Efficient syntheses of (10E,12Z,15Z)-9-oxo- and (9Z,11E,15E)-13-oxo-octadecatrienoic acids; two stress metabolites of wounded plants

Article abstract of DOI:10.1016/S0040-4020(02)00231-4

Configurationally pure 9-oxo-10E,12Z,15Z- and 13-oxo-9Z,11E,15E-octadecatrienoic acid are available from linolenic acid via regioselective functionalisation using lipoxygenases from soybean or tomato at specific pH conditions. Reduction of the resulting hydroperoxides followed by oxidation of the resulting allylic alcohols with Bobbitt's reagent yields the configurationally pure but labile ketotrienoic acids 4 and 5 without concomitant isomerisation.

Full text of DOI:10.1016/S0040-4020(02)00231-4

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 3271±3274
Ef®cient syntheses of (10E,12Z,15Z)-9-oxo- and
(9Z,11E,15E)-13-oxo-octadecatrienoic acids;
two stress metabolites of wounded plants
Thomas Koch, Michal Hoskovec and Wilhelm Bolandp
Max Planck Institute for Chemical Ecology, Winzerlaer Str. 10, D-07745 Jena, Germany
Received 19 November 2001; revised 6 February 2002; accepted 26 February 2002
AbstractÐCon®gurationally pure 9-oxo-10E,12Z,15Z- and 13-oxo-9Z,11E,15E-octadecatrienoic acid are available from linolenic acid via
regioselective functionalisation using lipoxygenases from soybean or tomato at speci®c pH conditions. Reduction of the resulting hydro-
peroxides followed by oxidation of the resulting allylic alcohols with Bobbitt's reagent yields the con®gurationally pure but labile
ketotrienoic acids 4 and 5 without concomitant isomerisation. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results
Oxylipins (oxidised fatty acid derivatives) play an important
role in plant biology as signal molecules for defence gene
expression.1 The biosynthetic pathway towards these bio-
active compounds starts with lipoxygenases which add
molecular oxygen to pentadienyl-units of unsaturated fatty
acids (linoleic- and, predominantly linolenic acid) yielding
hydroperoxides in a regio- and stereospeci®c fashion.
Products of hydroperoxide transformations typically
comprise a-ketols, g-hydroxy-enones, epoxyalcohols and
triols as their hydrolysis products.2 The hydroperoxides
13-HPOTE 2 and 9-HPOTE 3 serve as precursors for a
multitude of oxidation- and fragmentation products that
may act as selective signals for different defensive pathways
or represent themselves as defensive compounds acting
against invading (micro)organisms. The most prominent
terminal product of 13-HPOTE is jasmonic acid (JA), a
master switch controlling plant secondary metabolism.3 In
wounded leaves of Arabidopsis thaliana the hydroperoxides
2 or 3 are, among other transformations, reduced to the
corresponding allylic alcohols followed by oxidation to
the reactive oxotrienoic acids 13-KOTE 4 and 9-KOTE 5.4
To unravel the complexity of such signalling pathways
along with their characteristic molecules, an access to
con®gurationally de®ned octadecanoids such as 4 and 5
and other stress metabolites is of great importance. Of par-
ticular value are enzymatic methods, since they allow a
direct and regioselective functionalisation of the intact
skeleton of readily available unsaturated fatty acids.5 For
example, linolenic acid can be selectively functionalised
at C-9 or C-13 using either a 13-LOX from soybean or a
9-LOX from tomato.6±8 The resulting hydroperoxides are
readily reduced to con®gurationally stable allylic alcohols,
but their oxidation to the E,Z-ketotrienoic acids 4 or 5 is not
trivial and is often accompanied by excessive isomerisation
to the E,E-isomer. For example, oxidation of the allylic
alcohol 7 (Scheme 2; also known as didehydrochoriolic
acid)9 derived from 3 with MnO2 resulted in a low yield
of a complex mixture comprising the ketoacid 5 only as
minor compound. Decomposition of fatty acid hydro-
peroxides by Fe(III) provides an alternative access, but the
complexity of the product spectrum is not satisfying and
requires extensive puri®cation protocols.10 Previous
approaches to establish such a structural element in eicosa-
noids, e.g. 5-oxo-ETE (5-oxo-6E,8Z,11Z,14Z-eicosatetra-
enoic acid),11 utilised a dithianyl-protected building block
which was oxidatively deprotected after elaboration of the
fatty acid backbone. However, even mild deprotection
methods resulted in signi®cant isomerisation of the labile
E,Z-con®guration of the a,b-unsaturated carbonyl moiety.11
Here we report a direct and ef®cient process, utilising regio-
selective functionalisation of linolenic acid by lipoxygenases
from soybean and tomato, followed by reduction and oxida-
tion of the resulting allylic alcohols with Bobbitt's reagent
(4-acetylamino-2,2,6,6-tetramethylpiperidine-1-oxoammonium
Both compounds accumulate in leaves of A. thaliana
soon after wounding or infection with Pseudomonas
syringae and were shown to induce characteristic defence
reactions.4
Keywords: Bobbitt's reagent; hydroperoxides; linolenic acid; oxylipins;
9-KOTE; 13-KOTE; lipoxygenases.
p
Corresponding author. Tel.: 149-3641-571200; fax: 149-3641-571202;
e-mail: boland@ice.mpg.de
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00231-4

