Biosynthesis of sex pheromones in moths: Stereochemistry of fatty alcohol oxidation in Manduca sexta

Article abstract of DOI:10.1016/S0040-4020(02)01199-7

Six chiral deuterium labelled metabolic probes, (R) and (S)-enantiomers of [1,10,10-²H₃]-hexadecan-1-ol, (11E)-[1,10,10-²H₃]-hexadec-11-en-1-ol and (11Z)-[1,10,10-²H₃]-hexadec-11-en-1-ol, w

Full text of DOI:10.1016/S0040-4020(02)01199-7

Tetrahedron 58 (2002) 9193–9201
Biosynthesis of sex pheromones in moths: stereochemistry of fatty
alcohol oxidation in Manduca sexta
b
a
ˇa
Michal Hoskovec,a,b Anna Luxova, Ales Svatos and Wilhelm Boland
,
*
´
ˇ
aDepartment of Bioorganic Chemistry, Max Planck Institute of Chemical Ecology, Winzerlaer Str. 10, D-07745 Jena, Germany
bDepartment of Natural Products, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic (ACSR),
´
Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic
Received 26 June 2002; revised 30 August 2002; accepted 19 September 2002
Abstract—Six chiral deuterium labelled metabolic probes, (R) and (S)-enantiomers of [1,10,10-2H3]-hexadecan-1-ol, (11E)-[1,10,10-2H3]-
hexadec-11-en-1-ol and (11Z)-[1,10,10-2H3]-hexadec-11-en-1-ol, were synthesized to examine the stereospecificity of the fatty alcohol
oxidase from the female pheromone gland of the tobacco hawk moth (Manduca sexta, Sphingidae). Both in vitro and in vivo oxidations were
found to proceed by selective removal of the C1–HR hydrogen or deuterium atom (Re-specificity) to yield the corresponding aldehydes. (R)
and (S)-enantiomers of deuterium labelled salicyl alcohol and 2-thienyl-methanol, compounds entirely chemically diverse from the natural
pheromone precursors, were also oxidised Re-specifically to salicylaldehyde and 2-thiophenecarb-aldehyde, respectively. q 2002 Elsevier
Science Ltd. All rights reserved.
1. Introduction
exhibits pro-(R) C11–H and pro-(R) C12–H selectivity,
comparable to the more common D9-desaturase.6,7 In the
case of (E)-carbon–carbon double bond formation pro-(R)
C11–H and pro-(S) C12–H stereoselectivity was
observed.8
Pheromones are semiochemicals produced and perceived by
individuals belonging to the same species (interspecific
recognition). In Lepidoptera the most important semio-
chemicals are sex pheromones released by females to attract
conspecific males for mating.1
Another important chemical reaction is the transformation
of the functional group(s) of a pheromone. This affects both,
the perception by male antennae as well as the volatility of
the compound. One of these transformations, the oxidation
of primary alcohols to aldehydes,9 involves removal of one
of two hydrogen atoms from a prochiral –CH2OH group
(Scheme 1). In Lepidoptera, there is no precedent for the
stereoselectivity of this transformation, however, in
Coleoptera several investigations have been reported.10
Alcohol oxidase(s) from the leaf beetle Phaedon
armoraciae (Coleoptera: Chrysomelidae) are responsible
for the oxidation of geraniol to 8-oxo-geranial, the
immediate precursor of the two iridoids chrysomelidial
Biosynthesis of moth female sex pheromones is a part of
lipid metabolism in insects, where de novo biosynthesised
fatty acid precursors are desaturated and/or chain shortened
by b-oxidation. The resultant CoA-esters of predominantly
unsaturated acid, are transformed to hydrocarbons, alcohols,
esters or aldehydes by oxidation/reduction and esterification
enzymatic reactions.2,3 Fatty acid desaturation is performed
by specific D11-CoA-fatty acid desaturases, found only in
insects.4 The D11-desaturase from Trichoplusia ni
(Lepidoptera, Tortricidae) was recently cloned and
expressed in yeast.5 These investigations of Roelofs et al.
opened a way to a ‘molecular era’ in pheromone
biosynthesis studies. The D11-desaturases were character-
ized from a stereochemical point of view by several
authors.6–8 These studies revealed that the removal of the
two hydrogen atoms from prochiral C-centres is stereo-
specific and in the case of (Z)-double bond formation, it
Keywords: alcohols; aldehydes; biosynthesis; pheromones.
*
Corresponding author. Address: Department of Natural Products,
Institute of Organic Chemistry and Biochemistry, Academy of Sciences
´
of the Czech Republic (ACSR), Flemingovo nam. 2, CZ-166 10 Prague 6,
Czech Republic. Tel.: þ420-2-20183240; fax: þ420-2-24310177;
e-mail: hoskovec@uochb.cas.cz
Scheme 1.
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01199-7

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M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
and plagiodial. The oxidase catalyses the removal of the
C1– and C8–HR hydrogen atoms (Re-specificity).11 In
contrast to NADH dependent oxidoreductases from
plants,12 beetles use an oxygen-dependent enzyme. The
Re-specificity was documented with six additional leaf
beetles for related, iridoid-producing pathways.10 The
specificity is also the same with other types of substrates,
e.g. salicylaldehyde, a defensive allomone of leaf beetle
larvae (Phratora vitellinae), which is produced with
Re-specificity from the corresponding salicylalcohol by an
oxidase located in the poison gland.10 Furthermore, the
alcohol oxidase from the yeast Candida boidinii catalyzes
the oxidation of ethanol to acetaldehyde with removal of the
pro-R hydrogen atom (Re-specificity).13
The recent observation that the alcohol oxidases catalysing
the formation of the fatty aldehydes from fatty alcohols are
present in the pheromone glands of several moths14–16 has
suggested the possibility of determining the stereochemistry
of this transformation. For this study, the tobacco hawk
moth (Manduca sexta, Sphingidae) was selected as a model
lepidopteran species. This species is one of the most
thoroughly studied insect models used in pheromone
olfaction17–21 and biosynthesis7,22–25 research. Moreover,
the basic features of the M. sexta pheromone alcohol
oxidase producing aldehydes of various degree of unsatura-
tion were characterised by Fang et al.16
Scheme 2.
