Synthesis and use of deuterated palmitic acids to decipher the cryptoregiochemistry of a Δ13 desaturation

Article abstract of DOI:10.1021/jo061592s

(Chemical Equation Presented) The synthesis of two hexadeuterated palmitic acids differing in the position of the diagnostic labels, and their use to decipher the cryptoregiochemistry of a Δ¹³ desaturation are described. A dithiane and a triple bond functionalities were used to introduce the diagnostic (C 13 or C14) and tagging (C8 and C9) labels, respectively, in the palmitic acid skeleton. Using these probes, the cryptoregiochemistry of the Δ¹³ desaturation involved in the biosynthesis of Thaumetopoea pityocampa sex pheromone was studied by means of kinetic isotope effect determinations. Transformation of both (Z)-11-hexadecenoic and 11-hexadecynoic acids into (Z, Z)-11,13-hexadecadienoic and (Z)-13-hexadecen-11-ynoic acids, respectively, is initiated by abstraction of the hydrogen atom at the C13 position, followed by the fast elimination of the C14 hydrogen to give the double bond.

Full text of DOI:10.1021/jo061592s

Synthesis and Use of Deuterated Palmitic Acids to Decipher the
Cryptoregiochemistry of a ∆13 Desaturation
Jose´-Luis Abad,* Montserrat Serra, Francisco Camps, and Gemma Fabria`s
Departamento de Qu´ımica Orga´nica Biolo´gica, Instituto de InVestigaciones Qu´ımicas y Ambientales de
Barcelona, Consejo Superior de InVestigaciones Cient´ıficas, J. Girona 18, 08034, Barcelona, Spain
jlaqob@iiqab.csic.es
ReceiVed August 1, 2006
The synthesis of two hexadeuterated palmitic acids differing in the position of the diagnostic labels, and
their use to decipher the cryptoregiochemistry of a ∆13 desaturation are described. A dithiane and a triple
bond functionalities were used to introduce the diagnostic (C13 or C14) and tagging (C8 and C9) labels,
respectively, in the palmitic acid skeleton. Using these probes, the cryptoregiochemistry of the ∆13
desaturation involved in the biosynthesis of Thaumetopoea pityocampa sex pheromone was studied by
means of kinetic isotope effect determinations. Transformation of both (Z)-11-hexadecenoic and 11-
hexadecynoic acids into (Z, Z)-11,13-hexadecadienoic and (Z)-13-hexadecen-11-ynoic acids, respectively,
is initiated by abstraction of the hydrogen atom at the C13 position, followed by the fast elimination of
the C14 hydrogen to give the double bond.
Introduction
Besides these plants, some lepidopteran species, including the
moth Heterocampa guttiVitta3 and those belonging to the genus
Thaumetopoea, biosynthesize (Z,Z)-11,13-hexadecadienyl or (Z)-
13-hexadecen-11-ynyl acetates as their sex pheromone.4 The
biosynthetic pathways involve both ∆11 and ∆13 desaturations,
as well as an acetylenation reaction in the case of the enyne
structures (Figure 1).5
Introduction of double bonds into a saturated aliphatic chain
is an important biotransformation catalyzed by specific desatu-
rases, which are present in all eukaryotic cells. In addition to
the enzymes that act on saturated fatty acids to afford monoun-
saturated products, other desaturases that use unsaturated
substrates to give conjugated or non-conjugated polyunsaturated
fatty acids do also occur in nature. The several unsaturations
can be either methylene interrupted in cis-configuration, as found
in essential fatty acids such as linoleic and linolenic acids, or
conjugated in either cis- or trans-configuration. Within the
polyunsaturated compounds, enynes, either conjugated or not,
have also been reported in a few plant species, such as the
compositae Crepis alpina1 and the moss Ceratodon purpurea.2
The mechanistics of both the ∆11 desaturation6 and ∆11
acetylenation7 have been investigated by use of pheromone gland
metabolization studies with the properly deuterated probes. In
the first case, experimental evidence was presented that the ∆11
desaturase of Thaumetopoea pityocampa (T. pityocampa) trans-
(3) Silk, P. J.; Lonergan, G. C.; Allen, D. C.; Spear O’ Mara, J. Can.
Entomol. 2000, 132, 681-684.
(4) Fre´rot, B.; Demolin, G. Boll. Zool. Agrar. Bachic., Ser. 2 1993, 25,
33-40.
