Synthesis and use of probes to investigate the cryptoregiochemistry of the first animal acetylenase

Article abstract of DOI:10.1021/jo060789h

(Chemical Equation Presented) Thaumetopoea pityocampa pheromone glands contain an unusual Δ¹¹ acetylenase that produces an alkynoic fatty acid intermediate in the sex pheromone biosynthetic pathway of this species. In this article, we describe

Full text of DOI:10.1021/jo060789h

Synthesis and Use of Probes to Investigate the Cryptoregiochemistry
of the First Animal Acetylenase
Jose´-Luis Abad,* Sergio Rodr´ıguez, 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 April 13, 2006
Thaumetopoea pityocampa pheromone glands contain an unusual ∆11 acetylenase that produces an alkynoic
fatty acid intermediate in the sex pheromone biosynthetic pathway of this species. In this article, we
describe the synthesis and use of the deuterated (Z)-11-hexadecenoic acid probes required to decipher
the cryptoregiochemistry of this enzyme. The label in the olefinic bonds was introduced by Wittig reaction
between the appropriate deuterated reagents. Besides the vinyl deuterium atoms, for reliable GC-MS
analyses these compounds bear a tetradeuterium tag, which was introduced by deuteration of an alkyne
intermediate in the presence of the Wilkinson catalyst. Pheromone gland metabolization studies of these
probes provided experimental evidence that the transformation of (Z)-11-hexadecenoic acid into 11-
hexadecynoic 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.
Introduction
Enzymatic desaturation of fatty acids is a key process in moth
sex pheromone biosynthesis. This reaction is catalyzed by
distinctive desaturases that, in contrast to the common desatu-
rases of animal cells, afford a wide variety of unsaturated
products with different position and configuration of unsatura-
tions. Besides the desaturase-mediated formation of monoalkene
and polyene fatty acids, the introduction of triple bonds by
desaturases has also been reported in a few cases. Thus, some
lepidopteran species belonging to the genus Thaumetopoea (T.
jordana, T. piniVora, T. wilkinsoni or T. pityocampa), as well
as the moth Heterocampa guttiVitta,1 use (Z)-13-hexadecen-11-
ynyl acetate as their sex pheromone.2 This semiochemical is
biosynthesized by combined ∆11 and ∆13 desaturase reactions,
which transform palmitic acid into (Z)-13-hexedecen-11-ynoic
FIGURE 1. Desaturation reactions involved in the sex pheromone
biosynthetic pathways of the Thaumetopoea genus.
acid (IV) (Figure 1). The biosynthetic intermediate 11-hexa-
decynoic acid is produced by ∆11 desaturation of (Z)-11-
hexadecenoic acid (II), which in turn arises from ∆11 desatu-
* To whom correspondence should be addressed. Ph: + 34 93 400 6100.
Fax: + 34 93 204 5904.
(1) Silk, P. J.; Lonergan, G. C.; Allen, D. C.; Spear O’Mara, J. Can.
Entomol. 2000, 132, 681-684.
ration of palmitic acid. The final enyne (IV) is synthesized by
(2) Fre´rot, B.; Demolin, G. Boll. Zool. Agrar. Bachic. Ser. II 1993, 25,
33-40.
13
∆
desaturation of 11-hexadecynoic acid but not by ∆11
10.1021/jo060789h CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/08/2006
7558
J. Org. Chem. 2006, 71, 7558-7564

Acetylenase Cryptoregiochemistry
FIGURE 2. Tracers prepared to probe the cryptoregiochemistry of the ∆11 acetylenation.
SCHEME 1. Synthesis of Tracer 1aa
a Reagents and conditions: (a) BuLi/THF/HMPA, 90%; (b) ClCH2COOH/MeOH, 88%; (c) PDC/DMF, 71%; (d) BF3/MeOH complex, 91%; (e) LiAlD4/
diethyl ether, 98%; (f) Wilkinson catalyst/D2, 95%; (g) IBX/DMSO, 88%; (h) THF/BuLi/HMPA/ Ph3P+-C5H11Br- (Wittig reaction), 75%; (i) HCl/MeOH,
86%; (j) (1) IBX/DMSO; (2) CrO3/H2SO4/acetone, 85%.
SCHEME 2. Synthesis of Tracer 1ba
a Reagents and conditions: (a) Wilkinson catalyst/D2, 97%; (b) IBX/DMSO, 90%; (c) LiAlD4/diethyl ether, 88%; (d) PPh3/NBS/DMF, 82%; (e) PPh3/
CH3CN; (f) THF/HMPA/BuLi (Wittig reaction), 73%; (g) HCl/MeOH, 92%; (h) (1) IBX/DMSO; (2) CrO3/H2SO4/acetone, 87%.
