Antibacterial activity of halogenated sesquiterpenes from Malaysian Laurencia spp.

Article abstract of DOI:10.1016/j.phytochem.2008.06.015

During our studies on Malaysian Laurencia species, brominated metabolites, tiomanene, acetylmajapolene B, and acetylmajapolene A were isolated from an unrecorded species collected at Pulau Tioman, Pahang along with known majapolene B and majapolene A. Acetylmajapolene A was a mixture of diastereomers as in the case of majapolene A. Tiomanene may be a plausible precursor for acetylmajapolenes B and A. In addition, three known halogenated sesquiterpenes and two known halogenated C₁₅ acetogenins were found from other two unrecorded species collected at Pulau Karah, Terengganu and Pulau Nyireh, Terengganu, respectively. Some of these halogenated metabolites showed moderate antibacterial activity against some marine bacteria.

Full text of DOI:10.1016/j.phytochem.2008.06.015

Phytochemistry 69 (2008) 2490–2494
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Antibacterial activity of halogenated sesquiterpenes from Malaysian Laurencia spp.
Charles Santhanaraju Vairappan a, , Minoru Suzuki b,1, Takahiro Ishii b,1, Tatsufumi Okino b,
*
Tsuyoshi Abe c, Michio Masuda d
a Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia
b Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan
c The Hokkaido University Museum, Sapporo 060-0810, Japan
d Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2008
Received in revised form 1 May 2008
Available online 19 August 2008
During our studies on Malaysian Laurencia species, brominated metabolites, tiomanene, acetylmajapo-
lene B, and acetylmajapolene A were isolated from an unrecorded species collected at Pulau Tioman,
Pahang along with known majapolene B and majapolene A. Acetylmajapolene A was a mixture of diaste-
reomers as in the case of majapolene A. Tiomanene may be a plausible precursor for acetylmajapolenes B
and A. In addition, three known halogenated sesquiterpenes and two known halogenated C15 acetogenins
were found from other two unrecorded species collected at Pulau Karah, Terengganu and Pulau Nyireh,
Terengganu, respectively. Some of these halogenated metabolites showed moderate antibacterial activity
against some marine bacteria.
Keywords:
Laurencia sp.
Rhodomelaceae
Bioactive metabolites
Halogenated sesquiterpenes
Halogenated C15 acetogenins
Antibacterial activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
1974), and pacifenol (8) (Sims et al., 1971, 1973). Furthermore,
the sample-3 produced two known halogenated C15 acetogenins,
In our continuing studies on chemical compositions of the red
algal genus Laurencia from Malaysian waters (Masuda et al.,
1997b, 1999, 2002; Suzuki et al., 2001; Vairappan et al., 2001;
Vairappan and Phang, 2005), we examined three unrecorded spe-
cies collected at different locations; sample-1 from Pulau Tioman,
Pahang, sample-2 from Pulau Karah, Terengganu, and sample-3
from Pulau Nyireh, Terengganu. Sample-1 contained brominated
sesquiterpenes 1–5 with a 4-bromo-3,3-dimethylcyclohexyl group.
The structures of new compounds, tiomanene (1), acetylmajapo-
lene B (2), and acetylmajapolene A (3), were elucidated by spectral
and chemical methods. Compounds 4 and 5 were identified as maj-
apolene B and majapolene A, respectively, which have previously
been isolated from Laurencia majuscula (Harvey) Lucas collected
off Apo Island, near the southern tip of Negros Island, Central Visa-
yas, Philippines (Erickson et al., 1995). Acetylmajapolene A (3) was
found to be a mixture of diastereomeric isomers as in the case of
majapolene A (5). The sample-2 contained three known haloge-
nated sesquiterpenes, which were identified as prepacifenol (6)
(Sims et al., 1973), prepacifenol epoxide (7) (Faulkner et al.,
which were identified as chlorofucin (9) (Howard et al., 1980)
and a bromoallene (10) (Suzuki et al., 1989). In this paper we re-
port the isolation and structural elucidation of these new and
known halogenated metabolites as well as their antibacterial activ-
ities against marine bacteria.
2. Results and discussion
A combination of column and preparative thin-layer chroma-
tography of the methanol extract of sample-1 collected from Pulau
Tioman, Pahang gave three new brominated compounds, 1, 2, and
3, in 4.4% (based on the crude extract), 5.5%, and 3.8% yield, respec-
tively, along with known related compounds 4 (10.2%) and 5
(2.2%).
