Substrate-dependent stereochemical course of the (Z)-13-desaturation catalyzed by the processionary moth multifunctional desaturase

Article abstract of DOI:10.1021/ja0751936

The stereochemical course of the Δ¹³ desaturation involved in the biosynthesis of Thaumetopoea pityocampa sex pheromone was studied using stereotopically labeled and tagged palmitic acids as metabolic probes. In the synthetic pathway, a functionalized acetylene common synthon was used for introducing the four deuterium tags. Further coupling of the tetradeuterated synthon to the appropriated alkynol and a double chemoenzymatic strategy to resolve the alcohol functionality allowed one to obtain the enantiomerically enriched probes used in the mechanistic studies. Mass spectrometric analyses of extracts from tissues cultured with each probe revealed that removal of the C13 and C14 hydrogens in 11-hexadecynoate and (Z)-11-hexadecenoate are pro-(R)- and pro-(S)-specific syn-dehydrogenation processes, respectively. This finding constitutes the first example in the literature of an enzymatic (Z)-desaturation exhibiting a substrate-dependent stereochemical course.

Full text of DOI:10.1021/ja0751936

Published on Web 11/09/2007
Substrate-Dependent Stereochemical Course of the
(Z)-13-Desaturation Catalyzed by the Processionary Moth
Multifunctional Desaturase
Jose´-Luis Abad,* Francisco Camps, and Gemma Fabria`s
Contribution from the Departamento de Qu´ımica Orga´nica Biolo´gica, Instituto de
InVestigaciones Qu´ımicas y Ambientales de Barcelona, Consejo Superior de InVestigaciones
Cient´ıficas, 08034 Barcelona, Spain
Received July 12, 2007; E-mail: jlaqob@cid.csic.es
Abstract: The stereochemical course of the ∆13 desaturation involved in the biosynthesis of Thaumetopoea
pityocampa sex pheromone was studied using stereotopically labeled and tagged palmitic acids as metabolic
probes. In the synthetic pathway, a functionalized acetylene common synthon was used for introducing
the four deuterium tags. Further coupling of the tetradeuterated synthon to the appropriated alkynol and a
double chemoenzymatic strategy to resolve the alcohol functionality allowed one to obtain the enantio-
merically enriched probes used in the mechanistic studies. Mass spectrometric analyses of extracts from
tissues cultured with each probe revealed that removal of the C13 and C14 hydrogens in 11-hexadecynoate
and (Z)-11-hexadecenoate are pro-(R)- and pro-(S)-specific syn-dehydrogenation processes, respectively.
This finding constitutes the first example in the literature of an enzymatic (Z)-desaturation exhibiting a
substrate-dependent stereochemical course.
Introduction
depending on the substrate used. An even more extraordinary
case has been recently discovered in the processionary moth,
Desaturases are ubiquitous enzymes with a crucial role in
the homeostasis of cell membranes. Besides this important
function, desaturases are involved in cell signaling,1 as well as
in the biosynthesis of lipids and fatty acid-derived natural
products.2 In this last regard, fatty acyl CoA desaturases produce
the unsaturated intermediates involved in moth sex pheromone
biosynthetic pathways.3-5 In these biological systems, desatu-
rases catalyze the regio- and stereospecific introduction of
double bonds into fatty acyl chains to afford a number of
unsaturated fatty acids, both monoenoic and dienoic, either
conjugated or not. In all cases, besides the usual Z isomer, the
E isomer can also be produced. Because of their functional
diversity, desaturases of sex pheromone producing cells con-
stitute excellent models to gain insight into different aspects of
biological desaturation. An interesting feature of moth desatu-
rases is their multifunctionality, whereby a single enzyme can
lead to different desaturated products depending on the substrate
transformed. Thus, for example, the ∆11 desaturases of Bombyx
Thaumetopoea pityocampa. This species, along with other
members of the Thaumetopoeidae family, produce a conjugated
enyne acetate as its sex pheromone.9 Other species of this family
make a (Z,Z)-11,13-hexadecadienoate-derived aldehyde, alcohol,
or acetate as pheromone constituents.10 The (Z,Z)-11,13-
hexadecadienoate intermediate is also biosynthesized by T.
