Steroid compounds from two pacific starfish of the genus Evasterias

Article abstract of DOI:10.1134/S1068162009010166

Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retiferacollected from the Sea of Japan was identified as (20R, 22E)-3-O-(β-D

Full text of DOI:10.1134/S1068162009010166

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 1, pp. 123–130. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © E.V. Levina, A.I. Kalinovsky, P.V. Dmitrenok, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 1, pp. 134–141.
Steroid Compounds from Two Pacific Starfish
of the Genus Evasterias
E. V. Levina1, A. I. Kalinovsky, and P. V. Dmitrenok
Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences,
pr. Stoletiya Vladivostoka 159, Vladivostok, 690022Russia
Received March 14, 2008; in final form, March 28, 2008
Abstract—Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were
isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retifera collected from the
Sea of Japan was identified as (20R,22E)-3-O-(β-D-xylopyranosyl)-24-nor-5α-cholest-22-ene-
3β,6β,8,15α,26-pentaol 26-sulfate sodium salt. The structures of evasteriosides D and E from E. echinosoma
(collected from the Gulf of Shelichov, the Sea of Okhotsk) were established as (20R,24S)-24-O-(β-D-glucopy-
ranosyl)-5α-cholestane-3β,6α,8,15β,24-pentaol and (20R,24S)-3,24-di-O-(β-D-xylopyranosyl)-cholest-4-ene-
3β,6β,8,15α,24-pentaol, respectively. In addition, the known compounds pycnopodiosides A and C, luridoside
A, 5α-cholestane-3β,6α,8,15β,16β,26-hexaol. 5α-Cholestane-3β,6α,8,15β,24-pentaol 24-sulfate sodium salt
and marthasterone sulfate sodium salt were identified in E. echinosoma. The structures of the isolated com-
pounds were established on the basis of spectroscopic analyses, using 1D and 2D NMR techniques, mass spec-
trometry, and some chemical transformations.
Key words: Evasterias retifera, E. echinosoma, polyhydroxysteroids, starfish, steroid glycosides, FAB and
MALDI TOF mass spectra, NMR spectra
DOI: 10.1134/S1068162009010166
INTRODUCTION
RESULTS AND DISCUSSION
We have recently described [1] isolation of two new
steroid glycosides, evasteriosides A and B, from star-
fish Evasterias retifera Verrill Djakonov 1938 (order
Forcipulata, family Asteriidae).2 We continued the
studies of starfish metabolites [2, 3] from extracts of
two species of starfish from the genus Evasterias and
isolated three new glycosides: evasterioside C (I) from
E. retifera and evasteriosides D (II) and E (III) from
E. echinosoma along with six earlier described polar
steroids (IV)–(IX).
New steroid compounds (I)–(III) together with the
earlier known (IV)–(VIII) (scheme) and martasterone
sulfate, (20R)-6α-hydroxy-23-oxocholesta-9(11),24-
dien-3β-yl sodium sulfate (IX), were isolated from eth-
anolic extracts of starfish E. retifera and E. echinosoma
by a column chromatography on Polychrome, Florisil,
and silica gel with the subsequent purification by HPLC
on columns with reversed-phase sorbents Diasphere-
110-ë18 and Zorbax Bonus-RP by the techniques
described earlier [3]. Structural identification of the iso-
lated compounds and their derivatives was carried out
by spectral methods (NMR, FAB and MALDI TOF
MS) and comparison of their physical constants with
those described in literature.
All nine isolated compounds are typical secondary
metabolites of starfish, polyhydroxysteroids and their
glycosylated and sulfated derivatives.
1
Corresponding author; fax: (423-2) 31-4050; e-mail: levina@piboc.dvo.ru.
Abbreviations: MALDI TOF MS, matrix-assisted laser desorption/ionization mass spectrometry; HMBC, heteronuclear CH correlation
through multiple bonds; HSQC, heteronuclear single quantum coherence of the CH interaction through one bond; DEPT, Distortionless
2
Enhancement by Polarization Transfer, an experiment that allows one to differentiate CH, CH , and CH fragments according to the pos-
2
3
itive or negative polarity of signal; COSY, correlation spectroscopy, 2D correlations of shift through spin–spin coupling; TOCSY, total
correlation spectroscopy that reveals all the proton couplings of the structure fragments in addition to those detected by COSY.
123

124
LEVINA et al.