Compounds 4 and 5 were identified as majapolene B and maj-
apolene A, respectively, by independent structure elucidation
using 1D (1H and 13C NMR) and 2D (1H–1H COSY, HSQC, HMBC,
and NOESY) NMR techniques together with mass spectral analyses.
Majapolene B (4) and majapolene A (5), the latter of which was an
inseparable mixture of diastereomeric isomers (5a and 5b) (Fig. 1),
have previously been isolated from L. majuscula collected off Apo
Island, Philippines (Erickson et al., 1995).
* Corresponding author. Tel.: +60 88 320000x2384; fax: +60 88 320291.
E-mail address: csv@ums.edu.my (C.S. Vairappan).
1
One of the new metabolites, compound 2 was analyzed for
Present address: Institute for Tropical Biology and Conservation, Universiti
Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia.
C17H23BrO2 by HREIMS (m/z 338.0845 (
D
mmu ꢀ3.7) [M]). The 1H
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.06.015

C.S. Vairappan et al. / Phytochemistry 69 (2008) 2490–2494
2491
a prominent peak at m/z 342 and 340 [MꢀO2]+ and a base peak at
m/z 109 [C8H13]+. A facile loss of oxygen molecule (O2) revealed
that 3 contains a peroxide group as in the case of majapolene A
(5). The 1H and 13C NMR spectra of 3 were very similar to those
of majapolene A (5), strongly indicating that 3 is also a mixture
of diastereomers. Detailed comparison of the spectral data of 3
and 5 suggested that 3 is an acetyl derivative of 5. Treatment of
5 with acetic anhydride and pyridine yielded an acetylated prod-
uct, which was identical with 3.
H
14
9
11
13
S
7
12
R
10
S
Br
8
H
2
3
5
15
CH2X
1: X = OAc
11: X = H
4
1
R =
R =
R =
The third new metabolite, compound 1, designated as tioman-
ene, had
HRFDMS (m/z 340.1049 (
the presence of an acetoxyl group at
UV spectrum of 1 revealed a absorption maximum at
15,000), strongly indicating the presence of a 1,4-disubstituted
a
molecular formula of C17H25BrO2 established by
mmu +1.1) [M]). Its IR spectrum showed
max 1730 and 1230 cmꢀ1. The
max 262 nm
6
D
m
2: X = OAc
4: X = OH
m
CH2X
CH2X
(e
cyclohexa-1,3-diene moiety in the molecule. The 1H NMR spec-
trum (Table 2) of 1 showed signals due to two quaternary methyl
groups at dH 1.07 and 1.08 (3H, s, each), an acetoxyl group at dH
2.08, two methylene groups at dH 2.10 (2H, m) and 2.14 (2H, m),
which are probably allylic, a bromomethine group at dH 3.94 (1H,
dd, J = 12.7, 4.4 Hz), an acetoxymethyl group at dH 4.54 (2H, s),
and two olefinic protons at dH 5.62 and 5.88 (1H, d, J = 5.4 Hz,
each).
3a: X = OAc
5a: X = OH
O
O
O
3b: X = OAc
5b: X = OH
R =
CH2X
Detailed analysis of the 1H and 13C NMR spectra (Table 2) as
well as HSQC and 1H–1H COSY spectra revealed the presence of
partial structural units 1a—1e in the molecule (Fig. 2). Confirma-
O
Fig. 1. Halogenated metabolites from a Laurencia sp. from Pulau Tioman.
Table 2
NMR spectrum (Table 1) of 2 showed a close resemblance to that of
majapolene B (4), but distinct differences were observed; e.g., in
the spectrum of 2, a signal due to an acetoxymethyl group ap-
peared at dH 5.07 (2H, s) instead of a hydroxymethyl group at dH
4.66 (2H, s) in the spectrum of 4. Thus, it was strongly suggested
that compound 2 is an acetyl derivative of majapolene B (4). This
was confirmed by the following chemical correlation. Treatment
of 4 with acetic anhydride and pyridine yielded an acetylated prod-
uct, which was identical with 2 in all respects.
13C NMR (100 MHz, DEPT), 1H NMR (400 MHz), and HMBC dataa for tiomanene (1)
C
13Cb(d)
1H (d)
Multiplicity, (J in Hz)
Long Range Correlations
1
2
3
4
5
6
7
8
131.4
117.6
123.7
127.7
25.3
H-3, H2-5, H2-6
H-3, H2-6,
H2-5, H2-15
H-2, H2-15
H-3, H2-15
H-2
5.62
5.88
d (5.4)
d (5.4)
2.10
2.14
2.25
1.72
1.26
m
m
26.3
40.2
45.7
dddd (13.6, 13.6, 3.4, 3.4)
m
dd (13.6, 13.6)
H-2, H2-6
H3-13, H3-14
Compound 3 was found to have a molecular formula of
C17H25BrO4 by HRESIMS (m/z 395.0834 (D
mmu +0.1) [M+Na]+).
