Synthesis of gem-dideuterated tetradecanoic acids and their use in investigating the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid

Article abstract of DOI:10.1021/jo010560w

We report the preparation of the deuterated tetradecanoic acids [2,2,3,3-²H₄]-, [2,2,3,3,10,10-²H₆]-, and [2,2,3,3,13,13-²H₆]-tetradecanoic acids (1, 2, and 3, respectively) and their use to investigate the mechanism of the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid. Probes 2 and 3 were prepared from intermediate ketones 7 and 10, which were transformed into the labeled bromides 17 and 18 by reduction with NaBD₄, tosylation of the resulting alcohol, replacement of the tosyloxy group by deuteride with LiAlD₄, hydrolysis, and reaction with N-bromosuccinimide. The resulting bromides were converted into the α-acetylenic esters 21 and 22, respectively, and the additional deuterium labels were introduced by reduction of the conjugated triple bond with Mg in deuterated methanol. The same sequence of reactions starting with 11-bromoundecane afforded 27. Saponification of the labeled esters 23, 24, and 27 gave the deuterated acids 2, 3, and 1, respectively. The results of the biochemical experiments showed that C10-H removal, but not elimination of C13-H, was sensitive to deuterium substitution in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, which is consistent with the hypothesis that this desaturase reaction involves a first slow, C10-H bond cleavage, with probable formation of an unstable allylic intermediate, followed by a second fast C13-H bond removal and concomitant rearrangement.

Full text of DOI:10.1021/jo010560w

8052
J . Org. Chem. 2001, 66, 8052-8058
Syn th esis of gem -Did eu ter a ted Tetr a d eca n oic Acid s a n d Th eir Use
in In vestiga tin g th e En zym a tic Tr a n sfor m a tion of
(Z)-11-Tetr a d ecen oic Acid in to (E,E)-10,12-Tetr a d eca d ien oic Acid
Sergio Rodr´ıguez, Francisco Camps, and Gemma Fabria`s*
Departamento de Quı´mica Orga´nica Biolo´gica, IIQAB, CSIC, J ordi Girona 18, 08034-Barcelona, Spain
gfdqob@cid.csic.es
Received J une 4, 2001
We report the preparation of the deuterated tetradecanoic acids [2,2,3,3-2H4]-, [2,2,3,3,10,10-2H6]-,
and [2,2,3,3,13,13-2H6]-tetradecanoic acids (1, 2, and 3, respectively) and their use to investigate
the mechanism of the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-
tetradecadienoic acid. Probes 2 and 3 were prepared from intermediate ketones 7 and 10, which
were transformed into the labeled bromides 17 and 18 by reduction with NaBD4, tosylation of the
resulting alcohol, replacement of the tosyloxy group by deuteride with LiAlD4, hydrolysis, and
reaction with N-bromosuccinimide. The resulting bromides were converted into the R-acetylenic
esters 21 and 22, respectively, and the additional deuterium labels were introduced by reduction
of the conjugated triple bond with Mg in deuterated methanol. The same sequence of reactions
starting with 11-bromoundecane afforded 27. Saponification of the labeled esters 23, 24, and 27
gave the deuterated acids 2, 3, and 1, respectively. The results of the biochemical experiments
showed that C10-H removal, but not elimination of C13-H, was sensitive to deuterium substitution
in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, which is
consistent with the hypothesis that this desaturase reaction involves a first slow, C10-H bond
cleavage, with probable formation of an unstable allylic intermediate, followed by a second fast
C13-H bond removal and concomitant rearrangement.
In tr od u ction
The sex pheromone blend of the moth Spodoptera
littoralis is an especially interesting example, since it
contains two different conjugated diene systems: (Z,E)-
9,11-tetradecadienoate11,12 and (E,E)-10,12-tetradecadi-
enoate.13 Biosynthesis of these compounds takes place
from myristic acid, which is desaturated to both (E)- and
(Z)-11-tetradecenoic acid.12 The (E)-isomer is then con-
verted into the (Z,E)-9,11-tetradecadienoate,12 whereas
the (Z)-isomer is transformed into the (E,E)-10,12-tet-
radecadienoate.13 In the (E)-monoene desaturation, both
the location and stereochemistry of the substrate double
bond are maintained in the diene product, whereas the
(Z)-monoene desaturation involves the formation of a
second double bond with concomitant rearrangement of
the substrate monounsaturation.
Fatty acyl desaturases are non-heme iron-containing
oxygen-dependent enzymes involved in the regio- and
stereoselective introduction of double bonds in fatty acyl
aliphatic chains.1 Although most desaturases use satu-
rated fatty acyl derivatives, desaturases that transform
unsaturated substrates do also occur in nature. In the
resulting polyunsaturated fatty acids, double bonds tend
to be methylene-interrupted and in the (Z) configuration.
However, fatty acids containing conjugated olefinic bonds
with either (Z) or (E) double bonds have also been
reported in algae,2,3 plants, and insects. In plants, various
conjugated linolenic acid isomers accumulate in seeds;
examples include (Z,E,E)-(9,11,13)-octadecatrienoic acid,4
(Z,E,Z)-(9,11,13)-octadecatrienoic acid, and (Z,E,Z)-(8,-
10,12)-octadecatrienoic acid.5,6 In insects, moth phero-
mone glands contain desaturases that catalyze the
formation of conjugated dienoic acids, such as (E,Z)-10,-
12-hexadecadienoic acid,7,8 (Z,Z)-11,13-hexadecadienoic
acid,9 and (E,E)-8,10-dodecadienoic acid.10
In previous papers,14,15 we have reported on the cryp-
toregiochemistry14 and stereospecificity15 of the above (E)-
monoene desaturase. This reaction occurs by initial
abstraction of the pro-(R) hydrogen atom at C9 followed
by rapid elimination of the pro-(R) hydrogen atom at C10.
In this paper, we report on the cryptoregiochemistry of
the desaturation of (Z)-11-tetradecenoate into (E,E)-10,-
12-tetradecadienoate. The putative existence of a single
(1) Shanklin, J .; Cahoon, E. B. Annu. Rev. Plant Physiol. Mol. Biol.
1998, 49, 611-641.
(2) Burgess, J . R.; de la Rosa, R. I.; J acobs, R. S.; Butler, A. Lipids
1991, 26, 162-165.
(3) Wise, M. L.; Rossi, J .; Gerwick, W. H. Biochemistry 1997, 36,
2985-92.
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Physiol. 1990, 14, 47-56.
(4) Liu, L.; Hammond, E. G.; Nikolau, B. J . Plant Physiol. 1997,
113, 1343-1349.
(10) Lofstedt, C.; Bengtsson, M. J . Chem. Ecol. 1988, 14, 903-914.