R1
R1
27
OY
21
18
22
20
17
OSO3Na
25
26
23
12
11
OH
16
19
13
14
OH
1
4
9
6
R2
(II), (IIa), (IIIb)
HOH C
15
2
H
R2
10
5
7
H
XO
(I), (Ia)
3
H
R1
XO
H
OH
(I) R1 = H, R2 = αOH, X =
2
O
O
OH
(II) R1 = αOH, R2 = βOH, X = H, Y =
OH
OH
OH
OH
OH
OH
O
CH2OSO3Na
(Ia) R1 = CH3, R2 = αOH, X =
O
(IIa) R1 = αOH, R2 = βOH, X = H, Y =
OH
OH
OH
OH
OH
O
(IIIb) R1 = βOH, R2 = αOH, X = SO3Na, Y =
OH
OH
OY
OX
OH
OH
OH
OH
OH
OH
R2
OH
OH
H
H
H
XO
HO
HO
H
H
OH
(III), (IIIa)
R1
(IV)-(VI), (VIII)
OH
(VII)
O
(IV) R1 = αOH, R2 = βOH, X =
OH
O
OH
(III) X = Y =
OH
OH
CH2OSO3Na
OH
OH
O
O
(V) R1 = αOH, R2 = βOH, X =
(VI) R1 = αOH, R2 = βOH, X =
OH
OH
(IIIa) Y = SO3Na, X =
OH
OH
OH
OH
O
OH
NaO3SO
(VIII) R1 = αOH, R2 = βOH, X = SO3Na
OH
Scheme. Steroid compounds from starfish of genus Evasterias.
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STEROID COMPOUNDS FROM TWO PACIFIC STARFISH
125
Mass spectra MALDI TOF and LSI (anion registra- (δ 0.97 ppm) characteristic of steroids with such a con-
tion) of evasterioside C (I) from E. retifera displayed figuration.
peak of [M – Na]– ion at m/z 647. At spectra also there
An analysis of spectral data corresponding to the
were the fragmentation peaks at m/z 515 [M – Na –
steroid side chain and a comparison with the corre-
Xyl]– and 497 [M – Na – Xyl–H2O]–. In (+)-HR LSI
sponding data for the known (20R,22E)-24-nor-5α-
mass spectrum of (I), the peak of a pseudo-molecular
cholest-22-ene-3β,4β,6α,8,15β,26-hexaol (Ib) from
ion was observed at m/z 693.2863 [M + Na]+. These
Acodontaster conspicuus [7] showed that the signals of
side chains of (I) and (Ib) are close, except for the sig-
data together with the data of NMR spectra corre-
sponded to the molecular formula ë31H51O9SO3Na and
proved the presence of sulfate group and pentose as a
carbohydrate component of compound (I). In fact, an
nals of hydroxymethylene groups. Chemical shifts
ë26-H2 in NMR spectra of (Ib) are δH 3.43 and 3.30,
and δC 68.3 ppm [7] (whereas in spectra of (I), they are
downfield displaced to δH 3.88 and 3.74, and
δC 73.7 ppm), which corresponds to the shift of sulfa-
tion [8] and allows to assume the presence of sulfate
group at C26. The structure of the side chain of evaste-
rioside C was finally confirmed by cross peaks of H26'
(δH 3.74 ppm) and H26 (δH 3.88 ppm)/H25
(δH 2.43 ppm), and also H25 (δH 2.43 ppm)/27-H3
(δH 1.02 ppm) and H25/H23 (δH 5.31 ppm) in COSY
spectra.
13
analysis of a C NMR spectrum and DEPT specified
(Table 1) the presence in (I) of 31 carbon atom, includ-
ing 4 methyl, 9 methylene, and 15 methine groups,
including 2 olefin carbon atoms, and also three quater-
nary carbon atoms, including one connected with
oxygen.
In (−)-LSI mass spectrum, a more intensive peak at
m/z 233 than in other spectra was observed. It is charac-
teristic of steroid polyols containing no hydroxy groups
at C4 and C7 [4]. The presence in NMR spectra of (I)
of the signal of anomer carbon atom at (δ 103.2 ppm)
and a doublet of anomer proton (δç 4.35 ppm, J 7.5 Hz)
proves the presence of one monosaccharide residue in
which H1 and H2 occupy trans position. Values of
chemical shifts of C1−C21, C1'−C5', and also H3, H6,
At all, the spectra COSY, HMBC, and HSQC
allowed the assignment of signals of all atoms C and H
in NMR spectra. In the formula (I), the lines indicating
some fragments of molecule in which the sequences of
protons were established by COSY experiments. In
1
particular, in the spectrum H–1H-COSY, the presence
of spin coupling between protons çë3–ç2ë4–çë5–
çë6–ç2ë7 and çë14–çë15–ç2ë16–çë17 was
shown. On the basis of all the data, the structure of
sodium salt 26-sulfate of (20R,22E)-3-O-(β-D-xylopy-
ranosyl)-24-nor-5α-cholest-22-ene-3β,6β,8,15α,26-pen-
taol (I) was assigned to evasterioside C.