desaturation of (Z,Z)-11,13-hexadecadienoic acid (V), which is
a metabolic end product.3 Both the cryptoregiochemistry4 (site
of initial oxidation) and stereochemistry of the ∆11 desaturation
of palmitic acid have been reported in previous articles.5-7 To
gain some insight into the course of the acetylenation reaction,
in this report we describe the preparation of pentadeuterated
(Z)-11-hexadecenoic acids 1a and 1b, selectively monodeuter-
ated at the C-11 or C-12 olefinic positions (Figure 2), and their
use to investigate the cryptoregiochemistry of the acetylene
formation. The results obtained agree with those previously
reported on enzymes of the plant kingdom, specifically the
compositae Crepis alpina8 and the moss Ceratodon purpurea,9
which contain bifunctional enzymes with desaturase/acetylenase
activities.10
Results and Discussion
Preparation of the Labeled and Tagged Tracers. Most
studies for unveiling the site of initial oxidation in enzymatic
desaturations have relied on the use of substrates gem-dideu-
terated at each desaturation position. The amounts of desaturated
product formed from each substrate, relative to a metabolic
standard, indicate whether the desaturation rate is affected by
the presence of the mass labels, and it allows detecting the
isotope-sensitive cleavage step, which in turn reveals the site
of initial oxidation in the desaturation reaction. Following this
approach, the present investigation was carried out by using
the pentadeuterated substrates 1a and 1b. These (Z)-11-alkene
tracers bear a diagnostic deuterium atom attached at either the
C-11 or C-12 vinyl position, as well as four deuterium atoms
at C-7 and C-8 of the fatty acid chain. This last tetradeuterium
tag was required for GC-MS analytical differentiation of the
labeled compounds from the natural intermediates. The tetra-
deuterium moiety was introduced in a remote location from the
site of dehydrogenation to avoid interferences with the desatu-
ration process and the existence of secondary kinetic isotope
effects.
(3) Barrot, M.; Fabria`s, G.; Camps, F. Tetrahedron 1994, 50, 9789-
9796.
(4) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1996, 118, 6295-
6296.
(5) Abad, J.-L.; Fabria`s, G.; Camps, F. Lipids 2004, 39, 397-401.
(6) Ando, T.; Ikemoto, K.; Ohno, R.; Yamamoto, M. Arch. Insect.
Biochem. Physiol. 1998, 37, 8-16.
(7) Boland, W.; Fro¨bl, C.; Schottler, M.; Toth, M. J. Chem. Soc., Chem.
Commun. 1993, 1155-1157.
(8) 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.
(9) Sperling, P.; Lee, M.; Girke, T.; Za¨hringer, U.; Stymne, S.; Heinz,
E. Eur. J. Biochem. 2000, 267, 3801-3811.
(10) Carlsson, A. S.; Thomaeus, S.; Hamberg, M.; Stymne, S. Eur. J.
Biochem. 2004, 271, 2991-2997.
Substrates 1a and 1b were prepared as depicted in Schemes
1 and 2. Thus, trityl-protected compound 2 was coupled with
the methoxymethane derivative of 6-bromo-1-hexanol to afford
compound 3, which was selectively detritylated in acid media
to obtain the common intermediate 4a. This synthon had the
proper functionalities needed for introduction of both label and
J. Org. Chem, Vol. 71, No. 20, 2006 7559

Abad et al.
FIGURE 3. Deuterated probes and their desaturation products.
tag in the alcohol and alkyne positions, respectively, to prepare
compound 1a. Alcohol oxidation using PDC in DMF11 afforded
the corresponding acid 5 in high yields. However, to facilitate
its purification, this acid was transformed into methyl ester 6
using boron trifluoride-methanol complex. Deuterium introduc-
tion at the carbinol position in compound 4b was achieved by
reduction of the methyl ester with LiAlD4, which was trans-
formed into the corresponding pentadeuterated aldehyde 8a by
nonscrambling deuteration with the Wilkinson catalyst followed
by oxidation with IBX in DMSO. Wittig reaction with the
phosphonium salt of 1-bromopentane gave rise to the cis olefin
9a as the major product, with the aliphatic chain protected in
the final alcohol position. Methoxymethane deprotection of
derivative 9a was accomplished by acid treatment with HCl/
MeOH. The final acid 1a was obtained from the resulting
alcohol by IBX oxidation using DMSO as solvent to the
corresponding aldehyde, which was directly oxidized using
Jones conditions. These combined oxidation reactions afforded
a higher final yield than that obtained using direct Jones12 or
Corey13 conditions.
As shown in Scheme 2, a similar approach was attempted
for the synthesis of compound 1b. Thus, commercially available
ethyl valerate was reduced to the dideuterated 1-pentanol 12
with LiAlD4. Despite the different oxidation conditions used,
isolation of the deuterated valeraldehyde was very difficult;
therefore, we had to modify the strategy to obtain the labeled
olefin 9b. For this purpose, we transformed alcohol 12 into the
dideuterated 1-bromopentane 13, which was rapidly converted
into the corresponding phosphonium salt 14. Coupling of
compound 8b with 14 through a Wittig reaction allowed us to
obtain as a major component the cis isomer of compound 9b
(8:2 cis:trans). Methoxymethane deprotection and final oxidation
following the above conditions gave rise to acid 1b.
Characterization of the pentadeuterated compounds was
1
carried out by H and 13C NMR, as previously reported.5 The
number and the presence of the deuterium labels was the
expected as concluded from MS (the molecular ion mass was
in accordance with the presence of five deuterium atoms) and
13C NMR analyses (presence of two quintuplets for the CD2
groups with slight upfield chemical shift changes at the 30-
28.6 ppm range for compounds 7-10 and 1, and an additional
triplet for the olefinic CD in compounds 9, 10, and 1). In
addition, the presence of the olefinic deuterium atoms was also
evidenced in the 1H NMR spectra of 9, 10, and 1, which showed
simple olefinic hydrogen signals at ca. 5.3 ppm accounting for
only one hydrogen. Furthermore, the integration of the signal
at 1.25 ppm was in agreement with the presence of 2 CD2 groups
in the aliphatic chain.
