Production of octadienal in the marine diatom Skeletonema costatum

Article abstract of DOI:10.1021/ol034057c

Marine diatoms produce α,β,γ,δ-unsaturated aldehydes that have detrimental effects on the reproduction of their natural predators. The production of these defensive metabolites is suggested to involve enzymatic oxidation of polyunsaturated fatty acids. In this paper, feeding experiments with labeled precursor provide clear evidence in support of the origin of octadienals 1 and 2 from 6,9,12-hexadecatrienoic acid (5), thus proving the involvement of novel lipoxygenase/lyase activity for the oxidation of C₁₆ fatty acids.

Full text of DOI:10.1021/ol034057c

ORGANIC
LETTERS
2003
Vol. 5, No. 6
885-887
Production of Octadienal in the Marine
Diatom Skeletonema costatum
Giuliana d’Ippolito,†,‡ Giovanna Romano,‡ Tonino Caruso,§ Aldo Spinella,§
Guido Cimino,† and Angelo Fontana*
,†
Istituto di Chimica Biomolecolare (ICB) del CNR, Via Campi Flegrei 34,
80078 Pozzuoli (Napoli), Italy, Stazione Zoologica “A. Dohrn”, Villa comunale,
80121 Napoli, Italy, and Dipartimento di Chimica, Facolta` di Scienze,
UniVersity of Salerno, Via S. Allende, 84041 Baronissi (Salerno), Italy
afontana@icmib.na.cnr.it
Received January 13, 2003
ABSTRACT
Marine diatoms produce r,â,γ,δ-unsaturated aldehydes that have detrimental effects on the reproduction of their natural predators. The
production of these defensive metabolites is suggested to involve enzymatic oxidation of polyunsaturated fatty acids. In this paper, feeding
experiments with labeled precursor provide clear evidence in support of the origin of octadienals 1 and 2 from 6,9,12-hexadecatrienoic acid
(5), thus proving the involvement of novel lipoxygenase/lyase activity for the oxidation of C16 fatty acids.
Damaged or wounded marine diatoms produce R,â,γ,δ-
unsaturated aldehydes that have been suggested to induce
detrimental effects on the reproduction of planktonic preda-
tors, the herbivore copepods.1 In particular, we have recently
reported that damaged cells of Skeletonema costatum, one
of the main species forming algal blooms in world’s oceans,
produce four major compounds (1-4) responsible for the
abortive and teratogenic effects in zooplankton herbivores.2
In contrast to the great deal of interest on the ecological
role of these aldehydes,1,3 very little is known about the mo-
lecular mechanisms leading to their production in the micro-
alga. It is generally accepted that this family of oxylipins
may derive from breakdown of C20 or C18 polyunsaturated
fatty acids by lipoxygenase (LOX)/hydoperoxide lyase (HPL)
activity. The origin of octadienals 1 and 2 and octatrienal 3
of S. costatum, is, however, not a simple issue, since the
double-bond position of these compounds (belonging to ω-4
and ω-1 series) is not consistent with the composition of
LOX-sensititive C20 or C18 fatty acids in the diatom.2a,4
In this paper, we describe feeding experiments with deu-
terated precursors that substantiate the formation of 1 and
2 from 6,9,12-hexadecatrienoic acid (5) by lipoxygenase
pathway. To the best of our knowledge, formation of octa-
dienals in S. costatum is the first evidence for LOX-medi-
ated oxidation of C16 fatty acids under conditions very sim-
ilar to those occurring during wound-activated cellular
response.
† ICB.
‡ SZN.
§ University of Salerno.
(1) (a) Miralto, A.; Barone, G.; Romano, G.; Poulet, S. A.; Ianora, A.;
Russo, G. L.; Buttino, I.; Laabir, M.; Cabrini, M.; Giacobbe, M. G. Nature
1999, 402, 173. (b) Pohnert, G. Angew. Chem., Int. Ed. 2000, 39, 4352.
(2) (a) d’Ippolito, G.; Romano, G.; Iadicicco, O.; Miralto, A.; Ianora,
A.; Cimino, G.; Fontana, A. Tetrahedron Lett. 2002, 43, 6133. (b) d’Ippolito,
G.; Iadicicco, O.; Romano, G.; Fontana, A. Tetrahedron Lett. 2002, 43,
6137.
(3) (a) Wolfe, G.; Stinke, M.; Kirst, G. O. Nature 1997, 387, 894. (b)
Wolfe, G. W. Biol. Bull. 2000, 198, 225. (c) Pohnert, G. Plant Physiol.
2002, 129, 103. (d) Irigoien, X.; Harris, R. P.; Verheye, H. M.; Joly, P.;
Runge, J.; Starr, M.; Pond, D.; Campbell, R.; Shreeve, R.; Ward, P.; Smith,
A. N.; Dam, H. G.; Peterson, W.; Tirelli, V.; Koski, M.; Smith, T.; Harbour,
D.; Davidson, R. Nature 2002, 419, 387. (e) Pohnert, G. Mar. Ecol. Prog.
Ser. 2002, 245, 33.
10.1021/ol034057c CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/25/2003