(11) Dunkelblum, E.; Kehat, M. Insect Biochem. 1987, 17, 877-881.
(12) Martinez, T.; Fabrias, G.; Camps, F. J . Biol. Chem. 1990, 265,
1381-1387.
(5) Chisholm, M. J .; Hopkins, C. Y. Can. J . Biochem. 1967a , 45,
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(6) Chisholm, M. J .; Hopkins, C. Y. Can. J . Biochem. 1967b, 45,
1081-1086.
(13) Navarro, I.; Mas, E.; Fabrias, G.; Camps, F. Bioorg. Med. Chem.
1997, 5, 1267-1274.
(7) Ando, T.; Ikemoto, K.; Ohno, R.; Yamamoto, M. Arch. Insect
Biochem. Physiol. 1998, 37, 8-16.
(14) Abad, J . L.; Camps, F.; Fabrias, G. Angew. Chem., Int. Ed. 2000,
39, 3279-3281.
(8) Fang, N. B.; Teal, P.; Doolittle, R. E.; Tumlinson, J . H. Insect
Biochem. Mol. Biol. 1995, 25, 39-48.
(15) Abad, J . L.; Camps, F.; Fabrias, G. Insect Biochem. Mol. Biol.
2001, 31, 799-803.
10.1021/jo010560w CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/30/2001

Synthesis of gem-Dideuterated Tetradecanoic Acids
J . Org. Chem., Vol. 66, No. 24, 2001 8053
lation conditions, such as DABCO/xylene18 or lithium
chloride/dimethylsulfoxide.19,20
Ketone 10 was prepared from properly functionalized
dithiane 9 obtained by coupling reaction of 1-Bromo-7,9-
dioxadecane (5) with the anion of dithiane 8, generated
by reaction with BuLi.21 Final cleavage of 9 with NBS22
afforded ketone 10 (Scheme 1).
Introduction of deuterium label was carried out in
three steps (Scheme 2). Reduction of ketones 7 and 10
with NaBD4,23 followed by tosylation24 of the resulting
alcohols 11 and 12, respectively, and replacement of the
tosyloxy group by deuteride with LiAlD4.25 The alcohols
15 and 16 were recovered by hydrolysis26 and then
converted into the corresponding bromides27 17 and 18,
respectively. Reaction of bromoalkanes 17 and 18 with
lithium acetylide28 furnished the labeled terminal alkynes
19 and 20. The same reaction starting from 1-bromoun-
decane gave 1-tridecyne (25) (Scheme 3). Reaction of
methyl chloroformate with the lithium salts of terminal
alkynes29 19, 20, and 25 afforded the R-acetylenic esters
21, 22, and 26, respectively. The additional deuterium
labels were introduced by reduction of the conjugated
triple bond with Mg in deuterated methanol.30 Labeled
esters 23, 24, and 27 were thus obtained and were finally
saponified to the deuterated acids 2, 3, and 1, respec-
tively.
F igu r e 1. Deuterated myristic acids 1-3.
Sch em e 1. Syn th esis of Keton es 7 a n d 10a
The presence of deuterium in the probes synthesized
was confirmed by GC-MS analysis of the corresponding
methyl esters (M.+: 2 and 3, m/z 248; 1, m/z 246).
Furthermore, the presence of deuterium at C2 and C3
in all probes was confirmed by the multiplicity and
coupling constants of their corresponding 13C NMR
signals at 33.4 and 23.8 ppm, respectively (Table 1).
Likewise, the occurrence of a CD2 group at C13 of 2 was
assessed by the signal at 21.8 ppm (quintet, J ) 19.0 Hz)
and the existence of a CD2 moiety in 3 was confirmed by
the signal at 24.2 ppm (quintet, J ) 20.0 Hz) in the 13C
NMR spectra of these compounds.
Isotop e Effect Exp er im en ts. The intermolecular
primary kinetic isotope effects (KIE) are determined in
competitive experiments between one substrate and the
same substrate dideuterated at the appropriate sites of
desaturation. In this case, probes were labeled myristic
acids 1, 2, and 3, which, after activation to the corre-
a
Reagents and conditions: (a) Na/ethanol/ethyl acetoacetate,
reflux, then 4, reflux, 26 h (60%); (b) Na/ethanol, then ethanediol,
then 6/90 °C/30 min (60%); (c) BF3‚Et2O/AcOH/propanedithiol/
CHCl3, reflux, 16
Br(CH2)6OCH2OCH3 (5)/THF, -78 °C, 1 h (81%); (e) NBS, acetone/
water, -20 °C (85%).
h (93%); (d) BuLi -30 °C/90 min, then
(18) Huang, B.; Parish, E. J .; Miles, H. D. J . Org. Chem. 1974, 39,
2647-2648.
monoene desaturase that forms both the (Z,E)-9,11- and
the (E,E)-10,12-diene systems from the corresponding
(E)- and (Z)-11-monoene substrates is also discussed.
(19) Krapcho, A. P.; Lovey, A. J . Tetrahedron Lett. 1973, 12, 957-
960.
(20) Krapcho, A. P.; Weimaster, J . F.; Eldridge, J . M.; J ahngen, E.
G. E. J .; Lovey, A. J .; Stephens, W. P. J . Org. Chem. 1978, 43, 138-
146.
Resu lts
(21) Seebach, D.; Corey, E. J . J . Org. Chem. 1975, 40, 231-237.
(22) Corey, E. J .; Erickson, B. W. J . Org. Chem. 1971, 36, 3553-
3560.
Syn th esis of Deu ter iu m -La beled P r obes. The re-
quired deuterium-labeled compounds 1-3 (Figure 1) for
the competition experiments were prepared as depicted
in Schemes 2 and 3 from intermediate ketones 7 and 10,
obtained as shown in Scheme 1. Ketone 7 was obtained
by coupling of bromoalkylderivative 4 with the anion of
ethyl acetoacetate16 generated with sodium ethoxide,
afforded the â-ketoester 6. Treatment of 6 with sodium
ethoxide in ethylene glycol17 at 90 °C furnished the
expected ketone 7 in 60% yield. This procedure afforded
better yields than other attempted dealkyloxycarbony-
(23) Wai-si, E.; Prestwich, D. J . Lab. Comput. Radiopharm. 1988,
XXV, 627-633.
(24) Yoshida, Y.; Sakakura, Y.; Aso, N.; Okada, S.; Tanabe, Y.
Tetrahedron 1999, 55, 2183-2192.
(25) Brown, H. C.; Krishnamurthy, S. J . Org. Chem. 1980, 54, 849-
856.
(26) Auerbach, J .; Weinreb, S. M. J . Chem. Soc., Chem. Commun.
