Penasins A-E, long-chain cytotoxic sphingoid bases, from a marine sponge penares sp

Article abstract of DOI:10.1021/np1003565

Five sphingoid bases, penasin A (1), penasin B (2), and a mixture of penasins C-E (3-5), were identified from a marine sponge Penares sp. as cytotoxic constituents. The structure of the common polar head part was assigned by analysis of the NMR data, whereas the structures of the long aliphatic chains including the locations of double bond(s) and a branched methyl group were determined by analysis of tandem FABMS and ¹³C NMR data together with the GC-MS analysis of ozonolysis products. The absolute configuration of the headgroup was defined for the mixture of 3-5 by the modified Mosher method. Penasins exhibit moderate cytotoxicity against HeLa and P388 cells.

Full text of DOI:10.1021/np1003565

J. Nat. Prod. 2010, 73, 1947–1950
1947
Penasins A-E, Long-Chain Cytotoxic Sphingoid Bases, from a Marine Sponge Penares sp
Hideki Ando,† Reiko Ueoka,† Shigeru Okada,† Tsuyoshi Fujita,‡ Takashi Iwashita,‡ Takaaki Imai,§ Tomoya Yokoyama,§
Yuji Matsumoto,§ Rob W. M. van Soest,⊥ and Shigeki Matsunaga*,†
Laboratory of Aquatic Natural Products Chemistry and Laboratory of Wood Chemistry, Graduate School of Agricultural and Life Sciences,
The UniVersity of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan, Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho,
Osaka 618-8503, Japan, and Zoological Museum of the UniVersity of Amsterdam, P.O. Box 94766, 1090 GT Amsterdam, The Netherlands
ReceiVed May 27, 2010
Five sphingoid bases, penasin A (1), penasin B (2), and a mixture of penasins C-E (3-5), were identified from a
marine sponge Penares sp. as cytotoxic constituents. The structure of the common polar head part was assigned by
analysis of the NMR data, whereas the structures of the long aliphatic chains including the locations of double bond(s)
and a branched methyl group were determined by analysis of tandem FABMS and 13C NMR data together with the
GC-MS analysis of ozonolysis products. The absolute configuration of the headgroup was defined for the mixture of
3-5 by the modified Mosher method. Penasins exhibit moderate cytotoxicity against HeLa and P388 cells.
Sphingolipids play important structural roles in the plasma mem-
brane by comprising an integral part of lipid rafts.1 They also function
as signaling molecules to regulate cellular processes.2 In spite of the
extreme structural diversity of sphingolipids in higher organisms, the
structures of their sphingosine bases fall into only a few classes.3 On
the other hand, a wide variety of differentially modified sphingosine
bases have been isolated from marine sponges and tunicates.4 They
exhibit a variety of biological activities. One such compound, pachas-
trissamine, is the C18-anhydrophytosphingosine (1,4-anhydro-D-lyxo-
phytosphingosine) obtained from a marine sponge Pachastrissa sp.5
The same compound and the 4-hydroxybutyl aldehyde adduct were
reported under the name jaspines.6 In our study to discover antitumor
lead compounds from marine invertebrates, the extract of a marine
sponge Penares sp. exhibited cytotoxity against P388 murine leukemia
cells. Bioassay-guided fractionation afforded C29- or C30-anhydro-
phytosphingosine bases termed penasins (1-5). Marine sponges of
the genus Penares were the source of modified sphingosine bases
penaresidins and penazetidine A.7
Table 1. 1H NMR Data of 1, 2, the Mixture of 3-5, and the
Mixture of 6-8a
position
1b
2b
3-5b
6-8c
1a
1b
2
3
4
5
6
7
8
3.81
3.81
3.81
3.92
3.88
4.26
3.72
1.65
1.43
1.20-1.40
1.20-1.40
1.36
2.04
5.35
5.35
2.04
1.36
3.61
4.09
4.62
4.20
3.79
1.65
1.35, 1.47
1.20-1.40
1.20-1.40
1.35
2.03
5.36
5.36
2.03
1.36
1.20-1.40
3.91
3.88
4.25
3.72
1.67
1.42
1.43
2.06
5.36
5.36
2.06
2.06
5.36
5.36
2.06
1.44
1.20-1.40
3.91
3.88
4.26
3.71
1.65
1.47
1.42
2.10
5.42
5.33
2.3
9
10
11
12
13
14
15
16
17-28
28
29
30
27
28
29
δd
γ
2.36
5.52
2.16
1.39
1.20-1.40
The organic extract of the sponge was subjected to a solvent
partitioning process. The CHCl3 and n-hexane layers were combined
and fractionated by silica gel column chromatography, ODS flash
chromatography, and ODS-HPLC to afford penasin A (1), penasin
B (2), and an inseparable mixture of penasins C-E (3-5).
