Cytotoxic Glycosylated Fatty Acid Amides from a Stelletta sp. Marine Sponge

Article abstract of DOI:10.1021/acs.jnatprod.5b00795

We have discovered new glycosylated fatty acid amides, stellettosides, from a Stelletta sp. marine sponge. They were detected through LC-MS analysis of the extract combined with the cytotoxicity assay of the prefractionated sample. Their planar structures were determined by analyses of the NMR and tandem FABMS data. Stellettosides A1 and A2 (1 and 2) as well as stellettosides B1-B4 (3-6) were obtained as inseparable mixtures. Careful analysis of the NMR and tandem FABMS data of each mixture, along with comparison of the tandem FABMS data with that of a synthetic model compound, permitted us to assign the structure of the constituents in the mixture. The absolute configuration of the monosaccharide unit was determined by LC-MS after chiral derivatization. The relative configurations of the vicinal oxygenated methines in the fatty acid chains were assigned by the ¹H NMR data of the isopropylidene derivative. The mixture of stellettosides B1-B4 (3-6) exhibit moderate cytotoxic activity against HeLa cells with an IC₅₀ value of 9 ΜM, whereas the mixture of stellettosides A1 and A2 (1 and 2) was not active at a concentration of 10 ΜM.

Full text of DOI:10.1021/acs.jnatprod.5b00795

Article
pubs.acs.org/jnp
Cytotoxic Glycosylated Fatty Acid Amides from a Stelletta sp. Marine
Sponge
Victoria Peddie,† Kentaro Takada,*,† Shujiro Okuda,‡ Yuji Ise,§ Yasuhiro Morii,⊥ Nobuhiro Yamawaki,⊥
Tomohiro Takatani,⊥ Osamu Arakawa,⊥ Shigeru Okada,† and Shigeki Matsunaga*,†
†Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
‡Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8510, Japan
§Sugashima Marine Biological Laboratory, Nagoya University, Mie 517-0004, Japan
⊥Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki 852-8521, Japan
S
* Supporting Information
ABSTRACT: We have discovered new glycosylated fatty
acid amides, stellettosides, from a Stelletta sp. marine sponge.
They were detected through LC-MS analysis of the extract
combined with the cytotoxicity assay of the prefractionated
sample. Their planar structures were determined by analyses of
the NMR and tandem FABMS data. Stellettosides A1 and A2
(1 and 2) as well as stellettosides B1−B4 (3−6) were obtained
as inseparable mixtures. Careful analysis of the NMR and tandem FABMS data of each mixture, along with comparison of the
tandem FABMS data with that of a synthetic model compound, permitted us to assign the structure of the constituents in
the mixture. The absolute configuration of the monosaccharide unit was determined by LC-MS after chiral derivatization. The
1
relative configurations of the vicinal oxygenated methines in the fatty acid chains were assigned by the H NMR data of the
isopropylidene derivative. The mixture of stellettosides B1−B4 (3−6) exhibit moderate cytotoxic activity against HeLa cells with
an IC50 value of 9 μM, whereas the mixture of stellettosides A1 and A2 (1 and 2) was not active at a concentration of 10 μM.
mong many glycolipids isolated from marine sponges are a
limited number of glycosylated fatty acid amides:1,2
of the HPLC to sharpen the peaks. However, because the end
absorption of acetic acid obscured the end absorption of the
amide group in the stellettosides and because the stellettosides
gave broad HPLC peaks, we were not able to collect them as
distinct HPLC peaks. Therefore, we collected eluents at regular
intervals, and fractions exhibiting the same LC-MS data were
combined to afford the two fractions. Fraction 1 turned out to be
a mixture of two isomers, stellettosides A1 (1) and A2 (2),
whereas fraction 2 was presumably a mixture of four isomers,
stellettosides B1−B4 (3−6).
The molecular formula of C56H99N2O20 was assigned for the
constituents in fraction 1 on the basis of the HRESIMS data.
Even though fraction 1 appeared homogeneous in the LC-MS
analysis, it turned out to be a mixture of two isomeric compounds
differing in the structure of the alkyl chain. The planar structure
of each component was determined by interpretation of the
NMR data and tandem FABMS data.