1974, 298-299.
(27) Bates, H. A.; Farina, J .; Tong, M. J . Org. Chem. 1986, 51, 2637-
2641.
(28) Brandsma, L.; Verkruijsse, H. D. Synthesis of acetylenes, allenes
and cumulenes; Elsevier: New York, 1981.
(29) Hutzinger, M. W.; Oehlschlager, A. C. J . Org. Chem. 1995, 60,
4595-4601.
(30) Hutchins, R. O.; Suchismita; Zipkin, R. E.; Taffer, I. M.;
Sivakumar, R.; Monaghan, A.; Elisseou, E. M. Tetrahedron Lett. 1989,
30, 55-56.
(16) Marvel, C. S.; Hager, F. D. Org. Synthesis 1941, I, 248.
(17) Aneja, R.; Hollis, W. M.; Davier, A. P.; Eaton, G. Tetrahedron
Lett. 1983, 24, 4641-4644.

8054 J . Org. Chem., Vol. 66, No. 24, 2001
Sch em e 2. Syn th esis of Acid s 2 a n d 3a
Rodr´ıguez et al.
a
Reagents and conditions: (a) NaBD4/MeOH, 0 °C, 1 h (11, 95%; 12, 92%); (b) N(CH3)3-HCl/TEA/p-TsCl/CH2Cl2, 0 °C, 90 min (13,
73%; 14, 73%); (c) LiAlD4/Et2O, rt, 3 h (15, 78% 16, 80%), then hydrolysis; (d) NBS/DMF, 50 °C, 15 min (17, 70%; 18, 74%); (e) LiCtCH/
NH3/DMSO, then 17 or 18/DMSO, rt, 90 min (19, 60%; 20, 58%); BuLi/THF, -78 °C, 30 min, then methyl chloroformate/THF, rt, 1 h (21,
77%; 22, 78%); (g) Mg/CD3OD, rt, 24 h (23, 70%; 24, 72%); (h) KOH/MeOD, rt, 16 h (2, 70%; 3, 72%).
Sch em e 3. Syn th esis of Acid 1a
Ta ble 1. Tr a n sfor m a tion of 1-3 in to d 4 a n d d 6
(Z)-11-Tetr a d ecen oic Acid s a n d d 4 a n d d 5
(E,E)-10,12-Tetr a d eca d ien oic Acid s in th e Com p etitive
Exp er im en ts
a
Reagents and conditions: (a) LiCtCH/NH3/DMSO, rt, 90 min
(82%); (b) BuLi/THF, -78 °C, 30 min, then methyl chloroformate/
THF, rt, 1 h (87%); (c) Mg/CD3OD, rt, 24 h (68%); (d) KOH/MeOD,
rt, 12 h (74%).
a
Ratios between d4 and d6 probes in the mixture used and ratios
between d4 and d6 (Z)-11-tetradecenoic acids formed by the ∆11
desaturase (∆11) and d4 and d5 dienes formed by the monoene
desaturase (MD). b For the probes, the ratio corresponds to a single
determination with a BF3-MeOH-derivatized sample of the used
mixture. For the metabolites, data are mean ( standard deviation
of three different experiments. Product KIEs were calculated as
described in the Experimental Section. R ) (CH2)6(CD2)2COOH.
sponding CoA esters, are transformed, at equal rates
(Table 1), into the respective (Z)-11-tetradecenoic acid
derivatives, which are the actual enzyme substrates. The
base methanolyzed lipidic extracts, prepared after incu-
bation of the glands with approximately 1/1 mixture of
1 and each of the hexadeuterated substrates 2 or 3, were
analyzed by GC-MS under the selected ion monitoring
mode. Integration of peaks corresponding to the tetra-
deuterated and pentadeuterated methyl (E,E)-10,12-
tetradecadienoates formed from each mixture afforded
the data required to determine the KIEs and, subse-
quently, allowed to determine the site of initial oxidation
in this desaturation reaction. As shown in Table 1, a large
isotope effect was observed for the carbon-hydrogen bond
cleavage at C10, but negligible isotope discrimination
occurred in the removal of C13-H.
Discu ssion
The same methodology previously validated for other
desaturases14,31-33 was followed in this work to determine
the site of initial oxidation in the transformation of (Z)-
11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic

Synthesis of gem-Dideuterated Tetradecanoic Acids
J . Org. Chem., Vol. 66, No. 24, 2001 8055
Ta ble 2. 13C NMR Ch em ica l Sh ifts for P r obes 1-3
carbon atom
compd
C1
C2
C3
C4-C9, C11, C10
C12
C13
C14
1
2
3
180.5
180.2
180.6
33.4a
33.4c
33.4a
23.8a
23.8b
23.8a
29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 28.8
29.7, 29.6, 29.4, 29.1, 28.8
29.5, 29.4, 29.2, 29.1, 28.8
31.9
31.7
31.8
22.6
21.8b
22.6
14.1
13.8
14.1
a
b
Quintet, J ) 20.0 Hz. Quintet, J ) 19.0 Hz. c Quintet, J ) 18.0 Hz.
rate-determining step was the rupture of C9-H,14 fol-
lowed by formation of a homoallylic intermediate and
rapid loss of C10-H. The different cryptoregiochemistry
found for both monoene desaturation reactions may arise
from the proximity of either C9 or C10 to the oxidizing
iron cluster at the active site of the enzyme. The different
relationship of either C9 or C10 with respect to the iron
cluster could result from the different conformations
adopted by the substrates at the enzyme active site.
Assuming the existence of two discrete monoene desatu-
rases, the different substrate conformations would be
imposed by the specific amino acid sequences of each
enzyme. Nevertheless, it is also possible that the spatial
location of C9 and C10 with respect to the iron cluster is
determined by the geometry of the substrate double bond
and that a single monoene desaturase is sufficient for
the synthesis of both dienes. In this case, in the (E)-
isomer, C9 would be closer to the oxidizing iron center,
resulting in the formation of the homoallylic intermediate
upon C9 oxidation. The (Z,E)-diene would finally result
from elimination of C10-H. In contrast, (Z)-11-tetrade-
cenoate would be accommodated in the desaturase active
site in such a way that C10 would be near the iron cluster
with formation of the allylic intermediate upon oxidation.
