Total synthesis, proof of absolute configuration, and biosynthetic origin of stylopsal, the first isolated sex pheromone of strepsiptera

Article abstract of DOI:10.1002/chem.201204196

The asymmetric total synthesis of the diastereomers of stylopsal establishes the absolute configuration of the first reported sex pheromone of the twisted-wing parasite Stylops muelleri as (3R,5R,9R)-trimethyldodecanal. The key steps for the diastereo- and enantiodivergent introduction of the methyl groups are two different types of asymmetric conjugate addition reactions of organocopper reagents to α,β-unsaturated esters, whereas the dodecanal skeleton is assembled by Wittig reactions. The structure of the natural product was confirmed by chiral gas chromatography (GC) techniques, GC/MS and GC/electroantennography (EAD) as well as field tests. An investigation into the biosynthesis of the pheromone revealed that it is likely to be produced by decarboxylation of a 4,6,10-trimethyltridecanoic acid derivative, which was found in substantial amounts in the fat body of the female, but not in the host bee Andrena vaga. This triple-branched fatty acid precursor thus seems to be biosynthesized de novo through a polyketide pathway with two consecutive propionate-propionate-acetate assemblies to form the complete skeleton. The simplified, motionless and fully host-dependent female exploits a remarkable strategy to maximize its reproductive success by employing a relatively complex and potent sex pheromone. Love at first.i??-smell: An asymmetric total synthesis, gas chromatographic analyses, and field tests confirm the structure of the first isolated sex pheromone of a twisted-wing parasite Stylops muelleri (see figure). Its biosynthetic origin and complexity demonstrate the considerable efforts undertaken by the motionless and fully host-dependant female to attract the very short-lived free-living male, thus securing success in the perpetuation of the species. Copyright

Full text of DOI:10.1002/chem.201204196

FULL PAPER
DOI: 10.1002/chem.201204196
Total Synthesis, Proof of Absolute Configuration, and Biosynthetic Origin of
Stylopsal, the First Isolated Sex Pheromone of Strepsiptera
[a]
[a]
Roman Lagoutte, Petr Sebesta, Pavel Jirosˇ,[a] Blanka Kalinovꢀ,[a] Anna Jirosˇovꢀ,[a]
ˇ
ˇ
Jakub Straka,[b] Katerˇina Cernꢀ, Jan Sobotnꢁk,
Josef Cvacˇka,[a] and Ullrich Jahn*[a]
[b]
[a, c]
ˇ
Dedicated to Professor Dr. Ekkehard Winterfeldt on the occasion of his 80th birthday
Abstract: The asymmetric total synthe-
natural product was confirmed by
chiral gas chromatography (GC) tech-
niques, GC/MS and GC/electroanten-
nography (EAD) as well as field tests.
An investigation into the biosynthesis
of the pheromone revealed that it is
likely to be produced by decarboxyla-
acid derivative, which was found in
sis of the diastereomers of stylopsal es-
tablishes the absolute configuration of
the first reported sex pheromone of the
twisted-wing parasite Stylops muelleri
as (3R,5R,9R)-trimethyldodecanal. The
key steps for the diastereo- and
substantial amounts in the fat body of
the female, but not in the host bee An-
drena vaga. This triple-branched fatty
acid precursor thus seems to be biosyn-
thesized de novo through a polyketide
pathway with two consecutive propio-
enantio
divergent introduction of the
tion of
a
4,6,10-trimethyltridecanoic
nate-propioACTHUNGTERNNUnG ate-acetate assemblies to
methyl groups are two different types
of asymmetric conjugate addition reac-
tions of organocopper reagents to
a,b-unsaturated esters, whereas the do-
decanal skeleton is assembled by
Wittig reactions. The structure of the
form the complete skeleton. The sim-
plified, motionless and fully host-de-
pendent female exploits a remarkable
strategy to maximize its reproductive
success by employing a relatively com-
plex and potent sex pheromone.
Keywords: asymmetric synthesis ·
configuration determination · pher-
omones · total synthesis · Wittig re-
actions
Introduction
the male is a winged free-living insect with a very short life-
span of only a few hours.[3] The order Strepsiptera includes
more than 400 species, of which almost 100 belong to the
most diversified genus Stylops with all species being para-
sites of the bee genus Andrena.
The twisted-wing parasites (Strepsiptera) are a fascinating
insect order, with all species being endoparasites of various
insect taxa including silverfishes, bush-crickets, bees, wasps,
and others.[1] They display an extreme sexual dimorphism:[2]
the wingless larviform female is embedded in the hostꢀs ab-
domen with the cephalothorax protruding from it, whereas
Twisted-wing parasites belong to one of the least known
groups of insects; males are very difficult to collect owing to
their short lifespan, and the lack of helpful characteristics
on females makes their identification very difficult. Mate-lo-
cation is one of many unknowns; a very efficient signal must
operate because of the short lifespan of males, which possess
relatively large antennae.[4] Once hatched, males gather at
the hostꢀs nesting sites to find a mate, and efficiently recog-
nize host individuals with female parasites from a distance.[5]
We recently reported the isolation and partial configura-
tional assignment of the sex pheromone of Stylops muel-
leri.[6,7] These investigations revealed the presence of a
single-component pheromone, called stylopsal, having a con-
stitution consistent with (9R)-3,5-syn-3,5,9-trimethyldodeca-
nal (1; Figure 1). Because females of this insect produce 50–
150 ng of pheromone per individuum, the amount of natural
material available did not allow
+
[a] Dr. R. Lagoutte,+ Ing. P. Sebesta, Dr. P. Jiros, Dr. B. Kalinovꢁ,
ˇ
ˇ
ˇ
ˇ
ˇ
Dr. A. Jirosovꢁ, Dr. J. Sobotnꢂk, Dr. J. Cvacka, Dr. U. Jahn
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo nꢁmestꢂ 2, 166 10 Prague 6 (Czech Republic)
E-mail: jahn@uochb.cas.cz
ˇ
ˇ
[b] Dr. J. Straka, K. Cernꢁ
Department of Zoology, Faculty of Science
Charles University in Prague
Vinicnꢁ 7, 128 44 Prague 2 (Czech Republic)
ˇ
ˇ
[c] Dr. J. Sobotnꢂk
Czech University of Life Sciences
Faculty of Forestry and Wood Sciences
Kamyckꢁ 129, 165 21 Prague 6 (Czech Republic)
´
[+] These authors contributed equally.
further characterization. There-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204196. It contains experi-
fore, its relative and absolute
configuration had to be derived
from synthetic standards.
The proposed constitution of
1
mental procedures, analytical data, copies of H and 13C NMR spectra
of 1, 3, 6, 7, 10–14, and 20–22, comparison of the FAME content of
the fat bodies of female Stylops muelleri and the host bee Andrena
vaga, and chiral gas chromatograms of all diastereomers of 1.
