Flavusides A and B, antibacterial cerebrosides from the marine-derived fungus Aspergillus flavus

Article abstract of DOI:10.1248/cpb.59.1174

Flavusides A (1) and B (2), two new antibacterial cerebroside derivatives, and the previously described phomaligol A (3), kojic acid (4), methyl kojic acid (5), and dimethyl kojic acid (6) have been isolated from the extract of a marine isolate of the fungus Aspergillus flavus. The structure and absolute stereochemistry of two cerebrosides were assigned on the basis of NMR and Tandem FAB-MS/MS experiments. Compounds 1, 2, and 3 exhibited a mild antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus. The minimum inhibitory concentration (MIC) values for each strain are as follows: compounds 1 and 2 showed 15.6 μg/ml for S. aureus and 31.2 μg/ml for methicillin-resistant S. aureus and multidrug-resistant S. aureus, and compound 3 exhibited 31.2 μg/ml for S. aureus and methicillin-resistant S. aureus and 62.5 μg/ml for multidrug-resistant S. aureus.

Full text of DOI:10.1248/cpb.59.1174

1174
Note
Chem. Pharm. Bull. 59(9) 1174—1177 (2011)
Vol. 59, No. 9
Flavusides A and B, Antibacterial Cerebrosides from the Marine-Derived
Fungus Aspergillus flavus
Guohua YANG,a, # Louis SANDJO,a, # Keumja YUN,a Alain Simplice LEUTOU,a Gun-Do KIM,b
c
d
e
,a
*
Hong Dae CHOI, Jung Sook KANG, Jongki HONG, and Byeng Wha SON
b
a Department of Chemistry, Pukyong National University; Department of Microbiology, Pukyong National University;
c
Busan 608–737, Korea: Department of Chemistry, Dongeui University; Busan 614–714, Korea: d College of Dentistry,
Pusan National University; Yangsan, Gyeongnam 626–770, Korea: and e College of Pharmacy, Kyung Hee University;
Seoul 130–701, Korea. Received April 15, 2011; accepted June 7, 2011; published online June 8, 2011
Flavusides A (1) and B (2), two new antibacterial cerebroside derivatives, and the previously described
phomaligol A (3), kojic acid (4), methyl kojic acid (5), and dimethyl kojic acid (6) have been isolated from the ex-
tract of a marine isolate of the fungus Aspergillus flavus. The structure and absolute stereochemistry of two cere-
brosides were assigned on the basis of NMR and Tandem FAB-MS/MS experiments. Compounds 1, 2, and 3 ex-
hibited a mild antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and mul-
tidrug-resistant S. aureus. The minimum inhibitory concentration (MIC) values for each strain are as follows:
mg/ml for S. aureus and 31.2 mg/ml for methicillin-resistant S. aureus and
compounds 1 and 2 showed 15.6
multidrug-resistant S. aureus, and compound 3 exhibited 31.2 mg/ml for S. aureus and methicillin-resistant S.
aureus and 62.5 mg/ml for multidrug-resistant S. aureus.
Key words flavuside; cerebroside; antibacterial activity; marine-derived fungus; Aspergillus flavus
Marine microorganisms, particularly marine fungi, have
recently gained prominence as an important source of biolog-
ically active secondary metabolites.1) Among the marine
fungi, those living in association with marine algae are a par-
ticularly promising source of novel natural products due to
the special ecological niche in which they exist.2) As part of a
program to explore the bioactive metabolites produced by
fungi isolated from marine habitats, our efforts have focused
on microorganisms found in association with edible
seaweed.3) From the surface of the edible green algae,
Codium fragile (Korean name: CheongGak), we have iso-
lated a fungal strain Aspergillus flavus. When cultivated
under saline conditions, the broth and mycelium of a 1 l culti-
