Trofosides A and B and other cytostatic steroid-derived compounds from the far east starfish Trofodiscus ueber

Article abstract of DOI:10.1134/S1068162007030107

Three new polar steroids identified as trofoside A, 20R,24S)-24-O-(3-O- methyl-β-D-xylopyranosyl)-3β,6α,8,15β,24-pentahydroxy- 5α-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfooxy-(20R,24S)-5α-cholestane-3β,6β,8,15α, 24-pentaol sodium salt, were isolated fromTrofodiscus ueber starfish extracts collected in the Sea of Ohotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α- cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3β6α,8, 15β-tetrahydroxy-5α-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius(C _min) and terminating the cell division at the stage of the first division (C _min embr.), as well as the concentrations causing 50% immobilization of sperm cells (OC₅₀) and inhibiting their ability to fertilize egg-cells by 50% (IC₅₀) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono-and double chained glycosides with the monosaccharide fragment at C3 and fragments at C3 and C4 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity. Nauka/Interperiodica 2007.

Full text of DOI:10.1134/S1068162007030107

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 3, pp. 334–340. © Pleiades Publishing, Inc., 2007.
Original Russian Text © E.V. Levina, A.I. Kalinovsky, P.V. Andriyashchenko, N.I. Menzorova, P.S. Dmitrenok, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33,
No. 3, pp. 357–364.
Trofosides A and B and Other Cytostatic Steroid-Derived
Compounds from the Far East Starfish Trofodiscus über
E. V. Levina1 , A. I. Kalinovsky, P. V. Andriyashchenko, N. I. Menzorova, and P. S. Dmitrenok
Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences,
pr. 100-letiya Vladivostoka 159, Vladivostok, 690022 Russia
Received April 28, 2006; in final form, May 10, 2006
Abstract—Three new polar steroids identified as trofoside A, 20R,24S)-24-O-(3-O-methyl-β-D-xylopyrano-
syl)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sul-
fooxy-(20R,24S)-5α-cholestane-3β,6β,8,15α,24-pentaol sodium salt, were isolated from Trofodiscus über star-
fish extracts collected in the Sea of Ohotsk. Two known compounds, trofoside A aglycone, (20R,24S)-
3β,6α,8,15β,24-pentahydroxy-5α-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3β6α,8,15β-tet-
rahydroxy-5α-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of
the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation
of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (Cmin) and terminating the cell divi-
sion at the stage of the first division (Cmin embr.), as well as the concentrations causing 50% immobilization of
sperm cells (OC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the iso-
lated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a
sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3
and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected
the embryo development. The positions of monosaccharide residues in the core considerably influence the com-
pound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at
C3 and fragments at C3 and C4 atoms are active against sea-urchin sperm cells and embryos, whereas the C24
glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.
Key words: MALDI TOF mass spectra, NMR spectra, polyhydroxysteroids, starfish, Trofodiscus über
DOI: 10.1134/S1068162007030107
INTRODUCTION
Amberlite XAD-2, Sephadex LH-60, Florisil, and sil-
ica gel, followed by HPLC on Diaspher-110-ë18 and
YMC-Pack ODS-A reversed-phase columns according
to the earlier described procedures [4]. The structures
of the isolated compounds were determined by spectral
methods (NMR and MALDI TOF and EI mass spec-
trometry, see the table and the Experimental section).
Polar marine steroids belong to a large class of second-
ary metabolites that are structurally diverse and display
various types of physiological activities [1–2].2 We contin-
ued our multiyear studies of active steroid metabolites
from starfish [3] and isolated five polar steroids from the
Far East starfish Trofodiscus über extracts, including three
new compounds, and studied their cytostatic and spermio-
static activities in sea-urchin Strongylocentrotus interme-
dius embryos and sperm cells.
