Koshikamide B, a cytotoxic peptide lactone from a marine sponge Theonella sp

Article abstract of DOI:10.1021/jo801032n

(Chemical Equation Presented) Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual amino acid residues. The unusual amino acids are N^δ-carbamoylasparagine and 2-(3-amino-2-hydroxy-5- oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia cells and the human colon tumor (HCT-116) cell line with an IC₅₀ value of 0.45 and 7.5 μg/mL, respectively.

Full text of DOI:10.1021/jo801032n

Koshikamide B, a Cytotoxic Peptide Lactone from a Marine Sponge
Theonella sp
Takahiro Araki,†, Shigeki Matsunaga,*,† Yoichi Nakao,†,| Kazuo Furihata,‡
Lyndon West,§,# D. John Faulkner,§,O and Nobuhiro Fusetani†,3
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences,
The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, Graduate School of Agricultural and Life
Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, and Scripps Institution of
Oceanography, UniVersity of California, San Diego, 9500 Gilman DriVe, La Jolla, California 92037
assmats@mail.ecc.u-tokyo.ac.jp
ReceiVed May 15, 2008
Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the
major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic
amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual
amino acid residues. The unusual amino acids are Nδ-carbamoylasparagine and 2-(3-amino-2-hydroxy-5-
oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas
the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl
group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis
of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia
cells and the human colon tumor (HCT-116) cell line with an IC50 value of 0.45 and 7.5 µg/mL, respectively.
Introduction
viz. theonellamides3 and microsclerodermins (antifungal),4
cyclotheonamides (serine protease inhibition),5 polytheonamides
(cytotoxic),6 and papuamides (antiviral).7 Most of these peptides
contain amino acid residue(s) reminiscent of mixed nonribo-
somal peptide synthetase (NRPS)-polyketide synthase (PKS)
Marine sponges of the genus Theonella frequently contain
nonribosomal peptides with interesting biological activities,1,2
† Laboratory of Aquatic Natural Products Chemistry, Graduate School of
Agricultural and Life Sciences, The University of Tokyo.
‡ Graduate School of Agricultural and Life Sciences, The University of Tokyo.
§ Scripps Institution of Oceanography, University of California.
Present address: Mitsubishi Chemical Co., Ltd. Aoba-ku, Yokohama,
Kanagawa 227-8502, Japan.
(2) Bewley, C. A.; Faulkner, D. J. Angew. Chem., Int. Ed. 1998, 37, 2163–
2178.
(3) Matsunaga, S.; Fusetani, N. J. Org. Chem. 1995, 60, 1177–1181.
(4) Bewley, C. A.; Debitus, C.; Faulkner, D. J. J. Am. Chem. Soc. 1994,
116, 7631–7636.
(5) Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi, Y. J. Am.
Chem. Soc. 1990, 112, 7053–7054.
(6) Hamada, T.; Matsunaga, S.; Yano, G.; Fusetani, N. J. Am. Chem. Soc.
2005, 127, 110–118.
(7) Ford, P. W.; Gustafson, K. R.; McKee, T. C.; Shigematsu, N.; Maurizi,
L. K.; Pannell, L. K.; Williams, D. E.; de Silva, E. D.; Lassota, P.; Allen, T. M.;
van Soest, R. W. M.; ersen, R. J.; Boyd, M. R. J. Am. Chem. Soc. 1999, 121,
5899–5909.
| Present address: Department of Chemistry and Biochemistry, School of
Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo
169-8555, Japan.
# Present address: Department of Pharmaceutical and Biomedical Sciences,
The University of Georgia, Athens, GA 30602-2352.
3 Present address: Graduate School of Fisheries Sciences, Hokkaido Univer-
sity, Minato-cho, Hakodate 041-8611, Japan.
O Deceased on November 23, 2002.
(1) Matsunaga, S.; Fusetani, N. Curr. Org. Chem. 2003, 7, 945–966.
10.1021/jo801032n CCC: $40.75
Published on Web 09/24/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7889–7894 7889

Araki et al.