vation of A. flavus were found to exhibit mild antibacterial
activity against Staphylococcus aureus, methicillin-resistant
S. aureus, and multidrug-resistant S. aureus. This paper de-
scribes the isolation and structure elucidation of two antibac-
terial cerebrosides, flavusides A (1) and B (2), and identifica-
tion of four known phomaligol A (3), kojic acid (4), methyl
kojic acid (5), and dimethyl kojic acid (6).4)
13C-NMR spectrum revealed carbon resonances at dC 61.1
Flavuside A (1) was isolated as a colorless amorphous (CH2), 70.0 (CH), 73.4 (CH), 76.5 (CH), 76.9 (CH), and
solid and analyzed to reveal a molecular formula of 103.5 (CH), indicative of the presence of a b-glucopyra-
C43H81NO9 by high resolution fast atom bombardment mass noside.5) The coupling constant of the anomeric proton at dH
spectrometry (HR-FAB-MS) and 13C-NMR methods. The IR 4.11 (d, JH1–H2ꢁ7.8 Hz) and the chemical shift of the
spectrum of 1 showed bands characteristic of hydroxy anomeric carbon (dC 103.5) further confirmed the b configu-
(3333 cmꢀ1) and amide (1641 cmꢀ1) functionalities. Detailed ration of the glucose moiety (a-glucopyranoside: JH1–H2ꢁ
1
analysis of the H- and 13C-NMR spectra of 1, including 3.7 Hz; dC 98.5).6) The absolute configuration of the glucose
spectra from distortionless enhancement by polarization moiety was found to be D (vide infra). The 1H- and 13C-NMR
transfer (DEPT), correlation spectroscopy (COSY), hetero- spectra showed the presence of typical disubstituted D4 dou-
nuclear multiple quantum correlation (HMQC), and hetero- ble bond [dH 5.39 (dd, Jꢁ15.3, 6.5 Hz, H-4), dC 131.0 (CH,
nuclear multiple bond coherence (HMBC) experiments, re- C-4); dH 5.59 (dt, Jꢁ15.3, 6.0 Hz, H-5), dC 131.1 (CH, C-
vealed signals that were ascribable to a monosaccharide (an 5)]7) and an additional trisubstituted double bond [dH 5.08 (t-
anomeric proton at dH 4.11), an amide linkage (a nitro- like, Jꢁ6.2 Hz, H-9), dC 123.5 (CH, C-9); dC 134.9 (qC, C-
genated methine proton at dH 3.81 and a carbonyl carbon at 10)] in a sphingosine. The large vicinal coupling constant of
dC 173.7), and an aliphatic long chain (terminal methyl pro- the olefinic protons (JH4–H5ꢁ15.3 Hz) clearly indicated an E
tons at dH 0.85 and methylene protons at dH 1.22—1.43), in- geometry for the double bond,8) which was further supported
dicating that the compound was a glycosphingolipid. The from the chemical shift of the allylic carbons [dC 32.1 (C-6,
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: sonbw@pknu.ac.kr
These authors contributed equally to this work.
#

September 2011
1175
Fig. 2. Key FAB-CID Tandem Mass Fragmentations of the [MꢄNa]ꢄ Ions
of the FAMEs Derived from 1 and 2
Fig. 1. Key COSY and HMBC Correlations of Compounds 1 and 2
C-8)].7) The COSY correlations for H-1 through H-5 defined
the 2-amino-1,3-dioxigenated-4-ene moiety (Fig. 1). The anomeric mixture of glucose ([a]D ꢄ51), derived from acid
functional groups in 1 were identified and their connectivities hydrolysis (10% aq. H2SO4) of methyl glucopyranoside, with
elucidated using HMBC techniques. The key HMBC correla- that of the reported value (a/b-glucose: [a]D ꢄ52.7).14) On
tions, from H-1 to C-2, C-3, and C-1ꢂ, from 2-NH to C-1, C- the basis of the above data, the structure of flavuside A (1)
2, C-3, and C-1ꢃ, from H-3 to C-1 and C-5, from H-5 to C-3, was defined as 1-O-b-D-glucopyranosyl-(2S,3S,4E,9E)-2-
C-4, C-6, and C-7, from H2-7 to C-5 and C-9, from H2-8 to [(2ꢃR)-2ꢃ-hydroxyoctadecanoylamino]-10-methyl-octadeca-
C-9 and C-10, from H-9 to C-7, C-8, and C-19, from H3-19 4,9-dien-1,3-diol.
