Evasteriosides A and B and other sulfated steroids from the Pacific starfish Evasterias retifera

Article abstract of DOI:10.1007/s11172-008-0347-6

Two new polar steroidal glycosides identified as sodium (20R,22E,24R,25S)-3-O-(β-d-xylopyranosyl)-24-methyl-5α-cholest-22- ene-3β,6β,8,15α,26-pentol 26-sulfate (evasterioside A) and sodium (20R,22E)-24-O-(β-d-xylopyranosyl)-5α-cholest-22-ene-3β, 6β,8,15α,24-pentol 3-sulfate (evasterioside B) were isolated from the Pacific starfish Evasterias retifera collected in the Sea of Japan. Five known compounds, viz., coscinasterioside B, aphelasterioside A, marthasterone 3-sulfate and (20R)-cholest-7-en-3β-ol and cholesterol sulfates, were identified. The structures of the new natural compounds were established using their 2D NMR and mass spectra and some chemical transformations.

Full text of DOI:10.1007/s11172-008-0347-6

Russian Chemical Bulletin, International Edition, Vol. 57, No. 11, pp. 2431—2436, November, 2008
2431
Evasteriosides A and B and other sulfated steroids from
the Pacific starfish Evasterias retifera
E. V. Levina, A. I. Kalinovskii, P. V. Andriyashchenko, and P. S. Dmitrenok
Pacific Institute of Bioorganic Chemistry,
FarꢀEastern Branch of the Russian Academy of Sciences,
159 prosp. 100ꢀletya Vladivostoka, 690022 Vladivostok, Russian Federation.
Fax: +7 (423 2) 31 4050.Eꢀmail:levina@ piboc.dvo.ru
Two new polar steroidal glycosides identified as sodium (20R,22E,24R,25S)ꢀ3ꢀOꢀ
(βꢀDꢀxylopyranosyl)ꢀ24ꢀmethylꢀ5αꢀcholestꢀ22ꢀeneꢀ3β,6β,8,15α,26ꢀpentol 26ꢀsulfate (evaꢀ
sterioside A) and sodium (20R,22E)ꢀ24ꢀOꢀ(βꢀDꢀxylopyranosyl)ꢀ5αꢀcholestꢀ22ꢀeneꢀ3β,6β,8,15α,24ꢀ
pentol 3ꢀsulfate (evasterioside B) were isolated from the Pacific starfish Evasterias retifera
collected in the Sea of Japan. Five known compounds, viz., coscinasterioside B, aphelasterioside
A, marthasterone 3ꢀsulfate and (20R)ꢀcholestꢀ7ꢀenꢀ3βꢀol and cholesterol sulfates, were
identified. The structures of the new natural compounds were established using their 2D NMR
and mass spectra and some chemical transformations.
Key words: starfishes, Evasterias retifera, polyhydroxysteroids, sulfated polyhydroxysteroids,
glycosides, MALDI TOF mass spectra, NMR spectra.
Polyhydroxylated steroids, typical secondary metaboꢀ
lites of some marine invertebrates, are of considerable
interest owing to their structural diversity and biological
activities. Starfishes, which are exceptionally abundant
sources of such products unlike other echinoderms, conꢀ
tain mainly pentaꢀ, hexaꢀ, or octahydroxy compounds,
which are in some cases sulfated but more often glycoꢀ
sylated.1,2
In a continuation of the research into metabolites of
Far Eastern starfishes,3 we studied the steroid composiꢀ
tion of the starfish Evasterias retifera Verrill Djakonov,
1938 (Forcipulata order, Asteriidae family) gathered near
the Gamov Cape (Sea of Japan). All seven isolated comꢀ
pounds were monosulfates, in particular, two new and
two previously known monoglycosylated polyols, marthaꢀ
sterone sulfate frequently encountered in starfishes, and
two sterol sulfates. Four isolated glycosides contained a
βꢀDꢀxylopyranose residue, 3β,6β,8,15αꢀtetrahydroxyꢀ
cholestane core and differed from one another mainly in
the position of monosaccharide and the sulfate groups,
which were attached to C(3), C(15), or C(24) in all of
these compounds. Previously unknown glycosides, which
we called evasteriosides A and B, contained a transꢀdouble
bond C(22)=C(23) in the side chain.
the starfish E. retifera by column chromatography on Polyꢀ
chrome, Florisil, and silica gel followed by reversedꢀphase
HPLC on columns with Diasphereꢀ110ꢀC18,, Zorbax Boꢀ
nusꢀRP (4.6×250 mm, 5 μm), and YMCꢀPack ODSꢀA
using the previously reported procedures.3 Structural
identification of the isolated compounds and their derivaꢀ
tives was carried out by spectroscopic methods (NMR,
MALDIꢀTOF) and by comparing their physical paramꢀ
eters with the published in press.