Cryptoregiochemistry Studies. As shown in Figures 3 and
4, the labeled probes 1a and 1b follow two different pathways
in the processionary moth pheromone gland, ∆11 acetylenation
into 11-hexadecynoic acid and further ∆13 desaturation to (Z)-
13-hexadecen-11-ynoic acid, and ∆13 desaturation to (Z,Z)-11,-
13-hexadecadienoic acid. To determine the acetylenase cryp-
toregiochemistry, the elimination of the olefinic deuterium atoms
of both deuterated alkenes 1a and 1b was investigated by GC-
MS analysis. Accurate quantification of the resulting labeled
11-hexadecynoic acids produced from each probe was imprac-
ticable as a result of the low amounts of product occurring in
the tissue. Since the ∆13 desaturase activity was not substantially
affected by the presence of deuterium atoms in the probes, this
problem was circumvented by measuring the quantities of
labeled enynes, produced by ∆13 desaturation of the acetylenase
product. The slight sensitivity of the ∆13 desaturase activity to
the presence of labels was validated by measuring the levels of
pentadeuterated methyl (Z,Z)-11,13-hexadecadienoate, synthe-
sized by ∆13 desaturation of each probe. Thus, for equivalent
substrate incorporations, as assessed from the labeled to natural
methyl (Z)-11-hexadecenoate proportions (Table 1), similar
methyl d5/d0 dienoate ratios resulted from each deuterated
substrate (0.068 from 1a; 0.058 from 1b, Table 1). Therefore,
monitoring of the deuterated enynes, produced by ∆13 desatu-
ration of the acetylenase products, was considered appropriate
to ascertain the site of initial oxidation in the acetylenase
reaction. Furthermore, since conversion of both probes 1a and
1b into the dienoic acid was relatively insensitive to isotopic
substitution, the diene intermediate serves as a metabolic
standard for tracking the acetylenation of substrates bearing
either hydrogen or deuterium at the vinyl positions. A reliable
estimate of the KIE values can be obtained from the quotients
between the d5/d0 diene and the d4/d0 enyne ratios with each
probe. In all previous studies, the methodology for determining
intermolecular primary deuterium KIE in desaturase-catalyzed
As the Wittig reaction did not give the pure (Z)-stereoisomers,
a final purification step of the wanted acids 1 was necessary.
Removal of the (E)-stereoisomers was accomplished by prepa-
ration of the methyl esters and purification by column chro-
matography on silica gel impregnated with silver nitrate (10%).
Methyl esters were transformed into the final pure (Z)-acids 1a
and 1b by saponification with K2CO3/MeOH.
The final deuterium contents of the labeled probes were
determined by GC-MS analysis of their respective methyl esters
2
2
and were found to be as follows: 1a 13% H6, 68% H5, 10%
2H4, 7% 2H3 and 3% 2H2; 1b 14% 2H6, 65% 2H5, 10% 2H4, 8%
2
2H3 and 3% H2.
(11) Corey, E. J.; Schmidt, G. Tetrahedron Lett 1979, 399-402.
(12) Abad, J.-L.; Fabria`s, G.; Camps, F. J. Org. Chem. 2000, 65, 8582-
8588.
(13) Abad, J.-L.; Villorbina, G.; Fabria`s, G.; Camps, F. J. Org. Chem.
2004, 69, 7108-7113.
7560 J. Org. Chem., Vol. 71, No. 20, 2006

Acetylenase Cryptoregiochemistry
that no isotope discrimination occurs in the removal of C12-H
but the acetylenase reaction is sensitive to deuterium substitution
at C11. The results obtained in these experiments clearly support
a stepwise mechanism for the acetylenase-mediated dehydro-
genation of (Z)-11-hexadecenoate. The very strong vinyl C-H
bond at C-11 would be significantly perturbed first in an
energetically demanding and therefore isotopically sensitive step.
Loss of C12-H would occur next, although not necessarily after
complete abstraction of C11-H. It is possible that base assistance
is involved in the elimination of C12-H.
This cryptoregiochemistry is in accordance with that reported
for a ∆12 acetylenase of Crepis alpina, in which a large KIE of
14.615 was observed for the homologous carbon, and it conforms
to a general rule by which the desaturation is initiated at the
carbon atom closest to the acid functionality.
The Crepis enzyme has been shown to have bifunctional
oleate ∆12 desaturase and linoleate ∆12 acetylenase activity. An
enzyme with similar bifunctional ∆6 desaturase/∆6 acetylenase
activity has also been cloned from the moss Ceratodon
purpureus.9 The acetylenase studied here is the first acetylenase
reported in the animal kingdom. Whether it is also a bifunctional
desaturase/acetylenase is currently being investigated.
Conclusions
In summary, we have reported the synthesis of pentadeuter-
ared (Z)-11-hexadecenoic acids regiospecifically deuterated at
each vinyl position and bearing a tetradeuterium tag distant from
the site of desaturation. A novel methodological approach has
been described through which, by quantifying the conversion
of each probe into both tetradeuterated enyne and pentadeuter-
ated diene intermediates, the site of initial oxidation in the ∆11-
acetylenation has been elucidated. This reaction involves an
initial energetically demanding and thus isotope sensitive
perturbation of the strong vinyl C11-H bond followed by the
fast removal of the neighboring C12-H.
FIGURE 4. Metabolization of 1a and 1b in T. pityocampa pheromone
glands. The traces correspond to GC-MS chromatograms of a
methanolyzed lipid extract from T. pityocampa pheromone glands
incubated with either 1b (top), 1a (middle), or DMSO (control, bottom).