13
H15, 3 × H18, 3 × H19, 3 × H21, and ç2' –ç25' in ë-
spectra and a 1H NMR of glycoside (I) (Table 1) just as
spin coupling constants of the corresponding protons
practically coincided with the corresponding values in
the spectra of asteriidoside L (Ia) from a starfish of
family Asteriidae [5]. On this basis, a presumption was
made that glycoside (I) contains a similar 3β,6β,8,15α-
tetrahydroxycholestane nucleus with β-D-xylopyra-
nose residue at the C3 atom. In fact, an acidic hydroly-
sis of glycoside (I) led to D-xylose identified by TLC
and GLC methods (aldononitrile peracetates) and a
Marine 24-norsterols were found in phytoplankton
[9], therefore, the identification of polar steroids similar
to compound (I) with truncated oxidized side chains
proves the opportunity of oxidation and glycosidation
of these sterols at their getting up through a food chain
in organisms of starfish.
comparison of value of [α]2D0 with standard compound.
The (+)HR-LSI mass spectrum of isolated from the
Sea of Okhotsk starfish E. echinosoma evasterioside D
(II) exhibits a peak of pseudo-molecular ion at m/z
637.3887 [M + Na]+, which, in combination with the
data of 13ë and 1H NMR points out to the molecular for-
mula ë33H58O10. For example, spectra of 13C NMR and
DEPT (Table 1) indicate in the molecule (II) the pres-
ence of signals of 33 carbon atoms, including 5 methyl,
10 methylene, 15 methine groups, 2 quaternary carbon
atoms, and one such an atom connected to oxygen. The
chemical shift of anomeric proton δç 4.29 ppm (J 7.7 Hz),
anomeric carbon atom at δë 104.3 ppm, the atoms of
carbon connected to oxygen at δë 63.1, 67.7, 71.1, 72.0,
72.2, 75.6, 77.5, 77.7, 78.2, and 86.2 ppm proved the
presence in the molecule (II) of hexoside fragment with
trans arrangement of H1 and H2 and a pentahydroxy
substituted steroid aglycone with cholestane skeleton.
The bond of the monosaccharide residue to atom C3
was confirmed by correlation of anomeric proton
H1'/C3 in HMBC spectrum (Table 1). The arrangement
of hydroxy groups was also confirmed by HMBC- and
COSY-correlations.
The following signals of the side chain are present
in the NMR spectra of (I): two methyl doublets at δ 1.02
(J 6.8) and 0.97 (J 6.8) ppm (δë 20.9 and 17.1 ppm) and
signals of olefin protons at δç 5.31 and 5.27 ppm
(δë 130.5 and 138.2 ppm). The value of chemical shift
of C20 atom (δC 40.6 ppm) and spin coupling constant
J22, 23 of 15.3 Hz corresponded to a E-configuration of
double bond (for Z-isomer, the corresponding signal of
C20 is displayed at δC ~ 34.0 ppm [6]). These data and
the signals of hydroxymethylene protons at δç 3.88 (J
6.0 and 9.4 Hz) and 3.74 (J 7.4 and 9.4) ppm indicated
the ∆22E-24-nor-26-hydroxycholestane side chain. The
An analysis of 13ë and 1H NMR spectra of glycoside
R-configuration has been attributed to the chiral center (II) and their comparison with the spectra of pycnopo-
C20 on the basis of chemical shift of three H21 protons dioside C (IIa) from starfish Pycnopodia helianthoides
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

126
LEVINA et al.