The pseudomolecular peak [M + Na]+ at m/z 621 in
the MALDI TOF mass spectrum (positive ion registra-
1
tion) and 13ë and H NMR spectra of compound (I)
13
indicated the molecular formula of ë33H58O9. The C
NMR and DEPT spectra pointed out to the presence of
six methyl, 10 methylene, 14 methyne, and three pro-
ton-lacking C atoms in its molecule. The resonance sig-
nal at δ 105.0 ppm was ascribed to the anomeric carbon
of monosaccharide residue, whereas the resonances at
δ 66.7, 67.6, 71.0, 71.1, 72.2, 75.1, 77.5, 86.2, and
87.7 ppm were assigned to other carbons linked to oxy-
RESULTS AND DISCUSSION
Steroids (I)–(V) were isolated from the ethanol
extracts of starfish T. über by chromatography on
1
Corresponding author; fax: +7 (4232) 31-4050; e-mail:
levina@piboc.dvo.ru
2
1
Abbreviations: DEPT, distortionless enhancement by polarization
gen atoms. In the H NMR spectrum of glycoside (I)
1
1
transfer; H- H-COSY, correlated spectroscopy; HMBC, hetero-
nuclear multiple bond connectivity; HSQC, heteronuclear one
quantum connectivity; and MALDI TOF, matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry.
(table), doublet of an anomeric proton of the monosac-
charide residue at δç 4.23 (d, J 7.5 Hz) was observed,
whose chemical shift and coupling constant implied the
334

TROFOSIDES A AND B AND OTHER CYTOSTATIC
NMR spectra of steroids (I) and (II) (CD3OD), DRX-500, AC-250)
335
Atom
number
(I)
(II)
δC, DEPT δH (J, Hz)
HMBC
δC, DEPT δH (J, Hz)
HMBC
1
2
3
4
5
6
7
8
9
39.4 CH2 1.71 m, 0.96 m
31.4 CH2 1.72 m, 1.47 m
72.2 CH 3.48 m
41.4 CH2 1.71 m, 0.96 m
31.6 CH2 1.72 m, 1.50 m
72.4 CH 3.58 m
32.3 CH2 2.18 m, 1.20 m
53.8 CH 1.02 m
36.4 CH2 1.81 m, 1.58 m
48.9 CH 1.22 m
67.6 CH 3.69 td (10.8; 4.1)
74.2 CH 3.85 q (2.8)
C8, C10
C5, C6, C8, C9
49.4 CH2 2.36 dd (4.3; 12.5) 1.26 m C5, C6, C8, C9
44.7 CH2 2.53 dd (14.9, 3.1)
76.9 C
77.5 C
57.4 CH 0.82 m
56.9 CH 0.95 m
36.6 C
10 38.0 C
11 19.7 CH2 1.79 m; 1.48 m
12 43.4 CH2 1.97 m; 1.15 m
13 44.3 C
19.8 CH2 1.84 m, 1.56 m
42.7 CH2 1.97 m; 1.24 m
44.7 C
14 62.6 CH 1.00 d (5.5)
C13, C18
64.5 CH 1.37 d (9.7)
78.0 CH 4.90 dt (2.6, 9.5)
C13, C18
15 71.1* CH 4.40 ddd (2.1; 5.5; 7.6) C13
16 42.5 CH2 2.36 m; 1.37 m
C13
38.6 CH2 2.17 ddd (2.8; 8.5; 15.0)
1.92 m
17 57.9 CH 0.96 m
18 16.5 CH3 1.25 s
56.0 CH 1.31 m
15.3 CH3 0.98 s
C12, C13, C14,
C17
C12, C13, C14, C17
C1, C5, C9, C10
19 14.1 CH3 0.97 s
20 36.3 CH 1.52 m
C1, C5, C9, C10 15.9 CH3 1.16 s
36.2 CH 1.38 m
21 18.9 CH3 0.91 d (6.5)
22 32.6 CH2 0.98 m; 1.59 m
23 28.6 CH2 1.57 m; 1.35 m
24 86.2 CH 3.34 m
C17, C20, C22
18.9 CH3 0.92 d (6.5)
33.2 CH2 1.65 m
C17, C20, C22
31.6 CH2 1.53 m; 1.22 m
78.0 CH 3.20 m
25 31.8 CH 1.86 m
34.5 CH 1.64 m
26 18.3 CH3 0.93 d (7.0)
27 18.3 CH3 0.91 d (7.0)
OMe 60.9 CH3 3.62 s
1' 105.0 CH 4.23 d (7.5)
2' 75.1 CH 3.20 dd (7.5; 9.0)
3' 87.7 CH 3.00 t (8.9)
4' 71.0* CH 3.51 m
C24, C25, C27
C24, C25, C26
C3'
19.6 CH3 0.90 d (7.0)
17.4 CH3 0.88 d (7.0)
C24, C25, C27
C24, C25, C26
C24, C2'
C3'
OMe, C2', C4'
5' 66.7 CH2 3.79 dd (5.4; 11.4);
3.13 dd (10.3; 11.5)
C4', C3', C4', C3'
* Unambiguous assignment.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

336
LEVINA et al.