* To whom correspondence should be addressed. Phone: Int + 34 93 400
6100. Fax: Int + 34 93 204 5904.
(5) Barrot, M.; Fabria`s, G.; Camps, F. Tetrahedron 1994, 50, 9789-
9796.
(1) Lee, M.; Lenman, M.; Bana´s, A.; Bafor, M.; Singh, S.; Schweizer,
M.; Nilsson, R.; Liljenberg, C.; Dahlqvist, A.; Gummeson, P.-O.; Sjo¨dahl,
S.; Green, A.; Stymne, S. Science 1998, 280, 915-918.
(2) Sperling, P.; Lee, M.; Girke, T.; Za¨hringer, U.; Stymne, S.; Heinz,
E. Eur. J. Biochem. 2000, 267, 3801-3811.
(6) Abad, J.-L.; Villorbina, G.; Fabria`s, C.; Camps, F. J. Org. Chem.
2004, 69, 7108-7113.
(7) Abad, J.-L.; Rodr´ıguez, S.; Fabria`s, C.; Camps, F. J. Org. Chem.
2004, 71, 7558-7584.
10.1021/jo061592s CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/06/2007
760
J. Org. Chem. 2007, 72, 760-764

Cyrptoregiochemistry of a ∆13 Desaturation
SCHEME 1. Synthesis of Hexadeuterated Probes 1a,ba
FIGURE 1. Deuterated probes 1a-c and their consecutive desaturation
products: ∆13; ∆13desaturase; ∆11; ∆11desaturase/acetylenase.
forms palmitic acid into (Z)-11-hexadecenoic acid by removal
of the pro-(R)-hydrogen atoms from both C11 and C12, whereas
the transformation of (Z)-11-hexadecenoic acid into 11-hexa-
decynoic acid by the ∆11 acetylenase takes place by a stepwise
mechanism, in which a significant perturbation of the strong
vinyl C11-H bond occurs prior to a fast elimination of the vinyl
hydrogen at C-12. The products of those reactions, (Z)-11-
hexadecenoic and 11-hexadecynoic acid, are then transformed
a Reagents and conditions: (a) BuLi/THF/HMPA, 71%; (b) NBS/PPh3/
DMF, 87%; (c) BF3.Et2O/1,3-propanedithiol/CH3COOH/CHCl3, 88-92%;
(d) BuLi/THF, 69-73%; (e) NBS/H2O/acetone, 86%; (f) LiAlD4/Et2O, 95-
98%; (g) MsCl/Et3N/CH2Cl2, 92-95%; (h) LiAlD4/Et2O, 93%; (i) Wilkinson
catalyst/D2/benzene, 97%; (j) HCl/MeOH, 90%; (k) IBX/DMSO; (l) H2SO4/
CrO3/H2O/acetone, 85%.
oxidation of the resulting aldehyde.7 This two-step sequence
gave significantly higher yields than direct oxidation of the
alcohols to the acids.
into a conjugated diene or enyne fatty acid, respectively, by a
13
∆
desaturation. The cryptoregiochemistry of this reaction is
Finally, probe 1c used in the competition studies was prepared
by coupling of the dithiane 4a with Br(CH2)4CtC(CH2)6-
OMOM (3c) (Scheme 2). Cleavage of the resulting dithiane 5c
using the above conditions and reduction with LiAlH4 gave the
intermediate alcohol 7c, which was treated with mesyl chloride
to afford the corresponding ester 8c. Mesyl group displacement
with LiAlH4 gave rise to the alkyne 9c. The same final reaction
sequence used for preparing compounds 1a,b allowed obtaining
the tetradeuterated pure acid 1c.
reported in this paper. Likewise, the preparation of the deuterated
probes 1a-c (Figure 1) required for this investigation is also
described.
