Short synthesis of labeled and unlabeled 6Z,9Z,12Z,15-hexadecatetraenoic acid as metabolic probes for biosynthetic studies on diatoms

Article abstract of DOI:10.1016/j.chemphyslip.2004.04.011

We describe a short synthesis of the unusual polyunsaturated 6Z,9Z,12Z,15-hexadecatetraenoic acid found in marine and fresh water diatoms. Using a one pot reductive bis-Wittig-olefination, the homoconjugated tetraene backbone of the fatty acid can be gene

Full text of DOI:10.1016/j.chemphyslip.2004.04.011

Chemistry and Physics of Lipids 131 (2004) 159–166
Short synthesis of labeled and unlabeled
6Z,9Z,12Z,15-hexadecatetraenoic acid as metabolic
probes for biosynthetic studies on diatoms
Georg Pohnert∗, Sven Adolph, Thomas Wichard
Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany
Received 10 March 2004; received in revised form 26 April 2004; accepted 28 April 2004
Available online 28 July 2004
Abstract
We describe a short synthesis of the unusual polyunsaturated 6Z,9Z,12Z,15-hexadecatetraenoic acid found in marine and fresh
water diatoms. Using a one pot reductive bis-Wittig-olefination, the homoconjugated tetraene backbone of the fatty acid can
be generated from easy available precursors. Reductive olefination allows the non-statistical dissymmetrisation of a symmet-
rical bis-Wittig salt as key synthon. This short sequence was also applied to the generation of the corresponding 9,10-[2H2]
labeled fatty acid. If administered to cell fragments of Thalassiosira rotula 9,10-[2H2]-6Z,9Z,12Z,15-hexadecatetraenoic acid is
transformed oxidatively to the aldehyde 1,2-[2H2]-2E,4E/Z,7-octatrienal which is involved in the chemical defense of this alga.
Using the synthetic standard it could be shown that the C16:4 ␻1 fatty acid is released upon wounding of T. rotula cells. The
synthesis with the labeled bis-Wittig salt is of general use and can also be applied for the fast generation of other internally
labeled functionalized and non-functionalized polyunsaturated fatty acids. To our knowledge this represents the first synthesis
of 6Z,9Z,12Z,15-hexadecatetraenoic acid.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: One-pot synthesis; bis-Wittig reaction; Homoconjugated tetraenes; Deuterium labeled; Unsaturated fatty acids; Reductive
olefination
1. Introduction
therefore, be used as a trophic marker in the marine
food chain (Ackman and Tocher, 1968; Dunstan et al.,
1994). Recently, this fatty acid was also identified
from fresh water diatom biofilms as toxic princi-
ple against the anostracan grazer Thammocephalus
platyurus (Jüttner, 2001). Despite this importance as
yet no synthesis of this unusual fatty acid was re-
ported. This is at least partially due to the fact that the
synthesis of homoconjugated tetraene systems, like
it is found in C16:4 ␻1 requires long sequences in-
volving numerous coupling and protection steps (see
6Z,9Z,12Z,15-hexadecatetraenoic acid (C16:4 ␻1)
has initially been detected in fish oil (Silk and Hahn,
1954) and was later found as an important constituent
in phytoplankton samples (Klenk and Eberhagen,
1962). It is a common fatty acid in diatoms and, can
∗
Corresponding author. Tel.: +49-3641-571258;
fax: +49-3641-571256.
E-mail address: pohnert@ice.mpg.de (G. Pohnert).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.04.011

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G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
Durand et al., 2000 for a review). Here we show that
a one-pot bis-Wittig sequence, recently established in
our lab for the production of homoconjugated dienes
and trienes (Pohnert and Boland, 2000; Pohnert et al.,
1999) can be extended for the generation of the ho-
moconjugated tetraenoic acid C16:4 ␻1 (Scheme 1).
