Bioactive lipids from the sponge Spirastrella abata

Article abstract of DOI:10.1016/j.bmcl.2011.11.105

Three sphingosine 4-sulfates (1-3) and a lysophosphatidylglycerol (4) were isolated from the Korean sponge Spirastrella abata. The structures of these compounds were determined based on the combined results of spectroscopic analyses. Based on the results

Full text of DOI:10.1016/j.bmcl.2011.11.105

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1078–1081
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Bioorganic & Medicinal Chemistry Letters
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Bioactive lipids from the sponge Spirastrella abata
Kyoung Hwa Jang a, Yoonyeong Lee a, Chung J. Sim b, Ki-Bong Oh c, , Jongheon Shin a,
⇑
⇑
a Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Republic of Korea
b Department of Biological Science, College of Life Science and Nano Technology, Hannam University, 461-6 Jeonmin, Yuseong, Daejeon 305-811, Republic of Korea
c Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Three sphingosine 4-sulfates (1ꢀ3) and a lysophosphatidylglycerol (4) were isolated from the Korean
sponge Spirastrella abata. The structures of these compounds were determined based on the combined
results of spectroscopic analyses. Based on the results of combined synthesis and comparison of specific
rotation and circular dichroism, the absolute configurations of 1ꢀ3 were found to be enantiomeric to the
previously isolated metabolites. The configurations of 4 were also partially determined by similar
chemical and spectroscopic methods. The compounds exhibited significant cytotoxicity and weak
antimicrobial activity (1), as well as weak-to-moderate inhibitory activity against isocitrate lyase and
Na+/K+-ATPase. A structure–activity relationship was found for the sphingosine 4-sulfates.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 October 2011
Revised 25 November 2011
Accepted 28 November 2011
Available online 4 December 2011
Keywords:
Spirastrella abata
Sphingosine sulfates
Lysophosphatidylglycerol
Cytotoxicity
Antimicrobial activity
Sphingolipids and lysophospholipids are important membrane
components that function in cell recognition and cell regulation.1
Breakdownmetabolitesof sphingolipids, such as ceramide, sphingo-
sine, and sphingosine-1-phosphate, have been implicated in cell
regulation and exhibit a wide variety of activities related to signal
transduction as agonists or second messengers.1,2 These and
related metabolites are widely distributed among marine organ-
isms, such as algae, coelenterates, echinoderms, soft corals, tuni-
cates, and sponges,3,4 including those of the genus Spirastrella.5a–c
During the course of our search for bioactive metabolites from
Korean water organisms, we encountered the violet-colored sponge
Spirastrella abata (Order Hadromerida, Family Spirastrellidae), the
data, respectively. This linearity, in conjunction with the presence
of nitrogen in the mass data, suggested that 1 was a sphingosine,
consistent with the presence of signals for a linear double bond
and heteroatom-bearing methines in the NMR data (Experimental
Section). Additionally, the presence of a sulfate group was deduced
from the strong absorption band at 1250 cmꢀ1 in the IR spectrum,
OH
7
1
HO
HO
HO
n
7
-
NH3+ OSO3
1: n = 8
2: n = 7
organic extract of which exhibited significant lethality (LC50
=
51 ppm) towards brine-shrimp larvae. Bioassay-guided separation
of the crude extract using various chromatographic techniques
yielded several lipids. Here, we report the isolation and structural
determination of four new metabolites: three sulfated sphingosines
(1ꢀ3) and a lysophosphatidylglycerol (4) (Fig. 1). These compounds
exhibitedsignificant cytotoxicityand weak antimicrobialactivity, as
well as weak-to-moderate inhibitory activity against isocitrate lyase
and Na+/K+-ATPase.