to C-9 and C-10, from 2ꢃ-OH to C-1ꢃ, C-2ꢃ, and C-3ꢃ,
Flavuside B (2) was obtained in the form of a colorless
showed the connections, C1-O-C1ꢂ and C2-NH-C1ꢃ. Both amorphous solid. A molecular formula of C43H79NO9, which
connections were indicative of 1-O-glucopyransyl-2-amido- gave five degrees of unsaturation, was established by HR-
3,2ꢃ-dihydroxy-10-methyl-4,9-diene groups (Fig. 1). The FAB-MS and 13C-NMR methods. The general features of the
spectral features revealed that compound 1 had the general UV, IR, and NMR spectra closely resembled those of flavu-
structural components of cerebroside.9) The chemical shifts side A (1), except for the appearance of an additional disub-
of C-2 at 52.8 ppm and C-3 at 70.5 ppm supported a threo stituted double bond [dH 5.43 (1H, dd, Jꢁ15.5, 5.5 Hz, H-
C2–C3 configuration, in agreement with the configuration of 3ꢃ), dC 129.0 (CH, C-3ꢃ); dH 5.67 (1H, dt, Jꢁ15.5, 6.7 Hz,
D-glucosyl-L-threo-ceramide (dC 53.7, 70.0).10) The D-erythro H-4ꢃ), dC 130.9 (CH, C-4ꢃ)] in 2 (Table 1). The geometry of
isomers of these model compounds have been reported to an additional double bond was presumed to be E due to the
display C-2/C-3 chemical shifts at dC 53.8/72.6.10) Methanol- large vicinal coupling constant of the olefinic protons
1
ysis of 1 yielded a fatty acid methyl ester (FAME), a long (JH4–H5ꢁ15.5 Hz).8) Detailed analysis of the H- and 13C-
chain sphingosine base (LCSB), and an anomeric mixture of NMR spectra of 2, including the results from DEPT, COSY,
methyl glucoside. The FAME, methyl hydroxyoctadecanoate, HMQC, and HMBC experiments, suggested that 2 was a de-
and its molecular formula were established to be C19H38O3Na hydrogenated derivative of 1, and differed only in an addi-
([MꢄNa]ꢄ at m/z 337). The enhanced peak of the a-cleavage tional double bond on the lipid base or the lipid amide units.
of alcohol at m/z 112 supported the position of the 2-hydroxy The difference occurred in the fatty acid portion of 2, deter-
group in the fatty acid moiety (Fig. 2). The optical rotation of mined by the appearance of methyl (2R,3E)-2-hydroxyoc-
the ester ([a]D ꢀ62, CHCl3) identified it as the R isomer.9,11) tadec-3-enoate ([a]D ꢀ50, CHCl3) at m/z 335 (C19H36O3Na
Therefore, the fatty acid moiety was deduced to be (2R)-2- [MꢄNa]ꢄ) upon methanolysis of 2.9) The location of the new
hydroxyoctadecanoic acid from the foregoing data. The double bond was determined by FAB-collision induced dis-
LCSB was peracetylated using acetic anhydride in pyridine, sociation (CID)-MS/MS analysis of the [MꢄNa]ꢄ ion at m/z
giving the desired triacetyl derivative. Triacetyl LCSB 152. The enhanced peak corresponding to allylic cleavage at
showed the same sign of optical rotation ([a]D ꢄ4.2, CHCl3) m/z 152 indicated that the double bond was located at C-3ꢃ
as did threo-triacetylsphingosine ([a]D ꢄ8.43, CHCl3) (the (Fig. 2). Methanolysis of 2 also yielded LCSB and a mixture
erythro epimer: [a]D ꢀ12.9, CHCl3).12) The absolute config- of methyl a- and b-glucopyranosides. The data collected for
uration at C-2 of most natural cerebroside was all-2S.9,13) The LCSB, derived from 2, were in agreement with those of 1.
threo C2–C3 configuration, the specific rotation of the tri- The optical rotation of the mixture ([a]D ꢄ64, MeOH) iden-
acetyl LCSB, and the biogenetic pathway of the natural cere- tified glucose as the D-isomer.13) On the basis of these data,
broside suggested that 1 shared the 2S,2ꢃR,3S-configuration the structure of flavuside B (2) was established as 1-O-b-D-
at the asymmetric centers. The specific rotation of the methyl glucopyranosyl-(2S,3S,4E,9E)-2-[(2ꢃR,3ꢃE)-2ꢃ-hydroxyoc-
glucopyranoside ([a]D ꢄ64, MeOH) indicated a D-configura- tadec-3ꢃ-enoylamino]-10-methyl-octadeca-4,9-dien-1,3-diol.