pityocampa,11-14 although it is not further transformed into a
pheromone component.15 Both enyne and diene compounds are
biosynthesized from palmitic acid by consecutive desaturation
reactions, ∆11 desaturation, acetylenation, and ∆13 desaturation
(Figure 1).12-14
Molecular cloning and heterologous functional expression in
yeast have recently evidenced that the three desaturation
reactions are catalyzed by a single enzyme.17 A previous study
showed that the (Z)-11 desaturation takes places by removal of
both pro-(R) C11 and C12 hydrogen atoms from palmitate to
mori6 and Spodoptera littoralis7,8 exhibit a bifunctional ∆11/
10,12
(9) Guerrero, A.; Camps, F.; Coll, J.; Riba, M.; Einhorn, J.; Descoins, C.;
Lallemand, J. Y. Tetrahedron Lett. 1981, 22, 2013-2016.
(10) Frerot, B.; Demolin, G. Boll. Zool. Agrar. Bachic., Ser. 11 1993, 25, 33-
40.
∆
desaturase activity, producing both C11-monoenoic and
10,12-dienoic acids with different double bond geometries
(1) Nakamura, M. T.; Nara, T. Y. Annu. ReV. Nutr. 2004, 24, 345-376.
(2) Buist, P. H. Nat. Prod. Rep. 2004, 21, 249-262.
(11) Fabria`s, G.; Arsequell, G.; Camps, F. Insect Biochem. 1989, 19, 177-181.
(12) Arsequell, G.; Fabria`s, G.; Camps, F. Arch. Insect Biochem. Physiol. 1990,
14, 47-56.
(3) Jurenka, R. A. Top. Curr. Chem. 2004, 239, 97-131.
(4) Jurenka, R. A. Insect Pheromone Biochem. Mol. Biol. 2003, 53-80.
(5) Knipple, D. C.; Roelofs, W. L. Insect Pheromone Biochem. Mol. Biol. 2003,
81-106.
(13) Barrot, M.; Fabria`s, G.; Camps, F. Tetrahedron 1994, 50, 9789-9796.
(14) Fabria`s, G.; Barrot, M.; Camps, F. Insect Biochem. Mol. Biol. 1995, 25,
655-660.
(6) Moto, K.; Suzuki, M. G.; Hull, J. J.; Kurata, R.; Takahashi, S.; Yamamoto,
M.; Okano, K.; Imai, K.; Ando, T.; Matsumoto, S. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 8631-8636.
(7) Pinilla, A.; Camps, F.; Fabria`s, G. Biochemistry 1999, 38, 15272-15277.
(8) Serra, M.; Pin˜a, B.; Bujons, J.; Camps, F.; Fabria`s, G. Insect Biochem.
Mol. Biol. 2006, 36, 634-641.
(15) Quero, C.; Malo, E. A.; Fabria`s, G.; Camps, F.; Lucas, P.; Renou, M.;
Guerrero, A. J. Chem. Ecol. 1997, 23, 713-726.
(16) Abad, J.-L.; Rodr´ıguez, S.; Camps, F.; Fabria`s, G. J. Org. Chem. 2006,
71, 7558-7564.
(17) Serra, M.; Pin˜a, B.; Abad, J.-L.; Camps, F.; Fabria`s, G. Proc. Natl. Acad.
Sci. U.S.A., in press.
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J. AM. CHEM. SOC. 2007, 129, 15007-15012
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A R T I C L E S
Abad et al.
The probes needed to investigate the stereochemistry of the
13
∆
desaturation were prepared following the conventional
reactions depicted in Schemes 2 (compound 1a) and 3 (com-
pound 1b). Both synthetic pathways use protected [7,7,8,8-2H4]-
10-bromodecan-1-ol (4) as common intermediate. This com-
pound was obtained by coupling 3-butyn-1-ol with the
bromomethoxymethane derivative of 1,6-hexanediol, reduction
of the resulting alkynol with 2H2 using the Wilkinson catalyst,
and further bromination of the saturated tetradeuterated alcohol
(Scheme 1).