2. Results and discussion
The assignment of alcohol oxidase stereochemistry
(Scheme 1)† requires the use of chiral, deuterium labelled
precursors with defined stereochemistry on carbon C1. One
of the pro-R or pro-S hydrogen atoms on the C1 prochiral
carbon atom in the synthetic metabolic probes is replaced
with a deuterium atom. The fate of those C1-attached atoms
can be monitored by gas chromatography–mass spec-
trometry (GLC–MS).
2.1. Highly enantioselective synthesis of deuterium
labelled metabolic probes
A convenient synthetic route to the chirally labelled alcohols
1–3 is outlined in Scheme 3. Introduction of deuterium was
achieved byreduction of dimethyl sebacate (4) with LiAlD4.29
Theproductofthereaction,deuterateddiol5, wasconvertedto
10-bromodecan-1-ol30 6, and, after protection of the hydroxy
group, used for subsequent two-step alkylation of the
acetylene.31 This reaction sequence hand in hand with the
necessary protection–deprotection steps afforded the key
intermediate in the syntheses of desired chiral alcohols 1–3,
[1,1,10,10-2H4]-hexadec-11-yn-1-ol (8).
Twelve pheromone-like compounds have been identified in
solvent rinses of the M. sexta female pheromone gland.26,27
To determine the stereochemistry of the oxidation of all
active components of the pheromone blend would require
the preparation of (R) and (S)-metabolic probes for all of
them. However, the syntheses of optically active deuterated
dienals and the extremely unstable28 trienals might not be
necessary. A reasonable assumption is that the stereo-
selectivity of the enzymatic oxidation of C16-trienols/
dienols to the corresponding aldehydes is the same as for
monoenic and saturated C16-compounds.16
Hydrogenation of the akynol 8 using tris(triphenyl-
phosphine)rhodium bromide (Wilkinson’s catalyst32,33
)
gave hexadecanol 9. Reduction of 8 with sodium metal in
liquid ammonia34 (with protection–deprotection of the
hydroxy group) gave (E)-hexadecenol 10, and finally, cis-
semihydrogenation of 8 over P2–Ni catalyst35 afforded (Z)-
hexadecenol 11. The C16-alcohols 9–11 were converted to
the aldehydes 12–14 by simple oxidation with pyridinium
chlorochromate (PCC).36
Thus, six optically active [1,10,10-2H3]-C16-alcohols,
depicted in Scheme 2, were proposed as metabolic probes.
Here we present the synthesis of the proposed probes,
results of incubation experiments with them and the
assignment of the stereoselectivity of the M. sexta alcohol
oxidase.
The final step of the synthesis was stereospecific reduction
of the aldehydes 12–14 with (S) and (R)-B-isopino-
campheyl-9-borabicyclo[3.3.1]nonanes (Alpine-Boranesw;
Aldrich), which are known to be highly stereoselective
reducing agents.37,38 The enantiomeric purity of the
1
labelled chiral alcohols 1–3 was determined by H NMR
via their (1S)-camphanates39,40 according to the method
of Taguchi.38 The (R) and (S)-C16-alcohols 1–3 were
sufficiently enantiomerically pure (92–96%; see Table 1)
for the experiments with pheromone alcohol oxidase.
†
D was used as a symbol for deuterium atoms in Schemes 1–4 and plain
text. In chemical names symbol 2H was used due to IUPAC nomenclature
recommendations.

M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
9195
Scheme 3. (a) LiAlD4/ether; (b) HBr/benzene, reflux 8 h; (c) 3,4-dihydro-2H-pyran, PPTS/CH2Cl2; (d) LiHC2/NH3 (liquid), DMSO; (e) Dowex 50W
H%/MeOH; (f) (1) BuLi/THF, HMPA; (2) n-BuI/THF; (g) H2, Rh[Ph3P]3Br/benzene; (h) Na/NH3 (liquid), ether; (i) H2, P2–Ni/EtOH; (j) PCC,
NaOAc/CH2Cl2.
2.2. In vitro and in vivo oxidation of deuterium labelled
metabolic probes
solution of the substrate was shaken with a small volume of
phosphate buffer (pH 7.2) containing M. sexta pheromone
gland tissue. Mass spectroscopic analysis of the products
unambiguously demonstrated the stereospecificity of the
alcohol oxidase in M. sexta. The determination of the
In vitro oxidation experiments were carried out using 0.1%
solutions of the chiral C16-alcohols 1–3 in hexane. The
Table 1. In vitro oxidations of labelled chiral alcohols by M. sexta pheromone gland alcohol oxidase
Chiral
alcohol
ee
(%)
Conversiona,b
(%)
Aldehyde dominant ionsc
Oxidase
stereospecificity
(m/z and rel. abundanceb in%)
[Mþ1]þ
[M217]þ
[Mþ57]þ
(R)-1
(S)-1
(R)-2
(S)-2
(R)-3
(S)-3
(R)-16
(S)-17
(R)-18
(S)-18
96
95
93
94
93
92
.99
.99
.99
.99
65^12
70^8
77^10
68^11
94^9
91^15
90^7
88^14
100^0
100^0
243 (100)
244 (100)
241 (100)
242 (100)
241 (100)
242 (100)
122 (100)
127 (100)
113 (100)
114 (100)
225 (10)
226 (18)
223 (70)
224 (74)
223 (66)
224 (83)
104 (54)
110 (60)
95 (51)
299 (33)
300 (10)
297 (31)
298 (12)
297 (47)
298 (11)
178 (19)
183 (21)
169 (28)
170 (25)
Re
Re
Re
Re
Re
Re
Re
Re
Re
Re
96 (44)
a
Conversion of chiral alcohols to corresponding aldehydes was determined by GLC.
Average value from GLC–MS analyses of three experiments.