β configuration of the glycoside bond. Based on this corroboration of the glycoside (I) side chain, chemical
data, we assumed that this compound is composed of a
pentahydroxycholestane-derived aglycone e with a
methylated xylose-based residue in the side chain.
shifts of its resonances in NMR spectra [N17–N27,
N1'–N5', and H21–27, I1'–I5'] were compared with
those of (20R,24S)-24-O-(3-O-methyl-β-D-xylopyra-
nosyl)-3β,6α,8,15α,24-pentahydroxy-5α-cholestane
(borealoside C (VII) from the starfish Solaster borea-
lis), in which the stereochemistry of the chiral centers
of side chain was earlier established [6]. Their complete
agreement proves that 3-O-methylxylose is a monosac-
charide unit in trofoside A (table). The 20R,24S config-
uration, we assigned to the asymmetric centers of the
compound (I) side chain, is unique for polyols of this
The values of C1–C27 and I3–I27 chemical shifts
and the coupling constants of the corresponding pro-
tons in the 1H and 13C NMR (ëD3OD) spectra of glyco-
side (I) agreed with those of the earlier described sco-
parioside C (VI) from the Astropecten scoparius star-
fish [5]. It could be reasonable to think that their
aglycone es are identical and the aglycone e of glyco-
side (I) called by us trofoside A is 3β,6α,8,15β,24-pen-
tahydroxy-5α-cholestane glycosylated with a methyl-
xylopyranose residue in position C24. For the structure type isolated from starfish [7, 8].
OR3
R2
24
21
18
22
26
20
17
25
27
23
12
11
16
15
19
13
14
OH
1
4
9
2
R1
10
8
7
OSO3Na
R2
6
3
5
HO
HO
R1
OH
OH
(I), (III), (IV), (VI), (VII), (X)
(II), (VIII), (IX)
OR4
O
SO3Na
N
H
OH
OH
OH
R3
HO
R1O
R3
(V)
(XI), (XII)
OH
R2
OH
R1
R2
R4
(I)
β-OH
Xyl3Me
ΟΗ
(II)
Η
Η
(III) β-OH
Xyl3Me
ΟΗ
22(23)-dehydro
(IV)
(VI)
β-OH
β-OH
Xyl3Me, 4SO3
(VII) α-OH
(VIII) Η
Xyl3Me
ΟΗ
Xyl
Η
(IX)
(XI)
ΟΗ
Η
Xyl2Me
Η
Η
Ara
Η
(XII) Xyl2Me
Η
ΟΗ
The structure of compound (I) was confirmed by the mula of (I) were observed in the COSY-45 spectrum. In
NMR two-dimensional spectroscopy including 1ç-1ç- the HMBC spectra, the resonances of the side chain
ëOSY, HMBC, and HSQC experiments. Particularly, protons were observed as intensive cross-peaks (ç26)3
the sequences of (H2)2−(H7)2 and then H9–(H12)2 and and (ç27)3/ë25 (31.8 ppm) and /C24 (86.2 ppm),
H14−H17−H20−(H21)3 marked in boldface in the for- which confirms the presence in (I) of the side chain of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

TROFOSIDES A AND B AND OTHER CYTOSTATIC
337
the 24-hydroxycholestane type. The 2D NMR experi- sponding resonances of the side chain of the known
ments ensured the assignment of all the resonances of minutoside A (X) from the starfish Anasterias minuta
the glycoside carbohydrate fragment. The HMBC cor- [11], which contains a 24-é-(3-O-methyl-β-D-xylopy-
relation of H1/C24 (table) confirmed that the 3-é-Me-
β-D-xylopyranosyl residue was linked to the aglycone
at C24.