OH
-
NH3+ OSO3
3
O
1'
The molecular formula of compound 1 was deduced to be
1
C18H37NO6S by HRFABMS analysis. The linear nature of this com-
O
H
O
O
P
pound was evident from the presence of several signals in the re-
1"
gions of dC 30.8ꢁ30.4 and dH 1.36ꢁ1.28 in the 13C and 1H NMR
O
OH
O-Na+
OH
⇑
Corresponding authors. Tel.: +82 2 880 2484; fax: +82 2 762 8322 (J.S.); tel.: +82
2 880 4646; fax: +82 2 873 3112 (K.-B.O.).
4
E-mail addresses: ohkibong@snu.ac.kr (K.-B. Oh), shinj@snu.ac.kr (J. Shin).
Figure 1. Structures of compounds 1–4.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.105

K. H. Jang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1078–1081
1079
combined with the presence of a sulfur and several oxygens in the
mass data.
Given this information, the planar structure of 1 was determined
by a combination of 1H COSY, gHSQC, and gHMBC analyses. These
data readily defined a linear array of downfield NMR signals to form
indicated the 2R⁄, 3R⁄ relative configurations. Due to the free rota-
tion of the side chain, however, the relative configuration at the
sulfate-bearing C-4 remained unassigned at this stage.
Given this information, 1 was further derivatized to compare
spectroscopic data with known compounds. That is, treatment
with H2SO4/H2O/THF converted 1 to 2-amino-1,3,4-trihydroxyoc-
tadec-6-enoate (6). The 1H NMR spectrum and LC–ESIMS data
(m/z 317 [M+H]+) of the product confirmed the sulfate hydrolysis.
Hydrogenation of 6 with H2 with Pd/C furnished the phytosphingo-
sine (7). Comparison of the 1H NMR spectra of 7 with reported data
a
partial structure: –CH2–CH–CH–CH–CH2–CH@CH–CH2–. The
placement of heteroatoms in this framework was accomplished
by observing the chemical shifts and comparing the spectroscopic
data with those in the literature. That is, two hydroxy and an amine
group were placed at C-1, C-3, and C-2, respectively, by carbon
chemical shifts: dC 59.3 (C-1), 56.7 (C-2), and 70.6 (C-3). The down-
field shift of the C-4 methine (dH 4.30, dC 79.1) assigned the sulfate
group to this location. The remaining portion of the molecule, con-
sisting of one methyl and nine methylene groups, was linearly at-
tached at C-8 on the basis of proton and carbon chemical shifts,
thus forming a sulfated sphingosine, 2-amino-1,3-dihydroxyocta-
dec-6-ene-4-sulfate, for 1. The E configuration of the C-6 double
bond was assigned by the downfield shifts of allylic methylene car-
bons at dC 34.9 (C-5) and 33.8 (C-8), as well as cross peaks of H-5
and H-8 with olefinic ones in the NOESY data.
for commercial phytosphingosine (
stereomers ( -arabino, -lyxo, -xylo) showed identity between 7
and a phytosphingosine (
-ribo) 8.7 However, the specific rotations
D-ribo) 8 and its synthetic dia-
D
D
D
D
of 7 and its per-acetyl derivative 7a, prepared for the enhancement
of specific rotation, were the opposite of both phytosphingosine 8
and its per-acetyl derivative 8a (½a D25
ꢀ10.3, ꢀ4.0, +11.6, and +26.0
ꢂ
in CHCl3 for 7, 7a, 8, and 8a, respectively). Despite the remarkable
difference in the scalar values of optical rotations, these results as-
signed the 2R, 3R, and 4S configurations for 1.