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T. Koch et al. / Tetrahedron 58 (2002) 3271±3274
Scheme 1.
Scheme 2.
perchlorate)12 to give the title compounds 4 and 5 without
concomitant isomerisation.
and 7, were tested for their ability to induce volatile
biosynthesis in freshly cut plantlets of the Lima bean.14,15
While the two keto acids 4 and 5 failed to induce volatile
biosynthesis in the Lima bean, the two hydroperoxides 2 and
3 triggered a weak emission of volatiles.
Analogous to Scheme 1, linolenic acid 1 is regioselectively
functionalised at C(13) by using a commercial lipoxygenase
from soybean at pH 9 in borate buffer to generate
13-HPOTE 2.7,8 On the other hand, stirring of linolenic
acid with a homogenate of tomatoes in a phosphate buffer
(pH 5.7) along with simultaneous aeration yielded
9-HPOTE 3 without concomitant formation of 13-HPOTE.8
The crude hydroperoxides were isolated by extraction and
aliquots were directly reduced to the corresponding alcohols
6 or 7 (see Scheme 2) by treatment with P(OCH3)3.13
Stirring of the alcohols with 1 equiv. of Bobbitt's reagent
in CH2Cl2 in the presence of silica gel afforded the keto-
trienoic acids 4 or 5 in good yield (ca. 70%) and essentially
free from unwanted E,E-isomers.
3. Conclusions
In summary, by using the well established regioselective
functionalisation of linolenic acid with lipoxygenases and
®nal oxidation of intermediary allylic alcohols to the keto
acids, we have established an ef®cient and versatile route to
the con®gurationally pure ketotrienoic acids 4 and 5 which
can be used for elicitation studies in plants. It is to be
expected that analogous sequences may also be useful for
the synthesis of highly unsaturated keto acids in the ®eld of
eicosanoids.
The keto acids 4 and 5, as well as the two hydroxy acids 6

