Different enantiomers

_{Different enantiomers}

-

-

different species

different species

OH OH

(R)-ipsdienol (S)-ipsdienol

Ips avulsus Ips par aconfusus

Mori, Bioorg. Med. Chem., 15 (2007), 7505–7523

O O (+)-(S)-dominicalure 1 O O (-)-(R)-dominicalure 1 ACTIVE

NATURAL ACTIVESYNTHETIC

OH OH

(R)-seudenol (S)-seudenol (55 : 45)

+

ACTIVE NATURALRATIO ACTIVE

Introduction : chirality in

Introduction : chirality in

pheromones



Both enantiomers

_{Both enantiomers}

are

are

necessary

necessary



One enantiomer

_{One enantiomer}

is most active but a stereoisomer can enhance it

is most active but a stereoisomer can enhance it



One enantiomer

_{One enantiomer}

is active on

is active on

males

males

,

,

the other

the other

is active on

is active on

females

females



Only the

Only the

meso

meso

-isomer

-isomer

is active

is active

OH OH (35 : 65) + NATURAL RATIO ACTIVE (R)-sulcatol (S)-sulcatol INACTIVE INACTIVE O O (R)-olean O O (S)-olean MALE FEMALE Me(CH₂)₁₁ (CH₂)₉ (CH₂)₁₁Me (13R,23S)-13,23-dimethylpentatriacontane meso- ACTIVE O (4R,8R)-tribolure ACTIVE O tribolure mixture MORE ACTIVE

Collection : cold trapping volatiles

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.

Collection : SPME, body, flask

Collection : SPME, body, flask

Analysis

Analysis



_{GC-EAG (Electroantennogram)}

_{GC-EAG (Electroantennogram)}



_{GC-EAD (Electroantennographic}

_{GC-EAD (Electroantennographic}

Detector)

Detector)



_{GC-SCR (Single Cell Recording)}

_{GC-SCR (Single Cell Recording)}



_{GC-BB (Behavioural Bioassay)}

_{GC-BB (Behavioural Bioassay)}

Sensibility : 0.1 pg-100 ng

Sensibility : 0.1 pg-100 ng

GC-EAG : Electroantennogram

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

flush it toward an insect antennae coupled

to an amplifier to measure an electroantennogram (EAG).

GC-EAD : Electroantennographic Detector

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

GC-SCR : Single Cell Recording

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.

Structural characterization

Structural characterization



_{HPLC with or without derivatization (pg – ng)}

_{HPLC with or without derivatization (pg – ng)}



_{GC-FTIR (ng)}

_{GC-FTIR (ng)}



_{GC-MS (ng)}

_{GC-MS (ng)}



Microdegradation (H

_{Microdegradation (H}

, O

, O

, MeSSMe…) (μg)

, MeSSMe…) (μg)



_{Chiral columns (GC, HPLC…)}

_{Chiral columns (GC, HPLC…)}

Stereoisomeric

Stereoisomeric

characterization

characterization



_All

_All

_{possibles stereoisomers}

_{possibles stereoisomers}

_{are synthesized.}

_{are synthesized.}



_{They are tested on}

_{They are tested on}

_chiral-GC

_chiral-GC

_{with bioassay}

_{with bioassay}

techniques

techniques

to

_to

determine

_determine

the

_the

stereochemistry

stereochemistry

of the natural compound.

of the natural compound.



_{The blend of actives compounds is tested in}

_{The blend of actives compounds is tested in}

wind tunnel against living insects

wind tunnel against living insects

in

in

behavioural bioassay.

Behavioural bioassay in wind tunnel

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.

Pheromones in pest management

Pheromones in pest management



Population monitoring

_{Population monitoring}

:

:



Follow the

_{Follow the}

density

density

of a specific species with baited

of a specific species with baited

traps.

traps.



Mating disruption :

_{Mating disruption :}



F

_F

lood

lood

the air with synthetic pheromones.

the air with synthetic pheromones.

Prevent

Prevent

adult males and females finding each other to

adult males and females finding each other to

mate

mate

.

.



Lure and kill, mass trapping :

_{Lure and kill, mass trapping :}



A

_A

ttract

ttract

with pheromones and

with pheromones and

kill

kill

responding

responding

individuals with insecticides.

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.

Population monitoring and mating

Population monitoring and mating

disruption

disruption

Population monitoring of the light brown apple moth in New

Zealand before and after mating disruption with pheromones (P).

Different kinds of traps

Different kinds of traps

No pheromone

Pheromone

Philip McCabe's bee beard

Philip McCabe's bee beard

200 000 bees (27 kg) are attracted by swarm lures and

queen bee pheromone on Philip’s skin!