ranosyl)-22E-ene-cholestane side chain, namely, the
resonances of olefin carbons at δë 141.1 (N22) and
128.1 (N23) ppm and two doublets of doublets at δH
5.38 and 5.32 ppm (∆22-protons). The coupling con-
stant J22,23 of 15.3 Hz indicated the E-configuration of
the double bond in the side chain. In the 13C NMR spec-
trum (CD3OD)) of (III) + (I), along with the common
for both compounds C17–C28 resonances, dual reso-
nances of N1'-N5' were observed. Note that the hydrol-
ysis of the (III) + (I) mixture yielded only one
monosaccharide, 3-é-Me-D-xylose. Hence, compound
(III), we called trofoside B, differs from compound (I)
by only the presence of double bond in the side chain,
which probably affects chemical shift values of the
monosacchardie residue and induces dubling of the res-
onances. Based on this data, the structure of glycoside
(III) was identified as (20R,22E,24S)-24-O-(3-O-
methyl-β-D-xylopyranosyl)-3β,6α,8,15β,24-pentahy-
droxy-5α-cholest-22-ene.
These results allowed the identification of trofoside
A as (20R,24S)-24-O-(3-O-methyl-β-D-xylopyranosyl)-
3β,6α,8,15β,24-pentahydroxy-5α-cholestane (I).
The pseudomolecular peak [M + Na]+ at m/z 531 in
the MALDI TOF mass spectrum (registration of cat-
13
1
ions) and ë and H NMR spectra of compound (II)
pointed out to its molecular formula of
ë27H47O5SO3Na. According to C and DEPT spectra,
13
it contains 27 carbon atoms including the atoms of five
methyl, nine methyene, and 10 methyne groups and
three carbon atoms unlinked to protons.
The values of C1–C21 chemical shifts in the 13C
NMR spectra and the coupling constants of the corre-
1
sponding protons in the H NMR spectra coincided
with the corresponding values for coscinasteroside B
(VIII) from the starfish Coscinasterias tenuispina [9].
This allowed the suggestion on 3β,6β,8,15α-tetrahy-
droxy substitution and sulfation of the C15 hydroxy
group in the polycyclic part of (II). A comparison of the
spectra demonstrated the difference between the side
chains of (II) and (VIII). On the other hand, chemical
The EI mass spectrum of steroid (IV) contained a
peak at m/z 434 [M – H2O]–, which corresponds to the
loss of water by the molecular ion and, in combination
with the NMR data, points out to the ë27H48O5 molec-
13
ular formula. According to the data of C and DEPT
shifts of the C20–C27 side chain resonances and the spectra, it contains 27 carbon atoms including the
corresponding protons agreed with the corresponding atoms of five methyl, nine methylene, and 10 methyne
values for polyol with the known stereochemistry of the groups and three carbon atoms free of protons.
side chain, namely, 20R,24S-3β,5α,6β,15α,24-pentahy-
The values of chemical shifts and coupling con-
droxy-5α-cholestan-15-yl sulfate (IX) from the Luidia
1
13
stants in the H and C NMR spectra of the aglycone
part of trofoside A (I) as a rule coincided with the cor-
responding values for steroid (IV) (table), except for
the C24 resonance at δ 78.1 ppm, which was shifted as
compared with the corresponding resonance of (I) (δ
86.2 ppm) (table). Hence, one could assume that com-
pound (IV) is a trofoside A aglycone. The stereochem-
istry of asymmetric centers was determined similarly to
that of trofoside A (I). When spectral characterisitics of
steroid (IV) were compared with the corresponding
data for the known steroid polyol from the starfish
Gomophia watsoni [12], the complete coincidence of
the values was shown, which confirms its structure as
(20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane.
clathrata starfish [10]. The multiplicity of the H15 res-
onance and the coupling constants of H15 and H14
(J14,15 9.5 Hz) (table) also indicated the α-configuration
of the sulfoxy group, since the coupling constant value
of 15β-hydroxysteroids is within 5.3–6.5 Hz [9].