GLC–MS (positive CI; 2-methylpropane) of products of the chiral alcohols oxidations.
b
c

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M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
Figure 1. In vitro enzymatic oxidation of (1R,11E)-[1,10,10-2H3]-hexadec-11-en-1-ol, (R)-2. (A): section of the chromatogram obtained by GLC–MS (CI)
analysis of the reaction mixture after 48 h of incubation. (B): section of CI-MS specþtra of the aldehyde 15 with dominant [M217]þand [Mþ1]þions. (C):
section of CI-MS spectra of the chiral labelled alcohol (R)-2 with dominant [M217] and [Mþ1]þions.
enzyme specificity can be illustrated by the oxidations of
(1R,11E)-[1,10,10-2H3]-hexadec-11-en-1-ol [(R)-2] and
[Mþ57]þ ion at m/z 297 Da). This indicates the loss of the
deuterium atom from the labelled precursor (R)-2.
Incubation of (S)-2 resulted in aldehyde 13 with the
pseudo-molecular ion [Mþ1]þ at m/z 242 Da, [M217]þ
ion at m/z 224 Da and finally [Mþ57]þ ion at m/z 298 Da.
The data are consistent with a loss of one hydrogen atom
from the alcohol (S)-2.
(1S,11E)-[1,10,10-2H3]-hexadec-11-en-1-ol
[(S)-2].
Alcohol (R)-2 was oxidised to aldehyde 15 in vitro (Fig. 1)
with 77% conversion, while alcohol (S)-2 was oxidised to
aldehyde 13 (Fig. 2) with 68% conversion. The identities of
the oxidation products followed from their CI-MS spectra.
Both aldehydes displayed dominant (100%) pseudo-
molecular ions [Mþ1]þ accompanied by intense
[M217]þ and [Mþ57]þ ions. Incubations of pheromone
gland tissue with (R)-2 resulted in aldehyde 15 (100%
[Mþ1]þ ion at m/z 241 Da, [M217]þ ion at m/z 223 Da and
In vitro experiments with the metabolic probes (R)-1/(S)-1
and (R)-3/(S)-3 gave identical results. Thus, the alcohol
oxidase of the M. sexta pheromone gland has been shown to
remove selectively the C1–HR hydrogen/deuterium atom of
Figure 2. In vitro enzymatic oxidation of (1S,11E)-[1,10,10-2H3]-hexadec-11-en-1-ol, (S)-2. (A): section of the chromatogram obtained by GLC–MS (CI)
analysis of the reaction mixture after 48 h of incubation. (B): section of CI-MS spectra of the aldehyde 13 with dominant [M217]þand [Mþ1]þions. (C):
section of CI-MS spectra of the chiral labelled alcohol (S)-2 with dominant [M217]þand [Mþ1]þ ions.

M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
9197
aromatic/heterocyclic alcohols (R)-16, (S)-17, (R)-18 and
(S)-18 gave the same results, demonstrating that this
stereospecificty is intrinsically linked to the oxidation
process.
4. Experimental
4.1. Chemical Synthesis
Scheme 4.
NMR spectra were determined in CDCl3 solutions on a
Varian UNITY-500 spectrometer operating at 499.5 MHz
for 1H and at 125.7 MHz for 13C NMR, respectively.
Chemical shifts are expressed in d (ppm) scale relative to
tetramethylsilane for 1H and relative to CDCl3 signal
(77.00 ppm) for 13C NMR, respectively. Coupling constants
are reported in Hz. IR spectra (wavenumbers in cm21) were
recorded on a Bruker Equinox 55 FT-IR spectrometer in
CCl4 solutions. GLC–GLC–MS (EI; 70 eV) analyses were
performed on a Finnigan GCQ equipped with J and W
Scientific (DB-5 capillary column 30 m£0.25 mm, 0.25 mm
film thickness; with helium (linear velocity 30 mL s21) as
carrier gas). High-resolution MS (EI) data were obtained
using a Micromass MasSpec 2. GLC–MS (CI: chemical
ionisation in the positive ion mode with 2-methylpropane)
analyses were performed using a Hewlett Packard HP6890
gas chromatograph equipped with a J and W Scientific DB-5
capillary column (30 m£0.25 mm, 0.25 mm film thickness;
with helium (linear velocity 30 mL s21) as carrier gas)
interfaced to a Micromass MasSpec 2. Flash chromato-
graphy: Merck 60 silica gel (0.040–0.063 mm). Alcohols
1–3 were chromatographed on Merck 60 silica gel
impregnated with 5% silver nitrate for complete removal
of boron contaminants (from Alpine-Boranesw).42
chiral deuterium labelled alcohols during oxidation to the
corresponding aldehydes (Table 1). The Re-stereospecificity
of the alcohol oxidase was also observed by in vivo
incubations with saturated alcohols (R)-1 and (S)-1 carried
out on M. sexta females. The GLC–MS analyses of the
hexane extracts of excised pheromone glands gave identical
results to the corresponding above described in vitro
experiments.
Fang et al. demonstrated16 that the oxidase of M. sexta
converts alcohols of different chain length (C14–C17). We
were able to show that the specificity of this oxidase is even
broader and it can oxidise other primary alcohols,
including aromatic, allylic or heterocyclic compounds.41
This earlier work prompted in vitro incubation experiments
with chiral deuterium labelled salicylalcohol (R)-16/(S)-17
and 2-thienylmethanol (R)-18/(S)-18 (Scheme 4; syntheses
in Refs. 10,41). Although the structures of these compounds
are different from natural pheromone precursors, namely
saturated and unsaturated C16-fatty alcohols, the enzyme
produced the corresponding aldehydes and displayed
Re-stereospecificity in all cases (Table 1).
It is interesting, that all examined alcohol oxidases from
yeasts,13 plants12 or leaf beetles10,11 remove selectively the
pro-HR enantiotropic hydrogen atoms of the prochiral
–CH2OH groups during the oxidation of substrates to
aldehydes. The Re-stereospecificity of these enzymes is not
dependent on their basic features (O2 or NADHþ oxidation
system, substrate specificity, etc.). The question of whether
Re-stereospecificity is a common attribute of all lepi-
dopteran alcohol oxidases, or is limited to M. sexta remains
to be established. The herein prepared metabolic probes can
be used to determine the stereoselectivity of alcohol
oxidases in other lepidopteran species1 (for example:
diamondback moth (Plutella xylostella; Plutellidae), Eastern
spruce budworm (Choristoneura fumiferana; Tortricidae),
corn earworm (Heliothis virescens; Noctuidae), and tomato
fruitworm (Heliothis zea; Noctuidae)] covering a broader
phylogenetic range. Investigations into this question are in
progress and the first results will be reported soon.
