9-Methylgermacrene-B is confirmed as the sex pheromone of the sandfly Lutzomyia longipalpis from Lapinha, Brazil, and the absolute stereochemistry defined as S

Article abstract of DOI:10.1039/a907910f

The structure of the sex pheromone produced by the male sandfly Lutzomyia longipalpis, from the Lapinha Cave (Minas Gerais State) region of Brazil, previously proposed tentatively as the novel homosesquiterpene 9- methylgermacrene-B, is confirmed and the

Full text of DOI:10.1039/a907910f

9-Methylgermacrene-B is confirmed as the sex pheromone of the sandfly
Lutzomyia longipalpis from Lapinha, Brazil, and the absolute stereochemistry
defined as S
J. Gordon C. Hamilton,a Antony M. Hooper,b Helen C. Ibbotson,a Satoshi Kurosawa,c Kenji Mori,c Shin-etsu
Mutoc and John A. Pickett*b
a School of Life Sciences, University of Keele, Keele, Staffordshire, UK ST5 5BG
b IACR-Rothamsted, Harpenden, Hertfordshire, UK AL5 2JQ. E-mail: john.pickett@bbsrc.ac.uk
c Department of Chemistry, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan
Received (in Liverpool, UK) 29th September 1999, Accepted 13th October 1999
The structure of the sex pheromone produced by the male
sandfly Lutzomyia longipalpis, from the Lapinha Cave
(Minas Gerais State) region of Brazil, previously proposed
tentatively as the novel homosesquiterpene 9-methylgerma-
crene-B, is confirmed and the absolute stereochemistry
defined as S by comparing physical and biological properties
of the synthetic enantiomers with the natural product.
essentially the same as for racemic 1,†§ as expected, and with
[a]1D8 + 61.4 and [a]D20 261.3 (CHCl3), respectively. On chiral
GC‡, the R and S isomers of 1 eluted in that order (38.04, 38.20
min) and, when co-injected with racemic 1, the natural product
gave enhancement of the second peak, defining the absolute
stereochemistry of the pheromone as (S)-9-methylgermacrene-
B (S)-1. It should be noted, as explained previously,4 that the
pheromone is not stable and readily undergoes acid catalysed
cyclisation and a Cope rearrangement to the corresponding
elemene structure on heating, hence the requirement for cold
on-column injection or low injector port temperatures for
GC.‡
Bioassays¶ involving attraction of female L. longipalpis and
conducted in a Y-tube olfactometer resulted in racemic 1 giving
77% attraction compared with 72% (though not statistically
significantly different) for an equivalent amount of the natural
product. Attraction to (S)-1 was 67% compared with 56% to the
(R)-1 isomer. The response to (S)-1 was not significantly
different from that of the natural pheromone, whereas the
response to the (R)-1 isomer was significantly lower (c2 p =
The sandfly Lutzomyia longipalpis (Lutz and Neiva) (Diptera:
Psychodidae) is the vector of the protozoan parasite Leishmania
chagasi (Cunha and Chagas) (Kinetoplastida: Trypanosomati-
dae), the causative agent of visceral leishmaniasis in the New
World. Male L. longipalpis release a sex pheromone, from
glands on the tergites of the abdomen,1 which is highly
