Synthesis and use of stereospecifically deuterated analogues of palmitic acid to investigate the stereochemical course of the Δ11 desaturase of the processionary moth

Article abstract of DOI:10.1021/jo049320h

Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the immediate sex pheromone precursor. In this article, we describe the synthesis of different stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher the stereochemical course of the T. pityocampa Δ¹¹ desaturase. The synthesis of probes has been carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental evidence is presented that the Δ¹¹ desaturase of T. pityocampa transforms palmitic acid into (Z)-11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12.

Full text of DOI:10.1021/jo049320h

Syn th esis a n d Use of Ster eosp ecifica lly Deu ter a ted An a logu es of
P a lm itic Acid To In vestiga te th e Ster eoch em ica l Cou r se of th e ∆11
Desa tu r a se of th e P r ocession a r y Moth
J ose´-Luis Abad,* Gemma Villorbina, Gemma Fabria`s, and Francisco Camps
Departamento de Quı´mica Orga´nica Biolo´gica,
Instituto de Investigaciones Quı´micas y Ambientales de Barcelona,
Consejo Superior de Investigaciones Cientı´ficas, 08034, Barcelona, Spain
jlaqob@iiqab.csic.es
Received April 22, 2004
Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential
reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the
immediate sex pheromone precursor. In this article, we describe the synthesis of different
stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher
the stereochemical course of the T. pityocampa ∆11 desaturase. The synthesis of probes has been
carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed
resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond
simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows
the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental
evidence is presented that the ∆11 desaturase of T. pityocampa transforms palmitic acid into (Z)-
11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12.
In tr od u ction
Biosynthesis of Thaumetopoea pityocampa sex phero-
mone, (Z)-13-hexadecen-11-inyl acetate (IV), occurs from
palmitic acid by the combined action of different desatu-
rases.1 Thus, a ∆11 desaturase transforms palmitic acid
into (Z)-11-hexadecenoic acid and then this monoene is
converted into 11-hexadecynoic acid by a ∆11 acetylenase.
On the other hand, (Z)-11-hexadecenoic acid gives rise
to (Z,Z)-11,13-hexadecadienoic acid and 11-hexadecynoic
acid to (Z)-13-hexadecen-11-ynoic acid, the immediate
pheromone precursor, by the action of a ∆13 desaturase
(Figure 1).
F IGURE 1. Biosynthetic pathway of the sex pheromone of
the processionary moth T. pityocampa.
In-depth studies of the desaturase enzymes involved
in this biosynthetic pathway include their cloning and
functional expression, as well as the investigation of their
mechanistic features.
Besides insect cells, in which different ∆11 desaturases
have been cloned and sequenced,2 a ∆11 desaturase has
been identified recently in the microalga Thalassiosira
pseudonana.3 Interestingly, the latter enzyme has a
cytochrome b5 domain in its N-terminal region, whereas
this domain is not present in the insect ∆11 desaturases.
This represents a major primary structure difference
between the enzymes. Alignment of the desaturase
domain of T. pseudonana with the full sequence of insect
11
∆
desaturases showed an identity of 20%.
Several works have been published about the stereo-
chemical course of ∆11 desaturases.4-6 In addition, the
stereochemical course of other desaturases have recently
been reported.7-9 In all the reported examples, the
reactions occur by removal of both pro-(R)-hydrogen
atoms from the positions to be oxidized. This work was
undertaken to determine if the processionary moth ∆11
desaturase complies with this general rule.
* To whom correspondence should be addressed at Departamento
de Qu´ımica Orga´nica Biolo´gica, (IIQAB, CSIC), J ordi Girona 18-26,
08034 Barcelona, Spain. Phone int + 343 400 6100; fax int + 343 204
5904.
(1) Barrot, M.; Fabria`s, G.; Camps, F. Tetrahedron 1994, 50, 9789-
9796.
(2) Roelofs, W. L.; Rooney, A. P. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 9179-9184.
(3) Tonon, T.; Harvey, D.; Qing, R.; Li, Y.; Larson, T. R.; Graham,
I. A. FEBS Lett. 2004, 563, 28-34.
(4) Boland, W.; Fro¨ssl, C.; Schottler, M.; Toth, M. J . Chem. Soc.,
Chem. Commun. 1993, 1155-1157.