All reactions were run in oven-dried glassware under argon.
Tetrahydrofuran (THF), ether and benzene were distilled
from sodium benzophenone ketyl under argon. Dichloro-
methane was distilled from calcium hydride and stored over
molecular sieves. Hexamethylphosphoramide (HMPA) and
dimethylsulfoxide (DMSO) were dried over molecular
sieves. All other chemicals were used as purchased. NMR
spectra and high-resolution MS analyses of synthetic
compounds (.98% according to GLC) were fully consistent
with the proposed structures.
4.1.1. [1,1,10,10-2H4]-Decane-1,10-diol29 (5). Lithium
aluminium deuteride (LiAlD4; 7.39 g, 176 mmol) was
suspended in ether (300 mL). Dimethyl sebacate (4;
36.82 g, 160 mmol) in ether (50 mL) was added dropwise
over 1 h with stirring at 08C. After refluxing for 5 h, the
mixture was decomposed with ice-cold water (40 mL) and
25% sulphuric acid (100 mL). The organic phase was
separated, filtered through Celite and extracted with ether
(4£75 mL). The combined organic extracts were washed
with brine (2£100 mL) and dried (MgSO4). Removal of the
solvent in vacuo afforded crude diol which was recrystal-
lised from benzene to give pure product 5 (white crystalline
solid; 25.94 g, 91%), mp 72–738C (lit., 72.58C).29 Spectro-
scopic data were consistent with the literature.29
3. Conclusions
Using alkyne/borane chemistry we prepared a set of chiral
deuterium labelled metabolic probes 1–3, which were used
for both in vitro and in vivo determination of the
stereospecificity of M. sexta pheromone alcohol oxidase.
The data clearly established Re-stereospecificity for the
oxidation process. In vitro oxidations of non-natural chiral
4.1.2. 10-Bromo-[1,1,10,10-2H4]-decan-1-ol (6). Hydro-
bromic acid (46%; 16.4 mL, 140 mmol) was added to a

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M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
solution of diol 5 (25.0 g, 140 mmol) in benzene (300 mL),
and the mixture was heated under reflux for 8 h while
trapping the reaction water using a Dean–Stark water
separator. The mixture was washed with 20% NaOH
(150 mL), 10% hydrochloric acid (150 mL), water
(2£200 mL), brine (200 mL) and dried over MgSO4. The
solvent was removed in vacuo and the residue was purified
by flash chromatography (15% ethyl acetate in light
petroleum) to give 21.91 g (65%) of bromo alcohol 6 as a
70 mmol) over period of 30 min. The mixture was warmed to
08C over a period of 30 min, stirred at this temperature for
30 min, and kept at rt for 1 h. The mixture was cooled to
2208C and a solution of 1-iodobutane (12.9 g, 70 mmol) in
THF (20 mL) was added over a period 30 min. The red
reaction mixture was first stirred at 08C for 5 h, and then at rt
overnight. The reaction was quenched by saturated NH4Cl
solution (300 mL). Extraction with hexane:ether (2:1;
3£100 mL), drying (MgSO4) and removal of solvents
furnished 16.2 g of a red oil that was dissolved in methanol
(200 mL) and treated with Dowex 50W ion-exchange resin
(Hþ form, 20 g) for 24 h. The ion-exchange resin was filtered
off and the solvent removed in vacuo. Purification of the
residue by flash chromatography (15% ethyl acetate in light
petroleum)gave12.76 g (81%) ofalkynol 8 as a colourless oil.
1H NMR (CDCl3) d: 1.26 (3H, t, J¼7.2 Hz, CH3), 1.28–1.50
(18H, m, CH2[3–9,14,15]), 1.55 (2H, bt, J¼7.8 Hz, CH2[2]),
2.13(2H, t, J¼6.9 Hz, CH2[13]).13CNMR(CDCl3)d:13.6(q,
C16), 18.1 (m, JCD¼19.0 Hz, C10), 18.4 (t, C13), 21.9 (t, C15),
25.7(t),28.8(t),29.0(t),29.1(t), 29.4(t),29.4(t),29.5(t),31.3
(t), 32.59 (t), 62.4 (m, JCD¼21.5 Hz, C1), 80.1 (s, C11), 80.2 (s,
C12). FT-IR (CCl4, cm21): n (OH) 3638m; nAS (CD2) 2194w;
nS (CD2) 2096w; nS (C–O)þgS (CD2) 960s, 1164m; nAS
(CH3) 2958s. HRMS: for C16H26D4O calculated 242.2548;
found 242.2546.
1
pale yellow oil. H NMR (CDCl3) d: 1.28–1.46 (12H, m,
CH2[3–8]), 1.55 (2H, bt, J¼7.8 Hz, CH2[2]), 1.84 (2H, bt,
J¼7.3 Hz, CH2[9]). 13C NMR (CDCl3) d: 25.65 (t), 28.08
(t), 28.72 (t), 29.21 (m, JCD¼4.4 Hz, C2), 29.33 (t), 29.35
(t), 29.45 (t), 32.57 (t), 33.50 (m, JCD¼23.9 Hz, C10), 62.29
(t, JCD¼21.5 Hz, C1). FT-IR (CCl4, cm21): n (OH) 3638m;
n
AS (CD2) 2264w, 2196w; nS (CD2) 2161w, 2091w; nS (C–
O)þgS (CD2) 961s, 1165m; n (C–Br) 609w, 544m. HRMS:
for C10H17D749BrO calculated 240.1022; found 240.1020.