attractive to females.2 The sex pheromone gland of L.
longipalpis from the Lapinha region (Minas Gerais State) in
Brazil produces one principal volatile component that is
responsible for the attraction of females.3 It was proposed,
mostly on the basis of mass spectrometry on natural material
and on products from microchemical reactions, that the
pheromone comprised the novel homosesquiterpene 9-me-
thylgermacrene-B 1.4 The sex pheromones of other sympatric
and allopatric populations of L. longipalpis are different;5 for
example, that from the Jacobina region of Brazil (Bahia State)
has been confirmed as another novel sesquiterpene, 3-methyl-a-
himachalene, with relative stereochemistry defined as
1RS,3RS,7RS.6 The purpose of this work was to test the
proposed structure for the Lapinha L. longipalpis sex pher-
omone by synthesis of 9-methylgermacrene-B and to define the
absolute stereochemistry at C-9 by comparison between the
natural product and the synthetic enantiomers (R)-1 and (S)-1.
The overall synthetic approach adopted has recently been
developed for sesquiterpene germacrene-B and involves the
establishment of the cyclodecadiene ring system by an intra-
molecular alkylation before finally elaborating the isopropyli-
dene group.7 Initially, the racemic 9-methylgermacrene-B (1)
was prepared as in Scheme 1 from geraniol 2, in order to test the
initial structural hypothesis for this pheromone.4 The NMR data
from racemic 1† were similar to the limited data obtained
originally for the natural product.4 Co-injection on GC‡
(siloxane column) of racemic 1 with the natural product gave
one enhanced peak (18.22 min). The mass spectrum of synthetic
compound by GC-MS§ was similar to that published for the
natural product.4 Having confirmed the structure of the
pheromone as 9-methylgermacrene-B 1, the enantiomers were
then synthesised by the same cyclisation approach but with
methyl 3-hydroxy-2-methylpropionate 4 as chiral precursor, i.e.
(S)-4 to (R)-1 and (R)-4 to (S)-1 as in Scheme 2. The
enantiomers of 4 were obtained commercially and (R)-1 was
synthesised first, hence some improvements in yield for (S)-1.
The chiral syntheses required introduction of two stereogenic
double bonds at 5 and 6 which were both achieved with an E/Z
ratio of > 99% by NMR and the final products possessed an ee
of > 95%. Both (R)-1 and (S)-1 gave NMR and mass spectra
Scheme 1 Yields are for routes to (±)-1, and from compound 3 to 1 also are
quoted for routes to (R)-1 then (S)-1 in that order. Reagents and conditions:
i, TBSCl, imidazole, DMF, 96%; ii, MCPBA, CHCl3, 74%; iii, HIO4.2H2O,
Et2O, THF, 52%; iv, CHMeNCMeMgBr, THF, 55%; v, CH3C(OEt)3,
C2H5CO2H, 71% (E/Z: 98+2); vi, PPTS, MeOH, 87%; vii; Ph3P, CCl4; viii,
DIBAL-H, CH2Cl2; ix, TMSCN, KCN, 18-crown-6, then BnNMe3F, THF,
H2O; x, CH2NCHOEt, C6H6; xi, NaHMDS, THF, 19% (5 steps), 47%, 42%;
xii, PPTS, MeOH then aq. NaOH, Et2O, 18%, 51%, 50%; xiii, Me2CBr2,
Sm, CrCl3, Sml2, THF, 56%, 60%, 64%.
Chem. Commun., 1999, 2335–2336
This journal is © The Royal Society of Chemistry 1999
2335