Subsequent loss C13-H with rearrangement would af-
ford the (E,E)-10,12-diene system. For the time being,
there is no genetic evidence for the existence of a single
monoene desaturase in S. littoralis. However, it has been
demonstrated that both (Z)-11- and (E)-11-monoene fatty
acids can be afforded by a single ∆11 desaturase and that
the stereospecificity of this reaction depends on the
substrate chain length.33 Therefore, it is not unreasonable
to propose that a single monoene desaturase might also
be able to give both (Z,E)-9,11- and (E,E)-10,12-tetra-
decadienoates, depending on the geometry of the sub-
strate double bond. Should this be the case, S. littoralis
female moths would biosynthesize their complex phero-
mone blend by means of only two desaturases, a ∆11
desaturase, whose stereospecificity depends on the sub-
strate chain length, and a monoene desaturase, whose
cryptoregiochemistry, and hence the final reaction prod-
uct, would depend on the geometry of the double bond of
the substrate. In this way, the composition of the
pheromone blend would be regulated not by the existence
of several different desaturases, but by the supply of
specific substrates to only two desaturase enzymes.
Cloning and functional expression of the cDNA encoding
the monoene desaturase(s) of S. littoralis will be con-
ducted soon to test this hypothesis.
F igu r e 2. Mechanistic model for the formation of (E,E)-10,-
12-tetradecadienoic acid from (Z)-11-tetradecenoic acid (A).
The previously studied14 enzymatic transformation of (E)-11-
tetradecenoic acid into (Z,E)-9,11-tetradecadienoic acid is also
shown (B) for comparison.
acid. The results of the competitive experiments showed
that C10-H removal, but not elimination of C13-H, was
sensitive to deuterium substitution. In agreement with
the model proposed by Buist et al.,31,34 these results are
consistent with the hypothesis that this monoene de-
saturase reaction involves a first slow, isotope sensitive
C10-H bond cleavage, with probable formation of an
unstable allylic intermediate, followed by a second fast
C13-H bond removal and concomitant rearrangement
(Figure 2).
In contrast, the studies conducted on the (Z)-9 desatu-
ration of (E)-11-tetradecenoic acid had shown that the
(31) Buist, P. H.; Behrouzian, B. J . Am. Chem. Soc. 1996, 118, 6295-
6296.
Exp er im en ta l Section
(32) Buist, P. H.; Behrouzian, B. J . Am. Chem. Soc. 1998, 120, 871-
876.
(33) Pinilla, A.; Camps, F.; Fabrias, G. Biochemistry 1999, 38,
1
Gen er a l Meth od s. All H NMR spectra were acquired at
15272-15277.
300 MHz and 13C NMR spectra at 75 M Hz in recently
neutralized CDCl3 solutions, and chemical shifts are given in
(34) Buist, P. H.; Marecak, D. M. J . Am. Chem. Soc. 1992, 114,
5073-5080.

8056 J . Org. Chem., Vol. 66, No. 24, 2001
Rodr´ıguez et al.
ppm using as internal standards Si(CH3)4 for 1H and CDCl3
for 13C. Gas chromatography coupled to mass spectrometry
(GC-MS) analysis was performed by electron impact (EI) at
70 eV, using a nonpolar HP-1 capillary column (30 m × 0.20
mm i.d.). All IR spectra were run in film. Elemental analyses
were obtained in the Microanalysis Service of IIQAB-CSIC,
and they were conventional combustion analyses without
discrimination between hydrogen and deuterium contents.
NaBD4 and LiAlD4 (Deuterium content 99%) was obtained
from Aldrich Chemical Co. The final deuterium contents of
the labeled substrates were determined by GC-MS analysis
of their respective methyl esters and were found to be as
follows (%): 1, 90.2 H4, 8.5 H3, and 1.3 H2; 2, 89.2 H6, 7.9
2H5, and 2.9 2H4; 3, 91.0 2H6, 6.2 2H5, and 2.8 2H4. Organic
solutions were dried with anhydrous MgSO4. The purification
procedures were carried out by flash chromatography on silica
gel (230-400 mesh) and products were mostly obtained as oils
unless otherwise specified. Visualization of UV-inactive ma-
terials was accomplished with phosphomolybdic acid. Degree
of purity of all new compounds was established by elemental
analysis, except for the methyloxymethoxy derivatives 6, 7,
9, and 10-14, whose combustion analyses are not feasible, and
purity was determined by GC and carbon NMR.
29.4; 29.3; 29.2; 29.1; 26.1; 23.7. IR: 2929, 2856, 1716, 1465,
1359, 1147, 1110, 1045, 918 cm-1
.
2-Bu tyl-1,3-d ith ia n e (8). To a refluxing mixture of BF3‚
Et2O (2 mL), AcOH (4.1 mL), and CHCl3 (8.2 mL) was added
dropwise a solution of valeraldehyde (2.0 g, 23.5 mmol) and
propanedithiol (2.8 mL, 25.9 mmol) in CHCl3 (61 mL). Reflux
was continued for 16 h. The solution was cooled to room
temperature and washed sequentially with 10% KOH and
brine. The organic layers were extracted with dichloromethane,
and the combined organic layers were washed, dried, and
concentrated at reduced pressure. The residue was distilled
to obtain 3.7 g (21.0 mmol, 93%) of 2-butyl-1,3-dithiane. Bp )
122-123 °C/7.5 Torr.35 1H NMR δ: 4.0 (t, J ) 7.0 Hz, 1H); 2.8
(m, 4H); 2.1 (m, 2H); 1.2-1.8 (m, 6H); 0.8 (t, J ) 7.0 Hz, 3H).
13C NMR δ: 47.6; 35.1; 30.4; 26.0; 28.7; 22.2; 13.7. IR: 2954,
2
2
2
2
2931, 2898, 2858, 1465, 1421, 1274, 1242, 1182, 908 cm-1
.
2-Bu tyl-2-(7,9-d ioxa d ecyl)-1,3-d ith ia n e (9). A procedure
previously reported21 was followed. To a solution of dithiane
8 (0.370 g, 2.10 mmol) in dry THF (4 mL) and kept at -30 °C
was added, under argon, 2.5 mL of a 1.34 M solution of n-BuLi
in hexane. The mixture was stirred at -30 °C for 90 min and
cooled to -78 °C, and then 7,9-dioxa-1-bromodecane (5) (0.375
g, 1.69 mmol) dissolved in 1 mL of THF was added. After being
stirred for 1 h at -78 °C, the mixture was warmed to room
temperature, THF was removed under vacuum, water was
added, and the product was extracted with diethyl ether. The
combined organic layers were washed with brine and dried
and the crude resulting from evaporation of solvent was
purified by column chromatography (hexane/diethyl ether 92:
8) to afford 0.423 g (1.33 mmol, 81%) of dithioketal 9. 1H NMR
δ: 4.58 (s, 2H); 3.47 (t, J ) 6.5 Hz, 2H); 3.32 (s, 3H); 2.76 (m,
4H); 1.3-1.9 (m, 18H); 0.88 (t, J ) 7.0 Hz, 3H). 13C NMR δ:
96.3; 67.6; 55.0; 53.2; 38.0; 37.8; 29.6; 29.5; 26.1; 26.0; 25.9;
25.5; 23.9; 22.8; 13.9.