The (+)ꢀMALDIꢀTOF mass spectrum of evasterioside
A (1a) contained pseudomolecular ion peaks at m/z 721
[MNa + Na]+ and 737 [MNa + K]+ and an ion peak at
m/z 588 [(MNa – C5H9O4]+ corresponding to abstraction
of a pentose residue. These data, together with the NMR
spectra, corresponded to the molecular formula C33H55O12SNa
and attested to the presence of a sulfate group and a
pentose residue in 1a. Analysis of the 13C NMR and DEPT
spectra (Table 1) indicated that the molecule contained
33 carbon atoms: five methyl, nine methylene, sixteen
methine, in particular, two olefinic, and also two quaterꢀ
nary atoms and one tertiary atom linked to oxygen. The
chemical shifts of the anomeric proton (δH 4.35, d,
J = 7.5 Hz) and anomeric carbon atom (δ 103.2), and the
signals of nine carbon atoms linked to oxygen (δ 66.8,
69.9, 71.3, 72.5, 74.2, 75.0, 77.2, 77.9, and 80.0) indicate
that molecule 1a contains, apart from the βꢀpentoside
fragment, a pentasubstituted steroidal core. The 13C and
1H NMR chemical shifts for C(1)—C(21), C(1´)—C(5´),
and C(3)H, C(6)H, C(15)H, C(18)H3, C(19)H3, C(21)H3,
Results and Discussion
New steroidal compounds 1a and 2a and known comꢀ
pounds 3—7 were isolated from the ethanolic extract of
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2381—2386, November, 2008.
1066ꢀ5285/08/5711ꢀ2431 © 2008 Springer Science+Business Media, Inc.

2432
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 11, November, 2008
Levina et al.
Comꢀ
pound
R1
R2
R3
R4
R5
Compound
R1
R2
1a
1b
1c
OSONa
OH
H
Me
Me
2a
2b
3*
4*
SO3Na
H
SO3Na
H
βꢀOH
αꢀOH
βꢀOH
βꢀOH
βꢀOH
H
βꢀOH
βꢀOH
H
H
H
H
SO3Na
H
CH2OSO3Na
SO3Na
H
*Compounds 3 and 4: 22(23)ꢀsaturated
and C(2´)H—C(5´)H2 of glycoside 1a (see Table 1) as
well as the spinꢀspin coupling constants of protons were
in good agreement with those in the spectra of asteriidoside
L (1c) from starfishes of the Asteriidae family.4 Evidently,
1a contains a similar 3β,6β,8,15αꢀtetrahydroxycholestane
core with the βꢀDꢀxylopyranose residue at C(3). Indeed,
acid hydrolysis of 1a gave xylose, which was identified by
TLC and GLC (aldononitrile peracetates). The monosacꢀ
charide was assigned to the Dꢀseries by analogy with known
monoglycosylated polyols from starfishes containing a
βꢀDꢀxylopyranose carbohydrate residue1 and by comparꢀ
ing the specific rotation of the xylose obtained upon
downfield and observed at δH 3.73, 4.03 and δC 72.3).
Relying on the obtained data, the results of mass spectroꢀ
scopic study, and NMR spectroscopy, we suggested that
glycoside 1a has an Oꢀsulfate group at C(26). This
was confirmed by the cross peaks C(26´)H (δH 3.26)
and C(26)H (δH 3.57)/C(25)H (δH 1.52) and C(25)H
(δH 1.52)/C(27)H3 (δH 0.87) observed in the COSYꢀ90
spectrum of derivative 1b. The corresponding crossꢀ
peaks were also present in the spectrum of evasterioside
1a: C(26´)H (δH 3.73) and C(26)H (δH 4.03)/C(25)H
(δH 1.74) and C(25)H (δH 1.74)/C(27)H3 (δH 0.92).
The multiplets for the two olefinic protons in the
1H NMR spectrum of 1a (δH 5.21 and 5.24) were asꢀ
signed to the C(22)H = C(23)H fragment and the value
J22,23 = 15.3 Hz corresponded to the Eꢀconfiguration of
the side chain double bond. The chemical shift of the
C(20) atom (δC 40.7) also confirms the 22Eꢀconfiguraꢀ
tion of this double bond; this signal for the Zꢀisomer
appears at δC ≈ 35.0 (see Ref. 5). The 20R configuration
was assigned to the C(20) center in steroid 1a based on
the chemical shift of 21ꢀCH3 (δ 0.97).3
hydrolysis with the [α] values of Dꢀ and Lꢀxyloses. The
D
attachment of the monosaccharide residue to C(3) was
confirmed by correlation of C(3) and the anomeric HC(1´)
proton in the HMBC spectrum (see Table 1). The
arrangement of the hydroxy groups in the steroid nucleus
followed from 2D NMR experiments (see Table 1).
