Biosci. Biotechnol. Biochem., 69 (7), 1318–1322, 2005
Koshikamide A2 , a Cytotoxic Linear Undecapeptide Isolated from a Marine Sponge of Theonella sp.* Takahiro A RAKI, Shigeki M ATSUNAGA, and Nobuhiro F USETANIy Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received February 8, 2005; Accepted April 1, 2005
Koshikamide A2 (2) was isolated as a cytotoxic metabolite from a marine sponge of Theonella sp. Its structure was elucidated to be a linear undecapeptide by spectroscopic and chemical methods, together with enzymatic conversion to known koshikamide A1 (1). The new peptide moderately inhibited the growth of P388 murine leukemia cells. Key words:
cytotoxic metabolite; marine sponge; undecapeptide
A wide variety of bioactive non-ribosomal peptides containing unusual amino acids1) have been isolated from marine sponges of the Theonella genus, e.g., the cyclotheonamides, serine proteases-inhibitors,2) the theonellamides, antifungal peptides,3,4) and the highly cytotoxic polytheonamides.5) We have previously reported the isolation of the cytotoxic linear decapeptide, koshikamide A1 (1), from a sponge of Theonella sp. collected off the Koshiki-jima Islands of Japan.6) Further examination of the extract from the same sponge afforded a closely related peptide, koshikamide A2 (2) as a cytotoxic principle. We report in this paper the isolation and structural elucidation of koshikamide A2 (2).
Materials and Methods General procedures. Opitical rotation data were measured with a Jasco DIP-1000 digital polarimeter in MeOH. UV spectra were recorded by a Shimadzu BioSpec-1600 UV spectrometer in CH3 OH, and IR spectra by a Jasco FT/IR-5300 infrared spectrometer. NMR spectra were recorded by a Jeol A600 NMR spectrometer operating at 600 MHz for 1 H and 150 MHz for 13 C. 1 H- and 13 C-NMR chemical shifts were referenced to solvent peak: H 2.49 and C 39.5 for DMSO-d6 . FAB mass spectra were measured with a Jeol *
JMX-SX102/SX202 tandem mass spectrometer, using m-nitrobenzylalcohol as a matrix. The amino acid analysis was carried out with a Hitachi L 8500-A amino acid analyzer. Animal material. The sponge of Theonella sp. was collected by hand using SCUBA from a depth of 15 m off Shimo-koshiki-jima Island, Kagoshima (129 2440 N; 31 2440 E), immediately frozen, and kept at 20 C until being processed. A voucher specimen of the sponge (ZMA POR. 13011) has been deposited at the Zoological Museum of the University of Amsterdam. Extraction and purification. The frozen sponge (100 g-wet weight) was extracted with EtOH (3 200 ml). The combined extracts were concentrated and partitioned between CHCl3 and H2 O. The CHCl3 soluble layer was further partitioned between 90% MeOH and n-hexane. The aqueous MeOH fraction was separated by flash chromatography on ODS with an MeOH/H2 O system; the active fraction eluted with 80% MeOH was gel-filtered through Sephadex LH-20 [MeOH/CH2 Cl2 (1:1)] and then subjected to ODSHPLC in a Cosmosil AR II column (10 250 mm; 30% 1-PrOH containing 0.05% TFA; UV detection at 215 nm; flow rate 2.0 ml/min) to furnish koshikamide A2 (2, 8.7 mg) as a colorless solid. Koshikamide A2 (2): ½24 D 130:0 (c 0.1, MeOH); UV (MeOH) max 209 nm (" 19000); IR (film) max : 3417, 2964, 1682, 1455, 1203, 1136, 1006, 755, 666 cm1 ; see Table 1 for 1 H- and 13 C-NMR data; HRFABMS m=z 1457.8490 ½M þ Hþ (calcd. for C72 H113 N16 O16 , 1457.8487). Amino acid analysis of 2. A 0.1-mg portion of 2 was dissolved in 200 ml of 6 N HCl and heated at 110 C for 15 h. The solvent was then evaporated in a stream of N2 .