9
10
11
37.5
66.6
34.9
H-10, H3-13, H3-14
H2-8, H2-12, H3-13, H3-14
H-10
The EIMS of 3, however, showed no molecular ion peak and instead
3.94
2.19
2.16
1.75
1.29
1.07
1.08
4.54
2.08
–
dd (12.7, 4.4)
m
m
m
m
s
s
s
s
–
Table 1
12
33.4
13C NMR (100 MHz, DEPT), 1H NMR (400 MHz), and HMBC dataa for acetylmajapolene
B (2)
13
14
15
Ac
Ac
32.4
21.2
68.1
H3-14
H3-13
C
13Cb (d)
1H (d)
Multiplicity, (J in Hz)
Long range correlations
21.7
1
2
3
4
5
6
7
8
146.6
127.7
129.3
134.6
129.3
127.7
39.5
–
–
H-3, H-5, H-6
H-3, H-6,
H-5, H2-15
H-2, H2-15
H-3, H2-15
H-2
171.7
H2-15
7.18
7.28
–
7.28
7.18
2.84
1.84
1.52
–
4.04
2.26
2.16
1.88
1.53
1.09
1.16
5.07
2.09
–
d (8.3)
d (8.3)
–
d (8.3)
d (8.3)
dddd (12.6, 12.6, 3.4, 3.4)
ddd (12.6, 3.0, 3.0)
dd (12.6, 12.6)
–
dd (12.6, 4.4)
m
a
Measured in chloroform– d1.
Assigned by HSQC spectrum.
b
H-2, H-5, H-6
H3-13, H3-14
48.6
O
H
H
9
10
11
37.8
66.3
35.2
H-10, H2-8 H3-13, H3-14
H2-11, H2-12, H3-13, H3-14
H-7, H-10
H
2
H
H
3
O
12
H
11
10
4
7
1
15
CH3
dddd (12.9, 12.9, 12.6, 3.9)
H
8
12
36.1
m
m
s
s
s
Br
H
H
6
5
H
13
14
15
Ac
Ac
32.4
21.2
66.8
21.7
171.6
H-10, H3-14
H-10, H3-13
H-3, H-5
1a
1b
1c
1d
quaternary carbon x 1 1e
CH3 x 2
s
–
H2-15
a
Measured in chloroform – d1.
Assigned by HSQC spectrum.
b
Fig. 2. Partial structural units for 1.

2492
C.S. Vairappan et al. / Phytochemistry 69 (2008) 2490–2494
tion of the partial structural units and determination of their
connectivity were made by the HMBC spectrum. Mutual long-
range correlations between two tertiary methyls at dH 1.07 (dC
32.4) and 1.08 (dC 21.2) in unit 1d, which showed further
long-range correlations to the quaternary carbon at dC 37.5 (unit
Chemical composition of the species from Pulau Tioman,
Pahang (sample-1) was very similar to that of the Philippine
L. majuscula (Erickson et al., 1995).
The sample-2 collected from Pulau Karah, Terengganu gave
oxygenated chamigrane-type sesquiterpenes 6, 7, and 8 in 4.0%,
16.0%, and 10.0% yield, respectively. Compound 6 was identified
as prepacifenol by comparison of spectral data with those of the
authentic sample, which was isolated from prepacifenol race (pre-
pacifenol-producing strains) of Laurencia nipponica Yamada
(Masuda et al., 1997a). Prepacifenol has initially been isolated from
Laurencia pacifica Kylin and Laurencia filiformis (C. Agardh) Monta-
gne (Sims et al., 1973). Compound 7 was identified as prepacifenol
epoxide, which has initially been isolated from the sea hare Aplysia
californica (Faulkner et al., 1974). Furthermore, compound 8 was
identified as pacifenol, which has first been isolated from Laurencia
tasmanica Hooker et Harvey (Sims et al., 1971, 1973). The chemical
composition of the species from Pulau Karah (sample-2) was very
similar to that of Laurencia composita Yamada (Masuda et al., 1996)
from Japanese waters.
