2,4-Decadienals are produced via (R)-11-HPITE from arachidonic acid in marine green alga Ulva conglobata

Article abstract of DOI:10.1016/S0968-0896(03)00364-X

Marine green alga Ulva conglobata was investigated for the biogeneration of oxygenated products from exogenously added arachidonic acid (ARA). A crude enzyme from the alga afforded the detectable amount of a hydroperoxyicosatetraenoic acid (HPITE), which was identified as (R)-11-HPITE by HPLC and GC-MS. Headspace-SPME method indicated that ARA was selectively used to form 2,4-decadienals. These results showed that 2,4-decadienals are produced via (R)-11-HPITE from ARA exclusively.

Full text of DOI:10.1016/S0968-0896(03)00364-X

Bioorganic & Medicinal Chemistry11 (2003) 3607–3609
2,4-Decadienals are Produced via (R)-11-HPITE from
Arachidonic Acid in Marine Green Alga Ulva conglobata
Yoshihiko Akakabe,* Kenji Matsui and Tadahiko Kajiwara
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8515, Japan
Received 25 March 2003; revised 1 May2003; accepted 29 May2003
Abstract—Marine green alga Ulva conglobata was investigated for the biogeneration of oxygenated products from exogenously
added arachidonic acid (ARA). A crude enzyme from the alga afforded the detectable amount of a hydroperoxyicosatetraenoic acid
(HPITE), which was identified as (R)-11-HPITE byHPLC and GC–MS. Headspace–SPME method indicated that ARA was
selectivelyused to form 2,4-decadienals. These results showed that 2,4-decadienals are produced via ( R)-11-HPITE from ARA
exclusively.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
respectively.4 On the other hand, middle-chain alde-
hydes such as (2E,4Z)-2,4-decadienal and (2E,4E)-2,4-
decadienal were also detected in the oil. Recently, 2,4-
decadienals have been proposed to be formed via
11-hydroperoxyicosatetraenoic acid (11-HPITE) from
arachidonic acid (ARA) in marine diatom Thalassiosira
rotula.5 However, the asymmetric oxygenation of
lipoxygenase type in the diatom has not been fully
investigated, and the absolute configuration of the
hydroperoxides has remained unknown. Here the
author describes for the biosynthesis of 2,4-decadienals
via hydroxyeicosatetraenoic acids (HETEs) with a crude
enzyme of U. conglobata.
The aroma compounds produced bymarine algae have
been well characterized. Long-chain aldehydes such as
pentadecanal, (8Z)-8-heptadecenal, (8Z, 11Z)-8,11-hep-
tadecadienal, and (8Z, 11Z, 14Z)-8,11,14-heptadeca-
trienal are the most important flavor components
among a varietyof volatile compounds found in marine
green alga Ulva pertusa. The biosynthesis of the alde-
hydes is due to a-oxidation by a-oxygenase of long-
chain fattyacids into 2-hydroperoxyacids followed by
decarboxylation. We indicated that long-chain satu-
rated and unsaturated fattyacids such as palmitic, oleic,
linoleic, and linolenic acid are 2-hydroperoxylated with
a crude enzyme of U. pertusa to afford the correspond-
ing (R)-2-hydroperoxy acids with enantiomeric excess
(ee) of >99%.1À3 In the essential oil of Ulva conglobata,
short-chain aldehydes such as hexanal, (2E)-2-hexenal,
(3Z)-3-nonenal, (2E)-2-nonenal, and (2E, 6Z)-2,6-non-
adienal were found and identified byGC–MS. The bio-
generation was due to oxygenation by lipoxygenases of
linoleic or linolenic acid into regio- and stereo-specific
hydroperoxides, followed by enzymatic cleavage by
hydroperoxide lyases to produce the aldehydes. We
reported that linoleic and linolenic acid are oxygenated
with a crude enzyme of Ulva conglobata to produce (R)-
Results and Discussion
Confirmation and regioseparation of HPITEs
When ARA was incubated with a crude enzyme of
U. conglobata, one major peak of 9-anthrylmethyl ester
was found in RP-HPLC eluted with acetonitrile–water
(9/1). Furthermore, a portion of the extract was reduced
with triphenylphosphine (PPh3), the peak with the
retention time (tR) of 11.0 min disappeared whereas the
peak with tR of 11.5 min increased, indicating that the
product has a hydroperoxy group (Fig. 1). These com-
pounds were identified bycomparison with the
9-anthrylmethyl ester of authentic standards of HITEs
(Cayman Chemical Co Ltd.). Thus, incubation of ARA
with a crude enzyme U. conglobata led to the conversion
9-hydroperoxyoctadecadienoic
peroxyoctadecatrienoic acids with a high ee (>99%),
and
(R)-9-hydro-
*Corresponding author. Tel.: +81-83-933-5851; fax: +81-83-933-
5820; e-mail: akakabe@agr.yamaguchi-u.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00364-X