Figure 1. Constitution and con-
figuration of stylopsal as re-
stylopsal appeared surprising ported in ref. [6].
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considering the three methyl branches. Although phero-
mones promoting sexual attraction in both sexes of various
insects[8] display a relatively wide structural diversity, many
of them are derived from palmitoyl-CoA formed in the fatty
acid cycle and subsequently biosynthetically modified.[8,9,10]
Terpenoid-derived pheromones aside, sex pheromones with
methyl branching often contain one or two branches and are
biosynthesized by polyketide pathways incorporating
This approach could not subsequently be applied in a
fully asymmetric total synthesis of the natural product and
its diastereomers because efficient asymmetric Baeyer–Vil-
liger oxidation protocols did not exist at the beginning of
the work, and biocatalytic methods[13] were not available to
us. Kinetic resolution has been described[14] but would re-
quire a cumbersome synthetic sequence. After completion
of the syntheses, Feng reported a scandium-catalyzed asym-
metric Baeyer–Villiger oxidation, which could potentially be
used as an alternative.[15] In parallel to our synthetic effort,
propioACHTUNGTRENNUNG
nate, methylmalonate, or isovalerate units.[8,10] Excep-
tions exist, however; a few polyketide-derived insect female-
released sex pheromones contain up to six methyl
branches.[10,11]
the Tolasch group developed
a different asymmetric
approach to 1.[7]
To obtain as much information as possible on the consti-
tution and configuration of stylopsal, while minimizing the
synthetic effort, a partially racemic diastereodivergent syn-
thesis was previously pursued (Scheme 1).[6] Because a late
A different, fully asymmetric approach to both phero-
mone candidates had therefore to be devised. Here, the
enantioselective total synthesis of stylopsal is reported, es-
tablishing its absolute configuration unequivocally as
(3R,5R,9R)-3,5,9-trimethyldodecanal. The absolute configu-
ration (1) is supported by a variety of analytical techniques
and field tests. The likely biosynthetic origin of the phero-
mone is discussed on the basis of fatty acid components
found in the fat body of the parasite.
Results and Discussion
Asymmetric synthesis of stylopsal: The retrosynthetic dis-
connection of the pheromone candidate had to account for
the diastereodivergent generation of the C3 and C5 stereo-
centers based on a fixed 9R configuration in product 1
(Scheme 2). This is best achieved by sequential introduction
of those chiral centers. The C3 stereocenter is therefore in-
troduced by catalytic asymmetric conjugate addition to
a,b-unsaturated ester 6 using a protocol in which the cata-
Scheme 1. Stereodivergent partially racemic synthesis of synthetic stand-
ards 1 of stylopsal[6] (only one absolute configuration at C3 and C5 for
compounds 1 and 5 is shown for clarity).
stage unequivocal determination of the configuration at the
remote C9 center was felt impossible, the configuration at
C9 was fixed from the beginning by an enantioselective con-
jugate addition to methyl crotonate (2).[12] Racemic alde-
hyde 3,5-syn-5 was obtained in four steps from commercially
available cyclohexanol 4, whereas 3,5-syn/anti-5 was ob-
tained by partial epimerization of 3,5-syn-5. A Wittig cou-
pling of fragments 5 and 3 followed by catalytic hydrogena-
tion and functional group manipulations furnished four dif-
ferent diastereomers 1. Chiral GC/MS, GC/EAD and bioas-
says revealed that stylopsal (1) has a configuration consis-
tent with (9R)-3,5-syn-trimethyldodecanal. The mixture of
the two potential diastereomeric candidates (3R,5R,9R)-1
and (3S,5S,9R)-1 was separated on very small scale after de-
rivatization as mandelate esters, showing that only one of
the candidates was the actual pheromone; the absolute con-
figuration of the pheromone at C3 and C5 could, however,
not be established.
Scheme 2. Retrosynthesis for the pheromone candidates (for all inter-
mediates, the numbering in parentheses reflects that of the full backbone
of the pheromone).
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Characterizing Stylopsal
FULL PAPER
lyst was expected to determine the stereochemical outcome
as completely as possible with minimal impact of the
residing stereocenters at C5 and C9.[16]
Precursors 6 could be synthesized by a standard Wittig re-
action with the aldehydes derived from (R,R)- or (R,S)-7.
This compound will be approached by another Wittig cou-
pling of enantiomerically enriched phosphonium salt (R)-3
with enantiomerically pure aldehydes (R)-8 or (S)-8, which
are readily available from the corresponding chiral pool-de-
rived 2-methylbutanediols.
cause the only difference was the oxidation conditions, these
were reinvestigated. Indeed, it is known that the acetic acid
by-product in the Dess–Martin oxidation may promote eno-
lization of the aldehyde, and thereby epimerization.[19] At-
tempted buffering of the Dess–Martin oxidation with an or-
ganic base, such as pyridine,[20] did not suppress epimeriza-
tion (63% 11, 3R,7R/3S,7R=86:14). However, much less ep-
imerization occurred on addition of solid NaHCO3 (71% 11,
3R,7R/3S,7R=92:8).
Looking out for alternatives, tetrapropylammonium per-
ruthenate (TPAP)-mediated oxidation with its essentially
neutral conditions and very short reaction times seemed to
be a good choice. Moreover, Ley et al. reported a tandem
The synthesis of the diastereomeric C3–C12 fragments
(3R,7R)- and (3S,7R)-7 started from commercially available
(R)- and (S)-butanediols 9, but proved to be slightly more
challenging than expected (Scheme 3). The regioselective si-
TPAP oxidation/Takai olefination with 8 as a nonACTHNUTRGNEiUNG solated in-
termediate,[17d] and a tandem
TPAP oxidation/Wittig olefina-
tion protocol with different sub-
strates, including intermediate
a-chiral aldehydes for which no
epimerization was observed.[21]
This protocol led to higher
yields (90%), little epimeriza-
tion (3R,7R/3S,7R=90:10) and
essentially unchanged Z selec-
tivity (E/Z=10:90). Because of
the favorable diastereomeric
outcome, the better isolated
yields obtained, as well as the
Scheme 3. Preparation of the C3–C12 fragment (for all compounds, only one absolute configuration at C3 is
shown; the numbering in parentheses reflects that of the full backbone of the pheromone).
practical
convenience,
the
lylation of
9
by using tert-butyldiphenylsilyl chloride
[5.4.0]undec-
tandem TPAP oxidation/Wittig olefination protocol was ap-
plied on larger scale toward the target products.
(TBDPSCl) in the presence of 1,8-diazabicycloACHTUNGTRENNUNG
7-ene (DBU) as a base has been reported several times,[17]
with regioselectivities ranging from 16:1[17a] to 3:1.[17b] In our
hands, a result at the lower end of the selectivity scale was
initially observed. Unsatisfied with this outcome, a rapid re-
screening of the conditions improved the regioselectivity to
6.6:1 in favor of the desired silyl ethers 10. The key proved
to be a very slow dropwise addition of TBDPSCl in reac-
tions on 5 mmol scale, presumably to allow for better heat
transfer.
