Olfaction in Insects: A LookOlfaction in Insects: A Lookat the Chemistry and Biology of at the Chemistry and Biology of

Insect Sex PheromonesInsect Sex Pheromones

Jennifer N. Slaughter

OutlineOutline

I. Background

II. Bombykol

III. Chemical Characterization

IV. Synthesis1. Lineatin2. (+)-Grandisol3. Brazilian Stink Bug Pheromone4. Epianastrephin

V. Conclusions

IntroductionIntroduction

semiochemicals

pheromones allelochemicals

semiochemicals (Gk. semeon, meaning mark or signal)

pheromones (Gk. pherin, to carry and hormon, to excite)

Allelochemicals (Gk. allelon, of one another)

do Nascimento, R. R.; Morgan, E. D. Quim. Nova 1996, 19, 156-65.

Applications Applications

Biochemist: ideal models

Krieger J; Breer, H. Science 1999, 286, 720-23. Pheromones of Non-Lepidopteran Insects Associated with Agricultural Plants; Hardie, J. and Minks, A. K., Eds.; 1999.

Agricultural community: insect control (beetles)1. monitoring2. direct insect control3. in conjugation with microorganisms4. enhancing beneficial activity

Synthetic chemist: challenging targets

PheromonesPheromones

Insects largely perceive the world through molecular interactions.

As a result, their olfactory systems have evolved to an acute level of sensitivity and selectivity.

Moths with feathery antenna exhibit detection on molecular level.

>1600 insect species resulted in >300 unique chemical structures.

A newly hatched male gum emperor moth.

Roelofs, W. L. PNAS 1995, 92, 44-49

Stereochemistry and ActivityStereochemistry and Activity

roughly 10 categories

each combination represented

1. A single enantiomer is bioactive.

2. A bioactive pheromone is inhibited by the enantiomer

3. A bioactive pheromone is inhibited by the diastereomer

Mori, K. Chirality 1998, 10, 578-86.

O

O

exo-brevicomin(western pine beetle)

O disparlure(male gypsy moth)

OHO

serricornin(cigarette beetle)

Stereochemistry and ActivityStereochemistry and Activity

4. Stereoisomers of the natural pheromone are also active

5. The natural pheromone is an enantiomeric mixture; both are separately active

6. Different stereoisomers are employed by different species

Mori, K. Chirality 1998, 10, 578-86.

O

OH

H

H

(male spined citrus bug)

HOOH

+(female Douglas-fir beetle) 55:45 mix of R & S

OHOH

ipsdienol(Californina five-spined ips)

Stereochemistry and ActivityStereochemistry and Activity

8. One enantiomer is more active than the other

9. One enantiomer is active on males, while the other is active on females

Mori, K. Chirality 1998, 10, 578-86.

7. Both enantiomers are necessary for bioactivity; neither is separately active

OH OH

+sucatol, 35:65 mix(ambrosia beetle)

OH

(ant M. scabrinodis)

O

O

O

O olean(olive fruit fly)

(R) males (S) females

Insect Olfactory Receptor SystemInsect Olfactory Receptor System

male silkworm moth

How Do Pheromones Get from the How Do Pheromones Get from the Environment to the Receptor Cell?Environment to the Receptor Cell?

Pheromone TransportPheromone Transport

Pheromones are hydrophobic.

Water soluble PBPs pheromones at pore surfaces.

Elucidation of PBP-bombykol complex crystal structure

BombykolBombykol

OH

produced by the female silkworm moth Bombyx mori

structure elucidation by Butendant and co-workers in 1959

Techniques in Pheromone Research; Hummel, H. E. and Miller, T. A., Ed.; Springer-Verlag: New York, 1984.

Crystal Structure of PBP-bombykolCrystal Structure of PBP-bombykol

The 15.9 kDa PBP has approximate dimensions of 40 x 35 x 30 Å.

X-ray diffraction at 1.8Å resolution.

Sandler, B. H.; Nikonova, L.; Leal, W. S.; Clardy, J. Chemistry & Biology 2000, 7, 143-51.

BmPBP Binding PocketBmPBP Binding Pocket

Bombykol is found in a large flask-shaped cavity with a tiny opening at the surface.

Sandler, B. H.; Nikonova, L.; Leal, W. S.; Clardy, J. Chemistry & Biology 2000, 7, 143-51.

The only part of bombykol that is not surrounded by -helices is the hydroxyl end.

Bombykol Binding and ReleaseBombykol Binding and Release

BmPBP undergoes a pH-dependent conformational transition.

