Enantioselective formation of (R)-9-HPODE and (R)-9-HPOTrE in marine green alga Ulva conglobata.

Article abstract of DOI:10.1016/S0968-0896(02)00212-2

When linoleic and linolenic acid were incubated with a crude enzyme of marine green alga Ulva conglobata, the corresponding (R)-9-hydroperoxy-(10E, 12Z)-10, 12-octadecadienoic acid [(R)-9-HPODE] and (R)-9-hydroperoxy-(10E, 12Z, 15Z)-10, 12, 15-octadecatrienoic acid [(R)-9-HPOTrE] were formed with a high enantiomeric excess (>99%), respectively.

Full text of DOI:10.1016/S0968-0896(02)00212-2

Bioorganic & Medicinal Chemistry 10 (2002) 3171–3173
Enantioselective Formation of (R)-9-HPODE and (R)-9-HPOTrE
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 15 February 2002;accepted 7 June 2002
Abstract—When linoleic and linolenic acid were incubated with a crude enzyme of marine green alga Ulva conglobata, the corre-
sponding (R)-9-hydroperoxy-(10E, 12Z)-10, 12-octadecadienoic acid [(R)-9-HPODE] and (R)-9-hydroperoxy-(10E, 12Z, 15Z)-10,
1
#
2, 15-octadecatrienoic acid [(R)-9-HPOTrE] were formed with a high enantiomeric excess (>99%), respectively.
2002 Elsevier Science Ltd. All rights reserved.
3
In the intertidal zone of Japan, a variety of seaweeds, green
brown, and red algae are widely distributed. Particularly,
marine green alga Ulva spp. are widely distributed in the
temperate coastal waters throughout Japan and grows
in spring to summer to form ‘green tide’.
2-hydroperoxylate long-chain fatty acids. However, the
asymmetric oxygenation of lipoxygenase type in the
algae have not been fully investigated, and the absolute
configuration of the hydroperoxides has remained
unknown. In this paper, we describe highly enantio-
selective oxygenation of LA and LNA with a crude
enzyme of U. conglobata.
4
,5
An essential oil, which was prepared from marine green
alga Ulva conglobata by simultaneous distillation
extraction, was confirmed the odor components by GC–
MS. From this result, not only long-chain aldehydes
such as pentadecanal, (8Z)-8-heptadecenal, and
The alga was collected in the intertidal zone of Hikoshima,
Yamaguchi, Japan, in 2001 and was immediately frozen
ꢀ
at À20 C until being used. The tissue (25 g, fresh weight)
(
8Z,11Z,14Z)-8,11,14-heptadecatrienal but also short-
chain aldehydes such as hexanal, (2E)-2-hexenal, (3Z)-
-nonenal, (2E)-2-nonenal, and (2E,6Z)-2,6-nonadienal
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. The crude enzyme was incubated
3
were identified by comparison with authentic samples.
This result suggested that long-chain unsaturated
fatty acids such as linoleic (LA) and linolenic acid (LNA)
are oxidized to the corresponding 2-, 9-, and 13-hydro-
peroxides which subsequently are converted into C17,
C9, and C6 aldehydes, respectively.
ꢀ
with a substrate (0.2 mmol) at 0 C for 10 min. Then
(NH ) SO , NaCl, and tetrahydrofuran were added and
the mixture was centrifuged at 2000 g for 10 min for
separation of the organic layer. The layer was washed with
4
2
4
brine and dried over anhydrous MgSO . The extract was
4
concentrated 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
Recently, we reported that long-chain saturated and
unsaturated fatty acids such as palmitic, oleic acid, LA,
and LNA are 2-hydroperoxylated with a crude enzyme
of marine green alga Ulva pertusa to afford the corre-
sponding (R)-2-hydroperoxy acids with enantiomeric
ꢀ
for 10 min and the mixture was subjected to reversed-
phase (RP)-HPLC with Mightysil RP-18 GP column
(5 mm, 3.0 Â 250 mm) eluted with acetonitrile À0.1 M
CH CO NH (9:1, 1 mL/min). The oxygenated products
1
,2
excess (ee) of >99%. Furthermore, we found that not
only green algae but also brown and red algae can
3
2
4
were monitored with a fluorescence detector (excitation
at 365 nm and emission at 412 nm). The 9-anthrylmethyl
esters were also recorded after reduction to the alcohol
with triphenylphosphine (PPh ). With LA as a sub-
3
strate, one major peak of 9-anthrylmethyl ester was
*
5
Corresponding author. Tel.: +81-83-933-5851;fax: +81-83-933-
820;e-mail: akakabe@agr.yamaguchi-u.ac.jp
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00212-2