Synthetic overview

Synthetic overview



_{Domesticated silkmoth sex pheromone (1962 &}

_{Domesticated silkmoth sex pheromone (1962 &}

2012)

2012)



_{Pine moth sex pheromone (2012)}

_{Pine moth sex pheromone (2012)}



Tse-tse fly contact sex pheromone (2001)

_{Tse-tse fly contact sex pheromone (2001)}



Mosquito oviposition kairomone (2012)

_{Mosquito oviposition kairomone (2012)}



Lesser grain borer aggregation pheromone

_{Lesser grain borer aggregation pheromone}

(2006)

(2006)



_{Gypsy moth sex pheromone (2012)}

_{Gypsy moth sex pheromone (2012)}



Southern pine beetle aggregation pheromone

_{Southern pine beetle aggregation pheromone}

(2011)

Domesticated silkmoth (

Domesticated silkmoth (

Bombyx

Bombyx

mori

mori

) sex pheromone

) sex pheromone

HO



_{Bombykol :}

_{Bombykol :}

_{first pheromone}

_{first pheromone}

_isolated

_isolated

(1939-1959) (Butenandt)

(1939-1959) (Butenandt)



_{500 000}

_{500 000}

_{female scent}

_{female scent}

_glands

_glands

_{of B.}

_{of B.}

_mori

_mori

extracted

extracted



_{Male ‘}

_{Male ‘}

_{flutter dance}

_{flutter dance}

_{’ used as the detector}

_{’ used as the detector}



_{12 mg}

_{12 mg}

_{of bombykol obtained}

_{of bombykol obtained}



_{Structure elucidation}

_{Structure elucidation}

_{by chromatographic}

_{by chromatographic}

properties, derivatization,

properties, derivatization,

microdegradation (H

microdegradation (H

, KMnO

, KMnO

), UV, IR,

), UV, IR,

total synthesis and then mp

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

Domesticated silkmoth

pheromone

Domesticated silkmoth

Domesticated silkmoth

pheromone

pheromone

Flutter dance : male silkmoth (right) gets excited by the

female (left). She is releasing bombykol from her gland.

Ph

₃

P

Br

Br

HO

NaH

n-Pr-Br

EtMgBr

HCHO

PBr

₃

PPh

₃

A

B

C

D

E

Domesticated silkmoth

Domesticated silkmoth

pheromone

_pheromone

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

O O O O O O O Ph₃P Br + H₂N NH₂ O (E)-isomer urea inclusion complex E F G G (Z)-isomer (E)-isomer PhLi

Domesticated silkmoth

Domesticated silkmoth

pheromone

pheromone

Several recrystallisations

of the complex to get pure

(E)-isomer

Bombykol 1962 synthesis

HO O O LiAlH4 O O H2, Lindlar Bombykol G H

Domesticated silkmoth

Domesticated silkmoth

pheromone

pheromone

_{Bombykol 1962 synthesis}

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

(10E,12E) : 1

(10Z,12Z) : 10

(10Z,12E) : 10

(10E,12Z) : 10

O O O O NH O O O O O O O O Ph₃P Br KOtBu 0°C, 6h Br HNBn₂.TFA 0.8 eq NaHMDS A B C Ph₃P 1. 2. 10% aq oxalic acid RT 12 h 0.8 eq D HO Bombykol

De Figueiredo et al., J. Org. Chem., 72 (2007), 640-642

Domesticated silkmoth

Domesticated silkmoth

pheromone

pheromone

E-enals only

Z/E > 9:1

Bombykol 2012 synthesis

Pine moth (

Pine moth (

Dendrolimus

_Dendrolimus

spectabilis

spectabilis

) pheromone

_{) pheromone}

OH R O O R O O R Pine moth pheromone :

Mixture of

(5Z,7E)-dodecadien-1-ol

(5Z,7E)-dodecadien-1-yl acetate

Pine moth pheromone

Pine moth pheromone



_{Pine forests defoliating}

_{Pine forests defoliating}

insect

insect



_{100 000 ha of pine}

_{100 000 ha of pine}

trees in Korea infested

trees in Korea infested

in 2007

in 2007



_{Up to 2000 caterpillars}

_{Up to 2000 caterpillars}

(1 kg) / tree

(1 kg) / tree

Pine moth pheromone

Pine moth pheromone

NH

NH

K

H

H

Brown et al., J. Am. Chem. Soc., 97 (1975), 891–892

H

Acetylene Zipper Reaction

R

K

HO

KH

H

N

NH

HO

'KAPA'

B

A

Terminal alkyne

Internal alkyne

Pine moth pheromone

Pine moth pheromone

HO

O

O

Cl

Cl

O

O

_Cl

O

O

O

Pd

CuI

MgBr

Fe

H

B

C

D

E

Pine moth pheromone

Pine moth pheromone

O

O

HO

O

O

Zn

H

CSA

O

SO

H

CSA =

E

F

(5Z,7E)-dodecadien-1-ol

Z-alkene

Pine moth pheromone

Pine moth pheromone

OH R O

O

R O

O R

Pine moth pheromone : Mixture of

(5Z,7E)-dodecadien-1-ol (5Z,7E)-dodecadien-1-yl acetate

(5Z,7E)-dodecadien-1-yl propionate

Ac₂O, Py

EtCOOCOEt, Py

A blend of 100/3/25 : OH/OAc/OPr is the best attractant.

