5226 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Michaelakis et al.
solution of triphenylundecylphosphonium bromide (2.4 g, 5 mmol) in
THF at 0 °C and was stirred under argon. After 1 h at 0 °C, a solution
of the above lactol (0.2 g, 1.96 mmol) in dry THF (1 mL) was added.
The reaction mixture was allowed to reach room temperature. After 1
h of stirring, the reaction mixture was poured into a saturated solution
of NH4Cl and extracted with Et2O. The combined organic extracts were
washed with water and brine, dried over Na2SO4, and concentrated in
vacuo. Subjection of the crude mixture to flash chromatography (silica
gel, hexanes/EtOAc 6:4) yielded pure 5-hexadecenol (3, 4) (0.42 g,
89%) as a mixture of isomers with a ratio of Z/E > 9:1 (determined by
GC analysis). The mixture was used in the following reactions without
(7) when they reported that a Cx. quinquefasciatus egg raft acted
as an attractant to gravid Cx. pipiens biotype pipiens females.
The isolation and characterization of the (-)-(5R,6S)-6-
acetoxy-5-hexadecanolide from Cx. quinquefasciatus egg rafts
provided the impetus for several attempts toward its syntheses.
Sugars or amino acids (12), chiral auxiliaries (13), as well as
Sharpless asymmetric dihydroxylation (14) and epoxidation
reactions (15) have been used for the introduction of the
asymmetry (16) while Wittig coupling, Knoevenagel condensa-
tion, and Julia olefination have been applied for the construction
of the carbon skeleton. However, most of these syntheses suffer
from various drawbacks as they require either uncommon
reagents and/or usually expensive intermediates or entail many
steps giving low overall yields.
1
further purification. Rf ) 0.58 (hexanes/EtOAc 5:5). H NMR (250
MHz, CDCl3): δ 0.88 (t, J ) 6.7 Hz, 3 H, CH3), 1.1-1.5 (m, 18 H,
CH2), 1.5-1.75 (m, 2 H, CH2CH2OH), 1.9-2.2 (m, 4 H, CH2-
CHdCHCH2), 3.65 (t, J ) 6.25 Hz, 2 H, CH2OH), 5.25-5.5 (m, 2 H,
CHdCH).
Hwang et al. in 1987 (17) reported that the only enantiomer
bioactive to Cx. quinquefasciatus was the (-)-(5R,6S) isomer
whereas the other three were inactive but not repellant.
Sakakibara et al. in 1984 (18) first tested all four stereoisomers
with the molestus biotype and reported that they all showed a
significant bioactivity, but Clements in 1999 (19) stated that
these results could not be taken as proven, since there was not
a clear specification of their synthetic procedure.
The unclear evidence for the bioactivity possessed by all four
isomers in combination with the need to simplify the synthetic
procedure and reduce the cost led us to produce the racemic
pheromone in a simple, efficient, high yielding, and less
expensive way. Toward that, a synthetic racemic oviposition
pheromone (SRP) of Cx. quinquefasciatus was synthesized in
a five step procedure and its bioactivity was evaluated against
Cx. pipiens biotype molestus in laboratory bioassays.
5-(Z,E)-Hexadecenoic Acid. To a stirred solution of 5-(Z,E)-
hexadecenol (3 and 4) (0.42 g, 1.75 mmol) in acetone at 0 °C was
added portionwise a solution of Jones’ reagent until no alcohol was
traced by TLC. The reaction was then quenched with 2-propanol,
filtered through Celite, and poured in EtOAc/H2O 1:1. The organic
phase was separated, the aqueous phase was washed twice with EtOAc,
and the combined organic extracts were washed successively with water
and brine, dried over Na2SO4, and concentrated in vacuo. The crude
mixture was subjected to flash column chromatography (silica gel,
hexanes/EtOAc 6:4) to afford pure 5-(Z,E)-hexadecenoic acid (0.35 g,
1
80%) as a yellow oil. Rf ) 0.50 (hexanes/EtOAc 5:5). H NMR (250
MHz, CDCl3): δ 0.88 (t, J ) 6.7 Hz, 3 H, CH3), 1.05-1.45 (m, 16 H,
CH2), 1.55-1.85 (m, 2 H, CH2CH2COOH), 1.85-2.2 (m, 4 H, CH2-
CHdCHCH2), 2.35 (t, J ) 6.7 Hz, 2 H, CH2COOH), 5.2-5.55 (m, 2
H, CHdCH). IR (neat): ν˜ 3500-2500, 2920, 2856, 1710, 1460, 1410,
935 cm-1. HRMS (FAB): m/z calcd for C16H30O2 [M + H]+, 255.2324;
found, 255.2317.