The sample-3 collected at Pulau Nyireh, Terengganu gave two
halogenated C15 acetogenins 9 and 10 in 6.5% and 3.8% yield,
respectively. Compound 9 was identical with chlorofucin, which
has previously found in Laurencia snyderae Dawson collected from
La Jolla, California (Howard et al., 1980) and in Laurencia pannosa
Zanardini collected from An Thoi, Phu Quoc Island, Kien Giang
Province, Vietnam (Suzuki et al., 1996). (3Z)-Chlorofucin was also
isolated from the Malaysian L. pannosa collected at Pulau Talang-
Talang Kecil (1°5305000N, 109°4505900E), Kuching, Sarawak (Suzuki
et al., 2001). In addition, compound 10 was identical with a known
bromoallene previously found in Laurencia intricata Lamouroux
from Bikuni, Hokkaido, Japan (Suzuki et al., 1989) and in a Lauren-
cia sp. from Chinzei, Saga Prefecture, Japan (Suzuki et al., 2005)
(see Figs. 3 and 4).
1e), indicated that these methyls comprise
a gem-dimethyl
group. Moreover, this gem-dimethyl group showed long-range
correlations to the methine carbon at dC 66.6 (C-10) in unit 1a
and the methylene carbon at dC 45.7 (C-8) in unit 1b, thus con-
firming that the gem-dimethyl group can be inserted between
C-10 and C-8. Tiomanene, which possesses five degrees of unsat-
uration, must consist of an additional carbocyclic ring. Therefore,
C-11 in unit 1a and C-12 in unit 1b must be combined to form a
cyclohexane ring. In addition, long-range correlation between the
vinyl carbon C-2 (dC 117.6) in cyclohexadiene ring (unit 1c)
and the methine proton at dH 2.25 (H-7) confirmed the connec-
tion C-7 with C-1, thus completing a planar formula 1 for
tiomanene.
The relative stereochemistry of 1 was determined by the cou-
pling constants in the 1H NMR spectrum as well as the NOESY
experiment. The coupling constants of the methine proton (dd,
J = 12.7 and 4.4 Hz) at C-10 showed that the H-10 is axial in a
typical chair cyclohexane ring and hence the bromine atom adopts
equatorial configuration. Moreover, the J-value (dddd, 13.6, 13.6,
3.4, and 3.4 Hz) of H-7 indicated that the H-7, which is adjacent
to two axial and two equatorial protons, is also axial. Furthermore,
a NOE correlation between H-7 and H3-14 (dH 1.08) was observed
in the NOESY spectrum of 1, thus showing that tiomanene (1) has
the same relative stereochemistry at C-7 and C-10 as compounds,
2–5. It is noteworthy that tiomanene (1) partly changed into
acetylmajapolene B (2) but not into acetylmajapolene A (3) during
storage in a freezer.
To determine the absolute configurations of the isolated com-
pounds, acetylmajapolene B (2) and majapolene B (4) were sub-
jected to the vibrational circular dichroism (VCD). The chiroptical
technique, the VCD has been developed recently as a chiral method
of stereochemical analysis in which one measures the differential
absorption of left versus right circularly polarized IR radiation by
molecular vibrational transition (Nafie and Freedman, 2000;
Freedman et al., 2003; Polavarapu and He, 2004; Taniguchi et al.,
2004). In the VCD methods the absolute configuration is deter-
mined nonempirically by comparison between observed VCD spec-
tra with the simulated VCD spectra issued from the quantum
mechanical ab initio methods with density functional theory
(DFT) calculations.
The antibacterial activity for compounds obtained from Pulau
Tioman was tested against six marine bacteria isolated from algal
surface. The results of the paper disc diffusion assay are shown in
Table 3. Tiomanene (1) showed weak antibacterial activities against
four tested bacterial strains with MIC values of 40 l ,
g discꢀ1
respectively. Acetylmajapolene B (2) was inactive against all the
tested strains. Acetylmajapolene A (3), majapolene B (4), and maj-
apolene A (5) showed moderate antibacterial activities against all
the tested strains with MIC values between 10 and 40 l .
g discꢀ1
Detailed antibacterial activity of these compounds is shown in
Table 3.
As has previously been reported (Monde et al., 2006a), the re-
sults of the VCD methods indicated that the absolute configuration
is 7S and 10S in both compounds (2 and 4). In addition, the abso-
lute configuration of each diastereomer of endoperoxides (3a and
3b) was also established by the VCD application (Monde et al.,
2006b). The complete assignment for signals in the 1H and 13C
NMR spectra of each diastereomer are also reported in the previous
paper (Monde et al., 2006b).