Briand, L.; Nespoulous, C.; Huet, J.; Takahshi, M.; Pernollet, J. Eur. J. Biochem 2001, 268, 752-60. Horst, R.; Damberger, F.; Luginbuhl, P.; Guntert, P.; Peng, G.;Nikonova, L.; Leal, W. S.; Wuthrich, K. PNAS 2001, 98, 14374-79. Wojtaesk, H.; Leal, W. S. J. Bio. Chem. 1999, 274, 30950-56.

BmPBP dos not bind ligands below pH 5.

BmPBP undergoes a conformational change when mixed with model proteins.

Structure of BmPBP at Low pHStructure of BmPBP at Low pH

1H frequency of 750 MHz at 20ºC and pH 4.5. For comparison, the X-ray crystals were grown at pH 8.2.

The most pronounced difference between the BmPBP complex and BmPBPA is the appearance of helix -7.

Horst, R.; Damberger, F.; Luginbuhl, P.; Guntert, P.; Peng, G.; Nikonova, L.; Leal, W. S.; Wuthrich, K. PNAS 2001, 98, 14374-79.

Representations of the BmPBP Representations of the BmPBP Binding PocketBinding Pocket

BmPBP complex (pH 8.2)

Horst, R.; Damberger, F.; Luginbuhl, P.; Guntert, P.; Peng, G.; Nikonova, L.; Leal, W. S.; Wuthrich, K. PNAS 2001, 98, 14374-79.

BmPBP in solution (pH 4.5)

Isolation and CharacterizationIsolation and Characterization

Pheromones are obtained in small quantities as volatile oils.

The first step is the separation of insect parts.

In earlier years of pheromone research, three techniques were widely used in the initial isolation of pheromones.

These have been replaced due to large volumes of solvent and massive amount of insect material required.

GC, GC-MS, IR, GC-FTIR

do Nascimento, R. R.; Morgan, E. D. Quim. Nova 1996, 19, 156-65.

Determination of StereochemistryDetermination of Stereochemistry

Mori, K. Chirality 1998, 10, 578-86.

Stereochemical assignment by conventional analysis is not possible.

Enantioselective synthesis of a target pheromone

Compare chiroptical properties to natural pheromone

Mori and co-workers have demonstrated the utility this approach.

(+)-Acoradiene: Determination of (+)-Acoradiene: Determination of Absolute ConfigurationAbsolute Configuration

1

4 5

1R, 4R, 5S

Kurosawa, S.; Bando, M.; Mori, K. Eur. J. Org. Chem. 2001, 4395-99.

The structure above was proposed on the basis of NMR studies.

It is a major component of the aggregation pheromone of the broad-horned flour beetle.

unique spiro-sesquiterpene structure

Retrosynthetic StrategyRetrosynthetic Strategy

Kurosawa, S.; Bando, M.; Mori, K. Eur. J. Org. Chem. 2001, 4395-99.

1

4 5

OH

OH

OP

OP

ring-closingolefin metathesis

O

O

O

(R)-(+)-pulegone

(+)-Acoradiene(+)-Acoradiene

Kurosawa, S.; Bando, M.; Mori, K. Eur. J. Org. Chem. 2001, 4395-99.

O

1. Br2, AcOH

2. i) NaOMe, MeOH ii) KOH, then dil. HCl (60%-2 steps)

CO2H

(R)-pluegone

1. Br2, NaOH, H2O

2. KOtBu, tBuOH

(39%-2 steps)

O

O

1. H2, PtO2, EtOAc (99%)

2. i) LDA, THF ii) allyl iodide, HMPA (96%)

O

O

ClMg

1. DIBAL-H, CH2Cl2 (98%)

2. THF (99%)

OH

OH

1

4 5

Grubbs catalyst

CH2Cl2 (98%)

OH

OH

RuCl

Cl

PCy3

PCy3

ph

Absolute ConfigurationAbsolute Configuration

OH

OH

11

11

The X-ray structure reflects the major isomer from the cyclization (1R, 4S, 5R, 10S).

Kurosawa, S.; Bando, M.; Mori, K. Eur. J. Org. Chem. 2001, 4395-99.

The synthetic and natural pheromones are different.

Syntheses of Pheromones withSyntheses of Pheromones withInteresting Carbon SkeletonsInteresting Carbon Skeletons

LineatinLineatin

O

O

O O

1

45

7

1

4

5

7

1R, 4S, 5R, 7R (+)striped ambrosia beetle

Aggregation pheromone of female ambrosia beetle

Baeckstrom, P.; Li, L.; Polec, I.; Unelius, C. R.; Wimalasiri, W. R. J. Org. Chem. 1991, 56, 3358-62.