The structure of (II) was finally established using
COSY-45 and HMBC methods ensuring the assign-
ment of all the carbon and hydrogen atoms in its NMR
spectra (table). The proton sequences assigned by the
COSY-45 approach, namely, ç1–(ç7)2, H14–H7, and
ç20–(ç21)3–(ç27)3, particularly, multiplicities and
chemical shifts of the corresponding resonances con-
firmed the assumed structure. They are marked in bold-
face in the formula of compound (II). The stereochem-
istry of asymmetric centers was found similarly to that
of (I). Based on the all the data, sulfated polyoxysteroid
(II) was identified as 15-sulfoxy-(20R,24S)-5α-choles-
tane-3β,6β,8,15α,24-pentaol sodium salt.
The isolated (III) was contaminated with (I), and its
further HPLC purification failed. The MALDI TOF
mass spectrum (registration of positive ions) of this
mixture exhibited the pseudomolecular peaks [M +
Na]+ at m/z 619 and 621 in a 1 : 0.3 ratio. This suggested
the presence of approximately 30% of trofosideA (I) as
Polyoxysteroid
(V)
earlier
known
as
(20R,22E,24R,25S)-24-methyl-3β,6α,8,15β-tetrahy-
droxy-5α-cholest-22-ene-27-carboxylic acid (2-sulfo-
ethyl)amide (triseramide) was identified based on the
coincidence of its physical constants and MALDI TOF
(+) MS and NMR spectra with the corresponding
parameters of triseramide, we have earlier isolated from
the starfish Astropecten triseriatus [13]. For corrobora-
tion of steroid (V) structure, we also carried out its
acidic hydrolysis and isolated taurine, –NH2-
an impurity. In the NMR spectra of this mixture, there (CH2)2SO3Na identified by comparison of its spectral
were resonances completely coinciding with the corre- characteristics with the standard sample.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

338
LEVINA et al.
Minimal concentrations causing the cytostatic effect activity. Particularly, glycoside (XII) bearing one
on unfertilized ovules of the St. intermedius sea-urchin monosaccharide residue at C3 (I) was active in sperma-
(ëmin), minimal concentrations terminating the cell tozoon and sea-urchin embryo, whereas C24-glycosy-
division of fertilized cells at the first division stage (ëmin lated trofoside A (I) is inactive toward embryos and is
embr.), minimal concentrations inhibiting spermato- low spermiotoxic (éC50 and IC50 35 µg/ml).
zoon motilities of this sea-urchin species (éC50) by
50%, and minimal concentrations inhibiting spermato-
EXPERIMENTAL
zoon ability to fertilize egg-cells (IC50) were deter-
mined for compounds (I), (II), (IV), and (V) and refer-
ence granulatoside B (XI) and monoside (XII) from the
starfish Choriaster granulatus [14].
1ç and C NMR spectra (CD3OD, δ, ppm (J, Hz)
were registered on Brucker AC-250 and DRX-500
spectrometers with working frequencies for protons of
13
All the tested compounds were ineffective towards 250 and 500 MHz, respectively; an internal standard
unfertilized ovules at Cmin 50 µg/ml.
was tetramethylsilane. Optical rotations were measured
on a Perkin–Elmer 343 polarimeter. MALDI TOF mass
spectra were registered on a Biflex III mass spectrome-
ter (Bruker, Germany) with laser ionization/desorption
(N2 laser 337 nm). The samples were dissolved in meth-
anol (10 mg/ml) and 1-µ]l aliquots were analyzed using
α-cyanohydroxycinnamic acid as a matrix. The sodium
content was determined on a Nippon Jarrel Ash AA-
780 atomic absorptive flame-emission spectrophotom-
eter. HPLC was carried out on a Du Pont Model 8800
chromatograph using a refractometric detector and Dia-
spher 110-C18 (5 µm, 4 × 250 mm) and YMC-Pack
ODS-A (5 µm, 12 nm, 10 × 250 mm) columns. Melting
points were determined on a Leica VMTG hot plate.