3172
Y. Akakabe et al. / Bioorg. Med. Chem. 10 (2002) 3171–3173
found in RP-HPLC. Indeed, when a portion of the
extract was reduced with PPh , the peak with the reten-
tion time (R ) of 6.5 min disappeared whereas the peak
t
with R of 7.5 min increased (Fig. 1). These compounds
t
were identified by comparison with the 9-anthrylmethyl
ester of autoxidation products of LA.
Chiralcel OD-H column (5 mm, 4.6 Â 250 mm) eluted
with hexane–2-propanol (98:2, 1 mL/min). The hydroxy
ester was monitored with a UV detector at 234 nm.
After comparison with racemic 9-HODE obtained by
autoxidation of LA and (S)-9-HODE (Cayman Chemi-
cal Co Ltd.), the absolute configuration of the product
was predominantly (R)-9-HODE (>99% ee) as deter-
mined by NP(chiral)-HPLC (Fig. 3).
3
In the separate experiment, the extract was esterified
with diazomethane (CH N ) at 0 C for 10 min and the
ꢀ
resulting methyl ester was reduced with PPh at 0 C for
2
2
ꢀ
The hydroxy methyl ester was also analyzed by GC–MS
with a DB-WAX (0.25 mm  60 m) after reduction of
the double bonds with H /PtO and silylation of the
3
1
0 min. Then the solution was analyzed by normal-
phase (NP)-HPLC with a Mightysil Si 60 column (5 mm,
2
2
3
1
.0 Â 250 mm) eluted with hexane–2-propanol (98:2,
hydroxy
fluoroacetamide (BSTFA). The injector was set at
group
with
bis(trimethylsilyl)tri-
mL/min). The oxygenated products were monitored
ꢀ
with a UV detector at 234 nm. The NP-HPLC showed
one major peak with R of 3.5 min (Fig. 2). The reten-
240 C. The oven temperature was programmed from 80
ꢀ
ꢀ
to 230 C at a rate of 2 C/min. The carrier gas was
helium. The mass spectrum of the hydrogenated TMS
t
tion property of this peak was identical to that of race-
mic
+
9-hydroxy-(10E,12Z).12-octadecadienoic
9-HODE) obtained by autoxidation of LA.
acid
derivative was characterized by a molecular ion (M ) at
m/z 386 and by two intense ions at m/z 229 and 259
(
(Fig. 4). The characteristic mass fragments of the deri-
For identification of the absolute configuration of
-HODE, the hydroxy methyl ester was done with a
vative were consistent with the structure of the deriva-
tive of racemic 9-HODE obtained by autoxidation of
LA.
9
Similarly, oxygenated products of LNA were extracted
from the reaction mixture and separated by RP-HPLC
with a Mightysil RP-18 GP Aqua column (5 mm, 4.5 Â
250 mm) eluted with acetonitrile–water (9:1, 1.5 mL/
min). The 9-anthrylmethyl esters of the extract showed
one major peak with R of 8.1 min. When the ester was
t
reduced with PPh , the peak was eluted at 8.6 min.
3
Further determination of the regio and stereo-
selectivity was done with the Chiralcel OD-H column
eluted with hexane–2-propanol (96.5:3.5, 1 mL/min).
After comparison based on R s and co-chromato-
t
graphy with authentic standards such as racemic 13-
hydroxy-(9Z,11E,15Z)-9, 11, 15-octadecatrienoic acid
[
7
(Æ)-13-HOTrE] with R (R) of 6.6 and R (S) of
t
t
.5 min, racemic 9-hydroxy-(10E,12Z,15Z)-10, 12,
Figure 1. Confirmation of oxygenated products from a crude enzyme
of U. conglobata by RP-HPLC.
Figure 3. Enantioseparation of HODE from U. conglobata
NP(chiral)-HPLC.
Figure 2. Regioseparation of HODE from U. conglobata by
NP-HPLC.
Figure 4.