(+)-enantiomer is the naturally occurring pheromone

a member of the first class

Retrosynthetic StrategyRetrosynthetic Strategy

Key step in the synthesis of lineatin is the [2 + 2] cycloadditions to form cyclobutane ring

Baeckstrom, P.; Li, L.; Polec, I.; Unelius, C. R.; Wimalasiri, W. R. J. Org. Chem. 1991, 56, 3358-62.

a b

O

CO

LineatinLineatin

Baeckstrom, P.; Li, L.; Polec, I.; Unelius, C. R.; Wimalasiri, W. R. J. Org. Chem. 1991, 56, 3358-62.

OEtO

P OEtOEt

O O

1. , LHMDA (92%, E:Z 4:1)

2. 10% KOH, MeOH, reflux (95%, mixture)HO

O

O

Ac2O

NaOAc (62%)

1. LAH,Et2O (79%)

2. Ac2O, pyridine (96%)AcO

AcO

O

OMeOMe1. OsO4, NMO (83%)

2. H5IO6, Et2O (97%)3. pTsOH, MeOH, (82%)

MeMgBr, Et2O

then 10% HCl (76%) O

O1

45

7

(+)-Grandisol(+)-Grandisol

sex pheromone of male cotton boll weevils.

alkylation and [2 + 2] cycloaddition.

(+)-grandisol

HO HO

(+)-fraganol

100- to 200-timess less active

Retrosynthetic StrategyRetrosynthetic Strategy

based on work done with C2-symmetric bis(,-butenolides)

de March, P.; Figuerdo, M.; Font, J.; Raya, J. Org. Lett. 2000, 2, 163-65.

HO

O

O

HO

O O

O O

HO OH

[2 + 2] cycloadditionfollowed by oxidativecleavage

asymmetric induction during the photoaddition process.

HO

O

O

OH

OH

HO

(+)-Grandisol(+)-Grandisol

Figuerdo, M.; Font, J.; Virgill, A. Tetrahedron 1987, 43, 1881-86.de March, P.; Figuerdo, M.; Font, J.; Raya, J. Org. Lett. 2000, 2, 163-65.

HO

O

O

OH

OH

HOO

O

O

O

1. PhSeCHCO2-2, THF

2. AcOH

3. H2O2, AcOH, THF

(72% overall)

O O

O O

HO OH

1. TMSIm, THF (98%)

2. CH2N2, ether/THF 3. 1,4-dioxane, (85% overall)

O O

O O

TMSO OTMS

1. ethylene, acetone, hv

2. TBAF, THF (65% overall)

O O

OHHO

O O

1. Pb(OAc)4, EtOAc

2. NaBH4, EtOAc (72% overall)

O

O

HO

HO

Retrosynthetic StrategyRetrosynthetic Strategy

Monteiro, H. J.; Zuckerman-Schpector, J. Tetrahedron 1996, 52, 3879-88.

HO

SO2Ph

O

OMe

PhO2SO

N2

OMe

Rh intramolecularcarbenoid cyclization

OH(+)-citronellol

(+)-Grandisol(+)-Grandisol

Monteiro, H. J.; Zuckerman-Schpector, J. Tetrahedron 1996, 52, 3879-88.

1. Rh2(OAc)4, C6H6 (60%)

2. NaI, TMSCl, MeCN (71%)

SO2Ph

O

I

NaH, THF (94%)

O SO2Ph

HO

OH

1. NaH, MeI, DME

2. OsO4, CrO3, Me2CO3. MeOH, H2SO4, CH2Cl2 (81% overall)

MeO

OMe

O

1. PhSO2CH2Na, THF/DMSO (92%)

2. NaN3, NaOAc, MeOH

OMe

OPhO2S

N2

N ClB

F F

FF+

Brazilian Stink Bug PheromoneBrazilian Stink Bug Pheromone

O O

R S

structure was confirmed by synthesizing the racemic mixture

Kuwahara, S.; Hamade, S.; Leal, W. S.; Ishikawa, J.; Kodama, O. Tetrahedron 2000, 56, 8111-17.

enantiomers were synthesized and separated

Retrosynthetic StrategyRetrosynthetic Strategy

Kuwahara, S.; Ishikawa, J.; Leal, W. S.; Hamade, S.; Kodama, O. Synthesis 2000, 1930-35.

[TS-S]

[TS-R]

O

O

R

S

O

N

O

O

N

O

O

I+

Brazilian Stink BugBrazilian Stink Bug

Kuwahara, S.; Ishikawa, J.; Leal, W. S.; Hamade, S.; Kodama, O. Synthesis 2000, 1930-35.

N

O

O

1. s-BuLi, THF-HMPA (74%)

2. s-BuLi, MeI, THF (84%)3. Red-Al, THF

I

N

O

OH

1. (n-Bu)4NH2PO4, H2O-EtOH

2. K2CO3, t-BuOH (45%-3 steps)

O1. LDA, PhN(Tf)2, THF (59%)

2. Me2CuLi, THF (86%)

190oC, toluene

methylene blue

+

R S

Brazilian Stink BugBrazilian Stink Bug

Kuwahara, S.; Hamade, S.; Leal, W. S.; Ishikawa, J.; Kodama, O. Tetrahedron 2000, 56, 8111-17. Kuwahara, S.; Ishikawa, J.; Leal, W. S.; Hamade, S.; Kodama, O. Synthesis 2000, 1930-35.