Scheme 1. Synthesis of hexadecatrienoic-d6 Acid (5A)
S. costatum was grown in a 10 L tank for 1 week and
then harvested by centrifugation at 1436g. In agreement with
Gouygou,4 the diatom contained a large fraction of polyun-
saturated C16 fatty acids (65% of whole fatty acid content),
including 6,9,12-hexadecatrienoic acid (C16:3 ω-4, 26% of
C16 fatty acid content) and 6,9,12,15-hexadecatetraenoic acid
(C16:4 ω-1, 12% of C16 fatty acid content).2a Docosaesenoic
(C22:6 ω-3), eicosapentaenoic (C20:5 ω-3), and myristic
(C14:0) acids were the only other species significantly
present in the organism, whereas only a trace of C18 fatty
acids was detectable. For the biosynthetic experiments, frozen
samples of the diatom (1.5 × 106cell/g of wet weight) were
allowed to warm slowly to room temperature. Then, the cells
were suspended in 1 mL/g of distilled water, and [6,7,9,-
10,12,13-2H6]-6,9,12-hexadecatrienoic acid (5a) was added.5
The resulting suspensions were sonicated for 60 s and
extracted with acetone/H2O (1:1, three times). After partition
in a separatory funnel with CH2Cl2, the organic extracts were
concentrated at reduced pressure and derivatized with car-
betoxyethylidenetriphenylphosphorane (CET-triphenylphos-
phorane) as previously descibed.2 Purification on silica gel
column led to the mixture of CET-aldehydes 1a-4a that was
both analyzed by GC-MS and resolved by HPLC. The
hexadeuterated acid 5a was prepared as reported in Scheme
1, following a general procedure based on coupling of
propargyl halide with terminal alkyne in the presence of
cesium carbonate.6 Reduction of the resulting polyalkyne by
D2/Pd on BaSO4 as catalyst gave good yield (77%) of the
deuterated C16 ester, from which pure [6,7,9,10,12,13-2H6]-
6,9,12-hexadecatrienoic acid (5a, D6 91%) was quantitatively
obtained by basic hydrolysis.7
Figure 1. EI-MS spectrum of 2a from cells of S. costatum fed
with 5a.
+
C11H11D4O2 , m/z 183) and ethoxyl (natural C11H15O+, m/z
163; labeled C11H11D4O+, m/z 167) radicals in the mass
spectra of 1a and 2a (Figure 1).2 As expected, no trace of
labeling was detectable in the other compounds (3a and 4a),
supporting the prediction of a substrate-specific process.
The localization of the deuterium atoms, on the other hand,
was clearly established by NMR spectroscopy. In fact, the
2H NMR spectrum of 1a isolated from treated cells showed
2
2
three signals attributable to H-2 (δ 6.42), H-4 (δ 6.20),
2
and H-5 (δ 5.91) (Figure 2a). The resonance of the fourth
deuterium atom (2H-1) was only apparently missing, since
GC-MS analysis of the mixture of CET derivatives
obtained from cells treated with 5a showed clear labeling
of 1a and 2a.2 In fact, the MS spectra of these compounds
(Figure 1) showed significant M + 4 peaks (natural
+
+
C13H20O2 , m/z 208; labeled C13H16D4O2 , m/z 212) that well
agreed with the retention of four deuterium atoms. The
presence of these isotopomers was also confirmed by the
isotopic clusters associated to fragments generated by
+
diagnostic loss of ethyl (natural C11H15O2 , m/z 179; labeled
(4) Berge, J. P.; Gouygou, J. P.; Dubacq, J. P.; Durand, P. Phytochemistry
1995, 39, 1017.
(5) Feeding experiments with deuterated 6,9,12-hexadecatrienoic acid
were repeated several times using concentration of the precursor ranging
from 8.3 to 15.6 micromol/g of wet cells.
Figure 2. Downfield regions of 2H NMR (top) and 1H NMR
(bottom) of the CET derivatives of labeled (a) and commercially
available (b) trans,trans-2,4-octadienal (1a).
886
Org. Lett., Vol. 5, No. 6, 2003