Y. Akakabe et al. / Bioorg. Med. Chem. 10 (2002) 3171–3173
3173
1
8
5-octadecatrienoic acid [(Æ)-9-HOTrE] with R (R) of
barley18 LOXs convert LNA into (S)-9-HPOTrE, while
Hydra vulgaris LOX produces (R)-9-HPOTrE.
t
1
9
.0 and R (S) of 9.5 min, (S)-13-HOTrE constituted
t
of 0.5% (R)- and 99.5% (S)-form, and (S)-9-HOTrE
constituted of 3.1% (R)- and 96.9% (S)-form, the
methyl esters of the oxygenated products showed one
In this study, when LA and LNA were incubated with a
crude enzyme of U. conglobata, the corresponding
(R)-9-HPODE and (R)-9-HPOTrE were formed with a
high ee (>99%), respectively. This regio and stereo-
selective way differs from those found in plants. This is the
first time to find (R)-9-HPODE and (R)-9-HPOTrE in
marine algae.
major peak with R of 8.0 min. Thus, based on these
t
and on comparison with the GC–MS analysis of
authentic standard, the major peak of the extract was
identified as (R)-9-hydroperoxy-(10E,12Z,15Z)-10, 12,
1
5-octadecatrienoic acid [(R)-HPOTrE, >99% ee].
Quantification of the identified compounds was per-
formed by reference to 12-hydroxystearic acid as an
internal standard (IS). The oxygenated products were
expressed as meanÆSD of three samples. Prior to
extraction, the IS (100 nmol) was added and the extracts
were concentrated under reduced pressure. Following
derivatization with CH N , PPh , and BSTFA, the
References and Notes
1
. Akakabe, Y.;Matsui, K.;Kajiwara, T. Tetrahedron Lett.
1999, 40, 1137.
2. Akakabe, Y.;Matsui, K.;Kajiwara, T. Biosci. Biotechnol.
Biochem. 2000, 64, 2680.
. Akakabe, Y.;Matsui, K.;Kajiwara, T. Fisheries Sci. 2001, 67, 328.
. Kuo, J.-M.;Hwang, A.;Hsu, H. H.;Pan, B. S. J. Agric.
Food Chem. 1996, 44, 2073.
5. Kuo, J.-M.;Hwang, A.;Yeh, D.-B. J. Agric. Food Chem.
3
4
2
2
3
samples were analyzed by GC–MS. The oven tempera-
ture was programed from 80 to 200 C at a rate of
ꢀ
0 C/min. The area of the peaks on the ion chromato-
ꢀ
1
1
6
1
7
997, 45, 2055.
grams were calculated and compared to that observed
for the IS. When LA and LNA were added to the crude
enzyme, (R)-HODE and (R)-HOTrE were detected at
concentration of 16.98Æ0.72 and 3.51Æ0.23 nmol/g
. Siedow, J. N. Ann. Rev. Plant Physiol. Plant Mol. Biol.
991, 42, 145.
. Yamamoto, S. Biochim. Biophys. Acta 1992, 1128, 117.
8. Zimmerman, D. C.;Vick, B. A. Lipids 1973, 8, 264. Bene-
ytout, J. L.;Andrianarison, R. H.;Rakotoarisoa, Z.;Tixier,
M. Plant Physiol. 1989, 91, 367.
(
fresh weight), respectively. Other HODE and HOTrE
compounds were not detected by this GC–MS analy-
sis. Furthermore, with oleic acid and methyl ester of
LA, oxygenated products were not detectable in this
system.
9
1
. Shecher, G.;Grossman, S. Int. J. Biochem. 1983, 15, 1295.
0. Iny, D.;Pinsky, A.;Cojocoru, M.;Grossman, S. Int.
J. Biochem. 1993, 25, 1313.
1. Aarle, P. G. M. V.;Barse, M. M. J. D.;Veldink, G. A.;
Vliegenthart, J. F. G. FEBS Lett. 1991, 280, 159.
2. Kuhn, H.;Wiesner, R.;Lankin, V. Z.;Nekrasov, A.;
Alder, L.;Schewe, T. Anal. Biochem. 1987, 160, 24.
3. Poca, E.;Chable, H. R.;Moreau, J.C-.;Pages, M.;
Rigaud, M. Biochim. Biophys. Acta 1990, 1045, 107.
14. Ohta, H.;Ida, S.;Mikami, B.;Morita, Y. Plant Cell Phy-
siol. 1986, 27, 911.
5. Sanz, L. C.;Perez, A. G.;Rios, J. J.;Olias, J. M. J. Agric.
Food Chem. 1993, 41, 696. Sanz, L. C.;Perez, A. G.;Rios, J. J.;
Olias, J. M. Phytochemistry 1992, 32, 3381.
6. Galliard, T.;Phillips, D. R. Biochem. J. 1971, 124, 431.
¨
7. Regdel, D.;Ku hn, H.;Schewe, T. Biochim. Biophys. Acta
994, 210, 297.
8. Martini, D.;Buono, G.;Montillet, J.-L.;Iacazio, G. Tet-
rahedron/: Asymmetry 1996, 7, 1489.
19. DiMarzo, V.;Vardaro, R. R.;De Petrocellis, L.;Cimino,
G. Experientia 1996, 52, 120.
1
Lipoxygenases (EC 1.13.11.12, LOX) catalyze the oxy-
genation of fatty acids containing a (1Z, 4Z)-pentadiene
moiety in a regio and stereoselective way. These
1
1
6
7
enzymes are widely distributed in plants and mammals
and have further been demonstrated to occur in algae,
8
9
10
yeast, and bacteria. Among of these, the seeds of
barley, wheat, maize,13 and rice contain one major
1
1
12
14
1
LOX, which produces only (S)-9-hydroperoxy-
(10E,12Z).12-octadecadienoic acid [(S)-9-HPODE]
from LA. The seeds of legumes such as chickpea, kid-
1
1
1
1
1
5
ney, lentil, and pea bean contain two major LOXs.
Although these LOXs produce (R)-9-HPODE and
S)-13-HPODE from LA, respectively, the enantio-
selectivity of (R)-9-HPODE is relatively low. On the
(
1
6
17
other hand, potato tuber, tomato, and the seed of