4980 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Zou et al.
to ∼50 μL under a gentle stream of nitrogen while the vial was chilled in
an ice bath. Equivalent samples of the synthetic racemic, (R,R)-, and
(2.0 M in toluene, from Aldrich Chemical Co.), an ∼80:20
mixture of trans:cis isomers was obtained. However, when the
(
S,S)-enantiomers of the pheromone were submitted to the same proce-
reaction was repeated using Me Zn from a different source (1.2 M
2
dure to verify that no side products were generated that might interfere
with the subsequent analyses of the samples.
in toluene, from Alfa-Aesar), with the volume of THF being
adjusted to keep the ratio of THF to toluene constant, the trans-
isomer was obtained as >95% of the product mixture. The
reason for this improved selectivity is not clear.
Aliquots of the resulting samples were analyzed by splitless injections
on the chiral stationary phase Cyclodex B column, using the temperature
program described above. The sample from the insect-produced com-
pound was found to be an 85:15 mixture of the (R,R)-, and (S,S)-
enantiomers of R-necrodol. The results were confirmed by coinjection of
the insect-produced compound with synthetic racemic R-necrodol.
Field Bioassay of the Racemic and Chiral Pheromone. Stock
solutions of the racemic pheromone (1 mg/mL) and each of the enantio-
mers (0.5 mg/mL) were made up in hexane, and dispensed onto 11 mm
gray rubber septa (West Pharmaceutical Services, Lionville, PA) (25 μL/
septum, or 25 μg of racemic pheromone and 12.5 μg of each of the
enantiomers), allowing the solvent to evaporate and the pheromone to be
adsorbed into the septum matrix in a fume hood. Control lures were
treated with 25 μL of clean hexane. Each treatment was replicated 5 times,
The final carbonof the skeleton was attached bymethylenation
of ketone 6 in 67% yield, using the CH Br -Zn-TiCl reagent as
2
2
4
previously described (14), with the exception that the ketone was
added to the thick slurry of the preformed reagent rather than vice
versa because the thick slurry was difficult to transfer by cannula.
Reduction of ester 7 in almost quantitative yield with LiAlH in
4
ether then gave β-necrodol 8. The subsequent isomerization of the
exo double bond to give R-necrodol was carried out with the
lithium salt of ethylenediamine (8). The yellow solution of the salt
was most easily produced at elevated temperatures (90 °C); at
roomtemperature, a deep bluesolution of the dissolved metal was
obtained, rather than the strongly basic salt required for the
isomerization. The isomerization reaction also required careful
timing, because R-necrodol slowly isomerized further to the
tetrasubstituted γ-necrodol, which became the sole product after
15 h. The initial preference for formation of R-necrodol is
presumably due to the more facile removal of an allylic proton
with lures being deployed in sticky Delta traps (Trece Inc., Stillwater, OK),
´ ´
in each of two Crimson Seedless grape variety vineyards near Delano, CA.
One vineyard had a high density mealybug population (traps were
hung from 14 to 27 August, 2008), whereas the other had a relatively
low mealybug population level (traps were hung from 28 August to
2
6 September, 2008). At both sites, traps were spaced ∼10 m apart, with
the order of treatments being randomized in each block of four treatments.
Trap catch data were analyzed by analysis of variance (ANOVA),
followed by Tukey’s pairwise comparison to separate means (R = 0.05).
-
þ
on C(5) rather than on C(3), because the CH O Li group on
2
C(1) is cis to C(3)-H, making that proton less accessible than
C(5)-H. This first isomerization is then followed by the slower,
second isomerization to the thermodynamically most stable
tetrasubstituted alkene. The synthesis was completed by acylation
RESULTS AND DISCUSSION
The synthesis commenced with bromination of methyl aceto-
acetate to give methyl 4-bromoacetoacetate, using conditions
previously reported for the synthesis of the analogous ethyl
ester (16, 17). This procedure proved superior to those in earlier
reports (19, 20), which resulted in a higher percentage of side
products. The crude bromoester was coupled with the enolate of
ethyl acetoacetate, prepared from ethyl acetoacetate and NaH in
THF, and the resulting diketodiester underwent intramolecular
Knoevenagel condensation in the same pot, producing diester
of R-necrodol with isobutyryl chloride and Et N, with a catalytic
3
amount of N,N-dimethylaminopyridine. The overall yield for the
entire synthetic sequence was 7%.