T. Koch et al. / Tetrahedron 58 (2002) 3271±3274
3273
4. Experimental
removed in vacuo and compound 7 was puri®ed by chroma-
tography on silica gel using the same solvent as before.
Yield: 0.029 g (53%). Spectroscopic data of the products
obtained were in agreement with the literature.16
4.1. General
All reactions were run in ¯ame-dried glassware under argon.
1
Solvents and reagents were dried prior to use. H and 13C
4.1.3. (9Z,11E,15Z)-13-Oxooctadeca-9,11,15-trienoic acid
(4). A well stirred solution of the hydroxy acid resulting
from reduction of 2 (23 mg, 0.08 mM) in CH2Cl2 (300
mL) was treated at room temperature with Bobbitt's reagent
(0.027 g, 0.09 mM) and silica gel (5 mg) to catalyse the
oxidation. Stirring was continued for 30 min followed by
removal of solvent in a gentle stream of argon. The crude
residue was taken up in hexane/ethyl acetate/acetic acid
(1:1:0.1%, v/v/v) and puri®ed by column chromatography
on silica gel using the same solvent. Yield: 13.3 mg (58%).
1H NMR (500 MHz, CDCl3) d: 0.92 (t, Jˆ7.50 Hz, 3H),
1.15±1.4 (m, 8H), 1.50±1.62 (m, 2H), 2.02 (m, 2H),
2.20±2.33 (m, 4H), 3.24 (d, Jˆ6.91 Hz, 2H), 5.43±5.59
(m, 2H), 5.75±5.90 (m, 1H), 6.05 (t, Jˆ11.3 Hz, 1H),
6.13 (d, Jˆ15.3 Hz, 1H), 7.42±7.5 (dd, Jˆ11.6, 15.3 Hz,
1H). 13C NMR (125 MHz, CDCl3) d: 13.9, 20.9, 24.6,
27.2, 28.3, 28.6, 29.0, 29.4, 33.9, 40.1, 120.5, 126.9,
128.6, 135.3, 137.7, 142.8, 179.3, 198.7. MS (EI, 70 eV)
m/z: 292 (M1z, 7), 224 (16), 223 (100), 149 (29), 135 (10),
123 (12), 109 (15), 107 (17), 97 (14), 95 (27), 83 (17), 81
(64), 79 (19), 69 (24), 67 (30), 57 (23), 55 (45). IR (KBr,
neat): 3019, 2931, 2856, 1706, 1665, 1627, 1586, 1457,
1412, 1272, 1189, 996 cm21. HR-MS m/z calcd for
C18H28O3 292.2038, found 292.2039.
NMR: chemical shifts of H (500 MHz) and 13C NMR
1
(125 MHz) are given in ppm (d) down®eld relative to
TMS as internal standard. Thin layer chromatography was
performed with silica gel plates. Column chromatographic
separations were performed on silica gel (Merck, Kieselgel
60; 230±400 mesh). Soybean lipoxygenase was obtained
from Sigma±Aldrich, D-82024 Taufkirchen, Germany.
The con®gurational purity of the products was established
by gas chromatography on a capillary column, Econo-capq
EC-5 (SE 54), 15 m£0.25 mm (Alltech, Deer®eld), under
temperature programmed conditions. Free acids and
alcohols were derivatised with diazomethane and MSTFA
prior to analysis.
4.1.1. (13S,9Z,11E,15Z-Hydroxyoctadeca-9,11,15-trienoic
acid (6). Linolenic acid (1.0 g, 3.6 mM) was emulsi®ed by
sonication in a borate buffer (600 mL, 0.1 M, pH 9.0). To
the rapidly stirred emulsion (208C) was added lipoxygenase
from soybean (0.1 g, 5.3 U). After a few minutes the cloudy
solution became clear and stirring was continued for 1 h
followed by addition of dil. HCl (10 mL, 2 M). The crude
hydroperoxide was extracted with CHCl3 (3£500 mL) and
dried using anhydrous Na2SO4, followed by removal of
solvent at 208C. Puri®cation was achieved by chroma-
tography on silica gel using hexane/ethyl acetate/acetic
acid (600:400:1) for elution. Yield: 0.27 g (39%). An
aliquot of the hydroperoxide (60 mg, 0.20 mM) was reduced
to the alcohol 6 by stirring for 10 min at room temperature
with P(OCH3)3 (100 mL, 0.816 mM) in CH2Cl2 (8 mL).
Solvents and excess reagent were removed in vacuo and
the alcohol was puri®ed by chromatography on silica gel
using the same solvent as before. Yield: 0.029 g (53%). The
spectroscopic data of the compounds were in agreement
with the literature.9
4.1.4.
(10E,12Z,15Z)-9-Oxooctadeca-10,12,15-trienoic
acid (5). A well stirred solution of the hydroxy acid result-
ing from reduction of 3 (8.7 mg, 29.7 mM) in CH2Cl2
(300 mL) was treated at room temperature with Bobbitt's
reagent (10 mg) and silica gel (5 mg) to catalyse the oxida-
tion. Stirring was continued for 30 min followed by removal
of solvent in a gentle stream of argon. The crude residue was
taken up in hexane/ethyl acetate/acetic acid (1:1:0.1%,
v/v/v) and puri®ed by column chromatography on silica
1
gel. Yield: 6.0 mg (69%). H NMR (500 MHz, CDCl3) d:
0.92 (t, Jˆ7.48 Hz, 3H), 1.15±1.35 (m, 6H), 1.47±1.61 (m,
4H), 2.03 (m, 2H), 2.28 (t, Jˆ7.48 Hz, 2H), 2.5 (t, Jˆ
7.48 Hz, 2H), 3.0 (t, Jˆ7.48 Hz, 2H), 5.25 (m, 1H), 5.4
(m, 1H), 5.79 (m, 1H), 6.06 (t, Jˆ11.4 Hz, 1H), 6.12 (d,
Jˆ15.21 Hz, 1H), 7.45 (dd, Jˆ11.6, 15.21 Hz, 1H). 13C
NMR (125 MHz, CDCl3) d: 14.2, 20.6, 24.2, 24.5, 24.6,
26.5, 29.0, 29.1, 34.0, 41.0, 125.2, 126.8, 129.6, 133.3,
136.8, 140.2, 179.9, 201.1. IR (KBr, neat): 3015, 2962,
2928, 2856, 1695, 1620, 1589, 1468, 1408, 1306, 1226,
1196, 1113, 1068, 992, 966 cm21. MS (EI, 70 eV) m/z:
292 (M1z, 75), 274 (17), 223 (64), 171 (74), 149 (55), 135
(17), 125 (46), 121 (51), 107 (50), 97 (38), 95 (46), 93 (46),
83 (49), 81 (54), 79 (77), 77 (32), 69 (23), 67 (36), 55 (100).
HR-MS m/z calcd for C18H28O3 292.2038, found 292.2037.
4.1.2. (9S,10E,12Z,15Z-Hydroxyoctadeca-10,12,15-tri-
enoic acid (7). Tomatoes from the local market (1 kg)
were homogenized in a phosphate buffer (600 mL, 0.1 M,
pH 5.7) with simultaneous aeration. Then, linolenic acid
1 (1.0 g, 3.6 mM), previously emulsi®ed by sonication
(30 min) in the same buffer (300 mL) containing Tween
20 (1 mL), was added. Ef®cient stirring with simultaneous
aeration was maintained for 4 h and then the reaction was
stopped by addition of 0.2 M HCl (20 mL). The crude
hydroperoxide was extracted with ether (3£800 mL).
Separation of the phases was forced by centrifugation at
2500 rpm. Solid material accumulating at the interphase
was extracted with ether (3£100 mL) and the combined
extracts dried (anhydrous Na2SO4). Removal of solvent at
208C afforded 0.73 g (65%) of the crude hydroperoxide 3.
An aliquot of 0.10 g was puri®ed by preparative thin layer
chromatography using a ternary solvent mixture of hexane/
ethyl acetate/acetic acid (600:400:1) for elution. Yield:
0.058 g (58%). The hydroperoxide (58 mg, 0.19 mM)
was reduced to the alcohol 7 by stirring for 10 min at
room temperature with P(OCH3)3 (100 mL, 0.816 mM) in
CH2Cl2 (8 mL). Solvents and excess of reagent were
Acknowledgements
Support by the Fonds der Chemischen Industrie, Frankfurt,
and a fellowship from the Alexander von Humboldt Society
(M. Hoskovec) are gratefully acknowledged. We thank
R. Lauchli for proof reading the manuscript.

