Journal of Natural Products
Note
of the ODS flash column chromatography was further separated by
ODS flash column chromatography using a stepwise gradient elution
(50%, 70% MeOH, 70%, 90% MeCN, and MeOH). The 70% MeCN
fraction was purified by RP-HPLC (Cosmosil AR-II; 10 × 250 mm)
with a linear gradient elution of 50−70% MeCN containing 1% AcOH
(50 min) to afford ciliatamide A (2, 1.1 mg).
the absolute configurations of the two constituent amino acid
residues in ciliatamide A (2), both of which had been revised
from the L- to D-form by chemical synthesis.3b Fortunately
compound 2 that showed identical physicochemical properties
with those reported3a was isolated from the current sponge
extract. Because racemization during configurational analysis
had been pointed out as the source of misassignments,3b we
conducted acid hydrolysis of 2 for 2, 5, and 16 h, and each
hydrolysate was subjected to Marfey’s analysis to examine
whether racemization of the amino acids took place during acid
hydrolysis. The LC-MS data unambiguously demonstrated that
the two amino acids in the acid hydrolysates had the L-
configuration and that racemization did not occur during the
hydrolysis nor derivatization for Marfey’s analysis. Therefore,
we have concluded that our previous configurational assign-
ment of ciliatamide A (2) was correct.7
Ciliatamide D (1): white, amorphous powder; [α]24 +58 (c 0.035,
D
1
MeOH); UV (MeOH) λmax (log ε) 212 (3.7) nm; H and 13C NMR
data, see Table 1; HRESIMS m/z 464.2570 [M + Na]+ (calcd for
C22H39N3O4SNa, 464.2559).
Ciliatamide A (2): yellow oil; [α]22 +52 (c 0.05, MeOH);8 UV
D
1
(MeOH) λmax (log ε) 208 (3.9) nm; H NMR spectrum, see Figure
S7; HRESIMS m/z 464.2890 [M + Na]+ (calcd for C22H39N3O4SNa,
464.2889).
Preparation of N-Methyl-D- and N-Methyl-L-methionine
Sulfone and N-Methyl-D- and N-Methyl-L-phenylalanine. To a
solution of N-Boc-L-methionine (100 mg) in MeOH (2.5 mL) was
added Oxone (500 mg), and the solution was stirred at room
temperature for 2 h. The mixture was diluted with H2O and extracted
with CH2Cl2 to give N-Boc-L-methionine sulfone (m/z 282 [M +
H]+). To a solution of the above N-Boc-L-methionine sulfone in THF
(2 mL) was added NaH (50 mg) and MeI (330 μL), and the mixture
was stirred at room temperature for 24 h. The reaction mixture was
diluted with H2O and extracted with EtOAc. The aqueous layer was
acidified with 20% aqueous citric acid and extracted with EtOAc. The
combined organic layer was concentrated to afford N-Boc-N-methyl-L-
methionine sulfone (m/z 296 [M + H]+), which was dissolved in 6 N
HCl (1 mL) and heated at 110 °C for 2 h. The solution was
concentrated to provide N-methyl-L-methionine sulfone (m/z 196 [M
+ H]+). This material was used for the Marfey’s analysis. N-Methyl-D-
methionine sulfone was synthesized from N-Boc-D-methionine in the
same manner.
N-Methyl-D- and N-Methyl-L-phenylalanine were prepared from
commercially available N-Boc-D- and N-Boc-L-phenylalanine, respec-
tively, by N-methylation and deprotection as described above.
Oxidation of Ciliatamide D (1). To a solution of ciliatamide D
(1, 0.1 mg) in MeOH (0.5 mL) was added Oxone (5 mg in 100 μL of
H2O), and the mixture was stirred at room temperature for 2 h. The
solution was diluted with H2O, applied on InertSep RP-1 (GL
Science), washed with 10% MeOH, and eluted with MeOH to afford
ciliatamide D sulfone (m/z 458 [M + H]+).
Marfey’s Analysis (ref 6). Ciliatamide D sulfone was dissolved in
6 N HCl (150 μL) and heated at 110 °C for 2 h. The solution was
concentrated and redissolved in 50 μL of H2O. L-FDAA (1%) in
acetone (100 μL) and 1 M NaHCO3 (20 μL) were added to the
solution. The mixture was heated at 40 °C for 1 h. After cooling to
room temperature, the reaction mixture was quenched with 5 N HCl
(4 μL), concentrated, and redissolved in DMSO. D-Amino and L-
amino acid standards were treated with L-FDAA in the same manner.