The structure of the side chain was established using
mild solvolysis of glycoside 1a in a dioxane—pyridine
mixture (1 : 1) at 100 °C. This afforded compound 1b
whose NMR spectra exhibit signals for the hydroxymethyl
group protons, HC(26) and H´C(26), at δH 3.26, 3.57 and
δC 66.7 (in the spectra of 1a, these signals were shifted
The fact that the signals for the side chain recorded in
1
the H and 13C NMR spectra of glycoside 1a coincide
with those in the spectra of (22E,24R,25S)ꢀ24ꢀmethylꢀ

Steroids from the starfish Evasterias retifera
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 11, November, 2008 2433
Table 1. Data from NMR spectra of evasteriosides A(1) and B(2) (CD OD, DRXꢀ500)a
3
Atom
1a
2a
b
b
δ
δ
H (J/Hz)
HMBC
δ
δ
H (J/Hz)
HMBC
C
C
1
2
41.4 (CH2)
30.1 (СH2)
1.72 (m); 0.97 (m)
1.62 (m, Heq
41.4 (CH2) 1.01 (m), 1.75 (m)
29.2 (CH2) 2.00 (m), 1.64 (m)
)
1.82 (m, Hax)
3.69 (m)
1.74 (m), 1.84 (m)
1.19 (m)
3.85 (m)
1.56 (m); 2.36 (dd,
J = 3.2, J = 14.0)
3
4
5
6
7
80.0 (CH)
32.9 (CH2)
48.9 (CH)
74.2 (CH)
45.4 (CH2)
80.3 (CH)
33.9 (CH2) 1.87 (m), 1.95 (m)
4.33 (m)
С(3)
49.1 (СН)
73.9 (СН)
45.4 (CH2) 2.37 (dd, J = 4.8,
J = 11.6), 1.59 (m)
77.1 (СН)
1.27 (m)
3.86 (s)
С(8), С(10)
С(5), С(6),
С(8), С(9)
С(10), С(8)
С(5), С(8),
С(9)
8
77.2 (C)
9
57.2 (CH)
36.8 (C)
19.8 (CH2)
42.7 (CH2)
45.5 (С)
66.7 (CH)
69.9 (CH)
42.3 (CH2)
55.7 (CH)
15.5 (СH3)
0.96 (m)
57.1 (СН)
36.5 (С)
19.8 (СН2) 1.82 (m), 1.54 (m)
42.1 (СН2) 1.26 (m), 1.95 (m)
45.4 (С)
66.6(СН)
70.0 (СН)
42.1 (СН2) 1.58 (m), 1.89 (m)
55.6 (СН) 1.41 (m)
0.97 (m)
10
11
12
13
14
15
16
17
18
1.53 (m); 1.83 (m)
1.24 (m); 1.94 (m)
1.20 (d, J = 9.5)
4.25 (d.t, J = 3.0, J = 9.3)
1.58 (m); 1.89 (m)
1.36 (m)
С(7), С(15), С(18)
1.21 (d, J = 9.5)
4.25 (t.d, J = 9.4, J = 3.1)
С(15), С(7)
0.97 (s)
С(12), С(13)
С(14), С(17)
С(1), С(5),
15.6 (СН3) 0.98 (s)
С(14), С(17),
С(12), С(13)
С(1), С(5),
С(9), С(10)
19
15.8 (СH3)
1.16 (s)
15.7 (СН3) 1.17 (s)
С(9), С(10)
С(23)
20
21
40.7 (CH)
21.0 (СH3)
1.99 (m)
0.97 (d, J = 6.7)
40.4 (СН)
2.06 (m)
С(17), С(22), С(20)
21.0 (СН3) 0.99 (d, J = 5.5)
С(17), С(22),
С(20)
22
23
24
25
26
137.5 (CH)
133.1 (CH)
39.9 (CH)
39.6 (CH)
72.5 (СH)
5.21 (dd, J = 15.3, J = 7.5) С(24)
5.24 (dd, J = 15.3, J = 7.7) С(20)
141.0 (СН) 5.39 (dd, J = 15.4, J = 8.5) С(20), С(24)
128.1 (СН) 5.31 (dd, J = 15.4, J = 8.1) С(20)
2.11 (m)
С(22)
89.2 (СН)
33.8 (СН)
3.69 (dd, J = 6.0, J = 8.0)
1.82 (m)
1.74 (m)
3.73 (dd, J = 9.5, J = 7.6),
4.03(dd, J = 9.5, J = 5.5)
0.92 (d, J = 6.9)
С(25), С(27)
19.3 (СН3) 0.92 (d, J = 7.0)
С(24), С(25),
С(27),
27
14.3 (СH3)
С(24), С(25),
С(26)
18.3 (СН3) 0.86 (d, J = 7.0)
С(24),С(25),
С(26)
28
1´
2´
3´
4´
5´
17.4 (СH3)
103.2 (CH)
75.0 (CH)
77.9 (CH)
71.3 (CH)
66.8 (CH2)
0.95 (d, J = 6.9)
4.35 (d, J = 7.5)
3.13 (dd, J = 7.5, J = 9.0)
3.30c
С(24), С(25), С(23)
С(3)
С(1´)
104.3 (CH) 4.21 (d, J = 7.5)
С(24)
С(3´)
С(2´)
75.4 (CH)
78.1 (CH)
71.2 (CH)
3.17 (dd, J = 7.1, J = 9.0)
3.27 (t, 8.8)
3.45 (m)
3.46 (m)
3.83 (dd, J = 4.3, J = 11.4), C(4´), С(3´)
3.18 (t, J = 11.4) С(1´)
67.0 (CH2) 3.79 (dd, J = 5.2, J = 11.4), С(1´), С(3´),
3.13 (t, J = 12.6) C(4´)
a The signals were assigned using 1H–1H COSY, HMBC, and HSQC 2D NMR procedures.