Left, chromatograms obtained by selection of ion at m/z 269 (molecular
ion of methyl d4(Z)-13-hexadecen-11-ynoate) in the total ion current
chromatogram; right, chromatograms obtained by extraction of ion at
m/z 272 (molecular ion of d5(Z,Z)-11,13-hexadecadienoate) from the
total ion current chromatogram. The experiments were performed as
described in the Experimental Section. Asterisks indicate a labeled
compound (left, methyl d4(Z)-13-hexadecen-11-ynoate, and right,
methyl d5(Z,Z)-11,13-hexadecadienoate.)
Experimental Section
Preparation of (Pent-4-ynyloxy)triphenylmethane (2). To a
solution of 2.54 g (30 mmol) of 4-pentyn-1-ol in 60 mL of dry
pyridine was added 8.92 g (32 mmol) of trityl chloride. The reaction
mixture was stirred at room temperature for 36 h, and then 25 mL
of water was added. The mixture was extracted with CH2Cl2, the
organic layer was concentrated to dryness, and the residue was
purified by flash chromatography on silica gel (0-10% MTBE/
hexane) to give 9.2 g of a white solid (94% yield). Mp 79-80 °C;
IR 3305, 3060, 3020, 2930, 2875, 1490, 1445, 1220, 1070, 760,
705 cm-1; 1H NMR δ 7.46-7.41 (6H), 7.32-7.20 (9H), 3.16 (t, J
) 6.0 Hz, 2H), 2.39 (dt, J1 ) 7.0 Hz, J2 ) 2.5 Hz, 2H), 1.88 (t, J
) 2.5 Hz, 1H), 1.81 (t, J ) 7.0 Hz, 2H); 13C NMR δ 144.2 (C),
128.6 (CH), 127.6 (CH), 126.8 (CH), 86.3 (C), 84.0 (C), 68.3 (CH),
61.8 (CH2), 29.1 (CH2), 15.5 (CH2); MS m/z 326 (3, M•+), 283
(7), 271 (15), 249 (50), 243 (100), 167 (15). Anal. Calcd for
C24H22O: C, 88.31; H, 6.79. Found: C, 88.20; H, 6.68.
1,1,1-Triphenyl-2,14,16-trioxaheptadec-6-yne (3). To a mixture
of 2 (8.15 g, 25 mmol), 40 mL of dry HMPA, and 40 mL of
anhydrous THF was added dropwise a solution of butyllitium (1.6
M) in hexanes (18 mL, 29 mmol) at -15° C. The resulting pale
red mixture was stirred for 10 min, and a solution of BrCH2(CH2)4-
CH2OMOM (4.48 g, 20 mmol) in 10 mL of dry THF was added
dropwise at that temperature. Stirring was continued overnight at
TABLE 1. Conversion of Probes 1a and 1b into Biosynthetic
Intermediatesa
(d5diene/d0diene)/
(d5Z11/d0Z11)
(d4enyne/d0enyne)/
(d5Z11/d0Z11)
(d5diene/d0diene)/
(d4enyne/d0enyne)
1a
1b
0.068 ( 0.006
0.058 ( 0.001
0.009 ( 0.003
0.060 ( 0.007
7.55 ( 1.65
0.97 ( 0.13
a Data are expressed as the mean ( SD of three replicates. Labeled-to-
natural FAME ratios were determined from the areas of the M•+ + 1 ions
(d5Z11, 274; d0Z11, 269; d5diene, 272; d0diene, 267; d4enyne, 269; d0enyne,
265). Abbreviations are Z11, methyl (Z)-11-hexadecenoate; diene, methyl
(Z,Z)-11-13-hexadecadienonate; enyne, methyl (Z)-13-hexadecen-11-
ynoate.
reactions involved mass spectrometry analysis of the reaction
products derived from direct competition between the appropri-
ate regiospecifically dideuterated substrate and its nonlabeled
analogue, both with extra-labels for analysis.13,14 The different
approach followed in this article, based on the use of a metabolic
standard, avoids the need of synthesizing the competitor, as well
as the data corrections derived from the competitor’s use.
As summarized in Table 1, the (d5/d0 diene)/(d4/d0 enyne)
ratio was close to 1 from 1b and 7.5 from 1a, which indicates
(15) Reed, D. W.; Polichuk, D. R.; Buist, P. H.; Ambrose, S. J.; Sasata,
R. J.; Savile, C. K.; Ross, A. R.; Covello, P. S. J. Am. Chem. Soc. 2003,
125, 10635-10640.
(14) Behrouzian, B.; Saville, C. K.; Dawson, B.; Buist, P. H.; Shanklin,
J. J. Am. Chem. Soc. 2002, 124, 3277-3283.
J. Org. Chem, Vol. 71, No. 20, 2006 7561

Abad et al.