As shown in Scheme 2, crucial steps for the preparation of
pentadeuterated probes 1a were the double enzymatic resolutions
of 1-hexyn-3-ol with Candida antarctica lipase (CALB) to give
pure acetate (S)-6a and partially resolved alkynol (R)-5a (80%
ee).20 Likewise, a new enzymatic resolution with EP-100
immobilized Thermomyces (Humicola) lanuginosus (HLL)
afforded enantiomerically pure propargyl alcohol (R)-7a. Con-
ventional reactions such as non-scrambling deuteration with the
Wilkinson catalyst, mesylation, nucleophilic substitution with
LiAl2H4, methoxymethane deprotection, and two steps of
oxidation16 allowed the transformation of both alcohols (R)-7a
and (S)-7a into the corresponding acids (S)-1a and (R)-1a.
A similar synthetic approach for preparation of 1b enanti-
omers was unsuccessfully attempted by using 1-pentyn-3-ol as
starting material. However, as depicted in Scheme 3, com-
mercially available 5-hexyn-3-ol could be CALB resolved, and
the absolute configurations of the isolated products assigned
by NMR (see below) were in agreement with those predicted
by the Kazlauskas rule.21
The resolved enantiomers of 5-hexyn-3-ol were transformed
into the corresponding stereotopically deuterated fatty acids 1b
following the same sequence of reactions previously used in
the synthesis of compound 1a. The deuterated compounds were
characterized as previously reported for similar compounds.16,22,23
On the other hand, the absolute configuration and enantio-
meric excesses (ee) of the different alcohol intermediates were
determined by NMR,16,19 after derivatization with (R)-MPA ((R)-
(-)-methoxyphenylacetic acid).18,20,24 In this context, the sign
of the optical rotation of resolved (S)-(-)-1-hexyn-3-ol agreed
with that of similar propargylic alcohols (S)-(-)-1-octyn-3-ol
(commercially available) and (S)-(-)-1-heptyn-3-ol. These
compounds were synthesized in our laboratory by the same
methodology.18 Likewise, optical rotation signs of homoprop-
argylic 5-hexyn-3-ol stereoisomers agreed with previously
reported data.25 Number and location of the deuterium tags and
labels were as expected as concluded from the 1H and 13C NMR
analyses. The final deuterium contents of the acids 1 were
determined by GC-MS (EI) analysis of their respective methyl
esters and under the optimum synthetic conditions, mean ratios
of [M + 1]•+, [M]•+, [M - 1]•+, [M - 2]•+, and [M - 3]•+
fatty acids, were, respectively, 12.5, 78.4, 6.6, 2.1, and 0.4 for
compound 1a and 12.6, 78.4, 6.0, 2.2, and 0.8 for compound
1b, respectively.
Figure 1. Desaturases of the T. pityocampa sex pheromone biosynthetic
pathway. Bold hydrogens indicate first abstraction in the two-step desatu-
ration reactions.7,16 Among the different intermediates, only IV is reduced
and acetylated to the pheromone. Desaturation reactions are abbreviated
as: ∆11, (Z)-11-desaturation or 11-acetylenation, ∆13, (Z)-13-desaturation.
Figure 2. Pentadeuterated palmitic acids used in the stereochemistry study
of the T. pityocampa ∆13 desaturation.
give (Z)-11-hexadecenoate.18 Additionally, previous investiga-
tions reported that the three desaturations occur by initial
oxidation of the carbon atom located nearest to the carboxyl
functionality: C11 (∆11 desaturation and ∆11 acetylenation)7,16
and C13 (∆13 desaturation).19 To complete the mechanistic
studies on this unique desaturase, the stereochemical course of
the ∆13 desaturation of both (Z)-11-hexadecenoate and 11-
hexadecynoate was undertaken by determining the fate of
enantiomerically pure palmitic acids stereospecifically deuterated
at C13 and C14 (Figure 2). We report and discuss the novel
finding that the stereospecificity of this ∆13 desaturation depends
on the enzyme substrate used, even though a (Z)-13 double bond
is formed in both cases.