Results and Discussion
Preparation of Probes. The probes needed to investigate
the cryptoregiochemistry of the ∆13 desaturation were prepared
following the conventional reactions depicted in Scheme 1.7,8
Thus, properly functionalized alkynyldithianes 5 were obtained
by coupling reaction of bromoalkynyl derivatives 3 with the
anion of dithiane 4 generated by reaction with BuLi.9 Cleavage
of the dithiane functionality with N-bromosuccinimide (NBS)
afforded the corresponding ketones 6, which were reduced to
the monodeuterated alcohols 7 with LiAlD4. Mesylation of the
hydroxyl group and nucleophilic substitution with LiAlD4
produced dideuterated compounds 9. Introduction of the four
deuterium atoms for tagging at C8 and C9 positions was
achieved by nonscrambling deuteration of the protected hexa-
decanols 10 with the Wilkinson catalyst. Methoxymethane
protective group removal in acid media gave the corresponding
palmityl alcohols, which were oxidized to the final tri-gem-
hexadeuterated palmitic acids 1 in two steps, involving oxidation
with IBX in dimethyl sulfoxide (DMSO) and final Jones
The deuterated compounds were characterized as previously
reported for similar compounds.6-8 Thus, the gem-dideuterated
products showed IR symmetric and asymmetric stretching bands
at frequency ranges of 2080-2095 and 2180-2195 cm-1
,
respectively. On the other hand, the number and the presence
of the deuterium labels were those expected as concluded from
1
1
the analyses by H and 13C NMR. The characteristic H NMR
methyne hydrogen signals for compounds 7 (CHOH, ca. 3.60
ppm) and 8 (CHOMs, ca. 4.70 ppm) were not observed due to
the presence of deuterium in the carbinolic center. On the other
hand, 13C NMR of compounds 7 and 8 showed the presence of
a triplet at around 70 and 83 ppm, which corresponded to the
trisubstituted carbon atoms C13/C14 of alcohols 7 and mesylates
8, respectively (7a, 70.7 ppm; 7b, 72.0 ppm; 8a,b, 83.0 ppm).
These signals disappeared and became a quintuplet (CD2) for
compounds 9a,b-11a,b and 1a,b. In this case, 13C NMR
chemical shift differed between a and b series, being shifted at
lower field in the second case (C13, 28.6 ppm; C14, 31.0 ppm).
(8) Abad, J.-L.; Fabria`s, G.; Camps, F. J. Org. Chem. 2000, 65, 8582-
8588.
(9) Seebach, D.; Corey, E. J. J. Org. Chem. 1975, 40, 231-237.
J. Org. Chem, Vol. 72, No. 3, 2007 761

Abad et al.
SCHEME 2. Synthesis of Competitor 1ca
a Reagents and conditions: (d) BuLi/THF, Br(CH2)4-CtC-
(CH2)6OMOM (3c) 73%; (e) NBS/H2O/acetone, 88%; (f) LiAlH4/Et2O,
97%; (g) MsCl/Et3N/CH2Cl2, 92%; (h) LiAlH4/Et2O, 95%; (i) Wilkinson
catalyst/D2/benzene, 97%; (j) HCl/MeOH, 89%; (k) IBX/DMSO; (l) H2SO4/
CrO3/H2O/acetone, 85%.
FIGURE 2. Formation of 11-hexadecynoic (III), (Z)-13-hexadecen-
11-ynoic (IV), and (Z,Z)-11,13-hexadecadienoic (V) acids from 1a,b
in T. pityocampa pheromone glands. The traces correspond to GC-MS
chromatograms of methanolyzed lipidic extract from T. pityocampa
pheromone glands incubated with either 1a (A) or 1b (B) upon selection
of ions at m/z 270 (M•+ + 1 ion of d5-IV methyl ester), m/z 272 (M•+
+ 1 ion of d5-V methyl ester), and m/z 273 (M•+ + 1 ion of d6-III
methyl ester). The total ion current chromatogram (TIC) showing the
natural intermediates (III, IV, and V methyl esters) is depicted in the
bottom of each figure. The experiments were performed as described
in the Experimental Section. Compounds are the methyl esters of 11-
hexadecynoic acid (III), (Z)-13-hexadecen-11-ynoic acid (IV), and
(Z,Z)-11,13-hexadecadienoic acid (V). Labeled counterparts are indi-
cated with [xd2]dn, where n is the number of deuterium atoms and [xd2]
shows the diagnostic deuterium position.
The final deuterium contents of the acids 1 were determined
by GC-MS analysis of their respective methyl esters and under
the optimum synthetic conditions, mean ratios of d7, d6, d5, d4,
and d3 fatty acids were respectively 12.5, 78.4, 6.6, 2.1, and
0.4 for compounds 1a,b and 12.6, 78.4, 6.0, 2.2, and 0.8 for
compound 1c.
