Production of (5R,6S)-6-acetoxy-5-hexadecanolide, the mosquito oviposition pheromone, from the seed oil of the summer cypress plant, Kochia scoparia (Chenopodiaceae)

Article abstract of DOI:10.1021/jf990294b

The oviposition pheromone for the pathogen-vectoring mosquitoes in the genus Culex (Diptera: Culicidae), that is, (5R,6S)-6-acetoxy-5- hexadecanolide, is efficiently synthesized, in admixture with the inactive (5S,6R) enantiomer (~33% w/w), from the fixed oil extracted from the seeds of the summer cypress plant, Kochia scoparia (Chenopodiaceae), cultivated on an industrial scale. Oviposition bioassays using gravid females of Culex quinquefasciatus, a vector of filariasis in human beings, showed that the product was attractive, with activity comparable to that of a pure synthetic sample containing the same amount of the active enantiomer. Production of the pheromone in the form of a biologically active crude material via a cheap and renewable plant suitable for development as a new industrial crop provides the basis for control of Cx. quinquefasciatus and other congeneric vectors of pathogens in resource-poor areas of the world.

Full text of DOI:10.1021/jf990294b

J. Agric. Food Chem. 1999, 47, 3411−3415
3411
P r od u ction of (5R,6S)-6-Acetoxy-5-h exa d eca n olid e, th e Mosqu ito
Ovip osition P h er om on e, fr om th e Seed Oil of th e Su m m er Cyp r ess
P la n t, Koch ia scopa r ia (Ch en op od ia cea e)
Timothy O. Olagbemiro,†,‡ Michael A. Birkett,§ A. J ennifer Mordue (Luntz), and
J ohn A. Pickett*,§
Department of Chemistry, Abubakar Tafawa Balewa University, Private Mail Bag 0248, Bauchi, Nigeria,
Biological and Ecological Chemistry Department, IACR-Rothamsted, Harpenden,
Hertfordshire AL5 2J Q, United Kingdom, and Department of Zoology, University of Aberdeen,
Tillydrone Avenue, Aberdeen AB24 2TZ, United Kingdom
The oviposition pheromone for the pathogen-vectoring mosquitoes in the genus Culex (Diptera:
Culicidae), that is, (5R,6S)-6-acetoxy-5-hexadecanolide, is efficiently synthesized, in admixture with
the inactive (5S,6R) enantiomer (∼33% w/w), from the fixed oil extracted from the seeds of the
summer cypress plant, Kochia scoparia (Chenopodiaceae), cultivated on an industrial scale.
Oviposition bioassays using gravid females of Culex quinquefasciatus, a vector of filariasis in human
beings, showed that the product was attractive, with activity comparable to that of a pure synthetic
sample containing the same amount of the active enantiomer. Production of the pheromone in the
form of a biologically active crude material via a cheap and renewable plant suitable for development
as a new industrial crop provides the basis for control of Cx. quinquefasciatus and other congeneric
vectors of pathogens in resource-poor areas of the world.
Keyw or d s: Kochia scoparia; seed oil; Culex quinquefasciatus; (5R,6S)-6-acetoxy-5-hexadecanolide;
mosquito; oviposition; pheromone
INTRODUCTION
quinquefasciatus females use olfactory cues for the
location of suitable egg-laying sites. The main cue is a
volatile pheromone, released by mature egg rafts (Os-
good, 1971; Osgood and Kempsler, 1971; Starratt and
Osgood, 1973), which acts in combination with water-
based pollutants such as 3-methylindole produced, for
example, via bacterial fermentation [Millar et al., 1992;
Mordue (Luntz) et al., 1992]. The first step in developing
new strategies for control of Cx. quinquefasciatus using
such semiochemicals (behavior- or development-modify-
ing chemicals) was the identification of the oviposition
pheromone as (5R,6S)-6-acetoxy-5-hexadecanolide (1a )
Of the many blood-sucking and biting insects belong-
ing to the order Diptera, mosquitoes (Culicidae) pose
the greatest threat to public health because of their
ability to act as vectors of causative agents for diseases
such as malaria, dengue, yellow fever, encephalitis, and
filariasis, which afflict many millions of people world-
wide, particularly in sub-Saharan Africa (American
Association for the Advancement of Science, 1991; Giles
and Warrell, 1993; WHO/CTD, 1998). In terms of
numbers of affected individuals and level of mortality,
malaria and encephalitis are of greatest significance,
together affecting nearly one-third of the world’s popu-
lation (1.5 billion people) in some 90 countries, mostly
within Africa. For filariasis, it is estimated that, of the
450 million people that are susceptible, >15 million
people are currently infected, with 950000 new cases
occurring annually (Reeves and Milby, 1990). Efficient
techniques for vector surveillance and control are
therefore of paramount importance.