The catalytic hydrogenation of 11 also required an optimi-
zation effort. With palladium on charcoal, further epimeriza-
tion at C3 took place, as observed by an increase of a
second set of signals in the 13C NMR spectra, but the dia-
stereomeric ratio could not be quantified at this stage. Inter-
estingly, no epimerization had been observed during the
semiracemic synthesis.[6] Epimerization during catalytic hy-
drogenation of substrates with allylic chiral centers has been
studied and was proposed to occur through partial double
bond migration and subsequent hydrogenation of the result-
ing trisubstituted double bond, leading to overall epimeriza-
tion.[22] Based on Cramꢀs early report that Raney nickel
does not mediate this epimerization,[22a] the hydrogenation
of 11 proceeded very smoothly using these conditions, af-
fording silyl ethers 7 in high yields and with virtually no epi-
merization, as revealed by 13C NMR spectroscopic analysis.
Tetrabutylammonium fluoride (TBAF)-mediated desilyla-
tion afforded alcohols 12 in high yields (Scheme 4). These
were submitted to another tandem TPAP oxidation/Wittig
olefination reaction according to Leyꢀs procedure[19] to
afford a,b-unsaturated esters 6 in high yield and high E se-
lectivity (E/Zꢀ99:1). For the completion of the full C15
skeleton, the introduction of the methyl group at C3 re-
mained. Because this also had to be accomplished in a dia-
stereodivergent manner, a reagent-controlled approach was
mandatory, and an adaptation of the catalytic conjugate ad-
Aldehydes (R)- and (S)-8 have also been prepared numer-
ous times and under various oxidation conditions,[17a,b,d,e,18]
but have been described as unstable.[17d] The Dess–Martin
oxidation was initially chosen because of its mild conditions,
and it was decided to proceed with the Wittig olefination
immediately after work-up of the reaction mixture. The oxi-
dation of (R)-10 provided aldehyde (R)-8 as confirmed by
1H NMR spectroscopic analysis of the crude product. Wittig
olefination with the ylide derived from (R)-3, which was
prepared in four high-yielding steps by slight modifications
of the approach described before (see the Supporting Infor-
mation),[6] proceeded smoothly, resulting in 80% yield and
1
good Z selectivity (E/Z=10:90) of 11. However, H NMR
spectroscopic analysis revealed epimerization at C3 (3R,7R/
3S,7R=83:17). The Wittig reaction conditions did not seem
to be responsible for the epimerization because this was not
observed during the previous semiracemic synthesis.[6] Be-
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U. Jahn et al.
tion to the corresponding car-
boxylic acids as revealed by
GC/MS.
Confirmation of the structure
of stylopsal: In the preliminary
report, the relative configura-
tion of stylopsal was established
as that of (9R)-3,5-syn-1 by
comparison of the retention
times and coinjection of the
natural extract of a mixture of
both (9R)-3,5-syn-1 isomers
(Figure 2a).[6]
When
(3R,5R,9R)-1 and (3S,5S,9R)-1
were coinjected, they cleanly
separated (Figure 2b). Each
peak of the 92:8 (9R)-3,5-syn-1/
(9R)-3,5-anti-1 mixture was
identified by coinjection with
synthetic
(3S,5S,9R)-1,
(3R,5R,9R)-1, (3R,5S,9R)-1, and
(3S,5R,9R)-1 (Figure 2c–f). The
final proof for the identity of
stylopsal (Figure 2g) was fur-
nished by coinjection of the
natural extract and (3R,5R,9R)-
Scheme 4. Formation of the full C1–C12 backbone, and completion of the syntheses (for intermediates 7 and
12, the numbering in parentheses reflects that of the full backbone of the pheromone).
1
(Figure 2h). The retention
times matched perfectly and
dition protocol developed by Lum et al.[16] proved to be op-
timal to accomplish this task. Premixing CuBr·SMe2 with
(R)- or (S)-Tol-BINAP in dichloromethane generated the
active catalyst, which promoted the conjugate addition of
methylmagnesium bromide to the individual diastereomers
of 6 in anhydrous methyl tert-butyl ether (MTBE) in good
yields. It must be mentioned, however, that for reasons we
do not understand, no reaction occurred at scales lower than
0.2 mmol of starting a,b-unsaturated ester 6. The enantio-
and diastereoselectivity of the process was nonetheless to
some extent dependent on the configuration of the substrate
and ligand (catalyst). Best diastereomeric ratios of more
than 93:7 were obtained when the absolute configurations at
C5 and the ligand were matched, leading to the syn prod-
ucts. Substrates with mismatched absolute configurations at
C5 and the ligand gave markedly lower diastereoselectivi-
ties, indicating less favorable interactions in the transition
state of the conjugate additions.
The diastereomers of 13 were not separable and were sub-
jected to reduction by LiAlH4. Dess–Martin oxidation of the
resulting diastereomerically enriched alcohols 14 afforded
the four desired aldehydes 1, in good yields over two steps.
Because the aldehydes were well-resolved on chiral GC, the
diastereomeric ratios were determined and found to be ac-
ceptable for further study. These aldehydes are relatively
stable on storage in a freezer in the dark. However, over ex-
tended periods they slowly underwent spontaneous oxida-
the mass spectra were essentially identical. The chromato-
grams of the individual isomers also revealed that
(3R,5R,9R)-1 or (3S,5S,9R)-1 were not contaminated with
each other.
Based on this result, both synthetic (9R)-3,5-syn-isomers
1, namely (3R,5R,9R)-1 and (3S,5S,9R)-1, were subjected to
gas
chromatography/electroantennography
(GC/EAD)
measurements with male antennae (Figure 3). On an achiral
column, both (3R,5R,9R)-1 and (3S,5S,9R)-1 were found to
elute at the same time (tR =9.09 min; Figure 3). (3R,5R,9R)-
1 elicited a strong EAD response, whereas the response for
(3S,5S,9R)-1 was about 10 times weaker. Because
(3R,5R,9R)-1 or (3S,5S,9R)-1 were not contaminated with
each other (see above), the active pheromone is undoubted-
ly (3R,5R,9R)-1. This underlines the importance of the abso-
lute and relative configuration of the chiral centers in sty-
lopsal.[23] Coinjection of the two synthetic standards also led
to a clear response, suggesting that (3S,5S,9R)-1 has no
inhibitory activity.[23b]
With the identity of stylopsal established, field tests were
carried out with synthetic stylopsal (3R,5R,9R)-1 in the
Czech Republic. Ten sets of traps, each set consisting of
three traps with different pheromone loadings (14 mg, 140 mg
or none (control)), were prepared and exposed during the
only day of Stylops muelleri occurrence in 2012 on five dif-
ferent localities (Figure 4). Within three hours of exposition,
the combined catch was 50 (s.d.=6.148), 552 (s.d.=62.150),
and no males, respectively.