1.32
0.89
1.32
0.91
1.32
0.90
0.86
1.20-1.40
1.30
1.10, 1.30
1.39
1.10, 1.30
1.30
1.28
0.89
0.86
1.20-1.40
1.37
1.09, 1.26
1.28
1.09, 1.26
1.37
ꢀ
R
ꢀ
γ
δ
1.20-1.40
1.20-1.40
a JH,H in Hz: (1) tetrahydrofuran ring: 1a,1b ) 8.9; 1a,2 ) 4.8; 2,3 )
4.9; 3,4 ) 3.5; 4,5ab )7.5, 6.2: (2) methyl signals were split by 7 Hz.
b Chemical shift values in methanol-d4. c Chemical shift values in
CDCl3. d For the designations of the Greek letters, see Figure 2, unit b.
exhibited a cluster of four olefinic protons, five deshielded protons
(δ 3.7-4.3), a methylene envelope, and a terminal methyl group
(Table 1). Interpretation of the COSY and HSQC data (Table 2)
revealed that compound 1 contained a 2-amino-3-hydroxy-4-
alkyltetrahydrofuran ring, as found in pachastrissamine.5 The
molecular formula and the HSQC data suggested that penasin A
had an unbranched alkyl chain with two unsaturations. The double
bonds were located at C-9/C-10 and C-13/C-14 on the basis of the
tandem FABMS data (Figure 1). The geometries of the double
bonds were determined as 9Z,13Z on the basis of the chemical shifts
of 27.9 ppm for the allylic methylene carbons (C-8, C-11, C-12,
and C-15).8 The relative configuration of the tetrahydrofuran ring
was assigned to be identical with that of pachastrissamine by the
intense NOESY cross-peaks between H-2 and H-3 and between
Penasin A (1) had a molecular formula of C30H57NO2, which
was established by the HRESIMS data. The H NMR spectrum
1
* To whom correspondence should be addressed. Tel: 81-3-5841-5297.
Fax: 81-3-5841-8166. E-mail: assmats@mail.ecc.u-tokyo.ac.jp.
† Laboratory of Aquatic Natural Products Chemistry, The University of
Tokyo.
‡ Suntory Institute.
⊥ Laboratory of Wood Chemistry, The University of Tokyo.
§ University of Amsterdam.
10.1021/np1003565  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/15/2010

1948 Journal of Natural Products, 2010, Vol. 73, No. 11
Notes
Table 2. 13C NMR Data of 1, 2, the Mixture of 3-5, and the
Mixture of 6-8a
1b
2b
3-5b
6-8c
1
2
3
4
5
6
68.8
54.1
70.7
84.0
29.4
27.0
68.7
54.1
70.7
84.1
29.2
27.0
68.9
54.2
70.8
84.1
29.6
27.0
70.2
53.0
72.0
82.2
28.8
26.2
7
8
9
29-31
29.0-31.0
29.0-31.0
29.0-31.0
29.0-31.0
27.9
130.6
130.6
29.0-30.0
29.2d
29.6e
27.3
27.9
27.9
130.0-131.0
130.0-131.0
27.9
131.5
128.7
26.9
Figure 2. 13C NMR assignments for 6-8 on the basis of the HSQC-
TOCSY data.
10
11
12
13
14
15
16-26
25
26
27
28
29
30
δf
γ
130.0
130.0
27.3
27.9
40.2
130.0-131.0
130.0-131.0
27.9
135.1
126.9
29.2
27.9
29-31
135.1 ppm, which was in agreement with an sp2 carbon substituted
by a chlorine atom.9,10 The Z-geometry of the disubstituted olefinic
functionality was assigned via the chemical shifts of allylic carbons
C-8 and C-11, whereas the geometry of the trisubstituted double
bond was assigned as Z by the NOESY cross-peak between H-14
and H2-12. The latter assignment was supported by comparing the
carbon chemical shifts with those of model compounds.9,10 The
relative configuration of the tetrahydrofuran ring was assigned to
be identical with that of pachastrissamine by comparing the NMR
data. The positions of the double bonds were determined by the
tandem FABMS data (Figure 1).