(Laurence and Pickett, 1982, 1985; Laurence et al.,
1985). Laboratory and field trials in nine countries and
three continents, using synthetic material (Dawson et
al., 1990) containing an equal mixture of all four
possible stereoisomers, that is, (5R,6R), (5R,6S), (6R,5R),
(6R,6S), have demonstrated the efficacy of the phero-
mone in attracting Culex spp. mosquitoes (Otieno et al.,
1988). Despite the presence of three inactive unnatural
isomers, the biological activity of the naturally occurring
isomer is unaffected.
The characterization of the oviposition pheromone 1a
has provided the impetus for several asymmetric syn-
theses and large scale racemic routes, respectively, as
a challenge for synthetic chemists and to provide
material for field testing (Laurence and Pickett, 1982;
The mosquito Culex quinquefasciatus Say (Diptera:
Culicidae) is responsible for transmission of Wuchereria
bancrofti, the causative agent of human filariasis and
the St. Louis encephalitis virus and other arboviruses
in the United States (Reisen et al., 1992). Gravid Cx.
* Author to whom correspondence should be addressed (e-
mail J ohn.Pickett@bbsrc.ac.uk).
† Abubakar Tafawa Balewa University.
‡ Present address: IACR-Rothamsted, Harpenden, Hert-
fordshire, AL5 2J Q U.K.
§ IACR-Rothamsted.
University of Aberdeen.
10.1021/jf990294b CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/30/1999

3412 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Olagbemiro et al.
Sch em e 1a
MATERIALS AND METHODS
Gen er a l. 1H NMR spectra were recorded in deuteriochlo-
roform (CDCl3) with tetramethylsilane (TMS) as an internal
standard, at 400 MHz on a J EOL GNX spectrometer. 13C NMR
spectra were obtained from the J EOL spectrometer at 100
MHz also using TMS as the internal standard. Chemical shifts
(δH and δC) are reported in parts per million (ppm) from the
internal standard (TMS ) 0.00). Gas chromatography (GC)
was carried out using a Hewlett-Packard 5890 gas chromato-
graph equipped with
a cold on-column injector, a flame
ionization detector (FID), and a 50 m × 0.32 mm i.d. HP-1
capillary column. The oven temperature was maintained at
40 °C for 2 min and programmed at 10 °C/min to 250 °C. The
carrier gas was hydrogen. Gas chromatography/mass spec-
trometry (GC/MS) was performed using a Hewlett-Packard
5890 gas chromatograph equipped with a cold on-column
injector and a capillary column (50 m × 0.32 mm i.d., HP-1)
directly coupled to a mass spectrometer and integrated data
system (70-250 VG Analytical and VG Autospec, Fisons
Instruments). Ionization was by electron impact at 70 eV and
230 °C. The GC oven temperature was maintained at 30 °C
for 5 min and then programmed at 5 °C/min to 250 °C. The
carrier gas was helium. Tentative identifications made by GC/
MS were confirmed by comparison of the mass spectral data
with those of authentic samples and by peak enhancement
when co-injected with authentic compounds using GC. All
solvents were dried using anhydrous magnesium sulfate and
were evaporated using a Bu¨chi rotary evaporator. Solvents and
reagents were used as supplied except when specified.
a
Reagents: (a) 2 M NaOH, C6H12, reflux, then 6 M HCl;
(b) BF3‚MeOH, reflux; (c) t-BuOOH, OsO4 (cat.), 0-25 °C; (d)
p-TSA, C6H6, reflux; (e) Ac2O, C5H5N, 25 °C.