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Characterizing Stylopsal
FULL PAPER
Figure 3. GC/EAD responses of male antennae to diastereomeric syn-
thetic 1. The upper traces correspond to the GC/FID, whereas the lower
traces show the GC/EAD response. a) GC/EAD of (3R,5R,9R)-1, b) GC/
EAD of (3S,5S,9R)-1, and c) GC/EAD of coinjected (3R,5R,9R)-1 and
(3S,5S,9R)-1.
Figure 4. Photograph of
a trap loaded with 140 mg of synthetic
(3R,5R,9R)-1 during the field test.
Figure 2. Chiral GC chromatograms of a) a synthetic mixture of (9R)-3,5-
syn-1 and (9R)-3,5-anti-1 in a ratio of 92:8,[6] b) coinjected synthetic
(3R,5R,9R)-1 and (3S,5S,9R)-1, c) coinjection of (3S,5S,9R)-1 and (9R)-
3,5-syn-1, d) coinjection of (3R,5R,9R)-1 and (9R)-3,5-syn-1, e) coinjec-
tion of (3R,5S,9R)-1 and (9R)-3,5-syn-1, f) coinjection of (3S,5R,9R)-1
and (9R)-3,5-syn-1, g) natural stylopsal (tR =44.50 min), and h) coinjec-
tion of (3R,5R,9R)-1 and natural stylopsal. g,h) Additional peaks
(tR=42.60 and 45.70 min) are decomposition products formed on storage
of the natural extract.
lopsal with its three chiral centers as a single-component
pheromone. This may reflect the need for an extraordinarily
efficient mate-location system to efficiently attract the
short-living males. However, even the biosynthesis appeared
initially somewhat puzzling; the location of the methyl
groups in 3,5,9-positions strongly suggests that it is derived
from a polyketide pathway, but this is contradicted by their
positions at odd carbon linkages with respect to the carbonyl
function.[24] Indeed, the methyl groups would be expected at
C4, C6 and C10 or C2, C4 and C8, respectively, in a com-
pound resulting from a regular polyketide pathway. Thus, a
loss of a one-carbon linkage must occur during the biosyn-
thesis, a process that usually takes place by decarboxylation.
Biosynthesis of stylopsal: Considering the evolutionary sim-
plification of female Strepsiptera to a fully dependent orga-
ACHTUNGTRENNUNG
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U. Jahn et al.
contain only trace amounts of 1, which results in a loss of in-
terest of males.[27] This behavior as well as ultrastructural ob-
servations at the stylopsal-producing Nassonov gland in
virgin and mated females,[28] strongly suggest that sequestra-
tion of stylopsal occurs in the gland prior to mating. Careful
analysis of the chromatogram and mass spectra of the vari-
ous fatty acid methyl esters (FAME) obtained by transester-
ification of the triacyl glycerols of the fat body of Strepsip-
tera females revealed the presence of C15 ester 13 and C16
ester 20, representing respectively 0.5 and 1.1% of all the
FAME from the fat body extract (Figure 5).
Scheme 5. Possible pathways in the biosynthesis of stylopsal.
Figure 5. a) Two notable FAME found in the extract obtained by transes-
terification of the fat body contents of female Stylops muelleri. b) Gas
chromatogram of the FAME.
Two possible pathways can therefore be hypothesized for
this process (Scheme 5).
The structure of FAME (3R,5R,9R)-13 could be con-
firmed by comparison of retention times and mass spectra
with those of the corresponding intermediate of the total
synthesis (see above) (Figure 6). Coinjection of synthetic
(3R,5R,9R)-13 and the FAME from the fat body extract led
to a virtually unchanged mass spectrum over the whole
range of the coeluted peak.
As for the synthesis of FAME 20, the direct precursor to
the pheromone, alcohol (3R,5R,9R)-14, was iodinated under
Appel type conditions (Scheme 6). The resulting iodide 21
was converted into nitrile 22 using potassium cyanide under
standard Kolbe conditions. Hydrolysis of the nitrile function
In pathway (a), the biosynthesis commences with
a
propionyl-CoA starter unit. Elongation by methylmalonyl-
ACHTUNGTRENNUNG
CoA and malonyl-CoA, followed by a repetition of the se-
quence provides 15, which subsequently undergoes an a-oxi-
dation to 16, which decarboxylates to stylopsal (1). In path-
way (b), acetyl-CoA serves as the starter unit and the chain
would be elongated by two methylmalonyl-CoA units,
before repetition of the sequence produces 17. Subsequent
w-oxidation and decarboxylation would lead to alcohol 14
via 18, which is oxidized to pheromone 1. Remarkably, both
pathways exhibit a similar triad repeated twice. Although
w-oxidation of fatty acids is not uncommon,[25] three distinct
steps to the pheromone must take place in pathway (b)
whereas the a-oxidation with concomitant decarboxylation
in pathway (a) is an efficient one-step process.[26] It should
be noted that the decarboxylation/w-oxidation sequence de-
scribed for pathway (b) can in principle, also take place in
pathway (a). Moreover, the oxidation and decarboxylation
events starting from 15 or 17 may occur in reverse order.
This would lead to the formation of intermediate hydrocar-
bon 19 common to both pathways. Its further transformation
into 1 would, however, require selective enzymatic oxidation
of one of the two terminal methyl groups.
A steady release of stylopsal by mature Stylops muelleri
females has been observed until mating, which is followed
by a release of all remaining 1 at once.[27] Mated females
Scheme 6. Synthesis of FAME 20.
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Characterizing Stylopsal
FULL PAPER
Figure 6. Partial gas chromatograms of a) (3R,5R,9R)-13, b) coinjection
of (3R,5R,9R)-13 and FAME from the fat body extract, and c) FAME
from the fat body extract. Mass spectrum of d) synthetic (3R,5R,9R)-13
and e) the coeluted peak (tR =19.31 min) resulting from coinjection.
Figure 7. Partial gas chromatograms of a) (4S,6R,10R)-20, b) coinjection
of (4S,6R,10R)-20 and FAME from the fat body extract, and c) FAME
from the fat body extract. Mass spectrum of d) synthetic (4S,6R,10R)-20
and e) the coeluted peak (tR =21.03 min) resulting from coinjection.
and esterification of the resulting crude carboxylic acid
provided methyl ester 20.
is carried out entirely and de novo by the female (see the
Supporting Information).[29] From the information gathered,
a polyketide synthesis can be assumed to take place to form
a C16 carboxylic acid derivative 15, which is converted into
stylopsal (1) according to pathway (a). It may then be fur-
ther degraded to C15 carboxylic acid derivative 13. The
exact course of the events leading to the formation of sty-
lopsal, including the enzymatic apparatus needed for the for-
mation of precursors and end-products are, however, impos-
sible to claim at this stage. Further work is underway to
answer these questions and the results will be reported in
due course.