Table 1. The data of NMR spectra of evasteriosides C (I) and D (II) (CD3OD, DRX-500)1
(I)
(II)
Atom
num-
@δC,
δC, mult.2
δH (J, Hz)
HMBC
δH (J, Hz)
HMBC
ber
ÏÛθÚ.2
39.4, CH
1
2
3
4
5
6
7
41.4, CH2 1.72, m; 0.97, m
30.0, CH2 1.61, m; 1.82, m
80.0, CH 3.69, m
0.97, m; 1.71, m
31.5, CH2 1.47, m; 1.73, m
72.2, CH 3.47, m
32.3, CH2 1.20, m; 2.18, m
32.9, CH2 1.74, m; 1.82, m
48.8, CH 1.20, m
53.8, CH
67.7, CH
1.03, m
74.2, CH 3.86, m
C8, C10
C5, C6, C8, C9
3.69, dt (10.8; 4.1)
C8, C10
C5, C6, C8, C9
45.4, CH2 1.57, dd (3.0; 14.5)
2.36, dd (3.0; 14.7)
49.4, CH2 1.27, m; 2.37, dd (4.2; 15.6)
8
9
77.2, C
77.5, C
57.1, CH 0.96, m
57.4, CH
38.0, C
0.83, m
10
36.7, C
11
12
13
14
15
16
17
19.8, CH2 1.53, m; 1.83, m
42.7, CH2 1.25, m; 1.94, m
45.4, C
19.8, CH2 1.49, m; 1.81, m
43.4, CH2 1.16, m; 1.96, m
44.3, C
66.7, CH 1.20, d (9.5)
69.9, CH 4.24, dt (3.0; 9.3)
42.0, CH2 1.56, m; 1.87, m
55.6, CH 1.36, m
C13, C15, C18
62.6, CH
71.1, CH
1.00, m
C13, C18
4.40, Û¯.Ú@ (5.7)
42.4, CH2 1.39, m; 2.38, m
57.9, CH 0.99, m
C12, C13, C17,
C14
18
15.5, CH3 0.97, s
C12, C13, C14, C17 16.5, CH3 1.26, s
19
20
21
22
23
24
25
26
15.8, CH3 1.16, s
C1, C5, C9, C10
0.98, s
C1, C5, C9, C10
14.1, CH3
36.3, CH
40.6, CH 1.98, m
1.53, m
20.9, CH3 0.97, d (6.8)
138.2, CH 5.27, dd (15.4; 6.7)
130.5, CH 5.31, dd (15.3; 8.0)
C17, C20, C22
C20, C24
19.0, CH3 0.92, d (0.7)
C17, C20, C22
32.6, CH2 1.02, m; 1.61, m
28.6, CH2 1.42, m; 1.60, m
C20, C24, C27
86.2, CH
31.8, CH
3.43, m
1.87, m
37.7, CH 2.43, m
C22, C23, C26
C23, C24, C27
73.7, CH2 3.74, dd (7.4; 9.4)
3.88, dd (6.0; 9.4)
18.4, CH3 0.94, d (7.0)
C24, C25, C27
27
1'
2'
3'
4'
5'
17.5, CH3 1.02, d (6.8)
103.2, CH 4.35, d (7.5)
75.0, CH 3.14, dd (7.6; 9.0)
77.9, CH 3.303, m
C24, C26, C23
18.2, CH3 0.92, d (7.0)
C24, C25, C26
C24
C3
104.3, CH
75.6, CH
78.2, CH
72.0, CH
77.7, CH
4.29, d (7.7)
3.16, t (7.9)
3.333, m
C1'
71.3, CH 3.47, m
3.26, t (8.7)
3.23, m
66.9, CH 3.18, dd (10.5; 11.3)
3.82, dd (5.3; 11.6)
C4', C3', C1'
6'
63.1, CH2 3.84, dd (2.3; 11.7)
3.67, t (5.2; 11.7)
1
1
Notes: 1. Signal assignment is made with application of 2D NMR spectroscopy H- H-COSY, HMBC, and HSQC.
2. Multiplicity of signals was determined by DEPT technique.
3. Signal was overlaid with a signal of solvent.
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STEROID COMPOUNDS FROM TWO PACIFIC STARFISH
127
[10] showed that they practically coincide, except for showed that the spectra practically coincide, except for
some signals of the monosaccharide residue. At the some signals of a side chain of glycoside (III).
acidic hydrolysis of product (II), D-glucose was identi-
To confirm the structure of the side chain of (III),
which we named as evasterioside E, we compared the
spectral data with earlier known aphelasterioside A
(IIIb) from Aphelasterias japonica [12]. Full concur-
rence of chemical shifts of all carbon atoms and pro-
tons, together with the corresponding spin-coupling
constants confirmed the identity of their side chains and
localization in position C24 of the second xylose resi-
due.
fied by TLC and GLC methods (aldononitrile perace-
tates) and by a comparison of the value of [α]2D0 with
standard sample. For example, protons 6'–ç2 in the
NMR spectrum of (IIa) containing 6-O-sulfated β-D-
glucopyranose residue resonated under the influence of
sulfate group in a low field at δH 4.18 ppm (dd, J 5 and
12 Hz) and 4.34 (dd, J 2 and 12 Hz) ppm. The signals
of C6 atom have a chemical shift at δC 68.5 ppm [10].