Besides the direct toxicity of free polyunsaturated
fatty acids, also unsaturated aldehydes derived from
fatty acids are discussed to play a key role in di-
atom defense (Ianora et al., 2003; Miralto et al.,
1999). After cell disruption these unicellular algae
release a complex bouquet of saturated and unsat-
urated aldehydes (Pohnert, 2000). Among these,
the ␣,␤,␥,␦-unsaturated aldehydes act antiprolifera-
tively against the eggs of herbivorous copepods and
sea urchins (Adolph et al., 2003; d’Ippolito et al.,
2002b; Miralto et al., 1999). Most of the identi-
fied unsaturated aldehydes are derived from the
lipoxygenase-mediated transformation of eicosanoic
fatty acids (Pohnert and Boland, 2002), but recently,
2E,4E/Z-octadienal was reported as an exception to
be derived of ␻4-hexadecatrienoic acid (d’Ippolito
et al., 2003). d’Ippolito et al. suggested C16:4 ␻1
to be a possible precursor of the unusual metabolite
2E,4E/Z,7-octatrienal but experimental evidence is
still lacking. Our synthetic concept of bis-Wittig ole-
fination can be exploited for the generation of posi-
tion specific labeled metabolic probes that allowed to
show that 9,10-[2H2]-C16:4 ␻1 is the precursor of the
unusual metabolite 1,2-[2H2]-2E,4E/Z,7-octatrienal.
cal bis-Wittig salt 11 (Pohnert and Boland, 2000),
which was prepared from the mono THP-protected
(Z)-hex-3-ene-1,6-diol (9) the homoconjugated
tetraene was generated in a sequential one pot
procedure. To avoid the statistical distribution of
wanted unsymmetrical and unwanted symmetrical
side-products arising out of the reaction of the sym-
metrical bis-ylide 3, dissymmetrisation was required.
This can be achieved by generating the first aldehyde
equivalent in situ through diisobutyl aluminium hy-
dride (DIBALH) reduction of the corresponding ester.
We selected the commercial available methyl
3-butenoate (1) as first aldehyde equivalent which
also delivers one homoconjugated double bond. This
was reduced by slowly adding pre-cooled DIBALH
at −78 ◦C (Scheme 2). Meanwhile the bis-Wittig
salt 11 was deprotonated to the bis-ylide 3 with 2.2
equivalents of KN(SiMe3)2. The aluminate reaction
mixture was then transferred directly via a pre-cooled
cannula to the bis-ylide and the mixture was allowed
to warm slowly to 0 ◦C. During this procedure the
aluminum-complex 2 decomposes and releases slowly
but-3-enal that reacts preferentially with the more re-
active bis-ylide 3 compared to the mono-ylide 4. The
reaction mixture was re-cooled to −30 ◦C and 1.2
equivalents of the second aldehyde equivalent, ethyl
6-oxo-hexanoate 6 were added. After warming to room
temperature the reaction mixture was worked up as
described in the material and methods section to give
ethyl 6Z,9Z,12Z,15-hexadecatetraenoate (7) in 24%
yield. The symmetrical by-products were detected
in minor amounts of 1:0.35:0.05 (7:(4Z,7Z,10Z)-
tetradeca-1,4,7,10,13-pentaene (5):(6Z,9Z,12Z)-die-
thyl octadeca-6,9,12-triendioate (GC-MS)). 13C NMR
analysis showed that the newly generated double
bonds of 7 were of >95% Z-geometry, and the dou-
ble bond geometry of the bis-Wittig-salt 11 (>98%
Z) was retained during the reaction. This method
for the generation of homoconjugated tetraenes al-
lows thus the fast generation of highly unsaturated
products from simple starting materials in a one-pot
reaction. Compared to other known routes to re-
lated highly unsaturated products which are often
based on sequential alkyne coupling reactions fol-
lowed by a final reduction step of the generated
polyyne the present approach reduces the amount
of reaction steps and use of protecting groups to a
minimum.
2. Results and discussion
Based on a previously introduced synthesis of
homoconjugated dienes and trienes we developed a
short synthesis of C16:4 ␻1. Using the symmetri-
COOMe
+
O
+
COOEt
(D)H
(D)H
H(D)
P(Ph)3
P(Ph)3
one-pot
H(D)
COOEt
Scheme 1. Schematic representation of the formation of homo-
conjugated tetraenes using the bis-Wittig reaction.