TLC was carried out on Sorbfil alufolien with fixed sil-
icagel CTX-1A (5–17 µm, Russia, Krasnodar) in a
5 : 1 : 1 BuOH–EtOAc–H2O system. Column chroma-
tography was carried out on silicagel L (80–100 and
200–250 mesh, Chemapol, Czechia), polychrom (Rus-
sia), and florisil (200–250 mesh, Merck, Germany).
Animals. Species of starfish T. über were gathered
in August 2003 (the Sea of Okhotsk, Kuril Islands) dur-
ing the twenty-nineth voyage of “Academik Oparin”
research ship using a drag-net from the depth of 100–
200 m and identified by Prof. V.S. Levin (Pacific Insti-
tute of Bioorganic Chemistry, Far East Division, Rus-
sian Academy of Sciences).
Biological tests. For determination of biological
activity, 10% DMSO solutions of the isolated com-
pounds with starting concentrations of 1–2 mg/ml were
used.
Steroid (I) completely terminated the first and the
second embryo divisions at the concentrations (ëmin
embr) of 50 µg/ml, and glycoside (XII) caused multiple
(up to 60%) anomalies in ovule division followed by
death at the blastula stage. Other compounds did not
affect the embryo development up to the blastula stage
when compared with the control.
Minimal concentrations inhibiting spermatozoon
mobilities of this sea-urchin species (éC50) were 15,
12, and 25 µg/ml for compounds (II), (XI), and (XII),
respectively; for other compounds, (I), (IV), and (V),
they were considerably higher (35–50 µg/ml). It is
known that, unlike sperm cells, sea-urchin egg-cells are
less susceptible to majority of toxic compounds.
Spermiotoxicities of compounds (II), (XI), and
(XII) were expressed in 50% inhibition of the ability of
spermatozoa to fertilize egg-cells (the ECA test); the
IC50 values were 6, 5, and 15 µg/ml, respectively. The
other steroids were low toxic and did not affect the fer-
tilizing ability of spermatozoa at concentrations lower
than 50 µg/ml.
Therefore, the spermiotoxic compounds II), (XI),
and (XII) can affect the embryo development in differ-
ent manner. Particularly, the sulfated polyol (II) dis-
plays the highest toxicity toward sperm cells (OC50
12 µg/ml) and sea-urchin embryos (IC50 6 µg/ml) and
seems to block the activity of protein-synthesizing
apparatus in cell, which is elevated by several orders of
magnitude immediately after fertilization [15]. Granu-
latoside B (XI) is also spermiotoxic, but is inactive
toward the unfertilized ovules. Monoside (XII) is less
toxic toward spermatozoa (éC50 25, IC50 15 µg/ml), but
terminated embryo development at the blastula stage,
most probably, due to inhibition of transcription in the
sea-urchin ovules. It is known that the nuclear morpho-
genetic function starts running from the middle blastula
stage, since mRNA and major sea-urchin proteins are
synthesized during oogenesis [15].
(a) Cytostatic activity in tests with unfertilized
egg-cells and embryos from sea-urchin S. interme-
dius. Unfertilized egg-cells (6 × 103 cell/ml) were
stored in sea water at concentrations of the tested com-
pounds of 25 and 50 µg/ml for 14 h. Fertilized egg-cells
were stored under the same conditions until the blastula
developmental stage. The preparation of sex cells and
fertilization of egg-cells were carried out as described
in [16].