Cryptoregiochemistry Studies. As shown in Figure 1, the
cryptoregiochemstry of the ∆13 desaturation could be investi-
gated by determining the reaction rates of either the 11-
hexadecynoate (III) into (Z)-13-hexadecen-11-ynoate (IV)
biotransformation or that of (Z)-11-hexadecenoate (II) to (Z,
Z)-11,13-hexadecadienote (V).
In the first case, the transformation of the acetylene metabo-
lites [13d2]d6-III and [14d2]d6-III, with diagnostic labels at each
oxidation site, into the corresponding enynes [13d]d5-IV and
[14d]d5-IV was investigated. The experiments were carried out
with the saturated probes 1a ([14d2]d6-I) and 1b ([13d2]d6-I), which
were biotransformated in situ into the acetylene ∆13desaturase
substrates by subsequent ∆11desaturation and acetylenation
(Figure 1). The method of determining primary kinetic isotope
effects from data obtained in competition experiments between
each of the hexadeuterated probe ([14d2]d6-I and [13d2]d6-I) and
that without deuteriums in the diagnostic positions (d4-I) could
not be followed here, since their transformation into the
corresponding enynes (IV) was too low to allow reliable
integrations. Furthermore, this problem was worsened by the
presence of small impurities overlapping with the natural
isotopomeric (M•+ + 1, 269 for d4-IV and 270 for d5-IV) ions.
Therefore, an alternative procedure was used, which, while not
allowing the calculation of primary kinetic isotope effects, did
allow clear deciphering, in the case of the enyne, the site of the
initial oxidation of the ∆13 desaturation reaction. In this method,
the amounts of every labeled intermediate (d6-II, d6-III, and
d5-IV from any d6-I; d4-II, d4-III, and d4-IV from d4-I) relative
to the respective endogenous counterpart (d0-II, d0-III, and d0-
IV) were determined by GC/MS analysis of methanolyzed lipid
extracts (Figure 2) after treatment with each individual probe
1a ([14d2]d6-I), 1b ([13d2]d6-I), and 1c (d4-I), and the data were
corrected for the corresponding probe incorporation.
As shown in Figure 3, similar relative amounts of labeled II
were formed from the three probes. Likewise, proportions
between the relative amounts of labeled II, III, and IV were
similar after incubations with d4-I and [14d2]d6-I. These results
indicate that both d4-I and [14d2]d6-I are metabolized to labeled
IV with similar rates and, therefore, that the ∆13 desaturase
activity is not sensitive to the presence of deuterium at C14 in
[14d2]d6-I. In contrast, relative amounts of [13d2]d6-III found after
treatment with [13d2]d6-I were significantly higher than those
resulting from the other two probes. This result suggests that
[13d2]d6-III is desaturated to the corresponding enyne at a lower
rate than both [14d2]d6-III and d4-III. In agreement, the relative
amounts of [13d1]d5-IV produced from [13d2]d6-I were significantly
lower than those found from d4-I and [14d2]d6-I. Collectively,
these data indicate that hydrogen abstraction at C13 by the ∆13
desaturase is isotope-sensitive and, therefore, that this is the
rate-determining step of the desaturation reaction (C13 is the
site of initial oxidation). Thus, in accordance with the general
trend, conversion of acetylene III into enyne IV occurs by a
slow removal of the hydrogen atom nearest to the substrate
carboxy-terminal end (C13-H) followed by the fast elimination
of the neighboring C14-H.
This result was confirmed when the formation of dienes
[13d1]d5-V and [14d1]d5-V from their respective alkene precursors
[13d2]d6-II and [14d2]d6-II, which were in turn produced in situ
from the saturated probes [14d2]d6-I and [13d2]d6-I, respectively,
762 J. Org. Chem., Vol. 72, No. 3, 2007

Cyrptoregiochemistry of a ∆13 Desaturation
These results strongly suggest that dienoate production is
initiated by an energetically difficult and hence isotopically
sensitive hydrogen abstraction at C13 and completed by a second
facile and kinetically unimportant hydrogen abstraction at C14.
Both data indicate that enynoic IV and dienoic V acids are
produced via initial H-atom abstraction at C13 of an alkyne
and alkene substrates, respectively, and a second fast elimination
of hydrogens at C14 and support the hypothesis that this
transformation could represent a variation of the same enzyme
for two different substracts. Confirmation of this hypothesis
awaits cloning and functional expression of this enzyme, which
are currently ongoing in our laboratories.