29.8e
29.4d
29.0-31.0
29.0-31.0
29.0-31.0
31.8
23.6
14.3
20.0
29.6
32.0
22.7
14.2
19.8
29.0-31.0
29.0-31.0
31.8
23.6
14.3
29.0-31.0
29.0-31.0
31.8
23.6
14.3
29.0-31.0
28.0
38.0
33.6
38.0
28.0
29-31
29.6
27.1
37.2
32.8
37.2
27.1
29.6
ꢀ
R
ꢀ
Penasins C-E (3-5), isomeric compounds differing in the
position of a methyl branch, were obtained as an inseparable
mixture. The molecular formula of this fraction was determined as
γ
δ
a Chemical shifts were determined on the basis of the HSQC or
HSQC-TOCSY data. b Chemical shift values in methanol-d4. c Chemical
shift values in CDCl3. d Assignments may be interchanged. e As-
signments may be interchanged. f For the designations of the Greek
letters, see Figure 2, unit b.
1
C29H58NO2 by HRESIMS. The H NMR spectrum differed from
that of penasin A (1) in the intensity of the olefinic and allylic
proton signals (integrated to be 2H and 4H, respectively) and the
presence of a methyl doublet. The NMR data suggested that this
fraction also contained the common tetrahydrofuran unit (Tables 1
and 2). The Z-geometry of the double bond was established on the
basis of the chemical shifts of allylic carbons C-8 and C-13.8 The
position of the double bond in the side chain was assigned by
analysis of the tandem FABMS data (Figures S18 and S19). Even
though the 1H NMR spectrum is clean, the tandem FABMS of this
fraction did not give a pair of intense ions separated by 28 Da,
which was expected for a methyl branch. Instead, all the ions larger
than 262 Da were separated by 14 Da (Figure S18). By expecting
to get a better resolution of NMR spectra and to determine the
absolute configuration of the stereogenic centers of the tetrahydro-
furan ring, the mixture of 3-5 was converted to the (S)-MTPA
1
H-3 and H-4, together with the coincidence of the H and 13C
chemical shifts.
Penasin B (2) exhibited (M + Na)+ ions at m/z 498 and 500 in
a ratio of 3:1, suggesting the presence of a chlorine atom. The
molecular formula was determined to be C30H56NO2Cl by the
HRESIMS data. The NMR data showed the presence of one
disubstituted and one trisubstituted double bond each, a terminal
methyl group, and a methylene envelop in addition to the 2-amino-
3-hydroxytetrahydrofuran headgroup (Tables 1 and 2). Analysis of
the 2D NMR data demonstrated that the two double bonds (C-9,
C-10 and C-13, C-14) were separated by two methylene carbons
(C-11 and C-12). The position of the non-hydrogenated sp2 carbon
atom was assigned to be adjacent to one of the central methylene
carbons by analysis of the COSY spectrum. The HMBC data
showed the chemical shift of the non-hydrogenated carbon to be
1
amides (6-8, respectively), whose H NMR data of the tetrahy-
drofuran ring portion matched well with the corresponding data of
the (S)-MTPA amide of pachastrissamine,5 indicating the identical
absolute configuration for the headgroups. By taking advantage of
Figure 1. Tandem FABMS data for 1 and 2.

Notes
Journal of Natural Products, 2010, Vol. 73, No. 11 1949
respectively; HRESIMS m/z 498.4069 [(M
+
H)+, calcd for
the sharper 1H NMR signals of this derivative, the HSQC-TOCSY
spectrum was analyzed (Figure 2 and Table 2), which showed that
the chemical environment of the branched methyl group was
homogeneous: the C-10 and C-13 allylic carbons were both
connected to two or more methylene carbons (unit a); the methine
at the methyl branch was in the center of a C7 or longer methylene
chain (unit b), and the terminal methyl was located at one end of
an n-butyl or longer chain (unit c). Although it was not possible to
observe correlations among units a-c, the position of the methyl
branch was confined within the positions between C-19 and C-21.
The location of the methyl branch was determined by GC-MS
analysis of the ozonolysis products. The mixture of 3-5 was
subjected to ozonolysis followed by reduction and silylation with
TMSCl to afford the 1-OTMS ethers. The mixture was subjected
to GC-MS, which gave a broad peak with an intense (M - CH3)+
ion at m/z 327 as expected. Even though scant fragmentations were
observed, when the mass chromatograms of potential fragment ions
(m/z 171, 185, and 199) were generated, there appeared three peaks
within the broad peak (Figure S24). If we hypothesize that these
ions were generated due to the cleavage of the bond between the
branched methine and the methylene carbon at the side of the polar
group (Figure S24), it was possible to locate the methyl branch at
C-19, C-20, and C-21, consistent with the 13C NMR analysis. The
compounds within this group were named penasins C (3), D (4),
and E (5), respectively.