Results and Discussion
Synthesis of the Stereotopically Deuterated Tracers. As
carried out in our previous studies, the stereochemistry of this
∆
13 desaturation was analyzed through the use of multideuterated
substrates bearing both an analytical tag and a stereospecifically
deuterated stereogenic center of known configuration at the
diagnostic site (C13 and C14 in this case). The mass of the
resulting ∆13 desaturated products, as analyzed by GC/MS,
2
indicates whether H or H was lost to form the double bond.
As rationalized in previous studies, the use of the specific ∆13
desaturase substrates ((Z)-11-hexadecenoic and 11-hexadecynoic
acids) is not necessary, because these compounds are intracel-
lularly biosynthesized from the saturated pentadeuterated palm-
itic acid probes by using the tissue culture assay,19 thus
circumventing a more complex preparation of these labeled
unsaturated acid precursors.
(20) Abad, J.-L.; Soldevila, C.; Clape`s, P.; Camps, F. J. Org. Chem. 2003, 68,
5351-5356.
(21) Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A.
J. Org. Chem. 1991, 56, 2656-2665.
(22) Abad, J.-L.; Camps, F.; Fabria`s, G. Angew. Chem., Int. Ed. 2000, 39, 3279-
3281.
(18) Abad, J.-L.; Villorbina, G.; Fabria`s, G.; Camps, F. J. Org. Chem. 2004,
(23) Abad, J.-L.; Fabria`s, G.; Camps, F. Lipids 2004, 39, 397-401.
(24) Abad, J.-L.; Camps, F.; Fabria`s, G. Insect Biochem. Mol. Biol. 2001, 31,
799-803.
69, 7108-7113.
(19) Abad, J. L.; Serra, M.; Camps, F.; Fabria`s, G. J. Org. Chem. 2007, 72,
760-764.
(25) Roy, B. L.; Deslongchamps. Can. J. Chem. 1985, 63, 651-655.
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15008 J. AM. CHEM. SOC. VOL. 129, NO. 48, 2007

13
Stereochemistry of a
∆
Desaturation
A R T I C L E S
Scheme 1. Synthesis of Labeled Intermediate 4a
a Reagents and conditions: (a) BuLi/HMPA/THF/BrCH2(CH2)4CH2OMOM, 78%; (b) WC/D2/benzene, 75%; (c) PPh3/NBS/DMF, 79%.
Scheme 2. Synthesis of the Stereotopically Pentadeuterated Probes 1aa
a Reagents and conditions: (a) CALB/DIPE/AcOCHdCH2 (conversion 45% and 90%, two rounds, ee 96%); (b) K2CO3/MeOH, 82-93%; (c) BuLi/
HMPA/THF, 4, 71%; (d) HLL, DIPE, AcOCHdCH2, 78%; ee 96%; (e) WC/H2/benzene, 66%; (f) MsCl/NEt3/CH2Cl2, 92%; (g) LiAlD4/THF, 81%; (h)
HCl//MeOH, 85%; (i) (1) IBX/DMSO; (2) CrO3/H2SO4/acetone, 86%.
Scheme 3. Synthesis of the Stereospecifically Deuterated Probes 1ba
a Reagents and conditions: (a) CALB/DIPE/AcOCHdCH2 (conversion 45% and 90%, two rounds, ee 94%); (b) K2CO3/MeOH, 82-93%; (c) BuLi/
HMPA/THF, 4, 70-72%; (d) HLL, DIPE, AcOCHdCH2, 78-80%; ee 94% (acetate discarded); (e) WC/H2/benzene, 66%; (f) MsCl/NEt3/CH2Cl2, 90-
92%; (g) LiAlD4/THF, 81%; (h) (1) HCl//MeOH, 85%; (i) (1) IBX/DMSO; (2) CrO3/H2SO4/acetone, 80-85%.
