(2R,5S)-Theaspirane Identified as the Kairomone for the Banana Weevil, Cosmopolites sordidus, from Attractive Senesced Leaves of the Host Banana, Musa spp.

Article abstract of DOI:10.1002/chem.201800315

The principal active component produced by highly attractive senesced host banana leaves, Musa spp., for the banana weevil, Cosmopolites sordidus, is shown by coupled gas chromatography-electroantennography (GC-EAG), coupled GC-mass spectrometry (GC-MS),

Full text of DOI:10.1002/chem.201800315

A Journal of
Accepted Article
Title: (2R,5S)-Theaspirane Identified as the Kairomone for the Banana
Weevil, Cosmopolites sordidus, from Senesced Leaves of the
Host Banana, Musa spp.
Authors: Samson Abagale, Christine Woodcock, Antony Hooper, John
Caulfield, David Withall, Keith Chamberlain, Samuel Osafo-
Acquaah, Helmut van Emden, Haruna Braimah, john pickett,
and Michael A. Birkett
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To be cited as: Chem. Eur. J. 10.1002/chem.201800315
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10.1002/chem.201800315
Chemistry - A European Journal
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For: Chemistry – A European Journal
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(2R,5S)-Theaspirane Identified as the Kairomone for the Banana Weevil, Cosmopolites
sordidus, from Attractive Senesced Leaves of the Host Banana, Musa spp.
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Short title: Banana weevil attractant identification
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Samson A. Abagale,a,b Christine M. Woodcock,c Antony M. Hooper,d John C. Caulfield,c
David Withall,c Keith Chamberlain,c Samuel O. Acquaah,b Helmut Van Emden,d Haruna
Braimah,a* John A. Pickettf and Michael A. Birkett c*
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aCrops Research Institute, Council for Scientific and Industrial Research, P.O. BOX 3785,
Fumesua-Kumasi, Ghana. E-mail: braimah_haruna@yahoo.co.uk
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bDepartment of Chemistry, Kwame Nkrumah University of Science and Technology, PMB,
Kumasi, Ghana. E-mail: samnabgh@yahoo.com
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cDepartment of Biointeractions and Crop Protection, Rothamsted Research, Harpenden,
Hertfordshire, AL5 2JQ, UK. E-mail: mike.birkett@rothamsted.ac.uk
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dSchool of Biological and Chemical Sciences, Queen Mary University of London, London E1
4NS, United Kingdom. E-mail: a.hooper@qmul.ac.uk
eSchool of Agriculture, Policy and Development, The University of Reading, Earley Gate, PO
Box 237, Reading, Berkshire, RG6 6AR, UK. E-mail: h.f.vanemden@reading.ac.uk
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fSchool of Chemistry, Cardiff University, Cardiff, Wales CF10 3AT, United Kingdom. E-mail:
PickettJ4@cardiff.ac.uk
*Corresponding author
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The principal active component produced by highly attractive senesced host banana
leaves, Musa spp., for the banana weevil, Cosmopolites sordidus, is shown by coupled gas
chromatography-electroantennography (GC-EAG), coupled GC-mass spectrometry
(GC-MS), chemical synthesis and coupled enantioselective (chiral) GC-EAG to be
(2R,5S)-theaspirane. In laboratory behaviour tests, the synthetic compound is as
attractive as natural host leaf material and presents a new opportunity for pest control.
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The banana weevil, Cosmopolites sordidus Germar (Coleoptera, Curculionidae), is the most
important insect pest of bananas and plantains, Musa spp.1-3 throughout the world. Feeding
damage is caused by larvae of C. sordidus which are protected within the plant tissue, and so
management strategies target adult weevils. Pheromones and other semiochemicals (naturally-
occurring behaviour- or development-modifying chemicals) constitute important tools for
monitoring and detecting insect populations. A male-produced aggregation pheromone,
(1S,3R,5R,7S)-sordidin, has been identified for C. sordidus.4 For smallholder farmers in Ghana,
for whom banana and plantain provide staple food, (1S,3R,5R,7S)-sordidin is deemed to be too
expensive, and alternative semiochemical-based tools are urgently sought. Previous studies
have shown that host plant location by adult C. sordidus is influenced by a highly attractive
volatile kairomone from senesced banana leaves,5,6 which, if identified, could provide an
effective and affordable alternative lure for management of C. sordidus on smallholder farms.