One can presume that high activities of these com-
pounds result from the presence of such structural frag-
(b) The effect of the compounds on the mobility
ments as two monosaccharide chains localized at dif- of sea-urchin S. intermedius spermatozoa. Male
ferent ends of the molecule (at C3 and C24 in (XI)) and gametes (2 × 107 cell/ml) were stored in sea water (a
sulfated steroid nucleus [at C15 in (II)]. In addition, it control) and in sea water with the tested compounds at
is shown that localization of monosaccharide residues the corresponding concentrations for 1–4 h until com-
in single-chained monosaccharides can also affect the plete loss of their mobility was observed. The life-time
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

TROFOSIDES A AND B AND OTHER CYTOSTATIC
339
observation of the cell movements was conducted using fozide A (I), ë33H58O9, colorless crystals, mp
129−131oë (MeOH), [α]2D0 + 8.3 (Ò 0.36, MeOH).
an Amplival microscope (Carl Zeiss, Germany) and
30× magnification. The results of biotests were esti-
MALDI TOF MS: m/z 621 [M + Na]+ (I = 100 %). 1ç
mated by minimal concentrations of the tested com-
13
and C NMR (CD3OD) spectral data are given in the
pounds causing 50% spermatozoon immobility under
standard conditions (éC50) [17].
table.
(24S)-5a-cholestan-3b,6b,8,15a,24-pentaol 15-
(c) The effect on the fertilizing ability of sea-
urchin S. intermedius spermatozoa (the SFA tests).
Egg-cells (0.1 ml, 30000 cell/ml) were added to sea-
urchin S. intermedius spermatozoa (1.5 × 106 cell/ml)
preliminarily incubated for 20 min in 0.9 ml of sea
water containing the tested compounds. Then, the fer-
tilized cells with clearly separated envelope were
counted. The number of egg-cells fertilized by sperma-
tozoa following incubation in filtered sea water was
taken as 100% (a control). For the estimation of the
results of biotests, standard statistical methods were
used that ensure compound concentrations inhibiting
fertilization by 50% (IC50) [18].
sulfate sodium salt (II), ë27H47O5SO3Na, amorphous,
[α]2D0 + 20.4 (Ò 0.7, MeOH); MS(+), m/z 531 [M + Na]+
(I 100%); 1ç and 13C NMR (CD3OD) spectral data are
given in the table.
(20R,22E,24S)-24-O-(3-O-Methyl-b-D-xylopyra-
nosyl)-3b,6a,8,15b,24-pentahydroxy-5a-cholest-
22-ene (III), trofoside B; ë33H56O9, [α]2D0 + 8.9 (Ò
0.46, MeOH), contained (I) as an admixture; 1H NMR:
0.86 (3 H, d, J 6.8 Hz, H26); 0.92 (3 H, d, J 6.8 Ao,
H27); 5.38 (2 H, overlapped dd, H22 and H23); 13C
NMR: 57.6 (C17), 16.7 (C18), 14.0 (C19), 40.8 (C20),
20.8 (C21), 141.1 (C22), 128.1 (C23), 89.2 (C24), 33.8
(C25), 18.3 (C26), 19.3 (C27); MS, m/z 619 [M + Na]+
(I 100%), 621 [M + Na]+ (I 30%).
Extraction and isolation of total fractions.
Ground starfish T. über (animal mass of 0.108 kg) were
exhaustively extracted with 95% ethanol at room tem-
perature. The combined alcohol extract was evaporated
in a vacuum to dryness (25.4 g) and chromatographed
on a silica gel (50–100 mesh) column (6 × 25 cm) in
(20R,24S)-3b,6a,8,15b,24-Pentahydroxy-5a-
cholestane (IV); ë27H48O5, amorphous compound,
CHCl3–EtOH (100 : 0
50 : 50). Increasing the eluent
[α]2D0 + 10.8 (Ò 0.1, MeOH); EI MS: fragmentation
peaks corresponding to splitting of five H2O molecules
with m/z 434 (M – 4H2O)+, 416 (M – 2H2O)+, 398 (M –
polarity, two polyhydroxysteroid fractions: a less polar
fraction 1 (0.608 g) containing trofosides A (I) and B
(III) and compound (IV), and a more polar fraction 2
(0.253 g) composed of compounds (II) and (V) were
obtained.