Dimethyl sulfoxide and Grace’s medium were purchased
from Sigma.
Syn th esis of P r obes. P r ep a r a tion of Der iva tives 4 a n d
5. To a stirred solution of the bromo alcohol in dimethoxy-
methane (1.5 mL/mmol of bromo alcohol) were added LiBr (0.2
equiv) and p-toluenesulfonic acid (0.1 equiv). The mixture was
stirred at room temperature for 16 h. Brine was added, and
the product was extracted with hexane. The organic layers
were washed with brine and dried and concentrated at reduced
pressure to give the expected products as oils, which were
submitted to the following reaction without purification.
1-Br om o-9,11-d ioxa d od eca n e (4).13 Yield: 97% (12.9 g,
12,14-Dioxa -5-p en ta d eca n on e (10). To a solution of N-
bromosuccinimide (0.780 g, 4.38 mmol) in acetone/water 95:5
(13 mL) was added, at -20 °C, 180 mg of dithiane 9 (0.57
mmol) dissolved in the same solvent mixture (17 mL). Stirring
was continued at -20 °C for 5 min and warmed to room
temperature, and a 40% NaHSO3 water solution was added
until decoloration. Acetone was removed under vacuum, and
the resulting residue was extracted with diethyl ether. The
combined organic layers were washed with brine and dried,
and the solvent was removed to furnish 111 mg (0.48 mmol,
85%) of ketone 10. 1H NMR δ: 4.57 (s, 2H); 3.47 (t, J ) 6.5
Hz, 2H); 3.32 (s, 3H); 2.36 (t, J ) 7.5 Hz, 2H); 2.35 (t, J ) 7.5
Hz, 2H); 1.2-1.5 (m, 12H); 0.86 (t, J ) 7.0 Hz, 3H). 13C NMR
δ: 211.5; 96.3; 67.6; 55.0; 42.6; 42.5; 29.5; 28.9; 26.0; 25.9; 23.7;
22.3; 13.8. IR: 2923, 2864, 1714, 1465, 1379, 1147, 1110, 1045,
1
51.2 mmol). H NMR δ: 4.59 (s, 2H); 3.49 (t, J ) 6.5 Hz, 2H);
3.38 (t, J ) 7.0 Hz, 2H); 3.33 (s, 3H); 1.5-1.8 (m, 4H); 1.31 (br
s, 8H). 13C NMR δ: 96.3; 67.7; 55.0; 33.9; 32.7; 29.6; 29.2; 28.6;
28.0; 26.0. IR: 2931, 2856, 1465, 1440, 1386, 1215, 1147, 1110,
1047, 723 cm-1
.
1-Br om o-7,9-d ioxa d eca n e (5).13 Yield: 96% (0.350 g, 1.9
1
mmol). H NMR δ: 4.59 (s, 2H); 3.49 (t, J ) 6.5 Hz, 2H); 3.38
(t, J ) 7.0 Hz, 2H); 3.33 (s, 3H); 1.5-1.8 (m, 4H); 1.40 (m,
4H). 13C NMR δ: 96.3; 67.5; 55.0; 33.7; 32.7; 29.5; 27.9; 25.3.
IR: 2935, 2862, 1461, 1242, 1143, 1110, 1045 cm-1
.
Eth yl 2-(2,4-Dioxa d od ecyl)-3-oxobu ta n oa te (6). To a
solution of Na (46 mg, 2 mmol) in ethanol (1.3 mL) under argon
was added ethyl acetoacetate (0.252 mL, 2 mmol). The reaction
mixture was heated at reflux, 9,11-dioxa-1-bromooctane (0.504
g, 2 mmol) was added, and stirring was maintained at reflux
for 26 h. The mixture was cooled, saturated NH4Cl was added,
and the product was extracted with hexane. The combined
organic layers were washed with brine and dried. The crude
residue obtained upon solvent removal was purified by column
chromatography (hexane/diethyl ether 82:18) to obtain 0.360
g (1.2 mmol, 60%) of ketoester 6. 1H NMR δ: 4.57 (s, 2H); 4.15
(q, J ) 7.0 Hz, 2H); 3.46 (t, J ) 6.5 Hz, 2H); 3.34 (t, J ) 6.0
Hz, 1H); 3.31 (s, 3H); 2.17 (s, 3H); 1.5-1.8 (m, 4H); 1.24 (br s,
12H); 1.22 (t, J ) 7.0 Hz, 3H). 13C NMR δ: 200.3; 169.8; 96.3;
67.7; 61.2; 55.0; 28.6; 29.6; 29.2; 29.1; 28.1; 27.3; 26.0; 14.0.
919 cm-1
.
Red u ction of Keton es 7 a n d 10. To a 1 M solution of the
ketone in methanol was added, at 0 °C, NaBD4 (2 molar equiv).
The mixture was stirred at room temperature for 1 h and
cooled to 0 °C, water was carefully added, and the product was
extracted with dichloromethane. The organic layer was washed
with brine and dried (Na2SO4), and the solvent was removed
under vacuum to give the expected alcohols, which were
submitted to the following reaction without purification.
[2-2H]-12,14-Dioxa -2-p en ta d eca n ol (11). Yield: 95% (296
1
mg, 1.3 mmol). H NMR δ: 4.58 (s, 2H); 3.47 (t, J ) 6.5 Hz,
2H); 3.32 (s, 3H); 1.5-1.7 (m, 2H); 1.25 (br s, 17H); 1.13 (s,
3H). 13C NMR δ: 96.3; 67.8; 67.6 (t, J ) 21.5 Hz); 55.0; 39.2;
29.6; 29.3; 26.1; 25.6; 23.3. IR: 3440, 2927, 2856, 1465, 1373,
IR: 2931, 2856, 1741, 1716, 1465, 1147, 1112, 1043 cm-1
.
12,14-Dioxa -2-p en ta d eca n on e (7). To a solution of Na (56
mg, 2.4 mmol) in ethanol (1.8 mL) was added freshly distilled
ethanediol (4 mL) under argon. After 15 min at 85-90 °C,
ketoester 6 (200 mg, 0.66 mmol) was added, and stirring was
continued for 30 min. The mixture was cooled, aqueous
saturated NH4Cl was added, and the product was extracted
with hexane. The combined organic layers were washed with
brine and dried, and the solvent was removed to afford a crude
that was purified by column chromatography (hexane/ethyl
acetate 90:10) to give ketone 7 (90 mg, 0.39 mmol, 60%). 1H
NMR δ: 4.58 (s, 2H); 3.47 (t, J ) 6.5 Hz, 2H); 3.32 (s, 3H);
2.37 (t, J ) 7.0 Hz, 2H); 2.09 (s, 3H); 1.5-1.8 (m, 4H); 1.25 (br
s, 10H). 13C NMR δ: 209.3; 96.3; 67.8; 55.0; 43.7; 29.8; 29.6;
1149, 1112, 1045, 919 cm-1
.