The stereochemistry of 1 was also approached by another
chemical derivatization followed by CD measurements.8a,b As
shown in Scheme 1, in the first step, the natural product-derived
phytosphingosine (7) and commercial phytosphingosine (8) were
converted into N-naphthimide derivatives (7b and 8b), then ester-
ified to yield the pernaphthoate derivatives (7c and 8c, respec-
tively). Although the NMR data of these compounds were the
same, the CD spectra were opposite to each other [7c, extrema at
A literature survey revealed that the structure of 1 was identical
to that of a sulfated sphingosine previously isolated from the same
S. abata. However, the specific rotations measured for 1 and its per-
acetylated derivative 1a (½a D25
ꢀ16.8 and ꢀ20.4 for 1 and 1a,
ꢂ
respectively in CHCl3) were found to be the opposite of that re-
ported previously (½a D25
ꢂ
+26 in CHCl3 for the per-acetylated deriv-
ative).6a–c This prompted us to extensively investigate the
stereochemistry of this compound by diverse chemical derivatiza-
tions followed by measurements of specific rotation and CD.5c,7,8a,b
First, as shown in Scheme 1, the relative configurations at C-2 to
C-4 were approached by a ketal formation.4 Treatment of 1 with
2,2-dimethoxypropane and pyridinium p-toluenesulfonate (PPTS)
in acetone yielded the corresponding 1,3-cyclic ketal, 5. The vicinal
coupling constants (J1ax,2 = 5.8 Hz, J1eq,2 = 4.8 Hz, J2,3 = 8.0 Hz) anal-
ysis, aided by NOESY cross peaks at H-1ax (dH 3.65)/H-3, H-1ax/ke-
tal-Me (dH 1.41), H-3/ketal-Me, and H-1eq (dH 3.99)/ketal-Me (dH
1.36), positioned H-2 and H-3 in the anti orientation, and thus
240 nm (
D
e
ꢀ5.0), 248 nm (0.0), 263 nm (+2.2); 8c, 240 nm
(+5.1), 248 nm (0.0), 263 nm (ꢀ2.2)] (Fig. 2). Thus, the structure
of compound 1 was determined to be (E)-(2R,3R,4S)-2-amino-1,3-
dihydroxyoctadec-6-ene-4-sulfate by application of the Cotton ef-
fect.8b It is noteworthy that sphingosines from the same sponges
were enantiomeric to each other.5c
Spectroscopic analyses readily determined that the structure of
compound 2, having the molecular formula C17H35NO6S, was
analogous to its congener 1. Combined 2-D NMR experiments
revealed this compound to be a desmethylene derivative of 1.
4.8 Hz
OH
a
H
8.0 Hz
O
O
O
O
H
HO
8
5.8 Hz
NH3+ OSO3
-
+H3N
H
8
NH3+ OSO3
-
1
H
5
NOE
OH
OH
b
c
HO
HO
8
8
NH3+ OH
NH3+ OH
6
7
O
OH
O
O
OH
d
e
C14H29
C14H29
O
C14H29
HO
O
HO
OH
O
N
N
NH2 OH
O
O
O
O
L-ribo [2R, 3R, 4S] (7)
D-ribo [2S, 3S, 4R] (8)
7b, 8b
7c, 8c
Scheme 1. Reagents and conditions: (a) N2, 2,2-dimethoxypropane, PPTS, acetone, rt, 2 h; (b) H2SO4/H2O/THF, rt, 1 h; (c) H2, Pd/C, MeOH, rt, 10 h; (d) 2,3-
naphthalenedicarboxylic acid anhydride, pyridine, reflux, 15 h; (e) 2-naphthoylimidazole, DBU, MeCN, rt, 3 h.

1080
K. H. Jang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1078–1081
In addition to the sphingosine sulfates, compound 4 was iso-
lated as a colorless gum with the molecular formula C25H48O9PNa,
deduced from HRFABMS. The observation of a carbonyl carbon at
dC 175.5 in the 13C NMR spectra and an absorption band at
1760 cmꢀ1 in the IR spectrum were indicative of an ester linkage.
The NMR data displayed signals of six oxygenated carbons at dC
72.7ꢁ63.9 and their attached protons at dH 4.2ꢁ3.5. 1H COSY,
gHSQC, and gHMBC correlations among these signals revealed
the presence of two glycerol-type moieties. Several carbons and
protons showed additional splittings that differed from the antici-
pated findings based on their glycerol moieties; in conjunction
with the molecular formula, these were attributed to long-range
couplings with the phosphorous atom (2JCP 3JCP 3JHP), indicating
, ,
the insertion of a phosphate group between the glycerols.