OH
O
O
O
Br
Cl
Br
Cl
Br
Cl
Br
Br
Br
OH
OH
O
7
8
6
As mentioned above, since tiomanene (1) changed into acetyl-
majapolene B (2) during storage in a freezer, tiomanene has the
same (7S,10S)-configuration as acetylmajapolene B.
Fig. 3. Halogenated metabolites from a Laurencia sp. from Pulau Karah.
Erickson et al. (1995) have described in the literature that a
1,3-cyclohexadiene (11), which may presumably be derived from
bromonium ion-induced cyclization of a bisabolene system, is a
plausible immediate precursor to majapolenes A and B. Thus the
present finding of tiomanene (1) should confirm their proposed
biogenetic pathway. Acetylmajapolene B (2) may be derived from
tiomanene by dehydrogenation, whereas each diastereomer (3a
and 3b) of acetylmajapolene A may arise enzymatically or chemi-
cally from tiomanene by the 1,4-addition of oxygen molecule to
the conjugated diene system.
HO
Cl
H
Br
H
O
O
Br
C
C C
H
Br
O
O
H
Br
10
9
Fig. 4. Halogenated metabolites from a Laurencia sp. from Pulau Nyireh.

C.S. Vairappan et al. / Phytochemistry 69 (2008) 2490–2494
2493
343, 341 [M+H]+ (30:29), 342, 340 [M]+ (96:100); HR-FDMS m/z:
Table 3
340.1049. Calc. for C17H2579BrO, 340.1038 [M].
Antibacterial activities (MIC) of halogenated sesquiterpenes from Laurencia spp.
Test compounds (lg/disc)
3.5. Acetylmajapolene B (2)
1
2
3
4
5
Chromobacterium violaceum
Proteus mirabilis
Proteus vulgaris
Erwinia sp
Vibrio parahaemolyticus
Vibrio alginolyticus
40
40
40
–
40
–
–
–
–
–
–
–
20
20
40
20
30
30
20
20
20
10
20
20
10
30
40
30
30
30
Oil: ½a 2D5
ꢁ
ꢀ3.40° (CHCl3; c 0.22); IR mmax (neat) cmꢀ1: 1730,
1440, 1360, 1220, 1010, 960; for 1H and 13C NMR spectra, see,
Table 1; LR-EIMS m/z (rel. int.): 340, 338 [M]+ (65:65), 298, 296
(42:43) [MꢀCH2@C@O]+, 215 (46), 199 (92), 107 (100), 91 (28),
43 (34); HR-EIMS m/z: 338.0845. Calc. for C17H2379BrO2, 338.0882
[M].
Note: (–) No inhibition.
3.6. Acetylmajapolene A (3)
3. Experimental
Oil: ½a 2D5
ꢁ
ꢀ14.7° (CHCl3; c 0.18); IR mmax (neat) cmꢀ1: 1750,
3.1. General experimental procedures
1460, 1395, 1370, 1240, 1050, 970; 1H NMR, d 1.05 (3H, s, H3-
14), 1.06 (3H, s, H3-14), 1.06 (3H, s, H3-13), 1.08 (3H, s, H3-13),
1.18–1.30 (2H, m, Ha-8 and Ha-12), 1.41–1.55 (2H, m, Ha-5 and
Ha-6), 1.72–1.88 (2H, m, Hb-8 and Hb-12), ꢂ1.93 (1H, m, H-7),
1.98–2.16 (3H, m, Hb-5, Hb-6, and Ha-11a), 2.11 (3H, s, H3-Ac),
ꢂ2.20 (1H, m, Hb-11), 3.92 (1H, dd, J = 12.6, 4.2 Hz, H-10), 3.93
(1H, dd, J = 12.6, 4.2 Hz, H-10), 4.21 (1H, d, J = 12.5 Hz, Ha-15),
4.32 (1H, d, J = 12.5 Hz, Hb-15), 6.52 (2H, s, H-2 and H-3), 6.53
(2H, s, H-2 and H-3); 13C NMR, d 171.4 (COCH3), 134.6/134.2
(C2), 133.1/133.0 (C3), 79.9 (C1), 76.5 (C4), 65.8 (C10), 65.3
(C15), 41.1/41.0 (C8), 37.9/37.8 (C7), 37.3/37.2 (C9), 34.4 (C11),
32.4/32.3 (C13), 29.0/28.9 (C12), 25.9 (C6), 25.7/25.6 (C5), 21.4
(COCH3), 21.0/20.9 (C14); LR-ESIMS m/z: 397, 395 [M+Na]+; LR-
EIMS m/z (rel. int.): 342, 340 (3:3) [MꢀO2]+, 298, 296 (1:1), 282,
280 (3:3) [MꢀO2–CH3CO2H]+, 201 (2), 199 (2), 191, 189 (6:6),
109 (100), 92 (32), 67 (16), 55 (9), 43 (25); HR-ESIMS m/z:
395.0834. Calc. for C17H2579BrO4Na, 395.0833 [M+Na]+; HR-EIMS
m/z: 340.1033. Calc. for C17H2579BrO2, 340.1037 [MꢀO2] and m/z:
