A new steroidal glycoside phrygioside A and its aglycone from the starfish Hippasteria phrygiana

Article abstract of DOI:10.1007/s11172-005-0165-z

Three polyhydroxylated steroids were isolated from the Far-Eastern starfish Hippasteria phrygiana collected from the Sea of Okhotsk and were characterized as a new glycoside phrygioside A, viz., sodium (20R, 24S)-3β,4β, 7α,8,15β,24-hexahydroxy-24-O-[3-O-methyl-β-D-xylopyranosyl- (1→2)-α-L-arabinofuranosyl]-5α-cholestan-6α-yl sulfate, its aglycone, and the already known marthasterone sulfate.

Full text of DOI:10.1007/s11172-005-0165-z

2634
Russian Chemical Bulletin, International Edition, Vol. 53, No. 11, pp. 2634—2638, November, 2004
A new steroidal glycoside phrygioside A
and its aglycone from the starfish Hippasteria phrygiana
ꢀ
E. V. Levina, A. I. Kalinovskii, P. V. Andriyashchenko, P. S. Dmitrenok, E. V. Evtushenko, and V. A. Stonik
Pacific Institute of Bioorganic Chemistry of the FarꢀEastern Branch of the Russian Academy of Sciences,
159 prosp. 100ꢀletiya Vladivostoka, 690022 Vladivostok, Russian Federation.
Fax: +7 (423 2) 31 4050. Eꢀmail: levina@piboc.dvo.ru
Three polyhydroxylated steroids were isolated from the FarꢀEastern starfish Hippasteria
phrygiana collected from the Sea of Okhotsk and were characterized as a new glycoside
phrygioside A, viz., sodium (20R,24S)ꢀ3β,4β,7α,8,15β,24ꢀhexahydroxyꢀ24ꢀOꢀ[3ꢀOꢀmethylꢀ
βꢀDꢀxylopyranosylꢀ(1→2)ꢀαꢀLꢀarabinofuranosyl]ꢀ5αꢀcholestanꢀ6αꢀyl sulfate, its aglycone, and
the already known marthasterone sulfate.
Key words: starfishes, Hippasteria phrygiana, polyhydroxysteroids, sulfated polyhydroxyꢀ
steroids, glycosides, aglycones, FAB, MALDIꢀTOF, 1H NMR, and 13C NMR spectra.
As part of our continuing studies of metabolites from
Results and Discussion
starfishes of the FarꢀEastern seas,1 we established the
structures of steroids from the starfish Hippasteria
phrygiana belonging to the family Goniasteridae (the orꢀ
der Valvatida), whose chemistry is poorly studied. One of
these steroids is a bioside of polyhydroxylated steroid
monosulfate (1). Another polar steroid (2) appeared to be
identical to the aglycone of the aboveꢀmentioned glycoꢀ
side. We also identified marthasterone sulfate (3), viz.,
sodium (20R)ꢀ6αꢀhydroxyꢀ23ꢀoxocholestꢀ9(11),24ꢀdienꢀ
3βꢀyl sulfate, which is abundant in starfishes.2
The steroidal compounds were isolated from an
ethanolic extract of the starfish H. phrygiana according to
a procedure that we have used previously for related comꢀ
pounds.2 This involves chromatography on Polychrom,
Amberlite XADꢀ2, Florisil, and silica gel followed by reꢀ
versedꢀphase HPLC on ODSꢀA and DiasphereꢀC18. The
compounds were identified by spectroscopic methods
(NMR spectroscopy and MALDIꢀTOF and FAB mass
spectrometry).