Part 131 of the Bioactive Marine Metabolites series. Part 130: Matsunaga, S., Kobayashi, H., van Soest, R. W. M., and Fusetani, N., Novel bromotyrosine derivatives, which inhibit growth of the fish pathogenic bacterium Aeromonas hydrophyla, from a marine sponge Hexadella sp. J. Org. Chem., 70, 1893–1896 (2005). y To whom correspondence should be addressed. Tel: +81-3-5841-5299; Fax: +81-3-5841-8166; E-mail:
[email protected] Abbreviations: COSY, correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn; HMQC, heteronuclear multiple-quantum coherence; HMBC, heteronuclear multiple-bond coherence; ROESY, rotating frame Overhauser enhancement spectroscopy
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Table 1. NMR Data for Koshikamide A2 (2) in DMSO-d6 H (mult., J ¼ Hz)a trans-isomer MeOAc
Phe-1
MeVal-2
MeAsn-3
OMe CH2 CO CO C1 C2, 6 C3, 5 C4 NH CO 0 N-Me CO CONH2
Melle-4
MeVal-5
MeLeu-6
N-Me CO 0 N-Me CO 0 N-Me CO 0 N-Me
3.19 (s) 3.74
4.93 (m) 2.88 (m) 3.00 (dd, 13.4, 5.5) 7.28 7.25 7.18 8.03
(m) (m) (m) (d, 7.9)
4.89 2.15 0.57 0.78 2.88
(m) (m) (d, 6.5) (m) (s)
5.68 2.00 2.82 6.82 7.32 2.74
(dd, 9.7, 3.7) (dd, 14.8, 3.7) (m) (s) (s) (s)
5.05 2.08 0.64 0.98 1.21 0.80 2.79
(m) (m) (d, 6.5) (m) (m) (m) (s)
5.05 2.15 0.72 0.83 2.74
(m) (m) (m) (m) (s)
5.10 1.42 1.58 1.16 0.79 0.84 2.82
(m) (m) (m) (m) (m) (m) (s)
C 58.4 70.9 168.9 171.2 50.0 36.7
Asn-7
137.2 129.5 128.2 126.5
Phe-8
168.7 58.3 26.5 17.8 19.6 29.8 169.3 50.0 34.4
CO CONH2
Pro-9
NH CO C1 C2, 6 C3, 5 C4 NH CO
171.0 Pro-10 30.4 169.0 56.2 32.3 13.4 25.4
CO
Arg-11 10.5 29.3 170.1 57.6 26.5 17.8 19.4 29.4 170.2 53.4 36.2
CO guanidyl NH
H [mult., J (Hz)] trans-isomer
H [mult., J (Hz)] cis-isomer
4.55 2.24 2.32 6.95 7.27 7.71
4.40 2.14 2.18 6.91 7.16 7.53
(m) (m) (m) (s) (s) (d, 7.4)
(m) (m) (m) (s) (s) (d, 7.9)
4.72 (m) 2.74 (m) 2.88 (m)
4.26 (m) 2.74 (m) 2.74 (m)
7.04 7.21 7.16 7.98
(m) (m) (m) (d, 8.3)
7.15 7.24 7.18 8.40
(m) (m) (m) (d, 7.4)
4.48 1.74 1.98 1.65 3.18 3.50
(m) (m) (m) (m) (m) (m)
4.91 1.96 2.28 1.78 3.41 3.41
(m) (m) (m) (m) (m) (m)
4.52 1.90 2.02 1.90 3.49 3.66
(m) (m) (m) (m) (m) (m)
4.34 1.82 2.00 1.88 3.53 3.66
(m) (m) (m) (m) (m) (m)
4.17 1.59 1.75 1.53 3.09 7.62
(m) (m) (m) (m) (m) (br)
4.15 1.56 1.73 1.49 3.07 7.58
(m) (m) (m) (m) (m) (br)
8.20 (d, 7.9)
C transisomer
C cisisomer
170.1 49.6 37.8
170.8 49.6 37.0
171.4
171.2
168.3 51.7 38.0
169.3 51.7 38.0
138.1 128.9 128.2 126.2
136.8 129.5 128.2 126.5
— 57.6 28.0
— 58.0 30.0
24.0 46.4
22.0 46.4
171.6 59.2 28.8
171.7 59.0 29.8
24.4 46.2
24.4 46.4
173.5 51.8 28.0
173.5 51.5 28.2
25.1 40.3 156.5
25.0 40.3 156.5
8.09 (d, 7.9)
24.3 20.9 23.4 30.1
a1 H and 13 C chemical shifts from the N-terminus to MeVal-5 were identical between the trans- and cis-isomers. The 1 H chemical shifts of the cis-isomer for MeLeu-6 is as follows: 5.09 (m; H), 2.82 (3H, s; N-Me), 1.61 (m; H), 1.44 (m; H0 ), 1.16 (m; H), 0.84 (3H, d; H), and 0.79 (3H, d; H0 ). The 13 C chemical shifts for MeLeu-6 were identical between the trans- and cis-isomers.