Lin et al., CN 102613177 (2012)

Tse-Tse fly (

Tse-Tse fly (

Glossina austeni

Glossina austeni

)

)

contact sex pheromone

contact sex pheromone

Tse-Tse fly pheromone

Tse-Tse fly pheromone



_{Sleeping sickness caused by the parasite}

_{Sleeping sickness caused by the parasite}

Trypanosoma brucei gambiense

Trypanosoma brucei gambiense



_{50,000 to 70,000 people infected}

_{50,000 to 70,000 people infected}



_{Cost 1,000,000,000$/year in Africa}

_{Cost 1,000,000,000$/year in Africa}



_{Tse-Tse fly eradicated on Zanzibar in}

_{Tse-Tse fly eradicated on Zanzibar in}

1997 with the

1997 with the

Sterile Insect Technique

Sterile Insect Technique



_{Contact sex pheromone can be used to}

_{Contact sex pheromone can be used to}

rear competitive sterile males

rear competitive sterile males

O OH O OH HO OH O O O HO OR O HO OR O R = H or C₂H₅ or Pseudomonas putida

Stereospecific hydroxylation Pseudomonas putidaStereospecific hydration

Candida humicola

Stereoselective reduction

Gluconobacter roseus

Enantioselective oxidation

Roche ester by biotransformation

Roche ester by biotransformation

Tse-Tse fly pheromone

Tse-Tse fly pheromone

HO

O

O

O

I

O

HO

O

O

O

I

Si

(R)-Roche ester

A

B

Tse-Tse fly pheromone

Tse-Tse fly pheromone

O Si S O O O S O O O O Si O O O Si O I Si S O O CH₃ O I O Na(Hg) BuLi BuLi EtOH B C A D E

Tse-Tse fly pheromone

Tse-Tse fly pheromone

HO O O O O O S O O O O O O O Si TBAF TsCl, Py CH₂=CH(CH₂)₉MgBr Li₂CuCl₄ E F G H

Tse-Tse fly pheromone

Tse-Tse fly pheromone

OH O S O O O O TsOH, EtOH TsCl, Py Me(CH₂)₁₄MgBr Li₂CuCl₄ (13S,17R)-13,17-dimethyltritriacont-1-ene H I J

Southern House mosquito

Southern House mosquito

(

(

Culex quinquefasciatus

Culex quinquefasciatus

)

)

oviposition kairomone

oviposition kairomone



_{Vector of lymphatic filariasis and West Nile}

_{Vector of lymphatic filariasis and West Nile}

virus

virus



_{120 million people infected by filariasis}

_{120 million people infected by filariasis}

‘Elephantiasis’

‘Elephantiasis’



_{Ovitraps baited with the oviposition}

_{Ovitraps baited with the oviposition}

pheromone attract females to lay their eggs

pheromone attract females to lay their eggs



_{Water in the trap contain poison to larvae}

_{Water in the trap contain poison to larvae}

Mosquito oviposition

Mosquito oviposition

kairomone

kairomone

Mosquito oviposition kairomone

Mosquito oviposition kairomone

O O OH HO OH HO _HO O O O O OH N O O O O N H S O O O O O O O OH O 2,2-DMP TsOH acetone MeOH LiAlH₄ O O O O O NaIO₄ NH₂NHTs -gluconolactone _{A B} C D

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

Mosquito oviposition kairomone

Mosquito oviposition kairomone

N O O O O N S O O N O O O O N R R HO O O N O O O O N H S O O - N_2, - acetone - Ts 3 eq. n-C₈H₁₇MgBr D E

E-allyl alcohol

Chandrasekhar et al., Tetrahedron Lett., 36 (1995), 5071-5074 Das et al., Carbohydrate Research, 358 (2012), 7–11

Mosquito oviposition

Mosquito oviposition

kairomone

kairomone

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

Mosquito oviposition kairomone

Mosquito oviposition kairomone

O OH COOEt HO OH COOEt HO O O AcO O O O O COOEt BuLi, BF₃.OEt₂ H_2, Pd/C TsOH, benzene Ac₂O, DMAP (-)-(5R,6S)-6-acetoxy-5-hexadecanolide H I J K

