Monoglycerides from the brown alga Sargassum sagamianum: Isolation, synthesis, and biological activity

Article abstract of DOI:10.1016/j.bmcl.2008.05.008

Polyunsaturated fatty acid-derived monoglycerides were characterized from the marine brown alga Sargassum sagamianum, collected from Jeju Island, Korea. A new compound of this structural class was isolated and determined to be 1-octadecatetraenoyl glycerol, by combined spectroscopic methods. Based on the structures and bioactivity of these compounds, a series of monoglycerides were synthesized using glycerol and various fatty acids. Several compounds exhibited moderate to significant inhibition of phospholipase A₂ and cyclooxygenase-2.

Full text of DOI:10.1016/j.bmcl.2008.05.008

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3589–3592
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Monoglycerides from the brown alga Sargassum sagamianum: Isolation,
synthesis, and biological activity
Hyeun Wook Chang a, Kyoung Hwa Jang b,c, Doohyun Lee b, Hee Ryong Kang b, Tae-Yoon Kim d,
b,c,
Bong Ho Lee e, Byoung Wook Choi e, Sanghee Kim b,c, , Jongheon Shin
*
*
a College of Pharmacy, Youngnam University, 214-1, Dae, Gyeongsan, Gyeongsangbuk 712-749, Korea
b College of Pharmacy, Seoul National University, 599 Gwanakro, Sillim, Kwanak, Seoul 151-742, Korea
c Natural Products Research Institute, Seoul National University, 599 Gwanakro, Sillim, Kwanak, Seoul 151-742, Korea
d Department of Dermatology, The Catholic University of Korea, 505 Banpo, Seocho, Seoul 137-040, Korea
e Department of Biotechnology, Hanbat National University, San 61-1, Duckmyung, Yuseong, Daejeon 305-719, Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyunsaturated fatty acid-derived monoglycerides were characterized from the marine brown alga Sar-
gassum sagamianum, collected from Jeju Island, Korea. A new compound of this structural class was iso-
lated and determined to be 1-octadecatetraenoyl glycerol, by combined spectroscopic methods. Based on
the structures and bioactivity of these compounds, a series of monoglycerides were synthesized using
glycerol and various fatty acids. Several compounds exhibited moderate to significant inhibition of phos-
pholipase A2 and cyclooxygenase-2.
Received 4 December 2007
Revised form 5 April 2008
Accepted 1 May 2008
Available online 4 May 2008
Keywords:
Polyunsaturated fatt acid-derived
monoglycerides
Ó 2008 Elsevier Ltd. All rights reserved.
Brown alga
1-Octadecatetraenoyl glycerol
Synthesis
Bioactivity
Inhibition of phospholipase A2
Inhibition of cyclooxygenase-2
Marine brown algae (division Phaeophyta) produce a wide vari-
ety of secondary metabolites, including terpenoids, oxylipins,
phlorotannins, volatile hydrocarbons, and mixed biogenetic prod-
ucts.1 Several of these compounds exhibit a wide spectrum of phar-
macological activities such as cytotoxic, antimicrobial, and
antioxidant activities as well as inhibitory activity against various
enzymes. During the course of our search for anti-inflammatory
natural products from marine organisms from Korea, we encoun-
tered the brown alga Sargassum sagamianum, the crude extract of
which exhibited significant inhibitory activity (>45% at 100 lg/
mL) against cyclooxygenase-2 (COX-2).
Bioassay-guided separation of the crude extract using diverse
chromatographic methods yielded a fraction consisting primarily
of polyunsaturated fatty acid-containing monoglycerides. One
compound was successfully isolated and determined to be 1-octa-
decatetraenoyl glycerol (1, Fig. 1), a novel natural product. This
finding prompted us to design and synthesize a series of monoacyl
glycerides. Here, we report the synthesis, structural determination,
and bioactivity of fatty acid-derived monoacyl glycerides.
OH
O
OH
O
1
Figure 1. Structure of natural 1.
Sargassum sagamianum was collected in the intertidal zone off
of Jeju Island, South Korea, in April 2004. The specimens were
lyophilized (dry weight, 0.2 kg), macerated, and repeatedly ex-
tracted with MeOH (3ꢀ 2 L) and CH2Cl2 (2ꢀ 2 L). The combined
crude extract (10.2 g) was partitioned between 15% aqueous MeOH
and n-hexane. The aqueous MeOH layer (7.9 g) was separated by
C18 reversed-phase vacuum flash chromatography, using sequen-
tial mixtures of MeOH and H2O as eluents. The combined fractions
(270 mg), eluting with 20 and 10% aqueous MeOH, were separated
by C18 reversed-phase HPLC (YMC ODS-A column, 25% aqueous
MeCN) to yield four fractions active against COX-2 (48–96% inhibi-
tion at 12.5 lg/mL). Proton NMR analysis revealed that all of the
fractions consisted primarily of mixtures of monoglycerides bear-
ing polyunsaturated fatty acids at their side chains. Purification
of the most bioactive fraction was accomplished by reversed-phase
* Corresponding authors. Tel.: +82 2 880 2484 (J.S.).
E-mail address: shinj@snu.ac.kr (J. Shin).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.008