The remaining portion of the molecule, containing the ester and
all of the upfield carbons and protons, was found to form a long chain
by 1H COSY analysis and was connected to the C-1 of glycerol, based
on long-range correlations between the ester carbon and H-1 meth-
ylene protons in the gHMBC data. Thus, the gross structure of com-
pound 4 was defined as a sn1-lysophosphatidylglycerol.
Figure 2. CD spectra of N-naphthimide-O-trinaphthoate derivatives (7c, dotted
line; 8c, solid line) in MeOH.
The fatty acid chain of 4 possessed a cyclopropane (dC 16.8, 16.8,
11.6; dH 0.66, 0.66, 0.57, ꢀ0.33) that was placed at C-110 by con-
spicuous ion clusters, including those at m/z 476 and 408, attrib-
252
352
408
476
517 546
532
O
O
H
O
238
HO
O
P
uted to the characteristic
c-cleavage in FAB collision-induced
O
dissociation (FAB–CID) MS/MS data (Fig. 3). Significant differentia-
tions of the H-19 methylene protons (d 0.57 and ꢀ0.33) and large
vicinal coupling constants (J11,19 = J12,19 = 8.2 and 5.2 Hz) suggested
a cis-orientation for the cyclopropane.
OH
O-Na+
OH
194
Figure 3. FAB–CID–MS/MS fragmentations of compound 4.
Although a literature survey revealed that the structurally re-
lated lysophospholipid was isolated from S. abata, the stereochem-
istry at the asymmetric centers including the cyclopropane moiety
was not determined.5a The structure and stereochemistry of com-
pound 4 were further pursued by synthetic methods (Scheme 2).
Treatment with CH2I2 and Et2Zn converted the commercially avail-
able methyl cis-(9a) and trans-11-octadecenoate (9b) to the
corresponding cis-(10a) and trans-cyclopropane-containing ester
(10b), respectively. Significant differences in proton chemical
shifts in the cyclopropane portion (dH 0.64, 0.64, 0.57, ꢀ0.34 for
10a; dH 0.33, 0.33, 0.10, 0.10 for 10b) showed that 4 contained a
cis-cyclopropane with 11R⁄, 12S⁄ configurations at its fatty acid
chain. However, the absolute configurations remained undeter-
mined because the optical rotations of both cis-cyclopropane esters
from the natural and commercial products were zero. This could
have been because the natural and synthetic compounds were
mixtures with opposite absolute configurations (11R, 12S and
11S, 12R). Alternatively, the contribution to the optical rotation
Configurations identical to those of 1 were assigned based on chem-
ical shifts and coupling constants of key protons. And the specific
rotation of 2 was the opposite of previously reported sphingosine
from S.abata. (½a D25
ꢀ20.4 and +11.6 for 2 and reference, respectively
ꢂ
in CHCl3).5c Thus, the structure of 2 was (E)-(2R,3R,4S)-2-amino-1,3-
dihydroxyheptadec-6-ene-4-sulfate, another enantiomer of the
previously reported sphingosine from S. abata.
The molecular formula of 3 was found to be C18H37NO6S by HR-
FABMS. Spectroscopic data of this compound were very similar to
those of 1 and 2, with the appearance of a signal for a dimethyl
group (dH 0.87, 6 H, d, J = 6.6 Hz; dC 23.0) as the most significant
change in the NMR spectra. Mutual long-range correlations with
the C-16 methine (dH 1.51, dC 29.2) in the gHMBC data located
the dimethyl group at this position, determining the structure of
3 to be (E)-(2R,3R,4S)-2-amino-1,3-dihydroxyisooctadec-6-ene-4-
sulfate.