4.1.3. [1,1,10,10-2H4]-Dodec-11-yn-1-ol (7). 3,4-Dihydro-
2H-pyran (8.0 g, 95.0 mmol) was added dropwise to a
stirred solution of bromo alcohol 6 (21.7 g, 90.0 mmol) and
pyridinium p-toluenesulfonate (100 mg, 0.35 mmol) in
CH2Cl2 (250 mL) at 08C. After stirring for 8 h at 08C, the
mixture was diluted with ether (250 mL), washed with a
saturated solution of NaHCO3 (2£100 mL), brine (100 mL)
and dried over K2CO3. The solvents were evaporated and
the resulting THP-protected bromo alcohol (29.3 g) was
added dropwise to a stirred suspension of freshly prepared
lithium acetylide (120 mmol; the preparation is described in
lit.31) in liquid ammonia (400 mL) and anhydrous DMSO
(250 mL). After stirring for 4 h, the ammonia was allowed
to evaporate and brine (800 mL) was added. The mixture
was extracted with hexane (5£150 mL) and the combined
extracts dried over MgSO4. Evaporation of the solvents
furnished 24.2 g of a red oil that was dissolved in methanol
(300 mL) and treated with Dowex 50 W ion-exchange resin
(Hþ form, 10 g) for 24 h. The resin was removed by
filtration and the solvent removed in vacuo. Purification of
the residue by flash chromatography (15% ethyl acetate in
light petroleum) gave 12.20 g (73%) of alkynol 7 as a
4.1.5. [1,1,10,10-2H4]-Hexadecan-1-ol (9). A solution of
alkynol 8 (1.45 g, 6.0 mmol) in degassed benzene (5 mL)
was added to a solution of Rh(Ph3P)3Br (100 mg, 0.1 mmol)
in the same solvent (150 mL) and hydrogenated with
stirring (ultrasonic bath) at r.t. for 3 h. The solvent was
evaporated and the residue was purified by flash chroma-
tography (15% ethyl acetate in light petroleum) to give
1.35 g (91%) of alkanol 9 as a white crystalline solid (mp
1
47–48.58C). H NMR (CDCl3) d: 0.88 (3H, t, J¼7.0 Hz,
CH3), 1.21–1.38 (24H, m, CH2[3–9,11–15]), 1.55 (2H, bt,
J¼7.2 Hz, CH2[2]). 13C NMR (CDCl3) d: 14.1 (q, C16), 22.6
(t, C15), 25.6 (t), 28.8 (tt, JCD¼19.1 Hz, C10), 29.3 (t),
3£29.4 (t), 5£29.6 (t), 31.9 (t), 32.46 (t), 62.3 (tt,
JCD¼21.6 Hz, C1). FT-IR (CCl4, cm21): n (OH) 3639w;
nAS (CD2) 2179w; nS (CD2) 2096w; nS (C–O)þgS (CD2)
960w, 1166w; nAS (CH3) 2956m. HRMS: for C16H30D4O
calculated 246.2860; found 246.2858.
1
colourless oil. H NMR (CDCl3) d: 1.27–1.42 (12H, m,
CH2[3–8]), 1.51 (2H, m, CH2[9]), 1.55 (2H, bt, J¼7.8 Hz,
CH2[2]), 1.93 (1H, s, CH). 13C NMR (CDCl3) d: 17.8 (m,
JCD¼20.0 Hz, C10), 25.7 (t, C3), 28.3 (t), 28.7 (t), 29.0 (t),
2£29.4 (t), 29.5 (t), 32.6 (t), 62.3 (t, JCD¼22.0 Hz, C1), 68.0
(s, C12), 84.7 (s, C11). FT-IR (CCl4, cm21): n (OH) 3638m;
n (uCH) 3315s; n (CuC) 2120w; d (uCH) 633s; nAS
(CD2) 2198w; nS (CD2) 2096m; nS (C–O)þgS (CD2) 961m,
1168m. HRMS: for C12H18D4O calculated 186.1920; found
186.1922.
4.1.6. (11E)-[1,1,10,10-2H4]-Hexadec-11-en-1-ol (10). 3,4-
Dihydro-2H-pyran (0.59 g, 7.0 mmol) was added dropwise
to a stirred solution of alcohol 8 (1.45 g, 6.0 mmol) and
pyridinium p-toluenesulfonate (20 mg, 70 mmol) in CH2Cl2
(50 mL) at 08C. After stirring for 8 h at the same
temperature, the mixture was diluted with ether (100 mL),
washed with a saturated solution of NaHCO3 (2£50 mL),
brine (50 mL) and dried over K2CO3. The solvents were
evaporated, the O-protected alcohol 8 (2.0 g) was diluted
with ether (80 mL), and added dropwise to a stirred freshly
prepared solution of sodium (0.46 g, 20.0 mmol) in liquid
ammonia (200 mL). After stirring for 8 h, ammonia was
allowed to evaporate and the residue treated with brine
(200 mL). The mixture was extracted with ether (4£75 mL)
and the combined extracts dried over MgSO4. Evaporation
of the solvents furnished 1.9 g of a yellow oil that was
dissolved in methanol (50 mL) and treated with Dowex
50 W ion-exchange resin (Hþ form, 1 g) for 24 h. The ion-
exchange resin was filtered off and the solvent removed in
4.1.4. [1,1,10,10-2H4]-Hexadec-11-yn-1-ol (8). 3,4-Dihydro-
2H-pyran (5.89 g, 70 mmol) was added dropwise to a stirred
solution of alcohol 7 (12.1 g, 65 mmol) and pyridinium
p-toluenesulfonate (100 mg, 0.35 mmol) in CH2Cl2 (200 mL)
at 08C. After stirring for 6 h at the same temperature, the
mixture was diluted with ether (250 mL), washed with a
saturated solution of NaHCO3 (2£100 mL), brine (100 mL)
and dried over K2CO3. The solvents were evaporated and the
THP-protected alkynol (17.6 g) was dissolved in THF
(60 mL) and HMPA (20 mL). The alkyne was deprotonated
at 2508C with n-butyllithium in hexanes (2.5 M; 28 mL,

M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
9199
vacuo. Purification of the residue by flash chromatography
(15% ethyl acetate in light petroleum) gave 1.25 g (85%) of
(E)-alkenol 10 (98% isomeric purity, GLC) as a colourless
oil. 1H NMR (CDCl3) d: 0.90 (3H, t, J¼7.2 Hz, CH3), 1.24–
1.36 (18H, m, CH2[3–9,14,15]), 1.55 (2H, bt, J¼7.3 Hz,
CH2[2]), 1.95–1.99 (2H, m, CH2[13]), 5.34–5.42 (2H, m,
CHvCH). 13C NMR (CDCl3) d: 13.9 (q, C16), 22.2 (t, C15),
25.7 (t), 26.8 (tt, JCD¼19.2 Hz, C10), 29.4 (t), 4£29.5 (t),
29.6 (t), 31.9 (t, C2), 32.3 (t), 32.6 (t), 62.34 (tt,
JCD¼21.4 Hz, C1), 130.3 (d, C11), 130.4 (d, C12). FT-IR
(CCl4, cm21): n (OH) 3638m; nAS (vCH) 3016m; n (CvC)
1665w; g (vCH) 970s; nAS (CD2) 2187w; nS (CD2) 2095w;
nS (C–O)þgS (CD2) 960s, 1167w; nAS (CH3) 2957s.