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H. W. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3589–3592
HPLC (15% aqueous MeOH) to afford a single compound 1 as a col-
orless gum (8.4 mg). Compound 1 was determined to be C21H34O4
by combined HRFABMS and 13C NMR spectrometry: observed,
373.2350 (calculated for C21H34O4Na, 373.2355); UV kmax, no max-
imum; IR (KBr), 3450, 2930, 1740, and 1425 cmꢁ1. A carbonyl, four
double bonds, and three oxygenated carbons were revealed by sig-
nals at d 175.4 (1ꢀ C), 133–128 (6ꢀ CH), and 72–64 (2ꢀ CH2 and
1ꢀ CH), respectively, in the 13C NMR spectra and corresponding
signals in the 1H NMR spectra (Table 1).
O
OH
2a (
2b (
S
-form)
HO
O
+
*
R-form)
O
3
DCC, DMAP
CH2Cl2, rt
72-78%
O
4a (
4b (
R
S
-form)
-form)
O
O
*
Using this information, the structure of 1 was elucidated by
combined 2-D NMR analyses. The 1H COSY correlations among
the alkoxy protons in the region d 4.14–3.53, in conjunction with
the HSQC correlations of these protons with the carbons bearing them,
revealed the presence of a glycerol moiety. Long-range correlations of
a carbonyl carbon at d 175.4 with the oxymethylene protons at d
4.14 and 4.06 indicated the attachment of the carbonyl at C-1 of
the glycerol. A combination of the 1H COSY, TOCSY, and HMBC data
showed that a linear chain of four methylenes was directly at-
tached to the carbonyl carbon. In addition, an allylic ethyl group
was placed at the terminus of the molecule by combined 1H COSY
and HMBC analyses.
O
B(OH)3
CH3O(CH2)2OH, reflux
92-95%
OH
O
OH
*
O
1a (
1b (
R
-form)
S-form)
Scheme 1. Synthesis of 1-octadecatetraenoyl glycerol.
The remaining portion, four double bonds and three bis-allylic
methylenes, was alternately arranged in linear mode between
the alkyl chain and allylic ethyl group, on the basis of chemical
shifts of the allylic methylenes and the lack of an absorption max-
imum in the UV spectra. Thus, the structure of 1 was determined to
be 1-octadecatetraenoyl glycerol, a monoglyceride. Although fatty
acid-bearing glycerol compounds have frequently been isolated
from nature, to our knowledge, 1-octadecyltetraenoyl glycerol is
unprecedented.
To determine the absolute stereochemistry of natural mono-
glyceride 1, we synthesized each enantiomer of 1 from optically
pure 1,2-isopropylidene glycerol 2, according to a known proce-
dure.2 Octadecatetraenoic acid (ODTA, 3) was condensed with 2
in the presence of DCC and DMAP to give 4 in high yield (Scheme
1). The ketal protecting group of 4 was successfully removed by
treatment with boric acid in 2-methoxyethanol at reflux, to give
the desired 1-monoacylglycerols 1a and 1b in 92–95% yield and
sufficient purity (>95%). The impurity was most likely 2-monoacyl-
glycerol, a 1,2-acyl-migrated isomer. In this hydrolysis process,
acids other than boric acid, including TFA and Dowex, were not
useful, because the chemical yield was low and the chromato-
graphically isolated product was contaminated with significant
amounts of unidentified impurities and an acyl-migrated isomer.
The NMR spectroscopic data for the obtained 1a and 1b were iden-
tical with those of the isolated natural form. The optical rotation of
20
20
synthetic 1a was ½aꢂD ꢁ3.1 (c 1.1, MeOH) and that of 1b was ½aꢂD
+3.7 (c 0.93, MeOH). Although the optical rotation values of natural
20
1 f½aꢂD þ 1:09 ðc 0:14; MeOHÞg and synthetic 1b were slightly dif-
ferent, it seemed plausible that the absolute stereochemistry of the
natural monoglyceride 1 was S. The two enantiomers exhibited a
very similar degree of inhibitory activity against COX-2, with 35–
36% inhibition at 50 lM. This result suggests that the stereochem-
istry of the glycerol moiety is not important for biological activity.
The other COX-2-active fractions from S. sagamianum seemed to
contain various polyunsaturated fatty acid-containing 1-monogly-
cerides. However, the isolation and identification of those com-
pounds were not easy tasks, primarily due to their instability
under chromatographic conditions and low natural abundance. In-
stead of overcoming these isolation problems, we decided to syn-
thesize various 1-monoacylglycerols with fatty acids of different
chain lengths and unsaturation to find out which fatty acid resi-
due(s) was more effective biologically. Thus, we prepared 11
analogues of 1 in their racemic forms, according to the above-men-
tioned procedure. Most of the prepared monoacylglycerols have
been previously synthesized or isolated from natural sources.3
However, their biological functions have not been systematically
studied.
Table 1
1H and 13C NMR assignments for compound 1a,b
Position
1
dH
dC
66.5
4.14, dd (11.4, 4.4)
4.06, dd (11.4, 6.3)
3.81, m
3.56, dd (11.3, 5.3)
3.53, dd (11.3, 5.7)
CH2
2
3
71.2
64.12
CH
CH2
10
20
175.4
34.82
25.6
30.2
27.9
132.6
129.0c
26.6d
129.4c
129.0c
26.5d
129.3c
129.2c
26.4d
128.2
132.8
27.9
C
2.38, t (7.8)
1.64, tt (7.8, 7.3)
1.41, p (7.3)
2.09, m
5.39, m
5.38–5.32, m
2.83, m
5.38–5.32, m
5.38–5.32, m
2.83, m
5.38–5.32, m
5.38–5.32, m
2.83, m
5.31, m
5.38–5.32, m
2.08, m
CH2
CH2
CH2
CH2
CH
CH
CH2
CH
CH
CH2
CH
CH
CH2
CH
30
40
50
60
70
80
90
The prepared monoacylglycerols 5–15 were evaluated for inhib-
itory activity against COX-2 and group IIA secretory phospholipase
A2 (sPLA2-IIA). sPLA2 is a pivotal enzyme in the biosynthesis of
prostaglandins (PGs) and catalyzes the hydrolysis of glycerol-phos-
pholipids to release arachidonic acid.4 The released free arachi-
donic acid is metabolized by COX to PGH2, which is further
converted to various PGs. Several lines of evidence suggest that
the overproduction of PGs is involved in a variety of pathophysio-
logical processes, including inflammation, Alzheimer’s disease,
hypertension, heart failure, and carcinogenesis.5 Among the sPLA2
and COX members, sPLA2-IIA and COX-2, respectively, have
attracted the most interest over past decades and have been
100
110
120
130
140
150
160
170
180
CH
CH2
CH3
0.97, t (7.5)
14.6
a
Measured in CDCl3 solution.
b
Assignments were aided by a combination of 1H COSY, gHSQC, and gHMBC
experiments.
c,d
Interchangeable signals.