0 °C. The reaction mixture was then poured into saturated NaHCO3
(5 mL) and extracted with hexane (3 × 40 mL). Solvent was
evaporated, and the residue was purified by flash chromatography
on silica gel using a gradient hexane/MTBE (0-30%) to afford
8.46 g of the expected alcohol 3 in 90% yield. IR 3475, 3060, 3030,
2935, 2865, 1595, 1490, 1450, 1150, 1110, 1070, 1045, 915, 760,
[1,1-2H2]-12,14-Dioxapentadec-4-yn-1-ol (4b). A solution of
methyl ester 6 (1.54 g, 6 mmol) in 20 mL of Et2O, maintained
under nitrogen atmosphere and at room temperature, was treated
with 4.5 molar equiv of LiAlD4. The mixture was stirred until the
reaction was completed. After the usual workup by careful addition
of water, aluminum salts were filtered off, and the organic solution
was concentrated to a residue and then purified by flash chroma-
tography using as eluent hexanes/MTBE 75:25 to afford 1.35 g of
deuterated alcohol 4b (98% yield). IR 3340, 2935, 2865, 2210,
1
705 cm-1; H NMR (500 MHz) δ 7.46-7.41 (3H), 7.34-7.18
(12H), 4.60 (s, 2H), 3.51 (t, J ) 6.5 Hz, 2H), 3.36 (s, 3H), 3.14 (t,
J ) 6.0 Hz, 2H), 2.30 (m, 2H), 2.09 (m, 2H), 1.78 (t, J ) 2.5 Hz,
2H), 1.58 (m, 2H), 1.50-1.30 (6H); 13C NMR δ 144.2 (C), 128.6
(CH), 127.6 (CH), 126.7 (CH), 93.3 (CH2), 86.2 (C), 80.3 (C), 79.6
(C), 67.7 (CH2), 62.1 (CH2), 55.0 (CH3), 29.6 (CH2), 29.5 (CH2),
28.9 (CH2), 28.5 (CH2), 25.7 (CH2), 18.6 (CH2), 15.8 (CH2); MS
m/z 470 (2, M•+), 393 (5), 283 (5), 271 (9), 243 (100), 197 (10),
167 (70). Anal. Calcd for C32H38O3: C, 81.66; H, 8.14. Found: C,
81.47; H, 8.16.
2100, 1725, 1440, 1390, 1210, 1145, 1110, 1045, 965, 920 cm-1
;
1H NMR (500 MHz) δ 4.62 (s, 2H), 3.53 (t, J ) 6.5 Hz, 2H), 3.36
(s, 3H), 2.27 (m, 2H), 2.15 (m, 2H), 1.95 (d, J ) 7.0 Hz, 1H), 1.72
(t, J ) 7.0 Hz, 2H), 1.60 (quint, J ) 6.5 Hz, 2H), 1.49 (quint, J )
7.0 Hz, 2H), 1.45-1.34 (bb, 6H); 13C NMR δ 96.2 (CH2), 80.7
(C), 79.3 (C), 67.6 (CH2), 60.8 (CD2, quint, J ) 21.5 Hz), 55.0
(CH3), 31.3 (CH2), 29.4 (CH2), 28.8 (CH2), 28.4 (CH2), 25.6 (CH2),
18.5 (CH2), 15.2 (CH2); MS m/z 231 (2, M•+ + 1), 199 (100), 181
(24), 163 (15), 154 (14), 137 (13), 113 (35), 99 (18), 87 (15), 73
(20). Anal. Calcd for C13H222H2O3: C, 67.59; H, 10.51. Found:
C, 67.60; H, 10.49.
12,14-Dioxapentadec-4-yn-1-ol (4a). Trityl deprotection of 3
was achieved by treatment of 7.05 g (15 mmol) of the protected
alkyne diol with 200 mL of MeOH/Cl2HCCOOH solution (2%)
for 36 h at room temperature. The resulting mixture was neutralized
with saturated NaHCO3 aqueous solution and concentrated, and the
crude was extracted with CH2Cl2, dried, and concentrated to a
residue that was purified by flash chromatography on silica gel
using a gradient of 0-20% AcOEt in hexane to give rise 3.2 g of
an oil (88% yield) corresponding to alcohol 4. IR 3340, 2935, 2865,
Reduction with Wilkinson Catalyst. To a mixture of compound
4 and 0.1 equiv of RhCl(PPh3)3 (prepared according to the procedure
previously reported)13 was added degassed benzene under argon
atmosphere to get a reddish solution. The system was purged by
applying several times vacuum and then a stream of D2 through.
Finally a D2 atmosphere was kept from a balloon and the solution
was stirred for 48 h. The brown mixture was filtered through a
bed of Celite, and the solvent was evaporated. The residue was
purified by flash chromatography on silica gel (0-20% MTBE/
hexane) to obtain saturated alcohols 7 (95-98% yield).
1
1725, 1465, 1440, 1390, 1215, 1145, 1110, 1045, 920 cm-1; H
NMR δ 4.62 (s, 2H), 3.75 (t, J ) 6.0 Hz, 2H), 3.52 (t, J ) 6.5 Hz,
2H), 3.36 (s, 3H), 2.27 (m, 2H), 2.15 (m, 2H), 1.73 (m, 2H), 1.60
(t, J ) 7.0 Hz, 1H), 1.49-1.38 (bb, 8H); 13C NMR δ 96.3 (CH2),
80.9 (C), 79.4 (C), 67.7 (CH2), 61.9 (CH2), 55.1 (CH3), 31.5 (CH2),
29.5 (CH2), 28.9 (CH2), 28.5 (CH2), 25.7 (CH2), 18.6 (CH2), 15.4
(CH2); MS m/z 229 (3, M•+ +1), 197 (100), 179 (15), 161 (12)
135 (10), 111 (30) 97 (20), 85 (12), 71 (14). Anal. Calcd for
C13H24O3: C, 68.38; H, 10.69. Found: C, 68.47; H, 10.53.