HRMS: for C16H28D4O calculated 244.2704; found
244.2708.
(3H, t, J¼7.0 Hz, CH3), 1.22–1.38 (18H, m, CH2[3–
9,14,15]), 1.95–2.00 (2H, m, CH2[13]), 2.40 (2H, bt,
J¼7.2 Hz, CH2[2]), 5.36–5.43 (2H, m, CHvCH). 13C
NMR (CDCl3) d: 13.9 (q, C16), 22.1 (t, C15), 22.5 (t), 26.7
(tt, JCD¼19.1 Hz, C10), 2£29.4 (t), 29.5 (t), 3£29.6 (t), 29.7
(t), 32.1 (t), 43.8 (tt, JCD¼21.2 Hz, C2), 130.2 (d, C11), 130.3
(d, C12), 202.7 (t, JCD¼25.9 Hz, C1). FT-IR (CCl4, cm21): n
(CvO) 1719s; b (OCD) 1094w; nAS (vCH) 3016w; n
(CvC) 1667w; g (vCH) 971m; nAS (CD2) 2187w; nS
(CD2) 2103w; n (C–D) 2063w; nAS (CH3) 2957s. HRMS:
for C16H27D3O calculated 241.2485; found 241.2485.
4.1.10. (11Z)-[1,10,10-2H3]-Hexadec-11-enal (14). Alde-
hyde 14 (821 mg, 68%; colourless oil) was prepared from
(Z)-alkenol 11 (1.22 g, 5.0 mmol) using PCC (1.51 g,
1
7.0 mmol) as described for 12. H NMR (CDCl3) d: 0.90
4.1.7. (11Z)-[1,1,10,10-2H4]-Hexadec-11-en-1-ol (11). 1,2-
Diaminoethane (40 mL) and alkynol 8 (1.45 g, 6.0 mmol)
were added to a suspension of P2–Ni (prepared from 50 mg
of nickel(II) acetate and 200 mL of 1 M NaBH4 in EtOH) in
ethanol 30 mL and hydrogenated with stirring at rt. The
progress of the hydrogenation was monitored by GLC–MS.
The usual workup and flash chromatography (15% ethyl
acetate in light petroleum) of the crude product afforded
1.37 g (94%) of (Z)-alkenol 11 (98.5% isomeric purity,
(3H, t, J¼7.1 Hz, CH3), 1.22–1.36 (16H, m, CH2[3–9,14]),
1.62 (2H, m, CH2[15]), 2.41 (2H, bt, J¼7.3 Hz, CH2[2]),
2.0–2.04 (2H, m, CH2[13]), 5.33 (1H, bd, J¼11.2 Hz,
CHvCH), 5.36 (1H, dt, J¼2£5.8, 11.2 Hz, CHvCH). 13C
NMR (CDCl3) d: 14.00 (q, C16), 22.0 (t), 22.3 (t), 26.4 (tt,
JCD¼19.0 Hz, C10), 26.9 (t), 2£29.2 (t), 29.3 (t), 2£29.4 (t),
29.5 (t), 32.0 (t, C2), 43.7 (tt, JCD¼3.7 Hz, C2), 129.7 (d,
C11), 129.9 (d, C12), 202.6 (t, JCD¼25.9 Hz, C1). FT-IR
(CCl4, cm21): n (CvO) 1719s; b (OCD) 1094w; nAS
(vCH) 3006w; n (CvC) 1653w; b (vCH) 1404w; g
(vCH) 708w; nAS (CD2) 2190w; nS (CD2) 2102w; n (C–D)
2064w; nAS (CH3) 2957s. HRMS: for C16H27D3O calcu-
lated 241.2485; found 241.2485.
1
GLC) as a colourless oil. H NMR (CDCl3) d: 0.90 (3H, t,
J¼7.1 Hz, CH3), 1.22–1.38 (18H, m, CH2[3–9,14,15]),
1.55 (2H, bt, J¼7.9 Hz, CH2[2]), 1.97–2.01 (2H, m,
CH2[13]), 5.32–5.38 (2H, m, CHvCH). 13C NMR
(CDCl3) d: 14.00 (q, C16), 22.3 (t, C15), 25.7 (t), 26.5 (tt,
JCD¼19.2 Hz, C10), 26.9 (t), 29.2 (t), 29.4 (t), 29.5 (t),
3£29.6 (t), 32.0 (t, C2), 32.6 (t), 62.35 (tt, JCD¼21.5 Hz,
C1), 129.5 (d, C11), 129.9 (d, C12). FT-IR (CCl4, cm21): n
(OH) 3638w; nAS (vCH) 3005m; n (CvC) 1651w; b
(vCH) 1401w; g (vCH) 707w; nAS (CD2) 2193w; nS
(CD2) 2097w; nS (C–O)þgS (CD2) 961m, 1162m; nAS
(CH3) 2957s. HRMS: for C16H28D4O calculated 244.2704;
found 244.2702.