b Signal multiplicity was determined using the DEPT spectrum.
c The assignment is ambiguous.
5αꢀcholestꢀ22ꢀeneꢀ3β,5,6β,15α,26ꢀpentol 26ꢀsulfate (1c)
from the starfish Luidia clathrata6 confirmed that 1a and
1c have identical Δ(22E)ꢀ24ꢀmethylꢀ26ꢀsulfoxycholestane
side chains.
Thus, based on the data obtained altogether, the new
natural compound evasterioside A (1a) was identified
as sodium (20R,22E,24R,25S)ꢀ3ꢀOꢀ(βꢀDꢀxylopyranosyl)ꢀ
24ꢀmethylꢀ5αꢀcholestꢀ22ꢀeneꢀ3β,6β,8,15α,26ꢀpentol
26ꢀsulfate.
1
The H—1H COSYꢀ90, HMBC, and HSQC experiꢀ
ments allowed assigning the NMR signals for all protons
and carbon atoms of 1a (see Table 1). The bold lines
in the formula indicate the fragments where the proton
sequences were confirmed by 1H—1H COSY experiments.
The (–)ꢀMALDIꢀTOF mass spectrum of evasterioꢀ
side B (2a) contained a pseudomolecular ion peak at
m/z 661 [M – Na]– and the (+)ꢀMALDIꢀTOF spectrum
exhibited peaks at m/z 707 [MNa + Na]+ and 587

2434
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 11, November, 2008
Levina et al.
[(MNa + Na) – NaHSO4]+, which corresponds to the
molecular formula C32H53O12SNa and attests to the presꢀ
ence of a sulfate group. The 13C NMR and DEPT spectra
(see Table 1) exbited signals for 32 carbon atoms includꢀ
ing five methyl, eight methylene, and 14 methine groups,
two quaternary carbon atoms, one tertiary carbon atom
attached to oxygen, and two methine olefinic carbon
atoms. The signal at δ 104.3 was assigned to the anomeric
carbon atom of the monosaccharide residue and the sigꢀ
nals at δ 67.0, 70.0, 71.2, 73.9, 75.4, 77.1, 78.1 and 80.3,
89.2 were assigned to other carbon atoms linked to oxyꢀ
gen atoms. Glycoside 2a, like 1a, contained a βꢀxylose
fragment, this sugar being identified after acid hydrolysis
of 2a (see Experimental). The chemical shifts and the
corresponding spinꢀspin coupling constants of the proꢀ
tons and the chemical shifts of C atoms in the NMR
spectra of compound 2a almost coincided with those in
the spectrum of aphelasterioside A (3) from the starfish
Aphelasterias japonica,7 except for some signals for the
side chain containing a double bond.
The side chain structure of 2a was determined by comꢀ
paring its spectroscopic data with the corresponding
data for amurensoside C (2b) from the starfish Asterias
amurensis.8 The coincidence suggested identical side
chains in both glycosides containing a Δ(22E)ꢀ24ꢀβꢀDꢀxyloꢀ
pyranosyloxy fragment. As for compound 1a (see Table 1),
the chemical shifts and spinꢀspin coupling constants of
olefinic protons (δH 5.39 and 5.31, J22,23 = 15.4 Hz) corꢀ
responded to the transꢀconfiguration of the 22(23) double
bond in the side chain of glycoside 2a. The H(1´)/C(24)
correlation (see Table 1) in the HMBC spectrum conꢀ
firmed that the βꢀDꢀxylopyranose residue is attached to
the O(24) atom of the aglycone, and the 20Rꢀconfiguraꢀ
tion was ascribed to the asymmetric center C(20), as in
the case of glycoside 1a.