(5) Navarro, I., Universidad de Barcelona, 1997.
(6) Svatos, A.; Kalinova, B.; Boland, W. Insect Biochem. Mol. Biol.
1999, 29, 225-232.
(7) Behrouzian, B.; Buist, P.-H. Curr. Opin. Chem. Biol. 2002, 6,
577-582.
(8) Behrouzian, B.; Buist, P. H. Prostaglandins Leukot Essent Fatty
Acids 2003, 68, 107-112.
(9) Buist, P. H. Nat. Prod. Rep. 2004, 21, 249-262.
10.1021/jo049320h CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/14/2004
7108
J . Org. Chem. 2004, 69, 7108-7113

Deuterated Analogues of Palmitic Acid
sion to aliphatic chains. The approach is based on the
previous experimental evidence that unsaturated sub-
stituents in the hydroxymethine functionality enhanced
the enantioselectivity of some lipases.11-13 This synthetic
strategy, which starts with commercially available race-
mic mixtures of substituted secondary propargyl alcohols,
has been applied to the obtention of the enantiomerically
pure and tagged deuterated acids (R)-1a ,b and (S)-1a ,b
needed in this study (Schemes 1 and 2). According to
Scheme 1, a double enzymatic resolution (45% and 90%
conversions, respectively) of the propargyl alcohol 2 with
Candida antarctica lipase in anhydrous diisopropyl ether
in the presence of vinyl acetate afforded the enantio-
merically pure (>98% ee) acetate (S)-3 and partially
resolved alcohol (R)-2 (80% ee). The alkynyllithium salt
of this alcohol was then formed with n-butyllithium and
hexamethylphosphoramide (HMPA), and its coupling
with alkyl bromoderivative 5 afforded the elongated
intermediate alcohol (R)-6. A new enzymatic resolution
of (R)-6 with Thermomyces lanuginosus lipase immobi-
lized on EP-100 with the same reagents and conditions
as above afforded the enantiomerically pure acetate (R)-
7. Both acetates, (S)-3 and (R)-7, were saponified with a
methanolic potassium carbonate solution to their corre-
sponding enantiomerically pure alcohols, which were
further protected to the methoxymethyl derivatives under
standard conditions14 to give rise to compounds (S)-4 and
(R)-8. Finally, coupling of the alkynyllithium salt of (S)-4
with the alkyl bromoderivative 5 afforded (S)-8 in high
yields.
F IGURE 2. Enantiomerically pure deuterated probes and
their ∆11 desaturation products in T. pityocampa.
To carry out this investigation, the synthesis of chiral
deuterium-labeled fatty acids as probes was required. In
a previous article,10 we reported on a novel chemoenzy-
matic strategy for the synthesis of enantiomerically pure
secondary alcohols with sterically similar substituents.
The key step is the kinetic lipase-catalyzed resolution of
racemic mixtures of substituted propargylic alcohols. Two
strategies were tested. In the first one, racemic 1-alkynyl-
3-ols were efficiently resolved enzymatically with Can-
dida antarctica lipase B (CAL-B) and then elongated with
the suitable alkyl bromoderivative. Alternatively, disub-
stituted propargyl alcohols were resolved biocatalytically
with Thermomyces lanuginosus lipase (HLL). In this
article, these two strategies have been applied to prepare
isotopically tagged and stereotopically deuterated palm-
itic acid probes (Figure 2). Using these substrates, in the
present paper we show that the ∆11 desaturase of T.
pityocampa exhibits the same stereospecificity reported
for other ∆11 desaturase enzymes.
The presence of the alkyne functionality has simplified
the absolute configuration determination of the resolved
1
alcohols 2 and 6 by H NMR after derivatization of the
hydroxyl functionality with (R)-(-)-R-methoxy-R-pheny-
lacetic acid [(R)-MPA].
Furthermore, the acetylenic bond is susceptible to
being deuterated for isotopic tagging of the final probe.