CHART 1
pathways of microorganisms,8,9 suggesting the involvement of
symbiotic microorganisms in their production.10 Indeed, a
biosynthetic gene cluster likely to be involved in the biosynthesis
of onnamides, metabolites of a mixed NRPS-PKS pathway, was
isolated from the metagenome of T. swinhoei.11 In the course
of our search for novel cytotoxic metabolites from marine
invertebrates, we have isolated koshikamides A1 and A212,13
from a sponge of the genus Theonella. The extract of the same
sponge contained a more abundant and more potent cytotoxic
constituent named koshikamide B. In an independent investiga-
tion by the Scripps Institution of Oceanography group (SIO), a
collection of Theonella sp. from Palau also yielded koshikamide
B from the methanolic extract of the sponge. The isolation and
structure elucidation of koshikamide B is presented below.
HMBC spectra. From the analysis, eight standard amino acid
residues, Ala (×2), Thr, Phe, Asn (×3), and Ile, were identified.
Their presence was confirmed by the amino acid analysis. Seven
N-methylated amino acid residues, Sar (MeGly), MeVal (×2),
MeLeu, MeIle (×2), MeAsn, and a methoxyacetyl group were
also identified from 2D NMR data.
There were two unusual residues (residues X and Y, Figure
1). Residue X contained a pyrrolidone ring as identified by the
COSY, HMQC, HMBC, and 15N HMQC data. The C-3
quaternary carbon (δ 91.0) showed HMBC correlations with
exchangeable protons at δ 6.65 and 7.98. The 15N HMQC
spectrum revealed that the proton at δ 7.98 was attached to a
nitrogen at δ 137.0, wheras the proton at δ 6.65 was not
attached to a nitrogen, indicating that C-3 was connected to
an N-acyl and a hydroxyl group. A pair of methylene protons
(δ 1.82, 2.81; H2-5) were coupled to an amide proton at δ
8.88 and gave HMBC correlations with C-3 and a carbonyl
carbon (δ 177.3). A methine proton at δ 2.32 (H-2) was
substituted by a methyl group (δ 1.18, 3H, d) and correlated
to C-3 in the HMBC spectrum, allowing us to define the
structure of residue X as 2-(3-amino-2-hydroxy-5-oxopyr-
rolidin-2-yl)propionic acid (AHPP).
Results and Discussion
The EtOH extract of the sponge was partitioned between
CHCl3 and H2O, and the organic layer was evaporated and
further partitioned between 90% MeOH and n-hexane. The
aqueous MeOH fraction was subjected to ODS flash chroma-
tography, and the 90% MeOH eluate was separated by ODS
HPLC with 1-PrOH/H2O/TFA (30:70:0.05) to afford three
cytotoxic peaks. Because the three fractions gave indistinguish-
able NMR and mass spectral data, they were judged as identical
and named koshikamide B (1, 330 mg, Chart 1). A second
collection of the sponge Theonella also yielded 1 from the
methanolic extract of this sponge which was fractionated on
HP-20 followed by HPLC using a poly(styrene-divinylbenzene)
column (PRP-1).
Residue Y contained NMR signals for an Nδ-substituted
asparagine. Only one proton (δ 9.74) was attached to Nδ, and
a pair of additional exchangeable protons (δ 7.48, 7.88) were
observed. There was an unassigned carbon at δ 154.1, which
should be incorporated in this residue. The structural assignment
by NMR was hampered by the absence of HMBC correlations
from the carbon at δ 154.1. The 15N HMQC spectrum of 1
revealed that the protons at δ 7.48 and 7.88 were both connected
to a nitrogen at δ 89.0, while the proton at δ 9.74 was connected
to a nitrogen at δ 149.0, suggesting the presence of a ureido
moiety. In the COSY and TOCSY spectra protons at δ 7.48,
7.88, and 9.74 were correlated to each other. In order to compare
NMR data we prepared an Nδ-carbamoyl Asn derivative (2).
The COSY and TOCSY spectra of the synthetic compound (2)
Koshikamide B (1) exhibited the [M - H]- ion at m/z 2050
in the negative FAB-MS. The peptidic nature of 1 was inferred
from the 1H NMR spectrum, which exhibited signals for
R-protons between δ 4.0 and 5.6 and those for amide protons
between δ 7.8 and 8.9. The constituent amino acids of 1 were
assigned by interpretation of the COSY, TOCSY, HMQC, and
(8) Donadio, S.; Monciardini, P.; Sosio, M. Nat. Prod. Rep. 2007, 24, 1073–
1109.
(9) Finking, R.; Marahiel, M. A. Annu. ReV. Microbiol. 2004, 58, 453–488.