The L-FDAA derivatives were analyzed by LC-MS [Cosomosil 2.5C18-
MS-II (2.0 × 100 mm); flow rate, 0.5 mL/min; solvent, 10−50%
MeCN containing 0.5% AcOH (22 min)]. Retention time (tR) of each
amino acid was as follows: L-lysine (17.2 min), D-lysine (18.4), N-
methyl-L-methionine sulfone (9.5), and N-methyl-D-methionine
sulfone (10.2). The absolute configurations of the amino acids in 1
were determined as L-lysine (tR 17.2 min) and N-methyl-L-methionine
sulfone (tR 9.5 min). Ciliatamide A (2, 0.05 mg) was analyzed in the
same manner. The absolute configurations of the amino acids in 2
were determined as L-lysine (tR 17.2 min) and N-methyl-L-phenyl-
alanine (tR 16.2 min); retention times of standard N-methyl-L-
phenylalanine and N-methyl-D-phenylalanine were 16.2 and 18.3 min,
respectively.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP-1000 digital polarimeter. UV spectra
were determined in MeOH using a Shimadzu BioSpec-1600
spectrophotometer. NMR spectra were recorded on a JEOL delta
600 NMR spectrometer at 600 MHz for H and 150 MHz for 13C.
1
Chemical shifts were referenced to the solvent peaks of DMSO-d6:
2.50 ppm for 1H and 39.51 ppm for 13C. ESI mass spectra were
measured on a JEOL JMS-T100LC time-of-flight mass spectrometer.
FAB mass spectra were measured on a JEOL JMS-700T MStation.
Fluorescence for the enzyme inhibition assay was measured on a
Molecular Devices Spectra Max fluorescence spectrometer. Marfey’s
analyses were conducted with a Shimadzu Prominence UFLC
equipped with Bruker amaZon SL.
Animal Material. The sponge Stelletta sp. was collected by
dredging at a depth of 170 m at seamount Oshimashinsone (between
28°52.19′ N, 129°32.96′ E and 28°52.25′ N, 129°32.94′ E), southern
Japan, during a cruise of the T/S Nagasaki-Maru, on June 5, 2008.
Sponge description: subspherical with an optimally smooth surface; no
visible oscule; surface dark brown, choanosome brown; consistency
hard; cortex 4.0−5.0 mm thick, made of perpendicularly arranged
megascleres; megascleres orthotrianes in two size classes, anatrianes,
plagiotrianes, and oxeas in two size classes, with few protrianes;
microscleres tylasters. Stelletta naseana Thiele, 1898 originally reported
from Amami-Oshima has the closest geographical distribution to the
present species but clearly differs in having dichotriane and anthaster.
Other geographically comparable species are those reported by
Hoshino (1981) from Sadamisaki Peninsula, Stelletta atrophia
Hoshino, 1981, S. Schmidt, 1862, S. maxima Thiele, 1898, and those
from Uchinoura, S. japonica Lebwohl, 1914, S. subtilis (Sollas, 1888), S.
tetrafurcata Hoshino, 1981. Of these, S. atrophia, S. grubii, S. japonica,
S. maxima, and S. tetrafurcata lack anatriane, and S. subtilis has
dichotriane that is completely absent in the present species. The
specimen used for identification (NSMT-Po-1977) was deposited at
National Museum of Nature and Science, Tokyo.
Extraction and Isolation. The sponge (800 g, wet weight) was
homogenized and extracted with EtOH (2 L) three times. The
combined extracts were concentrated and partitioned between H2O
and CHCl3. The organic layer was partitioned between 90% MeOH
and n-hexane, and the 90% MeOH layer was partitioned between
CHCl3 and 60% MeOH. The latter CHCl3 layer was separated by
ODS flash column chromatography using stepwise gradient elution
(0−100% MeOH). The 70% MeOH eluate was chromatographed by
RP-HPLC (Inertsil ODS-3; 20 × 250 mm) with a linear gradient
elution of 50−100% MeOH containing 0.1% AcOH (60 min). The
fraction eluted between 23 and 25 min was further separated by RP-
HPLC (Cosmosil AR-II; 20 × 250 mm) with a linear gradient elution
of 45−60% MeOH containing 0.1% AcOH (75 min). The fraction
eluted between 68 and 72 min was purified by RP-HPLC (Cosmosil
AR-II; 10 × 250 mm) with a linear gradient elution of 30−50% MeCN
(50 min) to afford ciliatamide D (1, 0.8 mg). The 90% MeOH fraction
Biological Assays. See the Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
Biological assay procedures, 1H NMR data table for 2, a
photograph of the sponge specimen, 1D and 2D NMR spectra
1
of 1, H NMR spectrum of 2, LC-MS chromatogram charts of
757
dx.doi.org/10.1021/np300878b | J. Nat. Prod. 2013, 76, 755−758