The tagging strategy is necessary to avoid putative
interferences by the endogenous natural materials in the
gas chromatography/mass spectrometry (GC/MS) analy-
ses, which prevent the obtention of reliable data. Each
of the enantiomers of 8 was isotopically tagged and
transformed into the final probes as exemplified in
Scheme 2 for (R)-1a ,b. Thus, introduction of deuterium
into 8 to furnish compounds 9 was achieved by non-
scrambling deuteration of the triple bond by use of the
Wilkinson catalyst. It is worth mentioning that the
deuteration reaction had to be carried out with the
methoxymethyl-protected alcohol intermediates 8 since
the unprotected alcohols 6 rendered important amounts
of ketone10,15 and in order to reduce the deuterium
scattering.16 Under the optimum synthetic conditions,
mean ratios of d6, d5, d4, d3, and d2 fatty acids were,
Resu lts a n d Discu ssion
Syn th esis of P r obes. To study the stereospecificities
of fatty acid desaturases, the synthesis of chiral deuterium-
labeled fatty acids as probes is required. For preparation
of pure enantiomers of the monodeuterated acids, chiral
alcohols are suitable intermediates that afford the deu-
terated stereogenic centers by transformation of the
hydroxyl functionality into a good leaving group and its
subsequent replacement by the deuterium label. A good
approach to obtain the chiral alcohols is the kinetic
enzymatic resolution of the corresponding racemic pre-
cursors with lipases. However, biocatalytic racemic reso-
lutions become more difficult as the hydroxyl group is
displaced toward the middle position of the aliphatic
chain and the chain length is enlarged, making the
biocatalytic approaches useless for preparation of some
enantiomerically pure deuterated compounds. To solve
this problem, we have developed a general methodology
by combining the reduced steric requirements of the
acetylenic moiety for enzymatic chiral discrimination and
the chemical versatility of this group for its easy conver-
(11) Fukusaki, E.; Senda, S.; Nakazono, Y.; Omata, T. Tetrahedron
1991, 47, 6223-6230.
(12) Guanti, G.; Banfi, L.; Narisano, E. Tetrahedron: Asymmetry
1990, 1, 721-724.
(13) Naoshima, Y.; Kamezawa, M.; Tachibana, H.; Munakata, Y.;
Fujita, T.; Kihara, K.; Raku, T. J . Chem. Soc., Perkin Trans. 1 1993,
557-561.
(14) Gras, J .-L.; Kong, Y.-Y.; Chang, W.; Guerin, A. Synthesis 1985,
74-75.
(15) Mori, K.; Ohtaki, T.; Ohrui, H.; Berkebile, D. R.; Carlson, D.
A. Eur. J . Org. Chem. 2004, 2004, 1089-1095.
(16) Rakoff, H.; Rohwedder, W. K. Lipids 1992, 27, 567-569.
(10) Abad, J .-L.; Soldevila, C.; Clapes, P.; Camps, F. J . Org. Chem
2003, 68, 5351-5356.
J . Org. Chem, Vol. 69, No. 21, 2004 7109

Abad et al.
SCHEME 1. Syn th esis of (R)-8a ,b a n d (S)-8a ,ba
a
Reagents and conditions: (a) CAL-B/diisopropyl ether/vinyl acetate; (b) K2CO3/MeOH; (c) BrLi/TsOH/ dimethoxymethane; (d) BuLi/
THF/HMPA; (e) HLL/diisopropyl ether/vinyl acetate.
SCHEME 2. Tr a n sfor m a tion of (R)-8a ,b in P r obes (R)-1a ,ba
a
Reagents and conditions: (f) Wilkinson catalyst/D2/benzene; (g) HCl/MeOH; (h) TBMSCl/DBU/CH2Cl2; (i) NEt3/MsCl/CH2Cl2; (j) LiAlD4/
Et2O; (k) HCl/MeOH; (l) PDC/DMF.
respectively, 11.8, 78.4, 6.6, 2.4, and 0.8 (by GC/MS of
the corresponding fatty acid methyl esters).
revealing the replacement of five hydrogen atoms (2CH2
and CH) by deuterium, as well as some chemical shift
changes in the 30-28.6 ppm range in the 13C NMR
spectra.
The absolute configuration and enantiomeric excesses
(ee) of the different unsaturated and saturated alcohol
intermediates were determined by NMR, as in previous
articles,10,18 after derivatization with either (R)-MPA21 or
synthetic (R)-(-)-R-methoxy-R-(9-anthryl)acetic acid, [(R)-
ΑΜΑ, 97% ee], respectively.22 In the case of synthetic
series b, the absolute configuration was verified by (R)-
MPA derivatization of commercially available enanti-
omers (R)-2b and (S)-2b.