Ra w Ma ter ia ls. Seeds of the summer cypress, K. scoparia,
were produced by Colegrave Seeds Limited, West Adderbury,
Banbury, Oxon., U.K., by cultivation under glass using stand-
ard methodology. Seed oil was extracted by English Hop
Products Limited, Paddock Wood, Tonbridge, U.K. Carbon
dioxide was employed as extractant (Laws et al., 1977), and
the K. scoparia seeds (480 g) were extracted over a period of
7 h at 10 °C, 67 bar, and a flow rate of 6.5 kg of CO2/h to give
seed oil as a brown gum (39.9 g).
Fuganti et al., 1982; Mori and Otsuka, 1983; Sato et
al., 1984; Lin et al., 1985; Machiya et al., 1985; Ochiai
et al., 1985; Barua and Schmidt, 1986; J efford et al.,
1986; Ko and Eliel, 1986; Dawson et al., 1990; Coutrout
and Bomont, 1994; Gravierpelletier et al., 1995; Henkel
et al., 1997). Although the various synthesis routes cited
above can provide multigram quantities of 1a , the
economic circumstances of the less developed countries
make control strategies for Cx. quinquefasciatus based
on the synthetic pheromone unaffordable. Here we
describe a rapid, semi-biotechnological approach to
pheromone production, as a component of a crude but
chemically defined product, from the fixed oil extracted
from seeds of the summer cypress, Kochia scoparia
(Chenopodiaceae). Generally, seeds from the family
Chenopodiaceae are not rich in oil (Kleinman and Earle,
1972). However, the fatty acid composition of the oil
from some members of this family such as spinach
(Spinocea oleoracea), Russian thistle (Salsola pestifer),
Bassia hypsofolia, Kochia prostata, and Sieda setigera,
as well as K. scoparia, is reported to contain 0.2-5.0%
5-hexadecenoic acid (Kleiman and Earle, 1972). Al-
though K. scoparia seed oil does not possess the highest
amount (w/w) of the acid, the plant was chosen for
pheromone production because of its ease of cultivation
and prolific seed production [11000 kg of seed/hectare
(ha)]. It represents a good alternative as a grazing or
forage crop and can be baled and ensiled. K. scoparia
grows rapidly (1-2.5 cm/day) and vigorously; it tolerates
very dry conditions and is found in most countries
afflicted with Culex spp. mosquitoes (Garduno, 1993).
Three different approaches are utilized for the prepara-
tion of a common intermediate, (5R,6S)-6-hydroxy-5-
hexadecanolide (8), from glycerides of (Z)-5-hexadecen-
oic acid (2), with a final acetylation step to give the
desired enantiomerically enriched 6-acetoxyhexadecano-
lide (1a ) (see Scheme 1).
Ch em ica l Con ver sion . Synthesis of (5RS,6SR)-6-Hydroxy-
5-hexadecanolide: Route A. (Z)-5-Hexadecenoic Acid (3). To a
solution of K. scoparia seed oil (3.0 g) containing glycerides of
(Z)-5-hexadecenoic acid (2) in hexane (80 mL) was added dilute
sodium hydroxide (2 M, 100 mL), and the mixture was heated
under reflux overnight (18 h). The cooled solution was then
washed with hexane (100 mL), and the aqueous phase was
separated, cooled (ice bath), and then acidified using hydro-
chloric acid (6 M). Following extraction with ethyl acetate (3
× 100 mL), the combined organic layers were dried and
evaporated under reduced pressure to give a crude yellow-
green material (0.65 g). The presence of (Z)-5-hexadecenoic
acid (3) (4.25% w/w) was confirmed by treatment of the
material with boron trifluoride/methanol (Metcalfe and Schmitz,
1961) and location of the methyl ester by GC/MS analysis: m/z
268 (M+, 20%), 236 (16), 194 (20), 152 (13), 110 (8), 96 (55), 74
(100), 67 (55), 55 (74), 43 (60), and 41 (73).