Stereospecificity Studies. To determine the stereospecificity
of the ∆13 desaturation, the destiny of the C13 and C14
hydrogen/deuterium atoms of 1a and 1b, respectively, was
investigated. To this aim, pheromone glands of T. pityocampa
females were cultured with each one of the prepared probes,
tissues were extracted and methanolyzed, and the resulting
extracts were analyzed by chemical ionization GC/MS with
methane as ionization gas (Figure 3). This procedure affords
the [M + 1]•+ ion as the most abundant fragment in the mass
spectra (Table 1).
contrast, (S)-1b gave rise to methyl hexadecadienoate and methyl
hexadecaenynoate in which the most abundant [M + 1]•+ ions
in the clusters were those at m/z 271 and 270, respectively. These
results imply that conversion of (R)-1b into the enyne occurred
by loss of deuterium from C14, whereas diene formation took
place by hydrogen loss. Conversely, transformation of (R)-1b
into the enyne and the diene occurred by loss of C14-H and
C14-2H, respectively (Figure 4).
In parallel experiments, incubations with (R)-1a gave a major
methyl hexadecadienoate [M + 1]•+ isotopomer at m/z 272, but
deuterated methyl hexadecenynoate ([M + 1]•+ at m/z 269) was
not detected. On the other hand, (S)-1a afforded the main methyl
hexadecenynoate [M + 1]•+ isotopomer at m/z 270, whereas
In the experiments with (R)-1b, the [M + 1]•+ ion clusters
of the resulting methyl hexadecadienoate and methyl hexade-
cenynoate were centered at m/z 272 and 269, respectively. In
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A R T I C L E S
Abad et al.
Figure 4. Isotopomeric multidesaturation products of the pentadeuterated
palmitic acids used in this study. Numbers between brackets correspond to
the m/z values of the [M + 1]•+ ions. (a) ∆11, ∆11, and ∆13; (b) ∆11 and
∆13.
Figure 3. Isotopomers of (Z)-13-hexadecen-11-ynoic (IV) and (Z,Z)-11,-
13-hexadecadienoic (V) acids formed by ∆13 desaturation of (R)- and (S)-
1a, and (R)- and (S)-1b. The traces correspond to GC/MS chromatograms
of methanolyzed lipidic extracts from T. pityocampa pheromone glands
cultured without (none) or with the indicated probe upon selection of ions
at m/z 269, 270, 271, and 272 (see Table 1). The total ion current
chromatogram (TIC) showing the methyl esters derived from the natural
acylCoA biosynthetic intermediates (III, IV, and V, see Figure 1) is depicted
at the bottom of the figure. In the mass spectra of unlabeled IV and V,
abundance of ions are: IV m/z, 265, 100%; 266, 20%; 267, 12%; 268, 3%;
269, 5%; 270, 2%; 271, 2%; 272, 2%; 273, 1%; 274, 1%; 275, 1%; 276,
1%; V m/z, 267, 100%; 268, 18%; 269, 11%; 270, 3%; 271, 4%; 272, 2%;
273, 3%; 274, 1%; 275, 1%; 276, 1%. Shadowed peaks correspond to the
most abundant isotopomer formed from each probe; peaks marked with an
arrow indicate the retention time of isotopomers formed by loss of C13-
2H, which are barely detected because of the existence of primary isotope
effect.19 The results obtained by integration of ions are summarized in
Table 1.
Figure 5. Stereospecificity of the ∆13 desaturation.
diene and the enyne are formed by loss of C13-H and C13-
2H from (R)-1a and loss of C13-2H and C13-H from (S)-1a,
respectively.