The 13C NMR spectra (using DEPT experiments) of
compound 1 contain signals for six methyl groups, nine
methylene groups, twenty methine groups, and three C
atoms bearing no protons. These data together with the
negativeꢀion MALDIꢀTOF mass spectrum, which conꢀ
tains a pseudomolecular ion peak [M – H]– at m/z 841.48,
suggest the molecular formula C38H65O15SO3Na. In the
FABꢀ(–) mass spectrum, the peaks corresponding to sucꢀ
cessive abstraction of the Oꢀmethylpentose and penꢀ
tose residues at m/z 694.73 [(M – H) – C6H10O4]–,
676.71 [(M – H) – C6H12O5]–, and 544.84 [(M – H) –
C6H12O4 – C5H8O4]–, respectively, suggest the presence
of a disaccharide carbohydrate chain in compound 1. An
analogous fragmentation is also observed in the FABꢀ(+)
mass spectrum of 1 (see Experimental). Acid hydrolysis of
glycoside 1 afforded 3ꢀOꢀmethylxylose and arabinose,
which were identified by GLC as the corresponding
aldononitrile acetates and by paper chromatography usꢀ
ing comparison with authentic compounds. Judging from
the specific optical rotation of the resulting mixture of the
monosaccharides, arabinose and 3ꢀOꢀmethylxylose beꢀ
long to the L and D series, respectively.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2526—2529, November, 2004.
1066ꢀ5285/04/5311ꢀ2634 © 2004 Springer Science+Business Media, Inc.

A new steroidal glycoside from starfish
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 11, November, 2004 2635
In the 13C NMR spectrum of compound 1, the signals
at δC 104.9 and 107.4 were assigned to anomeric carbon
atoms, and the signals at δ 60.7, 62.1, 66.8, 69.2, 69.4,
70.0, 71.5, 74.1, 74.5, 74.7, 77.3, 78.9, 83.1, 84.3, 87.8,
and 92.3 were assigned to the carbon atoms bound to
oxygen in the monosaccharide residues and the aglycone.
The positions of the signals for C(1)—C(13) and C(19)
in the 13C NMR spectrum (C5D5N) of compound 1 are
identical to the corresponding values for sodium
3β,4β,7α,8,15β,16β,26ꢀheptahydroxyꢀ5αꢀcholestanꢀ6αꢀyl
sulfate isolated from the starfish Patiria pectinifera, which
we have characterized earlier.3 This suggests the
3β,4β,6α,7α,8,15βꢀhexahydroxy substitution and sulfation
of the hydroxy group at the C(6) atom in the polycyclic
moiety of the aglycone of glycoside 1. The chemical shifts
of the signals for the C atoms of the ring D, the side
chain, and the monosaccharide unit, C(15)—C(27) and
C(1´)—C(5´), are identical to the corresponding shifts
in the spectra of the known 24ꢀOꢀ[2,4ꢀdiꢀOꢀmethylꢀ
βꢀDꢀxylopyranosylꢀ(1→2)ꢀαꢀLꢀarabinofuranosyl]ꢀ5αꢀ
cholestaneꢀ3β,4β,6α,8,15β,24ꢀhexaol (culcitoside C1)
from Culcita novaeguineae.4 This indicates that comꢀ
pound 1 has the 15βꢀhydroxy functionality and that the
αꢀarabinofuranosyl residue of the carbohydrate chain is
linked to the hydroxy group at the C(24) atom.
The fact that the chemical shifts of the signals for the
second monosaccharide residue in the NMR spectra of
phrygioside 1 (C(1″)—C(5″)) are identical to those in the
spectra of borealoside C from Solaster borealis,5 which
contains the 3ꢀOꢀmethylxylopyranose carbohydrate chain,
as well as the data from FABꢀ(+) and FABꢀ(–) mass
spectrometry of 1 confirm that the 3ꢀOꢀmethylxylose
group is the terminal residue in the carbohydrate chain of
this glycoside. The coupling constant 3J1″,2″ (7.5 Hz) sugꢀ
gests the β configuration of the glycosidic bond.