H. W. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3589–3592
3591
considered as attractive drug targets.6 COX-2 is up-regulated by
sPLA2-IIA, and the two are functionally coupled. Thus, we tested
our synthesized compounds against COX-2 and sPLA2-IIA, accord-
ing to previously described procedures.7,8
The inhibitory potencies, expressed as percentage inhibition, of
the synthesized monoacylglycerols are compared with that of natu-
ral 1 shown in Table 2. These data reveal interesting trends in the
structural features of monoacylglycerols. The presence and position
of the double bond and the chain length were found to influence the
activity. The removal of the double bonds of ODTA of 1 resulted in
diminished inhibitory activity in the COX-2 assay (5–9). For exam-
ple, compounds containing 0–2 double bonds, such as 5–7, were less
active than 1, and those containing three cis double bonds (8 and 9)
were associated with increased COX-2 activity. A subset of 22-car-
bon polyunsaturated fatty acid-containing derivatives (13–15),
regardless of the number of cis double bonds, exhibited appreciable
COX-2 inhibitory activities, which were considerably higher than
that of 1. On the other hand, the COX-2 inhibitory activities of com-
pounds 10–12, which have fatty acid chains of 20 carbon atoms,
weresensitivetothenumberofcisdoublebonds. Compound12with
five cis double bonds showed high inhibitory activity, but com-
pounds 10 and 11 produced increased COX-2 activity.
Table 2
Enzyme inhibitory activity of natural 1 and synthetic compounds
Compound
COX-2 inhibition, %a (at 50 lM)
sPLA2-IIA inhibition, %a (at 10 lM)
OH
O
OH
1
35.6
26.1
O
O
O
O
OH
OH
5
16.5
24.7
32.8
60.6
38.1
32.0
38.8
53.9
33.6
37.4
37.9
37.4
41.7
OH
6
O
OH
O
OH
7
20.5
O
OH
O
OH
OH
8
ꢁ12.8
ꢁ6.5
ꢁ10.5
ꢁ20.5
71.9
O
O
OH
OH
OH
9
O
O
O
OH
OH
10
11
12
13
14
15
O
O
OH
O
OH
O
OH
O
OH
86.8
O
OH
O
OH
OH
92.9
O
O
OH
OH
74.2
O
a
Values are means of three experiments.