Methyl (Z)-5,6-Epoxyhexadecanoate. Methyl (Z)-5,6-hexa-
decanoate (5) (0.100 g, 0.0003 mol), obtained from a boron
trifluoride/methanol esterification of 3, was dissolved in hexane
(20 mL) and introduced into a 100 mL round-bottom flask
fitted with a magnetic bar and stirrer. To this solution was
added with stirring over a period of 10 min a 20 mL solution
of hexane containing m-chloroperbenzoic acid (0.5 g, 0.0003
mol). The combined solution was stirred at room temperature
for a period of 24 h. A white precipitate was observed, and at
the end of this period the precipitate was filtered and the
filtrate washed with a solution of sodium sulfite (10%), followed
by tests with starch iodide papers until the organic layer was
observed to be free of oxidized substances. The organic layer
was later washed with a (3 × 25 mL) solution of sodium
bicarbonate (5%) and water (3 × 25 mL), dried over anhydrous
magnesium sulfate, and filtered, and the solvent was removed
using a rotary evaporator to leave behind a yellow oily product
(0.065 g) containing the desired product: m/z 284 (M+, 2), 225

Production of Mosquito Pheromone
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3413
(8), 183 (60), 143 (65), 141 (28), 113 (70), 99 (80), 83 (70), 71
(85), 59 (100), 55 (72), 43 (80).
(20 mL) and then dried and evaporated to yield a viscous oil
(0.30 g). A sample of the oil (0.1 g) was washed with hexane
(3 × 15 mL), and the combined washings were evaporated
under reduced pressure to give a dark oil. GC/MS analysis of
the oil, followed by peak enhancement with an authentic
sample [prepared by sodium borohydride deacetylation of an
authentic sample of the oviposition pheromone, (5R,6S)-6-
acetoxy-5-hexadecanolide] confirmed the presence of (5RS,6SR)-
6-hydroxy-5-hexadecanolide (8) as a major component of the
oil: 13C NMR (CDCl3) δ 14.1, 18.5, 21.5, 25.6, 29.7, 31.9, 71.6,
83.7, 170.8; m/z 270 (M+, 0.04), 252 (0.7), 142 (30), 100 (100),
99 (85), 71 (36).
Using crude material (0.38 g) containing (5RS,6SR)-5,6-
dihydroxyhexadecanoic acid (4) (prepared via route A) and
methyl (5RS,6SR)-5,6-dihydroxyhexadecanoate (6) (prepared
via route B), 0.22 g of dark oil was recovered, containing in
each case 66 mg (30% w/w) of (8). Using crude material (0.47
g) containing (5RS,6SR)-5,6-dihydroxyhexadecanoic acid trig-
lyceride (7) (prepared via route C), 0.165 g of dark oil was
recovered, containing 74 mg (45% w/w) of 8.
(5RS,6SR)-6-Acetoxy-5-hexadecanolide ((1a ) and (1b)). To
a solution of crude material (0.5 g) containing (5RS,6SR)-6-
hydroxy-5-hexadecanolide (8) in dry pyridine (15 mL) was
added acetic anhydride (0.5 mL, 0.0004 mol), and the mixture
was allowed to stir at ambient temperature overnight (18 h).