109.1019. Calc. for C8H13, 109.1017 [MꢀC9H12BrO4]+.
IR spectra were measured on a JASCO A-102 spectrophotometer.
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
in CDCl3 with TMS as the internal standard by using a JEOL JNM-
EX-400 spectrometer. LREIMS and HREIMS were obtained on a
JEOL JMS-FABmate spectrometer. LRFDMS and HRFDMS were ob-
tained on a JEOL JMS-SX102A spectrometer. LRESIMS and HRESIMS
were obtained on a JEOL JMS-700TZ spectrometer. Optical rota-
tions were recorded on a JASCO DIP-140 polarimeter. Si gel (Merck,
Kieselgel 60, 70–230 mesh) was used for column chromatography.
Si gel plates (Merck, Kieselgel 60254S) were used for preparative
TLC. The known compounds were identified by comparison of
spectroscopic data with those of authentic samples or those re-
ported in the literature.
3.2. Collection
Three Laurencia species were gathered from different locations in
Malaysian waters: sample-1, Teluk Juara, Pulau Tioman (2°4605200N,
104°1203500E), Pahang, on 3rd June, 1999 (SAP 094621—094625);
sample-2, Pulau Karah (5°3504300N, 103°0305000E), Terengganu, on
23rd May, 1999 (SAP 094614—094616); sample-3, Pulau Nyireh
(4°5004600N, 103°3905500E), Terengganu, on 28th June, 1999 (SAP
094617—094620). The voucher specimens are deposited in the Her-
barium of Graduate School of Science, Hokkaido University (SAP).
3.7. Acetylation of majapolene B (4)
Acetylation of 4 (2 mg) was carried out with acetic anhydride
(1 mL) and pyridine (1 mL) at room temperature for 12 h in the
usual manner to afford the corresponding acetate in almost quan-
titative yield. The resulting acetate was identical with 2 in all
respect.
3.3. Extraction and isolation of the sample-1
3.8. Acetylation of majapolene A (5)
The partially dried alga (40 g) was extracted with MeOH. The
MeOH solution was concentrated in vacuo and partitioned be-
tween Et2O and H2O. The Et2O solution was washed with water,
dried over anhydrous Na2SO4, and evaporated to leave a dark green
oil (120 mg). The extract was then fractionated by Si gel column
chromatography with a step gradient (hexane and EtOAc). Fraction
eluted with hexane–EtOAc (9:1) was subjected to preparative TLC
with hexane–EtOAc (3:1) to give 1 (5.3 mg) and 2 (6.6 mg). Frac-
tions eluted in hexane–EtOAc (8:2), hexane–EtOAc (7:3) and hex-
ane–EtOAc (6:4) were further separated via preparative TLC with
Acetylation of 5 (2 mg) was carried out with acetic anhydride
(1 mL) and pyridine (1 mL) at room temperature for 12 h in the
usual manner to afford the corresponding acetate in almost quan-
titative yield. The resulting acetate was identical with 3 in all
respect.
3.9. Extraction and isolation of the sample-2
The dried alga (90 g) was extracted with MeOH. The MeOH
solution was concentrated in vacuo and partitioned between
Et2O and H2O. The Et2O solution was washed with water, dried
over anhydrous Na2SO4, and evaporated to leave a dark green oil
(400 mg). The extract was then fractionated by Si gel column chro-
matography with a step gradient (hexane and EtOAc). Fractions
eluted with hexane–EtOAc (9:1) and hexane–EtOAc (8:2) gave 6
(16 mg), whose spectral properties were identical with those of
the authentic prepacifenol from L. nipponica (Masuda et al.,
1997a). Fractions eluted with hexane–EtOAc (8:2) and hexane–
EtOAc (7:3) gave 7 (64 mg), whose spectral properties were
identical with those of the authentic prepacifenol epoxide from
L. composita (Masuda et al., 1996). Fraction eluted with hexane–
hexane–EtOAc (3:1) to give
3 (4.6 mg), 4 (12.2 mg), and 5
(2.6 mg). Compounds 4 (½a D25
ꢁ
ꢀ16.7° (CHCl3; c 0.20)) and 5 (½a D25
ꢁ
ꢀ23.0° (CHCl3; c 0.20)) were identified as majapolene B and maj-
apolene A, respectively, by comparison of spectral data with those
reported in the literature for majapolenes B and A (Erickson et al.,
1995).