tion.13) The D-configuration of the glucose moiety was further Compound 2 appears to be identical in its planar structure to
supported by comparison of the specific rotation of the the sphingosine A, (3ꢃE,4E)-1-(b-D-glucopyranosyloxy)-3-

1176
Vol. 59, No. 9
Table 1. 1H- and 13C-NMR Data for Flavusides A (1) and B (2)a)
Flavuside A (1)
Flavuside B (2)
Position
d
H (mult, J)
d
C (mult)
d
H (mult, J)
dC (mult)
1
3.52, dd (10.5, 3.8)
3.92, dd (10.5, 5.6)
3.81, m
3.98, ddd (6.5, 6.5, 6.0)
5.39, dd (15.3, 6.5)
5.59, dt (15.3, 6.0)
1.91, m
1.93, m
1.96, m
5.08, deformed t (6.2)
68.7, CH2
3.50, dd (10.2, 3.8)
3.93, dd (10.2, 5.4)
3.79, m
3.98, ddd (6.7, 6.7, 5.5)
5.36, dd (15.3, 6.7)
5.56, dt (15.3, 6.0)
1.91, m
1.93, m
1.95, m
5.09, deformed t (6.2)
68.6, CH2
2
3
4
5
6
7
8
9
52.8, CH
70.5, CH
52.9, CH
70.5, CH
131.0, CH
131.1, CH
32.1, CH2
27.2, CH2
32.1, CH2
123.5, CH
134.9, qC
39.0, CH2
27.4, CH2
28.7—31.3, CH2
22.1, CH2
13.9, CH3
15.7, CH3
130.9, CH
130.9, CH
32.1, CH2
27.3, CH2
31.3, CH2
123.5, CH
134.9, qC
39.3, CH2
27.4, CH2
28.7—31.3, CH2
22.1, CH2
13.9, CH3
15.7, CH3
10
11
12
13—16
17
1.90, t (7.3)
1.33, m
1.23, m
1.23, m
0.85, dd (7.5, 5.5)
1.53, s
1.90, m
1.32, m
1.23, m
1.23, m
0.84, dd (7.0, 6.5)
1.54, s
18
19
2-NH
3-OH
7.38, d (9.4)
4.91, d (6.0)
7.38, d (9.4)
4.94, d (5.5)
1ꢃ
2ꢃ
173.7, qC
71.0, CH
172.0, qC
71.9, CH
3.81, m
4.29, m
3ꢃ
4ꢃ
1.43, m
1.22, m
1.22, m
1.22, m
0.85, dd (7.5, 5.5)
5.51, d (5.1)
34.5, CH2
24.5, CH2
28.7—31.3, CH2
22.1, CH2
13.9, CH3
5.43, dd (15.5, 5.5)
5.67, dt (15.5, 6.7)
1.95, 1.23, m
1.23, m
0.84, dd (7.0, 6.5)
5.77, d (4.0)
129.0, CH
130.9, CH
31.6, 28.7—31.3, CH2
22.1, CH2
13.9, CH3
5ꢃ—16ꢃ
17ꢃ
18ꢃ
2ꢃ-OH
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
4.11, d (7.8)
2.95, ddd (8.5,8.0,4.0)
3.13, m
3.02, m
3.06, m
103.5, CH
73.4, CH
76.5, CH
70.0, CH
76.9, CH
61.1, CH2
4.11, d (7.8)
2.95, br dd (8.5, 8.0)
3.13, m
3.03, m
3.06, m
103.5, CH
73.4, CH
76.5, CH
70.0, CH
76.9, CH
61.1, CH2
3.44, m
3.44, m
3.66, dd (11.3, 5.9)
4.96, d (4.0)
4.93, d (5.6)
4.90, d (5.1)
4.51, dd (5.9, 5.7)
3.66, dd (11.5, 5.5)
4.98, m
4.98, m
4.94, d (5.4)
4.52, t (5.5)
2ꢂ-OH
3ꢂ-OH
4ꢂ-OH
6ꢂ-OH
a) Recorded in DMSO-d6 at 400 MHz (1H) and 100 MHz (13C).
hydroxy-2-[(2ꢃ-hydroxyoctadecanyloxy)amino]-10-methyl- cytotoxic, anti-HIV-1, neuritogenic, antihepatotoxic, im-
3ꢃ,4,9-octadecatriene, reported by Li et al.15) However, direct munosuppressive, immunomodulatory, cyclooxigenase-2 inhibi-
comparison of the stereostructures of 2 and sphingosine A tory, antifungal, antimicrobial, and antifouling activities.13)
remain unsolved because neither the full discussion for the Because cerebrosides have been reported to exhibit diverse
sphingosine A nor key data, including [a]D and MS, were biological activity, a detailed investigation of the biological
presented in the published paper.15)
activities of compounds 1 and 2 would be of interest.