+

1. OsO4, py, then aq. NaHSO3

2. NaIO4, H2O-Et2O3. K2CO3, t-BuOH (65%-4 steps)

O

O

R (30%)

S (35%)S

R

1. MeLi, Et2O

2. PCC, CH2Cl2 (62%-2 steps)

O

R

1. Me2CuLi, Et2O

2. PhSeCl, EtOAc, then Na2CO3, THF-H2O, aq H2O2 (50%-2 steps)

O

S

EpianastrephinEpianastrephin

sex pheromone mixture of the male Caribbean fruit fly

Schultz, A. G.; Kirincich, S. J. J. Org. Chem. 1996, 61, 5626-30.

O

O

(+)-epianastrephin

O

O

(-)-epianastrephin

natural pheromone contains 55:45 mixture of enantiomers

a member of class five

relative stereochemistry determined by crystallographic studies

absolute stereochemistry established by chemical synthesis

(-)-Epianastrephin: Retrosynthesis(-)-Epianastrephin: Retrosynthesis

Tadano, K.; Isshiki, Y.; Minami, M.; Seiichiro, O. J. Org. Chem. 1993, 58, 6266-79.

O

O

OH

OP

O

O

O

O

R'R

CO2R

O

R'R

SmI2 coupling

(-)-Epianastrephin(-)-Epianastrephin

Tadano, K.; Isshiki, Y.; Minami, M.; Seiichiro, O. J. Org. Chem. 1993, 58, 6266-79.

O

O1. LAH, THF

2. TBDPSCl, imidazole, DMF3. PCC, CH2Cl2, mol. sieves

O

OH

O

O

O

O

O

EtO

O

O

O

OR

H

O

CO2Et

SmI2, i-PrOH

THF-HMPA (53%)

O

O

OR

EtO2C

I2SmO

.

O O

O

O

O

O

O

(-)-Epianastrephin(-)-Epianastrephin

Tadano, K.; Isshiki, Y.; Minami, M.; Seiichiro, O. J. Org. Chem. 1993, 58, 6266-79.

PCC, CH2Cl2

mol. sieves (48%) O

O

1. m-CPBA, NaHCO3 CH2Cl2

2. LAH, THF (96% overall)

OH

OH

OH

OH

+

O

OHZn, CH2Br2,

TiCl4, THF(83%-4 steps)

OH

(+)-Epianastrephin: Retrosynthesis(+)-Epianastrephin: Retrosynthesis

Schultz, A. G.; Kirincich, S. J. J. Org. Chem. 1996, 61, 5626-30.Schultz, A. G.; Kirincich, S. J. J. Org. Chem. 1996, 61, 5631-34.

O

O

O

O

OMe

OMe

CO2R

iodolactonization

N

OMOM

O OMe

Birch reduction methylation

(+)-Epianastrephin(+)-Epianastrephin

Schultz, A. G.; Kirincich, S. J. J. Org. Chem. 1996, 61, 5626-30.

1. 10% Pd/C, H2, EtOAc (95%)

2. H2SO4, MeOH, H2O (84%)3. HC(OMe)3, MeOH, H2SO44. KOH, MeOH (86%)

CO2H

OMe

N

OMOM

O OMe

K, NH3, t-BuOH, THF

then piperylene, MeI(91%)

N

OMOM

O OMe

1. NaHCO3, H2O, THF I2, KI (85%)

2. AIBN, Bu3SnH, PhH (94%)

O

O

OMe

1. RuO4, NaIO4, CCl4 MeCN, H2O

2. KOH, MeOH, H2O (83%)3. (COCl)2, PhH, then Li(t-BuO)3AlH, THF4. SEMCl, DIPEA

O

O

OSEM

TPAP, NMO

CH2Cl2, MeCN(63%) O

O

1. DIBAL, CH2Cl2 (70%)

2. Ph3P=CH2, DMSO (85%)3. HOAc, MeOH, MeCN OH

OH

ConclusionsConclusions

insect olfactory system

pheromone transport (BmPBP)

isolation and characterization

synthetic challenges

AcknowledgementsAcknowledgements

Members of the Mecozzi Group:

Sandro Khanh Oana

Peers:

Whitney Erwin Valerie Keller Jason Pontrello Margaret Biddle Lisa Jungbauer John Campbell Erik Puffer Scott Petersen Matthias Brewer Nero Shah Konstantin Levitsky