G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
161
H(D)
H(D)
Al(O-iBu)2
ii) Ph3P
PPh3
O
O
i)
3
OMe
OMe
2
1
H(D)
H(D)
H(D)
H(D)
PPh3
+
minor amounts
5
4
O
H(D)
H(D)
OEt
O
4
6
OR
+ traces diester
7 (R = Et)
8 (R = H)
O
iii)
Scheme 2. Preparation of 6Z,9Z,12Z,15-hexadecatetraenoic acid (8). (i) DIBAL-H, (ii) from 11 by deprotonation with 2.2 equivalents of
KN(SiMe3)2, (iii) LiOH/THF/H2O.
Another advantage of this short sequence is that
isotope labels can be flexibly introduced into spe-
cific parts of the target molecule. We reasoned that
the deuterated bis-Wittig salt would be useful for
the introduction of internal labels in the homocon-
jugated polyene segment. The labeled bis-Wittig
salt was generated after a modified procedure re-
ported for the generation of the unlabeled analogue
(Pohnert and Boland, 2000). Mono THP-protected
hex-3-yne-1,6 diol (9) (Eya et al., 1990) was reduced
with D2 using in situ generated P-2-nickel as cata-
lyst (Brown and Ahuja, 1973). The resulting mono
the polar intermediate hex-3-ene-1,6-diol can be
avoided, but lower yields using the mono-protected
diol have to be considered. The labeled bis-Wittig
salt 11 was transformed as described above to ethyl
[9,10-2H2]-6Z,9Z,12Z,15-hexadecatetraenoate in 27%
yield.
The labeled bis-Wittig salt 11 can be obtained in
large batches and stored for a prolonged time. Since
it can be also employed to flexibly generate other
metabolic probes where internal label of (functional-
ized) unsaturated fatty acids is required, it provides
a useful synthetic tool. The tolerance of aldehydes
of varying chain lengths and degree of unsaturation
and of functional groups now extends the synthetic
approach beyond the generation of homoconjugated
trienes. Established syntheses of labeled homoconju-
gated polyenes involve often the deuteration of corre-
sponding polyynes (see, e.g. d’Ippolito et al., 2003).
THP-protected
[3,4-2H2]-(3Z)-hex-3-ene-1,6-diol
(10) was transformed directly without deprotection
into the labeled bis-Wittig 11 salt using Br2/PPh3
(Scheme 3). Compared to the published procedure
(Pohnert and Boland, 2000) this direct transfor-
mation bears the advantage that the work-up of
i)
HO
OTHP
HO
OTHP
D
D
9
10
ii)
Br-Ph3P+
P+Ph3Br-
D
D
11
Scheme 3. Preparation of [3,4-2H2]-(Z)-hex-3-enyl-1,6-bis-[triphenylphosphonium bromide] (11). (i) P2-Ni/D2, (ii) 1: Br2, PPh3; 2: PPh3
reflux.

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G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
This results in uniformly labeled double bonds, which
often make mass spectrometric investigations difficult.
In contrast, our synthesis provides the advantage, that
labels can be introduced in selected internal positions
of the homoconjugated polyene.
to determine if C10- or C8-unsaturated aldehydes
are preferentially generated as wound activated de-
fense by T. rotula. This is also reflected by the fact
that intact T. rotula, which does not contain any
2E,4E/Z,7-octatrienal (12) lacks also free C16:4 ␻1
(Fig. 1C). Only upon wounding of the alga C16:4 ␻1
is released from lipids and is available as precursor
for the unsaturated aldehyde (Fig. 1D). The wound
activated production of 2E,4E/Z,7-octatrienal (12) as
well as of the unsaturated C10 aldehydes (Pohnert,
2002) is thus controlled through the release of free
fatty acids upon cell disruption.
The availability of [9,10-2H2]-6Z,9Z,12Z,15-
hexadecatetraenoic acid (8) allowed to investigate
whether this fatty acid is a precursor for defen-
sive metabolites in the diatom Thalassiosira ro-
tula. This diatom is known to produce a variety
of ␣,␤,␥,␦-unsaturated aldehydes upon cell disrup-
tion, that contribute to its chemical defense (Adolph
et al., 2003; d’Ippolito et al., 2002a; Miralto et al.,
1999; Pohnert, 2000; Pohnert et al., 2002). In this
alga 2E,4Z-decadienal and 2E,4Z,7Z-decatrienal
are derived from the lipoxygenase-mediated trans-
formation of arachidonic and eicosapentaenoic
acid, respectively (Pohnert, 2002). To verify if
shorter chain length fatty acids are transformed to
␣,␤,␥,␦-unsaturated aldehydes as well, we applied
[9,10-2H2]-6Z,9Z,12Z,15-hexadecatetraenoic acid 8
to mechanically wounded T. rotula. Transforma-
tion products were enriched during the first 10 min
after wounding using solid phase microextraction
(SPME) and products were monitored with GC-MS
(Fig. 1).