3.4. Tiomanene (1)
Oil: ½a 2D4
ꢁ
ꢀ17.4° (CHCl3; c 0.47); UV kmax (EtOH) nm: 262 (
e
15,000); IR mmax (neat) cmꢀ1: 1730, 1440, 1360, 1230, 1010, 960;
for 1H and 13C NMR spectra, see, Table 2; LR-FDMS m/z (rel. int.):

2494
C.S. Vairappan et al. / Phytochemistry 69 (2008) 2490–2494
Faulkner, D.J., Stallard, M.O., Ireland, C., 1974. Prepacifenol epoxide, a halogenated
sesquiterpene diepoxide. Tetrahedron Lett., 3571–3574.
Freedman, T.B., Cao, X., Dukor, R.K., Nafie, L.A., 2003. Absolute configuration
determination of chiral molecules in the solution state using vibrational circular
dichroism. Chirality 15, 743–758.
EtOAc (7:3) gave 8 (40 mg), whose spectral properties were identi-
cal with those of the authentic pacifenol from L. composita (Masuda
et al., 1996).
Hindler, J.A., Gonzalez, A.H., Drake, T.A., 1990. Stability of viable-bacterium
counts in liquid media used for preparation of inocula and subsequent
impact on antimicrobial susceptibility test results. J. Clinl. Microb. 28, 1271–
1275.
Howard, B.M., Schulte, G.R., Fenical, W., Solheim, B., Clardy, J., 1980. Three new vinyl
acetylenes from the marine red alga Laurencia. Tetrahedron 36, 1747–1751.
Masuda, M., Abe, T., Suzuki, T., Suzuki, M., 1996. Morphological and
chemotaxonomic studies on Laurencia composita and Lokamurae (Ceramiales,
Rhodophyta). Phycologia 35, 550–562.
Masuda, M., Abe, T., Sato, S., Suzuki, T., Suzuki, M., 1997a. Diversity of halogenated
secondary metabolites in the red alga Laurencia nipponica (Rhodomelaceae,
Ceramiales). J. Phycol. 33, 196–208.
Masuda, M., Takahashi, Y., Okamoto, K., Matsuo, Y., Suzuki, M., 1997b. Morphology
and halogenated secondary metabolites of Laurencia snackeyi (Weber-van
Bosse) statNov. (Ceramiales, Rhodophyta).. Eur. J. Phycol. 32, 293–301.
Masuda, M., Kawaguchi, S., Takahashi, Y., Okamoto, K., Suzuki, M., 1999.
Halogenated secondary metabolites of Laurencia similis (Rhodomelaceae,
Rhodophyta). Bot. Marina 42, 199–202.
Masuda, M., Abe, T., Kogame, K., Kawaguchi, S., Phang, S.M., Daitoh, M., Sakai, T.,
Takahashi, Y., Suzuki, M., 2002. Taxonomic notes on marine algae from
MalaysiaVIII. Three species of Laurencia (Rhodophyceae).. Bot. Marina 45,
571–579.
Monde, K., Taniguchi, T., Miura, N., Vairappan, C.S., Suzuki, M., 2006a. Absolute
configurations of brominated sesquiterpenes determined by vibrational circular
dichroism. Chirality 18, 335–339.
Monde, K., Taniguchi, T., Miura, N., Vairappan, C.S., Suzuki, M., 2006b. Absolute
configurations of endoperoxides determined by vibrational circular dichroism
(VCD). Tetrahedron Lett. 47, 4389–4392.
Nafie, L.A., Freedman, T.B., 2000. Vibrational optical activity theory. In: Berova, N.,
Nakanishi, K., Woody, R.W. (Eds.), Circular dichroism: principles and
applications, second ed. John Wiley & Sons, New York, pp. 97–131.
Polavarapu, P.L., He, J., 2004. Chiral analysis using mid-IR vibrational CD
spectroscopy. Anal. Chem. 76, 61A–67A.