(10) Piel, J. Nat. Prod. Rep. 2004, 21, 519–538.
(11) Piel, J.; Hui, D.; Wen, G.; Buzke, D.; Platzer, M.; Fusetani, N.;
Matsunaga, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16222–16227.
(12) Fusetani, N.; Warabi, K.; Nogata, Y.; Nakao, Y.; Matsunaga, S.
Tetrahedron Lett. 1999, 40, 4687–4690.
(13) Araki, T.; Matsunaga, S.; Fusetani, N. Biosci. Biotechnol. Biochem. 2005,
69, 1318–1322.
FIGURE 1. Gross structures of residue X and residue Y
7890 J. Org. Chem. Vol. 73, No. 20, 2008

Koshikamide B
FIGURE 3. Equilibrium of the AHPP residue in 1.
FIGURE 2. Selected NOESY correlations of the AHPP residue in 1.
liberated L-Ala, as expected. Therefore, by default, MeaIle-4
was assigned as L.
displayed the correlations among the ureido protons as observed
in koshikamide B (1), and the 15N chemical shift values for the
ureido nitrogens in both compounds were almost identical.
Therefore, residue Y was assigned as Nδ-carbamoyl Asn.
The amino acid sequence of 1 was determined by interpreta-
tion of the constant time HMBC (CT-HMBC)14 and NOESY
data. The resolution of the 13C-axis of the HMBC spectrum is
reduced due to the f1-modulation caused by 1H-1H couplings.
The CT-HMBC spectrum is free of f1-modulation, which
facilitates the signal assignments in crowded regions. Inter-
residual HMBC cross peaks were observed across almost all of
the amide bonds, allowing us to determine the amino acid
sequence of 1. The methoxyacetyl group was placed at the
N-terminus, while the carboxyl group of the C-terminal MeaIle
was lactonized with Thr-8.
The stereochemistry of Phe, Thr, MeVal, MeAsn, and MeLeu
residues was determined to be D, L, L, L, and L, respectively, by
the Marfey method.15 The stereochemistry of Ile and Asp in
the hydrolysate was determined as D and L, respectively, by
chiral GC analysis with Chirasil-Val. Therefore, three Asn and
Nδ-carbamoylasparaginyl residues were all in the L-form. D- and
L-Ala were detected in a 1:1 ratio by chiral GC analysis. Because
it was not possible to completely separate the four isomers of
MeIle and MeaIle by chiral GC or Marfey’s method, the
stereochemical assignment of these residues was established in
two steps. First, the hydrolysate of koshikamide B (1) was
subjected to ODS-HPLC, and the peak eluting at the retention
time of MeIle and MeaIle, which coeluted, was isolated. The
1
isolated amino acid was identified as MeaIle by the H NMR
Stereochemistry of the AHPP residue was determined by a
combination of spectral and chemical methods. A NOESY cross
peak between OH-3 and H-4 showed that these protons were
on the same face of the five-membered ring. Cross peaks
between H-2 and 4-NH and between 2-CH3 and 7-NH allowed
us to define the relative stereochemistry of C-2 with respect to
the five-membered ring (Figure 2). Therefore the stereochemistry
of the AHPP residue was assigned as 2S*, 3R*, and 4S*.
Koshikamide B was susceptible to reduction with NaBH4 and
gave the alcohol 6. Interpretation of the NMR data of 6
demonstrated that the AHPP residue was converted to the
4-amino-5-carbamoyl-3-hydroxy-2-methylpentanoic acid resi-
due, indicating that C-3 in the AHPP residue was transiently
present as the ketone form (Figure 3). It is likely the reason for
the multiplication of HPLC peaks which was caused by an
spectrum. The Marfey analysis of the isolated MeaIle revealed
that it was a 1:1 mixture of D and L isomers. Consequently, 1
contained one residue each of D- and L-MeaIle.
Residue-specific stereochemical assignment of Ala and
MeaIle residues in 1 was accomplished by the fortuitous
isolation of fragment peptides containing only one of these
residues. Hydrolysis of 1 with 6 N HCl at room temperature
for 25 h followed by HPLC separation afforded fragment
peptides 3-5 (Figure 3), whose structures were assigned by a
combination of FABMS and amino acid analysis. Compounds
3 and 4 contained Ala-16 and MeaIle-17, whereas 5 contained
Ala-12. The Marfey analysis of the hydrolysates revealed the
presence of D-MeaIle and D-Ala in both 3 and 4. Therefore,
Ala-16 and MeaIle-17 were both shown to be D. Compound 5
J. Org. Chem. Vol. 73, No. 20, 2008 7891

Araki et al.