The resulting residue was redissolved in 0.02 N HCl and subjected to an amino acid analysis which indicated the presence of Asp, Pro, Phe, and Arg in the hydrolysate. HPLC analysis of the Marfey derivatives of 2. To the acid hydrolysate of koshikamide A2 (2, 0.5 mg) were added 50 ml of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide in acetone (10 mg/ml) and 100 ml of 1 M NaHCO3 . The mixture was kept at 80 C for 3 min. The reaction mixture was diluted with 50 ml of 2 N HCl
and 100 ml of 50% MeCN containing 0.05% TFA, and then analyzed by ODS HPLC in a Cosmosil MS column [(10 250 mm) linear gradient elution in 60 min from MeCN/H2 O/TFA (25:75:0.05) to MeCN/H2 O/TFA (55:25:0.05)] detected by UV absorption at 340 nm. Enzyme digestion of 2. To a MeOH solution of koshikamide A2 (2, 0.1 ml, 10 mg/ml) were added 1 ml of 1 M Tris–HCl containing 0.5 M NaCl (pH 7.5) and 10 units of carboxypeptidase B (Sigma Chem. Co.) con-
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taining 10% LiCl. The mixture was incubated at 37 C for 17 h and then inactivated by raising the temperature to 60 C. The reacton mixture was separated by ODSHPLC in a Cosmosil AR II column (10 250 mm; 30% PrOH containing 0.05% TFA; UV detection at 215 nm; flow rate 2.0 ml/min) to afford koshikamide A1 .
Results and Discussion The frozen sponge was extracted with EtOH, and the CHCl3 -soluble materials in the extract were partitioned between n-hexane and 90% MeOH. The aqueous MeOH layer was subjected to bioassay-guided fractionation by ODS flash chromatography, with subsequent gel-filtration through Sephadex LH-20 and ODS HPLC to afford koshikamide A2 (2; 0.003% yield based on wet sponge) as a colorless solid. Koshikamide A2 (2) had a molecular formula of C72 H112 N16 O16 , as determined by HR-FABMS. The 1 Hand 13 C-NMR spectra revealed its close structural similarity to koshikamide A1 (1). The COSY, HOHAHA, HMQC, and HMBC data allowed the identification of one residue each of Asn, Arg, MeAsn, MeIle and MeLeu, and two residues each of Pro, Phe and MeVal, which were all present in koshikamide A1 (1), except for Arg. The presence of a methoxyacetyl unit was confirmed by the HMQC and HMBC spectra. The amino acid sequence of 2 was mainly deduced from interpretation of the HMBC data which exhibited correlation between NH or an N-methyl proton signal and a carbonyl carbon through an amide bond, while the sequence from Phe-8 to Pro-10 was assigned on the basis of ROESY data (Fig. 1).
In the 1 H-NMR spectrum of 2, doubled signals for Asn-7, Phe-8, Pro-9, Pro-10, and Arg-11 were observed in the ratio of 1:1, suggesting the existence of conformational equilibrium (Fig. 2, Table 1). It was surmised that the two conformers arose from cis/trans isomerization of a prolyl peptide bond. Although the carbon chemical shifts of Pro-10 hardly fluctuated between the two conformers, those of Pro-9 differed significantly. The difference in chemical shifts of the - and -carbons of Pro-9 were 4.0 ppm for the trans isomer and and 8.0 ppm for the cis isomer,7,8) in agreement with the cis/trans isomerization of the Phe-8/Pro-9 amide bond. Interestingly, the amide bond between Pro-9 and Pro-10 adopted only the trans geometry. Similar isomerism has been observed in koshikamide A1 .6) The absolute configuration of 2 was determined by the combination of a Marfey analysis of the acid hydrolysate9) and enzymatic digestion of 2. The Marfey analysis of the acid hydrolysate of 2 demonstrated the presence of L-Arg, L-Asn, L-MeLeu, L-MeVal, D-Phe, and L-Pro. The absolute configuration of MeAsn and MeIle could not be determined by the Marfey analysis due to poor resolution of the isomers, so conversion of 2 to 1 was envisaged. Fortunately, des-[Arg-11]-koshikamide A2 could be obtained by treating koshikamide A2 (2) with carboxypeptidase B (CPB) that removed a basic C-terminal amino acid residue. The 1 H-NMR spectrum of des-[Arg-11]-koshikamide A2 was identical with that of 1. Thus, the absolute configuration of MeAsn and MeIle was concluded to be identical with that in 1, i.e. LMeAsn and L-MeaIle. Koshikamide A2 (2) exhibited moderate cytotoxicity against P388 cells with an IC50 value of 6.7 mg/ml. The
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Fig. 1. Key HMBC and ROESY Correlations for Koshikamide A2 (2).