[1,1,4,4,5,5-2H6]-12,14-Dioxapentadecan-1-ol (7a). This com-
pound (904 mg, 95% yield) was obtained from 915 mg (4 mmol)
of alkyne 4b. IR 3340, 2930, 2855, 2185, 2090, 1725, 1460, 1385,
1
1215, 1145, 1110, 1045, 965, 920 cm-1; H NMR (500 MHz) δ
4.62 (s, 2H), 3.51 (t, J ) 7.0 Hz, 2H), 3.36 (s, 3H), 1.56 (m, 5H),
1.36-1.26 (bb, 10H); 13C NMR δ 96.2 (CH2), 67.7 (CH2), 61.9
(CD2, quint, J ) 21.0 Hz), 54.9 (CH3), 32.4 (CH2), 29.6 (CH2),
29.4 (CH2), 29.3 (CH2), 29.1 (CH2), 28.4 (CD2, quint, J ) 18.5
Hz), 28.3 (CD2, quint, J ) 18.5 Hz), 26.0 (CH2), 25.3 (CH2); MS
m/z 239 (1, M•+ + 1), 235 (5, M•+ - 3), 207 (100), 177 (38), 156
(12). Anal. Calcd for C13H222H6O3: C, 65.50; H, 11.85. Found:
C, 65.60; H, 11.79.
12,14-Dioxapentadec-4-ynoic acid (5). According to the pro-
cedure reported by Corey and Schmidt,11 2.74 g (12 mmol) of the
previously obtained alcohol was stirred in a solution of 180 mL of
DMF containing 3 equiv of PDC for 3 days. After this time, the
reaction mixture was treated with a Na2S2O3 solution to give a green
color mixture, and then 12 mL of HCl (1 M) was added, extracted
with CH2Cl2, dried, and concentrated to a residue that was purified
by flash chromatography on silica gel using hexane/MTBE 85:15
to give 2.05 g (71% yield) of the corresponding acid. IR 2935,
Aldehydes Preparation. These compounds were prepared by
using the procedure reported by Frigerio et al.16 To a solution of
compound 7 (3 mmol), in 20 mL of DMSO was added 3 equiv of
freshly prepared IBX17 with vigorous stirring under nitrogen
atmosphere at room temperature for 4 h. Then 50 mL of water
was added, and the organic mixture was extracted with hexane/
MTBE 1:1 (3 × 20 mL). The combined organic fractions were
dried, concentrated to dryness, and purified by flash chromatography
on silica gel (0-10% MTBE/hexane) obtaining the pure aldehyde
(88-91% yield).
2860, 1740, 1465, 1440, 1360, 1250, 1165, 1115, 1045, 995 cm-1
;
1H NMR (500 MHz) δ 4.64 (s, 2H), 3.53 (t, J ) 6.5 Hz, 2H), 3.37
(s, 3H), 2.55 (m, 2H), 2.47 (m, 2H), 2.13 (m, 2H), 1.60 (m, 2H),
1.47 (m, 2H), 1.38 (bb, 4H); 13C NMR δ 177.4 (C), 96.2 (CH2),
81.1 (C), 77.9 (C), 67.7 (CH2), 55.0 (CH3), 33.8 (CH2), 29.4 (CH2),
28.7 (CH2), 28.4 (CH2), 25.6 (CH2), 18.5 (CH2), 14.5 (CH2); Anal.
Calcd for C13H22O4: C, 64.44; H, 9.15. Found: C, 64.59; H, 9.00.
Methyl 12,14-Dioxapentadec-4-ynoate (6). To a solution of 2.05
g of acid 5 (8.5 mmol) in 40 mL of methanol was added 10 mL of
boron trifluoride methanol complex (20%). The reaction mixture
was vigorously stirred until reaction was completed (TLC monitor-
ing) and then neutralized with saturated NaHCO3 aqueous solution,
concentrated at reduced pressure, and extracted with hexane, and
the crude obtained after evaporation of the solvent was purified by
flash chromatography on silica gel using a gradient hexane/MTBE
(5%) to give 1.98 g (91% yield) of the corresponding ester. IR
[1,4,4,5,5-2H5]-12,14-Dioxapentadecanal (8a). In this case 620
mg (88% yield) of aldehyde 8a were obtained from 715 mg (3
mmol) of initial alcohol 7a. IR 2930, 2855, 2190, 2085, 1715, 1455,
1
1390, 1215, 1150, 1110, 1045, 920 cm-1; H NMR (500 MHz) δ
4.62 (s, 2H), 3.52 (t, J ) 6.5 Hz, 2H), 3.36 (s, 3H), 2.41 (t, J )
7.5 Hz, 2H), 1.62-1.57 (m, 4H), 1.36-1.27 (bb, 8H); 13C NMR δ
202.6 (CD, t, J ) 26.0 Hz), 96.3 (CH2), 67.7 (CH2), 55.0 (CH3),
43.6 (CH2, t, J ) 3.5 Hz), 29.6 (CH2), 29.4 (CH2), 29.3 (CH2),
29.0 (CH2), 28.4 (CD2, quint, J ) 18.5 Hz), 28.3 (CD2, quint, J )
18.5 Hz), 26.1 (CH2), 21.7 (CH2); MS m/z 235 (3, M•+), 234 (5,
M•+-1), 215 (25), 187 (100), 169 (25), 151 (15), 137 (10), 123
(15). Anal. Calcd for C13H212H5O3: C, 66.34; H, 11.14. Found:
C, 66.25; H, 11.05.
1
2930, 2855, 1735, 1455, 1440, 1360, 1250, 1190 cm-1; H NMR
(500 MHz) δ 4.61 (s, 2H), 3.69 (s, 3H), 3.51 (t, J ) 6.5 Hz, 2H),
3.36 (s, 3H), 2.49 (m, 4H), 2.12 (t, J ) 7.0 Hz, 2H), 1.59 (m, 2H),
1.47 (m, 2H), 1.37 (bb, 4H); 13C NMR (125 MHz) δ 172.6 (C),
96.3 (CH2), 81.0 (C), 78.0 (C), 67.7 (CH2), 55.0 (CH3), 51.6 (CH3),
33.8 (CH2), 29.6 (CH2), 28.8 (CH2), 28.5 (CH2), 25.7 (CH2), 18.6
(16) Frigerio, M.; Santagostino, M.; Sputore, S.; Palmisano, G. J. Org.