In our initial report of the identification of the grape mealybug
pheromone, an authentic sample of the pheromone was prepared
in milligram amount by acylation of a sample of R-necrodol
obtained as a gift (4), and thought to be the (S,S)-enantiomer (3).
The resulting isobutyrate ester gave a single peak when analyzed
on a chiral phase Cyclodex B GC column, but without having
either the racemate or the (R,R)-enantiomer available for com-
parison, it was not possible to conclusively determine the en-
antiomeric purity of the synthesized pheromone. In a small-scale
trial in a greenhouse, this material appeared to be attractive to
male grape mealybugs (3). Because of the uncertainty with regard
to the enantiomeric purity of the synthetic pheromone sample, we
decided that an unequivocal synthesis of the two enantiomers
would be prudent to verify these preliminary results. Fortunately,
both enantiomers were readily accessible via a known synthesis of
the enantiomers of β-necrodol that employed an enzyme-
catalyzed kinetic resolution (14) of alcohol 11 derived from
racemic ketoester 6 (Figure 2). Furthermore, the relative and
absolute configurations of the enantiomers of 11 had been
unequivocally demonstrated by reaction of the (þ)-enantiomer
with (-)-(1S,4R)-camphanic acid chloride, followed by determi-
nation of the crystal structure of the resulting camphanate ester
by X-ray crystallography (14).
Thus, racemic ketoester 6 was stereoselectively reduced to
(()-alcohol 11, which was then subjected to lipase-assisted kinetic
resolution with Amano AK lipase and vinyl acetate as previously
described (14). Galano et al. had reported that this reaction
resulted in 44% conversion after 8 h, whereas in our hands, only
40% conversion was achieved after 12 h. Thus, longer reaction
time, or use of a higher proportion of lipase was required in order
to get the reaction to go to completion. After adjustment of the
reaction conditions, satisfactory yields of unreacted (-)-11
(>98% ee) and the acetate (-)-12 (>98% ee) were obtained.
4
(18, 19). Fortuitously, after the condensation reaction was
complete, the weakly acidic diester product was obtained as the
sodium salt of the enolate, which was relatively insoluble in ether.
Thus, removal of the THF solvent followed by trituration of the
resulting solids with ether effectively removed both the neutral
organic side products and the mineral oil from the NaH disper-
sion. The remaining solidswere then taken up in 1 M HCl, and the
neutralized diester was readily extracted with ether, as a mixture
of the keto and enol forms. The mixture was then decarboxylated
with NaI and AcOH in refluxing diglyme (19), giving ketoester 5
in 28% purified yield over three steps. In the above reaction
sequence, the relative positions of the methyl and ethyl esters were
critically important; during optimization of the reaction para-
meters, it was found that if the methyl and ethyl ester components
were switched by starting the sequence with ethyl 4-bromoacetate
and methyl acetoacetate respectively, the decarboxylation reac-
tion gave only a complicated mixture of products.
Conjugate addition of a methyl group to ketoester 5 using
dimethylzinc with nickel catalysis occurred smoothly, but the
conditions for methylation of the resulting enolate in the same pot
with methyl iodide proved to be critically dependent on solvent.
Methylation occurred readily using HMPA as cosolvent (14),
whereas use of the less toxic DMPU in place of HMPA resulted in
no methylation of the enolate after the conjugate addition. The
stereochemical course of the reaction was also very sensitive to the
reaction conditions. In the literature report of this reaction
sequence (14), the desired trans-isomer was apparently obtained
as a single product. In our hands, using commercial Me Zn
2