(6 R,10 S)-pallantione: The first ketone identified as sex pheromone in stink bugs

Article abstract of DOI:10.1021/ol400413w

This work describes the structural elucidation of the sex pheromone of the soybean stink bug, Pallantia macunaima. The biological activity of the synthetic pheromone was demonstrated by behavioral and EAD experiments. Furthermore, the absolute configuration of the natural pheromone was determined as (6R,10S)-6,10,13-trimethyltetradecan-2-one. This is the first ketone identified as a male-produced sex pheromone in Pentatomidae, and the trivial name, pallantione, was assigned to this novel pheromone molecule.

Full text of DOI:10.1021/ol400413w

ORGANIC
LETTERS
2013
Vol. 15, No. 8
1822–1825
(6R,10S)‑Pallantione: The First Ketone
Identified as Sex Pheromone in Stink Bugs
†
†
‡
§
ꢀ
Carla F. Favaro, Rafael A. Soldi, Tetsu Ando, Jeffrey R. Aldrich, and
Paulo H. G. Zarbin*,†
ꢀ
Universidade Federal do Parana, 81531-990 Curitiba-PR, Brazil, Tokyo University
of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, and University of
California - Davis, 519 Washington Street, Santa Cruz, California 95060, United States
pzarbin@ufpr.br
Received February 12, 2013
ABSTRACT
This work describes the structural elucidation of the sex pheromone of the soybean stink bug, Pallantia macunaima. The biological activity of the
synthetic pheromone was demonstrated by behavioral and EAD experiments. Furthermore, the absolute configuration of the natural pheromone
was determined as (6R,10S)-6,10,13-trimethyltetradecan-2-one. This is the first ketone identified as a male-produced sex pheromone in
Pentatomidae, and the trivial name, pallantione, was assigned to this novel pheromone molecule.
To date, pheromones identified for pentatomid species
(stink bugs) exhibit a wide range of molecular structures,
such as saturated hydrocarbons, terpenes and terpenoids,
and methyl esters.1ꢀ6
soybean pest in Brazil.7 We now report the structural
elucidation, synthesis, and absolute configuration of the
sex pheromone for this species, including electrophysiolog-
ical and behavioral activity.
Stink bugs produce two types of volatile chemicals,
defensive compounds (allomones) and pheromones.
Previously, we identified the allomones produced by
adults and nymphs of Pallantia macunaima, an important
The GC analysis of volatiles produced by males and
females revealed the presence of a male-specific compound
that elicited a strong electrophysiological response only
from the antennae of females of the species (Figure 1).
Kovats indices for this compound were calculated on three
different chromatographic columns, RTX-5, EC-WAX,
and EC-1, with values of 1754, 2035, and 1735, respectively.
Bioassays showed that male aeration extracts are sig-
nificantly more attractive to females than were the controls
[treatment 30 (65%), control 16 (35%), N = 46, P < 0.05];
however, the same extract was not attractive to males,
suggesting the existence of a male-produced sex pheromone.
The male-specific compound was analyzed by GC-
FTIR, and the infrared spectrum (Figure 2A) showed
†
ꢀ
Universidade Federal do Parana.
‡ Tokyo University of Agriculture and Technology.
§ University of CaliforniaꢀDavis.
(1) Soldi, R. A.; Rodrigues, M. A. C. M.; Aldrich, J. R.; Zarbin,
P. H. G. J. Chem. Ecol. 2012, 38, 814.
(2) Moraes, M. C. B.; Pareja, M.; Laumann, R. A.; Borges, M.
Neotrop. Entomol. 2008, 37, 489.
(3) Leal, W. S.; Kuwahara, S.; Shi, X.; Higuchi, H.; Marino, C. E. B.;
Ono, M.; Meinwald, J. J. Chem Ecol. 1998, 24, 1817.
(4) Aldrich, J. R.; Lusby, W. R.; Marron, B. E.; Nicolaou, K. C.;
Hoffmann, M. P.; Wilson, L. T. Naturwissenschaften 1989, 76, 173.
(5) Borges, M.; Mori, K.; Costa, M. L. M.; Sujii, E. R. J. Appl.
Entomol. 1998, 122, 335.
ꢀ
ꢀ
(6) Zarbin, P. H. G.; Favaro, C. F.; Vidal, D. M.; Rodrigues, M. A.