The labeled fatty acid was transformed with
high efficiency, resulting in strongly increased lev-
els of [1,2-2H2]-2E,4E/Z,7-octatrienal (12). The
specific labeling of the fatty acid combined with
the analysis of the fragmentation pattern of the
mass spectrum allowed to show, that the intact
C9–C16-terminus of the fatty acid is incorporated
into [1,2-2H2]-2E,4E/Z,7-octatrienal without loss of
the label at C9. This product formation is in ac-
cordance with a lipoxygenase/lyase mechanism in
which molecular oxygen is introduced in 9-position
of the fatty acid, followed by the action of a lyase
that releases 2E,4E/Z,7-octatrienal (12) and a sec-
ond yet unidentified C8-fragment (Fig. 1). The di-
atom T. rotula has thus the ability to transform both,
polyunsaturated C20 and C16 fatty acids. In contrast,
Skeletonema costatum which was shown to produce
octadienal from 6Z,9Z,12Z-hecodecatrienoic acid
lacks the activity for the formation of C10 aldehydes
(d’Ippolito et al., 2002a; d’Ippolito et al., 2003). Since
fatty acid addition results in a pronounced increase of
labeled metabolites, the substrate availability seems
3. Materials and methods
3.1. General remarks
Reactions were performed under Ar. Solvents were
dried according to standard methods. 1H and 13C
NMR: Bruker Avance DRX 500 or AV 400 spectrom-
1
eter. Chemical shifts of H and 13C NMR are given
in ppm (δ) downfield relative to TMS. GC-MS: Finni-
gan Trace MS equipped with an Alltech EC5 column
(i.d.: 0.25 mm, 0.25 ␮m film thickness, 15 m), Helium
as carrier. HR-MS: Micromass MasSpec (Micromass,
Manchester, UK). Preparative column chromatogra-
phy was performed on Florisil (Sigma), or SiO2 (ICN)
32–63, 60 Å. Reagents and solvents were purchased
from Aldrich, Merck and Fluka.
3.1.1. Ethyl 6Z,9Z,12Z,15-Hexadecatetraenoate (7)
3.1.1.1. Preparation of the bis-ylide. 0.5 g (0.65
mmol) (Z)-hex-3-enyl-1,6-bis-[triphenylphosphonium
bromide] (11) was suspended in 25 ml THF and
cooled to −78 ◦C before addition of 1.4 ml of 28 ml
a 0.5 M solution KN(SiMe3)2 (1.4 mmol) in hex-
ane. The suspension was warmed to −20 ◦C until
no further deepening of the dark red color could
be observed (20 min). The solution was re-cooled
to −78 ◦C.
3.1.1.2. Preparation of the aluminate. To a cold
(−78 ◦C) solution of methyl but-2-enoate (1) (65 mg,
0.65 mmol) in ether (3 ml), pre-cooled (−78 ◦C)
DIBALH (0.65 ml of a 1 M solution in hexanes,
0.65 mmol) was added drop wise.

G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
163
Fig. 1. Transformation of [9,10-2H2]-6Z,9Z,12Z,15-hexadecatetraenoic acid (8) by suspensions of damaged T. rotula in seawater.
Above: Mechanistic suggestion for the lipoxygenase-mediated transformation of [9,10-2H2]-6Z,9Z,12Z,15-hexadecatetraenoic acid (8).
(A) Gas chromatogram of SPME extracted volatiles from damaged T. rotula (I: 2E,4E/Z-heptadienal; II: 2E,4E/Z-decadienal and
2E,4E/Z,7Z-decatrienal, 2E,4E/Z-octadienal at RT, 7.4 min) and EI/MS of 2E,4E/Z,7-octatrienal (12). (B) as (A) after addition of 100 ␮g
[9,10-2H2]-6Z,9Z,12Z,15-hexadecatetraenoic acid (8). (C) Free fatty acids in intact T. rotula IS: [2H27]-myristic acid. (D) Free fatty acids
in wounded T. rotula.