Ster eosp ecificity Stu d ies. For the unequivocal de-
termination of the stereospecificity of the ∆11 desaturase,
the fate of the individual hydrogen/deuterium atoms
bound to C11 and C12 of both enantiomers of 1a and 1b
was investigated. The pheromone glands of T. pityocampa
females were incubated with each probe, and tissues were
extracted and methanolyzed. The fatty acid methyl ester
extracts thus obtained were analyzed by chemical ioniza-
tion GC/MS with methane as ionization gas. This mode
affords the M• + + 1 ion as the most abundant fragment
in the mass spectra (Figure 3). From incubations with
(R)-1a , the resulting labeled methyl dienoate displayed
a M• + + 1 isotopic cluster centered at m/z 272. This
implicates the loss of two deuterium atoms from the
saturated, deuterium-labeled precursor (R)-1a . Con-
versely, after incubations with (S)-1a , the M• + + 1 ion
of the corresponding methyl dienoate was centered on
Deprotection of the hydroxyl groups in acidic media
gave rise to the corresponding diols 10, which could be
regioselectively protected in the primary alcohol group
with tert-butyldimethylsilyl chloride (TBDMSCl) in the
presence of DBU, thus affording compounds 11.17 The
stereotopic deuterium label was introduced in two steps
with absolute configuration inversion of the stereogenic
center by mesylation of the secondary hydroxyl group,
followed by reaction of 12 with LiAlD4.18,19 Finally,
treatment with HCl/MeOH and further PDC oxidation
with N,N-dimethylformamide (DMF)20 as solvent af-
forded each one of the enantiotopically deuterated palm-
itic acids 1a ,b, which also bear a tetradeuterium tag for
unambiguous GC/MS investigation of their metabolic
fate.
The number and the presence of the deuterium labels
was as expected from the analyses of 13C NMR (presence
of two quintuplets for CD2 in compounds 9-12 and an
additional triplet for CHD in compounds 13, 14, and 1)
and mass spectra. In addition, NMR comparison of the
deuterated compounds 1a ,b and 14a ,b with the corre-
sponding nondeuterated counterparts (palmitic acid and
cetyl alcohol, respectively) showed some differences owing
to the presence of the deuterium atoms. Thus, the most
important dissimilarities include the minor integration
of the signal at 1.25 ppm in the 1H NMR spectra,
(17) Aizpurua, J . M.; Palomo, C. Tetrahedron Lett 1985, 26, 475-
476.
(18) Abad, J .-L.; Fabria`s, G.; Camps, F. J . Org. Chem. 2000, 65,
8582-8588.
(21) Trost, B. M.; Belletire, J . L.; Godleski, S.; McDougal, P. G.;
Balkovec, J . M. J . Org. Chem. 1986, 51, 2370-2374.
(22) Seco, J . M.; Quin˜oa´, E.; Riguera, R. Chem. Rev. 2004, 104, 17-
117.
(19) Krishnamurthy, S.; Brown, H. C. J . Org. Chem. 1980, 45, 849-
856.
(20) Corey, E. J .; Schmidt, G. Tetrahedron Lett 1979, 399-402.
7110 J . Org. Chem., Vol. 69, No. 21, 2004

Deuterated Analogues of Palmitic Acid
F IGURE 3. Isotopomers of the M• + + 1 ion clusters corresponding to the methyl (Z)-11-hexadecenoates present in the fatty acid
methyl ester extracts from pheromone glands of T. pityocampa cultured in the presence of the indicated enantiomerically pure
deuterated probes 1a ,b.
m/z 273, indicating the loss of the C11 deuterium atom
along with the C12 hydrogen from the saturated precur-
sor. In the experiments with (R)-1b, the M• + + 1 ion
cluster of the resulting dienoate was centered at m/z 273,
whereas (S)-1b gave rise to a dienoate in which the most
abundant M• + + 1 isotopomer was that at m/z 274. This
implies the loss of either deuterium or hydrogen atoms
from (R)-1b and (S)-1b, respectively. These overall
results indicate that the ∆11 desaturation of palmitic acid
in the moth T. pityocampa occurrs with removal of both
pro-(R)-hydrogen atoms from C11 and C12 of the sub-
strate.