A
erylusamines from Erylus placenta are antagonists for the IL-6
receptor;3 pachymosides from Pachymatisma johnstonia are
active in the type III secretion system assay against Gram-
negative pathogenic bacteria;4 and there are related metabolites
from a Red Sea Erylus sponge.5 In our continuing search for
cytotoxic metabolites from marine invertebrates, we imple-
mented LC-MS analysis and prefractionation combined with
bioassay to expedite the detection of new cytotoxic metabolites.
The analysis showed that fractions of the extract of a Stelletta sp.
marine sponge contained metabolites with molecular weights of
1119 and 1133 (Figure 1a), which were associated with cytotoxic
activity (Figure 1b). We embarked on the isolation and structure
elucidation of the active constituents from the extract.
RESULTS AND DISCUSSION
■
Initial analysis of the NMR data of fraction 1 showed the
presence of four acetyl groups, one dimethylamino group, three
anomeric carbons, 11 oxygenated methines, three oxygenated
methylenes, one ketone carbonyl, and one carboxyamide
together with a long aliphatic chain (Table 1). Heterogeneity
was observed in the signals of the aliphatic methyl region: two
The MeOH extract of the sponge was partitioned between H2O
and CHCl3, and the aqueous phase was extracted with n-BuOH.
The CHCl3 fraction was partitioned between MeOH−H2O
(9:1) and n-hexane, and the resulting aqueous MeOH fraction
was combined with the n-BuOH fraction. The combined organic
layers were fractionated by ODS flash chromatography followed
by ODS-HPLC to afford fraction 1 and fraction 2, which
exhibited [M + H]+ ions at m/z 1120 and 1134 in the ESIMS
spectrum, respectively. Acetic acid was added to the mobile phase
Received: September 3, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. (a) Retention time vs molecular weight diagram of the lipid-soluble fraction of a Stelletta sp. sponge (x-axis: retention time, y-axis: m/z) and
(b) the cytotoxicity of fractions separated by ODS-HPLC into a 96-well plate. The colored fractions showed cytotoxicity, with red-colored wells being
more potent than the pink-colored wells. Fractions 52 and 58 contained stellettosides A1 and A2 and stellettosides B1−B4 as the major components,
respectively. Due to the scarce amounts of materials, we were not able isolate other active constituents.
trisaccharide unit. Ring A was connected to an oxygenated
methine (C-22) in the fatty acid chain, as shown by the HMBC
data. H-22 was coupled to another oxygenated methine at δH
5.01 (H-21), demonstrating the presence of vicinal oxygenated
methines.
The positions of the ketone carbonyl and vicinal oxygenated
methines, which were separately placed in the middle of the alkyl
chain, were left unassigned. We anticipated that charge-remote
fragmentations in the tandem FABMS would be observed with a
positive charge on the dimethylamino group. FABMS of fraction
1 afforded an intense fragment ion at m/z 598.1 corresponding to
the product ion formed by deglycosylation. This ion was selected
as the precursor in the tandem FABMS analysis (Figure S9),
which showed the presence of ions separated by 56 Da at m/z
297 and 353, permitting us to locate the ketone carbonyl at C-13
(Figures 2a and S9). There was a report on the tandem FABMS
of cationized oxofatty acids,6 in which prominent cleavages were
observed around the oxo position: (1) between the oxo position
and the adjacent carbon (α carbon) proximal to the charge
(α ion) and (2) between the second (β carbon) and the third
carbon (γ carbon) distal to the charge (γ′ ion). Because ions
derived from other modes of cleavages around the oxo site were
very weak, a notable gap of 56 Da was observed. In order to
confirm whether similar fragmentations occur in our system, we
synthesized N-(4-(dimethylamino)butyl)-14-oxohexacosana-
mide (7) and subjected this compound to the tandem FABMS
doublet methyls (stellettoside A1) and a triplet methyl
(stellettoside A2) were present in a ratio of 2:2:1, suggesting
that stellettosides A1 and A2 were present in a ratio of 2:1. The
above structural units were reminiscent of glycosylated fatty acid
amides such as the erylusamines.3
analysis (Figures 2b and S27). Product ions were observed
at m/z 311 (α ion) and 367 (γ′ ion), as expected. In aliphatic
compounds with vicinal oxygenated methines, the bond between
the oxymethine carbons is prone to cleavage.7 In the tandem
FABMS of fraction 1 (Figures 2a and S9), an ion at m/z 453
assignable to such a cleavage was observed, indicating the
presence of oxygenated methines at C-21 and C-22.8
The structure of the terminal alkyl group was determined
by interpretation of the 2D NMR data. In stellettoside A1 (1) a
pair of doublets were both correlated to a methine (δC 29.6) and
a methylene (35.8) in the HMBC spectrum, suggesting the
presence of a methyl branch at the penultimate carbon, whereas
in stellettoside A2 (2) the HMBC correlation from the triplet
methyl to methylene carbons at δC 23.7 and 33.2 indicated an
unbranched alkyl terminus.11
The amine portion was shown to be N,N-dimethylputrescine
on the basis of the 2D NMR data. The COSY and TOCSY
spectra demonstrated the presence of two contiguous methyl-
enes (C-2′ and C-3′) placed between two nitrogen-substituted
methylene carbons (Table 1). One of the nitrogen-substituted
methylene carbons (C-4′) was correlated to the NMe2 protons,
whereas the protons on the other (C-1′) were coupled to the
amide carbonyl carbon (C-1) in the HMBC spectrum.