3H2O)+, 380 (M – 4H2O)+, 362 (M – 5H2O); 1ç and 13C
NMR spectra (CD3OD) of steroid (IV agreed with
those for polyoxysteroid from G. watsoni starfish ear-
lier reported in [12].
Isolation of steroid derivatives (I)–(V). Fraction 1
was dissolved in water and separated into fractions on
a Polychrom column (3 × 12 cm) by eluting the column
with water and 50% ethanol. The water–ethanol eluate
was concentrated in a vacuum to a brown gum residue
(0.41 g), which was successively chromatographed on
a Florisil column (1.5 × 20 cm, 200–300 mesh) in a
CHCl3–EtOH system (9 : 1 to 6 : 4) and then on a silica
gel column (3 × 10 cm) in a CHCl3–EtOH system (9 : 1
to 7 : 3). The fraction containing steroid (IV) with small
amount of admixtures (2.9 mg) was purified by HPLC
on a Diaspher 110-C18 column in a MeOH–H2O
(70 : 30) system to give (IV) (2.5 mg, 0.002% of the
animal weight). Glycoside (I) was isolated by HPLC in
the same system on a Diaspher 110-C18 column to give
trofoside A (I) (5.5 mg, 0.005%) and a mixture of tro-
fosides B (III) and A (I) (5 mg).
Fraction 2 was chromatographed on Polychrom in a
similar manner. The water–ethanol fraction was evapo-
rated to dryness (0.160 g) and successively chromato-
graphed on a Florisil column in a CHCl3–EtOH system
(8 : 2 to 5 :5) and then by HPLC on a YMC-Pack
ODS-A column in aqueous MeOH (60%) to give sul-
fated polyol (II) (0.005%) and triseramide (V) (19 mg,
0.01%).
(20R,22E,24R,25S)-24-Methyl-3b,6a,8,15b-tet-
rahydroxy-5a-cholest-22-en-27-oic acid (2-sulfoet-
hyl)amide Na-salt (V), triseramide; mp 182–183.5°C
(MeOH); [α]2D0 –13.4 (Ò 1.7, MeOH). MALDI TOF (+)
MS; 1ç and 13C NMR spectra (pyridine-d5) agreed with
those for the corresponding steroid conjugate from A.
triseriatus starfish earlier reported in [13].
Hydrolysis of triseramide (V) and taurin identifi-
cation. A solution of (V) (3 mg) in 2 M HCl (1 ml) was
heated on a water bath for 2 h. Taurin was identified in
the hydrolyzate by comparison with the authentic sam-
ple by TLC (silica gel, butanol–ethyl acetate–water
5 : 1 : 1; detected with ninhydrin); 1H NMR (pyridine-
d5): 4.20 (2 H, m, H29); 3.45 (2 H, t, J 6.8 Hz, H30);
13
8.42 (1 H, t, J 5.0 Hz, NH). C NMR (pyridine-d5):
36.1 (C29), 51.5 (C30). The spectral data agreed with
those for the reference sample of taurin.
ACKNOWLEDGMENTS
The authors are thankful to Academician
V.A. Stonik for discussions and fruitful advice.
(20R,24S)-24-O-(3-O-Methyl-b-D-xylopyrano-
The work was supported by the Russian Foundation
syl)-3b,6a,8,15b,24-pentahydroxy-5a-cholestane, tro- for Basic Research, project no. 05-04-48246, the Pres-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

340
LEVINA et al.
8. D’Auria, M.V., Fontana, A., Minale, L., and Riccio, R.,
ident grant NSh-6491.2006.4; the grant of Russian
Academy of Sciences, Far East Division, project
no. 06-III-A-05-121; and the program of Presidium of
Russian Academy of Sciences of 2006 on Molecular
and Cell Biology.
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