SCHEME 1
IC50 values of 0.45 and 7.5 µg/mL, respectively. Koshikamide
B (1) represents a new class of the peptide lactone possessing
two unique moieties: the carbamoylated Asn and AHPP residues.
A ureido moiety is present in several peptides from the sponge
Theonella swinhoei, e.g., konbamide, keramamide A, and
mozamides, where the ureido moiety participates in the linkage
between the N-terminal residue and another amino acid.1,2
Although Nδ-carbamoyl Asn itself was discovered in urine of
rats fed ozonated casein,18 1 is the first example of Nδ-carbamoyl
Asn to be present as a constituent of a natural peptide. The
AHPP unit is a new amino acid residue. Pyrrolidone-containing
amino acid residues have only been found in microscleroder-
mins.4 The AHPP residue in 1 may be biosynthesized by Claisen
condensation of a propionate and Asn, followed by spontaneous
cyclization. The present study demonstrated the transient
presence of the ketone form 1a. It is interesting to note that the
structures of koshikamides A1, A2, and B are identical from
the N-terminus until residue-7.12,13
FIGURE 4. J-based configurational analysis of 6.
equilibrium among the ketone, hemiacetal, and cyclized aminal
forms in this residue (Figure 3). Compound 6 was used for
further analysis of the stereochemistry of the AHPP residue by
the application of J-based configuration analysis (Figure 4).16
2,3
3JH,H and
J
C,H
values were measured by 1D TOCSY and
J-resolved HMBC spectra,1718 respectively. A small coupling
constant between H-2 and H-3 (0.8 Hz) suggested that they were
3
gauche. H-2 exhibited a small J coupling constant with C-4,
and H-3 displayed a large 3J coupling constant with C-1,
indicating that H-2 and C-4 were gauche, while H-3 and C-1
were antiperiplanar. An intense ROESY cross peak between
H-3 and 2-CH3 was consistent with 2,3-anti stereochemistry with
C-1 and C-4 in the gauche relationship (Figure 4a). On the other
hand, the large coupling constant between H-3 and H-4 (10.4
Hz) and an intense ROESY cross peak between H-2 and 4-NH
indicated 3,4-anti stereochemistry (Figure 4b). Therefore, the
relative stereochemistry of C-2, C-3, and C-4 in 6 was assigned
as 2S*,3R*,4S*.
Experimental Section
Animal Material. The sponge was collected by scuba at a depth
of 15 m off Shimokoshiki Island, Kagoshima prefecture (129°244’N,
31°244’E), immediately frozen, and kept at -20 °C until processed.
A voucher specimen (ZMA POR. 13011) was deposited at the
Zoological Museum of the University of Amsterdam. The SIO
collection of Theonella was hand-collected by Pat Colin from a
depth of 85 m at Palau using a mixed gas rebreathing apparatus.
The specimen was immediately frozen and kept at -20 °C until
extraction. A voucher specimen has been deposited in the SIO
Benthic Invertebrate Collection (nos. 99-411).
Extraction and Purifcation. The sponge (280 g; wet weight)
was chopped into small pieces and extracted with EtOH (3 × 200
mL). The combined extracts were concentrated and partitioned
between CHCl3 and H2O. The CHCl3 layer was evaporated and
partitioned between n-hexane and 90% MeOH. The 90% MeOH
layer was fractionated by flash chromatography on ODS with
MeOH/H2O system; the active fraction eluted with 90% MeOH
was further separated by reversed-phase HPLC on an ODS column
with 30% 1-PrOH containing 0.05% TFA to furnish koshikamide
B (1, 330 mg) as a white powder. The extract also afforded the
known koshikamides A1 and A2. The SIO sponge (372 g; wet
weight) was extracted with MeOH (2 × 750 mL) for 24 h. The
second and then the first extracts were passed through a column of
HP-20 (5 × 20 cm). The combined eluents was repassed through
the column. Finally, the eluent was concentrated to 500 mL and
diluted with H2O (1.5 L) and passed again through the column.