eters with those of known (20R,22E,24R,25S)ꢀ24ꢀOꢀ
(βꢀDꢀxylopyranosyl)ꢀ5αꢀcholestaꢀ3β,6β,8,15α,24ꢀpentol
15ꢀsulfate (coscinasterioside B) from the starfish Coscinaꢀ
sterias tenuispina.9
The (ꢀ)ꢀMALDIꢀTOF mass spectrum of steroid 5a
exhibited peaks of the pseudomolecular ion with m/z 488
–
[M(SO3Na)]– and an ion with m/z 465 [M(SO3 )]. Toꢀ
gether with NMR data, this pointed to the presence of a
sulfate group in the hydroxycholestene core of the molꢀ
ecule and suggested the molecular formula C27H45OSO3Na
for steroid 5a. In its NMR spectra, the protons H3C(18)
and H3C(19) are manifested as threeꢀproton singlets at
δ 0.56 and 0.82, and the doublets at δ 0.94 and 0.87 (6H)
corresponded to the H3C(21), H3C(26), and H3C(27)
methyl groups. The C(3)H proton resonates in the
1H NMR spectrum as a multiplet at δ 4.24 and the correꢀ
sponding C(3) carbon atom is manifested in the 13C NMR
spectrum at δ 79.3. This means that the sulfate group is
attached to C(3) and the width of the H(3) multiplet
attests to the βꢀconfiguration of the substituent at C(3).
The NMR spectra of steroid 5a also contain a oneꢀproton
singlet at δH 5.16 and the signals of carbon atoms of trisubꢀ
stituted double bond at δC 140.6 and 118.5, which can be
assigned to the Δ7 double bond.10 The solvolytic desulfation
of 5a affords 5αꢀcholestꢀ7ꢀenꢀ3βꢀol (5b), which was
identified by comparing its spectral characteristics with
those of a known compound.11 The 20Rꢀconfiguration
was ascribed to the C(20) asymmetric center, as for glycoꢀ
sides 1a, 2a. The proton sequences established from the
COSYꢀ90 spectrum are marked in bold (see structural
formula 5a). The 7(8)ꢀposition of the double bond was
confirmed by the absence of crossꢀpeaks of the H3C(18)
and H3C(19) methyl groups with the carbon atoms at the
double bond (δC 140.6 and 118.5) in the HMBC spectrum
of sulfate 5a. Based on these results, compound 5a was
identified as sodium (20R)ꢀcholestꢀ7ꢀenꢀ3βꢀyl sulfate.
The previously known (20R)ꢀcholestꢀ5ꢀenꢀ3βꢀol
3ꢀsulfate (6) and (20R)ꢀ6αꢀhydroxyꢀ23ꢀoxocholestaꢀ
9(11),24ꢀdiene 3ꢀsulfate (7) (marthasterone) were identiꢀ
fied in the starfish E. retifera by comparing their spectral
characteristics with those of authentic samples.12,13
Thus, the starfish E. retifera accumulates, in the polar
fraction of sulfated sterols, not only free Δ5 sterols arriving
with food, but also mainly Δ7 components. Intense
sulfation of sterols, steroidal polyols, and their glycosides
may have a certain biological meaning. It is known2 that
toxic glycosides of starfishes interact with membrane choꢀ
lesterol and other Δ5 sterols to form pores in membranes.
Therefore, cholesterol and its homologs are toxic for the
cells of starfishes themselves, while their sulfation or
replacement of cholesterol in membranes by Δ9(11) and
Δ7 sterols and stanols protects the animal cells from the
action of inherent toxic glycosides. Usually Δ7 sterols and
stanols predominate in the starfish fractions of free sterols
without being sulfated. The reason for this selectivity in
The structure of 2a was established by 2D NMR specꢀ
1
troscopy including the H—1H COSYꢀ90, HMBC, and
HSQC experiments, which allowed us to assign the sigꢀ
nals of all protons and carbon atoms of the aglycone and
the carbohydrate moiety in the spectra of 2a (see Table 1)
and to show the presence of the proton sequences that are
marked in bold (see structural formulas). Based on the
data obtained altogether, the new natural product evaꢀ
sterioside B (2a) was identified as sodium salt (20R,22E)ꢀ
24ꢀOꢀ(βꢀDꢀxylopyranosyl)ꢀ5αꢀcholestꢀ22ꢀeneꢀ3β,6β,8,15α,24ꢀ
pentol 3ꢀsulfate. Thus, evasterioside B is the Δ(22)E analog
of the previously known aphelasterioside A.7
Glycoside 3 isolated from the total glycoside fraction
was identified by comparison with a known compound, as
its physical parameters and the (+)ꢀMALDIꢀTOF and
NMR spectra coincided with the corresponding data for
aphelasterioside A from the starfish Aphelasterias japonica.7
Glycoside 4, which differed from 3 in the position of
the sulfate group at C(15), was identified from coinciꢀ
dence of its spectral characteristics and physical paramꢀ

Steroids from the starfish Evasterias retifera
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 11, November, 2008 2435
Table 2. Sulfated steroidal compounds from the Pacific starfish E.retifera
b
c
Compound
m/mg a
[α]20
Mass spectrum
Source
D
MALDIꢀTOF, m/z
Glycosides
1a
2a
16.3
15.7
–4.5
721 [MNa + Na]+, 737 [MNa + K]+,
588 [(M + NaC5H9O4]+
661 [M – Na]–, 707 [MNa + Na]+,
587 [M + Na – NaHSO4]+
663 [M – Na]–, 709 [M + Na]+
709 [M + Na]+
E. retifera
E. retifera
–10.5
3
4
6
7.5
+6.5
+7.2
Coscinasterias tenuispina9
Aphelasterias japonica7
Sulfated steroids
5a
6
7
29
19
5.6
+40.6
–5.1
–8.3
488 [M(SO3Na)]–, 465 [M(SO3–)]
465 [M(SO3–)]
Eupentacta fraudatrix11
Pteraster obscurus12
Lysastrosoma anthosticta
13
a Isolated from 826 g of the ethanolic extract obtained by extraction from 4.1 kg of animals (crude weight).