[5-2H]-12,14-Dioxa -5-p en ta d eca n ol (12). Yield: 92% (255
1
mg, 1.1 mmol). H NMR δ: 4.57 (s, 2H); 3.47 (t, J ) 6.5 Hz,
2H); 3.32 (s, 3H); 1.3-1.5 (m, 17H); 0.86 (t, J ) 7.0 Hz, 3H).
13C NMR δ: 96.3; 71.3 (t, J ) 21.0 Hz); 67.7; 55.0; 37.2; 37;0;
29.6; 29.4; 27.7; 26.1; 25.5; 22.7; 14.0. IR: 3438, 2931, 2858,
1465, 1380, 1217, 1151, 1112, 1045, 919 cm-1
.
P r ep a r a tion of Tosyl Ester s 13 a n d 14. A 0.5 M solution
of the alcohol in dichloromethane was treated with trimethyl-
(35) Seebach, D.; Wilka, E. Synthesis 1976, 476-477.

Synthesis of gem-Dideuterated Tetradecanoic Acids
J . Org. Chem., Vol. 66, No. 24, 2001 8057
amine hydrochloride (1 molar equiv) and anhydrous triethyl-
amine (5 molar equiv). The mixture was cooled to 0 °C, and a
0.5 M solution of p-toluenesulfonyl chloride (1.5 molar equiv)
in dichloromethane was added. The mixture was stirred at 0
°C for 90 min, and the product was extracted with ethyl
acetate. The organic layers were washed with 1 N HCl and
brine, dried, and concentrated to dryness to afford a crude that
was purified by column chromatography (hexane/ethyl acetate
86:14) to obtain the expected products.
18.0 Hz); 14.1. IR: 2958, 2923, 2854, 1463, 1178, 1110, 1056
cm-1. Anal. Calcd for C14H212H2Br: C, 55.93; H + D, 9.74.
Found: C, 55.82; H, 9.81.
Obten tion of Ter m in a l Alk yn es 19 a n d 20. A suspension
of lithium acetylide in liquid ammonia, prepared from lithium
(2 molar equiv) and acetylene, was dissolved in dry dimethyl
sulfoxide (0.5 mL/mmol Li). After evaporation of NH3, the
acetylide was treated with a 3 M solution of the bromoalkane
in dry dimethyl sulfoxide. The mixture was stirred at room
temperature for 90 min, cooled to 0 °C and saturated NH4Cl
was added. The product was extracted with hexane, and the
combined organic layers were washed with brine and dried.
Removal of solvent afforded a crude that was purified by
column chromatography (hexane).
[2-2H]-12,14-Dioxa-2-pen tadecyl p-tolu en su lfon ate (13).
1
Yield: 73% (325 mg, 0.84 mmol). H NMR δ: 7.76 (d, J ) 8.5
Hz, 2H); 7.29 (d, J ) 8.0 Hz, 2H); 4.58 (s, 2H); 3.48 (t, J ) 6.5
Hz, 2H); 3.32 (s, 3H); 2.41 (s, 3H); 1.21 (s, 3H); 1.2-1.4 (m,
18H). 13C NMR δ: 144.3; 134.5; 129.6; 127.6; 96.3; 80.2 (t, J
) 22.0 Hz); 67.8; 55.0; 36.3; 29.7; 29.3; 29.2; 29.0; 26.1; 24.7;
21.5; 20.6. IR: 3031, 2929, 2856, 1598, 1463, 1359, 1178, 1110,
[12,12-2H2]-1-Tr id ecyn e (19). Yield: 60% (56 mg, 0.31
mmol). 1H NMR δ: 2.16 (dt, J 1 ) 2.5 Hz, J 2 ) 6.5 Hz, 2H);
1.91 (t, J ) 2.5 Hz, 1H); 1.83 (m, 2H); 1.23 (br s, 14H); 0.84 (s,
3H). 13C NMR δ: 84.6; 67.9; 31.7; 29.6; 29.5; 29.3; 29.1; 28.7;
28.5; 21.8 (quintet, J ) 19.0 Hz); 18.3; 13.8. IR: 3315, 2925,
2854, 1465, 1163, 721 cm-1. Anal. Calcd for C13H222H2: C,
87.71; H + D, 13.18. Found: C, 87.60; H, 13.31.
1045, 908 cm-1
.
[5-2H]-12,14-Dioxa-5-pen tadecyl p-tolu en su lfon ate (14).
1
Yield: 73% (0.238 g, 0.61 mmol). H NMR δ: 7.76 (d, J ) 8.5
Hz, 2H); 7.29 (d, J ) 8.0 Hz, 2H); 4.58 (s, 2H); 3.45 (t, J ) 6.5
Hz, 2H); 3.33 (s, 3H); 2.41 (s, 3H); 1.2-1.5 (m, 16H); 0.78 (t, J
) 6.5 Hz). 13C NMR δ: 144.3; 134.7; 129.6; 127.6; 96.3; 84,0
(t, J ) 21.5 Hz); 67.7; 55.0; 33.8; 33.6; 29.5; 29.0; 26.7; 25.9;
24.5; 22.3; 21.5; 13.8. IR: 2935, 2864, 1598, 1465, 1359, 1178,
[9,9-2H2]-1-Tr id ecyn e (20). Yield: 58% (0.163 g, 0.89
mmol). 1H NMR δ: 2.15 (dt, J 1 ) 2.5 Hz, J 2 ) 7.0 Hz, 2H);
1.90 (t, J ) 2.5 Hz, 1H); 1.84 (m, 2H); 1.23 (br s, 14H); 0.85 (t,
J ) 7.0 Hz, 3H). 13C NMR δ: 84.7; 67.9; 31.8; 29.5; 29.4; 29.2
(quintet, J ) 18.0 Hz); 29.1; 28.4; 22.6; 18.3; 14.0. IR: 3315,
2954, 2923, 2854, 1465, 721 cm-1. Anal. Calcd for C13H222H2:
C, 87.71; H + D, 13.18. Found: C, 87.58; H, 13.35.
1110, 1045, 900 cm-1
.