3608
Y. Akakabe et al. / Bioorg. Med. Chem. 11 (2003) 3607–3609
of the latter one (1.1%). Thus, the absolute configur-
ation of the 11-HITE was (R)-form and the optical
puritywas estimated as 97.8% ee.
Detection of 2,4-decadienals by headspace-SPME
Quantification of 2,4-decadienals was performed by
reference to dodecanal as an internal standard (IS). The
aldehydes were expressed as meanÆSD of three sam-
ples. The area of the peaks on the ion chromatograms
were calculated and compared to that observed for the
IS. When ARA was added to the crude enzyme,
(2E,4Z)-decadienal and (2E,4E)-decadienal were detec-
ted at concentration of 1.97Æ0.15 nmol/g and
0.34Æ0.07 nmol/g fresh weight (fw), respectively( Fig.
4). Other aldehydes were not confirmed by this GC–MS
analysis. On the other hand, when the incubation was
performed in the presence of trimethyl phosphite as a
reducing agent of hydroperoxy group, the formation of
aldehydes is effectively inhibited (0.44Æ0.02 nmol/g fw),
whereas the detectable amount of (R)-11-HITE was
observed byNP (chiral)-HPLC analysis. Furthermore,
with linoleic acid, the amounts of 2,4-decadienals were
not significantlyincreased in this system.
Figure 1. Confirmation of oxygenated products from a crude enzyme
of U. conglobata byRP-HPLC.
of the exogenous fattyacid into mainlyone HPITE.
The RP-HPLC analysis eluted with acetonitrile–water
(75/25) showed one major peak with tR of 57.0 min
(Fig. 2). The retention propertyof this peak was iden-
tical to that of racemic 11-HITE (Cayman Chemical Co
Ltd.). On the other hand, the mass spectrum of the
hydrogenated TMS derivative was characterized by a
molecular ion (M+) at m/z 414 and bytwo intense ions
at m/z 229 and 287 (Fig. 3). The characteristic mass
fragments of the derivative were consistent with the
structure of the derivative of racemic 11-HITE. This
HPITE obtained from the alga was characterized as
11-HPITE bymeans of RP-HPLC and GC–MS techni-
ques. Furthermore, a trace amount of 15-HPITE in the
alga was also suggested on the basis of GC–MS data.
The dioxygenated products of ARA by lipoxygenase
isolated from hairyroot cultures of Solanum tuberosum
treated with a fungal elicitor were investigated, and
11-HPITE was the most abundant oxygenated pro-
duct.6 In whole homogenates from hydroid species, the
homogenates from Hydra oligactis and Halocordyle dis-
ticha produced measurable amounts of a metabolite
which was identified as 11-HITE.7 Particularly, incu-
bation of ARA with homogenates of Hydra vulgaris
converted into mainlytwo metabolites. These were
characterized as 11-HPITE and 11-HITE. NP (chiral)-
HPLC analysis of 11-HITE revealed that this metabo-
lite was composed mainlyof the ( R)-enantiomer.8 In
this paper, the following findings were obtained: (1) the
crude enzyme with ARA showed a high preference for
Enantioseparation of HITEs
Comparison based on tRs and co-chromatographywith
authentic standards such as racemic 11-HITE with tR
(R) of 28.5 and tR (S) of 31.5 min, (S)-11-HITE (Cay-
man Chemical Co Ltd.) revealed that the 11-HITE from
the alga was constituted of the former peak (98.9%) and
Figure 2. Regioseparation of HODE from U. conglobata byNP-
HPLC. Rt, retention time.
Figure 4. Enantioseparation of HODE from U. conglobata NP
(chiral)-HPLC.
Figure 3.