The overall results obtained in the biochemical studies
with the enantiomerically pure deuterated probes 1a and
1b are a clear indication of a syn-elimination process
occurring with removal of both pro-(R)-hydrogen atoms
from C11 and C12 of the precursor acid. The observed
stereochemistry corresponds to the previously established
stereochemistry of the desaturation step in Mamestra
brassicae,4 Spodoptera littoralis,23 Bombyx mori,6 and
Manduca sexta.6 Thus, the present work provides a new
example of the stereospecificity course of ∆11 desaturases
from moth females.
Such a steric course probably arises from the confor-
mation adopted by the substrate at the enzyme active
site in that the hydrophobic chain of the fatty acid is
twisted around the carbon atoms to be oxidized to form
a U-shape. This particular orientation of the substrate
exposes the two pro-(R)-hydrogen atoms to the enzyme
diiron center, thus allowing the syn-elimination of both
hydrogen atoms and the formation of the Z isomer of the
resulting olefinic product.
It is worth mentioning that the relative amounts of
deuterated olefinic products formed from either (R)-1a ,
(S)-1a , and (R)-1b were similar and approximately 6
times lower than those produced from the (S)-1b precur-
sor. This result suggests that the abstraction of C11-H
is sensitive to isotopic substitution and it occurs with a
primary isotope effect of ca. 6. This result agrees with
the mechanism found for the ∆11 desaturase present in
S. littoralis, in which the rate-limiting step is oxidation
at C11.24 A more detailed study of the cryptoregiochem-
istry (site of initial oxidation)25 of this ∆11 desaturase is
currently under way.
Con clu sion s
In summary, we have reported a procedure to obtain
enantiomerically pure deuterated palmitic acids with a
tetradeuterium tag and their use to decipher the ste-
reospecificity of the processionary moth ∆11 desaturase.
The synthesis of probes has been carried out by applica-
tion of a chemoenzymatic strategy previously reported,
in which the key step is the kinetic lipase-catalyzed
resolution of racemic mixtures of secondary propargylic
alcohols. The presence of the acetylenic bond simplifies
the absolute configuration determination of the resolved
alcohols and, on the other hand, allows the introduction
of the isotopic tag by deuteration. Using the substrates
thus prepared, we show that the ∆11 desaturation of
palmitic acid in T. pityocampa occurs with removal of
both pro-(R)-hydrogen atoms from C11 and C12 of the
substrate and, therefore, it has the same stereospecificity
reported for other (Z)-11-desaturase enzymes.
Exp er im en ta l Section
En zym e Dep osition a n d Absor p tion . C. antarctica was
supplied as an immobilized preparation on a macroporous
acrylic resin. Adsorption of T. lanuginosus lipase onto polypro-
pylene support (EP100) was carried out following the meth-
odology described by Gitlesen et al.26 The lipase (10 g) was
dissolved in aqueous phosphate buffer (200 mL, 0.1 M, pH 7.0)
and mixed with the solid support EP100 (10 g), containing
ethanol/water (30 mL), 24:1 v/v. The mixture was reciprocally
shaken (120 rpm) for 24 h at 25 °C. After this period, the
enzyme-support preparation was filtered off and dried under
vacuum until a constant weight was obtained.
(24) Pinilla, A.; Camps, F.; Fabrias, G. Biochemistry 1999, 38,
15272-15277.
(25) Buist, P. H.; Behrouzian, B. J . Am. Chem. Soc. 1996, 118, 6295-
6296.
(23) Navarro, I.; Font, I.; Fabrias, G.; Camps, F. J . Am. Chem. Soc.
1997, 119, 11335-11336.
(26) Gitlesen, T.; Bauer, M.; Adlercreutz, P. Biochim. Biophys Acta
1997, 1345, 188-196.
J . Org. Chem, Vol. 69, No. 21, 2004 7111

Abad et al.