The insignificant levels of labeled methyl (Z)-13-hexadecen-
11-yonate and methyl (Z,Z)-11,13-hexadecadienoate formed
from (R)-1a and (S)-1a, respectively, were not unexpected in
the light of the cryptoregiochemistry found for the ∆13 desatu-
ration, which occurs with the slow initial oxidation of C13 and
subsequent fast elimination of C14-H.19 As an experimental
proof of this mechanism, the ∆13 desaturation is sensitive to
isotope substitution at C13, but not C14, and, therefore, the loss
of C13-2H should occur at a lower rate than that of the geminal
hydrogen atom, which should have a final influence on the levels
of the resulting desaturation products. Furthermore, it is
noteworthy that the ratios of labeled to natural methyl (Z)-13-
hexadecen-11-yonate resulting from either (R)-1b or (S)-1b were
similar and approximately 6 times lower than that produced from
(S)-1a. Because the desaturation rate of (S)-1a to the diene is
lowered by the kinetic isotope effect, the pathway is shifted
toward formation of the enyne. In agreement, the ratios of
labeled to natural methyl (Z,Z)-11,13-hexadecadienoate resulting
from either (R)-1b or (S)-1b were similar and approximately 8
times lower than that produced from (R)-1a. In that case, the
isotope effect affects the enyne formation, and desaturation to
the diene is favored.
Table 1. Stereochemistry of the ∆13 Desaturationsa
isotopomer [M
d4
+
1]•+ mass
probe
product
d5
d5/d4 ratio
(R)-1a
V
IV
V
IV
V
IV
V
271
269
271
269
271
269
271
269
272
270
272
270
272
270
272
270
8.3 ( 2.7
bg
bg
9.1 ( 4.9
(S)-1a
(R)-1b
(S)-1b
11.1 ( 3.7
0.1 ( 0.0
0.1 ( 0.0
8.3 ( 2.7
IV
a Percentages of isotopomers correspond to the mean ( standard deviation
of two different experiments with duplicates and have been corrected for
the abundance of the [M + 1]•+ and [M - 1]•+ ions in the probes, in which
the mean d5/d4 ratios are 11.9 (acid 1a) and 13.1 (acid 1b). V refers to
methyl (Z,Z)-11,13-hexadecadienoate and IV to methyl (Z)-13-hexadecen-
11-ynoate. bg, not determined because peak abundances are not different
from background.
The overall data obtained in the above biochemical studies
indicate that the ∆13 desaturation studied here follows different
sterochemical courses depending on the substrate converted.
Thus, whereas the ∆13 desaturation of 11-hexadecynoic occurs
with removal of both pro-(R) C13-H and C14-H, that of (Z)-
11-hexadecenoic acid takes place with loss of both pro-(S)
levels of labeled methyl hexadecadienoate ([M + 1]•+ at m/z
271) were negligible in this case. These results imply that the
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13
Stereochemistry of a
∆
Desaturation
A R T I C L E S
Figure 6. Transformation of palmitic acid into both (Z,Z)-11,13-hexadecadienoic (A) and (Z)-13-hexadecen-11-ynoic (B) acids. In the models, the hydrogen
atoms removed to give the unsaturations are syn and facing toward the reader, wherein the oxidizing site is considered to reside. Rotation of substrates
occurs whenever necessary to place the hydrogens for removal in the right orientation (i.e., ii f iii, ii f iv, and iv f v). In (A), the stereochemical outcome
of the overall conversion is determined by the transoid conformation of the palmitate C10-C15 bonds at the enzyme active site (i). In the enyne case (B),
rotation of the hexadecenoate substrate (ii f iv) locates the two sp2 hydrogens in the appropriate spatial arrangement for elimination. The resulting acetylene
adopts a conformation v similar to that initially assumed by palmitate (i), leaving the two pro-(R) C13-H and C14-H properly oriented for ∆13 desaturation.
In the models, light gray indicates C, and red shows oxygen. The pro-(R) and pro-(S) hydrogen atoms at C11, C12, C13, and C14 are shown in green and
orange, respectively. The models were obtained with the ChemBats3D 9.0 program. The dihedral angles C10-C11-C12-C13 and C11-C12-C13-C14
were set to 0°.
