Rhizochalin A, a novel two-headed sphingolipid from the sponge Rhizochalina incrustata

Article abstract of DOI:10.1021/np049710z

Rhizochalin A (4), the fourth representative of two-headed glycosphingolipids, was isolated as its peracetate from the sponge Rhizochalina incrustata. Its structure has been established as the 2-ethyl carbamoate of rhizochalin on the basis of spectroscopic data and chemical transformations.

Full text of DOI:10.1021/np049710z

J. Nat. Prod. 2005, 68, 255-257
255
Rhizochalin A, a Novel Two-Headed Sphingolipid from the Sponge
Rhizochalina incrustata
Tatyana N. Makarieva,*,† Alla G. Guzii,† Vladimir A. Denisenko,† Pavel S. Dmitrenok,† Elena A. Santalova,†
Evgenii V. Pokanevich,† Tadeusz F. Molinski,‡ and Valentin A. Stonik†
Laboratory of the MaNaPro Chemistry, Pacific Institute of Bioorganic Chemistry of the Russian Academy of Sciences,
690022 Vladivostok-22, Russia, and Department of Chemistry, University of California, One Shields Avenue,
Davis, California 95616
Received August 31, 2004
Rhizochalin A (4), the fourth representative of two-headed glycosphingolipids, was isolated as its peracetate
from the sponge Rhizochalina incrustata. Its structure has been established as the 2-ethyl carbamoate
of rhizochalin on the basis of spectroscopic data and chemical transformations.
Table 1. 1H and 13C NMR Data for Compounds 4a and 5
Sphingolipids appear to play an important role in cellular
regulation. Unusual sphingolipid analogues may act on the
(CDCl3, TMS)a
metabolism of normal sphingolipids, or as agonists or
antagonists interacting with sphingolipid recognition sites
in regulatory processes.1
4a
δH (m, Hz)
1.11, d, 6.8
5
atom no.
δC
18.8
48.8
76.4
31.5
25.2
29.1-29.8
23.9
42.8
δH (m, Hz)
δC
1
2
3
4
5
1.11, d, 6.8 18.8
Two-headed sphingolipids from marine sponges are
striking because of their rare R,ω-bifunctionalized struc-
tures and high biological activity. Since the discovery, in
1989, of rhizochalin (1), the first member of this series,2
only two additional members of this group have been
described. One of these compounds, oceanapiside (2), exhi-
bits significant antifungal activity against the pathogenic
fluconazole-resistant yeast Candida glabrata.3 Rhizochalin
(1) shows antibacterial activity against Staphylococcus
aureus and cytotoxic activity against mouse Ehrlich car-
cinoma cells (IC50 10 µg/mL).2 Another analogue of 1,
calyxoside (3), is a selective DNA-damaging agent, but
lacks inhibitory activity against topoisomerase I or II.4
3.92, m
3.90, m
4.85 m
49.0
76.4
31.7
25.3
29.1-29.8
24.0
4.84 td, 3.5, 6.7
1.55, m
6-8, 14-23 1.25, bs
9
1.55, m
2.37, t, 7.5
1.55, m
10
11
12
13
24
25
26
2.37, t, 7.5 42.9
211.6
2.36, t, 7.5 42.8
1.55, m
211.6
42.7
2.36, t, 7.5
1.55, m
25.3
30.7
1.42
3.50, dt, 2.7, 6.2, 82.5
4.85, m
4.20, m
76.6
47.3
6.2
27
28
2-NH
27-NH
1′
4.10, m
46.7
18.6
1.17, d, 6.8
4.71, d, 9.7
5.81, d, 8.5
1.10, d, 6.8 18.5
4.71, d, 8.5
5.51, d, 8.7
156.2
60.8
14.6
100.4
69.2
2′
3′
4.10, m
4.11, q, 7.0 60.9
1.25, t, 6.8 14.7
1.25, t, 6.8
4.48, d, 7.8
5.16, dd, 7.8 10.5
1′′
2′′
3′′
5.04, dd, 3.4, 10.5 70.7
5.39, dd, 1.1, 3.4 67.0
3.91, dt, 1.11, 6.6, 70.7
6.6
4.10, dd, 6.6, 11.2; 61.3
4.19, dd, 6.6, 11.2
4′′
5′′
6′′
a All 1H NMR experiments were performed at 300 and 500 MHz;
13C NMR experiments were performed at 75 and 125 MHz in
CDCl3.