The purpose of this work was to identify the active component(s) from volatile material
collected from senesced leaves, using coupled gas chromatography-electroantennography
(GC-EAG) recordings from the antennae of adult female C. sordidus, and confirm the
attractiveness of the identified compound(s), thereby providing the quality assurance for using
senesced banana leaves as an ethnobotanically-based locally produced material in C. sordidus
management.
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Coupled GC-EAG (ESI) analysis with natural volatile material collected from senesced banana
leaf material confirmed that the attractiveness of the material was caused by a very minor
component with highly significant EAG activity (Figure 1). The 70eV EI mass spectrum of the
unknown EAG-active component (Figure 2) showed a base peak at m/z 138, an additional
diagnostic fragment at m/z 179 and a molecular ion at m/z 194. Comparison of this spectrum
with the literature7,8 suggested a theaspirane isomer 1, the base peak being rationalised by loss
formally of isobutene (C4H8) via a retro Diels-Alder rearrangement (Figure 2 inset). The
presence of two stereocentres (at the 2- and 5- positions) gives four possible stereoisomers,
produced initially as the mixture, by chemoenzymatic synthesis from dihydro--ionone 2
(Scheme). To approach resolution of the natural EAG active isomer, initial reduction of 2 with
sodium borohydride in a non-stereospecific manner gave a mixture of the (R) and (S)-isomers
of dihydro--ionol in overall 100% yield. The mixture of ionol isomers was resolved
chemoenzymatically using lipase-mediated acetylation (Pseudomonas cepaciae lipase Amano
PS-C, vinyl acetate, 99.2% ee R, 94.8% ee S). By adjusting incubation time, it was possible to
obtain 99.1% ee S. Following separation of the (R)-ionol acetate and the (S)-ionol by silica gel
liquid chromatography, the ionol then underwent intramolecular 5-exo-trig cyclisation upon
heat treatment with selenium dioxide in dioxan to generate a diastereomeric pair of theaspirane
isomers ((2S,5S)-1, (2S,5R)-1) (ESI), overall 35% yield over 2 steps). Cleavage of the (R)-
acetate (using potassium hydroxide in aqueous methanol) followed by similar treatment of the
(R)-ionol with selenium dioxide in dioxan furnished the other diastereomeric pair of
theaspirane isomers ((2R,5R)-1, (2R,5S)-1) (ESI) in overall 41% yield over 2 steps. The
diastereoisomers were difficult to separate on silica gel (4% diethyl ether in petroleum ether)
due to their lack of polarity and so the isolated diastereomeric excesses were variable and mixed
fractions reduced recovery. However, a purified enantiomer of the synthetic natural product,
(2R,5S)-1, was obtained in 98.7% ee, 99.5% de. To verify the relative stereochemistry, nuclear
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Overhauser experiments on the (2R,5S)-1 showed a nOe correlation between the 6-Me groups
and the H-2 proton showing this proton must be on the face of the tetrahydrofuran moiety
facing to the C-6 gem-dimethyl group (ESI). Complementary verification was observed by
analysing (5R,2R)-1 in which a nOe correlation was observed between the 2-Me group and the
C-6 gem-dimethyl group. Coupled enantioselective (chiral) GC-EAG (ESI) analysis using a
mix of all four synthetic isomers revealed the relative GC retention times of the isomers (Figure
3, upper trace), and comparison with coupled enantioselective GC-EAG analysis using the
natural volatile material collected from senesced banana leaf material revealed matching GC
retention times for the (2R,5S)-isomer and the natural theaspirane isomer (Figure 3 lower trace),
thus confirming the identity of the electrophysiologically active naturally-occurring isomer to
be (2R,5S)-1.