Lesser grain borer (

Lesser grain borer (

Rhyzopertha

Rhyzopertha

dominica

dominica

) aggregation pheromone

_{) aggregation pheromone}

O

O



_The

_The

_{biggest pest of stored}

_{biggest pest of stored}

grain in Australia

grain in Australia



_{Thousands of tonnes of}

_{Thousands of tonnes of}

stored grain are lost each

stored grain are lost each

year in Australia alone

year in Australia alone



_{Use of dominicalure baited}

_{Use of dominicalure baited}

trap to detect, monitor and

trap to detect, monitor and

control infection

control infection

Lesser grain borer pheromone

Lesser grain borer pheromone

Baylis - Hillman

O

O

O

O

O

OH

O

O

OH

O

O

O

(+)-(S)-dominicalure 1

N

N

OH

NaBH

CuCl

MeOH

NaOH,

MeOH

1.SOCl

2.

A

B

C

D

E

(S)-pentan-2-ol

E-acrylate

Biswanath et al., Helvetica Chimica Acta, 89 (2006), 876-883

Lesser grain borer pheromone

HOOC COOH NH₂ O O O O CH₂I OH HO OH HNO₂ BH₃.Me₂S O OH O O OH O O O S O O (S)-pentan-2-ol TsCl, Py NaI acetone LiAlH₄ OH O TsCl, Py S O O LiAlH₄ E (R)-glutamic acid

Williams et al., J. Chem. Ecol., 7 (1981), 759

Lesser grain borer pheromone

Lesser grain borer pheromone

O N N O HN N O N N O NH N Mn Cl Br Mn Cl

‘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

Complex (1 mol%), PhI(OAc)₂ CH2Cl2 : H2O/KBr, 20°C ) + 95% ee OH OH (S)-pentan-2-ol E O

IL-bridged Salen Complex

:

Oxidative Kinetic Resolution (OKR)

Lesser grain borer

Lesser grain borer

pheromone

Gypsy moth (

Gypsy moth (

Lymantria dispar

_{Lymantria dispar}

)

₎

sex

sex

pheromone

_pheromone

O



_{Introduced in 1869 in US}

_{Introduced in 1869 in US}

and infestation is expanding

and infestation is expanding



_{In 1981, a record 52,200 km}

_{In 1981, a record 52,200 km}

were defoliated

were defoliated



_{1.2 million ha treated by}

_{1.2 million ha treated by}

mating disruption

mating disruption



_{37.5 g (}

_{37.5 g (}

_rac

_rac

_{)-disparlure is}

_{)-disparlure is}

used by ha

used by ha

Gypsy moth

Gypsy moth

pheromone

_pheromone

Thorpe, Entomologia Experimentalis et Applicata, 125 (2007), 223–229

‘Moderate enantioselectivity of

Sharpless epoxidation for

(Z)-allylic alcohol has been

observed. Fortunately epoxy is

solid and could be enriched.’

O

OH

OH

OH

OH

BuLi (2 eq.)

n-C

H

Br

H

Lindlar

Ti(i-PrO)

D-(-)-DIPT,

t-BuOOH, CaH

SiO

CH

Cl

-25°C, 72h

80% ee

after one recristallisation :

yield 60%, e.e. 98%

A

B

C

D

Zhigang et al., Chin. J. Chem., 30 (2012), 23-28

Gypsy moth

O

(+)-(7R,8S)-disparlure

O

O

OTf

O

OH

Tf

O, Et

N

BuLi

H

Pd/C

D

E

F

e.e. 98%

Zhigang et al., Chin. J. Chem., 30 (2012), 23-28

Gypsy moth

Southern pine beetle (

Southern pine beetle (

Dendroctonus

_Dendroctonus

frontalis

frontalis

) aggregation pheromone

_{) aggregation pheromone}

O

O

1

₅



_{Most destructive insect pest of pine in the}

_{Most destructive insect pest of pine in the}

southern US

southern US



_{900,000,000$ of damage to pine forests}

_{900,000,000$ of damage to pine forests}

from 1960 through 1990

from 1960 through 1990



_{Frontalin attract the pest to tree treated}

_{Frontalin attract the pest to tree treated}

with insecticide

with insecticide



₍

₍

_rac

_rac

_{)-frontalin is half as active as (-)-}

_{)-frontalin is half as active as}

(-)-frontalin

frontalin

Southern pine beetle

Southern pine beetle

pheromone

pheromone

25% overall yield

Rac. - Gram scale

50% activity of (-)-frontalin

Protective

group

free

Mori, Biosci. Biotechnolo. Biochem., 75 (2011), 976-981

Southern pine beetle

Southern pine beetle

pheromone

Thank you !

Thank you !

…. Any questions ?