(5,6)-Dihydroxyhexadecanoic Acid (5-8). A solution of K3Fe(CN)6
(1.94 g, 5.91 mmol), K2CO3 (0.816 g, 5.91 mmol), and K2OsO2(OH)4
(7.36 mg, 0.02 mmol) in t-BuOH/H2O/acetone 4.2 mL:6 mL:1.5 mL
was cooled to 0 °C. 5-(Z,E)-Hexadecenoic acid (0.5 g, 1.97 mmol)
was added, and the reaction mixture was stirred vigorously at 0 °C for
12 h. After the addition of Na2SO3, the mixture was allowed to warm
to 25 °C and ethyl acetate was added. The organic layer was washed
with saturated NH4Cl and saturated NaCl, dried over Na2SO4, filtered,
and concentrated under reduced pressure. Purification by recrystalli-
zation afforded (5,6)-dihydroxyhexadecanoic acid (5-8) (0.52 g, 90%)
MATERIALS AND METHODS
General. All chemical reactions were carried out under anhydrous
conditions and argon atmosphere using dry, freshly distilled solvents,
unless otherwise noted. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone, dichloromethane (CH2Cl2) was distilled from
CaH2, and pyridine was first dried over solid sodium hydroxide and
distilled through an efficient fractionating column. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous
materials, unless otherwise stated. All reagents were purchased at the
highest commercial quality and used without further purification, unless
otherwise mentioned. All reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm Merck silica gel plates
(60 F254) by using UV light as the visualizing agent and ethanolic
phosphomolybdic acid or p-anisaldehyde solution followed by heat as
developing agents. Merck silica gel (60; particle size, 0.040-0.063 mm)
was used for flash column chromatography. NMR spectra were recorded
on a Brucker AC-250 instrument at 25 °C. IR spectra were recorded
on a Perkin-Elmer 1600 series FT-IR. Gas chromatograms were
recorded on a Fisons GC 8000 instrument, and high-resolution mass
spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer
under fast atom bombardment (FAB) conditions.
Synthesis of Racemic Pheromone. δ-Valerolactol. To a solution
of δ-valerolactone (2) (4 g, 40 mmol) in CH2Cl2 (60 mL) cooled at
-78 °C was added dropwise DIBAL-H (44 mL, 44 mmol, 1 M in
hexane) over 15 min. After 15 min at -78 °C, the reaction mixture
was quenched with MeOH. EtOAc (130 mL) and saturated sodium
potassium tartrate (10 mL) were added, and the whole mixture was
stirred at room temperature for 3-4 h until complete decomposition
of the aluminum complex. The granular precipitate of alumina was
filtered off through Celite, and the product was purified by flash column
chromatography (silica gel, using ether as eluent) to afford δ-valero-
lactol as a colorless oil (3.6 g, 88%). The volatile lactol was used in
the following reaction without further analysis. Rf ) 0.23 (hexanes/
EtOAc 8:2).
as a white solid. Rf ) 0.58 (hexanes/EtOAc 5:5); mp 135-138 °C. 13
C
NMR (62.5 MHz, acetone-d6): δ 13.9, 21.8, 22.8, 26.2, 28.7, 29.0,
29.4, 32.0, 32.1, 32.8, 33.9, 74.5, 74.7, 174.4. IR (KBr): ν˜ 3650-
3000, 2922, 2848, 1600, 1467, 1417, 1404, 1069 cm-1
.
6-Acetoxy-5-hexadecanolide (1). To a stirred solution of (5,6)-
dihydroxyhexadecanoic acid (5-8) (0.10 g, 0.35 mmol) in dry pyridine
(6 mL) at 0 °C was added Ac2O (1.18 mL, 12.5 mmol) (16b). The
reaction mixture was allowed to stir at 0-5 °C for 3 h. EtOAc was
added to the reaction mixture, and the organic layer was washed with
saturated CuSO4, NaHCO3, water, and brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. Purification by flash
column chromatography (silica gel, hexanes/EtOAc 6:4) afforded
6-acetoxy-5-hexadecanolide (1) (0.087 g, 80%) as a clear yellow oil.
Rf ) 0.50 (hexanes/EtOAc 6:4). 1H NMR (250 MHz, CDCl3): δ 0.88
(t, J ) 6.7 Hz, 3 H, CH3), 1.1-1.4 [m, 16 H, (CH2)8CH3], 1.5-2.0
[m, 6 H, CH2CH(O-) and CH2CH2C(O)], 2.1 (s, 3 H, Ac), 2.35-2.7
[m, 2 H, CH2C(O)], 4.3-4.42 [m, 1 H, CH(OAc)], 4.95-5.05 [m, 1
H, CH(O-)]. 13C NMR (62.5 MHz, CDCl3): δ 14.0, 18.1, 20.8, 22.5,
23.4, 25.1, 29.2, 29.3, 29.4, 31.8, 74.3, 80.5, 170.0, 170.6; major peaks
correspond to erythro (5R,6S and 5S,6R) isomers whereas less than
10% of the mixture, according to the spectrum, corresponds to threo
(5S,6S and 5R,6R) isomers. IR (neat): ν˜ 2920, 2860, 1740, 1460, 1370,
1230, 1040 cm-1. HRMS (FAB): m/z calcd for C18H32O4 [M + H]+,
313.2379; found, 313.2369.
Mosquito Rearing. A Cx. pipiens biotype molestus colony that was
maintained in the laboratory of Benaki Phytopathological Institute
(Kifissia, Greece) for more than 2 decades was used. Adults were kept
in wooden framed cages (33 cm × 33 cm × 33 cm) with 32 × 32
5-(Z,E)-Hexadecenol (3 and 4). A solution of n-butyllithium (1.6
M solution in hexane, 2.94 mL, 4.7 mmol) was added to a stirred