The activity of both immobilized lipase preparations was
measured by use of the acylation of 1-dodecanol in organic
media.10 The specific activities (units per milligram of im-
mobilized preparation) of the immobilized preparations were
5.05 for CAL-B, and 0.30 for HLL [where 1 unit corresponds
to the amount of enzyme that converts 1 µmol of dodecyl
acetate from 1-dodecanol and vinyl acetate (0.2 mmol) in
anhydrous diisopropyl ether at 25 °C].
(15), 140 (20), 133 (75), 127 (25), 115 (25), 99 (30); (S)-(+)-11a
[R] ) +0.6 (c ) 3, CHCl3); (R)-(-)-11a [R] ) -0.6 (c ) 3,
CHCl3).
Red u ction of Mesyla tes 12: Gen er a l P r oced u r e. The
mesyl derivative was dissolved in Et2O (4 mL) and treated with
LiAlD4 (8 molar equiv) for 16 h at 20 °C (TLC monitoring).
H2O was added dropwise to the crude reaction mixture and
the resulting white precipitate was filtered through Celite and
concentrated to give a residue that, after purification by flash
chromatography on silica gel with a gradient of 0-10% MTBE
in hexane, gave the corresponding pure deuterated products
13 with 61-68% yields. In this reaction some desilylated
alcohol was obtained (20-28%) that could be combined with
what came from the next reaction.
[10,10,11,11,12-2H 5]-1-O-(t Bu t yd im et h ylsilyl)h exa d e-
ca n e (13a ). Compounds (S)-13a (161 mg, 68%) and (R)-13a
(138 mg, 62%) were obtained from 290 mg of (S)-12a and 272
mg of (R)-12a : IR 2955, 2925, 2855, 1465, 1380, 1360, 1255,
1100, 835, 775 cm-1; 1H NMR δ 3.60 (t, J ) 6.5 Hz, 2H), 1.57-
1.44 (2H), 1.40-1.18 (21H), 0.89 (s, 9H), 0.88 (t, J ) 7 Hz,
3H), 0.05 (s, 6H); 13C NMR δ 63.4 (CH), 32.9 (CH2), 31.9 (CH2),
29.7 (CH2), 29.5 (CH2), 29.2 (CH2), 29.2 (t, J ) 19 Hz, CHD),
28.7 (quint, J ) 19 Hz), 28.6 (quint, J ) 19 Hz), 26.0 (CH2),
25.8 (CH2), 22.7 (CH2), 18.4 (C), 14.1 (CH3), -5.3 (CH3); MS
m/z 390 (M• + + 18, 10), 360 (M‚+-2, 90), 346 (80), 304 (100),
228 (30), 133 (45), 127 (35), 115 (10), 99 (10).
P r ep a r a tion of Ca r boxylic Acid s 1: Gen er a l P r oce-
d u r e. According to the procedure reported by Corey and
Schmidt,20 alcohols obtained previously were stirred in a
solution of PDC (3 equiv) in DMF (0.2 M) for 3 days. At this
time, the reaction mixture was treated with Na2S2O3 solution
until a green solution was obtained; then 2 mL of HCl (1 M)
was added and the mixture was extracted with CH2Cl2, dried,
and concentrated to a residue that was purified by flash
chromatography on silica gel with hexane/MTBE 85:15 to give
the corresponding acids in 65-70% yields.
Deu ter a tion of Meth oxym eth a n e-P r otected Alk yn ols
8: Gen er a l P r oced u r e. Preparation of Wilkinson’s cata-
lyst:27 A mixture of 2.1 g (8 mmol) of triphenylphosphine and
0.25 g (1.2 mmol) of RhCl3 in 60 mL of ethanol was refluxed
under an argon atmosphere. Heating was continued for 5 days
until red crystals precipitated; the mixture was then allowed
to cool to room temperature. The precipitate was filtered out,
washed with methanol and ether, and dried at reduced
pressure to afford 1.08 g (1.16 mmol, 98% yield) of Wilkinson’s
catalyst that was stored at 4 °C at dark under argon atmo-
sphere. The product prepared under these conditions has been
active after 1 year of storage.
To a mixture of 1.71 g of 8 (5 mmol) and 275 mg (0.3 mmol)
of RhCl(PPh3)3 was added 20 mL of degassed benzene under
an argon atmosphere to get a reddish solution. The system
was purged by passing a stream of D2 through, then the D2
atmosphere was kept from the balloon and the solution was
stirred for 36 h. The mixture was filtered through a bed of
Celite and the solvent was evaporated. Residue was purified
by flash chromatography on silica gel (0-3% MTBE/hexane)
to give product 9 (92-96% yield).