The structures of the three monosaccharide units (rings A−C)
were studied by interpretation of the COSY, TOCSY, and
1
HSQC spectra and H−1H coupling constants, which showed
that all of them were arabinopyranose with an axial anomeric
proton (Table 1). H-2, H-3, and H-4 in ring C were deshielded,
indicating that they were on carbons substituted by acetoxy
groups. HMBC correlations between H-1C and C-3B and
between H-1B and C-4A showed the mode of linkage of the
We studied the position of glycosylation by interpretation of
the NMR data. The chemical shift of 5.01 ppm for one of the
B
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. 1H and 13C NMR Data (600 MHz, CD3OD) for 1 and 3
a
b
1
3
c
c
position
δC
δH (J in Hz)
2.16, t (7.5)
position
δC
δH (J in Hz)
2.16, t (7.5)
1
176.0, C
1
176.3, C
2
37.1, CH2
27.0, CH2
30.4, CH2
24.8, CH2
43.4, CH2
214.2, C
2
37.2, CH2
27.0, CH2
30.4, CH2
25.0, CH2
43.6, CH2
214.4, C
3
1.59, m
3
1.58, m
4−10
11
1.30, m
4−10
11
1.30, m
1.53, m
1.52, m
12
2.43, t (7.3)
12
2.43, t (7.4)
13
13
14
43.4, CH2
24.8, CH2
30.4, CH2
30.4, CH2
76.6, CH
81.1, CH
31.3, CH2
35.8, CH2
29.6, CH
22.5, CH3
22.9, CH3
39.8, CH2
28.1, CH2
24.8, CH2
59.7, CH2
44.7, C
2.43, t (7.3)
1.53, m
14
43.6, CH2
25.0, CH2
30.4, CH2
30.4, CH2
76.8, CH
80.8, CH
31.4, CH2
26.5, CH2
30.4, CH2
32.8, CH2
23.5, CH2
14.2, CH3
39.7, CH2
27.9, CH2
25.0, CH2
59.7, CH2
44.6, CH3
173.1, C
2.43, t (7.4)
1.52, m
15
15
16−19
20
1.30, m
16−19
20
1.30, m
1.65, m, 1.59, m
5.01, dt (9.6, 3.2)
3.69, m
1.65, m, 1.58, m
5.01, dt (9.6, 3.3)
3.71, m
21
21
22
22
23
1.50, m
23
1.49, m
24
1.38, m, 1.24, m
1.52, m
24
1.33, m
25
25
1.30, m
26
0.88, d (6.6)
0.89, d (6.6)
3.18, t (6.7)
1.51, m
26
1.28, m
27
27
1.30, m
1′
2′
3′
4′
28
0.90, t (7.1)
3.18, t (6.7)
1.50, m
1′
2′
3′
4′
1.53, m
2.58, t (7.2)
2.37, s
1.52, m
4′NMe2
21-OAc
21-OAc
1A
2.52, m
172.6, C
4′NMe2
21-OAc
21-OAc
1A
2A
3A
4A
5A
2.38, s
21.1, CH3
103.8, CH
72.9, CH
73.6, CH
79.0, CH
65.9, CH2
2.05, s
4.33, d (6.5)
21.1, CH3
103.7, CH
72.9, CH
73.9, CH
79.3, CH
66.1, CH2
2.05, s
2A
3.53, m
4.33, d (6.3)
3A
3.61, dd (2.9, 8.7)
3.87, m
3.52, dd (6.5, 8.7)
3.62, dd (3.6, 8.7)
3.87, m
4A
5A
4.07, dd (3.4, 12.4)
3.50, dd (1.9, 12.4)
4.35, d (7.9)
4.07, dd (3.5, 12.4)
3.49, dd (1.9, 12.3)
4.34, d (7.4)
1B
2B
3B
4B
5B
107.1, CH
71.0, CH
83.5, CH
69.9, CH
67.0, CH2
3.64, m
1B
2B
3B
4B
5B
107.1, CH
71.9, CH
83.7, CH
70.0, CH
67.0, CH2
3.58, dd (3.4, 9.7)
3.93, m
3.67, m
3.58, dd (3.4, 9.6)
3.93, m
3.85, dd (2.1, 12.9)
3.54, dd (1.9, 13.0)
4.79, d (7.4)