4.1.11. (1R)-[1,10,10-2H3]-Hexadecan-1-ol [(R)-1]. (S)-
Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
3.73 mmol) was added to a solution of aldehyde 12
(300 mg, 1.23 mmol) in THF (10 mL). The mixture was
stirred for 72 h at rt and then cooled in an ice-bath.
Acetaldehyde (300 mL) was added to the mixture, which
was then stirred at rt for 1 h to decompose excess borane and
finally evaporated in vacuo. The residue was dissolved in
ether (50 mL), ethanolamine (300 mL) was added at rt and
stirring was continued for 1 h. The insoluble precipitate was
filtered off, and the organic layer was washed with 1 M HCl
(2£25 mL), a saturated solution of NaHCO3 (2£25 mL),
brine (2£25 mL) and dried over MgSO4. Evaporation of the
solvents gave an oily residue, which was extracted with
hexane. The extract was evaporated and the residue purified
by flash chromatography (AgNO3 impregnated42 silica gel;
15% ethyl acetate in light petroleum) to give 251 mg (83%)
of the target alcohol (R)-1 as a white crystalline solid (mp
48–498C). 1H NMR (CDCl3) d: 0.88 (3H, t, J¼7.0 Hz,
CH3), 1.22–1.38 (24H, m, CH2[3–9,11–15]), 1.56 (2H, bq,
J¼7.1 Hz, CH2[2]), 3.62 tt (1H, J¼2£1.5, 2£6.6 Hz, CHD).
13C NMR (CDCl3) d: 14.1 (q, C16), 22.7 (t, C15), 25.7 (t),
28.8 (tt, JCD¼18.8 Hz, C10), 2£29.4 (t), 2£29.5 (t), 4£29.6
(t), 29.7 (t), 31.9 (t), 32.7 (t), 62.7 (tt, JCD¼21.7 Hz, C1). FT-
IR (CCl4, cm21): n (OH) 3639w; n (C–OH) 1063w; nAS
(CD2) 2176w; n (C–D) 2148w; nS (CD2) 2101w; nAS (CH3)
2956m. HRMS: for C16H31D3O calculated 245.2798; found
245.2795.
4.1.8. [1,10,10-2H3]-Hexadecanal (12). Alkanol 9 (1.23 g,
5.0 mmol) was injected into a stirred suspension of
pyridinium chlorochromate (PCC; 1.51 g, 7.0 mmol) and
anhydrous sodium acetate (200 mg) in dichloromethane
(10 mL). The mixture was stirred at rt for 90 min, poured
into ether (50 mL) and filtered through a combined layer of
neutral alumina/charcoal/Celite. Evaporation of solvents
and flash chromatography (3% diethyl ether in light
petroleum) afforded 937 mg (77%) of aldehyde 12 as a
1
colourless oil. H NMR (CDCl3) d: 0.88 (3H, t, J¼7.0 Hz,
CH3), 1.22–1.35 (24H, m, CH2[3–9,11–15]), 2.41 (2H, t,
J¼7.4 Hz, CH2[2]), 1.63 (2H, m, CH2[3]). 13C NMR
(CDCl3) d: 14.10 (q, C16), 22.1 (t), 22.7 (t), 28.8 (m,
JCD¼14.2 Hz, C10), 29.2 (t), 3£29.4 (t), 2£29.5 (t), 3£29.6
(t), 31.9 (t), 43.7 (tt, JCD¼3.7 Hz, C2), 202.7 (td,
JCD¼26.1 Hz, C1). FT-IR (CCl4, cm21): n (CvO) 1719s;
b (OCD) 1094w; nAS (CD2) 2178w; nS (CD2) 2100w; n (C–
D) 2063w; nAS (CH3) 2956s. HRMS: for C16H29D3O
calculated 243.2641; found 243.2640.
4.1.9. (11E)-[1,10,10-2H3]-Hexadec-11-enal (13). Alde-
hyde 13 (845 mg, 70%; colourless oil) was prepared from
(E)-alkenol 10 (1.22 g, 5.0 mmol) using PCC (1.51 g,
4.1.12. (1S)-[1,10,10-2H3]-Hexadecan-1-ol [(S)-1]. Alcohol
(S)-1 (233 mg, 77%; white crystalline solid, mp 47.5–498C)
was prepared from aldehyde 12 (300 mg, 1.23 mmol) and
(R)-Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
1
7.0 mmol) as described for 12. H NMR (CDCl3) d: 0.89

9200
M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
3.73 mmol) following the procedure for (R)-1. NMR and
FT-IR data are identical with (R)-1. HRMS: for C16H31D3O
calculated 245.2798; found 245.2797.
brine (2£5 mL) and dried over MgSO4. Evaporation of the
solvents gave an oily residue, which was extracted with
hexane. The hexane extracts were combined, evaporated
and the oily residue purified by flash chromatography (5%
ethyl acetate in light petroleum) to give the ester as a white
wax. Yields of camphanates: 90–95%.
4.1.13. (1R,11E)-[1,10,10-2H3]-Hexadec-11-en-1-ol [(R)-
2]. Alcohol (R)-2 (240 mg, 80%; colourless oil) was
prepared from aldehyde 13 (300 mg, 1.24 mmol) and (S)-
Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
3.73 mmol) following the procedure for (R)-1. d: 0.89
(3H, t, J¼7.1 Hz, CH3), 1.23–1.38 (18H, m, CH2[3–
9,14,15]), 1.55 (2H, m, CH2[2]), 1.94–1.99 (2H, m,
CH2[13]), 5.37–5.40 (2H, m, CHvCH), 3.62 tt (1H,
J¼2£1.5, 2£6.6 Hz, CHD). 13C NMR (CDCl3) d: 14.0 (q,
C16), 22.2 (t, C15), 25.7 (t), 26.9 (tt, JCD¼19.3 Hz, C10), 29.1
(t), 29.4 (t), 3£29.5 (t), 29.6 (t), 31.8 (t, C2), 32.3 (t), 32.7 (t),
62.7 (tt, JCD¼21.5 Hz, C1), 130.3 (d, C11), 130.4 (d, C12).