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In behaviour assays with female C. sordidus conducted in a linear three chamber olfactometer
(ESI), senesced banana leaf material and collected volatile organic compounds (VOCs) were
significantly more attractive (P = 0.013 and 0.001 respectively) than controls and were equally
attractive in dual-choice assays. A mixture of the natural (2R,5S)-1 and non-natural (2S,5R)-1
isomers was behaviourally active at a dose of 0.2 g and 0.02 micrograms (g) (Students’ t-
test; P < 0.003, P<0.01 respectively). A mixture of the non-natural (2S,5S)-1 and (2R,5R)-1
isomers was shown to have behavioural activity only at a dose of 0.2 g (P = 0.04), in spite of
the observed EAG activity for (2S,5S)-1. A mixture of all four isomers of 1 was behaviourally
active at all doses tested ie. 2 (tested twice), 0.2 and 0.02 g (P = 0.001, 0.017, 0.001 and 0.002
respectively). When tested in combination with commercially available sordidin (Cosmolure),
a mixture of (2R,5S)-1 and (2S,5R)-1 at a dose of 0.05 g synergised the activity of the
pheromone (P = 0.04). The EAG data suggests that antennal detection of the theaspiranes
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requires a particular structural motif, ie. 5S stereochemistry, but that a specific overall 3D
structure of the compound, ie (2R,5S), is required to elicit the behavioural response in adult
female C. sordidus. Our data suggest that the newly identified compound (2R,5S)-1, present in
minor quantities in senesced banana leaf material, is responsible for the attraction of adult
female C. sordidus and is therefore the major kairomone component. The identification
provides the quality assurance for the deployment of readily available senesced banana leaf
material, or locally-produced extracts thereof, as a lure component of affordable trapping
technology that can manage C. sordidus on smallholder banana and plantain farms.
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This work was supported by a Royal Society Leverhulme Africa Award, which supported S.
Abagale during his PhD. Rothamsted Research receives grant-aided support from the United
Kingdom Biotechnology and Biological Sciences Research Council (BBSRC).
1. H.E. Ostmark, Ann. Rev. Entom. 1974, 19, 161.
2. D.F. Waterhouse and K.R. Norris, Biological Control: Pacific Prospects. Inkata Press.
Melbourne. 1987, 454 pp.
3. C.S. Gold, P.R. Speijer, E.B. Karamura and N.D. Rukazambuga, Proc. Banana
Nematode/Borer Weevil Conference. Kuala Lumpur, 1994, 170.
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4. J. Beauhaire, P.H. Ducrot, C. Malosse and D. Rochat, Tetrahedron Lett. 1995, 36, 1043.
5. H. Braimah, PhD Thesis, 1997, University of Reading, UK.
6. H. Braimah and H. F. van Emden, Bull. Ent. Res. 1999, 89, 485.
7. G. Ohloff, Fortschr. Chem. Org. Naturst. 1978, 35, 431.
8. H. Masuda and S. Mihara, Agric. Biol. Chem. 1985, 49, 861.
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Figure 1. Coupled GC-EAG responses of adult C. sordidus to natural volatile material collected
from senesced banana leaves volatile material collected by headspace collection, on a non-
polar DB-1 GC column. The annotated peak is a minor component with major consistent EAG
activity.
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EAG
FID
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Figure 2. 70eV EI mass spectrum of EAG-active compound identified from natural volatile
material collected from senesced banana leaves (upper), identified as a theaspirane isomer 1
and NIST-MS of theaspirane (lower). Inset: retro-Diels-Alder re-arrangement of parent ion
from 1.
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Figure 3. Enantioselective (chiral) coupled gas chromatography-electroantennography (GC-
EAG) analysis of the four synthesized theaspirane isomers (upper traces) and natural volatile
material collected from senesced banana leaves (lower traces), showing alignment of the
(2R,5S)-isomer 1 with the natural theaspirane isomer and the single EAG peak for the natural
isomer.
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EAG
(2S,5R)-1
(2S,5S)-1
FID
(2R,5S)-1
(2R,5R)-1
EAG
(2R,5S)-1
FID
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Scheme. Chemoenzymatic synthesis of theaspirane isomers.
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