chloroform. Chloroform-soluble materials were further
separated by column chromatography on Polychrome-1
(powdered Teflon) eluting with EtOH-H2O, 1:1, then on
silica gel (CHCl3-EtOH-H2O, 3:2:0.2) to obtain a crude
mixture containing 1 and 4. To obtain structural informa-
tion on the new compound, the mixture was acetylated
(Ac2O/pyr), and products were purified by preparative TLC
followed by HPLC (YMC-Pack ODS-A column) to provide
rhizochalin A peracetate (4a, 0.0006%, based on dry weight
of sponge).
The molecular formula C49H84N2O16 of 4a was obtained
from a high-resolution mass measurement of the [M + Na+]
ion in HRMALDI-TOF-MS and consideration of FABMS
and EIMS data. The 1H and 13C NMR data of 4a (Table 1)
showed signals typical of a pseudo-dimeric amino alcohol
In the course of our continuing studies on marine natural
products,5 we have found additional two-headed sphin-
golipids, including the new compound 4, which we have
named rhizochalin A. In this report we describe the
isolation and structure elucidation of 4 containing a rare
N-substituted carbamoyl group from the sponge Rhizoch-
alina incrustata (order Haplosclerida, family Phloeodicty-
diae).
The EtOH extract of the lyophilized sponge was concen-
trated and sequentially partitioned with hexane and
* To whom correspondence should be addressed. Tel: 7 (4232) 31-11-68.
Fax: 7 (4232) 31-40-50. E-mail: makarieva@piboc.dvo.ru.
† Laboratory of the MaNaPro Chemistry.
‡ University of California.
10.1021/np049710z CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/03/2005

256 Journal of Natural Products, 2005, Vol. 68, No. 2
Notes
unidentified biosynthetic intermediates of divergent car-
bamoyl or carbonate transferase reactions, followed by
solvolytic interception.
Experimental Section
General Experimental Procedures. Optical rotations
were measured using a Perkin-Elmer 343 polarimeter. The
NMR experiments were performed with Bruker DPX-300 and
Bruker DRX-500 spectrometers. FAB and EI mass spectra
were obtained on an AMD-604S mass spectrometer (AMD-
Intectra, Germany). MALDI-TOF mass spectra were obtained
on a Bruker Biflex III laser desorption mass spectrometer
coupled with delayed extraction using an N2 laser (337 nm)
on R-cyano-4-hydroxycinnamic acid as matrix.
Low-pressure column liquid chromatography was performed
using Polichrom-1 (powder Teflon, Biolar, Latvia), Sephadex
LH-20 (Sigma, Chemical Co.), and silica gel L (40/100 µm,
Chemapol, Praha, Czech Republic); silica gel plates of 4.5 ×
6.0 cm (5-17 µm, Sorbfil, Russia) were used for thin-layer
chromatography.
Animal Material. The sponge Rhizochalina incrustata
(Porifera, class Demospongiae, subclass Ceratinomorpha, order
Haplosclerida, family Phloeodictydiae) was collected using
scuba (depth 3-12 m) during the third scientific cruise of R/V
Akademik Oparin (November 1986, Seychelles Islands, 4°26′45′′
N, 54°54′75′′ E) and identified by Prof. V. M. Koltun (Zoological
Institute, St. Petersburg, Russia). A voucher specimen (03-
297) was deposited in the collection at the Pacific Institute of
Bioorganic Chemistry, Vladivostok, Russia.
Extraction and Isolation. The fresh collection of the
sponge R. incrustasta was immediately lyophilized and kept
at -20 °C until required. The lyophilized material (400 g) was
extracted with EtOH (1 L × 3). The ethanolic extract after
evaporation in vacuo was redissolved with EtOH-H2O (9:1).
The n-hexane-soluble fraction was extracted three times by
partitioning with equal volumes of hexane. The water content
of the aqueous EtOH extract was adjusted to 7:1 EtOH-H2O.