Compounds 1, 2, and 3 exhibited a mild antibacterial ac-
The known phomaligol A (3), kojic acid (4), methyl kojic
tivity against Staphylococcus aureus, methicillin-resistant S. acid (5), and dimethyl kojic acid (6) were also obtained in
aureus, and multidrug-resistant S. aureus. The minimum in- this investigation. They were identified by inspecting their
hibitory concentration (MIC) values to each strain are as fol- NMR spectra and comparing these data with literature val-
lows: compounds 1 and 2 exhibited 15.6 mg/ml for S. aureus ues.4)
and 31.2 mg/ml for methicillin-resistant S. aureus and mul-
Experimental
tidrug-resistant S. aureus, and compound
3 showed
General Optical rotation was determined on a Perkin Elmer model 341
polarimeter. UV/visible spectra were measured on a Hitachi U-2001 UV/Vis
spectrometer. IR spectra were recorded on a Bruker Fourier transform (FT)-
IR model IFS-88 spectrometer. 1H- (400 MHz) and 13C-NMR (100 MHz)
spectra were obtained on a JEOL JNM-ECP 400 NMR spectrometer, using
TMS or solvent peaks [DMSO-d6: 1H (d 2.50) and 13C (d 39.5)] as reference
standard. FAB-mass spectra were recorded with a JMS-700 Mstation mass
spectrometer (JEOL, Tokyo, Japan) using a MS-MP9020D data system.
31.2 mg/ml for S. aureus and methicillin-resistant S. aureus
and 62.5 mg/ml for multidrug-resistant S. aureus.
Cerebrosides are glycosphingolipids, consisting of a ce-
ramide and a single sugar residue (glucose or galactose) at C-
1. The hydrophobic ceramide portion involves a sphingoid
base and an amide-linked fatty acyl chain. These amphi-
pathic molecules have been reported to exhibit antitumor/ HPLC was performed on a YOUNG LIN-ACME HPLC system using a re-

September 2011
1177
eluted with distilled water (100 ml) and MeOH (100 ml), successively. The
elute with MeOH was concentrated to afford methyl glucopyranoside
(2.2 mg). The optical rotation recorded for methyl glucopyranoside isolated
in this study was [a]D ꢄ64 (cꢁ0.22, MeOH), which showed methyl D-glu-
copyranoside in 1 (ref 13): [a]D20 ꢄ70.8). The obtained methyl D-glucopyra-
noside was refluxed with 10% aq. H2SO4 (1 ml) for 2 h, and the reaction
mixture was neutralized with NaHCO3 and concentrated in vacuo. The reac-
tion solid was dissolved in H2O (10 ml), and then purified by Diaion HP-20
column, as described above, to yield D-glucose (1.1 mg) as a colorless syrup:
[a]D ꢄ51 (cꢁ0.1, H2O) [ref. 14): [a]D20 ꢄ52.7].
Methanolysis of 2 The methnolysis of 2 was carried out similarly to 1,
and furnished a FAME (methyl-2-hydroxyocadec-3-enoic acid, 1.0 mg), tri-
acetyl LCSB, and methyl glucopyranoside. The optical rotation and FAB-
MS of FAME were [a]D ꢀ50 (cꢁ0.06, CHCl3); FAB-MS m/z 335 [MꢄNa]ꢄ
(100), 317 (0.30), 305 (0.12), 291 (0.15), 277 (0.18), 263 (0.2), 249 (0.21),
235 (0.18), 221 (0.14), 207 (0.12), 191 (0.16), 177 (0.16), 165 (0.26), 152
(0.76). The physicochemical data for methyl glucopyranoside and triacetyl
LCSB were identical with those of 1.
versed-phase analytical column (Gemini C18, 4.6ꢅ250 mm, 5 mm) with UV
detection.
