Isolation of Ciliatamide D from a Marine Sponge Stelletta sp. and a

Mar 6, 2013 - Misaki Marine Biological Station, The University of Tokyo, Miura, Kanagawa 238-0225, Japan. §. Faculty of Fisheries, Nagasaki Universit...
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Isolation of Ciliatamide D from a Marine Sponge Stelletta sp. and a Reinvestigation of the Configuration of Ciliatamide A Yasufumi Imae,† Kentaro Takada,† Shigeru Okada,† Yuji Ise,‡ Hiroshi Yoshimura,§ Yasuhiro Morii,§ and Shigeki Matsunaga*,† †

Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan ‡ Misaki Marine Biological Station, The University of Tokyo, Miura, Kanagawa 238-0225, Japan § Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan S Supporting Information *

ABSTRACT: A new lipopeptide, ciliatamide D (1), was isolated from a marine sponge Stelletta sp., collected at Oshimashinsone, together with the known compound ciliatamide A (2). Ciliatamide D (1) is a congener of 2, in which N-Me-Phe is replaced by N-Me-Met(O). Marfey’s analysis of the acid hydrolysate of 1 demonstrated that the two constituent amino acids were both in the L-form. This result prompted us to carefully investigate the configuration of 2, resulting in the assignment of the L-form for both residues.

M

arine sponges contain a wide variety of secondary metabolites with unique structures and potent biological activities.1 In the course of our exploration of bioactive secondary metabolites from sponges, we fractionated the extract of a marine sponge Stelletta sp. on the basis of cathepsin B inhibitory activity.2 After HPLC separation of an active fraction, we isolated a new compound named ciliatamide D (1), which was not associated with the activity. Compound 1 is a congener of ciliatamides A (2), B, and C, previously isolated from the marine sponge Aaptos ciliata.3a We have determined the structure of 1 to be a lipopeptide consisting of two L-amino acid residues. This raised a question regarding the validity of the structural revision of 2 by chemical synthesis.3b Therefore, from the current extract of Stelletta sp. we have isolated 2, which exhibited identical properties with those previously reported for ciliatamide A. We have conducted a thorough investigation on the configuration of 2, allowing us to conclude that our initial assignment was correct.3a The EtOH extracts of Stelletta sp. (800 g, wet weight) were combined and fractionated by solvent partitioning, ODS flash chromatography, and RP-HPLC to furnish ciliatamide D (1, 0.8 mg). Ciliatamide D (1) had a molecular formula of C22H39O4N3S as determined by HRESIMS. An analysis of the 1H NMR data in combination with the HSQC spectrum showed the presence of two amide protons, one of which appeared in a 3:1 ratio, two α-protons and an N-methyl signal both emerging in a 3:1 ratio, one finely split S-methyl, one terminal vinyl group, three low© 2013 American Chemical Society and American Society of Pharmacognosy

field methylenes, and 10 other methylenes (Table 1 and Figure 1). The doubling of some signals implied the presence of a chemical equilibrium or an inseparable mixture, or both. Interpretation of the COSY and TOCSY data demonstrated a spin system from doubled signals for NH-18 (δH 7.57, 7.58) to another amide NH-22 (δH 7.86) via an α-methine and four contiguous methylenes, indicating the presence of a lysine residue or derivative. HMBC correlations from methylene proton H-22 (δH 3.05) to the amide carbonyl carbon (C-17, δC 173.8) and from NH-22 to the α-carbon (C-18, δC 51.3) suggested that the lysine residue forms an ε-caprolactam. The COSY data showed another spin system from H-12 (δH 5.12, Received: December 18, 2012 Published: March 6, 2013 755

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Table 1. 1H and 13C NMR Data for 1 in DMSO-d6 (1H 600 MHz, 13C 150 MHz) 1 (major conformer)

a

position

δC

1 2 3 4−6 7 8 9 10a 10b 11 12 13a 13b 14a 14b 15 16 17 18 NH-18 19a 19b 20a 20b 21a 21b 22a 22b NH-22

173.3, C 32.4, CH2 24.2, CH2 28.5, CH2 28.0, CH2 32.9, CH2 138.5, CH 114.5, CH2 168.5, C 54.4, CH 20.5, CH2 49.8, CH2 37.6, CH3 30.5, CH3 173.9, C 51.3, CH 30.5, CH2 27.4, CH2 28.5, CH2 40.3, CH2