The retention times and mass spectra of (4S,6R,10R)-20
matched those of the natural FAME from the fat body ex-
tract (Figure 7). No change in the spectrum was observed
over the whole range of the coeluted peak upon coinjection
of 20 and the FAME from the fat body extract. Although
the amounts of FAME of interest in the extract were suffi-
cient for detection, the relative quantities with respect to
the other components in the extract made it technically im-
possible to analyze the contents by chiral GC for confirma-
tion of the absolute configuration of the detected FAME. It
may however be assumed that both detected FAME present
the same absolute configuration as that of the biogenetically
related stylopsal.
Conclusion
It is noteworthy that alcohol 14 was not found within the
different extracts. Moreover, no biosynthetically related
compounds were found in extracts of the fat body of the
host bees, suggesting that the biosynthesis of the pheromone
The absolute configuration of stylopsal, the first identified
sex pheromone of a twisted-wing parasite, was unequivocal-
ly established by total synthesis, GC analyses, and bioassays.
Chem. Eur. J. 2013, 19, 8515 – 8524
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8521

U. Jahn et al.
With Raney nickel:
A
mixture of olefin (3S,4Z,7R)-11 (200 mg,
Stylopsal and three configurational isomers were prepared
in 11 steps in the longest linear sequence in 16–21% overall
yield. Gas chromatographic investigations showed that the
natural material has 3R,5R,9R configuration. This is sup-
ported by GC/EAD investigations in which this isomer elic-
0.47 mmol) and activated Raney nickel (200 mL) in anhydrous ethanol
(4 mL) was stirred at RT under hydrogen (10 bar) in an autoclave for 5 h.
Upon completion of the reaction as indicated by GC, the mixture was fil-
tered over a pad of silica gel and the solvent was removed in vacuo.
Flash column chromatography (hexane/EtOAc, 50:1) afforded (3R,7R)-7
1
as a colorless oil (192 mg, 96%). [a]2D0 =À2.3 (c=0.310, CHCl3); H NMR
ACHTUNGTRENNUNGited a strong response at the male antennae, whereas the
(400 MHz, CDCl3): d=7.72–7.67 (m, 4H; HAr), 7.45–7.35 (m, 6H; HAr),
corresponding (3S,5S,9R)-diastereomer was much less active.
In field tests, the synthetic (3R,5R,9R)-diastereomer was
highly attractive to male Stylops muelleri, thus further sup-
porting the structure of stylopsal. Analytical and synthetic
studies revealed that the pheromone is likely to be biosyn-
thesized through a polyketide pathway to a 4,6,10-trimethyl-
tridecanoic acid derivative as a central intermediate. Over-
all, as simple as the life of this entirely host-dependent
female organism appears, it undertakes the effort to produce
a relatively complex stereochemically defined potent female
sex pheromone to maximize success of reproduction. Fur-
ther research concerning the biosynthesis of the pheromone
and the peculiar biology of Strepsiptera is underway.
3.77–3.65 (m, 2H; H-1), 1.68–1.55 (m, 2H; H-2), 1.46–1.06 (m, 12H; H-3,
H-4, H-5, H-6, H-7, H-8, H-9), 1.07 [s, 9H; CACTHNURGTNEG(UN CH3)3], 0.89 (t, J=7.1 Hz,
3H; H-10), 0.85 (d, J=6.6 Hz, 3H; CH3-3), 0.83 ppm (d, J=6.6 Hz, 3H;
CH3-7); 13C NMR (100 MHz, CDCl3): d=135.6 (CAr), 134.2 (CAr), 129.5
(CAr), 127.6 (CAr), 62.3 (CH2-1), 39.7 (CH2-2), 39.4 (CH2-8), 37.4 (CH2-6),
37.3 (CH2-4), 32.5 (CH-7), 29.4 (CH-3), 26.9 [C
ACHTNUGTRNEN(NUG CH3)3], 24.4 (CH2-5),
20.2 (CH2-9), 19.8 (CH3-3), 19.7 (CH3-7), 19.2 [C(CH3)3], 14.4 ppm (CH3-
AHCTUNGTRENNUNG
10); IR (film): n˜max =2956, 2927, 2858, 1462, 1428, 1378, 1108, 823, 736,
700, 614 cmÀ1; MS (ESI+): m/z (%): 447 (100), 359 (13), 315 (11), 301
(7), 273 (5), 217 (10), 153 (5); HRMS (ESI+): m/z calcd for
[C28H44ONaSi]+: 447.3054; found: 447.3055.
AHCTUNGTREG(NNUN 3R,7R)-3,7-Dimethyldecan-1-ol, (3R,7R)-12: TBAF (1m in THF,
1.28 mL, 1.28 mmol) was added to a solution of silyl ether (3R,7R)-7
(360 mg, 0.85 mmol) in THF (2 mL) at RT. After stirring for 2 h, the mix-
ture was diluted with Et2O (5 mL), filtered over a pad of silica gel, and
concentrated in vacuo. Purification by flash column chromatography
(hexane/EtOAc, 5:1) afforded alcohol (3R,7R)-12 as a pungent colorless
oil (152 mg, 96%). [a]D20 = +2.7 (c=1.31, CHCl3) {lit.: [a]2D0 = +1.90
(c=1.86, CHCl3)}. The spectral data matched those reported in the litera-
ture.[30]
Experimental Section
AHCTUNGERTG(NNUN 2E,5R,9R)-Methyl 5,9-dimethyldodec-2-enoate, (2E,5R,9R)-6: NMO
For general analytical and experimental conditions see the Supporting In-
formation.