Following removal of the solvent under reduced pressure, the
residue was taken up in ethyl acetate (15 mL) and then
washed with dilute hydrochloric acid (3 M, 2 × 20 mL) and
saturated sodium bicarbonate solution (15 mL) and brine (10
mL). The organic layer was dried and evaporated under
reduced pressure to yield a yellow oil (0.35 g). GC/MS analysis
of the oil showed the presence of erythro and threo diastereo-
isomers of 6-acetoxy-5-hexadecanolide (1): m/z 312 (M+, 2%),
142 (20), 100 (52), 99 (100), 71 (28), 55 (33), 43 (85), 41 (24);
1H NMR δ 0.88 (3H, t, CH3), 1.25 [16H, m, (CH2)8], 1.60 (2H,
m, CH2), 1.71 (2H, m, CH2), 2.0 (2H, m, CH2), 2.08 (3H, s, CH3),
2.53 (2H, m, CH2), 4.30 (1H, m, CH), 4.98 (1H, m, CH); 13C
NMR δ 14.1, 18.2, 21.0, 22.7, 23.5, 25.1, 29.3-29.8 (seven
signals), 31.9, 74.3, 80.5, 170.6, 170.8. Using an internal
standard (tetracosane, C24H50), the amount of natural phero-
mone (i.e., the 5R,6S isomer) in the oil using (5RS,6SR)-6-
hydroxy-5-hexadecanolide prepared following routes A, B, and
C was estimated by GC analysis to be approximately 8, 12,
and 16.5% (w/w), respectively.
Ovip osition Bioa ssa ys. Cage bioassays with Cx. quinque-
fasciatus followed the method of Mordue (Luntz) et al. (1992).
Test bowls contained either distilled water (100 mL) plus the
test material (3 µL of either plant oil containing the oviposition
pheromone or authentic oviposition pheromone). Materials
were tested at a dose of 3 µg of pheromone per experiment.
Control bowls contained distilled water (100 mL) plus hexane
(3 µL). The number of egg rafts in the test bowls was recorded
at the end of each experiment and converted to percentages
of the total number of rafts in both bowls (control and test)
for each cage, with differences between the test and control
trials being determined by Student’s t test.
(5RS,6SR)-Dihydroxyhexadecanoic Acid (4). To a solution
of the crude yellow-green material (0.5 g) containing (Z)-5-
hexadecenoic acid (3) in acetone (60 mL) was added a solution
of tert-butylhydroperoxide (2 mL, 0.014 mol) in methanol
(50%), followed by tetraethylammonium acetate‚4H2O (0.2 g,
0.007 mol). The solution was allowed to stir at ambient
temperature for 0.75 h and then cooled (ice bath) and treated
with osmium tetraoxide (0.002 g, 0.00001 mol). After 1 h, the
mixture was stirred at ambient temperature overnight (18 h)
and then recooled (ice bath). Ethyl acetate (20 mL) and dilute
sodium bisulfite solution (10%, 10 mL) were then added, the
mixture was then stirred at ambient temperature for 1 h, and
the resultant golden brown reaction was filtered with suction.
The combined filtrate and washings were dried and evaporated
to give an oil (0.38 g), which was utilized directly in the
cyclization step.
Synthesis of (5RS,6SR)-6-Hydroxy-5-hexadecanolide: Route
B. Methyl (Z)-5-Hexadecenoate (5). A solution of K. scoparia
seed oil (1.0 g) in boron trifluoride/methanol (50%, 30 mL) was
refluxed for 2 h, and the cooled reaction mixture was diluted
with distilled water (100 mL) and then extracted with ethyl
acetate (2 × 50 mL). The combined organic layers were washed
with dilute sodium bicarbonate solution (10%, 40 mL) and then
dried and evaporated under reduced pressure to give a dark
green oil (0.68 g). Dissolution of the oil in ethyl acetate (20
mL), followed by treatment with decolorizing charcoal and
removal of the solvent, yielded a yellow, sweet-smelling oil
(0.61 g). The presence of methyl (Z)-5-hexadecenoate (5) (4.5%
w/w) in the oil was confirmed by GC/MS analysis: m/z 268
(M+, 20%), 236 (16), 194 (20), 152 (13), 110 (18), 96 (50), 74
(100), 67 (52), 55 (71), 43 (62), 41 (86).