After being stirred for 10 min (GC control) the cold
(−78 ◦C) aluminate 2 was transferred quickly via
a pre-cooled cannula to the bis-ylide reaction mix-
ture. The mixture was allowed to warm to RT over
a period of 60 min, and stirring was continued for
30 min before re-cooling to –78 ◦C. Then, a solution
of ethyl 6-oxo-hexanoate (6) (110 mg, 0.7 mmol) in
5 ml THF was added. The mixture was allowed to

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G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
3.1.3. [3,4-2H2]-(Z)-Hex-3-enyl-1,6-bis-
[triphenylphosphonium bromide] (11)
reach room temperature and stirred for 30 min. Hy-
drolysis with HCl (2 N), extraction with ether, drying
over Na2SO4, and flash chromatography on silica gel
gave ethyl 6Z,9Z,12Z,15-hexadecatetraenoate (7) in
24% yield. 1H NMR (400 MHz, CDCl3) δ = 1.25
(t, J = 7.05 Hz, 3H, O–CH2CH3); 1.38 (quint., J
= 7.7 Hz, 2H, H–C3); 1.6 (quint., J = 7.7 Hz, 2H,
H–C4); 2.1 (dt., J = 7.4, 7.2 Hz, 2H, H–C5); 2.3 (t, J
= 7.5 Hz, 2H, H–C2); 2.8 (m, 6H, C8, C11, C14); 4.1
(quart., J = 7.1 Hz, 2H, O–CH2CH3); 5.0 (dquart.,
J = 10.0 Hz, 1.6, 1H, C16); 5.05 (dquart., J = 17.2,
1.7 Hz, 1H, C16); 5.3–5.4 (m, 6H, C6, C7, C9, C10,
C12, C13), 5.8 (m, 1H, C15). 13C NMR (100 MHz,
CDCl3) δ = 14.66, 25.02, 25.97, 26.03, 27.28, 29.49,
31.92, 34.66, 60.6, 115.15, 127.46, 128.39, 128.51,
128.76, 129.37, 130.1, 137.14, 174.11. The signals
at 131.06 and 129.1 were used to determine the con-
figurational purity of the product. EI-MS (70 eV):
276 (0.7), 247 (1), 235 (3), 208 (5), 189 (7), 147
(20), 133 (31), 119 (27), 105 (45), 91 (60), 79 (100),
67 (40).
A
suspension of triphenylphosphine (27.3 g,
104 mmol) in benzene (300 ml) was treated at 0 ◦C
with bromine (16.6 g, 104 mmol). After being stirred
for 10 min at room temperature, the suspension is
recooled to 0 ◦C and [3,4-2H2]-(Z)-1-(tetrahydro-2H-
pyran-2-yloxy)hex-3-en-6-ol (10) (10 g, 50 mmol)
are added. After additional stirring for 2 h at room
temperature, the reaction mixture was poured into
petrol ether and triphenylphosphine oxide was fil-
tered off. Crude [3,4-2H2]-(Z)-1,6-dibromo-hex-3-ene
that was transferred without further purification into
a solution of triphenylphosphine (29 g, 110 mmol)
in acetonitrile (200 ml). After reflux for 5 days the
mixture was poured in toluene (500 ml) and the re-
sulting bis-Wittig-salt was filtered off. Recrystalliza-
tion from methanol–ether afforded pure crystals of
[3,4-2H2]-(Z)-hex-3-enyl-1,6-bis-[triphenylphosphon-
ium bromide] (11) in 39% yield. 1H NMR ([D4]
MeOH, 400 MHz): δ = 2.2 (m, 4H), 3.41 (m, 4H),
7.98–7.13 (m, 2H), 7.6–7.83 (m, 28H). 13C NMR
([D4] MeOH, 100 MHz): δ = 21.8 (d, J = 15.4 Hz),
22.8 (d, J = 49.9 Hz), 119.9 (d, J = 86.6 Hz), 139.9
(d, J = 71.9 Hz), 131.9 (d, J = 13.2 Hz), 135.2 (d, J
= 10.2 Hz), 136.9 (d, J = 2.9 Hz). IR (KBr) ν: 2897,
3.1.2. [3,4-2H2]-(E)-1-(Tetrahydro-2H-pyran-
2-yloxy)hex-3-en-6-ol (10)
2.5 g (10.1 mmol) Ni(Ac)4·(H2O)4 are suspended
in 81 ml ethanol (95%) and the reaction vessel was
filled with deuterium. The suspension was treated
with 382 mg (10.1 mmol) NaBH4 in 10 ml ethanol.