3.85, dd (1.9, 12.7)
3.54, d (12.6)
4.79, d (7.4)
1C
2C
3C
4C
5C
104.0, CH
70.7, CH
71.6, CH
69.6, CH
64.6, CH2
5.14, dd (7.3, 10.0)
5.10, dd (3.5, 10.0)
5.26, m
1C
2C
3C
4C
5C
104.2, CH
70.9, CH
71.9, CH
69.5, CH
64.6, CH2
5.15, dd (7.4, 10.0)
5.10, dd (3.5, 10.0)
3.96, dd (2.5, 13.3)
3.76, dd (1.2, 13.3)
2.11, s
5.26, ddd (1.4, 2.5, 3.7)
3.96, dd (2.6, 13.3)
3.76, dd (1.4, 13.3)
2.11, s
Ac
Ac
Ac
20.8, CH3
171.5, C
Ac
Ac
Ac
20.6, CH3
171.9, C
20.8, CH3
171.7, C
2.07, s
1.97, s
20.6, CH3
171.9, C
2.07, s
1.97, s
20.5, CH3
171.5, C
20.6, CH3
171.6, C
a
NMR data for 2, which are different from those of 1, are as follows: δC/δH 80.7/3.71 (m) (C-22); 31.3/1.50 (2H, m) (C-23); 26.6/1.34 (C-24);
b
32.9/1.28 (2H, m) (C-25); 23.4/1.31 (2H, m) (C-26); 14.2/0.90 (3H, t, J = 7.0 Hz) (C-27). NMR data of 3−6 were indistinguishable. Carbon
numbering of stellettoside B1 (3) was used for the sake of simplicity. The carbon chemical shifts were determined on the basis of the HSQC and
c
HMBC spectra.
oxymethine protons (H-21) indicated acetylation of C-21,
whereas the H-22 oxymethine (δH/δC 3.71/80.8) was glycosy-
lated as described above. TOCSY correlations were observed
from the terminal methyl doublets to H-22 but not to H-21 in
stellettoside A1, and a corresponding correlation was observed in
stellettoside A2, demonstrating that the glycosylation occurred
on the oxygenated methine proximal to the terminal methyls
(Figure S4).
C
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
381, and 395 were small (Figure S27), demonstrating that the
product ion derived from the γ′ cleavage (m/z 367) represents
the position of the ketone carbonyl. Therefore, the ratio of ions at
m/z 353 and 367 approximately reflects the ratio of 13-oxo and
14-oxo derivatives. On the other hand the ion at m/z 453 arose
from the 21,22-diol but not from the 22,23-diol, whereas the ion
at m/z 467 is due to the 22,23-diol and not from the 21,22-diol,
suggesting that the ratio of these ions reflects the composition of
the positional isomers of the two diols. In the tandem FABMS of
fraction 3, the ratio of the γ′ ions at m/z 353 and 367 was
approximately half of the ratio of the ions at m/z 453 and 467,
demonstrating that the ratios of 13-oxo and 14-oxo derivatives
and that of the 21,22-diol and the 22,23-diol derivatives were
different in fraction 3. This phenomenon cannot be accounted
for by the presence of one or two isomers. Therefore, we
tentatively propose that fraction 2 was a mixture of four isomers,
stellettosides B1−B4 (3−6).