Methyl (5RS,6SR)-5,6-Dihydroxyhexadecanoate (6). To a
solution of the yellow oil (0.32 g) containing methyl (Z)-5-hexa-
decenoate (5) in acetone (40 mL) was added a solution of tert-
butylhydroperoxide (2 mL, 0.014 mol) in methanol (50%),
followed by tetraethylammonium acetate‚4H2O (0.2 g, 0.0007
mol). The solution was allowed to stir at ambient temperature
for 0.75 h and then cooled (ice bath) and treated with osmium
tetraoxide (0.002 g, 0.00001 mol). After 1 h, the mixture was
allowed to stir at ambient temperature overnight (18 h) and
then recooled (ice bath). Ethyl acetate (20 mL) and dilute
sodium bisulfite solution (10%, 10 mL) were then added, the
mixture was then allowed to stir at ambient temperature for
1 h, and the resultant mixture was filtered with suction. The
combined filtrate and washings were dried and evaporated to
give an oil (0.28 g), which was used directly in the cyclization
step.
Synthesis of (5RS,6SR)-6-Hydroxy-5-hexadecanolide: Route
C (5RS,6SR)-5,6-Dihydroxyhexadecanoic Acid Triglyceride (7).
To a solution of K. scoparia seed oil (1.0 g) in acetone (40 mL)
was added a solution of tert-butylhydroperoxide (2 mL, 1.4
mmol) in methanol (50%), followed by tetraethylammonium
acetate‚4H2O (200 mg, 0.7 mmol). The mixture was allowed
to stir at ambient temperature for 0.75 h and then cooled (ice
bath) and treated with osmium tetraoxide (2 mg, 0. 01 mmol).
After 1 h, the mixture was allowed to stir at ambient
temperature overnight (18 h) and then recooled (ice bath).
Ethyl acetate (20 mL) and dilute sodium bisulfite solution
(10%, 10 mL) were then added, and the mixture was then
allowed to stir at ambient temperature for 1 h. Using the same
workup procedure as in 6 above gave a yellow oil (0.87 g),
which was used directly in the cyclization step.
RESULTS AND DISCUSSION
Prior to the planned synthesis of the oviposition
pheromone 1a from (Z)-5-hexadeconoic acid, the first
step was to confirm that K. scoparia seed oil contained
triglycerides of (Z)-5-hexadecenoic acid (2). The mono-
unsaturated esters in the epoxidized seed oil after
treatment with m-chloroperbenzoic acid and analysis of
the resulting oil by GC/MS confirmed the presence of
characteristic mass fragment ions for the epoxide of the
5-hexadecenoic acid methyl ester.
Having confirmed the presence of the (Z)-5-hexa-
decenoic acid, pheromone production was undertaken
using three approaches depending upon the stage at
which triglyceride cleavage was performed (see
Scheme 1). The synthetic routes A and B involved initial
(5RS,6SR)-6-Hydroxy-5-hexadecanolide (8), General Proce-
dure. To a solution of the crude material containing either
(5RS,6SR)-5,6-dihydroxyhexadecanoicacid (4),methyl(5RS,6SR)-
5,6-dihydroxyhexadecanoate (6), or (5RS,6SR)-5,6-dihydroxy-
hexadecanoic acid triglyceride (7) in dry benzene (60 mL) was
added p-toluenesulfonic acid (0.03 g, 0.0002 mol), and the
mixture was allowed to stir under reflux for 2.5 h. After cooling
to ambient temperature, the solvent was removed under
reduced pressure and the dark brown oily residue taken up
in ethyl acetate (30 mL). The organic layer was washed with
saturated sodium bicarbonate solution (2 × 25 mL) and brine

3414 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Olagbemiro et al.
cleavage to give oils containing either the (Z)-5-hexa-
decenoic acid (3) or its methyl ester 5, respectively, for
further manipulation (Scheme 1, routes A and B). Ester
preparation was accomplished via direct treatment of
the triglycerides with boron trifluoride/methanol (Met-
calfe and Schmitz, 1961). The oils containing 3 or 5 were
treated with a catalytic amount of osmium tetraoxide
(OsO4) in the presence of tert-butylhydroperoxide (t-
BOOH) (Akashi et al., 1978), to give oils containing
(5RS,6SR)-dihydroxyhexadecanoic acid (4) or its methyl
ester 6, respectively. In general, catalytic osmylation of
olefins in the presence of peroxides is a very effective
method for the synthesis of 1,2-dihydroxy compounds.