After 1 min at RT 2 ml ethylenediamine (30.3 mmol)
are added. To the catalyst mixture 16 g (80.7 mmol)
1-(tetrahydro-2H-pyran-2-yloxy)hex-3-yn-6-ol (9)
was added and stirred over night under D2 atmo-
sphere. The reaction mixture was filtered over celite
and the filtrate was diluted with water. After extrac-
tion with Et2O, drying over Na2SO4, removal of the
solvent and column chromatography on silica gel
[3,4-2H2]-(Z)-1-(tetrahydro-2H-pyran-2-yloxy)hex-3-
2857, 2751, 1584, 1482, 1434, 1319, 1111, 992 cm−1
.
3.1.4. [9,10-2H2]-Ethyl
6Z,9Z,12Z,15-hexadecatetraenoate (7)
Prepared as described for the unlabeled 7 from
[3,4-2H2]-(Z)-hex-3-enyl-1,6-bis-[triphenylphospho-
1
nium bromide] (11) 1 g (1.3 mmol). Yield: 27%. H
NMR (400 MHz, CDCl3) δ = 1.24 (t, J = 7.05 Hz,
3H, O–CH2CH3); 1.38 (quint., J = 7.7 Hz, 2H,
H–C3); 1.6 (quint., J = 7.7 Hz, 2H, H–C4); 2.1 (dt.,
J = 7.4, 7.2 Hz, 2H, H–C5); 2.3 (t, J = 7.5 Hz, 2H,
H–C2); 2.8 (m, 6H, C8, C11, C14); 4.1 (quart., J
= 7.1 Hz, 2H, O–CH2CH3); 5.0 (dd, J = 10.0 Hz,
1.6, 1H, C16); 5.05 (dquart., J = 17.2 Hz, 1.7, 1H,
C16); 5.3–5.4 (m, 4H, C6, C7, C12, C13), 5.7 (m,
1H, C15). 13C NMR (100 MHz, CDCl3) δ = 14.66,
25.02, 25.97, 26.03, 27.28, 29.49, 31.92, 34.66,
60.6, 115.15, 127.46, 128.51, 129.37, 130.1, 137.14,
174.11. EI-MS (70 eV): 278 (0.8), 249 (1), 237 (3),
210 (7), 191 (9), 149 (40), 135 (49), 121 (56), 107
(71), 92 (90), 80 (100), 67 (87).
1
en-6-ol (10) was obtained in 82.1% yield. H NMR
(400 MHz, CDCl3) δ = 1.4–1.65 (m, 5H, H–C2ꢀ,
2H–C3ꢀ, 2H–C4ꢀ); 1.75–1.85 (m, 1H, H–C2ꢀ); 2.24
(t, J = 6.42 Hz, 2H, H–C5); 2.32 (t, J = 6.79 Hz, 2H,
H–C2); 3.63–3.82 (m, 2H, H–C6); 3.28–3.45 (m, 2H,
C1); 3.45–3.52 (m, 2H, H–C5ꢀ); 3.38 (s, 1H, –OH)
4.55 (t, J = 3.21 Hz, 1H, C1ꢀ). 13C NMR (100 MHz,
CDCl3) δ = 20.3, 26.7, 29.0, 31.7, 32.3, 62.1, 62.6,
67.6, 99.2, (127.9, 128.2, 128.3, 128.4, 128.5, 128.7,
128.8, C3, C4). EI-MS (70 eV): 202 (0.1), 172 (1),
101 (8), 85 (100), 82 (10), 67 (7), 57 (11).