The position of glycosylation in 3−6 was deduced by
interpretation of the NMR data. The chemical shifts of the
vicinal oxygenated methines of stellettosides B1−B6 (3−6)
coincided well with those of stellettosides A1 (1) and A2 (2),
suggesting that the trisaccharide moiety was linked to the
oxygenated methine proximal to the terminal methyl. This idea
was supported by the TOCSY correlation between the terminal
methyl and the protons attached to the methylene carbon
adjacent to the site of glycosylation.
Figure 2. Assignments of product ions in the tandem FAB mass spectra.
(a) Mixture of stellettosides A1 and A2 by using the deglycosylation
product as the presursor. (b) Compound 7 by using the [M + H]+ ion as
the presursor. (c, d) Fraction 3 by using the [M + H]+ ion as the
presursor.
The relative configuration of the vicinal oxygenated methines
1
was assigned by the analysis of the H NMR data of the
isopropylidene derivative of the aglycone. Mild acid hydrolysis
of the mixture of 3−6 followed by treatment with 2,2-
dimethoxypropane in the presence of pyridinium p-toluenesul-
fonate (PPTS) gave a mixture of the acetonides. Although the
products were not purified due to the paucity of material, two
singlet methyl signals of the acetonide group were observed at
δH 1.31 and 1.41, which was in accordance with the data reported
for the cis-acetonides.12−14 Hence, the 1,2-diol in stellettosides
B1−B4 was deduced to be erythro. This assignment was
Fraction 2 exhibited similar NMR spectra to fraction 1 and
had a molecular formula larger than that of fraction 1 by one
CH2 unit. Interpretation of the 2D NMR data revealed that
compounds in fraction 2 had the same sets of partial structures
found in stellettosides A1 (1) and A2 (2) and that the alkyl
terminus was unbranched. A large fragment ion at m/z 611.8 was
observed in the FABMS spectrum as a result of deglycosylation
(Figure S17). Tandem FABMS using this ion peak as the
precursor was more complex than the corresponding spectrum
obtained for fraction 1 (Figure S18). In order to facilitate the
interpretation of the tandem FABMS data, we sought to obtain
the corresponding data of the aglycones of the compounds in
fraction 2. Fraction 2 was subjected to acid hydrolysis, and the
product was separated by HPLC. The eluate was collected in
short intervals, and each fraction was analyzed by ESIMS to
detect the aglycone. Two fractions (fractions 3 and 4) gave the
ion corresponding to the aglycone. These two fractions eluted
from the HPLC side by side, indicating that the peaks were not
completely separated, as evidenced from the tandem FABMS
analysis described below. In the tandem FABMS spectrum of
fraction 3, product ions were observed at m/z 297, 311, 353, and
367 (Figure S19); the difference of 56 Da between the ions
at m/z 297 and 353 as well as the ions at m/z 311 and 367
showed that fraction 3 was a mixture of 13-oxo and 14-oxo
derivatives. In this spectrum the ions at m/z 453 and 467 were
considered to arise from the cleavage of the bond between the
two carbinol carbons, indicating the presence of both 21,22-diol
and 22,23-diol in this fraction. Interpretation of the tandem
FABMS data of fraction 4 revealed that it was also a mixture of
positional isomers of the ketone carbonyl and the vicinal
diol albeit with different composition, as inferred from the
intensity ratios of product ions at m/z 353, 367, 453, and 467
(Figure S20). The tandem FABMS data of compound 7 showed
an intense product ion at m/z 367, but product ions at m/z 353,
1
supported by the H NMR data of the acetonides of erythro-
and threo-dodecane-5,6-diol. Because the NMR data of the
contiguous oxygenated methines of the mixture of stellettosides
A1 and A2 are almost identical with those of the mixture of
stellettosides B1−B4, we consider that the relative configuration
of the 1,2-diol in stellettosides A1 and A2 is also erythro.
Unfortunately, conversion of the mixture of the diols to the
MTPA esters was unsuccessful. Therefore, the absolute
configuration of the aglycone was not determined.
The absolute configuration of the arabinose residue was
determined by LC-MS of the acid hydrolysate after chiral deriva-
tization. Fraction 1 and fraction 2 were separately subjected to
acid hydrolysis and solvent partitioning to afford fractions
enriched with monosaccharides. The residue was converted to
the methyl 3-phenylthiocarbamoylthiazoline-4(R)-carboxylate
derivative and analyzed by LC-MS (Figure 3),15,16 which
demonstrated the L-configuration of all of the arabinose residues
in the stellettosides.