b Solutions in MeOH.
c Biological source from which this compound was isolated for the first time.
extracts were concentrated to a syrup and chromatographed on
the content of the fraction of E. retifera sulfated sterols
(Table 2) is most probably in the unusual composition of
the glycosides of this starfish.
a column (6Ѕ25 cm) with silica gel (50—100 μm) in a chloroꢀ
form—ethanol system (100 : 0 → 45 : 55 v/v). This gave: 2.3 g of
fraction I comprising steroidal glycosides 1a—4 (elution with
20% ethanol in chloroform), 0.5 g of a more polar fraction II
(elution with 35% ethanol) composed of steroid sulfates 5a—7,
and 1.58 g of a polar lipid fraction III (elution with 55—60%
ethanol).
Experimental
1H and 13C NMR spectra were recorded on Bruker DPXꢀ300
and Bruker DRXꢀ500 spectrometers operating at 300 and 500 MHz,
respectively, for protons with SiMe4 as the internal standard.
The specific optical rotation was measured on a PerkinꢀElmer 343
polarimeter, MALDI TOF mass spectra were run on a Bruker
Biflex III mass spectrometer (Gеrmany) with laser ionization/
desorption (N2 laser, 337 nm). A sample was dissolved in
methanol (10 mg mL–1) and a 1 μL aliquot was analyzed using
αꢀcyanohydroxycinnamic acid as the matrix. The sodium content
was determined by a Nippon Jarrel Ash AAꢀ780 atomic adsorpꢀ
tion flame emission spectrophotometer. HPLC was carried out
on a Du Pont Model 8800 chromatograph using a refractometric
detector and columns packed with Diasphereꢀ110ꢀC18 (5 μm,
4×250 mm), ZorbaxꢀBonus (4×250 mm, 5 μm, 12 nm), and
YMCꢀPack ODSꢀA (5 μm, 12 nm, 10×250 mm). The melting
points were determined on a Leica VMTG hot stage. TLC was
performed on Sorbfil plates with a STKhꢀ1A silica gel (5—17 μm,
Russia, Krasnodar) layer fixed to foil and BuOH—EtOAc—H2O,
5 : 1 : 1, as the eluent. Column preparative chromatography was
performed on silica gel L (80—100 and 200—250 mesh, Chemapol,
Czech Republic), Polychrome (Russia), and Florisil (200—250 mesh,
Merck, Germany).
Animals. Specimens of the starfish E. retifera were collected
in August 2003 by divers near the Gamov Cape (Sea of Japan),
from 60—150 m depth and identified by V. C. Levin (Pacific
Institute of Bioorganic Chemistry, FarꢀEastern Branch of the RAS).
Extraction and isolation of overall fractions. The disintegrated
starfishes E. retifera (the weight of crude animals was 4.1 kg)
were exhaustively extracted with 95% ethanol at room temperaꢀ
ture. The combined ethanolic extract was concentrated in vacuo
to a resinous residue (0.826 kg). This was dissolved in water and
extracted three times with butanol. The combined butanolic
Isolation of steroidal compounds 1a—7. Fraction I was
dissolved in water and passed through a column with Polychrome
(3×12 cm), the column being washed with water, 50% aqueous
ethanol, and anhydrous ethanol. The water—ethanolic and
ethanolic eluates were concentrated in vacuo to brown resinous
residues (0.36 and 0.45 g), the combined residues were succesꢀ
sively chromatographed on columns with Florisil (1.5×20 cm,
200—300 mesh) in a CHCl3–EtOH system (9 : 1 → 8 : 2) and with
silica gel (1.3×15 cm) in a CHCl3–EtOH system (7.5 : 2.5 → 7 : 3).