Conclusions
FIGURE 3. Conversion of tracers 1a-c into biosynthetic intermedi-
ates. Data correspond to the mean ( SD of three replicates. Labeled
natural fatty acid methyl ester ratios were determined from the areas
of the specific M•+ + 1 ions in the GC-MS chromatograms (labeled:
[13d2]d6-I and [14d2]d6-I, 277; [13d2]d6-II and [14d2]d6-II, 275; d4-II, 273;
[13d2]d6-III and [14d2]d6-III, 273; d4-III, 271; [13d1]d5-V and [14d1]d5-V, 272;
[13d1]d5-IV and [14d1]d5-IV, 270; d4-IV, 269; natural and II, 269; III and
V, 267; IV, 265). The resulting data were corrected for the respective
levels of incorporation of each specific probe, which were administered
individually (probe/Σ(II + III + IV + V): 1a, 1.3 ( 0.21; 1b, 1.4 (
0.22; 1c, 0.8 ( 0.12). Compounds are the methyl esters of (Z)-11-
hexadecenoic acid (II), 11-hexadecynoic acid (III), (Z)-13-hexadecen-
11-ynoic (IV), and (Z,Z)-11,13-hexadecadienoic acids (V). Isotopic
labeling is indicated with [xd2]dn, where n is the number of deuterium
atoms and [xd2] shows the diagnostic deuterium position.
In summary, we have reported the preparation of novel
deuterated palmitic acids and their use to decipher the site of
initial oxidation of a ∆13 desaturation reaction that transforms
11-hexadecynoic and (Z)-11-hexadecenoic acids into (Z)-13-
hexadecen-11-ynoic and (Z,Z)-11,13-hexadecadienoic acids,
respectively. In agreement with other previously reported acyl
CoA desaturase reactions, both substrates are first oxidized at
the position closest to the acid functionality (C13) in the rate-
determining step of the reaction, which is then followed by the
fast removal of the hydrogen atom located at the neighboring
position (C14).
Experimental Section
TABLE 1. Transformation of 1a-c into d4 and d5
(Z,Z)-11,13-Hexadecadienoic Acids in Competitive Experiments
Preparation of Deuterated Alcohols 7. The reaction was
accomplished according to a previously reported procedure.8 Thus,
treatment of ketone 6 with LiAlD4 in Et2O, under argon atmosphere
and at room temperature, followed by the usual workup, allowed
one to obtain an oily residue that was purified by flash chroma-
tography on silica gel using 80:20 hexane/MTBE to give the
corresponding pure deuterated alcohols 7 in 96-98% yields.
[4-2H]-17,19-Dioxaicos-8-yn-4-ol (7a). This deuterated alcohol
was isolated (882 mg, 98% yield) starting from 3 mmol (888 mg)
of the corresponding ketone. IR 3425, 2935, 2860, 2210, 1465,
substrates (ratio)a
products (ratio)a
KIEb
1c/1a (1.1)
1c/1b (1.2)
d4-V/[13d2]d5-V (7.4 ( 1.1)
d4-V/[14d2]d5-V (1.4 ( 0.2)
6.7 ( 1.2
1.2 ( 0.1
a Ratios between d4 and d6 probes in the mixture used and ratios between
d4 and d5 (Z,Z)-11,13-hexadecadienoic acids produced. For the probes, the
ratio corresponds to a single determination with a BF3-MeOH-derivatized
sample of the mixture used. For the dienes, data are mean ( standard
deviation of four different experiments. b Product KIEs were calculated using
the equation: [% d4(product)/% d5(product)]/[% d4(substrate)/% d6(substrate)].
1
1410, 1145, 1110, 1045, 940, 920 cm-1; H NMR δ 4.62 (s, 2H),
3.52 (t, J ) 6.5 Hz, 2H), 3.36 (s, 3H), 2.18 (m, 2H), 2.14 (m, 2H),
1.28-1.68 (19H), 0.93 (t, J ) 7 Hz, 3H); 13C NMR 96.3 (CH2),
80.5 (C), 79.9 (C), 70.7 (CD, t, J ) 21 Hz), 67.8 (CH2), 55.1 (CH3),
39.5 (CH2), 36.4 (CH2), 29.6 (CH2), 29.0 (CH2), 28.9 (CH2), 28.7
(CH2), 26.1 (CH2), 25.2 (CH2), 18.8 (CH2), 18.7 (CH2), 18.7 (CH2),
14.1(CH3); MS m/z 300 (2, M•+ + 1), 298 (5, M•+ - 1), 268 (90),
250 (100), 238 (28), 232 (25), 220 (20), 194 (15), 164 (10), 150
(15), 124 (20), 109 (25); Anal. Calcd for C18H332HO3: C, 72.19;
H, 11.44. Found: C, 72.18; H, 11.39.