The structure of glycoside 1 was also confirmed
by the COSYꢀ45, HMBC, and HSQC spectra, which
allowed us to make the assignment of the signals
for all carbon and hydrogen atoms in the NMR spectra
(Table 1). In particular, the 1H—1H COSYꢀ45 specꢀ
trum shows coupling in the system of the protons
H2C(2)—HC(3)—HC(4)—HC(5)—HC(6)—HC(7). The
multiplicities of the protons and the chemical shifts of the
signals for the H(6) atom confirm the presence of the
sulfo group. Two lowꢀfield protons give crossꢀpeaks with
the highꢀfield protons. They were assigned to H(24)
(δ 3.59, the signal characteristic of 24ꢀOꢀglycosylated steꢀ
roids from starfishes)4 and H(15) (δ 5.05). The chemical
shifts of the signals and the coupling constants of the
protons H2(16) (J15,14 ≈ 4.0 Hz, the coupling constant
was measured from the crossꢀpeak) confirm the 15βꢀOH
configuration.
The HMBC correlations H(1´)/C(24) and H(1″)/C(2´)
support the fact that the αꢀLꢀarabinofuranosyl residue
is linked to O(24) of the aglycone and is glycosylated
with the 3ꢀOꢀmethylꢀβꢀDꢀxylopyranosyl group at posiꢀ
tion 2 (see Table 1). We assigned the (R) configuraꢀ
tion to the C(20) center of compound 1 based on the
chemical shift of the protons H3C(21) (CD3OD, δ 0.93)
taking into account the results of the earlier study of a
series of model steroids6 where it was demonstrated
that the signal for the protons H3C(21) in the 20S isomer
(δ 0.83) is shifted upfield compared to the correspondꢀ
ing signal in the spectrum of the 20R isomer by apꢀ
proximately 0.1 ppm. Finally, the chemical shifts of the
signals for the protons and C atoms in the side chain of
compound 1 (CD3OD) appeared to be identical to the
corresponding shifts in the spectra of borealoside C,5 for
which the stereochemistry of the chiral centers of the side
chain has been established earlier. Hence, taking into
account the aforesaid, we assigned the structure of soꢀ
dium (20R,24S)ꢀ3β,4β,7α,8,15β,24ꢀhexahydroxyꢀ24ꢀ
Oꢀ[3ꢀOꢀmethylꢀβꢀDꢀxylopyranosylꢀ(1→2)ꢀαꢀLꢀarabinoꢀ
furanosyl]ꢀ5αꢀcholestanꢀ6αꢀyl sulfate (1) to phrygioꢀ
side A.
The positiveꢀion MALDIꢀTOF mass spectrum of steꢀ
roid 2 shows a pseudomolecular ion peak [M + Na]+
at m/z 585, which suggests the molecular formula
C27H46O7SO3Na. According to the 13C NMR spectroꢀ
scopic data (see Table 1), molecule 2 contains 27 C atoms,
including carbon atoms of five methyl groups, seven meꢀ
thylene groups, twelve methine groups, and three C atoms
bearing no protons.
The chemical shifts of the signals for the C(1)—C(13)
and C(19) atoms in the NMR spectrum (CD3OD) of
compound 2 are virtually the same as those in the specꢀ
trum of sodium (25S)ꢀ3β,4β,7α,8,15α,16β,26ꢀheptaꢀ
hydroxyꢀ5αꢀcholestanꢀ6αꢀyl sulfate from the starfish
Oreaster reticulatus,7 which confirms that the correꢀ
sponding fragments in both compounds are identical.
Sulfation of the hydroxy group at the C(6) atom leads to a
downfield shift of the signal for H(6) by 0.7 ppm (δ 5.05)
compared to the chemical shift of the analogous proꢀ
ton (δ 4.32) in the spectrum of nonsulfated 5αꢀcholeꢀ
stanꢀ3β,4β,6α,7α,8,15β,16β,26ꢀoctaol from Asterina
pectinifera.8 A comparison of their 13C NMR spectra also
revealed the characteristic shifts associated with the αꢀ and
βꢀeffects of sulfation of the hydroxy group at the C(6)
atom.7 The chemical shifts of the C(15)—C(27) and C(18)
atoms in the spectrum of compound 2 are identical to the
corresponding chemical shifts for (24S)ꢀ5αꢀcholestaneꢀ
3β,6α,8,15β,24ꢀpentaol from Comophia watsoni.9 Based
on these data, we suggested that compound 2 is the aglyꢀ
cone of phrygioside A (1) and has the structure of sodium
(20R,24S)ꢀ3β,4β,7α,8,15β,24ꢀhexahydroxyꢀ5αꢀcholeꢀ
stanꢀ6αꢀyl sulfate.