3.10. Extraction and isolation of the sample-3
The dried alga (27 g) was extracted with MeOH. The MeOH
solution was concentrated in vacuo and partitioned between
Et2O and H2O. The Et2O solution was washed with water, dried
over anhydrous Na2SO4, and evaporated to leave a dark green oil
(137 mg). The extract was then fractionated by Si gel column chro-
matography with a step gradient (hexane and EtOAc). Fraction
eluted with hexane–EtOAc (8:2) gave 9 (8.9 mg; ½a D25
ꢀ18.5°
ꢁ
(CHCl3; c 0.80)), whose spectral properties were identical with
those of the authentic chlorofucin (Suzuki et al., 1996). Fraction
eluted with hexane–EtOAc (7:3) gave 10 (5.2 mg; ½a D25
ꢁ
+121°
(CHCl3; c 0.50)), whose spectral properties were identical with
those of the authentic bromoallene from L. intricata (Suzuki et al.,
1989).
3.11. Antibacterial bioassay
The antibacterial bioassay for compounds obtained from Pulau
Tioman was carried out using six species of marine bacteria iso-
lated from algal surface collected in the Malaysia waters. These
bacteria are Chromobacterium violaceum, Proteus mirabilis, Proteus
vulgaris, Erwinia sp., Vibrio parahaemolyticus, and Vibrio
alginolyticus.
One loopful of each organism was precultured in 20 mL of pep-
tone water (3% NaCl) overnight. The turbidity of the culture was
adjusted to an optical density (OD) McFarland 0.5 (Sonnerwirth
and Jarett, 1980; Hindler et al., 1990). Then 0.1 ml of the precul-
tured bacterial suspension was used to seed Nutrient Agar plates
(3% NaCl). Paper discs (Whatman, 6 mm) impregnated with various
amounts of the respective isolated compound were placed on the
seeded agar plates and the diameters of the inhibitory zones were
measured after the plates were incubated at 28°C for 24 h.
Sims, J.J., Fenical, W., Wing, R.M., Radlick, P., 1971. Marine natural products. I.
Pacifenol, a rare sesquiterpene containing bromine and chlorine from the red
alga, Laurencia pacifica. J. Am. Chem. Soc. 93, 3774–3775.
Sims, J.J., Fenical, W., Wing, R.M., Radlick, P., 1973. Marine natural products IV.
Prepacifenol, a halogenated epoxy sesquiterpene and precursor to pacifenol
from the red alga, Laurencia filiformis. J. Am. Chem. Soc. 95, 972.
Sonnerwirth, C.A., Jarett, L., 1980. Gradwohl’s Clinical Laboratory Methods and
Diagnosis, eighth ed. The C. V Mosby Company, St. Louis, pp. 1959–1970.
Suzuki, M., Sasage, Y., Ikura, M., Hikichi, K., Kurosawa, E., 1989. Structure revision of
okamurallene and structure elucidation of further C15 non-terpenoid
bromoallenes from Laurencia intricata. Phytochemistry 28, 2145–2148.
Suzuki, M., Takahashi, Y., Matsuo, Y., Masuda, M., 1996. Pannosallene,
a
Acknowledgements
brominated C15 nonterpenoid from Laurencia pannosa. Phytochemistry 41,
1101–1103.
Suzuki, M., Daitoh, M., Vairappan, C.S., Abe, T., Masuda, M., 2001. Novel halogenated
metabolites from the Malaysian Laurencia pannosa. J. Nat. Prod. 64, 597–
602.
Suzuki, M., Kawamoto, T., Vairappan, C.S., Ishii, T., Abe, T., Masuda, M., 2005.
Halogenated metabolites from Japanese Laurencia spp.. Phytochemistry 66,
2787–2793.
Taniguchi, T., Miura, N., Nishimura, S.-I., Monde, K., 2004. Vibrational circular
dichroism: chiroptical analysis of biomolecule. Mol. Nutr. Food Res. 48, 246–
254.
This research was supported by International Foundation of Sci-
ence (IFS) (F/4286-1) in Cooperation with the Organization for the
Prohibition of Chemical Weapon. CSV is also thankful for the sup-
port and encouragement given by the Director of Institute for Trop-
ical Biology and Conservation, Universiti Malaysia Sabah during
the course of this study.
Vairappan, C.S., Phang, M., 2005. Morphology and halochamigrene metabolites in
Spratly Island species of Laurencia majuscula (Rhodomelaceae, Ceramiales).
Malaysian J. Sci., 24:21–27.
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