δH (J in Hz)

1 (minor conformer)

HMBC

2.34, t (7.6) 1.50, m 1.2b 1.34, m 2.0, dt (7.4, 7.0) 5.79, ddt (10.1, 17.1, 6.6) 4.93, dd (1.8, 10.1) 4.99, ddt (1.8, 17.1, 3.7)

1, 3 1, 2

3, 16 2, 16

8, 7, 7, 8 8,

8 7, 10b 10a 9 8

5.12, 5.13,a t (5.0) 1.88, m 2.18, m 2.54, m 2.63, m 2.516, 2.517,a s 2.85, 2.86,a s

1, 11, 13, 14, 16

4.33, 7.57, 1.35, 1.79, 1.62, 1.86, 1.27, 1.73, 3.05, 3.15, 7.86,

4.35,a dd (6.4, 1.2) 7.58,a d (6.4) m m m m m m m m t (5.9)

9 9, 10 8 9

15 14 1, 12 11, 11, 17, 17,

δC

NOESY

172.5, C 32.4, CH2 24.2, CH2 28.5, CH2 28.0, CH2 32.9, CH2 138.5, CH 114.5, CH2 168.3, C 57.9, CH 20.6, CH2

13ab, 14ab, 16, NH-18 12, 13b, 14ab 12, 13a, 14ab 12, 13ab 12, 13ab

49.8, CH2

2, 3, 12, NH-18

17, 19, 20 17, 18 21 21

NH-18, 19ab, 20ab, NH-22 12, 16, 18, 19ab 18 18 18, 20b 18, 20a

37.6, CH3 28.0, CH3 173.9, C 51.3, CH 30.5, CH2 27.4, CH2 28.5, CH2

20 17, 20 20 18, 22

22ab 21b, NH-22 21b, NH-22 18, 22ab

40.3, CH2

δH (J in Hz) 2.34, t (7.6) 1.50, m 1.2b 1.34, m 2.0, dt (7.4, 7.0) 5.79, ddt (10.1, 17.1, 6.6) 4.93, dd (1.8, 10.1) 4.99, ddt (1.8, 17.1, 3.7) 4.63, 4.64,a t (5.6) 1.91, m 2.14, m 2.54, m 2.63, m 2.546, 2.547,a s 2.736, 2.737,a s 4.39, 7.97, 1.35, 1.79, 1.62, 1.86, 1.27, 1.73, 3.05, 3.15, 7.86,

4.41,a brd (6.8) d (6.8) m m m m m m m m t (5.9)

Indicates R/S diastereomers at stereogenic S atom. bSignals were overlapped.

and H-18 (δH 4.33, 4.35) to C-11 (δC 168.5). HMBC crosspeaks from H-12, H3-16, and H2-2 to C-1 suggested the connection between N-methyl methionine sulfoxide and the 9decenoyl group, completing the planar structure of 1. The differences of 13C chemical shifts between the major and the minor conformer were mainly observed around C-1, C-12, and C-16, which implied geometrical isomerism of the amide bond between C-1 and N-12. In the NOESY data for the major conformer a cross-peak between H3-16 and methylene protons H2-2 was observed, suggesting that the amide bond was Z, while the amide bond in the minor conformer was E, as deduced from the NOESY cross-peak between H-12 (δH 4.63, 4.64) and H2-2 (δH 2.34). The absolute configurations of the amino acid residues in 1 were determined by Marfey’s method.6 Standard N-methyl-Dand N-methyl-L-methionine sulfone were prepared from commercially available N-Boc-D- and N-Boc-L-methionine, respectively. Compound 1 was converted to the sulfone, which was more stable toward acid hydrolysis conditions, and hydrolyzed with 6 N HCl to provide lysine and Nmethylmethionine sulfone. The standard amino acids and the hydrolysates of 1 were derivatized with L-FDAA followed by LC-MS analysis, which showed that the two amino acids had the L-configuration. Ciliatamide D (1) did not show inhibitory activity against cathepsin B at 50 μg/mL nor cytotoxicity against HeLa cells at 50 μg/mL. With the configurations of the amino acid residues in ciliatamide D (1) securely determined, a question arose as to

Figure 1. Selected COSY, HMBC, and NOESY correlations for 1.