(53 mg, 0.52 mmol), activated powdered molecular sieves 4 ꢄ (50 mg),
and TPAP (9 mg, 0.05 mmol) were successively added portionwise to a
solution of alcohol (3R,7R)-12 (93 mg, 0.5 mmol) in anhydrous CH2Cl2
(5 mL), resulting in the formation of a deep-green mixture. After stirring
for 15 min, methoxycarbonylmethylenetriphenylphosphorane (200 mg,
0.6 mmol) was added to the black solution in one portion as a solid and
stirring was continued overnight. Filtration over a pad of silica gel, con-
centration in vacuo, and flash column chromatography afforded a,b-unsa-
turated ester (2E,5R,9R)-6 as a colorless oil (107 mg, 89%). 1H NMR
(400 MHz, CDCl3): d=6.95 (dt, J=15.4, 7.5 Hz, 1H; H-3), 5.81 (d, J=
15.6 Hz, 1H; H-2), 3.72 (s, 3H; OCH3), 2.20 (m, 1H; H-4a), 2.07–1.98
(m, 1H; H-4b), 1.68–1.53 (m, 1H; H-5), 1.44–1.00 (m, 11H; H-6, H-7, H-
8, H-9, H-10, H-11), 0.89 (d, J=6.6 Hz, 3H; CH3-5), 0.87 (t, J=7.3 Hz,
3H; H-12), 0.83 ppm (d, J=6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz,
CDCl3): d=167.0 (C-1), 148.6 (CH-3), 121.9 (CH-2), 51.3 (OCH3), 39.7
(CH2-4), 39.3 (CH2-10), 37.2 (CH2-6), 36.9 (CH2-8), 32.5 (CH-5), 32.4
(CH-9), 24.4 (CH2-7), 20.1 (CH2-11), 19.6 (CH3-5), 19.5 (CH3-9),
14.4 ppm (CH3-12); IR (film): n˜max =2955, 2926, 2872, 1727, 1657, 1460,
1436, 1379, 1321, 1269, 1195, 1171, 1131, 1043, 983 cmÀ1. MS (EI): m/z
(%): 240 (6), 209 (13), 142 (15), 100 (100), 85 (34), 71 (28), 57 (41), 43
(46); HRMS (EI): m/z calcd for [C15H28O2]+: 240.2089; found: 240.2088.
tert-Butyl-[(3S,4Z,7R)-3,7-dimethyldec-4-enyloxy]diphenylsilane,
(3S,4Z,7R)-11: nBuLi (1.6m in hexanes, 655 mL, 1.05 mmol) was added
dropwise to a suspension of phosphonium salt (R)-3 (537 mg, 1.1 mmol)
in anhydrous THF (5 mL) cooled to À788C under a nitrogen atmosphere,
and the resulting deep-orange mixture was stirred at this temperature for
30 min. In a separate flask, to a solution of alcohol (S)-10 (342 mg,
1.0 mmol) in anhydrous CH2Cl2 (5 mL) were successively added N-meth-
ylmorpholine N-oxide (NMO; 106 mg, 1.05 mmol), activated powdered
molecular sieves 4 ꢄ (100 mg), and TPAP (18 mg, 0.05 mmol) portion-
wise, resulting in the formation of a deep-green mixture. After stirring
for 15 min, the black solution was transferred into the phosphorane solu-
tion by using a cannula. The cooling bath was removed, and stirring was
continued for 30 min. Dilution with Et2O (10 mL), filtration over a pad
of silica gel, concentration in vacuo, and flash column chromatography
(hexane/EtOAc, 50:1) afforded olefin (3S,4Z,7R)-11 as a colorless oil
(385 mg, 91%; E/Z=10:90). 1H NMR (400 MHz, CDCl3): d=7.67 (dd,
J=6.6, 1.3 Hz, 4H; HAr), 7.43–7.33 (m, 6H; HAr), 5.29 (dt, J=10.9,
7.3 Hz, 1H; H-5), 5.12 (dd, J=10.9, 9.7 Hz, 1H; H-4), 3.64 (t, J=6.5 Hz,
2H; H-1), 2.73–2.61 (m, 1H; H-3), 2.05 (dddd, J=14.3, 7.5, 6.3, 1.5 Hz,
1H; H-6a), 1.83 (dtd, J=14.3, 7.3, 1.5 Hz, 1H; H-6b), 1.60–1.38 (m, 3H;
H-7, H-2), 1.38–1.19 (m, 4H; H-8, H-9), 1.04 [s, 9H; C
G
ACHTUNGTNER(NUNG 3R,5R,9R)-Methyl 3,5,9-trimethyldodecanoate, (3R,5R,9R)-13: A solu-
J=6.7 Hz, 3H; CH3-3), 0.87 (t, J=6.8 Hz, 3H; H-10), 0.82 ppm (d, J=
6.7 Hz, 3H; CH3-7); 13C NMR (100 MHz, CDCl3): d=136.4 (CH-4),
135.6 (CAr), 134.2 (CAr), 129.5 (CAr), 127.5 (CAr), 127.4 (CH-5), 62.2
(CH2-1), 40.2 (CH2-6), 38.9 (CH2-2), 34.7 (CH-3), 33.2 (CH-7), 28.1
tion of (R)-Tol-BINAP (30.6 mg, 0.045 mmol) and CuBr·SMe2 (6.2 mg,
0.03 mmol) in anhydrous CH2Cl2 (0.7 mL) was stirred for 20 min and an-
hydrous tBuOMe (2 mL) was added. The bright-yellow solution was
cooled to À208C and MeMgBr (3m in Et2O, 0.130 mL, 0.39 mmol) was
(CH2-8), 26.9 [CACHTUNGTRENNUNG(CH3)3], 21.1 (CH2-9), 20.2 (CH3-7), 19.6 (CH3-3), 19.2
added. After stirring for 5 min,
a solution of (2E,5R,9R)-6 (75 mg,
[CACHTUNGTRENNUNG(CH3)3], 14.3 ppm (CH3-10); IR (film): n˜max =2956, 2929, 2858, 1462,
0.3 mmol) in tBuOMe (1 mL) was added dropwise over 30 min. Stirring
was continued for 1 h, and the reaction mixture was quenched with
MeOH (0.5 mL) and a saturated NH4Cl solution (2 mL). The layers were
separated and the aqueous layer was extracted with Et2O (3ꢅ3 mL). The
combined organic layers were dried over Na2SO4, filtered, and concen-
trated in vacuo. Flash column chromatography (pentane/Et2O, 50:1) af-
forded ester (3R,5R,9R)-13 as a colorless oil (69 mg, 86%, dr=96:4).
1H NMR (400 MHz, CDCl3): d=3.66 (s, 3H; OCH3), 2.31 (m, 1H; H-
2a), 2.07 (m, 2H; H-2b, H-3), 1.45 (m, 1H; H-5), 1.38 (m, 1H; H-9),
1.35–1.16 (m, 8H; H-4a, H-6a, H-7, H-8a, H-10a, H-11), 1.11–0.95 (m,
4H; H-4b, H-6b, H-8b, H-10b), 0.92 (d, J=6.4 Hz, 3H; CH3-3), 0.88 (t,
J=7.0 Hz, 3H; H-12), 0.86 (d, J=6.6 Hz, 3H; CH3-5), 0.84ppm (d, J=
1428, 1110, 823, 737, 701, 613 cmÀ1; MS (EI): m/z (%): 365 (100), 287
(27), 281 (25), 225 (18), 203 (36), 199 (74), 183 (64), 135 (18); HRMS
(ESI+): m/z calcd for [C28H42ONaSi]+: 445.2897; found: 445.2893.
tert-Butyl-[(3R,7R)-3,7-dimethyldecyloxy]diphenylsilane,
(3R,7R)-7:
With palladium on charcoal: Olefin (3S,4Z,7R)-11 (380 mg, 0.9 mmol)
was dissolved in degassed EtOAc (3 mL) and hydrogenated over 10%
Pd/C (38 mg, 10% w/w) under hydrogen (10 bar) in an autoclave at RT
for 1.5 h. Upon completion of the reaction as indicated by GC, the mix-
ture was filtered over a pad of silica gel and the solvent was removed in
vacuo. Flash column chromatography (hexane/EtOAc, 50:1) afforded
(3R,7R)-7 as a colorless oil (363 mg, 95%).