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H. W. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3589–3592
In the sPLA2-IIA assay, all the tested compounds exhibited
21st Century Frontier Research Program, funded by the Ministry
of Science and Technology of the Korean government.
inhibitory activity similar to or higher than 1, with oleic acid-de-
rived compound 6 having the most appreciable inhibitory activity.
Compounds 5 and 7, which differ from 6 by only one cis double
bond in the fatty acid moiety, were about half as active as 6. The
polyunsaturated fatty acid-containing compounds generally exhib-
ited modest activity, regardless of the number of cis double bonds.
However, dihomo-c-linolenic acid-derived compound 10 showed
inhibitory activity against sPLA2-IIA comparable to that of 6.
These results suggest that the appropriate presentation of the
cis double bond may be important in the inhibitory activity of
monoacylglycerols against COX-2 and sPLA2-IIA. Although our re-
sults do not provide conclusive information on the structure-activ-
ity relationships, they suggest the possibility of developing potent
and well-balanced dual inhibitors of both enzymes, as well as spe-
cific inhibitors of selected PG biosynthetic enzymes. The develop-
ment of such compounds is likely to be feasible, considering that
compounds 12–15 exhibited appreciable inhibitory potency
against both COX-2 and sPLA2-IIA, whereas 10 showed inhibitory
activity against only sPLA2-IIA.
In conclusion, we isolated and determined the structure of
monoglycerides from the brown alga S. sagamianum. Based on
the COX-2 inhibitory activity of a novel compound, 1-octadeca-
tetraenoyl glycerol, we synthesized a series of monoglycerides.
Bioactivity tests revealed that several compounds of this struc-
tural class had moderate to significant inhibitory activity to-
ward COX-2 and sPLA2-IIA. It is well recognized that the
polyunsaturated fatty acids are chemically and metabolically
unstable. Therefore, the monoglycerides would not be good po-
tential lead compounds for drug development. However, the
information obtained may facilitate the design of novel inhibi-
tors of COX-2 and sPLA2-IIA.
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the medium were determined in triplicate using
a commercially available
enzyme immunoabsorbent assay (EIA) kit (Cayman Chemical Company, Ann
Arbor, MI), following the manufacturer’s protocol. The assay was performed in a
total volume of 150 lL, with the following components added in 50 lL volumes:
standards or biological samples, enzymatic tracer, and specific antiserum. After
overnight incubation at 4 °C, the plates were washed, and 200 lL of Ellman’s
reagent was added to each well. After 1–3 h, the absorbance of each well was
measured at 414 nm. A standard curve with values of 50–0.39 pg/mL was used
to evaluate the concentrations.
Acknowledgments
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J.; Garcia-Carmona, F.; Perez-Gilabert, M. Anal. Biochem. 2003, 319, 131.
Phospholipase A2 activity was determined by means of a coupled enzyme
assay using DL-PC as a substrate and lipoxygenase as a coupling enzyme. The
linoleic acid released by phospholipase activity was oxidized by lipoxygenase,
giving rise to the corresponding hydroperoxide derivative. The phospholipase
activity was followed by measuring the absorbance at 234 nm.
The authors thank J.T. Yoon, National Fisheries Research &
Development Institute, Yeosu, Korea for the identification of the al-
gal specimen. This research was supported in part by a Grant
(PF06218-01) from the Plant Diversity Research Center of the