Fungal Isolation and Culture The fungal strain, Aspergillus flavus,
was isolated from the surface of the edible green algae, Codium fragile (Ko-
rean name: CheongGak), collected in GeoMun Island, Yeosu, Korea and
identified based on 18S ribosomal RNA (rRNA) analyses (SolGent Co.,
Ltd., Daejeon, Korea), identity of 99%. A voucher specimen is deposited at
Pukyong National University with the code MFA500. The fungus was cul-
tured in 2.8-l Fernbach flasks (10ꢅ1 l) at first, and then additionally cultured
two times more in culture medium consisting of soytone (0.1%), soluble
starch (1.0%), and seawater (100%). The cultures were incubated at 29 °C
for 20 d on the static condition.
Extraction and Isolation Each culture was filtered through cheesecloth
to yield broth and mycelium residue, and the resulted broth and mycelium
were extracted with EtOAc and CH2Cl2–MeOH (1 : 1) to afford broth extract
(1.9 g) and mycelium extract (2.7 g), respectively. Both extracts exhibited an-
tibacterial activity against S. aureus, methicillin-resistant S. aureus, and mul-
tidrug-resistant S. aureus. So, we combined both extracts, and subjected to
Si gel flash chromatography. Elution was performed with CH2Cl2–MeOH
(stepwise, 0—100% MeOH) to yield eight fractions. Fractions 2 and 7,
which exhibited antibacterial activity against S. aureus, methicillin-resistant
S. aureus, and multidrug-resistant S. aureus, were separated by medium-
pressure liquid chromatography (MPLC) [octadesyl silica (ODS)] using a
H2O–MeOH gradient elution to afford crude compound 3 and compounds 1
and 2, respectively. They were further purified by recycling HPLC (Gemini
C18, 4.6ꢅ250 mm, 5 mm, 1 ml/min) utilizing a 30 min gradient program of
50 to 100% MeOH in H2O to furnish 1 (16.5 mg), 2 (12 mg), and 3 (15 mg),
respectively. Compounds 4 (105 mg), 5 (15 mg), and 6 (21 mg) were isolated
from fraction 5 by the same chromatographic method above.
Antibacterial Assay The in vitro antibacterial activity of the extract and
purified samples were evaluated by a conventional 2-fold serial dilution
method using S. aureus, methicillin-resistant S. aureus, and multidrug-resist-
ant S. aureus as indicator strains. A 5 ml suspension containing 105 cells per
ml was used as inoculum of the test organism. The MIC values were deter-
mined after the inoculation for 18 h at 37 °C.16)
Acknowledgment This research was supported by the National Re-
search Foundation of Korea Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF-2008-314-F00048).
Mass spectral data were kindly provided by the Korea Basic Science Insti-
tute. Pukyong National University in the 2010 Post-Doc. Program to LS and
the second Brain Korea 21 Graduate Fellowship Grant to students are grate-
fully acknowledged (09B2519).
Flavuside A (1): was obtained as a colorless amorphous solid; [a]D20 ꢄ5
(cꢁ0.55, MeOH); IR (neat) nmax 3333, 2921, 2851, 1733, 1641, 1541, 1468,
1278, 1248, 1079, 1029, 966, 721 cmꢀ1; UV lmax (MeOH) (log e) 199 (4.1)
1
nm; H-NMR (DMSO-d6, 400 MHz) and 13C-NMR (DMSO-d6, 100 MHz):
see the Table 1; LR-FAB-MS m/z: 778 [MꢄNa]ꢄ (100), 760
[MꢄNaꢀH2O]ꢄ (0.4), 496 [MꢄNaꢀfatty acid]ꢄ (5), 396 (5), 348 (2), 244
(0.3), 203 (0.1), and 185 (0.3); HR-FAB-MS m/z: 778.5823 [MꢄNa]ꢄ
(Calcd for C43H81NO9Na, 778.5809) (D ꢄ1.8 ppm).
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Flavuside B (2): was obtained as a colorless amorphous solid; [a]D20 ꢀ8.3
(cꢁ0.4, MeOH); IR (neat) nmax 3327, 2923, 2853, 1739, 1651, 1538,
1467, 1378, 1241, 1078, 1032, 966, 721 cmꢀ1; UV lmax (MeOH) (log e)
199 (4.1) nm; 1H-NMR (DMSO-d6, 400 MHz) and 13C-NMR (DMSO-d6,
100 MHz): see the Table 1; LR-FAB-MS m/z: 776 [MꢄNa]ꢄ (100), 758
[MꢄNaꢀH2O]ꢄ (0.7), 522 (0.1), 496 [MꢄNaꢀfatty acid]ꢄ (3), 346 (2),
348 (2), 244 (0.5), 203 (1), and 185 (0.3); HR-FAB-MS m/z: 776.5644
[MꢄNa]ꢄ (Calcd for C43H79NO9Na, 776.5653) (D ꢀ1.1 ppm).