C13-H and C14-H (Figure 5). In the first case, the stereo-
chemistry is the same reported for other (Z)-desaturation
reactions, including those catalyzed by the ubiquitous (Z)-9
desaturase,26,27 several moth (Z)-11 desaturases,28-30 as well as
a monoene desaturase.24
However, the ∆13 desaturation of (Z)-11-hexadecenoate
represents the first case of rupture of the stereochemical trend
of (Z)-desaturation. Taking into account that desaturation
reactions are syn elimination processes, the different stereo-
chemical courses of this ∆13 desaturation can be explained
assuming that, as occurring in multifunctional enzymes with
different active site domains,31 the consecutive reactions occur
without dissociation of each product into the bulk solvent.32 In
that case, the palmitate substrate would adopt a transoid (Z,Z)-
diene producing conformation (i, Figure 6A), in which the
hydrogen atoms lost from C11/C12 and C13/C14 have opposite
configurations (ii,iii, Figure 6A). Because the ∆11 desaturation
occurs with removal of the two pro-(R) hydrogen atoms from
C11 and C12,18 then the two hydrogen atoms lost from C13
and C14 must have a pro-(S) configuration.
In the enyne case (Figure 6B), rotation of the hexadecenoate
substrate (ii f iv, Figure 6B) is required to locate the two sp2
hydrogens in the appropriate spatial arrangement for elimination,
after which the resulting acetylene conformer that best fits into
the enzyme active site for ∆13 desaturation is that having the
two pro-(R) C13-H and C14-H facing forward (v, Figure 6B).
This conformer is similar to that initially adopted by palmitate
(i, Figure 6A). In the acetylene case, the presence of the triple
bond shortens the distance between C13 and the carboxylate
end and makes C13 accessible to the oxidizing site.
(26) Morris, L. J.; Harris, R. V.; Kelly, W.; James, A. T. Biochem. J. 1968,
109, 673-678.
(27) Schroepfer, G. J., Jr.; Bloch, K. J. Biol. Chem. 1965, 240, 54-63.
(28) Boland, W.; Froessl, C.; Schoettler, M.; Toth, M. J. Chem. Soc., Chem.
Commun. 1993, 1155-1157.
Conclusions
In summary, we have extended the chemoenzymatic strategy
previously reported to obtain enantiomerically enriched deu-
(29) Navarro, I.; Font, I.; Fabria`s, G.; Camps, F. J. Am. Chem. Soc. 1997, 119,
11335-11336.
(30) Svatos, A.; Kalinova, B.; Boland, W. Insect Biochem. Mol. Biol. 1999, 29,
225-232.
(32) The possibility that intermediates are released cannot be ruled out with the
current data. Isotope dilution studies are necessary to distinguish between
the two possibilities (see ref 31).
(31) Huang, X.; Holden, H. M.; Raushel, F. M. Annu. ReV. Biochem. 2001, 70,
149-180.
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A R T I C L E S
Abad et al.
terated palmitic acids with a tetradeuterium tag and use them
as probes to decipher the stereochemistry of the ∆13 desaturation
involved in the sex pheromone biosynthetic pathway of the
processionary moth. The key step in the synthesis of probes is
the kinetic lipase-catalyzed resolution of racemic mixtures of
secondary propargylic alcohols. The presence of the acetylenic
triple bond not only simplifies the absolute configuration
determination of the resolved alcohols but also improves the
lipase-mediated resolution of the alcohols assayed. Using the
pentadeuterated palmitic acids thus prepared, we have demon-
strated that the stereochemical course of the (Z)-13 desaturation
is substrate-dependent, as the removal of hydrogen atoms in
11-hexadecynoate and (Z)-11-hexadecenoate are pro-(R)- and
pro-(S)-specific syn-dehydrogenation processes, respectively.
Acknowledgment. This work was supported by grants from
Generalitat de Catalunya (2005SGR01063), CICYT, and FED-
ER (AGL2001-0585). J.-L.A. thanks Spanish MEC for a Ramo´n
y Cajal contract. We also acknowledge Rodolfo Herna´ndez from
Laboratorio de Sanidad Forestal (Gobierno de Arago´n, Mora
de Rubielos, Teruel) for providing T. pityocampa female pupae.
Supporting Information Available: General experimental
1
section and copies of H and 13C NMR and DEPT spectra for
compounds 2-4 and 6-12. NMR spectra of (R)-MPA deriva-
tives of compounds 5 and 7 used to determine the absolute
configuration. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA0751936
9
15012 J. AM. CHEM. SOC. VOL. 129, NO. 48, 2007