it fell under the large peak of CDCl3 (δ 7.26).8 The assign-
ment was rigorously proved by comparison with the 1H NMR
spectrum of a commercially available sample of derivatized
t,t-octadienal (1a) (Figure 2b). The labeling pattern is fully
consistent with the mechanism summarized in Scheme 2 that
hexadecatrienoic acid (5), whereas trans,trans-octadienal (1)
is probably derived by a nonenzymatic process involving
the isomerization of the C-4/C-5 double bond of 2.
In previous papers, it has been shown that another marine
diatom, Thalassiosira rotula, is able to produce C10 aldehydes
by oxidation of polyunsaturated eicosanoic fatty acids.3c Our
experiments and the structural differences of 1-4 seem to
indicate that S. costatum2a possesses, at least, two lipid-
oxidizing systems differing for the substrate affinity. In fact,
in analogy with the formation of aldehydes in T. rotula,3c
heptadienal (3, C7:2 ω-3) is probably derived by LOX-
dependent peroxidation of eicosapentaenoic acid (C20:5
ω-3), and whereas the production of octadienals (1 and 2,
C8:2 ω-4) and octatrienal (3, C8:3 ω-1) is fulfilled by
oxidation of 6,9,12-hexadecatrienoic acid (C16:3 ω-4) or
6,9,12,15-hexadecatetraenoic acid (C16:4 ω-1), respectively.
In agreement with a wound-activated response, it has been
reported that the LOX-dependent formation of aldehydes in
T. rotula is retained in seawater over several minutes.3c
Analogously, oxidation of 5 and production of C8 aldehydes
continued for a few minutes under the conditions used during
our experiments. On these grounds, since only trace levels
of free fatty acids are found in intact cells,9 the mobilization
of endogenous 5 from lipid reserves may be the limiting step
in the formation of toxic aldehydes at sea. In this regard, it
is significant that the aldehyde content nearly resembles the
composition of polyunsaturated fatty acids. However, the
factors that can elicit the process might be various and further
studies are required to understand the molecular mechanism
leading to the production of these toxic compounds.
Scheme 2. Biosynthesis of Deuterated Octadienals 1 and 2 in
Damaged Cells of S. costatum
involves the oxidation of 6,9,12-hexadecatrienoic acid (5)
by LOX/HPL activity. As previously reported,1a,2a,3e no
detectable level of octadienal was found in intact cells of S.
costatum. Analogously, octadienal production was totally
inhibited when the acid 5a was added to a boiled sample of
the diatom, confirming that the aldehydes are not formed
by a spontaneous, auto-oxidative process.
In conclusion, this study proves that damaged cells of S.
costatum are able to produce octadienals 1 and 2 from 6,9,-
12-hexadecatrienoic acid by lipoxygenase pathway. As
outlined in the biogenetic proposal (Scheme 2), trans,cis-
octadienal (2) is the former product of the oxidation of
Acknowledgment. This research was partially supported
by a PharmaMar (Madrid, Spain) grant. The authors are
grateful to the personnel of the “Servizio NMR dell’ Istituto
di Chimica Biomolecolare” for the technical assistance.
(6) (a) Spinella, A.; Caruso, T.; Martino, M.; Sessa, C. Synlett 2001, 12,
1971-1973.; (b) Spinella, A.; Caruso, T.; Coluccini, C. Tetrahedron Lett.
2002, 43, 1681-1683. (c) Caruso, T.; Spinella, A. Tetrahedron: Asymmetry
2002, 13, 2071-2073.
(7) 6,9,12-Hexadecatriynoic acid methyl ester (0.82 mmol) was added
to 10 mL of MeOH containing a catalytic amount of 5% Pd on BaSO4 and
quinoline. The reaction was vigorously stirred under D2 atmosphere for 16
h. After filtration on paper, the clear solution was evaporated. The reaction
mixture was purified on silica gel and then hydrolyzed by 10% NaOH in
EtOH to give [6,7,9,10,12,13-2H6]-6,9,12-hexadecatrienoic acid (5a, 77%).
(8) The intrinsic limits of the 2H NMR spectroscopy (e.g., fast relaxation
delay) do not allow reduction of the line broadening of the signals. The
observed CDCl3 resonances are due to the natural abundance of deuterium
in CHCl3.
Supporting Information Available: NMR and MS
spectra of the biosynthetic precursor and target compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL034057C
(9) The level of the free fatty acids in nonstressed cells was established
after gentle centrifugation of the diatom culture and subsequent extraction
of the fresh pellet with hot methanol.
Org. Lett., Vol. 5, No. 6, 2003
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