The COSYꢀ45 spectrum of compound 1 also shows
coupling between the protons of the carbohydrate chain.

2636
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 11, November, 2004
Levina et al.
Table 1. Parameters of the NMR spectra (δ, J/Hz) of phrygioside A (1) and steroid (2)
Atom C,
group
1 (C5D5N)
HMBC
2 (CD3OD)
δ
(m)
δ
δ
C (m)
δ
H
C
H
1
2
3
4
5
6
7
8
38.9 (CH2)
26.8 (CH2)
71.5 (CH)
69.2 (CH)
46.2 (CH)
74.7 (CH)
74.1 (CH)
78.9 (C)
1.13, 1.84 (both m)
2.16 (m, Hax); 1.80 (m, Heq
3.86 (m)
39.5 (CH2)
26.6 (CH2)
73.2 (CH)
69.4 (CH)
46.7 (CH)
76.6 (СH)*
75.1 (CH)
79.4 (С)
)
1.97 (m, Hax); 2.13 (m, Heq
3.53 (m)
)
5.12 (m)
4.26 (br.s, W1/2 = 9)
1.76 (dd, J = 11.5, J = 2.5)
5.09 (dd, J = 11.5, J = 2.5)
4.30 (d, J = 3.0)
2.49 (dd, J = 11.9, J = 2.0)
6.01 (dd, J = 11.9, J = 2.5)
5.24 (d, J = 2.5)
9
50.4 (CH)
37.6 (C)
18.2 (CH2)
42.0 (CH2)
43.3 (C)
55.6 (CH)
69.4 (CH)
42.3 (CH2)
56.9 (CH)
16.3 (CН3)
1.62 (m)
51.2 (CH)
38.4 (С)
18.9 (CH2)
42.9 (CH2)
44.2 (C)
56.4 (CH)*
71.0 (СH)
42.7 (CH2)
57.9 (CH)
16.4* (CH3)
10
11
12
13
14
15
16
17
18
1.55, 2.11 (both m)
1.26, 2.07 (both m)
2.02 (m)
5.05 (m)
2.62, 1.73 (both m)
1.10 (m)
1.62 (s)
1.41 (d, J = 5.5)
4.81 (td, J = 3.3, J = 10.0)
1.95, 2.4 (both m)
1.00 (m)
С(12), С(13),
С(14), С(17)
С(1), С(5),
С(9), С(10)
1.26 (s)
19
16.8 (CН3)
1.76 (s)
16.8* (CH3)
1.23 (s)
20
21
22
23
24
25
26
27
OMe
1´
35.4 (CH)
18.7 (CH3)
31.8 (CH2)
27.9 (CH2)
83.1 (CH)
30.4 (CH)
17.9* (СH3)
18.0* (СH3)
60.7 (CH3)
107.4 (CH)
92.3 (CH)
77.3 (CH)
84.3 (CH)
62.1 (CH2)
1.65 (m)
36.5 (CH)
19.5 (CH3)
33.3 (CH2)
31.5 (CH2)
78.1 (CH)
34.5 (CH)
17.5 (CH3)
19.5 (CH3)
1.52 (m)
0.94 (d, J = 7.0)
1.02 (d, J = 6.2)
1.20, 1.86 (both m)
1.55, 1.73 (both m)
3.59 (m)
С(17), С(20), С(22)
1.53, 1.20 (both m)
3.57 (m)
1.97 (m)
0.95 (d, J = 6.9)
0.99 (d, J = 6.9)
3.90 (s)
5.65 (br.s)
4.90 (br.s)
С(24), С(25)
С(24), С(25)
0.98 (d, J = 6.8)
0.98 (d, J = 6.8)
С(24), С(2´)
С(3´), C(4´)
2´
3´
4´
5´
4.91 (m)
4.73 (m)
4.37 (dd, J = 3.0,
J = 12.3); 4.21 (m)
5.07 (d, J = 7.5)
3.96 (t, J = 8.5)
3.60 (m)
4.14 (m)
3.60 (m); 4.24 (dd,
J = 5.4, J = 11.0)
1″
2″
3″
4″
5″
104.9 (CH)
74.5 (CH)
87.8 (CH)
70.0 (CH)
66.8 (CH2)
С(2´)
С(2″), С(4″)
С(4″)
* Assignments may be interchangable.