The column was eluted with 600 mL fractions of (1) H2O, (2) 25%
Me2CO/H2O, (3) 50% Me2CO/H2O, (4) 75% Me2CO/H2O, and (5)
Me2CO. Fraction 3 (500 mg) was then subjected to reversed-phase
HPLC (Hamilton PRP-1; 21.5 × 250 mm; 10 mL/min; 30-50%
CH3CN/H2O over 50 min) to give koshikamide B (1, 383 mg).
Koshikamide B (1): [R]D -120 (c 0.1, 1-PrOH-H2O, 4:6); UV
(1-PrOH-H2O, 4:6) 266 nm (ꢀ 5.9 × 103); IR (film) νmax 3850,
It was reported that the pyrrolidone-containing amino acid
residue in microsclerodermin A gave a dehydration product by
treatment with acid, and the product was used to determine the
absolute stereochemistry of this unit.4 The aminal hydroxyl
group did not dehydrate either under acidic conditions or in
the presence of dehydrating agents such as POCl3. Under these
reaction conditions, the products were always a complex
mixture. Eventually, we found that 1 underwent dehydration
on heating at 75 °C in EtOH and the product was the desired
olefin 7. Ozonolysis of 7 followed by a reductive workup
furnished imide 8 which was hydrolyzed with 6 N HCl and the
acid hydrolysate was subjected to the amino acid analysis and
GC analysis, revealing that 8 afforded 5 equiv of L-Asp. The
additional equivalent of L-Asp arose from the imide moiety in
8 which should have the 4S-stereochemistry (Scheme 1).
Considering the relative stereochemistry mentioned above, the
absolute configuration of the AHPP residue was concluded to
be 2S,3R,4S.
Koshikamide B (1) exhibited cytotoxicity against P388 murine
leukemia cells and human colon tumor (HCT-116) cell line with
(14) Furihata, K.; Seto, H. Tetrahedron Lett. 1998, 39, 7337–7340.
(15) Marfey, P. Carlsberg. Res. Commun. 1984, 49, 591–596.
(16) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K.
J. Org. Chem. 1999, 64, 866–876.
(17) Furihata, K.; Seto, H. Tetrahedron Lett. 1999, 40, 6271–6275.
(18) Kasai, T.; Kiriyama, S. Agric. Biol. Chem. 1990, 54, 2663–2667.
7892 J. Org. Chem. Vol. 73, No. 20, 2008

Koshikamide B
TABLE 1. 1H and 13C NMR Data of Koshikamide B (1) in DMSO-d6
assignment
δH
δC
HMBC (C no.)a
assignment
Asn-9
δH
δC
HMBC (C no.)a
methoxyacetyl
1
2
168.8
71.0 OMe, 1
58.2 2
1
2
3
171.8
3.75
3.22
5.18 (m)
2.28 (m)
2.58 (m)
44.6 1, 3, 4, (Thr-8)-1
37.5 1, 2, 4
OMe
Phe-1
1
2
3
171.2
1, 2, 4
4.95 (m)
2.89 (m)
3.02 (dd, 13.5, 5.8)
50.0 1, 3, (methoxyacetyl)-1
36.7 1, 2, 1′, 3′, 5′
1, 2, 1′, 3′, 5′
4-CONH2 6.82 (s)
170.6
3
7.26 (s)
8.42 (d, 8.8)
NH
1
2
(Thr-8)-1
1′
137.0