C30H57NO235Cl, 448.4072].11
Penasins C-E (3-5): colorless oil; 1H NMR (600 MHz, methanol-
d4) and 13C NMR (150 MHz, methanol-d4) data, see Tables 1 and 2,
respectively; HRESIMS m/z 452.4460 [(M + H)+, calcd for C29H58NO2,
452.4462].12
GCMS Analysis. O3 was bubbled into a solution of the mixture of
3-5 (0.2 mg) in MeOH (0.5 mL) at -78 °C for 5 min. The solution
was flashed with N2 gas. To the solution was added 1 mg of NaBH4 in
0.5 mL of MeOH, and the reaction mixture was left at rt for 5 min.
The reaction mixture was concentrated and partitioned between H2O
and EtOAc. The organic phase was concentrated, dissolved in N,O-
bis(trimethysilyl)trifluoroacetamide (BSTFA) (0.1 mL), and left at rt
for 1 h. The reaction mixture was subjected to GC-MS analysis: column
TC-5 (GL Science), 0.32 mm × 60 m; 50 °C for 1 min; raised to 210
at 10 °C/min; raised to 240 at 5 °C/min; and held at 240 °C for 10
min.
MTPA Amides (6-8). To a solution of a mixture of 3-5 (0.3 mg)
and DMAP (1 mg) in CH2Cl2 was added (-)-MTPACl (5 µL), and the
reaction mixture was left at rt for 5 min. The solution was diluted with
CHCl3 (2 mL) and washed with 0.1 M NaHCO3 (2 mL × 3), 0.1 N
HCl (2 mL × 2), and H2O (2 mL × 2). The organic layer was
concentrated and purified by preparative silica gel TLC developed with
n-hexane/EtOAc (8:2) to afford the mixture of 6-8.
1
6-8: H NMR (CDCl3) and 13C NMR data, see Tables 1 and 2,
respectively; ESIMS m/z 690 (M + Na)+, 668 (M + H)+.
Cytotoxicity Assay. Cytotoxicity of compounds 1, 2, and the mixture
of 3-5 against P388 and HeLa cells was determined as previously
described.13 P388 murine leukemia cells (JCRB17) were cultured in
RPMI-1640 medium (Wako Pure Chemical Industries, Ltd.) containing
10% fetal bovine serum, 2 µg/mL of antibiotic-antimicotic (a mixture
of 10 000 units of penicillin, 10 mg of streptomycin, and 25 µg of
amphotericin B per mL), and 10 µg/mL of 2-hydroxyethyldisulfide at
37 °C under an atmosphere of 5% CO2. To each well of the 96-well
microplate containing 100 µL of tumor cell suspension (1 × 104 cells/
mL) was added 100 µL of test solution dissolved in RPMI-1640
medium, and the plate was incubated in a TABAI BNA-111 CO2
incubator (Espec Co., Tokyo, Japan) at 37 °C for 96 h. After addition
of 50 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) saline solution (1 mg/mL) to each well, the plate was
incubated for 3 h under the same conditions to stain live cells. After
the incubation, the plate was centrifuged, the supernatants were
removed, and the cells were dissolved in 150 µL of DMSO to determine
the IC50 values. HeLa human cervical cells were cultured in Dulbecco’s
modified Eagle’s medium (Wako Pure Chemical Industries, Ltd.)
containing 10% fetal bovine serum, 2 µg/mL gentamycin, and 2 µg/
mL antibiotic-antimicotic 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 test solution after the 24 h
preincubation, and the plate was incubated for 72 h. To determine the
IC50 values, the plate was processed as described for P388 cells. The
experiments were duplicated, but statistical analyses for the IC50 values
have not been conducted.
Penasin A (1), pensin B (2), and the mixture of penasins C-E
(3-5) exhibit moderate cytotoxic activity against HeLa cells (with
IC50 values of 10, 10, and 2 µg/mL, respectively) and weak
cytotoxic activity against P388 cells (IC50 value of 50 µg/mL each).
It is interesting to note that another class of modified sphingosine
bases, termed penaresidines, have been isolated from a marine
sponge Penares sp.11
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-1000 digital polarimeter. NMR spectra were recorded
on a JEOL alpha 600 NMR spectrometer at 300 K. Chemical shifts
were referenced to solvent peaks: δH 3.31 and δC 49.15 for methanol-
d4. ESI mass spectra were measured on a JEOL JMS-T100LC. GC-
MS was conducted with a Shimadzu GC-2010/Parvum 2 gas chro-
matograph mass spectrometer.