For evasterioside D, these signals are shifted upfield to
δH 3.67 and 3.84 ppm, and δC to 63.1 ppm. Taking into
account the differences in chemical shifts of these
atoms and the data [10] published earlier, we assumed
that evasterioside D (II) differs from pycnopodioside C
only by the absence of sulfate group at C6' in monosac-
charide residue.
The (20R,24S)-configurations for chiral centers of
the side chain were accepted by analogy to other (24S)-
hydroxyxylosides from starfish [12].
The HMBC-correlations H1'/C3 and H1''/C24
(Table 2) confirmed that evasterioside E (III) contains
two β-D-xylopyranose residues located at C3 and C24.
The methods of bidimentional spectroscopy with the
help of HMBC, 1H–1H-COSY, 1D-TOCSY, and HSQC
spectra allowed us to establish finally for evasterioside
E the structure of (20R,24S)-3,24-di-O-(β-D-xylopyra-
nosyl)-cholest-4-ene-3β,6β,8,15α,24-pentaol (III).
Such a structure of glycoside (II) was confirmed by
the methods of bidimentional spectroscopy with the
1
help of HMBC, H–1H-COSY and HSQC spectra,
which allowed us to assign all the carbon and hydrogen
atoms in NMR spectra (Table 1). A spin-coupling
between the protons of carbohydrate chain H1'–H2'–
H3'–H4'–H5'–2 × H6' is observed in the spectrum
COSY-90 of (II) and the HMBC-correlation H1'/C24
confirms that β-D-glucopyranose residue is attached to
C24 of aglycone (see Table 1). The R-configuration of
chiral center C20 was attributed on the basis of size of
chemical shift of protons 21-ç3 (ëD3OD, δ 0.92 ppm),
and, for chiral center C24, the S-configuration by anal-
ogy to compound (IIa) is accepted. Thus, the new ste-
roid glycoside (II) has the structure (20R,24S)–24-O-(β-
D-glucopyranosyl)-5α-cholestane-3β,6α,8,15β,4-pen-
taol. In the formula (II), the bonds specifying the key
fragments of molecule in which sequences of protons
are established by COSY experiments are marked.
The identification of the before known polyoxys-
teroid (IV) was executed on the basis of coincidence of
its spectral characteristics with the corresponding data
for the earlier known pycnopodioside A [10]. Glyco-
side (V) was identified similarly as pycnopodioside C
[10], and glycoside (VI) differed from (IV) by the pres-
ence of sulfate group at 4' in xylose residue and was
also identified as earlier known luridoside A from star-
fish Cosmasterias lurida [13]. Compounds (VII)–(IX)
were
identified
as
(25S)-5α-cholestane-
3β,6α,8,15β,16β,26-hexaol
(VII), 5α-cholestane-
3β,6α,8,15β,24-pentaol 24-sulfate (VIII), and marthas-
terone sulfate (20R)-6α-hydroxy-23-oxocholesta-
9(11),24-dien-3β-yl sodium sulfate (IX) by comparison
of their 1H and 13C NMR spectra with the correspond-
ing data for these compounds we earlier isolated [14,
15], and also by a direct comparison of their chromato-
graphic behavior with available samples (TLC, HPLC).
The peak of a pseudo-molecular ion at m/z 737 [M +
Na]+ and also the fragmentation peaks corresponding to
the loss of pentoside fragment and a water molecule
were seen in MALDI TOF and HR-LSI mass spectra
(registration of cations) of steroid (III) (see the Exper-
imental section). These data correspond to molecular
formula ë37H62O13. The presence in the NMR spectra of
signals of two anomeric carbon atoms (δ 104.9 and
104.3 ppm) and two doublets of anomeric protons with
spin-coupling constants J of 7.6 and 7.5 Hz indicate the
existence of two monosaccharide residues with trans
arrangement of H1 and H2. At acid hydrolysis of evas-
terioside E, only one monosaccharide, D-xylose, was
obtained; it was identified by TLC and GLC methods
(aldononitrile peracetates) and by a comparison of
Thus, we isolated from two species of starfish of the
same genus earlier unknown glycosides named evaste-
riosides C, D, and E, which differ from each other by
both monosaccharide residues, and their localization.
They have an identical arrangement, but different stere-
ochemistry of hydroxyl functions in the steroid
nucleus. Evasteriosides C and D are monoglycosylated
polyhydroxysteroids, and evasterioside E belongs to
the group of 3,24-diglycosylated steroid polyols.