The structure of 2 was completely confirmed by
2D NMR spectroscopy (HMBC, HSQC, and COSYꢀ45)
analogously to compound 1 (see Table 1).
A comparison of the spectroscopic characteristics of
compound 3 with the corresponding data for marthaꢀ
sterone sulfate, which we have isolated earlier from the
starfish Lysastrosoma anthosticta,2 demonstrated that they
are completely identical. Hence, we identified steroid 3
as sodium (20R)ꢀ6αꢀhydroxyꢀ23ꢀoxocholestaꢀ9(11),24ꢀ
dienꢀ3βꢀyl sulfate.
Phrygioside A (1) can be related to glycosylated
steroids containing the 3β,4β,6α,8,15βꢀpentahydroxyꢀ
cholestane fragment sulfated at the O(6) atom. Most of
these steroids were isolated from the Antarctic starfish
Acodontaster conspicuous.10 The isolation of the new comꢀ
pounds added to a knowledge of the chemical diversity of

A new steroidal glycoside from starfish
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 11, November, 2004 2637
column with Diaspherꢀ110ꢀC18; the yield of marthasterone sulꢀ
fate 3 was 6.2 mg (0.0057%).
secondary metabolites and various biosynthesis and meꢀ
tabolism processes of natural steroids occurring in starꢀ
fishes.
Fraction II (0.98 g) was passed through Polychrom and
consecutively purified on a column with Florisil using the
CHCl3—EtOH system (6 : 4 → 1 : 9) and on a column with
YMCꢀPack ODSꢀA using the EtOH—H2O mixture (43 : 57) as
the eluent. Glycoside 1 was obtained in a yield of 7.4 mg
(0.0068%).
Experimental
The 1H and 13C NMR spectra were recorded on Bruker
ACꢀ250 (250 MHz for 1H and 62.9 MHz for 13C) and Bruker
DPXꢀ300 (300.13 MHz for 1H and 75.5 MHz for 13C) specꢀ
trometers with tetramethylsilane as the internal standard. The
optical rotation was measured on a Perkin—Elmer 343 polarimꢀ
eter. The MALDIꢀTOF mass spectra were obtained on a Bruker
Biflex III matrixꢀassisted laser ionization/desorption mass specꢀ
trometer (N2 laser, 337 nm). A sample was dissolved in methaꢀ
nol (10 mg mL–1), and an aliquot (1 µL) was analyzed using
αꢀcyanohydroxycinnamic acid as the matrix. The sodium conꢀ
tent was determined on a Nippon Jarrel Ash AAꢀ780 atomicꢀ
absorption flameꢀemission spectrophotometer. Studies by HPLC
were carried out on a DuPont Model 8800 chromatograph
equipped with a refractometer detector and columns packed
with Diasphereꢀ110ꢀC18 (5 µm, 4×250 mm) and YMCꢀPack
ODSꢀA (5 µm, 12 nm, 10×250 mm). The melting points were
determined on a Leica VMTG hotꢀstage apparatus. The TLC
analysis was carried out on Sorbfil plates with a layer of silica gel
CTXꢀ1A (5—17 µm, Krasnodar, Russia) fixed on aluminum
foil using the 5 : 1 : 1 BuOH—EtOAc—H2O system. Column
chromatography was performed on silica gel L (80—100 and
200—250 mesh, Chemapol, Czech Republic), Polychrom (Rusꢀ
sia), and Florisil (200—250 mesh, Merck, Germany).