(7) Favaro, C. F.; Rodrigues, M. A. C. d. M.; Aldrich, J. R.; Zarbin,
C. M. J. Chem. Ecol. 2012, 38, 825.
P. H. G. J. Braz. Chem. Soc. 2011, 22, 58.
r
10.1021/ol400413w
Published on Web 04/01/2013
2013 American Chemical Society

Figure 1. Gas chromatograms of volatiles produced by P.
macunaima males (2) and females (3) and the antennal response
of a female to the male-specific compound (1) (∼50 ng).
absorption at 1716 cmꢀ1 and 1168 cmꢀ1, related to CdO
and the CꢀCꢀC stretching characteristic of a ketone
functionality. The intense symmetrical (1378 cmꢀ1) and
asymmetrical (1466 cmꢀ1) bands indicative of methyl
groups were also observed, suggesting a chemical structure
containing at least two methyl branches. The mass spectrum
(Figure 2B) had a molecular ion at m/z 254, and a fragment
at m/z 236 (Mþ ꢀ18) due to loss of a water molecule.
The base peak at m/z 58, attributed to McLafferty
rearrangement,8 suggested the presence of a carbonyl group
at C-2. Neither mass nor infrared spectra showed any
indication of carbonꢀcarbon double bonds in the molecule.
Therefore, the empirical formula of the molecule is C17H34O.
GCꢀMS analysis of a silylated alcohol derivative
(obtained by reaction with LiAlH4 followed by TMSCl)
established the carbonyl position at C-2, since a base peak
was observed at m/z 117 due to a cleavage R to the siloxy
group (Figures S1 and S2, Supporting Information [SI]).9
To further verify the structural elucidation, a catalytic
hydrogenation of geranyl acetone (6,10-dimethylundecan-2-
one) was performed because the product would, theoretically,
exhibit a structural pattern similar to that of the natural
pheromone (a carbonyl at C-2 and two methyl branches). As
expected, the hydrogenated geranyl acetone exhibited a base
peak at m/z 58, and a fragment at m/z 180 due to loss of a
molecule of water (Mþ ꢀ18). A similarity in the intensity of
fragments at m/z 113 and m/z 95 was also observed for the
geranyl acetone product, comparable to the corresponding
fragments in the mass spectrum of the natural product for
a methyl branch at C-6 in the carbon chain. This strong
similarity suggested the chemical structure of the male-specific
compound also has a methyl branch at the same position as
that in the hydrogenation product of geranyl acetone.
The relative intensity of the fragment at m/z 165
(m/z 183; Mþ ꢀ18) in the MS of the natural compound,
suggested the presence of a second methyl branch at C-10.
On the basis of the above data, the proposed chemical
structure was determined to be 6,10-dimethylpentadecan-
2-one (1).
Figure 2. Infrared (A) and electron impact mass spectra (B) of
the male-specific P. macunaima compound.
Compound 1 was synthesized as a mixture of all possible
stereoisomers, employing geranyl acetone (2) as the start-
ing material (Figure 3). The initial step was carbonyl
protection with ethylene glycol, which led to the ketal 3.
An allylic oxidation of ketal 3 was performed using a
mixture of selenium dioxide (SeO2) and tert-butylhydro-
peroxide (t-BuOOH) resulting in the allyl alcohol, which
was readily oxidized with PCC to the aldehyde 4.10
The product of the Wittig reaction of phosphonium salt 5
(obtained from n-butyl bromide and triphenylphosphine)
with the aldehyde 4led to formation of compound 6.11 Ketal
6 was submitted to catalytic hydrogenation over Pd/C, and
the hydrogenated product was deprotected with oxalic acid
as catalyst,12 resulting in the desired ketone 1 in 30.3%
overall yield.
Figure 3. Synthetic route for the 6,10-dimethylpentadecan-2-one (1).
(10) McMurry, J. E.; Dushin, R. G. J. Am. Chem. Soc. 1990, 112,
6942.
(11) Li, J. J.; Limberakis, C.; Pflum, D. A. Modern Organic Synthesis
in the Laboratory: A Collection of Standard Experimental Procedures;
Oxiford University Press: New York, 2007.
(12) Babler, J. H.; Malek, N. C.; Coghlan, M. J. J. Org. Chem. 1978,
43, 1821.
(8) Silverstein, R. M.; Webster, F. X.; Kiemle, D. J., Spectrometric
Identification of Organic Compounds; John Wiley & Sons: New York, 2005.