The mixture of stellettosides B1−B4 displayed cytotoxic
activity against HeLa cells with an IC50 value of 9 μM. However,
the mixture of stellettosides A1 and A2 did not show cytotoxic
activity at 10 μM. The stellettosides are congeners of the
erylusamines3 with some structural differences: the stellettosides
contain an N,N-dimethylputrescine moiety as the amine
component, whereas the erylusamines contain N,N-dimethylca-
daverine; the stellettosides have the erythro dioxygenation of the
D
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Portions of the solution were subjected to RP-HPLC on an ODS
column (Cosmosil 5C18-AR-II, 20 × 250 mm, 8 mL/min, 40 °C,
210 nm) with MeCN/H2O/acetic acid (40:60:0.1 to 100:0:0.1 over
50 min) to give a total of 2 mg of fraction 1 and 2.5 mg of fraction 2.
Mixture of stellettosides A1 (1) and A2 (2): colorless oil; [α]28D −22
(c 0.06, MeOH); UV (MeOH) λmax (log ε) 211 (3.52); 1H and
13C NMR data (CD3OD), Table 1; HRESIMS m/z 1119.6759 [M + H]+
(calcd for C56H99N2O20, 1119.6791).
Mixture of stellettosides B1−B4 (3−6): colorless oil; [α]28 −24
D
(c 0.08, MeOH); UV (MeOH) λmax (log ε) 211 (3.41); 1H and
13C NMR data (CD3OD), Table 1; HRESIMS m/z 1133.6911 [M + H]+
(calcd for C57H101N2O20, 1133.6948).
Figure 3. LC-MS of the arabinose residue after converting into the
methyl 3-phenylthiocarbamoylthiazoline-4(R)-carboxylate derivative:
(a) the hydrolysate from the mixture of 3−6; (b) L-arabinose;
(c) D-arabinose; (d) co-injection of D- and L-arabinose.
Prefractionation. An approximately 10 g portion of the sponge was
ground and extracted with MeOH (20 mL). The extract was
concentrated in vacuo and partitioned between H2O (10 mL) and
CHCl3 (2 × 10 mL). The organic layers were combined, concentrated
in vacuo, dissolved in MeOH, and passed through a short ODS column,
which was washed with MeOH. The eluate was concentrated in vacuo
and redissolved in 100 μL of MeOH. A half-portion of the solution
was subjected to RP-HPLC on an ODS column (Cosmosil 5C18-AR-II,
10 × 250 mm, 1.2 mL/min, 40 °C, 210−450 nm (photodiode array
detector)) with MeCN/H2O/acetic acid (20:80:1 to 100:0:1 over
95 min). The effluents from the HPLC were collected every minute with
a fraction collector into a 96-well plate. The plate was dried in vacuo, and
to each well was added 30 μL of DMSO. The cytotoxicity of the solution
in each well against HeLa cells was evaluated using an MTT assay.
Isopropylidene Derivative. The mixture of 3−6 (100 μg) was
dissolved in 10% HCl in MeOH (100 μL) and heated at 90 °C for 2 h.
The solvent was evaporated (stream of N2), and the product was
partitioned between H2O (500 μL) and EtOAc (500 μL). The EtOAc
layer was concentrated (stream of N2) to give the crude aglycone. To the
crude product was added 2,2-dimethoxypropane (100 μL) and
pyridinium p-toluenesulfonate (catalytic amount). DMF was then
added dropwise until the PPTS dissolved. The reaction was stirred for
48 h and then partitioned between H2O (500 μL) and EtOAc (500 μL).
The organic layer was concentrated (stream of N2) to give the crude
fatty acid moiety, whereas the erylusamines have the threo
dioxygenation; the stellettosides have L-arabinose as the sugar
components, whereas the erylusamines contain L-arabinose and
D-xylose.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP-1000 digital polarimeter. UV spectra were
measured on a Shimadzu Biospec 1600. NMR spectra were recorded on
a JEOL alpha 600 NMR spectrometer at 300 K. Chemical shifts were
referenced to solvent peaks: δH 7.24 and δC 77.0 for CDCl3; δH 3.30 and
δC 49.0 for CD3OD. ESI mass spectra were measured on a JEOL
JMS-T100LC. FABMS and tandem FABMS were measured on a JEOL
JMS-700T. HPLC was carried out on a Shimadzu LC 20AT with an
SCL-10Avp controller and a SPD-10Avp detector. LC-MS was
conducted on an Amazon SL mass spectrometer with UFLC performed
on a Shimadzu LC 20AT with an SPD-M20A detector.