Preparation of Mesyl Esters 8. General Procedure. These
products were prepared by the procedure described by Abad et al.8
A solution of alcohol (2 mmol) and Et3N (830 µL, 6 mmol) in 20
mL of CH2Cl2 was treated with CH3SO3Cl (170 µL, 2.2 mmol),
and the mixture was stirred under argon for 2 h at room temperature.
Then, the reaction mixture was washed with H2O (2 × 20 mL),
dried, concentrated, and purified by flash chromatography on silica
gel using 85:15 hexane/MTBE to give the expected products in
95% yields.
were investigated (Figure 3). In this case, we were able to probe
the mechanism using kinetic isotope effect (KIE) measurements
from data obtained in competitive experiments with probe 1c,
which is labeled with the tetradeuterium tag, but lacks the
diagnostic dideuterium units. As shown in Figure 3, the three
compounds are transformed at equal rates into the respective
(Z)-11-hexadecenoic acids, which are the actual ∆13 desaturation
substrates. The base methanolyzed lipidic extracts obtained after
incubation of tissues with an approximately 1:1 mixture of 1c
and each of the hexadeuterated substrates 1a or 1b, were
analyzed by GC-MS under the selected ion monitoring mode.
Integration of peaks corresponding to the tetradeuterated and
pentadeuterated methyl (Z, Z)-11,13-hexadecadienoates formed
from each mixture afforded the data required to determine the
KIEs. As shown in Table 1, a large isotope effect was observed
for the carbon-hydrogen bond cleavage at C13, while the
C14-H bond breaking step was shown to be essentially
insensitive to deuterium substitution.
[4-2H]-17,19-Dioxaicos-8-yn-4-yl methanesulfonate (8a). (685
mg, 95% yield). IR 2935, 2860, 1465, 1355, 1175, 1145, 1115,
1045, 910 cm-1; 1H NMR δ 4.62 (s, 2H), 3.52 (t, J ) 6.5 Hz, 2H),
3.36 (s, 3 H), 3.01 (s, 3H), 2.20 (t, J ) 7 Hz, 2H), 2.13 (t, J ) 7
Hz, 2H), 1.52-1.86 (10H), 1.28-1.52 (12H), 0.95 (t, J ) 7 Hz,
3H); 13C NMR δ 96.3 (CH2), 83.0 (CD, t, J ) 22.5 Hz), 81.0 (C),
The magnitude of the obtained KIE effects correlates well
with others previously reported in other biological models.10,11
(10) Behrouzian, B.; Fauconnot, L.; Daligault, F.; Nugier-Chauvin, C.;
Patin, H.; Buist, P. Eur. J. Biochem. 2001, 268, 3545-3549.
(11) Reed, D. W.; Savile, C. K.; Qiu, X.; Buist, P. H.; Covello, P. S.
Eur. J. Biochem. 2002, 269, 5024-5029.
J. Org. Chem, Vol. 72, No. 3, 2007 763

Abad et al.
79.1 (C), 67.7 (CH2), 55.0 (CH3), 38.7 (CH3), 36.4 (CH2), 33.3
(CH2), 29.6 (CH2), 28.9 (CH2), 28.9 (CH2), 28.8 (CH2), 26.1 (CH2),
24.3 (CH2), 18.6 (CH2), 18.4 (CH2), 18.2 (CH2), 13.8 (CH3).
Reduction of Mesylates 8. General Procedure. A solution of
the mesyl derivative 8 (650 mg, 1.8 mmol) in Et2O (20 mL) was
treated with LiAlD4 (6 molar equiv) for 16 h at 20 °C. H2O was
added dropwise to the crude reaction mixture, and the resulting
white precipitate was filtered through a bed of Celite. Solvent was
concentrated to give a residue that, after purification by flash
chromatography on silica gel using a gradient of 0-10% MTBE
in hexane, gave the corresponding pure deuterated products in 90-
93% yields.