MeGly-10
Asn-11
168.1
2′, 6′
3′, 5′
4′
7.25 (m)
7.28 (m)
7.18 (m)
128.0 1′, 2′, 6′
129.1 3′, 4′, 5′
126.4 3′, 5′
3.31 (m)
4.41 (m)
3.18 (s)
50.4 1, N-Me, (Asn-9)-1
1, N-Me
37.3 2, (Asn-9)-1
172.1
50.5 1, 3, 4, (MeGly-10)-1
36.7 1, 4
N-Me
NH
1
2
3
4
7.91 (d, 8.1)
2, 3, (methoxyacetyl)-1
168.8
58.0 1, 3, 4, 5, N-Me, (Phe-1)-1
26.5 2, 5
17.8 2, 3, 5
19.2 2, 3
1
2
3
MeVal-2
MeAsn-3
4.43 (m)
2.28 (m)
2.28 (m)
4.90 (m)
2.17 (m)
0.58 (d, 6.9)
0.78 (m)
2.88 (s)
4-CONH2 6.90 (s)
170.4
3
5
6.98 (s)
8.10 (d, 6.2)
N-Me
29.8 2, (Phe-1)-1
169.3
NH
1
2
3
NH
1
(MeGly-10)-1
Ala-12
Ile-13
172.1
48.5 1, 3
5.68 (dd, 10.0, 4.6) 50.0 1, 3, N-Me, 4
2.05 (dd, 15.4, 4.2) 34.5 1, 2, 4
4.05 (m)
1.28 (d, 6.9) 17.8
8.64 (m)
2
2.82 (m)
4-CONH2 6.77 (s)
1, 2, 4
3
(Asn-11)-1
170.9
172.1
7.28 (s)
2.76 (s)
2
3
4
4.19 (m)
1.57 (m)
1.05 (m)
1.38 (m)
0.72 (m)
0.72 (m)
8.38 (d, 9.6)
56.0 1, 3, 4
35.0
24.2 3, 5
N-Me
30.3 2, (MeVal-2)-1
169.0
56.2 1, 3, 4, 6, N-Me
32.2
MeIle-4
1
2
3
4
5.08 (m)
2.10 (m)
0.98 (m)
1.22 (m)
0.81 (m)
0.68 (d, 6.9)
2.81 (s)
5
6
NH
1
2
3
4
9.5 3, 4
14.8 2, 3, 4
25.0 3, 6
(Ala-12)-1
5
6
10.5 3, 4
13.2 2, 3, 4
29.2 2, (MeAsn-3)-1
170.1
AHPP-14
177.3
2.32 (m)
4.16 (m)
38.8 1, 3, 7
91.0
53.5 5, 6
N-Me
1
MeVal-5
MeLeu-6
2
3
4
5
5.09 (m)
2.17 (m)
0.74 (m)
0.86 (m)
2.76 (s)
57.8 1, 3, 4, 5, N-Me
26.5 2, 5
17.6 2, 3, 5
19.3 2, 3, 4
29.2 2, 1, (MeIle-4)-1
170.0
5
1.82 (d, 6.5) 35.8 3, 4, 6
2.81 (m) 3, 4, 6
176.5
1.18 (d, 7.3) 14.0 1, 2, 3
6
7
N-Me
6-NH
OH
4-NH
1
2
3
7.98 (s)
6.65 (s)
3, 4, 5, 6
2, 3
1
2
3
5.11 (m)
1.50 (m)
1.67 (m)
1.20 (m)
0.80 (m)
0.88 (m)
2.85 (s)
53.5 1, 3, N-Me
36.2 2, 5, 6
8.88 (d, 8.1)
(Ile-13)-1
carbamoyl Asn-15
169.2
4.98 (m)
2.45 (m)
3.21 (m)
49.0 1, 3, 4, (AHPP-14)-1
38.8 1, 2, 4
4
5
6
24.2
21.0 4, 6
23.1 4, 5
30.0 2, (MeVal-5)-1
170.0
49.7 1, 3, 4
36.2 1, 2, 4
1, 2, 4
4-CONH 9.74 (s)
5-CONH2 7.48 (s)
NH
1
2
3
NH
1
2
3
172.8
155.0
4
N-Me
Asn-7
Thr-8
1
2
3
7.88 (s)
7.85 (d, 8.9) 174.2
5.0 (m) 44.7 1, 3
1.34 (d, 7.3) 16.0 1, 2
(AHPP-14)-1
4.47 (m)
2.38 (m)
2.38 (m)
Ala-16
4-CONH2 6.75 (s)
171.8
8.61 (m)
(carbamoyl Asn-15)-1
7.25 (s)
7.72 (d, 6.9)
MeIle-17
168.8
NH
1
2
3
4
(MeLeu-6)-1
167.6
56.4 1, 3, NH
72.0 1, 2
3.38 (m)
2.31 (m)
1.05 (m)
1.78 (m)
0.85 (m)
0.73 (m)
3.18 (s)
67.5 1, 3, 4, N-Me
33.8 2, 4, 6
27.2 3, 6
4.32 (t, 9.2)
4.60 (m)
0.61 (d, 6.5)
7.67 (d, 8.8)
4
16.7 2, 3
5
6
11.5 3, 4
14.8 2, 3, 4
39.2 2, (Ala-16)-1
NH
(Asn-7)-1
N-Me
a For intraresidual correlations, residue numbers are not specified.