[6,6,7,7-2H 4]-5-Me t h oxym e t h yloxy-17,19-d ioxa e ico-
sa n e (9a ). Compounds (S)-9a (1.61 g, 92%) and (R)-9a (1.63
g, 93%) were obtained from compounds (S)-8a and (R)-8a ,
respectively: IR 2925, 2855, 1465, 1150, 1110, 1045, 920 cm
1
-1; H NMR δ 4.65 (s, 2H), 4.62 (s, 2H), 4.65-4.55 (1H), 3.52
(t, J ) 6.5 Hz, 4H), 3.38 (s, 3H), 3.36 (s, 3H), 1.65-1.53 (2H),
1.53-1.20 (18H), 0.90 (t, J ) 7 Hz, 3H); 13C NMR δ 96.3 (CH2),
95.2 (CH2), 77.2 (CH), 67.8 (CH2), 55.3 (CH3), 55.0 (CH3), 33.9
(CH2), 33.5 (quint, J ) 18 Hz), 29.7 (CH2), 29.5 (CH2), 29.4
(CH2), 27.4 (CH2), 26.1 (CH2), 24.2 (quint, J ) 18 Hz), 22.8
(CH2), 14.1 (CH3); MS m/z 349 (M• + - 1, 1), 317 (2), 289 (25),
275 (10), 257 (100), 239 (15), 217 (10), 187 (10), 169 (5), 141
(10), 113 (12), 95 (20), 85 (20); (S)-(-)-9a [R] ) -2.0 (c ) 2,
CHCl3); (R)-(+)-9a [R] ) +1.9 (c ) 2, CHCl3).
[10,10,11,11,12-2H5]Hexa d eca n oic Acid (1a ). Compounds
(S)-[10,10,11,11,12-2H5]hexadecanoic acid [(S)-1a ] (56 mg, 68%)
and (R)-[10,10,11,11,12-2H5]hexadecanoic acid [(R)-1a ] (59 mg,
70%) were obtained from 78 mg of (S)-14a and 80 mg of (R)-
14a : mp 59-61 °C; IR 2920, 2855, 1705, 1460, 1415, 1295,
1230, 910, 735 cm-1; 1H NMR δ 2.35 (t, J ) 7.5 Hz, 2H), 1.70-
1.55 (2H), 1.40-1.14 (21H), 0.88 (t, J ) 7 Hz, 3H); 13C NMR
δ 180.4 (CO), 34.1 (CH2), 29.6 (CH2), 29.4 (CH2), 29.2 (CH2),
29.1 (CH2), 29.0 (t, J ) 19 Hz, CHD), 28.6 (quint, J ) 19 Hz),
28.5 (quint, J ) 19 Hz), 24.7 (CH2), 22.7 (CH2), 14.1 (CH3).
Anal. Calcd for C16H272H5O2: C, 73.50; H, 12.34. Found: C,
73.53; H, 12.12.
In Vitr o Gla n d Cu ltu r e P r oced u r e: Deter m in a tion of
Ster eosp ecificity. These experiments were carried out in
round-bottom 96-well plates. In these experiments, 1-day-old
virgin T. pityocampa females were briefly anesthetized on ice
and pheromone glands were everted and excised, carefully
cleaned, and immersed (1 gland/well). To each well, a 10 µL
drop of incubation medium was added. The incubation medium
consisted of the commercial Grace’s insect medium (135 µL)
and a dimethyl sulfoxide (DMSO) solution (15 µL) of stere-
oespecifically deuterated probe 1 (10 mg/mL each) for treated
tissues or a DMSO solution for controls. Plates were sealed
with adherent plastic covers, and incubations proceeded for 3
h at 25 °C. After this time, to obtain the methyl ester
derivatives of the gland lipids for analysis, pheromone glands
were soaked in chloroform/methanol (2:1) at 25 °C for 1 h and
base-methanolized in 0.5 M KOH for 30 min, and then the
organic solution was neutralized with 1 N HCl and extracted
with hexane containing methyl pentadecanoate (10 ng/gland)
as internal standard for quantification. Five glands were used
for each sample.