The reaction of (Z)-hexadecenoic acid (3) with a catalytic
amount of osmium tetraoxide leads exclusively to the
erythro-dihydroxyhexadecanoic acid (4) or its methyl
ester 6 as a result of the formation of an intermediate
osmiate ester, which when hydrolyzed gives exclusively
the cis-dihydroxy product. The tetraethylammonium
salt is used to regenerate the OsO4, allowing glycoliza-
tion to proceed and give the desired stereoisomeric
products, 4 and 6, in their enantiomeric erythro forms.
Subsequent cyclization and acetylation on either mate-
rial gave crude materials, which were shown by GC/
MS to contain the 6-acetoxy-5-hexadecanolide. Peak
enhancement using an authentic sample showed that
mainly the erythro (5RS,6SR) isomers of the pheromone
1a and 1b were in the material, with little sign of the
threo (5RS,6RS) isomers. Using an internal alkane
standard (tetracosane, C24H50) for quantification, the
materials were shown to contain approximately 16%
(route A) and 24% (route B) (w/w) of the pheromone and
its enantiomer. The other approach to the pheromone
(Scheme 1, route C) involved direct treatment of the
triglycerides in the seed oil with a catalytic amount of
osmium tetraoxide in tert-butylhydroperoxide to obtain
the dihydroxytriglyceride 7. Cyclization and acetylation,
as above, afforded a material containing the desired
6-acetoxy-5-hexadecanolide as confirmed by GC/MS.
Peak enhancement using an authentic sample again
showed the major presence of the erythro (5RS,6SR)
isomers 1a and 1b and little sign of the threo isomers
(Scheme 1, route C). The crude material was shown by
the internal standard method to contain ∼33% (w/w) of
the pheromone and its enantiomer. Other identified
extraneous plant materials and byproducts, which were
carried over from the successive chemical conversion of
K. scoparia seed oil to the crude reaction product
containing the active pheromone 6-acetoxy-5-hexade-
canolide, included hexadecanoic acid, octadecanoic acid,
and 6-acetoxy-5-octadecanolide in varying amounts and
quantities.
F igu r e 1. Oviposition behavior of gravid female Cx. quin-
quefasciatus in the presence of crude material (prepared using
route C) containing 3 µg of oviposition pheromone (n ) 8) and
synthetic oviposition pheromone (n ) 8). Vertical lines ) (SE.
pheromone and, more significantly, neither does the
presence of extraneous plant material.
On the basis of the yields obtained here, that is, 0.04
g of oviposition pheromone/g of K. scoparia seed oil, the
estimated recurring cost of synthesizing the oviposition
pheromone from seed oil ($3.00/g) compares favorably
with that from conventional synthetic materials [e.g.,
$15.00/g using the route in Dawson et al. (1990)].
Furthermore, the high seed yields (11000 kg/ha) suggest
that the pheromone yield would be in excess of 3 kg/ha.
Thus, production of the pheromone via a renewable
plant resource, which grows in developing countries,
represents an important development in sustainable,
cheap, and efficient production of this semiochemical.
The ability of developing countries to grow K. scoparia
as a crop for the production of the materials required
for pheromone synthesis would provide the basis for the
development of Cx. quinquefasciatus control strategies
in resource-poor regions of the world. This would most
likely involve a “lure-and-kill” approach, whereby the
oviposition pheromone is used along with oviposition
site derived attractants, for example, skatole (3-meth-
ylindole), to attract gravid females to sites containing
either a highly selective larvicide or a larvae specific
pathogen (Pickett and Woodcock, 1996).
ACKNOWLEDGMENT
We thank Brenda King for assistance with the ovi-
position bioassays.
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Received for review March 23, 1999. Revised manuscript
received J une 10, 1999. Accepted J une 14, 1999. This work
was supported by a Wellcome Trust Travelling Fellowship [Ref.
No. 054532/Z/98/Z/MEP/RC/CRD (T.O.O.)]. IACR receives
grant-aided support from the Biotechnology and Biological
Sciences Research Council (BBSRC) of the United Kingdom.
J F990294B