Animal Material. A Stelletta sp. sponge was collected by dredging at
a depth of 170 m at the seamount “Oshima-Shinsone” (between
28°52.19′ N, 129°32.96′ E and 28°52.25′ N, 129°32.94′ E) near
Amami-oshima Island, southern Japan, during a cruise of T/S Nagasaki-
maru, June 5, 2008. Sponge description: intact external morphology
unknown because the analyzed sample was broken; surface hispid
because of numerous protruding oxeas; consistency hard; surface dark
brown in life, ocher in EtOH; choanosome ocher in ethanol;
megascleres oxeas, plagiotriaenes, and anatriaenes; microscleres oxy-
asters and spheroxyasters; oxeas, abundant, wide range in size, length
1350 (703−1878) μm; width 49.6 (20−84) μm; plagiotriaenes, rare,
rhabdome length 410.8 (357−547) μm; rhabdome width 38.8 (28.8−
50) μm; clad length 133.3 (127−140); anatriaenes, rare, rhabdome
length 667 to more than 1200 μm (usually broken); rhabdome width 3
(2−4) μm; clad length 15.3 (12−20) μm; oxyasters, common, diameter
16 (12−20) μm; spheroxyasters, abundant, diameter 10.1 (9−13) μm.
Up to now, 21 species have been reported as valid for the genus Stelletta
from Japanese waters (Ise, submitted). Of these, no species can be
compared to our specimen because of the unique combination of
megascleres that are oxea, plagiotriaene, and anatriaene. The specimen
used for the identification was deposited at National Museum of Nature
and Science, Tokyo, under the museum identification number NSMT
Po-2485.
Extraction and Isolation. The sponge (880 g, wet weight) was
ground and extracted with MeOH (3 × 1 L). The combined extracts
were concentrated in vacuo and partitioned between H2O (1 L) and
CHCl3 (3 × 1 L). The H2O layer was further extracted with n-BuOH
(1 L), while the combined CHCl3 layers were concentrated and
partitioned between 90% MeOH (500 mL) and n-hexane (3 × 500 mL).
The n-BuOH phase was concentrated and combined with the 90%
MeOH fraction. The combined fraction (5.94 g) was then subjected to
ODS column chromatography and eluted with (1) 20% MeCN/H2O,
(2) 40% MeCN/H2O, (3) 60% MeCN/H2O, (4) 80% MeCN/H2O,
(5) MeCN, (6) MeOH, and (7) CHCl3/MeOH (1:1). Fractions 6 and 7
were combined (734 mg yield) and dissolved in a mixture of MeCN/
H2O/acetic acid (40:60:0.1, 3 mL) to remove the insoluble material.
1
acetonide, which was used for the H NMR analysis. Selected data:
1H NMR (CDCl3, 600 MHz) δ 1.41 (3H, s, CH3CCH3), 1.31 (3H, s,
CH3CCH3); ESIMS m/z 609.7 [M + H]+.
Determination of the Absolute Configurations of the
Arabinose Residues. Fraction 1 (100 μg) was dissolved in 10%
HCl in MeOH (100 μL) and heated at 90 °C for 2 h. The solvent was
evaporated with a stream of N2, and the residue dissolved in H2O
(500 μL) was extracted with EtOAc (500 μL) and three times with
CHCl3 (500 μL). To the aqueous phase was added a solution of
L-cysteine methyl ester hydrochloride in pyridine (2 mg/mL; 100 μL).
The solution was heated at 60 °C for 1 h; then 5 μL of phenyl
isothiocyanate was added and the solution was heated for a further hour
at 60 °C. The solvent was evaporated, and the crude product was
dissolved in MeOH (100 μL) and analyzed by LC-MS (10:90:05 to
15:85:05 MeCN/H2O/AcOH over 60 min) (Cosmosil 2.5C18-MS-II,
2.0 × 100 mm, 0.2 mL/min, 40 °C). L- and D-Arabinose and the
hydrolysate of fraction 2 were treated in the same manner. The retention
times (tR) are as follows: D-(−)-arabinose derivative, 36.6 min;
L-(+)-arabinose derivative, 35.5 min; monosaccharide derivative
from 1, 35.5 min.