P r im a r y Alcoh ol P r otection : Gen er a l P r oced u r e. tBu-
tyldimethylsiloxy (TBDMS) protecting group was regioselec-
tively introduced at the primary alcohol by the procedure
reported by Aizpurua and Palomo with minor modifications.17
Thus, a mixture of the diol 10 (525 mg, 2 mmol), tert-
butyldimethylsilyl chloride (TBDMSCl) (360 mg, 2.4 mmol),
and DBU (456 mg, 3 mmol) in dry CH2Cl2 (20 mL) was stirred
under an argon atmosphere for 1 h. Then the resulting mixture
was poured into 10 mL of an aquous solution saturated with
CO2 gas and extracted with CH2Cl2 (2 × 3 mL), and the
combined organic fractions were evaporated to obtain a residue
that was purified by column chromatography (hexanes/MTBE
10:1) on silica gel to afford the silylated product 11 (84-91%
yield).
[6,6,7,7-2H4]-16-O-(ter t-Bu tyld im eth ylsilyl)h exa d eca n -
5-ol (11a ). Compounds (S)-11a (639 mg, 85%) and (R)-11a (677
mg, 90%) were obtained from 525 mg of (S)-10a and 525 mg
of (R)-10a : IR 3355, 2955, 2925, 2855, 1465, 1255, 1100, 835
cm-1 1H NMR δ 3.60 (t, J ) 6.5 Hz, 4H), 1.66-1.20 (20H),
;
0.91 (t, J ) 7 Hz, 3H), 0.90 (s, 9H), 0.05 (s, 6H); 13C NMR δ
71.8 (CH), 63.3 (CH), 37.1 (CH2), 36.4 (quint, J ) 19 Hz, CD2),
32.8 (CH2), 29.6 (CH2), 29.4 (CH2), 27.8 (CH2), 25.9 (CH2), 25.8
(CH2), 24.6 (quint, J ) 19 Hz), 22.7 (CH2), 18.3 (C), 14.1 (CH3),
-5.3 (CH3); MS m/z 377 (M• + + 1, 20), 359 (100), 342 (45),
319 (35), 301 (50), 273 (10), 245 (15), 227 (35), 173 (10), 156
In str u m en ta l An a lysis of th e Biologica l Extr a cts. GC/
MS analysis of extracts was performed by chemical ionization
(CI) with methane as ionization gas. The system was equipped
with a nonpolar capillary column (30 m × 0.25 mm i.d., 0.25
(27) Osborn, J . A.; J ardine, F. H.; Young, J . F.; Wilkinson, G. J .
Chem. Soc. A 1966, 1711-1732.
7112 J . Org. Chem., Vol. 69, No. 21, 2004

Deuterated Analogues of Palmitic Acid
µm stationary phase thickness) and used the following pro-
gram: from 120 to 180 °C at 5 °C/min and then to 260 °C at
2 °C/min after an initial delay of 2 min. Analyses were carried
out on methanolyzed lipidic extracts from pheromone glands
with the equipment and conditions described above. Ste-
reospecificity was determined from the abundance of ions in
the range m/z 270-275, which afforded a cluster of ions in
which the most abundant corresponded to the molecular ion
of the resulting isotopomer of ∆11-16-acid, analyzed as methyl
ester, formed from each probe.
J .-L.A. thanks Spanish MCYT for a Ramo´n y Cajal
contract. We are indebted to Dr. Pere Clape´s for the
helpful advice in the lipase-catalyzed reactions. We also
acknowledge Rodolfo Herna´ndez from Laboratorio de
Sanidad Forestal (Gobierno de Arago´n, Mora de Rubie-
los, Teruel) for T. pityocampa female pupae.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
1
tal section and copies of H and 13C NMR and DEPT spectra
for compounds 1b, 2-4, and 6-14 (PDF). This material is
Ack n ow led gm en t . We gratefully acknowledge
CICYT and FEDER (AGL2001-0585) and Generalitat
de Catalunya (2001SGR 00342) for financial support.
available free of charge via the Internet at http://pubs.acs.org.
J O049320H
J . Org. Chem, Vol. 69, No. 21, 2004 7113