Rea ction of Tosyla tes 13 a n d 14 w ith LiAlD4. The tosyl
derivative was dissolved in dry diethyl ether (2 mL/mol) and
treated with LiAlD4 (3 molar equiv) dissolved in diethyl ether
(1 mL/mol) for 3 h at room temperature. Water was added
dropwise to the crude reaction mixture, and the product was
extracted with hexane. The combined organic layers were
washed with saturated NaHCO3 and brine and dried. Evapo-
ration of solvent afforded a crude that was dissolved in HCl
10% in methanol and stirred at room temperature for 16 h.
The solvent was removed under vaccum, water was added, and
the product was extracted with dichloromethane. The com-
bined organic layers were treated as above and concentrated
to give a crude that was purified by column chromatography
(hexane/ethyl acetate 88:12) to afford the pure alcohols 15 and
16.
1-Tr id ecyn e (25).36 Yield: 82% (500 mg, 2.73 mmol). 1H
NMR δ: 2.15 (dt, J 1 ) 2.5 Hz, J 2 ) 7.0 Hz, 2H); 1.91 (t, J )
2.5 Hz, 1H); 1.84 (m, 2H); 1.24 (br s, 16H); 0.85 (t, J ) 7.0 Hz,
3H). 13C NMR δ: 84.8; 67.9; 31.9; 29.6; 29.4; 29.3; 29.1; 28.7;
28.5; 22.6; 18.3; 14.1. IR: 3315, 2956, 2925, 2854, 1465, 721
cm-1
.
Syn th esis of Ester s 21, 22, a n d 26. A 0.5 M solution of
the terminal alkyne in dry THF was treated, at -78 °C under
argon, with a 1.6 M solution of BuLi in hexane (1.2 molar
equiv) and stirred at -78 °C for 30 min. Methyl chloroformate
(1 molar equiv) was added, the reaction mixture was warmed
to room temperature and quenched with water, and the
product was extracted with diethyl ether. The crude obtained
upon solvent removal was purified by column chromatography
(hexane/diethyl ether 97:3).
[10,10-2H2]-1-Un d eca n ol (15). Yield: 78% (108 mg, 0.62
mmol). 1H NMR δ: 3.61 (t, J ) 6.5 Hz, 2H); 1.54 (m, 3H); 1.23
(br s, 14H); 0.83 (s, 3H). 13C NMR δ: 62.7; 32.6; 31.6; 29.6;
29.5; 29.4; 29.2; 21.8 (quintet, J ) 19.0 Hz); 13.7. IR: 3344,
2925, 2854, 1461, 1377, 1348, 1163, 1056, 721 cm-1. Anal.
Calcd for C11H222H2O: C, 75.86; H + D, 13.79. Found: C, 75.60;
H, 13.71.
Meth yl [13,13-2H2]-2-Tetr a d ecyn oa te (21). Yield: 77%
1
(35 mg, 0.14 mmol). H NMR δ: 3.73 (s, 3H); 2.30 (t, J ) 7.0
Hz, 2H); 1.54 (m, 2H); 1.23 (br s, 14H); 0.83 (s, 3H). 13C NMR
δ: 154.1; 89.8; 72.7; 52.4; 31.6; 29.5; 29.3; 29.2; 28.9; 28.7; 21.8
(quintet, J ) 19.0 Hz); 18.5; 13.8. IR: 2952, 2927, 2854, 2237,
1722, 1434, 1253, 1076 cm-1. Anal. Calcd for C14H242H2O2: C,
73.68; H + D, 11.40. Found: C, 73.42; H, 11.62.
[7,7-2H2]-1-Un d eca n ol (16). Yield: 80% (0.286 g, 1.64
mmol). 1H NMR δ: 3.61 (t, J ) 6.5 Hz, 2H); 1.54 (m, 2H); 1.23
(br s, 15H); 0.85 (t, J ) 7.0 Hz, 3H). 13C NMR δ: 62.8; 32.6;
31.8; 29.4; 29.3; 29.0; 28.7 (quintet, J ) 19.0 Hz); 25.7; 22.6;
Meth yl [10,10-2H2]-2-Tetr a d ecyn oa te (22). Yield: 78%
14.0. IR: 3360, 2956, 2923, 2854, 1460, 1178, 1110, 1056 cm-1
.
1
(71 mg, 0.29 mmol). H NMR δ: 3.73 (s, 3H); 2.30 (t, J ) 7.0
Anal. Calcd for C11H222H2O: C, 75.86; H + D, 13.79. Found:
C, 75.72; H, 13.51.
Hz, 2H); 1.54 (m, 2H); 1.23 (br s, 14H); 0.85 (t, J ) 7.0 Hz).
13C NMR δ: 154.2; 90.0; 72.7; 52.5; 31.8; 29.3; 29.2 (quintet, J
) 18.0 Hz); 29.1; 29.0; 28.8; 27.4; 22.6; 18.6; 14.1. IR: 2954,
2925, 2856, 2237, 1718, 1434, 1253, 1076, 752 cm-1. Anal.
Calcd for C14H242H2O2: C, 73.68; H + D, 11.40. Found: C,
73.29; H, 11.72.
Syn th esis of Br om oa lk a n es 17 a n d 18. To a 0.5 M
solution of the alcohol in dry dimethylformamide was added,
under argon, N-bromosuccinimide (2 molar equiv). The mix-
ture was heated at 50 °C for 15 min. Methanol (1 mL/mmol of
alcohol) was added to remove the excess reagent, and stirring
was maintained for 5 min. The product was extracted with
diethyl ether, and the combined organic layers were washed
with 1 N HCl and brine and dried. Evaporation of solvent
afforded a crude that was purified by column chromatography
(hexane).
Meth yl 2-Tetr a d ecyn oa te (26).37 Yield: 87% (115 mg, 0.48
1
mmol). H NMR δ: 3.73 (s, 3H); 2.30 (t, J ) 7.0 Hz, 2H); 1.54
(m, 2H); 1.23 (br s, 14H); 0.85 (t, J ) 7.0 Hz, 3H). 13C NMR δ:
154.2; 90.0; 72.7; 52.5; 31.8; 29.5; 29.39; 29.30; 28.9; 28.8; 27.4;
22.6; 18.6; 14.1. IR: 2952, 2925, 2854, 2237; 1720; 1434, 1253,
1076, 752 cm-1
.
[10,10-2H2]-1-Br om ou n d eca n e (17). Yield: 70% (127 mg,
0.66 mmol). 1H NMR δ: 3.38 (t, J ) 7.0 Hz, 2H); 1.83 (m, 2H);
1.23 (br s, 14H); 0.84 (s, 3H). 13C NMR δ: 34.0; 32.8; 31.6;
29.6; 29.5; 29.4; 29.2; 28.7; 28.1; 21.8 (quintet, J ) 19.0 Hz);
13.8. IR: 2958, 2923, 2854, 1461, 1163, 721 cm-1. Anal. Calcd
for C14H212H2Br: C, 55.93; H + D, 9.74. Found: C, 55.60; H,
9.71.