EXPERIMENTAL
1
13
Spectra ç and C NMR were registered on spec-
trometers Bruker DPX-300 and DRX-500 with work-
ing frequencies of 300 and 500 MHz for protons, an
value [α]2D0 with standard compound. An analysis of
1
13ë and H NMR of glycoside (III) and a comparison internal standard was ëD3OD/δH 3.30 ppm, δë 49.0 ppm.
with the corresponding spectral data for known pisas- Specific optical rotation was measured on a Perkin-
teroside D (IIIa) from starfish Pisaster giganteus [11] Elmer 343 polarimeter; MALDI TOF MS, on a Bruker
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

128
LEVINA et al.
Table 2. The data of NMR spectra of evasterioside E (III) (CD3OD, DRX-500)1
Atom
Atom
δC, mult.1
δH (J, Hz)
HMBC
δC, mult.1
δH (J, Hz)
0.89, d (6.6)
HMBC
number
number
1
2
39.7, CH2 1.79, m
21
22
19.0, CH3
32.8, CH2
C17, C20, C22
27.7, CH2 1.76, m; 1.96, m
1.58, m; 0.97, m
1.58, m; 1.35, m
3.34, m
3
4
5
6
7
77.1, CH 4.19, m
127.0, CH 5.67, brs
148.3, C
23
24
25
26
27
28.6, CH2
86.2, CH
31.9, CH
18.22, CH3 0.93, d (6.5)
18.32, CH3 0.91, d (6.5)
C2, C6, C10
C8, C10
1.87, m
76.4, CH 4.30, brt (3.0)
C24, C25, C27
C24, C25, C26
44.5, CH2 2.52, dd (3.0; 15.0); C6, C9
1.51, m
8
9
76.5, C
1' 104.3, CH
4.36, d (7.5)
3.13, dd (7.8; 9.0)
3.303
C3
57.8, CH 1.03, dd (2.5; 13.0)
37.7, C
2'
3'
4'
75.0, CH
78.0, CH
71.22, CH
10
11
19.7, CH2 1.84, m; 1.50, m
3.46, m
3.82, dd (5.0; 11.3)
3.19, t (11.0)
12
13
42.8, CH2 1.97, m; 1.22, m
5'
66.9, CH2
45.4, C
1'' 104.9, CH
4.22, d (7.6)
C24
14
15
16
17
66.5, CH 1.15, m
2''
3''
4''
5''
75.4, CH
78.0, CH
71.42, CH
66.8, CH2
3.14, dd (7.4; 9.1)
3.27, t (8.8)
3.46, m
70.1, CH 4.28, td (9.6; 3.2)
41.6, CH2 2.00, m; 1.70, m
55.9, CH 1.31, m
3.81, dd (5.3; 11.3)
3.14, t (11.0)
18
15.3, CH3 0.96, s
C12, C13, C14,
C17
19
20
22.6, CH3 1.35, s
36.2, CH 1.34, m
C1, C5, C9, C10
1
1
Notes: 1. Signal assignment is made with application of 2D NMR spectroscopy H- H-COSY, HMBC, and HSQC.
2. Equivocal assignment of signals.
3. Signal was overlaid with a signal of solvent.
Biflex III spectrometer (Germany) with laser ioniza- column chromatography was carried out on a silica
gel L (80–100 and 200–250 mesh, Chemapol, Cze-
chia), Polychrome (Russia), and Florisil (200–
250 mesh, Merck, Germany).
Animals. Samples of starfish E. retifera were col-
lected in August, 2003 by divers at Gamov cape, Sea of
Japan, from the depth of 60–150 m and were identified
by Professor V.S. Levin (Pacific Institute of Bioorganic
Chemistry, Pacific Division, Russian Academy of Sci-
tion/desorption (the N2-laser at 337 nm). A sample was
dissolved in methanol (10 mg/ml) and aliquot of 1 µl
was analyzed, using cyanohydroxycinnamic acid as a
matrix. LSI (–) and (+)-mass spectra of the high resolu-
tion were obtained on an Intectra AMD-604S mass
spectrometer (Germany) at the energy of cesium ions of
8 keV with glycerol as a matrix. The content of sodium
was determined) on an atomic adsorption Nippon Jarrel
Ash AA-780 spectrophotometer. HPLC was carried out ences). Starfish E. echinosoma were collected by expe-
dition of TINRO CENTER in August, 2004 during sail-
ing of NIS Professor Kaganovsky (Shelikhov bay, Sea
of Okhotsk) by a trawl from the depth of 100–200 m.