(9) Attygalle, A. B. In Methods in Chemical Ecology; Millar, J. G.,
Haynes, K. F., Eds.; Chapman & Hall: New York, 1998; Vol. 1, p 207.
Org. Lett., Vol. 15, No. 8, 2013
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The fragmentation pattern of the mass spectrum of
synthetic 1 was very similar to that of the natural product
(Figure S3 in SI). However, there were huge differences in
the Kovats indices (1790/RTX-5; 2085/EC-WAX; 1772/
EC-1) of synthetic 1 vs those of the natural product.
The differing retention times of the synthetic standard
and natural compound suggested that the natural product
should contain three methyl branches in a C14 carbon chain
instead of two methyl branches in a C15 carbon chain.
To better evaluate the branch positions, a derivatization of
the natural product was performed with tosylhydrazine to
obtain the tosylhydrazone product. Subsequently, the prod-
uct was reduced with LiAlH4 and LiAlD4 to obtain the
basic carbon skeleton with two hydrogens or deuterons in
place of oxygen.13 In addition, the deuterated and hydro-
genated products were analyzed by GCꢀMS with lower
ionization energy (20 eV); at lower energies, the more stable
fragments (which are the diagnostic fragments from cleavage
of the bonds adjacent to branches) are usually enhanced.8
The resulting hydrocarbon mass spectrum (Figure 4A),
showed that the relative intensities of the fragments m/z 99
and m/z 169 are quite prominent; these fragments were
associated with the methyl branches at positions C-6 and
C-10. The deuteride spectrum confirmed the methyl branch
at the C-6 position, since an increase in the intensity of the
fragments at m/z 100/101 was observed.1 Also, in the deute-
ride spectrum, the relative intensity of the m/z 169 fragment
decreased by about 50% relative to that of the corresponding
hydrogenated product, plus an m/z 171 ion was present,
confirming the C-10 branch position (Figure 4B).
this fragment did not differ from the deuteride spectrum,
indicating that this ion is derived from the part of the
molecule that does not have deuterium.
The m/z 57 ion could be associated with a sec- or an iso-
butyl fragment, both of which are more stable than n-butyl
and, thereby, justify the large relative intensity (Figure 4A
and B). From this observation, we deduced the presence of
a third methyl branch at positions C-12 or C-13. Thus, two
other chemical structures for the natural product were
proposed: 6,10,12-trimethyltetradecan-2-one (7) and 6,10,
13-trimethyltetradecan-2-one (8).
Compounds 7 and 8 were synthesized as a mixture of all
possible isomers using the same methodology previously
employed in the synthesis of ketone 1. In these two synthetic
routes, the phosphonium salts 9 and 11 were employed to
obtain the ketals 10 and 12 by a Wittig reaction with
aldehyde 4. After hydrogenation and deprotection, ketones
7 and 8 were obtained from 4 in 64.0% and 69.9% yield,
respectively (Figure 5).
Ketone 7eluted as two barely resolved peaks by GC due to
separation of the diasteroisomers, with Kovats indices close
to those for the natural product. The mass spectrum of 7 was
also similar to that for the natural product (Figure S4, SI).
However, when synthetic standard 7 was co-injected with the
natural product, the natural compound eluted between the
synthetic diasteroisomers (Figure S5A in SI).
On the other hand, synthetic ketone 8 and the natural
product co-eluted on three different GC columns tested
(Figure S5B, SI; EC-WAX and EC-1 data are not shown).
In addition, mass and infrared spectra of the synthetic
compound (p S16, SI) were identical to those for the
natural product, confirming the chemical structure of the
P. macunaima male-specific compound as that of 6,10,13-
trimethyltetradecan-2-one (8).
Another fragment of notable relative intensity in the
hydrocarbon spectrum was the m/z 57 of much higher
intensity than that for unbranched hydrocarbons. However,
Figure 5. Synthetic route for the 6,10,12-trimetyltetradecan-2-
one (7) and 6,10,13-trimetyltetradecan-2-one (8).
Synthetic ketone 8 showed the same bioactivity for anten-
nae of females by GC-EAD as that of the natural compound.
In addition, of 34 females tested in a olfactometer, 25 (76%)
chose the source containing 8 (P < 0.001). However, only
Figure 4. Mass spectra (20 eV) of LiAlH4 (A) and LiAlD4 (B)
hydrocarbon derivatives from the natural Pallantia macunaima
male-specific compound.