Y. Akakabe et al. / Bioorg. Med. Chem. 11 (2003) 3607–3609
3609
the formation of 11-HPITE byRP-HPLC and GC–MS;
(2) NP (chiral)-HPLC analysis of the 11-HITE obtained
from the extract showed the predominance of (R)-enan-
tiomer, indicating that 11-HPITE was produced enzy-
matically; (3) SPME method showed that ARA was
selectivelyused to form 2,4-decadienals and the forma-
tion of aldehydes could be inhibited with trimethyl
phosphite. These results supported that 2,4-decadienals
are produced via (R)-11-HPITE from ARA exclusively
and stronglysuggested the presence of a lipoxygenase
and a hydroperoxide lyase in U. conglobata.
DB-WAX (0.25 mmÂ60 m) after reduction of the dou-
ble bonds with H2/PtO2 and silylation of the hydroxy
group with bis(trimethylsilyl)trifluoroacetamide (BSTFA).
The injector was set at 240ꢀC. The oven temperature was
programmed from 120 to 210 ꢀC at a rate of 10ꢀC/min.
The carrier gas was helium.
NP (chiral)-HPLC analyses of HPITEs
For identification of the absolute configuration of
HITEs, the 9-anthrylmethyl esters were done with a
Chiralcel OD-H column (5 mm, 4.6Â250 mm) eluted
with hexane-2-propanol (96.5/3.5, 1.0 mL/min). The
HITEs were monitored with a fluorescence detector
(excitation at 365 nm and emission at 412 nm).
Experimental
Materials
Headspace–SPME
The alga was collected in the intertidal zone of Hikosh-
ima, Yamaguchi, Japan, in 2002 and was immediately
frozen at À20 ꢀC until being used.
An SPME holder (Supelco) was used to perform the
experiments. A fused-silica fiber, coated with a 65 mm lay er
of polydimethylsiloxane-divinylbenzene (PDMS-DVB),
was chosen to absorb the volatile components of the flows.
For the headspace–SPME process, the crude enzyme (25
mL) was added in the flask. ARA (0.5 mmol) was admi-
nistered to the flask and the mixture was stirred at rt. After
stirred for 90 min, IS (dodecanal, 230 nmol) was added to
the mixture. The fiber was exposed in the headspace of the
stirred mixture at rt for 30 min and then it was removed
from the headspace and introduced into the GC injector
where the thermal desorption at 240 ꢀC for 10 min was
carried out. The volatile compounds were analyzed by
GC–MS with the DB-WAX. The oven temperature was
programmed from 50 to 230 ꢀC at a rate of 10 ꢀC/min.
Preparation of a crude enzyme of U. conglobata
The tissue (25 g, fresh weight) was homogenized with
0.1 M phosphate buffer (125 mL, pH 6.0) containing
0.1% Triton X-100 in a blender. After the homogenate
was filtered, the filtrate was used for the enzyme assay.
Recovery of HPITEs
The crude enzyme was incubated with a substrate (0.5
mmol) at 0 ꢀC for 10 min. Then (NH4)2SO4, NaCl, and
tetrahydrofuran were added and the mixture was cen-
trifuged at 2000g for 10 min for separation of the
organic layer. The layer was washed with brine and
dried over anhydrous MgSO4. The extract was con-
centrated under reduced pressure and the residue was
diluted with t-butyl methyl ether. A portion of the
solution was treated with 9-anthryldiazomethane at 0 ꢀC
for 10 min.
Acknowledgements
This work was supported in part bya grant-in-aid for
scientific research from the Ministryof Education, Cul-
ture, Sports, Science, and Technology, Japan (No
14760134).
RP-HPLC analyses of HPITEs
For confirmation of HPITEs, the mixture was subjected
to RP-HPLC with a Mightysil RP-18 GP Aqua column
(5 mm, 4.5Â250 mm) eluted with acetonitrile–water (9/1,
1.0 mL/min). The HPITEs were monitored with a
fluorescence detector (excitation at 365 nm and emission
at 412 nm). The 9-anthrylmethyl esters were also recor-
ded after reduction to the hydroxy forms with PPh3.
Determination of the regioselectivityof HITEs was
done with the Mightysil RP-18 GP Aqua column eluted
with acetonitrile–water (75/25, 1.5 mL/min).
References and Notes
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1999, 40, 1137.
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Biochem. 2000, 64, 2680.
3. Akakabe, Y.; Matsui, K.; Kajiwara, T. Fisheries Sci. 2001,
67, 328.
4. Akakabe, Y.; Matsui, K.; Kajiwara, T. Bioorg. Med. Chem.
2002, 10, 3171.
5. Pohnert, G. Plant Physiol. 2002, 129, 103.
6. Reddy, G. R.; Reddanna, P.; Reddy, C. C.; Curtis, W. R.
Biochem. Biophys. Res. Commun. 1992, 189, 1349.
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Villani, G.; Cimino, G. Comp. Biochem. Physiol., Part B: Bio-
chem. Mol. Biol. 1993, 106B, 901.
GC–MS analyses of derivatized HITEs
The extract was esterified with diazomethane (CH2N2)
at 0 ꢀC for 10 min and the resulting methyl ester was
reduced with PPh3 at 0 ꢀC for 10 min. The hydroxy
methyl ester was also analyzed by GC–MS with a
8. Di Marzo, V.; De Petrocellis, L.; Gianfrani, C.; Cimino, G.
Biochem. J. 1993, 295, 23.