The fractions obtained (40.2 mg) that contained sulfated
glycosides, according to TLC data, Rf = 0.8—0.7 in a butanol—
ethyl acetate—water system (5 : 1 : 1), were purified by HPLC
on a column with Diasphereꢀ110ꢀC18 (elution with 50% aqueous
methanol) and reꢀchromatographed on a ZorbaxꢀBonus column
(4×250 mm, 5 μm, 12 nm), elution with 65% methanol. More
polar fraction II was chromatographed under similar conditions
and reꢀchromatographed on a Diasphereꢀ110ꢀC18 column (5 μm,
4×250 mm) to give 29 mg of sulfate 5a (5.8% of the weight of the
overall fraction II), 18 mg (3.6%) of sulfate 6, and 5.6 mg (1.12%)
of marthasterone sulfate 7. The results of chromatographic
isolation of compounds 1—7 are summarized in Table 2.
Desulfation of evasterioside A (1a). Glycoside 1a (4.5 mg)
was dissolved in a dioxane—pyridine mixture (1 : 1) (2 mL) and
heated for 1 h at 100°C. The product was concentrated in vacuo
to dryness and chromatographed on a column with Florisil
(1×5 cm), elution with a chloroform—methanol system (8 : 2)
to give 3.2 mg of compound 1b.
Hydrolysis of glycosides 1a, 2a and identification of monoꢀ
saccharides. Glycosides 1a and 2a (2 mg each) were dissolved in
2 M HCl (2 mL) and the solutions were heated for 2 h on a
boiling water bath. Xylose was identified in hydrolyzates by GLC

2436
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 11, November, 2008
Levina et al.
(aldononitrile peracetates) and by paper chromatography (butanol—
pyridine—water system, 10 : 3 : 3, Whatman No. 1). The absolute
Dꢀconfiguration of the monosaccharide was determined from
the following optical rotation values: [α]D +30 (c 0.38, H2O)
(for glycoside 1a) and +21 (c 0.12, H2O) (for glycoside 2a). For
This work was supported by the Russian Foundation
for Basic Research (project no. 05ꢀ04ꢀ48246), Council on
grants at President of the Russian Federation (Program
for the State Support of Leading Scientific Schools of
the Russian Federation, Project NSh 2813.2008.4), and
the Russian Academy of Sciences (Program of the Preꢀ
sidium of the RAS “Molecular and Cell Biology”).
20
20
20
Dꢀxylose, [α]D +35 (c 0.68, H2O); [α]
+92 → +19.0 (H2O)
D
20
(Ref. 14); for Lꢀxylose, [α]D –18.6 (c 9.94) (Ref. 14).
Evasterioside A (1a), C33H55O12SNa, amorphous compound,
[α]D20 –9 (c 0.22 MeOH). MALDIꢀTOF MS, m/z (Irel (%)): 721
[MNa + Na]+ (100), 737 [MNa + K]+ (30), 588 [(MNa — C5H9O4]–
(25). The 1H and 13C NMR (MeOH) spectra are summarized in
Table 1 and discussed in the text.
References
1. L. Minale, R. Riccio, F. Zollo, in Progress in the Chemistry of
Organic Natural Products, Eds H. Herz, H. G. Kirby, R. Moore,
W. Steglich, Ch. Tamm, SpringerꢀVerlag, NewꢀYork, 1993,
2, 75—308.
2. V. A. Stonik, Usp. Khim., 2001, 70, 763 [Russ. Chem. Rev.,
2001, 70, 673].
3. E. V. Levina, A. I. Kalinovskii, V. A. Stonik, P. S. Dmitrenok,
Izv. Akad. Nauk. Ser. Khim., 2003, 1539 [Russ. Chem. Bull.,
Int. Ed., 2003, 52, 1623].
4. S. De. Marino, M. Iorizzi, E. Palagiano, F. Zollo, C. Roussakis,
J. Nat. Prod., 1998, 61, 1319.
5. L. C. Wright, A. G. Shimizu, S. Smith, J. A. Walter, D. Idler,
W. Khalil, Can. J. Chem., 1978, 56, 1898.
6. M. Iorizzi, P. Bryan, J. McClintock, L. Minale, E. Palagiano,
S. Maurelli, R. Riccio, F. Zollo, J. Nat. Prod., 1995, 58, 653.
7. E. Finamore, F. Zollo, L. Minale, T. Yasumoto, J. Nat.
Prod., 1992, 55, 767.
8. R. Riccio, M. Iorizzi, L. Minale, Y. Oshima, T. Yasumoto,
J. Chem. Soc., Perkin Trans. 1, 1988, 1337.
9. R. Riccio, M. Iorizzi, L. Minale, Bull. Soc. Chim. Belg.,
1986, 95, 869.
