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 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.
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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. 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
mong many glycolipids isolated from marine sponges are a limited number of glycosylated fatty acid amides:1,2 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 Gramnegative 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 implemented 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.
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RESULTS AND DISCUSSION 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 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 3, 2015
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DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
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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-oxohexacosanamide (7) and subjected this compound to the tandem FABMS 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 We studied the position of glycosylation by interpretation of the NMR data. The chemical shift of 5.01 ppm for one of the
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 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 methylenes (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 HSQC spectra and 1H−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 B
DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Data (600 MHz, CD3OD) for 1 and 3 1a position
δC
1 2 3 4−10 11 12 13 14 15 16−19 20 21 22 23 24 25 26 27 1′ 2′ 3′ 4′ 4′NMe2 21-OAc 21-OAc 1A 2A 3A 4A 5A
176.0, C 37.1, CH2 27.0, CH2 30.4, CH2 24.8, CH2 43.4, CH2 214.2, C 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 172.6, C 21.1, CH3 103.8, CH 72.9, CH 73.6, CH 79.0, CH 65.9, CH2
1B 2B 3B 4B 5B
107.1, CH 71.0, CH 83.5, CH 69.9, CH 67.0, CH2
1C 2C 3C 4C 5C
104.0, CH 70.7, CH 71.6, CH 69.6, CH 64.6, CH2
Ac
20.8, CH3 171.5, C 20.8, CH3 171.7, C 20.5, CH3 171.5, C
Ac Ac
c
3b δH (J in Hz) 2.16, t (7.5) 1.59, m 1.30, m 1.53, m 2.43, t (7.3) 2.43, t (7.3) 1.53, m 1.30, m 1.65, m, 1.59, m 5.01, dt (9.6, 3.2) 3.69, m 1.50, m 1.38, m, 1.24, m 1.52, m 0.88, d (6.6) 0.89, d (6.6) 3.18, t (6.7) 1.51, m 1.53, m 2.58, t (7.2) 2.37, s 2.05, s 4.33, d (6.5) 3.53, m 3.61, dd (2.9, 8.7) 3.87, m 4.07, dd (3.4, 12.4) 3.50, dd (1.9, 12.4) 4.35, d (7.9) 3.64, m 3.58, dd (3.4, 9.7) 3.93, m 3.85, dd (2.1, 12.9) 3.54, dd (1.9, 13.0) 4.79, d (7.4) 5.14, dd (7.3, 10.0) 5.10, dd (3.5, 10.0) 5.26, m 3.96, dd (2.5, 13.3) 3.76, dd (1.2, 13.3) 2.11, s
position
δC
1 2 3 4−10 11 12 13 14 15 16−19 20 21 22 23 24 25 26 27 28 1′ 2′ 3′ 4′ 4′NMe2 21-OAc 21-OAc 1A 2A 3A 4A 5A
176.3, C 37.2, CH2 27.0, CH2 30.4, CH2 25.0, CH2 43.6, CH2 214.4, C 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 21.1, CH3 103.7, CH 72.9, CH 73.9, CH 79.3, CH 66.1, CH2
1B 2B 3B 4B 5B
107.1, CH 71.9, CH 83.7, CH 70.0, CH 67.0, CH2
1C 2C 3C 4C 5C
104.2, CH 70.9, CH 71.9, CH 69.5, CH 64.6, CH2
Ac
20.6, CH3 171.9, C 20.6, CH3 171.9, C 20.6, CH3 171.6, C
2.07, s Ac 1.97, s Ac
c
δH (J in Hz) 2.16, t (7.5) 1.58, m 1.30, m 1.52, m 2.43, t (7.4) 2.43, t (7.4) 1.52, m 1.30, m 1.65, m, 1.58, m 5.01, dt (9.6, 3.3) 3.71, m 1.49, m 1.33, m 1.30, m 1.28, m 1.30, m 0.90, t (7.1) 3.18, t (6.7) 1.50, m 1.52, m 2.52, m 2.38, s 2.05, s 4.33, d (6.3) 3.52, dd (6.5, 8.7) 3.62, dd (3.6, 8.7) 3.87, m 4.07, dd (3.5, 12.4) 3.49, dd (1.9, 12.3) 4.34, d (7.4) 3.67, m 3.58, dd (3.4, 9.6) 3.93, m 3.85, dd (1.9, 12.7) 3.54, d (12.6) 4.79, d (7.4) 5.15, dd (7.4, 10.0) 5.10, dd (3.5, 10.0) 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 2.07, s 1.97, s
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); 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). bNMR data of 3−6 were indistinguishable. Carbon numbering of stellettoside B1 (3) was used for the sake of simplicity. cThe carbon chemical shifts were determined on the basis of the HSQC and HMBC spectra. a
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).
oxymethine protons (H-21) indicated acetylation of C-21, whereas the H-22 oxymethine (δH/δC 3.71/80.8) was glycosylated as described above. TOCSY correlations were observed from the terminal methyl doublets to H-22 but not to H-21 in C
DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
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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. The relative configuration of the vicinal oxygenated methines was assigned by the analysis of the 1H NMR data of the isopropylidene derivative of the aglycone. Mild acid hydrolysis of the mixture of 3−6 followed by treatment with 2,2dimethoxypropane in the presence of pyridinium p-toluenesulfonate (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 supported by the 1H NMR data of the acetonides of erythroand 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 derivatization. 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-dimethylcadaverine; the stellettosides have the erythro dioxygenation of the
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.
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, D
DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
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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 13 C 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]28D −24 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 211 (3.41); 1H and 13 C NMR data (CD3OD), Table 1; HRESIMS m/z 1133.6911 [M + H]+ (calcd for C57H101N2O20, 1133.6948). 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 acetonide, which was used for the 1H NMR analysis. Selected data: 1 H 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.
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.
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.
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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 Nagasakimaru, 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 oxyasters 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. E
DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00795. 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)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail (K. Takada):
[email protected]. *Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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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.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.5b00795 J. Nat. Prod. XXXX, XXX, XXX−XXX