The CHCl3-soluble fraction was extracted three times by
partitioning with equal volumes of CHCl3. The CHCl3 extracts
were evaporated in vacuo at 50 °C to give a brown oil, which
was separated over a Polichrome I (powder Teflon, Biolar,
Latvia) by elution with a gradient of H2O f 50% EtOH f
EtOH. The two-headed sphingolipid fraction (ninhydrin posi-
tive) eluted with 50% EtOH. The latter was further separated
over a SiO2 column using CHCl3 f CHCl3-EtOH (10:1 f 3:2)
and CHCl3-EtOH-H2O (3:2:0.2) mixtures as eluents to give
a mixture (50 mg) of the known rhizochalin (1)2 and crude
4.
Figure 1. Rhizochalin A (4) and substructures from HMBC spectra.
glycoside, reminiscent of 1. These included signals for two
secondary methyl groups (δH 1.11, 1.17; δC 18.8, 18.6), two
N-substituted CH carbons (δH 3.92, 4.10; δC 48.8, 46.7), two
oxymethines (δH 4.84, 3.50; δC 76.4, 82.5), and one ketone
carbonyl group (δC 211.6), flanked by two R-CH2 groups
(δH 2.37, 2.36; δC 42.8 and 42.7). The remainder of the
signals was assigned to long CH2 chains (δH 1.25; δC 29.1-
29.8). 1H NMR data of 4a were similar to those of
rhizochalin peracetate except that the amide doublet (C2-
NH) was shifted upfield from δ 5.63 to δ 4.71.2 Conse-
quently, the structure of 4 was formulated as an analogue
of rhizochalin with a modification at C-2 that was subse-
quently revealed by analysis of 13C NMR, COSY, and
HMBC data. The 13C NMR spectrum of 4a revealed signals
due to an ethoxyl group (OCH2CH3: δH 1.25, t, J ) 6.8 Hz,
3H; 4.10 m, 2H; δC 14.6, q; 60.8, t). The balance of the
formula indicated a CdO group whose 13C chemical shift
(δ 156.2, s) was consistent only with a carbamoyl group.
HMBC correlations (Figure 1) placed this NH(CO)OCH2-
CH3 group at C-2 in 4. The galactopyranosyl group in 4
has the â-configuration at the anomeric carbon, as revealed
by the H1′′ coupling constant (δ 4.48, d, J ) 7.8 Hz). The
cross-peak with C-26 (δ 82.5) in the HMBC spectrum
established the attachment of monosaccharide to this
position. Hydrolysis of 4a (6 N HCl, 100 °C, 2.5 h) liberated
D-galactose and two aglycone-derived compounds, which
were peracetylated (Ac2O/pyr, 1:1) and separated by silica
chromatography. The earlier-eluting compound was identi-
fied as the peracetate 5, and the second product proved to
be peracetylaglycone, 6, identical to that derived from
rhizochalin (1) by NMR, EIMS, and [R]D data.2 Therefore,
the keto groups in 1 and 4 are located at the same position
(C-11), and the absolute configuration of 4 is the same as
that of 1 (2R,3R,26R,27R).6
Peracetate Derivatives, 1a and 4a. A sample of the
mixture of 1 and 4 (50 mg) was dissolved in pyridine (1.0 mL)
and acetic anhydride (1.0 mL) and allowed to stand at 25 °C
for 18 h. Removal of the volatile material gave a residue (55
mg) containing 1a and 4a. The latter was separated on a SiO2
column using EtOAc to give 1a (30 mg) and a fraction
containing less polar compounds (10.7 mg), which was purified
by preparative TLC (SiO2, EtOAc) to give 4a (5.3 mg).
Preparative HPLC (YMC-Pack ODS-A, 80:20 EtOH-H2O)
gave rhizochalin A peracetate (4a) (2.3 mg; 0.0006%, based
on dry weight of sponge).