Animal Material. A marine sponge Penares sp. was collected by
hand using scuba off Shikine Island, Tokyo Prefecture, Japan. A
voucher specimen was deposited at the Zoological Museum of the
University of Amsterdam (ZMAPOR19855).
Extraction and Isolation. The sponge was frozen after collection
and kept frozen until extraction. The sponge (400 g, wet weight) was
homogenized and extracted with MeOH (2 L × 2) and CHCl3/MeOH
(1:1) (2 L × 1). The combined extracts were concentrated, and the
residue was partitioned between H2O and CHCl3. The CHCl3 layer was
concentrated and partitioned between 90% MeOH and n-hexane. The
90% MeOH layer was diluted with H2O to afford a solution of 60%
MeOH and extracted with CHCl3. The CHCl3 and n-hexane layers were
combined and subjected to silica gel column chromatography eluting
with CHCl3, CHCl3/MeOH (98:2), CHCl3/MeOH (95:5), CHCl3/MeOH
(9:1), and CHCl3/MeOH/H2O (8:2:0.1). The CHCl3/MeOH (95:5 and
9:1) eluates were concentrated and fractioned by ODS flash chroma-
tography with 50% MeOH, 70% MeOH, 90% MeOH, 100% MeOH,
and CHCl3/MeOH/H2O (7:3:0.5). The fractions eluted with 90% MeOH,
100% MeOH, and CHCl3/MeOH/H2O (7:3:0.5) were combined and
purified by ODS HPLC with a gradient elution of 50-80% 1-PrOH
containing 1% HOAc followed by ODS-HPLC with either 80% MeCN
containing 0.05% TFA or 55% 1-PrOH containing 0.05% TFA to afford
penasin A (1, 2.3 mg), penasin B (2, 0.7 mg), and a mixture of penasins
C-E (3-5, 2.7 mg).
Acknowledgment. This work was partly supported by Grants-in-
Aid for Scientific Research on Priority Area 16073207 from Ministry
of Education, Culture, Sports, Science, and Technology, Japan.
Supporting Information Available: NMR and tandem FABMS data
for penasins, HSQC-TOCSY spectrum of MTPA amides, and GC-MS
of ozonolysis products. This material is available free of charge via
the Internet at http://pubs.acs.org.
References and Notes
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2006, 5, 200–208.
(3) Pruett, S. T.; Bushnev, A.; Hagedorn, K.; Adiga, M.; Haynes, C. A.;
Sullards, M. C.; Liotta, D. C.; Merrill, A. H., Jr. J. Lipid Res. 2008,
49, 1621–1639.
(4) Shier, W. T.; Shier, A. C. J. Toxicol. Toxin ReV. 2000, 19, 189–246.
(5) Kuroda, I.; Musman, M.; Ohtani, I. I.; Ichiba, T.; Tanaka, J.; Gravalos,
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(6) Ledroit, V.; Debitus, C.; Lavaud, C.; Massiot, G. Tetrahedron 2003,
44, 225–228.
Penasin A (1): colorless oil; [R]24D +11 (c 0.12, MeOH); 1H NMR
(600 MHz, methanol-d4) and 13C NMR (150 MHz, methanol-d4) data,
see Tables 1 and 2, respectively; HRESIMS m/z 464.4460 [(M + H)+,
calcd for C30H58NO2, 446.4462].
Penasin B (2): colorless oil; 1H NMR (600 MHz, methanol-d4) and
13C NMR (150 MHz, methanol-d4) data, see Tables 1 and 2,

1950 Journal of Natural Products, 2010, Vol. 73, No. 11
Notes
(7) Pruett, S. T.; Bushnev, A.; Hagedorn, K.; Agida, M.; Haynes, C. A.;
Sullards, M. C.; Liotta, D. C.; Merrill, A. H., Jr. J. Lipid Res. 2008,
49, 1621–1639.
(8) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH:
Weinheim, 1990; pp 192-199.
(9) Coleman, M. M.; Tabb, D. L.; Brame, E. G. Rubber Chem. Technol.
1977, 50, 49–62.
(11) Kobayashi, J.; Tsuda, M.; Cheng, J. F.; Ishibashi, M.; Takikawa, H.;
Mori, K. Tetrahedron Lett. 1996, 37, 6775–6776.
(12) Optical rotation was not determined due to the paucity of the
material.
(13) Ueoka, R.; Ito, A.; Izumikawa, M.; Maeda, S.; Takagi, M.; Shin-ya,
K.; Yoshida, M.; van Soest, R. W. M.; Matsunaga, S. Toxicon 2009,
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(10) For natural products with the chlorinated olefinic group, see: Ueda,
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NP1003565