The collector of the material is N.V. Bekova, samples
were identified by Professor A.V. Smirnov (Zoological
Institute, Russian Academy of Sciences).
on a Du Pont Model 8800 chromatograph using a
refractometric detector and columns packed with Dias-
phere-110-C18 (5 µm, 4 × 250 mm) and Zorbax Bonus-
RP (4.6 × 250 mm, 5 µm) in systems of 45 and 55%
methanol–water (V = 1 ml/min), respectively. Melting
points were determined on a Leica VMTG microtable.
TLC was carried out on Sorbfil plates with a silica gel
CTX-1A layer fixed on a foil (5–17 µm, Russia, Krasn-
Extraction and isolation of total fractions.
Crushed starfish E. retifera were exhaustively extracted
odar) in 5 : 1 : 1 BuOH–EtOAc–H2O system. Preparative with 95% ethanol at room temperature. The combined
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

STEROID COMPOUNDS FROM TWO PACIFIC STARFISH
129
hydrolyzate of (II): [α]2D0 + 45 (Ò 0.10, H2O). Lit. [16]:
D-glucose [α]2D0 +111.2 to +52.5 (H2O).
alcohol extract was evaporated in a vacuum up to a gum
residue (0.826 kg), dissolved in water, and three times
extracted with butanol. The combined butanol extracts
were evaporated to a syrup and chromatographed on a
silica gel (50–100 µm) column (6 × 25 cm) in chloro-
Evasterioside C (I), amorphous, [α]2D0 – 1 (Ò 0.1,
form–ethanol system (100 : 0
45 : 55). The fraction
MeOH). In (+)-HR LSI-MS of (I), peak of pseudo-
molecular ion at m/z 693.2863 [M + Na]+ (calc. for
ë31H51O9SO3Na, 693.2897); MS (+)-LSI: peak at m/z
233 [calc. for ë15H21O2, splitting of C8–C14, C13–C17]
[4]. 1H NMR spectra and 13C NMR spectra (MeOH) are
given in Table 1 and in the text.
of steroid glycosides was obtained (2.3 g); it was eluted
with 20 vol % ethanol in chloroform. The fraction was
dissolved in water and was passed through a column
with Polychrome (3 × 12 cm). The column was washed
with water and 50% water ethanol. The water ethanol
eluate was concentrated in a vacuum and chromato-
graphed on a column with Florisil (200 mesh, 1.5 ×
19 cm) in system chloroform–methanol (100 : 0
85 : 15). We obtained an enriched fraction (25 mg) con-
taining glycoside (I). After the extraction of starfish
E. echinosoma, the dry ethanolic extract (49 g) was sep-
arated on a column with Polychrome at eluted with 40,
50 and 100% ethanol–water; fractions 1 (0.5 g),
2 (0.615 g), and 3 (1.3 g) were obtained. From apolar
fraction 1, chromatography on a silica gel column (50–
100 µm) in a gradient chloroform–ethanol (100 : 0 to
80 : 20) gave 15 mg of the fraction containing com-
pound (IX). A successive chromatography on silica gel
columns eluted with a gradient chloroform–methanol
(95 : 5 to 40 : 60), and then on a Florisil column (200–
300 mesh, 2 × 50 cm) eluted with chloroform–methanol
(95 : 5 to 65 : 35) gave from a more polar fraction 2 two
fractions of steroid compounds: A (II)–(V) and B (VI)–
(VIII).
Evasterioside D (II), amorphous, [α]2D0 + 18.6
(Ò 0.16, MeOH); (+) HR-LSI: m/z 637.3887 [M + Na]+
(calc. for ë33H58O10 637.3927), m/z 653 [M + K]+. (−)-
1
13
LSI MS: m/z 613 [M – H]–; H and C NMR spectra
(MeOH) are given in Table 1 and in the text.
Evasterioside E (III), amorphous, [α]2D0 – 2.7
(Ò 0.26, MeOH); (+) HR-LSI-MS, m/z: 737.4035 [M +
Na]+ (calc. for ë37H62O13 737.4088), 753 [M + K]+, 605
[(M + Na)–Xyl]+ and 587 [(M + Na) – Xyl – H2O]+; (–)-
LSI MS: m/z 713 [M – H]–, 581 [(M – H) – Xyl]–, 563
[(M – H) – Xyl – H2O]–. 1H and 13C NMR (MeOH) are
given in Table 2 and discussed in the text.