Chem. 1995, 60, 7272-7276.
(17) Frigerio, M.; Santagostino, M.; Sputore, S. J. Org. Chem. 1999,
64, 4537-4538.
(CH2), 14.6 (CH2); MS m/z 284 (15, M•+ + 29), 257 (100, M•+
+
1), 225 (32). Anal. Calcd for C14H24O4: C, 65.60; H, 9.44. Found:
C, 65.47; H, 9.42.
7562 J. Org. Chem., Vol. 71, No. 20, 2006

Acetylenase Cryptoregiochemistry
Preparation of [1,1-2H2]-1-Pentanol (12) A solution of 5.2 g
(40 mmol) of ethyl valerate (11) in 50 mL of dry Et2O, maintained
under argon and at room temperature, was treated with 1.52 g of
LiAlD4 (40 mmol), and the mixture was stirred until the reaction
was completed (TLC monitoring). After careful addition of deu-
terated water to the reaction mixture, the white solid was filtered
off and the solvent carefully evaporated to dryness and then the
residue obtained was purified by flash chromatography on silica
gel using a gradient of pentane/diethyl ether (0-50%) to give 3.24
g of the pure dideuterated alcohol 12 (88%). IR 3345, 2925, 2855,
Methoxymethane Deprotection. General Procedure. Products
9 were deprotected to the corresponding alcohols 10 by treat-
ment with a MeOH/HCl solution (0.5 M) for 24 h at room
temperature. Solvent was concentrated, and the crude was neutral-
ized with a saturated NaHCO3 aqueous solution, extracted with
CH2Cl2, dried, and concentrated to a residue that was finally
purified by flash chromatography on silica gel using a gradient
of AcOEt in hexanes (10-25%) to obtain an oil (86-92%
yield).
[7,7,8,8,11-2H5]-(Z)-Hexadec-11-en-1-ol (10a). This alcohol
1
2190, 2090, 1460, 1170, 1135, 1085, 1065, 970 cm-1; H NMR
was isolated (106 mg, 86%) starting from 145 mg (0.5 mmol) of
9a. IR 3350, 2925, 2855, 2185, 2090, 1725, 1460, 1045 cm-1
;
(500 MHz) δ 1.56 (t, J ) 6.5 Hz, 2H, CH2), 1.46 (sa, 1H), 1.38-
1.30 (4H, CH2), 0.91 (t, J ) 6.5 Hz, 3H, CH3); 13C NMR (125
MHz) δ 62.1 (CD2, quint, J ) 21 Hz), 32.3 (CH2), 27.8 (CH2),
22.4 (CH2), 14.0 (CH3).
1H NMR (500 MHz) δ 5.34 (t, J ) 7.0 Hz, 1H), 3.63 (t, J ) 6.5
Hz, 2H), 2.01 (m, 4H), 1.56 (m, 2H), 1.43 (s, 1H), 1.35-1.25
(bb, 14H), 0.89 (t, J ) 7.5 Hz, 3H); 13C NMR δ 129.6 (CH),
129.5 (CD, t, J ) 23.0 Hz), 62.9 (CH2), 32.7 (CH2), 31.9 (CH2),
29.5 (CH2), 29.4 (CH2), 29.4 (CH2), 29.3 (CH2), 28.4 (CD2, quint,
J ) 18.5 Hz), 28.2 (CD2, quint, J ) 18.5 Hz), 27.0 (CH2),
26.8 (CH2), 25.7 (CH2), 22.3 (CH2), 13.9 (CH3); MS m/z 246
(100, M•+ + 1), 226 (35), 202 (15), 184 (10), 170 (12), 156 (20),
142 (30), 128 (42), 114 (50), 99 (70), 85 (50), 71 (40). Anal.
Calcd for C16H272H5O: C, 78.29; H, 13.15. Found: C, 78.23; H,
13.07.
Preparation of [1,1-2H2]-1-Bromo-pentane (13) This reaction
was accomplished with minor modifications according to the
procedure described by Bates et al.18 A solution of NBS (36 mmol,
6.1 g in 18 mL of DMF) was added dropwise at 0 °C to a stirred
DMF solution (24 mL) containing 3.24 g (35.2 mmol) of alcohol
12 and 9.47 g (36 mmol) of triphenylphosphine. Stirring was
continued for 1 h, and 250 µL of methanol was added to the pale
yellow mixture to quench the reagent excess. After 5 min, water
was added (240 mL), the mixture was extracted with pentane (3 ×
20 mL), and the organic layers were combined and distilled to obtain
4.17 g (78%) of the dideuterated bromoderivative 13 (bp 129 °C).
Carboxylic Acids Preparation (1). These compounds were
prepared by reaction of alcohol 10 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 was 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 solution of H2SO4/CrO3. Chromatography on
silica gel using hexane/MTBE 85:15 afforded the corresponding
acids in 85-87% yield.
[7,7,8,8,11-2H5]-(Z)-Hexadec-11-enoic Acid (1a). This product
(88 mg, 85%) was isolated as oil from 98 mg (0.4 mmol) of 10a.