Rhizochalin A peracetate (4a): amorphous solid, [R]18
D
+15° (c 0.22 EtOH); HRMALDI m/z (M + Na+) 979.5759 (cald
for C49H84N2O16Na 979.5719); EIMS m/z 956 (M+), 911, 897,
841, 798, 782, 609, 563; FABMS m/z 957 (M + H)+; FABMS
Rhizochalin A is the first example of a natural product
among known sphingolipids, including the family of two-
headed sphingolipids that contains the rare N-alkyl car-
bamoyl group. N-Carbamates have also been detected in
other marine alkaloids,7-9 while the polyketides discoder-
molide A10 and kabiramide C11 contain O-alkyl carbamoyl
groups. Since the latter compounds were obtained from
MeOH extracts and the specimen of R. incrustata used in
this study was stored in ethanol, it is possible that 4 and
naturally derived O-Me carbamates originate from as-yet-
1
m/z 955 (M+ - H)-; H NMR (CDCl3, see Table 1); 13C NMR
(CDCl3, see Table 1).
Hydrolysis of Rhizochalin A Peracetate (4a). A solution
of 4a (2.2 mg) in 6 N HCl (1 mL) was heated at 100 °C for 2.5
h. The mixture was cooled and treated with Dowex ion-
exchange resin (HCO3- form) and extracted with n-BuOH. The
aqueous solution was separated and concentrated to afford
D-galactose (0.4 mg). The n-BuOH layer was concentrated, and

Notes
Journal of Natural Products, 2005, Vol. 68, No. 2 257
References and Notes
a residue was dissolved in pyridine (0.5 mL) and acetic
anhydride (0.5 mL) and allowed to stand at 25 °C for 18 h.
Removal of the volatile material gave a residue (1.6 mg)
containing a mixture of 5 and 6. The latter was separated on
a SiO2 column with SiO2, EtOAc f EtOAc-EtOH (100:3) to
give pure 5 (0.3 mg) and 6 (1.1 mg).
(1) Shier, W. T.; Shier, A. C. J. Toxicol. 2000, 19, 189-246.
(2) Makarieva, T. N.; Denisenko, V. A.; Stonik, V. A.; Milgrom, Yu. N.;
Rashkes, Ya. W. Tetrahedron Lett. 1989, 30, 6581-6584.
(3) Nicholas, G. M.; Hong, T. W.; Molinski, T. F.; Lerch, M. L.; Cancilla,
M. T.; Lebrilla, C. B. J. Nat. Prod. 1999, 62, 1678-1681.
(4) Zhou, B. N.; Mattern, M. P.; Johnson, R. K.; Kingston, D. G. I.
Tetrahedron 2001, 57, 9549-9554.
(5) Makarieva, T. N.; Rho, J.-R.; Lee, H.-S.; Santalova, E. A.; Stonik, V.
A.; Shin, J. J. Nat. Prod. 2003, 66, 1010-1012.
(6) Molinski, T. F.; Makarieva, T. N.; Stonik, V. A. Angew. Chem., Int.
Ed. 2000, 39, 4076-4079.
(7) Pina, I. C.; Gautschi, J. T.; Wang, G. Y. S.; Sanders, M. L.; Schmitz,
F. J.; France, D.; Cornell-Kennon, S.; Sambucetti, L. C.; Remiszewski,
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3866-3873.
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Compound 5: amorphous solid; [R]18D +30° (c 0.03 CHCl3);
MALDI m/z 691 (M + Na+); 707 (M + K+); EIMS m/z 623 (M+
- 45), 535, 519, 450, 434, 350, 340, 294, 280; FABMS m/z 669
(M+ + H); H NMR (CDCl3, see Table 1).
1
Compound 6: amorphous solid; [R]18D +35° (c 0.11 CHCl3);
EIMS m/z 638 (M+); 595, 578, 553, 535, 519, 493, 450, 434,
1
350, 340, 294, 280; H NMR (CDCl3, see Table 1).
Acknowledgment. This investigation was supported by
the Fogarty International Center and NIH (TWOO6301-01).
The research described in this publication was made possible
in part by the Program of Presidium of RAS “Molecular and
Cell Biology”, Grant N SS-725.2003.4 from the President of
RF.
(9) Ovenden, S. P. B.; Capon, R. J. J. Nat. Prod. 1999, 62, 1246-1249.
(10) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Koseki, K.; Noma, M.
J. Am. Chem. Soc. 1986, 108, 847-849.
(11) Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G. K. J.
Org. Chem. 1990, 55, 4912-4915.
NP049710Z