3287, 2964, 2876, 1681, 1633, 1556, 1538, 1504, 1415, 1283, 1100,
1002, 755, 665 cm-1; HRFABMS m/z 2074.0837 [M + Na]+ (calcd
for C93H150N24O28Na 2074.0725); 1H, 13C, and 15N NMR data
(DMSO-d6), see Table 1.
Amino Acid Analysis of Koshikamide B (1). A 1 mg portion
of koshikamide B (1) was dissolved in 200 µL of 6 N HCl and
heated at 110 °C for 15 h. The solvent was evaporated under a
stream of N2, and the residue was redissolved in 0.02 N HCl and
subjected to amino acid analysis.
asparagine, which was dissolved in dioxane (50 mL) and treated
with monochloroacetyl isocyanate (600 µL, 6 mmol). The reaction
mixture was stirred for 3 h at room temperature. After additions of
methanol (4 mL) and Zn dust (1.0 g), the suspension was stirred
for 3 h at room temperature. Zn dust was then removed by filtration,
and the filtrate was concentrated. The product was purified by
column chromatography on silica gel with CHCl3/MeOH (98:2) to
give benzyl NR-benzyloxycarbonyl-Nδ-carbamoyl-L-asparagine (2,
122.3 mg, 82%). 2: 1H NMR (DMSO-d6) δ 10.15 (s, 1H), 7.83 (d,
1H, J ) 8.1 Hz), 7.59 (s, 1H), 7.35-7.32 (m, 10H, ArH), 7.15 (s,
1H), 5.12 (s, 2H, CH2Ph), 5.03 (s, 2H, CH2Ph), 4.54 (m, 1H, H-2),
2.88 (dd, H-3), 2.71 (dd, H-3).
Synthesis of Benzyl Nr-Benzyloxycarbonyl-Nδ-carbamoyl-L-
asparagine (2). To a solution of NR-benzyloxycarbonyl-L-asparagine
(100 mg, 0.376 mmol) in DMF (300 µL) was added benzyl bromide
(500 µL, 4.21 mmol) in the presence of a small amount of K2CO3.
After being stirred for 3 h at room temperature, the reaction mixture
was quenched with water. The mixture was extracted with ethyl
acetate (100 mL) to give crude benzyl NR-benzyloxycarbonyl-L-
NaBH4 Reduction of Koshikamide B (1). To a solution of
koshikamide B (1, 10 mg) in MeOH (0.5 mL) was added NaBH4
(5 mg), and the mixture was stirred at room temperature for 30
min. The reaction was quenched by addition of water, the MeOH
J. Org. Chem. Vol. 73, No. 20, 2008 7893

Araki et al.
was removed under high vacuum, and the residue was dissolved
in aqueous 1-PrOH (1 mL) and purified by ODS HPLC [10 × 250
mm; 30% 1-PrOH containing 0.05% TFA (2 mL/min) detection
with absorption at 210 nm] to obtain 6 (7.2 mg).
and the hydrolysate was neutralized with 5 N NaOH and purified
by ODS HPLC [10 × 250 mm × 2; elution with a linear gradient
from 15% to 60% MeCN containing 0.05% TFA (2 mL/min);
detection with absorption at 220 nm]; peaks between tR 35 and 65
min were further purified by ODS HPLC [10 × 250 mm × 2;
elution with a linear gradient from 3% to 35% MeCN containing
0.05% TFA (2 mL/min); detection with absorption at 220 nm], to
afford the peptide fragments 3, 4, and 5. 3: FAB-MS/MS [m/z 942
(M + H)+, 789 (M - pyrrolidone unit)+, 675 (M - pyrrolidone
unit - Asp)+, 604 (M - pyrrolidone unit - Asp-Ala)+]. 4: FABMS
[m/z 790 (M + H)+]. 5: FABMS [m/z 829 (M + H)+]. Constituent
amino acids of 3-5 were analyzed by amino acid analysis, Marfey’s
analysis, and chiral GC analysis.