R ed u ct ion of Acet ylen ic E st er s 21, 22, a n d 26 a n d
Sa p on ifica tion . A mixture of the starting ester and Mg (5
molar equiv) in CD3OD (10 mL/mmol of ester) was stirred at
room temperature for 24 h. After this time, 37% HCl was
added under stirring until complete solubilization of the
suspension, and the product was extracted with diethyl ether.
The combined organic layers were washed with brine and dried
[7,7-2H2]-1-Br om ou n d eca n e (18). Yield: 74% (286 mg,
1.48 mmol). 1H NMR δ: 3.38 (t, J ) 7.0 Hz, 2H); 1.83 (m, 2H);
1.23 (br s, 14H); 0.86 (t, J ) 7.0 Hz, 3H). 13C NMR δ: 34.0;
32.8; 31.8; 29.4; 29.3; 29.1; 28.7; 28.1; 22.6; 29.2 (quintet, J )
(36) Elsner, P. J . Chem. Soc. 1951, 893-895.
(37) Zehnter, R.; Gerlach, H. Liebigs Ann. Org. Bioorg. Chem. 1995,
12, 2209-2220.

8058 J . Org. Chem., Vol. 66, No. 24, 2001
Rodr´ıguez et al.
and solvent was removed under vacuum. The resulting crude
was stirred at room temperature for 16 h in 2.5 M KOH in
deuterated methanol (0.25 mL/mmol of ester). Solvent was
removed, and the product was extracted with dichloromethane.
The organic layers were washed with brine and dried. Solvent
removal afforded a crude that was purified by column chro-
matography (dichloromethane/methanol 98:2).
incubations proceeded for 3 h at 25 °C. After this time,
pheromone glands were collected and soaked in chloroform/
methanol (2:1) at 25 °C for 1 h. The lipidic extracts thus
obtained were base methanolyzed as described elsewhere12 to
obtain the fatty acid methyl esters.
The extracts were analyzed by GC-MS, at 70 eV, on a
Fisons gas chromatograph (8000 series) coupled to a Fisons
MD-800 mass selective detector. The system was equipped
with a nonpolar Hewlett-Packard HP-1 capillary column (30
m × 0.20 mm I. D.) using the following temperature program:
from 120 °C 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 by monitoring the ions 238, 239, 240, 241, 242, 243, 244,
245, 246, 247, 248, 249, and 250. Dwell was set at 0.02 and
mass span at 0.5.
Kinetic isotope effects (KIEs) were calculated from the ratios
of product formed from unlabeled substrate and that produced
from the deuterated analogues and were based on the abun-
dance of the respective molecular ions of the various iso-
topomers of methyl (E,E)-10,12-tetradecadienoate (d4, 242; d5,
243). Isotope effects were corrected for the exact proportion of
unlabeled and labeled substrates administered, which was
determined by GC-MS analysis of a BF3‚MeOH derivatized
sample of the applied mixture. Corrections were also made for
incomplete deuterium incorporation in the substrates and for
the natural abundance of carbon and oxygen isotopes in the
ions monitored. These latter values were obtained from the
GC-MS chromatograms of extracts of tissues incubated with
the individual substrates.
[2,2,3,3,13,13-2H6]-Tetr a d eca n oic Acid (2). Yield: 70% (9
1
mg, 0.03 mmol). Mp: 50-52 °C. H NMR δ: 9.01 (br s, 1H);
1.23 (br s, 18H); 0.84 (s, 3H). 13C NMR δ: 180.2; 33.3 (quintet,
J ) 18 Hz); 31.7; 29.7; 29.6; 29.4; 29.1; 28.7; 23.8 (quintet, J
) 20 Hz); 21.9 (quintet, J ) 19 Hz); 13.8. IR: 2923, 2854, 1739,
1458, 1282, 1091 cm-1. Anal. Calcd for C14H222H6O2: C, 71.76;
H + D, 12.04. Found: C, 71.60; H, 11.71.
[2,2,3,3,10,10-2H6]-Tetr a d eca n oic Acid (3). Yield: 72%
1
(20 mg, 0.085 mmol). Mp: 50-52 °C. H NMR δ: 11.02 (br s,
1H); 1.23 (br s, 18H); 0.85 (t, J ) 7.0 Hz, 3H). 13C NMR δ:
180.6; 33.4 (quintet, J ) 20 Hz); 31.8; 29.5; 29.4; 29.2; 29.1;
28.8; 24.2 (quintet, J ) 20 Hz); 23.8 (quintet, J ) 20 Hz); 22.6;
14.1. IR: 2954, 2916, 2850, 1699; 1463, 1393, 1215, 908, 757
cm-1. Anal. Calcd for C14H222H6O2: C, 71.76; H + D, 12.04.
Found: C, 72.06; H + D, 12.25.
[2,2,3,3-2H4]-Tetr a d eca n oic Acid (1). Yield: 74% (20 mg,
0.085). Mp: 51-53 °C. 1H NMR δ: 10.99 (br s, 1H); 1.23 (br s,
18H); 0.85 (t, J ) 7.0 Hz, 3H). 13C NMR δ: 180.5; 33.4 (quintet,
J ) 20 Hz); 31.9; 29.7; 29.6; 29.5; 29.4; 29.3; 29.1; 28.8; 23.8
(quintet, J ) 20 Hz); 22.6; 14.1. IR: 2953, 2918, 2850, 1696,
1466, 1305, 950 cm-1. Anal. Calcd for C14H242H4O2: C, 71.37;
H + D, 12.14. Found: C, 71.41; H + D, 12.05.
Kin et ic Isot op e E ffect E xp er im en t s. Cryptoregio-
chemistry was determined in vitro following the previously
reported procedure.14 The experiments were carried out using
round-bottom-96-well plates. To each well, a 5 µL drop of
incubation medium was added. The incubation medium con-
sisted of Grace’s saline (135 µL) and a dimethyl sulfoxide
solution (15 µL) of a 1:1 mixture of 2 and 1 or 3 and 1 (10
mg/mL each). S. littoralis pheromone glands were excised,
carefully cleaned, and immersed into a drop of the incubation
medium. Plates were sealed with adherent plastic covers and
Ack n ow led gm en t. We thank CAICYT (Grant No.
AGF98-0844), Generalitat de Catalunya (Grant No.
99SGR-00187), and SEDQ S.A. for financial support and
the Ministry of Education and Culture for a predoctoral
fellowship to S.R. We thank Dr. J esu´s J oglar for
critically reading the manuscript and Germa´n La´zaro
for rearing the insects used in this study.
J O010560W