Carboxylic Acids Preparation (1). These compounds were
prepared by reaction of alcohol with a 0.2 M (6 equiv) solution of
IBX in DMSO at room temperature for 12 h to afford the aldehyde
intermediate. After this time (4 × DMSO volume) mL of H2O were
added, and the reaction mixture was extracted with diethyl ether/
hexanes mixture (1:1), dried, and concentrated to a residue that
was solubilized in 10 mL of acetone and then treated dropwise
with a 1 M aqueous solution of H2SO4/CrO3. Chromatography on
silica gel using 85:15 hexane/MTBE afforded the corresponding
acids 1 in 85-87% yields.
[8,8,9,9,13,13-2H6]-1-Hexadecanoic acid (1a). This product (88
mg, 85%) was isolated from 98 mg (0.4 mmol) of 11a. IR 3000,
2955, 2915, 2850, 2180, 2075, 1700, 1460, 1410, 1315, 1090, 940
[4,4-2H2]-17,19-Dioxa-8-icosyne (9a). (475 mg, 93% yield). IR
1
2930, 2855, 2180, 2100, 1440, 1145, 1110, 1050, 920 cm-1; H
1
cm-1; H NMR δ 2.35 (t, J ) 7.5 Hz, 2H), 1.63 (quint, J ) 7.5
NMR δ 4.62 (s, 2H), 3.52 (t, J ) 6.5 Hz, 2H), 3.36 (s, 3H), 2.14
(m, 4H), 1.59 (quint, J ) 7.0 Hz, 2H), 1.48 (m, 4H), 1.20-1.42
(12H), 0.88 (t, J ) 6.5 Hz, 3H); 13C NMR δ 96.3 (CH2), 80.3 (C),
80.1 (C), 67.8 (CH2), 55.0 (CH3), 31.5 (CH2), 29.7 (CH2), 29.1
(CH2), 29.0 (CH2), 28.9 (CH2), 28.7 (CH2), 28.6 (CH2), 28.0 (CD2,
quint, J ) 19 Hz), 26.1 (CH2), 22.5 (CH2), 18.7 (CH2), 18.7 (CH2),
14.1 (CH3); MS m/z 285 (15, M•+ + 1), 253 (100), 235 (30); Anal.
Calcd for C18H322H2O2: C, 76.00; H, 12.05. Found: C, 75.95; H,
11.99.
Hz, 2H), 1.18-1.38 (18H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR δ
180.6 (C), 34.1 (CH2), 31.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4
(CH2), 29.4 (CH2), 29.3 (CH2), 29.3 (CH2), 29.1 (CH2), 28.6 (CD2,
quint, J ) 19.0 Hz), 28.5 (CD2, quint, J ) 19.0 Hz), 24.7 (CH2),
22.6 (CH2), 14.1 (CH3); MS m/z (methyl ester), 305 (100, M•+
+
28), 277 (100, M•+ + 1); Anal. Calcd for C16H262H6O2: C, 73.22;
H, 12.29. Found: C, 73.13; H, 12.19.
In Vivo Gland Culture Procedure. In these experiments, newly
emerged virgin T. pityocampa females were briefly anesthetized
on ice and pheromone glands were everted and impregnated (1 µL
every 3 h × 4 times) with the DMSO solutions of the corresponding
deuterated probes 1 (10 mg/mL each). The in ViVo incubation
proceeded for 36 h. To obtain the methyl ester derivatives of the
gland lipids for analysis, the pheromone glands were excised and
soaked in chloroform methanol (2:1) at 25 °C for 1 h and base
methanolized in 0.5 M KOH for 1 h. After this time, the organic
solution was neutralized with 1 N HCl, washed with saturated
NaHCO3 solution, and extracted with hexane. Ten glands were used
for each assay.
Deuteration of Compounds 9. To a mixture of 426 mg (1.5
mmol) of 9 and 93 mg (0.1 mmol) of RhCl(PPh3)3,12 20 mL of
degassed benzene was added under argon atmosphere to get a
reddish solution. The system was purged by a combination of
vacuum and passing a D2 stream throughout, then D2 atmosphere
was kept from a balloon, and the solution was stirred for 36 h. The
mixture was filtered through a bed of Celite and the solvent
evaporated. Residue was purified by flash chromatography on silica
gel (0-3% MTBE/hexane) to give product 10.