Samples of the starfish Hippasteria phrygiana were trawled
up from a depth of 100—200 m in the Sea of Okhotsk (Kuril
Islands) in August 2002 during the expedition No. 27 of the
research ship "Akademik Oparin" and were identified by Prof.
V. S. Levin (Pacific Institute of Bioorganic Chemistry of the
FarꢀEastern Branch of the Russian Academy of Sciences).
Extraction and isolation of total fractions. Samples of the
starfish H. phrygiana (0.108 kg) were milled and exhaustively
extracted with 95% ethanol at room temperature for 3 days. The
combined ethanolic extracts were concentrated in vacuo to a
crude oily residue, which was chromatographed on a column
(6×25 cm) with silica gel (80—100 mesh) using the chloroꢀ
form—ethanol system (75 : 25 → 50 : 50). The following two
fractions of polyhydroxysteroids were eluted as the polarity of
the eluent was increased: less polar fraction I (0.55 g) containing
the known sulfate 3 and steroid 2 and more polar fraction II
(0.98 g) containing compound 1.
Sodium
(20R,24S)ꢀ3β,4β,7α,8,15β,24ꢀhexahydroxyꢀ
24ꢀOꢀ[3ꢀOꢀmethylꢀβꢀDꢀxylopyranosylꢀ(1→2)ꢀαꢀLꢀarabinoꢀ
furanosyl]ꢀ5αꢀcholestanꢀ6αꢀyl sulfate (phrygioside A) (1),
C
38H65O15SO3Na, colorless crystals, m.p. 129—131 °C (from
20
MeOH), [α]D
–10.9 (с 0.33, EtOH). MS FABꢀ(+),
m/z (Irel (%)): 887.38 [MNa + Na]+ (70), 767.46 [(MNa + Na) –
NaHSO4]+ (100), 723.35 [(MNa + Na) – C6H12O5]+ (40), 621.25
[767.46 – C6H10O4]+, 489.29 [621.25 – C5H8O4]+ (10), 471.14
[489.29 – H2O]+ (17). The 1H and 13C NMR spectra (CD3OD)
of 1 were used for comparing with the published data5 and are
1
mentioned in the text. The H and 13C NMR spectra (C5D5N)
are given in Table 1.
Sodium (20R,24S)ꢀ3β,4β,7α,8,15β,24ꢀhexahydroxyꢀ5αꢀ
cholestanꢀ6αꢀyl sulfate (2), C27H46O7SO3Na, an amorphous
compound, [α]D20 +14.1 (с 0.25, EtOH). MS MALDIꢀTOFꢀ(+)
(Irel (%)): m/z 585 [M + Na]+ (100). The 1H and 13C NMR
spectra (CD3OD) are given in Table 1.
Sodium (20R)ꢀ6αꢀhydroxyꢀ23ꢀoxocholestaꢀ9(11),24ꢀdienꢀ
3βꢀyl sulfate (3), C27H41NaO6S, colorless crystals, m.p.
20
126—128 °C (from MeOH—EtOAc), [α]D +2.6 (с 0.12,
1
EtOH). The H and 13C NMR spectra (CD3OD, C5D5N) were
identical to those documented for marthasterone sulfate from
the starfish Lysastrosoma anthosticta.2
Hydrolysis of glycoside 1 and identification of monosacchaꢀ
rides. Glycoside (2 mg) was dissolved in 2 M HCl (2 mL) and
heated at 100 °C for 2 h. 3ꢀOꢀMethylꢀDꢀxylose and Lꢀarabinose
were identified in the hydrolyzate by GLC (aldononitrile
peracetates) and paper chromatography (nꢀbutanol—pyriꢀ
dine—water, 6 : 4 : 40). For a mixture of monosaccharides
20
prepared from 1, [α]D +57.5 (с 0.08, H2O); for an equimolar
20
mixture of the authentic samples [α]D +53.4 (с 0.35, H2O)
(cf. lit. data11: [α]D20 +61 (3ꢀOꢀmethylꢀDꢀxylose + Lꢀarabinose),
[α]D20 –44 (3ꢀOꢀmethylꢀDꢀxylose + Dꢀarabinose)).