FT-IR (CCl4, cm21): n (OH) 3638w; n (C–OH) 1067m; nAS
(vCH) 3016w; n (CvC) 1666w; g (vCH) 971s; nAS (CD2)
2182w; n (C–D) 2148w; nS (CD2) 2099w; nAS (CH3) 2957s.
HRMS: for C16H19D3O calculated 243.2641; found
243.2642.
4.2. Insects
M. sexta pupae were obtained from the Institute of Organic
Chemistry and Biochemistry (Prague, Czech Republic)
laboratory colony. They were sexed and the female pupae
were held in cages at L/D 16:8 photoperiod regime (24–
258C, 40–50% relative humidity) until adults emerged.
Virgin females 1 day old were used for experiments. To
avoid interference by naturally produced pheromone, in
vivo experiments and gland exenterations for in vitro
experiments were performed during the photophase, when
the female pheromone glands do not produce the pheromone
C16-aldehydes.
4.3. In vitro oxidations
4.1.14. (1S,11E)-[1,10,10-2H3]-Hexadec-11-en-1-ol [(S)-
2]. Alcohol (S)-2 (217 mg, 72%; colourless oil) was
prepared from aldehyde 13 (300 mg, 1.24 mmol) and (R)-
Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
3.73 mmol) following procedure for (R)-1. NMR and FT-
IR data are identical with (R)-2. HRMS: for C16H19D3O
calculated 243.2641; found 243.2642.
The stereochemistry of the alcohol oxidase from M. sexta
was studied with the intact epidermis of the abdominal tips
of females remaining after removing the muscles and all
other internal tissues. The epidermis from a single female
was used for each experiment. In vitro assays were
conducted by incubating the tissue with a deuterated alcohol
dissolved in hexane (50 mL of 1 mg mL21 stock solution)
and phosphate buffer (5 mL, pH 7.2) in a 2 mL standard vial.
After 48 h of shaking (promote the absorbtion of air) at 258C
the tissue was removed from the vial and solution was
analysed by GLC–MS (CI). The incubation experiment was
repeated three times for each tested compound.
4.1.15. (1R,11Z)-[1,10,10-2H3]-Hexadec-11-en-1-ol [(R)-
3]. Alcohol (R)-3 (220 mg, 73%; colourless oil) was
prepared from aldehyde 14 (300 mg, 1.24 mmol) and (S)-
Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
1
3.73 mmol) following the procedure for (R)-1. H NMR
(CDCl3) d: 0.90 (3H, t, J¼7.1 Hz, CH3), 1.22–1.39 (18H,
m, CH2[3–9,14,15]), 1.56 (2H, bq, J¼7.4 Hz, CH2[2]),
2.00–2.04 (2H, m, CH2[13]), 5.32–5.38 (2H, m, CHvCH),
3.62 (1H, tt, J¼2£1.5, 2£6.6 Hz, CHD). 13C NMR (CDCl3)
d: 14.00 (q, C16), 22.3 (t, C15), 25.7 (t), 26.5 (tt,
JCD¼18.7 Hz, C10), 26.9 (t), 29.2 (t), 29.4 (t), 2£29.5 (t),
2£29.6 (t), 32.0 (t, C2), 32.7 (t), 62.8 (tt, JCD¼21.7 Hz, C1),
129.8 (d, C11), 129.9 (d, C12). FT-IR (CCl4, cm21): n (OH)
3638w; n (C–OH) 1065m; nAS (vCH) 3005m; n (CvC)
1651w; b (vCH) 1413m; g (vCH) 700w; nAS (CD2)
2187w; n (C–D) 2150w; nS (CD2) 2102w; nAS (CH3) 2957s.
HRMS: for C16H19D3O calculated 243.2641; found
243.2640.
4.4. In vivo oxidations
M. sexta females were anaesthetised with carbon dioxide
and their abdomen was gently squeezed by wooden alligator
clips until the abdominal tip with pheromone gland
extruded.22 Females were introduced into an anaesthetisa-
tion chamber and their pheromone gland was treated twice
at interval of 20 min with the solution of 20 mg of labelled
metabolic probe (R)- or (S)-1 in 1 mL of DMSO.22 After the
solution of metabolic probe was absorbed into the gland
(45 min), clips were removed and the moths were returned
to their cage. After 6 h, the pheromone glands were excised
and extracted with 50 mL of hexane and the extracts were
consecutively analysed by by GLC–MS (CI).
4.1.16. (1S,11Z)-[1,10,10-2H3]-Hexadec-11-en-1-ol [(S)-
3]. Alcohol (S)-3 (237 mg, 79%; colourless oil) was
prepared from aldehyde 14 (300 mg, 1.24 mmol) and (R)-
Alpine-Boranew (0.5 M solution in THF; 7.5 mL,
3.73 mmol) following the procedure for (R)-1. NMR and
FT-IR data are identical with (R)-3. HRMS: for C16H19D3O
calculated 243.2641; found 243.2642.
Acknowledgements
M. H. thanks the Alexander von Humboldt Stiftung (Bonn)
for a fellowship. The project was also supported in part by
research project Z4-055-905 from Academy of Sciences of
the Czech Republic. Special thanks are addressed to Dr Neil
J. Oldham (mass spectrometry, MPICE Jena), Dr David
4.1.17. (1S)-Camphanates of alcohols 1–3. Alcohol
(30 mg, ca. 125 mmol), (1S)-camphanic acid chloride
(28 mg, 140 mmol) and anhydrous pyridine (100 mL) in
dichloromethane (400 mL) were stirred at rt overnight. The
reaction mixture was diluted with ether (10 mL), washed
with 0.5 M HCl (2£5 mL), saturated NaHCO3 (2£5 mL),
ˇ
Saman (NMR spectrometry, IOCB Prague), Dr Pavel
Fiedler (FT-IR spectrometry, IOCB Prague) and Dr Robert
E. Doolittle (Gainesville, Florida) for proofreading of the
manuscript.

M. Hoskovec et al. / Tetrahedron 58 (2002) 9193–9201
9201
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23. Fang, N. B.; Teal, P. E. A.; Tumlinson, J. H. Arch. Insect
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