8522
www.chemeurj.org
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 8515 – 8524

Characterizing Stylopsal
FULL PAPER
6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz, CDCl3): d=174.00 (C=O),
51.52 (CH3O), 44.91 (CH2-4), 41.78 (CH2-2), 39.60 (CH2-10), 37.62 (CH2-
8), 37.27 (CH2-6), 32.74 (CH-9), 30.31 (CH-5), 28.13 (CH-3), 24.46 (CH2-
7), 20.64 (CH3-3), 20.36 (CH2-11), 20.33 (CH3-5), 19.93 (CH3-9),
14.62 ppm (CH3-12); IR (film): n˜max =2926, 1741, 1460, 1192, 1009,
740 cmÀ1; MS (CI+): m/z (%): 257 (100), 255 (42), 241 (14), 225 (8);
HRMS (CI+): m/z calcd for [C16H33O2]+: 257.2481; found: 257.2478.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(3S,5S,9R)-Methyl 3,5,9-trimethyldodecanoate, (3S,5S,9R)-13: Following
the same procedure with (2E,5S,9R)-6 (75 mg, 0.3 mmol) and (S)-Tol-
BINAP as the chiral ligand, (3S,5S,9R)-13 was obtained as a colorless oil
(55 mg, 69%, dr=93:7). 1H NMR (400 MHz, CDCl3): d=3.66 (s, 3H;
OCH3), 2.31 (m, 1H; H-2a), 2.07 (m, 2H; H-2b, H-3), 1.45 (m, 1H; H-5),
1.38 (m, 1H; H-9), 1.35–1.16 (m, 8H; H-4a, H-6a, H-7, H-8a, H-10a, H-
11), 1.11–0.95 (m, 4H; H-4b, H-6b, H-8b, H-10b), 0.92 (d, J=6.4 Hz, 3H;
CH3-3), 0.88 (t, J=7.0 Hz, 3H; H-12), 0.86 (d, J=6.6 Hz, 3H; CH3-5),
0.84 ppm (d, J=6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz, CDCl3): d=
174.01 (C=O), 51.52 (CH3O), 44.98 (CH2-4), 41.80 (CH2-2), 39.73 (CH2-
10), 37.54 (CH2-8), 37.18 (CH2-6), 32.70 (CH-9), 30.27 (CH-5), 28.13
(CH-3), 24.44 (CH2-7), 20.61 (CH3-3), 20.38 (CH2-11), 20.27 (CH3-5),
19.83 (CH3-9), 14.62 ppm (CH3-12); IR (film): n˜max =2926, 1741, 1460,
1192, 1009, 740 cmÀ1; MS (CI+): m/z (%): 257 (100), 255 (40), 241 (15),
225 (8); HRMS (CI+): m/z calcd for [C16H33O2]+: 257.2481; found:
257.2482.
ACHTUNGTRENNUNG(3R,5R,9R)-3,5,9-Trimethyldodecan-1-ol, (3R,5R,9R)-14: Anhydrous
THF (1.5 mL) and a solution of ester (3R,5R,9R)-13 (66 mg, 0.257 mmol)
in THF (1 mL) were added to a solution of LiAlH4 (2m in THF,
0.129 mL, 0.257 mmol) cooled to 08C, under an inert atmosphere. The
cooling bath was removed and stirring was continued at ambient temper-
ature for 2 h. The flask was again immersed in an ice-water bath and
methanol (1.5 mL) was added in small portions followed by aqueous
H2SO4 (1m, 5 mL). The resulting mixture was transferred to a separation
funnel, the layers were separated, and the aqueous layer was extracted
with Et2O (2ꢅ3 mL). The combined organic layers were washed with sa-
turated NaHCO3 solution (2ꢅ3 mL), dried over anhydrous MgSO4, and
concentrated. Flash column chromatography (pentane/Et2O, 3:1) afford-
ed alcohol (3R,5R,9R)-14 as a colorless oil (45 mg, 77%; dr=96:4).
1H NMR (400 MHz, CDCl3): d=3.71 (ddd, J=10.4, 7.7, 5.8 Hz, 1H; H-
1a), 3.66 (ddd, J=10.4, 7.3, 6.7 Hz, 1H; H-1b), 1.65 (m, 1H; H-3), 1.62
(m, 1H; H-2a), 1.49 (m, 1H; H-5), 1.38 (m, 1H; H-9), 1.35–1.14 (m,
10H; H-2b, H-4a, H-6a, H-7, H-8a, H-10a, H-11, OH), 1.11–1.00 (m, 3H;
H-6b, H-8b, H-10b), 0.97 (m, 1H; H-4b), 0.89 (d, J=6.7 Hz, 3H; CH3-3),
0.88 (t, J=7.0 Hz, 3H; H-12), 0.85 (d, J=6.5 Hz, 3H; CH3-5), 0.84 ppm
(d, J=6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz, CDCl3): d=61.43 (CH2-
1), 45.54 (CH2-4), 40.04 (CH2-2), 39.59 (CH2-10), 37.65 (CH2-8), 37.39
(CH2-6), 32.74 (CH-9), 30.24 (CH-5), 27.18 (CH-3), 24.52 (CH2-7), 20.49
(CH3-3), 20.46 (CH3-5), 20.35 (CH2-11), 19.93 (CH3-9), 14.61 ppm (CH3-
12); IR (film): n˜max =3322, 2925, 1460, 1377, 1157, 741 cmÀ1; MS (CI+):
m/z (%): 228 (16), 227 (100), 211 (50), 155 (47), 141 (66), 127 (74), 113
(73), 99 (71), 97 (89), 85 (76), 71 (57); HRMS (CI+): m/z calcd for
[C15H31O]+: 227.2375; found: 227.2376.