IR 3210, 2925, 2855, 2190, 2090, 1725, 1460, 1270, 965 cm-1; 1H
NMR (500 MHz) δ 5.34 (t, J ) 7.0 Hz, 1H), 2.34 (t, J ) 7.5 Hz,
2H), 2.01 (m, 4H), 1.63 (m, 2H), 1.32-1.26 (bb, 12H), 0.89 (t, J
) 7.0 Hz, 3H); 13C NMR (125 MHz) δ 179.7 (C), 129.7 (CH),
129.5 (CD, t, J ) 23.0 Hz), 33.9 (CH2), 31.9 (CH2), 31.5 (CH2),
29.4 (CH2), 29.1(CH2), 29.1 (CH2), 29.0 (CH2), 28.2 (CD2, quint,
J ) 19.0 Hz), 27.0 (CH2), 26.9 (CH2), 26.8 (CH2), 24.6 (CH2),
22.3 (CH2), 14.0 (CH3); MS m/z (OCH3), 302 (20, M•+ + 29), 274
(100, M•+ + 1), 242 (32). Anal. Calcd for C16H252H5O2: C, 74.07;
H, 11.66. Found: C, 74.13; H, 11.59.
In ViWo 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.
Instrumental Analysis of the Biological Extracts. The GC-
MS analysis of 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 that afforded a cluster of ions,
analyzed as methyl ester, and are based in the abundance of the
1
IR 2925, 2855, 1720, 1460, 1375 cm-1; H NMR δ 1.85 (t, J )
6.5 Hz, 2H, CH2), 1.41 (m, 2H, CH2), 1.35 (m, 2H, CH2), 0.92 (t,
J ) 6.5 Hz, 3H, CH3); 13C NMR δ 33.4 (CD2, quint, J ) 23 Hz),
32.3 (CH2), 30.2 (CH2), 21.8 (CH2), 13.8 (CH3).
Wittig Reaction. A mixture of (455 mg, 3 mmol) of 1-bro-
mopentane 13 and 0.9 g (3.25 mmol) of triphenylphosphine was
refluxed in 20 mL of anhydrous CH3CN under argon atmosphere
for 36 h. Solvent was evaporated, and residue was suspended in
dry pentane (3 × 20 mL), decanted, and dried at reduced pressure
to give a white solid corresponding to the triphenylphosphonium
bromide 14.
To a mixture of the triphenylphosphonium salt 14 (650 mg, 1.5
mmol) previously obtained, 5 mL of anhydrous THF, and 1 mL of
dry HMPA was added 1.2 mL of a butyllitium solution in hexane
(1.2 equiv, 1.5 M) dropwise at -50°C under nitrogen atmosphere
to afford a pale orange color suspension. The reaction mixture was
stirred at that temperature for 30 min and then cooled at -78 °C.
Stirring was continued for 10 additional min, and aldehyde 8 (1
mmol, ca. 234 mg) dissolved in 4 mL of anhydrous THF was added
dropwise and stirred for 1 h at -78 °C and 3 h at room tempera-
ture. The reaction mixture was poured into water and extracted
with hexane. The combined organic fractions were dried, concen-
trated to dryness, and purified by flash chromatography on silica
gel impregnated with AgNO3 (10%) using as eluent a gradient
hexane/MTBE (5%) to give a 9/1 mixture of Z/E isomers (70-
75% yield).
[6,9,9,10,10-2H5]-(Z)-17,19-Dioxaeicos-5-ene (9a). This com-
pound was isolated (219 mg, 75% yield) starting from 235 mg of
aldehyde 8a. IR 2925, 2855, 2185, 2090, 1725, 1460, 1385, 1145,
1
1105, 1045, 965, 920 cm-1; H NMR (500 MHz) δ 5.35 (t, J )
7.5 Hz, 1H), 4.62 (s, 2H), 3.52 (t, J ) 7.0 Hz, 2H), 3.36 (s, 3H),
2.01 (m, 4H), 1.59 (m, 2H), 1.35-1.28 (bb, 14H), 0.89 (t, J ) 7.0
Hz, 3H); 13C NMR δ 129.6 (CH), 129.4 (CD, t, J ) 22.5 Hz),
96.3 (CH2), 67.8 (CH2), 54.9 (CH3), 31.9 (CH2), 29.7 (CH2), 29.5
(CH2), 29.4 (CH2), 29.4 (CH2), 29.2 (CH2), 28.2 (CD2, quint, J )
18.0 Hz), 27.0 (CH2), 26.8 (CH2), 26.1 (CH2), 22.3 (CH2), 13.9
(CH3); MS m/z 290 (15, M•+ + 1), 258 (100), 240 (25), 214 (10).
Anal. Calcd for C18H312H5O2: C, 74.68; H, 12.54. Found: C, 74.53;
H, 12.37.
(18) Bates, H. A.; Farina, J.; Tong, M. J. Org. Chem. 1986, 51, 2637-
2641.
J. Org. Chem, Vol. 71, No. 20, 2006 7563

Abad et al.
respective molecular ions in the range m/z 265-272 in which the
most abundant corresponded to the M•+ + 1 of the resulting
isotopomers.
(Gobierno de Arago´n, Mora de Rubielos, Teruel) for providing
T. pityocampa female pupae.
Supporting Information Available: 1H and 13C NMR and
DEPT spectra for compounds 1-10 and 12-13. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We gratefully acknowledge CICYT
and FEDER (AGL2001-0585) and Generalitat de Catalunya
(2001SGR 00342) for financial support. J.-L.A. thanks Spanish
MEC for a Ramo´n y Cajal contract. The authors also acknowl-
edge Rodolfo Herna´ndez from Laboratorio de Sanidad Forestal
JO060789H
7564 J. Org. Chem., Vol. 71, No. 20, 2006