G. Pohnert et al. / Chemistry and Physics of Lipids 131 (2004) 159–166
165
3.1.5. 6Z,9Z,12Z,15-Hexadecatetraenoic acid (8)
The free acid was prepared from the ethyl ester
7 (10 mg, 0.036 mmol) by stirring in 4 ml of a 3:1
mixture THF:H2O in the presence of LiOH (2.4 mg,
0.1 mmol) over night at room temperature. THF was
removed under reduced pressure and pH was adjusted
to 1 with dil. HCl. Extraction with Et2O (3×) and
column chromatography on SiO2 gave 8 in 84% yield.
1H NMR (400 MHz, [D4] MeOH) δ = 1.32 (quint., J
= 7.4 Hz, 2H, H–C3); 1.51 (quint., J = 7.7 Hz, 2H,
H–C4); 2.1 (dt, J = 6.6, 7.5 Hz, 2H, H–C5); 2.2 (t, J
= 7.3 Hz, 2H, H–C2); 2.72 (m, 4H, C8, C11); 2.83 (m,
2H, C14), 5.0 (dd, J = 10.2, 2.0 Hz, 1H, C16); 5.1 (dd,
J = 16.7, 2.2 Hz, 1H, C16); 5.2–5.4 (m, 4H, C6, C7,
C12, C13), 5.7 (m, 1H, C15). 13C NMR (100 MHz,
[D4] MeOH) δ = 26.11, 28.27, 30.26, 30.55, 31.3,
32.88, 35.21, 115.47, 128.42, 129.09, 129.6, 129.75,
130.42, 131.46, 138.31, 177.92.
gen, Germany). The samples were directly sealed
after sonication and a polydimethylsiloxane-coated
(100 ␮m) SPME fiber (Supelco, Bellefonte, PA) was
introduced in the headspace over the medium. Extrac-
tion was performed for 10 min at room temperature.
Evaporation of the volatiles from the fiber was di-
rectly performed within the injection port (250 ◦C)
of the GC-MS (T program: 50 ◦C [2 min, splitless]
ramped with 10 ◦C min−1 to 200 ◦C and then with
30 ◦C min−1 to 280 ◦C). Unsaturated aldehydes were
identified as described (Adolph et al., 2003). Free
fatty acids in intact and wounded T. rotula were
determined as described elsewhere (Pohnert, 2002),
6Z,9Z,12Z,15-hexadecatetraenoic acid was identified
by co-injection with the synthetic standard; GC-MS
(T program: 60 ◦C [2 min, split 1:20] ramped with
5 ◦C min−1 to 300 ◦C (2 min)).
3.1.6. [9,10-2H2]-6Z,9Z,12Z,15-Hexadecatetraenoic
acid (8)
Acknowledgements
[9,10-2H2]-8 was prepared as described for 8 from
[9,10-2H2]-ethyl 6Z,9Z,12Z,15-hexadecatetraenoate
(7) (10 mg, 0.036 mmol) in 82% yield. 1H NMR
(400 MHz, [D4] MeOH) δ = 1.31 (quint., J = 7.6 Hz,
2H, H–C3); 1.52 (quint., J = 7.7 Hz, 2H, H–C4); 2.0
(dt, J = 6.5, 7.4 Hz, 2H, H–C5); 2.2 (t, J = 7.3 Hz,
2H, H–C2); 2.7 (m, 6H, C8, C11, C14); 4.9 (d, J
= 10.2 Hz, 1H, C16); 4.95 (d, J = 17.0 Hz, 1H, C16);
5.2–5.4 (m, 6H, C6, C7, C12, C13), 5.8 (m, 1H,
C15). 13C NMR (100 MHz, [D4] MeOH) δ = 26.09,
28.22, 30.26, 30.54, 31.3, 33.87, 35.21, 117.91,
128.41, 129.09, 129.6, 131.46, 138.34, 177.96. EI-MS
(70 eV): 250 (0.3), 209 (5), 182 (7), 149 (10), 135
(14), 121 (21), 107 (35), 92 (74), 80 (100), 67 (52).
We thank Prof. Dr. W. Boland for the support of this
work and the helpful discussion during the preparation
of the manuscript. The Deutsche Forschungsgemein-
schaft and the Max-Planck-Gesellschaft is acknowl-
edged for financial support. We thank Dr. Christoph
Beckmann for the supply of 1-(tetrahydro-2H-pyran-
2-yloxy)hex-3-yn-6-ol.
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