(13) Witte, V.; Abrell, L.; Attygalle, A. B.; Wu, X.; Meinwald, J.
Chemoecology 2007, 17, 63.
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Org. Lett., Vol. 15, No. 8, 2013

Figure 7. GC partial separation of the (6R,10S)- and (6S,10R)-
6,10,13-trimethyltetradecan-2-one (8) enantiomers by their
acetylated derivatives.
Figure 6. GC partial separation of all the four stereoisomers of
6,10,13-trimethyltetradecan-2-one (8).
36% of males tested chose the odor source containing the
synthetic pheromone, meaning that 6,10,13-trimethyltetra-
decan-2-one (8) is a male-produced sex pheromone of the
stink bug Pallantia macunaima (Figure S6 in SI).
By comparison of the retention time of the 2-(R/S)-acety-
lated pheromone with the isomers discussed, the absolute
configuration of the sex pheromone is the enantiopure
(6R,10S)-6,10,13-trimethyltetradecan-2-one, which was
confirmed by co-injection (Figure 7D,E).
To determine the chirality of the natural product, the four
stereoisomers of synthetic ketone 8 were synthesized by
coupling with two chiral blocks, which were synthesized start-
ing from (S)- and (R)-propylene oxides, by applying stereo-
specific inversion of secondary tosylates as a key reaction.14
The resolution of all stereoisomers were tested by em-
ploying several chiral GC and HPLC stationary phases;
the β-DEX 325 column (Supelco) was partially effective,
separating a mixture of (6R*,10S*)- from (6S*,10S*)-8.
(Figure 6AꢀC). By comparing the retention times of
natural pheromone and synthetic stereoisomers, it was
clear that the natural configuration was restricted to the
(6R,10S)- or (6S,10R)-8 isomers, which was confirmed by
co-injection (Figure 6DꢀE).
In summary, the structural elucidation for the P. macunai-
ma sex pheromone was determined. The biological activity of
the racemic synthetic pheromone was demonstrated by
behavioral and EAD experiments. Furthermore, the abso-
lute configuration of the natural pheromone was determined.
To date, attractant pheromones have been identified for
more than 30 stink bug species, but this is the first ketone
identified as a male-produced sex pheromone in Pentato-
midae. The trivial name, pallantione, was assigned to this
novel pheromone molecule. Further experiments employ-
ing enantiopure, as well as racemic, pallantione are under-
way in the laboratory and the field; these results will be
described in due course.
Due to the difficulty in separating these enantiomers, we
decided to introduce an acetate group at C-2, generating a
third chiral center in the molecule, since it is well-known
that secondary acetates usually promote resolution on
chiral GC columns.15 So, the enantiomers (6R,10S)- and
(6S,10R)-8 and the natural pheromone were reduced to the
corresponding alcohols, employing LiAlH4, and acety-
lated with Ac2O and pyridine.
Acknowledgment. We thank Conselho Nacional de De-
ꢀ
´
fico e Tecnologico (CNPq, Brazil) and
senvolvimento Cientı
Instituto Nacional de Ciencias e Tecnologia de Semioquı
^
´
-
micos na Agricultura for supporting our research. We are
also grateful to CNPq for funding the visit of Dr. Jeffrey R.
Aldrich (University of California - Davis, U.S.A.) to the
The 2-(R/S)-acetate obtained from a mixture of the
(6R*,10S*)-8 resulted in four peaks upon chiral GC, but
the enantiomers were also resolved (Figure 7A). A base-
line separation was achieved for the 2-(R/S)-acetates
of (6R,10S)- and (6S,10R)-8 isomers (Figure 7B,C).
ꢀ
Federal University of Parana (visiting researcher; Proc.:
401604/2009-8).
Supporting Information Available. Experimental pro-
cedures, characterization data, chiral chromatograms,
and copies of 1H and 13C NMR. This material is available
free of charge via the Internet at http://pubs.acs.org.
(14) Muraki, Y.; Taguri, T.; Yamamoto, M.; Zarbin, P. H. G.; Ando,
T. Eur. J. Org. Chem. 201310.1002/ejoc.201201688.
(15) Vidal, D. M.; Fonseca, M. G.; Zarbin, P. H. G. Tetrahedron
Lett. 2010, 51, 6704.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 8, 2013
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