AHCTUNGTERG(NNUN 3S,5S,9R)-3,5,9-Trimethyldodecanal, (3S,5S,9R)-1: Following the same
procedure with (3S,5S,9R)-14 (15 mg, 0.066 mmol), (3S,5S,9R)-1 was ob-
tained as a colorless oil (12 mg, 81%; dr=93:7). 1H NMR (400 MHz,
CDCl3): d=9.76 (dd, J=2.5, 1.9 Hz, 1H; H-1), 2.39 (m, 1H; H-2a), 2.18
(m, 1H; H-2b), 2.15 (m, 1H; H-3), 1.47 (m, 1H; H-5), 1.37 (m, 1H; H-
9), 1.35–1.21 (m, 8H; H-4a, H-6a, H-7, H-8a, H-10a, H-11), 1.13–1.02 (m,
4H; H-4b, H-6b, H-8b, H-10b), 0.96 (d, J=6.5 Hz, 3H; CH3-3), 0.88 (t,
J=7.0 Hz, 3H; H-12), 0.87 (d, J=6.6 Hz, 3H; CH3-5), 0.84 ppm (d, J=
6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz, CDCl3): d=203.36 (CH-1),
51.21 (CH2-2), 45.13 (CH2-4), 39.73 (CH2-10), 37.54 (CH2-8), 37.24 (CH2-
6), 32.70 (CH-9), 30.28 (CH-5), 25.99 (CH-3), 24.47 (CH2-7), 20.85 (CH3-
3), 20.38 (CH2-11), 20.21 (CH3-5), 19.84 (CH3-9), 14.63 ppm (CH3-12); IR
(film): n˜max =2925, 2710, 1727, 1460, 740 cmÀ1; MS (CI+): m/z (%): 227
(100), 207 (19), 183 (22), 182 (24), 153 (12), 139 (24), 125 (36), 111 (43),
97 (39), 83 (23); HRMS (CI+): m/z calcd for [C15H31O]+: 227.2375;
found: 227.2379.
(4S,6R,10R)-Methyl 4,6,10-trimethyltridecanoate, (4S,6R,10R)-20:
A
solution of H2SO4 (1m, 0.5 mL) was added to a solution of nitrile 22
AHCTUNGTRENNUNG
(15 mg, 0.063 mmol) in acetic acid (0.2 mL) and the temperature was
maintained at 708C for 4 h. After cooling to RT, the reaction mixture
was extracted with EtOAc (3ꢅ1 mL) and the organic layer was dried
over MgSO4 and concentrated in vacuo. The intermediate crude carbox-
ylic acid (15 mg) was dissolved in benzene (0.3 mL) and methanol
(0.09 mL), followed by addition of a trimethylsilyldiazomethane solution
(2m in Et2O, 0.06 mL, 0.117 mmol). The resulting solution was stirred at
RT for 1 h. The reaction mixture was quenched with saturated NaHCO3
solution (0.5 mL) and extracted with Et2O (3ꢅ1 mL). The organic layer
was dried over MgSO4 and concentrated in vacuo. Flash column chroma-
tography (pentane/Et2O, 20:1) afforded methyl ester 20 (11 mg, 60%) as
a colorless oil. 1H NMR (400 MHz, CDCl3): d=3.67 (s, 3H; OCH3), 2.37
(ddd, J=15.6, 9.7, 5.9 Hz, 1H; H-2a), 2.29 (ddd, J=15.6, 9.4, 6.4 Hz, 1H;
H-2b), 1.69 (m, 1H; H-4), 1.51 (m, 2H; H-6, H-10), 1.42–1.14 (m, 10H;
H-3, H-5a, H-7a, H-8, H-9a, H-11a, H-12), 1.12–1.00 (m, 3H; H-7b, H-
9b, H-11b), 0.96 (m, 1H; H-5b), 0.90–0.84 ppm (m, 12H; CH3-4, CH3-6,
CH3-10, H-13); 13C NMR (100 MHz, CDCl3): d=174.79 (C=O), 51.66
(CH3O), 44.99 (CH2-5), 39.61 (CH2-11), 37.67 (CH2-9), 37.47 (CH2-7),
32.75 (CH-10), 31.99 (CH2-3), 31.90 (CH2-2), 30.23 (CH-6), 29.96 (CH-4),
24.50 (CH2-8), 20.39 (CH3-4), 20.36 (CH2-12), 20.12 (CH3-6), 19.93 (CH3-
10), 14.62 ppm (CH3-13); IR (film): n˜max =2925, 1743, 1460, 1435, 1378,
1253, 1193, 1170, 1118 cmÀ1; MS (CI+): m/z (%): 299 (20), 271 (100), 269
(51), 255 (15), 197 (5); HRMS (CI+): m/z calcd for [C17H35O2]+:
271.2637; found: 271.2645.
ACHTUNGTRENNUNG
a
(400 MHz, CDCl3): d=3.71 (ddd, J=10.5, 7.7, 5.8 Hz, 1H; H-1a), 3.66
(ddd, J=10.5, 7.3, 6.7 Hz, 1H; H-1b), 1.65 (m, 1H; H-3), 1.62 (m, 1H;
H-2a), 1.50 (m, 1H; H-5), 1.38 (m, 1H; H-9), 1.35–1.18 (m, 10H; H-2b,
H-4a, H-6a, H-7, H-8a, H-10a, H-11, OH), 1.12–1.01 (m, 3H; H-6b, H-
8b, H-10b), 0.98 (m, 1H; H-4b), 0.89 (d, J=6.7 Hz, 3H; CH3-3), 0.88 (t,
J=7.0 Hz, 3H; H-12), 0.85 (d, J=6.6 Hz, 3H; CH3-5), 0.84 ppm (d, J=
6.6 Hz, 3H; CH3-9); 13C NMR (100 MHz, CDCl3): d=61.46 (CH2-1),
45.61 (CH2-4), 40.12 (CH2-2), 39.74 (CH2-10), 37.58 (CH2-8), 37.31 (CH2-
6), 32.70 (CH-9), 30.21 (CH-5), 27.18 (CH-3), 24.51 (CH2-7), 20.48 (CH3-
3), 20.41 (CH3-5), 20.37 (CH2-11), 19.84 (CH3-9), 14.62 ppm (CH3-12); IR
(film): n˜max =3322, 2925, 1460, 1377, 1157, 741 cmÀ1; MS (CI+): m/z (%):
228 (16), 227 (100), 211 (86), 155 (36), 141 (63), 127 (72), 113 (68), 99
(75), 97 (72), 85 (75), 71 (52); HRMS (CI+): m/z calcd for [C15H31O]+:
227.2375; found: 227.2381.
Chem. Eur. J. 2013, 19, 8515 – 8524
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8523

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Acknowledgements
We thank the Academy of Sciences of the Czech Republic (RVO:
61388963) and the Grant Agency of the Czech Republic (P506/10/1466)
ˇ
for financial support. J.S. thanks the Internal Grant Agency of the Facul-
ty of Forestry and Wood Sciences, Czech University of Life Sciences
(Specific research project 20124364). We are grateful to Prof. Mark Laut-
ens, Prof. Steven Ley, and Dr. Antoine FranÅais for fruitful discussions
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Received: November 23, 2012
Revised: March 15, 2013
Published online: April 29, 2013
8524
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ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 8515 – 8524