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T. Koch et al. / Tetrahedron 58 (2002) 3271±3274
9. Yadav, J. S.; Deshpande, P. K.; Sharma, G. V. M. Tetrahedron
1992, 48, 4465±4474.
References
10. Spiteller, P.; Spiteller, G. Biochim. Biophys. Acta 1998, 1392,
23±40.
1. Walling, L. L. J. Plant Growth Reg. 2000, 19, 195±216.
2. Noordermeer, M. A.; Veldink, G. A.; Vliegenthart, J. F. G.
ChemBioChem 2001, 2, 494±504.
11. Khanapure, S. P.; Shi, X.-X.; Powell, W. S.; Rochach, J.
J. Org. Chem. 1988, 63, 337±342 and 1998, 63, 8976±8982.
12. Bobbitt, J. M. J. Org. Chem. 1998, 63, 9367±9374.
13. Knowles, W. S.; Thompson, Q. E. J. Org. Chem. 1960, 25,
1031±1033.
3. Reymond, P.; Farmer, E. E. Curr. Opin. Plant Biol. 1998, 1,
404±411 and references cited therein.
4. Vollenweider, S.; Weber, H.; Stolz, S.; Chetelat, A.; Farmer,
E. E. Plant J. 2000, 24, 467±476.
14. SchuÈler, G.; Goerls, H.; Boland, W. Eur. J. Org. Chem. 2001,
9, 1663±1668.
5. Feussner, I.; KuÈhn, H. In Enzymes in Lipid Modi®cation,
Bornscheuer, U. T., Ed.; Wiley-VCH: Weinheim, 2000;
pp. 309±336.
15. Koch, T.; Krumm, T.; Jung, V.; Engelberth, J.; Boland, W.
Plant Physiol. 1999, 121, 153±162.
6. Yadav, J. S.; Nanda, S.; Bhaskar Rao, A. Tetrahedron:
Asymmetry 2001, 12, 2129±2135.
16. Shimura, M.; Mase, S.; Iwata, M.; Suzuki, A.; Watanabe, T.;
Sekizawa, Y.; Sasaki, T.; Furihata, K.; Seto, H.; Otake, N.
Agric. Biol. Chem. 1983, 47, 1983±1990.
7. Hamberg, M.; Samuelsson, B. J. Biol. Chem. 1967, 242, 5329.
8. Matthew, J. A.; Chan, H. W. S.; Galliard, T. Lipids 1977, 12,
324±326.