10. W. K. Wilson, R. M. Sumper, J. J. Warren, P. S. Rogers,
B. Ruan, G. J. Schroepfer, Lipid Res., 1996, 37, 1529.
11. T. N. Makarieva, V. A. Stonik, I. I. Kapustina, V. M.
Boguslavsky, A. S. Dmitrenok, V. I. Kalinin, M. L. Cordeiro,
C. Djerassi, Steroids, 1993, 58, 508.
12. E. V. Levina, A. I. Kalinovskii, P. S. Dmitrenok, Bioorgan.
khimiya, 2007, 33, 365 [J. Bioorg. Chem., 2007, 33 (Engl.
Transl.)].
13. E. V. Levina, P. V. Andriyashchenko, A. I. Kalinovskii, V. A.
Stonik, P. S. Dmitrenok, N. G. Prokof´eva, Izv. Akad. Nauk.
Ser. Khim., 2002, 497 [Russ. Chem. Bull., Int. Ed., 2002,
51, 535].
14. F. Micheel, A. Klemer, Chemie der Zucker und Polysacchaꢀ
ride, Academische verllagsgesellschaft Geest, Leipzig, 1956.
Desulfated evasterioside A (1b), amorphous compound, was
20
prepared by solvolytic desulfation of 1a; [α]D –37.5 (c 0.16
MeOH). MALDIꢀTOF MS, m/z (Irel (%)): 619 [M + Na]+(100).
1
Some data from the H NMR and 13C NMR spectra (CD3OD)
are given in the Results and Discussion section.
Evasterioside B (2a), C32H53O12SNa, amorphous compound,
[α] 20 –36.3 (c 0.11 MeOH). MALDIꢀTOF MS, m/z (Irel (%)): 661
D
[M – Na]– (70), 707 [MNa + Na]+, 587 [(MNa + Na) – NaHSO4]+.
The 1H and 13C NMR spectra (MeOH) are summarized in
Table 1 and discussed in the text.
Aphelasterioside A, sodium (20R)ꢀ24ꢀOꢀ(βꢀDꢀxylopyranosyl)ꢀ
20
5αꢀcholestaneꢀ3β,6β,8,15αꢀpentol 3ꢀsulfate (3), [α]D –6.1 (c
0.16 MeOH). MALDIꢀTOF MS, m/z (Irel (%)): 663 [M – Na]–
(70), 709 [MNa + Na]+ (100). The compound was identified
by comparing the spectral characteristics and physical paramꢀ
eters with those described for the known compound from the
Aphelasterias japonica starfish.7
Coscinasterioside B, sodium (20R,22E,24R,25S)ꢀ24ꢀOꢀ
(βꢀDꢀxylopyranosyl)ꢀ5αꢀcholestaneꢀ3β,6β,8,15αꢀpentol 15ꢀ
20
sulfate (4), [α]D + 7.5 (c 0.2 MeOH). MALDIꢀTOF MS, m/z
(Irel (%)): 709 [MNa + Na]+ (100). The compound was identified
by comparing the spectral characteristics and physical parameters
with those described for the known compound from the starfish
Coscinasterias tenuispina.9
Sodium (20R)ꢀcholestꢀ7ꢀenꢀ3βꢀyl sulfate (5a), C27H47SO3Na,
[α]D20 +40.6 (c 0.59 MeOH); m.p. 110—113 °C (from methanol).
MALDIꢀTOF MS, m/z (Irel (%)): 488 [M(SO3Na)]– (100), m/z
465 [M(SO3–)] (50). 1H NMR, (CD3OD), δ: 0.94 (d, 3 H,
C(21)H3, J = 5.5 Hz); 0.56 (s, 3 H, C(18)H3); 0.82 (s, 3 H,
C(19)H3); 0.87 (d, 6 H, C(26)H3, C(27)H3, J = 7.0 Hz); 4.24
(m, 1 H, C(3)H; 5.17 (m, 1 H, C(7)H). 13C NMR (CD3OD),
δ: 21.3 C(18); 13.4 C(19); 19.4 C(21); 22.9 C(26), 23.0 C(27);
118.5 C(7), 140.6 C(8).
The product was identified by comparing the spectral
characteristics of the desulfation product of 5b with the paramꢀ
eters of the known compound.11
Sodium (20R)ꢀcholestꢀ5ꢀenꢀ3βꢀyl sulfate (6), C27H47SO3Na,
identified by comparing the spectral characteristics with
those described for the known compound from the starfish
P. obscurus.12
Sodium (20R)ꢀ6αꢀhydroxyꢀ23ꢀoxocholestaꢀ9(11),24ꢀdienꢀ
3βꢀyl sulfate) (marthasterone sulfate) (7) was identified by
comparing the physical parameters and NMR spectra with the
characteristics of the known compound from the Lysastrosoma
anthosticta starfish.13
Received August 2, 2007;
in revised form December 25,2007