O
O
O
a
b
O
c
9
5
O
O
O
O
BnO
H
OH
9
5
9
5
5
11
9a: cis
10a: cis
O
O
5
9
9
9b: trans
10b: trans
O
O
O
O
H
P
O
H
OH
d
HO
HO
O
4
12
O
OH
O-Na+
OH
Scheme 2. Reagents and conditions: (a) CH2I2, Et2Zn, benzene, 70 °C, 6 h; (b) 2-O-benzylglycerol, lipase, CHCl3,30 °C, 6 days; (c) H2, Pd/C, MeOH, rt, 24 h; (d) phospholipase C,
Tris–HCl buffer, CaCl2, rt, 22 h.

K. H. Jang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1078–1081
1081
Table 1
Results of bioactivity test
Compound
MIC(lg/mL)
K562
ICL
Na+/K+ꢀAPTase
Gram(+) bacterium
Gram(ꢀ) bacterium
Fungus
G
LC50
(lM)
IC50
(
lM)
IC50 (lM)
A
B
C
D
E
F
H
I
J
1
2
3
4
50
6
25
100
>100
100
NT
25
25
100
>100
1
>100
>100
>100
NT
100
>100
>100
>100
NT
50
50
8
7
8
4
2
25
21
21
4
>100
>100
>100
1
12
100
NT
2
100
100
NT
2
>100
>100
>100
>100
>100
NT
>100
>100
13
20
24
87
Ampicillin
Amphotericin B
Doxorubicin
3-NPa
2
6
1
2
2
1
7
1
Ouabain
4
A: Staphylococcus aureus (ATCC 6538p), B: Bacillus subtilis (ATCC 6633), C: Micrococcus luteus (IFO 12708), D: Salmonella typhimurium (ATCC 14028), E: Proteus vulgaris (ATCC
3851), F: Escherichia coli (ATCC 35270), G: Aspergillus fumigatus (HIC 6094), H: Trichophyton rubrum (IFO 9185), I: Trichophyton mentagrophytes (IFO 40996), J: Candida albicans
(ATCC 10231).
a
3-Nitropropionic acid. NT: Due to the limited amounts of isolated materials, test was not performed.
of the cyclopropane in the middle part of the fatty acid chain could
be minimal.
cytotoxicity and Na+/K+-ATPase inhibition than the sphingosine
sulfates.
The configuration at the C-2 of the glycerol moiety was also ap-
proached by chemical reactions. The lipase from Pseudomonas
cepacia (lipase PS)-catalyzed transesterification of 10a with 2-O-
benzylglycerol in CHCl3 yielded the monoester (11). Based on the
stereoselectivity of this enzyme-catalyzed reaction, the configura-
tion at C-2 was assigned as S.9 Then, palladium-catalyzed deprotec-
tion of 11 under a H2 atmosphere gave the desired compound 12a.
Also, 4 was hydrolyzed to 12b by phospholipase C and CaCl2.10 The
1H NMR spectrum and specific rotation of the synthetic 12a were
Acknowledgments
We are grateful to the Basic Science Research Institute in Daegu,
Korea, for providing mass data. This study was partially supported
by National Research Foundation of Korea (NRF) grants, funded by
the Korean government (MEST, Ministry of Education, Science and
Technology) (Nos. 20100028430 and 20110018562).
very similar to those of natural product-derived 12b (½a D25
ꢀ5.2
ꢂ
Supplementary data
and ꢀ6.1 in CHCl3 for 12a and 12b, respectively). Thus, an absolute
configuration of S (R for 4) was assigned at C-2 of 12b. The config-
uration at the remote C-200 remained unassigned since several at-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.105.
tempts to synthesize
4 from 12a or 12b with asymmetric
glycerol derivatives were unsuccessful. Overall, the structure of
compound 4 was determined to be 1-O-(cis-11,12-methyleneocta-
decanoyl)-syn-glycero-3-phosphoglycerol.
References and notes
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ids have been reported to exhibit diverse bioactivities.5a–c In our
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