5.13) to H2-14 (δH 2.54 and 2.63). An HMBC correlation from a doubled singlet peak for H3-15 (δH 2.516, 2.517) to C-14 (δC 49.8) as well as 13C chemical shift values of C-14 and C-15 (δC 37.6) indicated that C-14 and C-15 could be linked via a sulfur atom of a sulfoxide group, suggesting the presence of a methionine sulfoxide residue, which was in agreement with a 1:1 doubling of NMR signals such as H-12 (δH 5.12 and 5.13) or H3-15 (δH 2.516 and 2.517) due to the asymmetry of the sulfur atom.4,5 An HMBC correlation from the N-methyl protons [H3-16 (δH 2.85, 2.86)] to C-12 (δC 54.4) and a correlation from H-12 to C-16 (δC 30.5) indicated that the methionine sulfoxide residue was N-methylated. The remaining signals consisted of one carbonyl carbon (δC 173.4), seven methylenes, and a terminal monosubstituted olefin, indicating the presence of a 9-decenoyl group. The linkage between the cyclized lysine and N-methyl methionine sulfoxide was established by the HMBC correlations from lysine NH-18 756

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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). Ciliatamide D (1): white, amorphous powder; [α]24D +58 (c 0.035, MeOH); UV (MeOH) λmax (log ε) 212 (3.7) nm; 1H 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]22D +52 (c 0.05, MeOH);8 UV (MeOH) λmax (log ε) 208 (3.9) nm; 1H 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-Lmethionine 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-Dmethionine 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, respectively, 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 Lamino acid standards were treated with L-FDAA in the same manner. The L-FDAA derivatives were analyzed by LC-MS [Cosomosil 2.5C18MS-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), Nmethyl-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-phenylalanine (tR 16.2 min); retention times of standard N-methyl-Lphenylalanine and N-methyl-D-phenylalanine were 16.2 and 18.3 min, respectively. Biological Assays. See the Supporting Information.

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 Lconfiguration and that racemization did not occur during the hydrolysis nor derivatization for Marfey’s analysis. Therefore, we have concluded that our previous configurational assignment of ciliatamide A (2) was correct.7

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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 1H and 150 MHz for 13C. 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. tetraf urcata 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 RPHPLC (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

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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 of 1, 1H NMR spectrum of 2, LC-MS chromatogram charts of 757

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derivatives of lysine and N-methylphenylalanine, and the optical rotation data for 2. This material is available free of charge via the Internet at http://pubs.acs.org.

L-FDAA

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AUTHOR INFORMATION

Corresponding Author

*Tel: +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 We appreciate the crew of the T/S Nagasaki-Maru and Prof. Osamu Arakawa of Nagasaki University for their assistance in collecting the sponge specimen. This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

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REFERENCES

(1) Blunt, J. W.; Munro, M. H. Dictionary of Marine Natural Products; Chapman & Hall/CRC: Boca Raton, 2008. (2) Manson, S. D.; Joyce, J. A. Trends Cell Biol. 2011, 21, 228−237. (3) (a) Nakao, Y.; Kawatsu, S.; Okamoto, C.; Okamoto, M.; Matsumoto, Y.; Matsunaga, S.; van Soest, R. W. M.; Fusetani, N. J. Nat. Prod. 2008, 71, 469−472. (b) Lewis, J. A.; Nathan Daniels, R.; Lindsley, C. W. Org. Lett. 2008, 10, 4545−4548. (4) (a) Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.; Paul, V. J. J. Nat. Prod. 1999, 62, 655−658. (b) Nogle, L. M.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2001, 64, 716− 719. (c) Matthew, S.; Ross, C.; Paul, V. J.; Luesch, H. Tetrahedron 2008, 64, 4081−4089. (5) The 1:1 NMR signal doubling was abolished after converting 1 to the corresponding sulfone by treatment with Oxone. (6) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (7) Even if racemization did occur under the hydrolysis or derivatization conditions, a 50:50 mixture of D- and L-forms would be expected, not inversion to the opposite configuration. We tried to obtain synthetic ciliatamide A for comparison, but the request was unsuccessful because the sample was no longer available. (8) The specific rotation of ciliatamide A (2) was reported to be [α]20D +40 (c 0.05, MeOH) in the original paper.3a (9) Hoshino, T. J. Sci. Hiroshima Univ. Ser. B. Div. 1 (Zoology) 1981, 29, 207−289.

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