Cytotoxicity Assay. The cytotoxicities of fractions 1 and 2 against
HeLa cells were evaluated by an MTT assay. HeLa human cervical
cancer cells were cultured in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum, 2 μg/mL gentamycin, and 2 μg/mL
antibiotic−antimicotic (Gibco) at 37 °C under an atmosphere
of 5% CO2. To each well of a 96-well microplate containing 200 μL
of tumor cell suspension (1 × 104 cells/mL) was added a sample
after 24 h preincubation, and the plate was incubated for 72 h. After
addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) saline solution (1 mg/mL, 50 μL) to each well, the
plate was incubated for 3 h. After the incubation, the supernatant was
discarded and DMSO (150 μL) was added. The absorbance was
measured to determine IC50 values. In this assay adriamycin was used
as a positive control, which exhibited an IC50 value of 1.3 μM.
E
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.5b00795.
■
S
NMR spectra and tandem FABMS data for a mixture of
stellettosides A1 and A2 and a mixture of stellettosides B1−
B4 and the experimental part of the synthesis of 7 and the
acetonides of erythro- and threo-dodecane-5,6-diol (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail (K. Takada): atakada@mail.ecc.u-tokyo.ac.jp.
*Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166.
E-mail: assmats@mail.ecc.u-tokyo.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
Products” and JSPS KAKENHI Grant Numbers 25252037,
25712024, and 25660163 from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. V.P. thanks the
Japan Society for the Promotion of Science for funding through
their Postdoctoral Fellowship Program for Foreign Researchers.
REFERENCES
■
(1) Dembitsky, V. M. Lipids 2005, 40, 641−660.
(2) Kalinin, V. I.; Ivanchina, N. V.; Krasokhin, V. B.; Makarieva, T. N.;
Stonik, V. A. Mar. Drugs 2012, 10, 1671−1710.
(3) Sata, N.; Asai, N.; Matsunaga, S.; Fusetani, N. Tetrahedron 1994,
50, 1105−1110.
(4) Warabi, K.; Zimmerman, W. T.; Shen, J.; Gauthier, A.; Robertson,
M.; Finlay, B. B.; van Soest, R.; Andersen, R. J. Can. J. Chem. 2004, 82,
102−112.
(5) Goobes, R.; Rudi, A.; Kashman, Y.; Ilan, M.; Loya, Y. Tetrahedron
1996, 52, 7921−7828.
(6) Cheng, C.; Giblin, D.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1998,
9, 216−224.
(7) Griffiths, W. J.; Yang, Y.; Sjoval, J.; Lindgren, J. A. Rapid Commun.
Mass Spectrom. 1996, 10, 183−196.
(8) The number of methylene carbons between the terminal methyl
and C-22 was shown to be four or more than four on the basis of the
carbon chemical shift of 33.0 ppm for the third carbon from the methyl
terminus. If the number of methylene carbons is three, the relevant
carbon appears at ca. 28 ppm due to the γ-effect of the hydroxy group;9
glycosylation shifts of carbohydrates toward the γ-carbon are less than 1
ppm.10
(9) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH
Publishers: Weinheim, 1990; pp 206−209.
(10) Tori, K.; Seo, S.; Yoshimura, Y.; Arita, H.; Tomita, Y. Tetrahedron
Lett. 1977, 18, 179−182.
(11) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH
Publishers: Weinheim, 1990; pp 183−186.
(12) Miyata, Y.; Matsunaga, S. Tetrahedron Lett. 2008, 49, 6334−6336.
(13) Takada, K.; Uehara, T.; Nakao, Y.; Matsunaga, S.; van Soest, R. W.
M.; Fusetani, N. J. Am. Chem. Soc. 2004, 126, 187−193.
(14) McCulloch, M. W. B.; Bugni, T. S.; Concepcion, G. P.; Coombs,
G. S.; Harper, M. K.; Kaur, S.; Mangalindan, G. C.; Mutizwa, M. M.;
Veltri, C. A.; Virshup, D. M.; Ireland, C. M. J. Nat. Prod. 2009, 72, 1651−
1656.
(15) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem.
Pharm. Bull. 2007, 55, 899−901.
(16) Wang, Y.-H.; Avula, B.; Fu, X.; Wang, M.; Khan, I. A. Planta Med.
2012, 78, 834−837.
F
DOI: 10.1021/acs.jnatprod.5b00795
J. Nat. Prod. XXXX, XXX, XXX−XXX