Phomaligol A (3), kojic acid (4), and methyl kojic acids (5, 6): spectro-
scopic data were virtually identical to those reported in the literature.4)
Methanolysis of 1 A solution of 1 (10.0 mg) in 9% HCl in MeOH
(2.0 ml) was refluxed at 90 °C for 10 h (N2 atmosphere). The reaction mix-
ture was neutralized with Ag2CO3 and filtrated. The residue, obtained by re-
moval of the solvent, was partitioned into MeOH (10 ml) and hexane (10 ml)
to give the hexane phase and the MeOH phase. The hexane layer was evapo-
rated under N2, and purified by Si gel flash chromatography. Elution was
performed with hexane–EtOAc (stepwise, 0—100% EtOAc) yielded the
FAME of 1 (1.0 mg): [a]D ꢀ62 (cꢁ0.1, CHCl3); FAB-MS m/z 337
[MꢄNa]ꢄ (100), 321 (0.17), 307 (0.15), 293 (0.22), 279 (0.28), 265 (0.24),
251 (0.22), 237 (0.24), 223 (0.25), 209 (0.22), 195 (0.20), 181 (0.16), 167
(0.22), 153 (0.1), 139 (0.11), 125 (0.35), 112 (0.62). The MeOH layer was
concentrated, and the residue was partitioned into EtOAc and H2O. The
EtOAc portion was dried, and peracetylated by using acetic anhydride
(0.5 ml) in pyridine (1 ml) at r.t. for 10 h (N2 atmosphere). The reaction mix-
ture was dried, and subjected to Si gel flash chromatography [hexane–EtOAc
(stepwise, 0—100% EtOAc)] to yield triacetyl LCSB of 1 (3.1 mg): [a]D
9) Wang W., Wang Y., Tao H., Peng X., Liu P., Zhu W., J. Nat. Prod., 72,
1695—1698 (2009).
10) Sarmientos F., Schwarzmann G., Sandhoff K., Eur. J. Biochem., 146,
59—64 (1985).
11) Sugimura H., Yoshida K., J. Org. Chem., 58, 4484—4486 (1993).
12) Tkaczuk P., Thornton E. R., J. Org. Chem., 46, 4393—4398 (1981).
13) Mansoor T. A., Shinde P. B., Luo X., Hong J., Lee C.-O., Sim C. J.,
Son B. W., Jung J. H., J. Nat. Prod., 70, 1481—1486 (2007), and refer-
ences therein.
14) Budavari S., O’Neil M. J., Smith A., Heckelman P. E., Kinneary J. F.
(eds.), “The Merck Index, an Encyclopedia of Chemicals, Drugs, and
Biologicals,” Twelfth Ed., Merck & Co., New Jersey, 1996, p. 4465.
15) Li H., Lin Y., Liu X., Zhou S., Vrijmoed L. L. P., Marine Sciences, 26,
57—59 (2002).
1
ꢄ4.2 (cꢁ0.3, CHCl3); H-NMR (CDCl3) dH 0.87 (3H, t, Jꢁ6.7 Hz, H3-18),
1.25 (14H, br s), 1.57 (3H, s, H3-19), 2.00—2.15 (6H, m), 2.01, 2.04, 2.07
(each 3H, s, three Ac), 3.99 (1H, m), 4.19 (1H, m), 4.32 (1H, m), 4.83—
4.87 (1H, m), 5.40 (1H, t, Jꢁ7.0 Hz), 5.46 (1H, dd, Jꢁ16.0, 9.5 Hz), 5.97
(1H, dt, Jꢁ16.0, 7.0 Hz). The H2O portion was applied to a Diaion HP-20
column (Mitsubishi Kasei, 20 ml of resin, 10ꢅ250 mm). The column was
16) Li Y., Li X., Son B. W., Choi H. D., Korean J. Pharmacogn., 34, 142—
144 (2003).