[12,12,13,13,17,17-2H6]-2,4-Dioxaicosane (10a). (429 mg, 98%
yield). IR 2920, 2855, 2185, 2100, 1465, 1150, 1110, 1050, 920
1
cm-1; H NMR δ 4.62 (s, 2H), 3.52 (t, J ) 6.5 Hz, 2H), 3.36 (s,
Instrumental Analysis of the Biological Extracts. The GC-
MS analysis of biological extracts was performed by Chemical
Ionization (CI) using methane as ionization gas. The system was
equipped with a nonpolar HP5-MS capillary column (30 m × 0.25
mm i.d., 0.25 µm stationary phase thickness) using the following
program: from 120 to 180 °C at 5 °C/min and then 260 °C at 2
°C/min after an initial delay of 2 min. Analyses were carried out
on methanolyzed lipidic extracts from pheromone glands using the
equipment and conditions described above. KIEs were calculated
from the ratios of formed products from each probe, which afforded
a cluster of ions, analyzed as methyl ester, and are based in the
abundance of the respective molecular ions in the range m/z 265-
272 in which the most abundant corresponded to the molecular
ion of the resulting isotopomers.
3H), 1.59 (quint, J ) 6.5 Hz, 2H), 1.20-1.40 (20H), 0.88 (t, J )
7.0 Hz, 3H); 13C NMR δ 96.3 (CH2), 67.8 (CH2), 55.0 (CH3), 31.7
(CH2), 29.7 (CH2), 29.6 (CH2), 29.6 (CH2), 29.4 (CH2), 29.4 (CH2),
28.7 (CD2, quint, J ) 19 Hz), 28.6 (CD2, quint, J ) 19 Hz), 26.2
(CH2), 22.6 (CH2), 14.1 (CH3); MS m/z 293 (5, M•+ + 1), 291
(30, M•+ - 1), 261 (100), 259 (90), 247 (25), 231 (40); Anal. Calcd
for C18H322H6O2: C, 73.90; H, 13.10. Found: C, 73.92; H, 13.02.
Alcohol Deprotection. General Procedure. The methoxymethane
protecting group of alcohols 11 were removed by acid treatment
with a MeOH/HCl solution (1 M) for 36 h at room temperature.
Solvent was evaporated, and the crude product was washed with
saturated NaHCO3 solution, extracted with CH2Cl2, dried, and
purified by flash chromatography on silica gel using a gradient of
0-35% MTBE in hexane, for obtaining the corresponding pure
deuterated alcohols 11 in 83-90% yields.
[8,8,9,9,13,13-2H6]-1-Hexadecanol (11a). Starting from 146 mg
(0.5 mmol) of protected alcohol 10a, this compound was isolated
as a white solid (108 mg, 88% yield). mp 48-50 °C; IR 3250,
Acknowledgment. We gratefully acknowledge CICYT and
FEDER (Grant AGL2001-0585) and Generalitat de Catalunya
(Grant 2005SGR00726) for financial support. J.-L.A. thanks
Spanish MEC for a Ramo´n y Cajal contract. The authors also
acknowledge Rodolfo Herna´ndez from Laboratorio de Sanidad
Forestal (Gobierno de Arago´n, Mora de Rubielos, Teruel) for
providing T. pityocampa female pupae.
1
2955, 2915, 2850, 2180, 2090, 1465, 1215, 1065, 760 cm-1; H
NMR δ 3.64 (t, J ) 6.5 Hz, 2H), 1.57 (quint, J ) 8.0 Hz, 2H),
1.18-1.44 (21H), 0.88 (t, J ) 7 Hz, 3H); 13C NMR δ 62.9 (CH2),
32.8 (CH2), 31.7 (CH2), 29.6 (CH2), 29.6 (CH2), 29.6 (CH2), 29.4
(CH2), 29.4 (CH2), 28.6 (CD2, quint, J ) 19 Hz), 28.5 (CD2, quint,
J ) 19 Hz), 25.7 (CH2), 22.6 (CH2), 14.1 (CH3); MS m/z 247 (35,
M•+ - 1), 231 (100), 191 (25), 177 (20), 163 (28), 151 (25), 137
(30), 123 (35), 109 (45), 97 (60); Anal. Calcd for C16H282H6O: C,
77.34; H, 13.80. Found: C, 77.21; H, 13.82.
Supporting Information Available: 1H and 13C NMR and
DEPT spectra for compounds 1-3 and 5-11. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Abad, J.-L.; Fabria`s, G.; Camps, F. Lipids 2004, 39, 397-401.
JO061592S
764 J. Org. Chem., Vol. 72, No. 3, 2007