sponge. This work was partly supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1)
2)
3) Fig. 2. Amide Region of the 1 H NMR Spectrum of 2.
distinguishing feature of koshikamides A1 (1) and A2 (2) is the presence of a high proportion of N-methyl amino acid residues. Linear peptides rich in N-methyl amino acid residues have been reported from marine organisms; for instance, pupukeamide has been isolated from the cephalaspidean mollusk, Philinopsis speciosa,10) and apramide A from the marine cyanobacterium, Lyngbya majuscula.11) However, the presence of five consecutive N-methyl amino acid residues in the sequence is rare. Koshikamide A2 (2) is present as an equilibrated mixture of conformers in solution due to the presence of the proline residues. A similar observation has been reported for patellin 2, isolated from the Fijian marine tunicate, Lissoclinum patella, which exists in two conformers due to cis/trans isomerization of the ValPro amide bond, with the trans isomer being predominant.12) Koshikamide A2 (2) is a homolog of 1 by extension with the C-terminal Arg residue, as in the case of criamide and hemiasterlin, linear peptides isolated from the Papua New Guinean sponge, Cymbastela sp.13)
4)
5)
6)
7)
8)
9)
10)
Acknowledgment We are indebted to the crew of R/V Toyoshio-maru of Hiroshima University for assistance in collecting the
11)
Matsunaga, S., and Fusetani, N., Nonribosomal peptides from marine sponges. Curr. Org. Chem., 7, 945–966 (2003). Fusetani, N., Matsunaga, S., Matsumoto, H., and Takebayashi, Y., Cyclotheonamides, potent thrombin inhibitors, from a marine sponge Theonella sp. J. Am. Chem. Soc., 112, 7053–7054 (1990). Matsunaga, S., Fusetani, N., Hashimoto, K., and Wa¨lchli, M., Theonellamide F, a novel antifungal bicyclic peptide from a marine sponge Theonella sp. J. Am. Chem. Soc., 111, 2582–2588 (1989). Matsunaga, S., and Fusetani, N., Theonellamides A–E, cytotoxic bicyclic peptides, from a marine sponge Theonella sp. J. Org. Chem., 60, 1177–1181 (1995). Hamada, T., Matsunaga, S., Yano, G., and Fusetani, N., Polytheonamides A and B, highly cytotoxic, linear polypeptides with unprecedented structural features, from the marine sponge, Theonella swinhoei. J. Am. Chem. Soc., 127, 110–118 (2005). Fusetani, N., Warabi, K., Nogata, Y., Nakao, Y., and Matsunaga, S., Koshikamide A1 , a new cytotoxic linear peptide isolated from a marine sponge, Theonella sp. Tetrahedron Lett., 40, 4687–4690 (1999). Pihlaja, K., and Kleinpeter, E., ‘‘Carbon-13 NMR Chemical Shifts in Structural and Stereochemical Analysis’’, VCH Publishers, New York, p. 325 (1994). Dorman, D. E., and Borvey, F. A., Carbon-13 magnetic resonance spectroscopy. Spectrum of proline in oligopeptides. J. Org. Chem., 38, 2379–2383 (1973). Marfey, P., Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun., 49, 591–596 (1984). Nakao, Y., Yoshida, W. Y., and Scheuer, P. J., Pupukeamide, a linear tetrapeptide from a cephalaspidean mollusk Philinopsis speciosa. Tetrahedron Lett., 37, 8993–8996 (1996). Luesch, H., Yoshida, W. Y., Moore, R. E., and Paul, V. J., Apramides A–G, novel lipopetides from the marine
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cyanobacteriumlyngbya majuscula. J. Nat. Prod., 63, 1106–1112 (2000). Zabriskie, T. M., Foster, M. P., Stout, T. J., Clardy, J., and Ireland, C. M., Studies on the solution- and solidstate structure of patellin 2. J. Am. Chem. Soc., 112,
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8080–8084 (1990). Coleman, J., de Silva, E. D., Kong, F., Andersen, R. J., and Allen, T. M., Cytotoxic peptides from the marine sponge Cymbastela sp. Tetrahedron, 51, 10653–10662 (1995).