putative biosynthetic terms to the C-9 methyl of the homoge-
rmacrene 1 and the relative stereochemistry has already been
established,6 but this will be reported separately when the
hypothesis has been fully tested.
Notes and references
† Selected NMR data for (±)-1: Spectra were recorded using a JEOL JNM-
LA 500 MHz spectrometer. dH(CDCl3, 500 MHz) 1.04 (3H, d, J = 7.0),
1.44 (3H, s), 1.55 (3H, s), 1.68 (3H, s), 1.70 (3H, s), 2.4–1.8 (8H, m), 3.06
(1H, br d, J 14.0), 4.39 (1H, br d, J 11.0), 4.72 (1H, dd, J 12.0, 3.0);
dC(CDCl3, 125 MHz) 11.1, 16.4, 20.4, 20.7, 20.9, 25.3, 34.4, 38.9, 40.7,
46.3, 125.7, 127.0, 128.7, 130.9, 133.7, 140.2.
‡ GC conditions: HP-5 (a siloxane) 0.32 mm id 3 30 m 3 25 mm film
thickness, 40 to 150 °C at 5 °C min21; chiral GC (b-cyclodextrin) 0.25 mm
id 3 30 m 3 25 mm film thickness, 40 to 180 °C at 3 °C min21
.
§ GC-MS: 0.32 mm id 3 50 m HP-1 (a siloxane 3 0.52 mm film thickness,
30 °C on-column injection, then 30 to 200 °C at 5 °C min21; EI at 70 eV,
250°C (VG-Autospec, Fisons Instruments). Selected data for (±)-1: m/z 121
(100%), 93 (75), 107 (59), 41 (57), 67 (51), 135 (47), 91 (35), 55 (34), 79
(30), 105 (30), 119 (28), 53 (26), 77 (24), 43 (23), 81 (22), 39 (21), 133 (19),
136 (18), 29 (17), 147 (17), 71 (16), 175 (16), 95 (14), 122 (13), 203 (13),
161 (11), 218 (10, M+).
¶ Virgin female sandflies were removed from larval rearing pots within 10
h after eclosion to ensure that they were unmated. They were provided with
a saturated sugar solution on cotton wool and subsequently maintained for
5–6 days in Barraud cages (18 3 18 3 18 cm). Bioassays were conducted
in a glass (9 mm internal diameter) Y-tube olfactometer. Zero grade air was
passed (2 ml min21) through two charcoal filters into the test and control
arms (10 cm long). The olfactometer was connected to the air supply by
Teflon tubing. A filter paper disk (1.5 cm diameter) was inserted into the
Teflon tubing at the connection with the olfactometer test and control arms.
During bioassays, pheromone extracts or synthetic chemicals in hexane
were placed on one of the filter paper disks and hexane in the same quantity
as for the test arm was placed on the other filter paper disk. The female
sandfly was introduced into the third arm (10 cm long) of the olfactometer
and its response observed for 5 min.
Scheme 2 Yields are quoted for routes to (R)-1 then (S)-1 in that order.
Reagents and conditions: i, TBSCl, imidazole, DMF, quant., 99%; ii,
DIBAL-H, CH2Cl2, 85%, 94%; iii, TsCl, Py; iv, NaCN, DMSO, 79%, 84%,
2 steps; v, DIBAL-H, CH2Cl2; vi, NaBH4, EtOH, 58%, 65%, 2 steps; vii,
TBDPSCl, imidazole, DMF, 89%, 99%; viii, AcOH, THF, H2O, 84%, 86%;
ix, Swern oxidation; x, PPh3, CBr4, CH2Cl2, 87%, 93%, 2 steps; xi, BuLi,
Et2O, 98%, 98%; xii, Me3Al, Cp2ZrCl2, CH2Cl2, H2O; xiii, BuLi, hexane;
xiv, (CH2O)n, THF, 80%, 80%, 3 steps; xv, PPh3, CCl4; xvi, NaSO2Ph,
DMF, 76%, 83%, 2 steps; xvii, BuLi, THF, HMPA then (E)-TBSOCH2C-
MeNCHCH2Cl,8 78%, 87%; xviii, AcOH, THF, H2O, 85%, 81%; xix,
[PdCl2(dppp)], Super-Hydride, THF, 82%, 81%; xx, Ac2O, Py, 87%, 95%;
xxi, TBAF, THF, 90%, 83%; xxii, Dess–Martin oxidation, 75%, 87%; xxiii,
TMSCN, KCN, 18-crown-6; xxiv, BnMe3NF, THF, H2O; xxv,
CH2NCHOEt, TsOH, C6H6, quant., 94%, 3 steps; xxvi, H2CO3, MeOH,
87%, 86%; xxvii, LiCl, MsCl, DMF, s-collidine, 95%, 90%.
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0.009). Thus, while the S isomer of 1 is highly active, the R
isomer is itself also active but does not appear to interfere with
the activity of its antipode. This will reduce the need to create
highly stereochemically pure (S)-1 for field development of the
pheromone in control of these important disease vectors.
The demonstration that the sex pheromone produced by male
L. longipalpis from the Lapinha region is (S)-9-methylgerma-
4 J. G. C. Hamilton, G. W. Dawson and J. A. Pickett, J. Chem. Ecol., 1996,
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533.
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Chem. Commun., 1999, 355.
7 S. Muto, Y. Nishimura and K. Mori, Eur. J. Org. Chem., in the press.
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crene-B,
(S)-(E,E)-7-isopropylidene-4,9,10-trimethylcyclo-
deca-1(10),4-diene (S)-1, would suggest that the absolute
stereochemistry of that from Jacobina would be (1S,3S,7S)-
3-methyl-a-himachalene, since the C-3 methyl is analogous in
Communication 9/07910F
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Chem. Commun., 1999, 2335–2336