Dehydration of Koshikamide B (1) Followed by Ozonolysis. A
solution of 1 (5 mg) in ethanol (1 mL) was heated at 75 °C for
17 h. The reaction mixture was purified by HPLC [10 × 250 mm;
30% 1-PrOH containing 0.05% TFA (2 mL/min) detection with
absorption at 270 nm] to afford the olefin 7 (2.8 mg). Compound
7 gave the (M -H)- ion peak at m/z 2032 in the FABMS. A stream
of ozone was bubbled into a solution of 7 (2 mg) in MeOH (1 mL)
at room temperature for 30 min. The solution was dried under a
stream of nitrogen, and the residue was reduced by addition of Me2S
(100 µL). The solution was kept at room temperature for 1 h, and
the excess reagent was removed under high vacuum. The reaction
product was hydrolyzed and subjected to GC analysis as described
above.
HPLC Analysis of the Marfey Derivatives. To the acid
hydrolysate of koshikamide B (1, 1 mg) were added 50 µL of
1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide in acetone (10 mg/
mL) and 100 µL of 1 M NaHCO3, and the mixture was kept at 80
°C for 3 min. To the reaction mixture were added 50 µL of 2 N
HCl and 100 µL of 50% MeCN containing 0.05% TFA and the
mixture analyzed by ODS HPLC [10 × 250 mm: linear gradient
elution from MeCN/H2O/TFA (25:75:0.05) to MeCN/H2O/TFA (55:
45:0.05) in 60 min; at a flow rate of 1.0 mL/min] peaks were
detected with UV absorption at 340 nm. Each peak was identified
by comparing the retention time with that of the FDAA derivative
of standard amino acids. The amino acid standards showed the
following retention times: D-MeAsp 18.0 min, L-Thr 19.8 min,
L-allo-Thr 20.6 min, L-MeAsp 22.2 min, D-allo-Thr 26.6 min, D-Thr
27.2 min, L-MeVal 38.1 min, L-Phe 38.8 min, D-MeVal 40.8 min,
L-MeLeu 41.2 min, D-Phe 43.4 min, D-MeLeu 43.4 min.
Chiral GC Analysis of the Acid Hydrolysate. Koshikamide B
(1, 500 µg) was heated with 6 N HCl (200 µL) at 105 °C for 15 h.
After evaporation of the solvent under a stream of nitrogen, 10%
HCl-MeOH (100 µL) was added, and the sealed vial was heated
at 105 °C for 45 min. After evaporation of the reagent under a
stream of nitrogen, TFAA (30 µL) in CH2Cl2 (50 µL) was added
to the residue, and the sealed vial was heated at 105 °C for 10
min. The product was again dried under a stream of nitrogen and
redissolved in CH2Cl2 (100 µL), and the solution was subjected to
GC analysis. The retention time of the standard amino acids were
as follows: D-Ala 9.6 min, L-Ala 11.0 min, L-Ile 14.6 min, D-Ile
15.3 min, D-allo-Ile 15.8 min, L-allo-Ile 16.2 min, D-Asp 21.0 min,
L-Asp 21.5 min.
Acknowledgment. We are indebted to the crews of R/V
Toyoshio-maru of Hiroshima University for assistance in
collecting the sponge. We thank Professor R. W. M. van Soest,
University of Amsterdam, for the identification of the sponge.
This work was supported by Grant-in-Aids for Scientific
Research 09306014 from JSPS and on Priority Areas 16073207
from MEXT. We thank the Government of the Republic of
Palau for research permits and Patrick L. Colin (Coral Reef
Research Foundation, Palau) for collecting the sponge.
Isolation of MeaIle. Koshikamide B (1, 9.2 mg) was heated
with 6 N HCl (150 µL) at 105 °C for 24 h, and the hydrolysates
were purified by ODS-HPLC [10 × 250 mm; elution with H2O
containing 0.05% TFA (2 mL/min); detection with absorption at
210 nm]. The peak eluted at the retention time of authentic samples
of MeIle and MeaIle, which were coeluted, was collected. Isolated
MeaIle: 1H NMR (D2O) δ 3.70 (br s), 2.72 (3H, s), 2.08 (m), 1.56
(sep, J ) 6.5 Hz), 1.28 (m), 1.06 (3H, d, J ) 6.9 Hz), 0.98 (3H, t,
J ) 7.3 Hz).
Supporting Information Available: Spectral data of 1, 2
and 6-8. This material is available free of charge via the Internet
at http://pubs.acs.org.
Peptide Fragments 3-5. Koshikamide B (1, 10 mg) was
hydrolyzed with 6 N HCl (1 mL) at room temperature for 25 h,
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7894 J. Org. Chem. Vol. 73, No. 20, 2008