Pycnopodioside A (IV), amorphous, ë32ç56O9,
[α]2D0 + 3.8 (Ò 0.5, MeOH); (+) MALDI TOF, m/z: 607
[M + Na]+, FAB (–): m/z 583 [M – H]–. It was identified
by comparison of spectral characteristics with those
given in the literature [10, 14].
Isolation of steroid compounds (I)–(IX). Fraction
Ä consisting according to TLC of polar compounds,
Rf = 0.8–0.7 in system butanol–ethyl acetate–water (5 : 1 : 1)
was purified by HPLC on a Diasphere 110-C18 column
under elution with 45% methanol–water and rechro-
matography on a Zorbax Bonus-RP column in system
of 55% methanol–water (V = 1 ml/min). Under similar
conditions, HPLC was carried out of the fraction
mainly consisting of glycoside (I); there was obtained
pure evasterioside C (3.5 mg). A less polar fraction B
and the fraction containing compound (IX), was
rechromatographed under similar conditions, using
55% methanol, and, for rechromatography, 65% meth-
anol. There were obtained 4 mg of (II), 3.5 mg of (III),
3.1 mg of (IV), 3 mg of (V), 2.3 mg of (VI), and 3 mg
of each (VII) and (VIII).
Pycnopodioside C (V), amorphous, ë33ç57O13SNa,
[α]2D0 + 4 (Ò 0.5, MeOH); H and C NMR data and
1
13
MALDI TOF (+) spectrum were identical with those
for standard compound from L. anthosticta [14].
Luridoside A (VI), amorphous, ë32ç55O12SNa,
[α]2D0 + 3.1 (Ò 0.2, MeOH); MALDI TOF (+): m/z 709
[MNa + Na]+ (100). Spectral characteristics are identical
to those in literature [13].
(25S)-5a-cholestane-3b,6a,8,15b,16b,26-hexaol
(VII); colorless crystals (from MeOH), mp 260–262°ë,
ë27ç48O6, [α]2D0 + 38.1 (Ò 0.1, MeOH); FAB (–): m/z 467
[M – H]–. It is identified by a comparison of spectral
characteristics with known steroid we earlier isolated
from starfish L. anthosticta [14].
Hydrolysis of glycosides (I), (II), and (III) and
identification of monosaccharides. Solutions of (I),
(II), and (III) (2 mg of each compound) in 2 ml 2 M
HCl were heated for 2 h on a boiling water bath. Xylose
was identified in hydrolyzates of (I) and (III) by GLC
(as aldononitrile peracetates) and paper chromatogra-
phy (butanol–pyridine–water system, 10 : 3 : 3, Whatman
no. 1). There were determined [α]2D0 +20 (from (I) (Ò 0.10,
H2O) and +26 (Ò 0.11, H2O) (from (III)). For D-xylose,
(24S)-5a-cholestane-3b,6a,8,15b,24-pentaol sul-
fate sodium salt (VIII), amorphous, ë27ç47SO3Na,
[α]2D0 + 8.1 (Ò 0.1, MeOH); FAB (–) m/z 531 [M – H]−.
It was identified by comparison of constants and spec-
tral characteristics with standard compound [15].
Marthasteron sulfate, (20R)-6a-hydroxy-23-oxo-
cholesta-9(11),24-dien-3b-yl sodium sulfate (IX),
was identified by analogy to known compound [14].
lit. [α]2D0 + 92 to19.0 (H2O); for L-xylose [α]2D0
18.6 (ç2é) [16]. D-Glucose was similarly identified in
–
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

130
LEVINA et al.
7. De Marino, S., Iorizzi, M., and Zollo, F., Minale, L.,
ACKNOWLEDGMENTS
Amsler, C.D., Baker, B.J., and McClintock, J.B., J. Nat.
Prod., 1997, vol. 60, pp. 959–966.
This work was financially supported by the Russian
Federal Property Fund (grant 08-04-00599-a), the grant
of President of the Russian Federation SS2813.2008.4
(Scientific School), and the program of Presidium of
the Russian Academy of Sciences Molecular and Cel-
lular Biology.
8. Iorizzi, M., Bryan, P., McClintock, J.B., Minale, L.,
Palagiano, E., Maurelli, S., Riccio, R., and Zollo, F., J.
Nat. Prod., 1995, vol. 58, pp. 653–671.
9. Ferezou, J.P., Devys, M., Allais, J.P., and Barbier, M.,
Phytochemistry, 1974, vol. 13, pp. 593–598.
10. Bruno, I., Minale, L., and Riccio, R., J. Nat. Prod., 1989,
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