We thank V. S. Levin (Pacific Institute of Bioorganic
Chemistry of the FarꢀEastern Branch of the Russian Acadꢀ
emy of Sciences) for identifying the species of the starfish.
This study was financially supported by the Russian
Foundation for Basic Research (Project No. 02ꢀ04ꢀ
49491), the Foundation of the President of the Russian
Federation (Programs for Support of Leading Scientific
Schools, Grant NShꢀ725.2003.4), and the Presidium of
the Russian Academy of Sciences (Program "Molecular
and Cellular Biology").
Isolation of steroid derivatives 1, 2, and 3. Fraction I was
dissolved in water and passed through a column (3×12 cm) with
Polychrom and eluted with water and 50% aqueous ethanol.
The aqueousꢀethanolic eluate was concentrated in vacuo to
a brown resinous residue (0.41 g), which was successively
chromatographed on columns with Florisil (1.5×20 cm,
200—300 mesh) using the CHCl3—EtOH system (9 : 1 → 6 : 4).
The resulting fractions were chromatographed on SiO2 (1.3×15)
with the CHCl3—EtOH system (7.5 : 2.5 → 7 : 3). The fraction
containing steroid 2 with minor impurities (5.2 mg) was purified
by HPLC on a column with Diasphereꢀ110ꢀC18 using the
MeOH—H2O mixture (75 : 25) as the eluent. Sulfate 2 was
obtained in a yield of 2.1 mg (0.0019 % of the starfish weight).
Steroid 3 was isolated by HPLC using the same system on a
References
1. E. V. Levina, A. I. Kalinovskii, V. A. Stonik, and P. S.
Dmitrenok, Izv. Akad. Nauk, Ser. Khim., 2003, 1539 [Russ.
Chem. Bull., Int. Ed., 2003, 50, 1623].

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Russ.Chem.Bull., Int.Ed., Vol. 53, No. 11, November, 2004
Levina et al.
2. E. V. Levina, P. V. Andriyashchenko, A. I. Kalinovskii, and
V. A. Stonik, Izv. Akad. Nauk, Ser. Khim., 2001, 300 [Russ.
Chem. Bull., Int. Ed., 2001, 50, 313].
3. A. A. Kicha, A. I. Kalinovskii, and E. V. Levina, Khim.
Prirod. Soedin., 1984, 6, 738 [Chem. Nat. Compd., 1984, 6
(Engl. Transl.)].
4. A. A. Kicha, A. I. Kalinovskii, E. V. Levina, and P. V.
Andriyashchenko, Khim. Prirod. Soedin., 1985, 6, 801 [Chem.
Nat. Compd., 1985, 6 (Engl. Transl.)].
5. M. Iorizzi, L. Minale, R. Riccio, and T. Yasumoto, J. Nat.
Prod., 1992, 55, 866.
8. M. Iorizzi, L. Minale, and R. Riccio, Gazz. Chim. Ital.,
1990, 120, 147.
9. R. Riccio, M. D´Auria, M. Iorizzi, L. Minale, D. Laurent,
and D. Duhet, J. Nat. Prod., 1985, 115, 405.
10. S. De Marino, M. Iorizzi, F. Zollo, L. Minale, C. D. Amsler,
B. J. Baker, and J. B. McClintock, J. Nat. Prod., 1997,
60, 959.
11. F. Micheel and A. Klemer, Chemie der Zucker und Polyꢀ
saccharide, Academische Vellagsgesellschaft Geest,
Leipzig, 1956.
6. L. Minale, C. Pizza, A. Plomitallo, R. Riccio, F. Zollo, and
F. A. Mellon, Gazz. Chim. Ital., 1984, 114, 143.
7. R. S. Correa, R. Riccio, L. Minale, and C. Duque, J. Nat.
Prod., 1985, 48, 751.
Received May 17, 2004;
in revised form September 7, 2004