Isolation and structures of erylosides from the carribean sponge Erylus formosus

Article abstract of DOI:10.1021/np060364q

Nine new triterpene glycosides, erylosides F₁-F₄ (1-4), M (5), N (6), O (7), P (8), and Q (9), along with previously known erylosides F (10) and H (11) were isolated from the sponge Erylus formosus collected from the Mexican Gulf (Puerto Morelos, Mexico). Structures of 1-4 were determined as the corresponding biosides having aglycons related to penasterol with additional oxidation patterns in their side chains. Erylosides 5-9 contain new variants of carbohydrate chains with three (5, 6), four (7), and six (8, 9) sugar units, respectively. Erylosides 5, 7, 8, and 6, 9 contain 14-carboxy-24-methylenelanost-8(9)-en-3β-ol and penasterol as aglycons, respectively. In contrast with its epimer 2, the compound 3 induced the early apoptosis of Ehrlich carcinoma cells at a concentration of 100 μg/mL, while 1 and 10 activated the Ca²⁺ influx into mouse spleenocytes (130% of the control) at the same doses.

Full text of DOI:10.1021/np060364q

J. Nat. Prod. 2007, 70, 169-178
169
Isolation and Structures of Erylosides from the Carribean Sponge Erylus formosus
Alexandr S. Antonov,† Anatoly I. Kalinovsky,† Valentin A. Stonik,*,† Shamil S. Afiyatullov,† Dmitry L. Aminin,†
Pavel S. Dmitrenok,† Ernesto Mollo,‡ and Guido Cimino‡
Laboratory of the MaNaPro Chemistry, Pacific Institute of Bioorganic Chemistry, VladiVostok-22, Russian Federation, and
Instituto di Chimica Biomoleculare (ICB-CNR), Via Campi Flegerei, 80078 Pozzuoli, Napoly, Italy
ReceiVed July 24, 2006
Nine new triterpene glycosides, erylosides F1-F4 (1-4), M (5), N (6), O (7), P (8), and Q (9), along with previously
known erylosides F (10) and H (11) were isolated from the sponge Erylus formosus collected from the Mexican Gulf
(Puerto Morelos, Mexico). Structures of 1-4 were determined as the corresponding biosides having aglycons related to
penasterol with additional oxidation patterns in their side chains. Erylosides 5-9 contain new variants of carbohydrate
chains with three (5, 6), four (7), and six (8, 9) sugar units, respectively. Erylosides 5, 7, 8, and 6, 9 contain 14-
carboxy-24-methylenelanost-8(9)-en-3â-ol and penasterol as aglycons, respectively. In contrast with its epimer 2, the
compound 3 induced the early apoptosis of Ehrlich carcinoma cells at a concentration of 100 µg/mL, while 1 and 10
activated the Ca2+ influx into mouse spleenocytes (130% of the control) at the same doses.
The triterpene glycosides erylosides have been reported from
several marine sponges belonging to the genus Erylus.1-6 Some
glycosides of this structural group were described using other names,
for example, nobiloside from Erylus nobilis,7 formoside from Erylus
formosus,8 and feroxosides A and B from Ectyoplasia ferox.9 There
are two series of erylosides differing from each other by the aglycon
structure, namely, (a) glycosides derived from penasterol or
congeners carrying additional substituents at C-24, C-25, or both
and (b) glycosides derived from 4R-methyl-5R-cholesta-8,14-dien-
3â-ol or related nortriterpenoids. Penasterol or 14-carboxylanosta-
8,24-dien-3â-ol was originally isolated from the sponges Penares
sp.10 and Penares incrustans11,12 and showed potent antileukemic
activity and inhibitory action against IgE-dependent histamine
release from rat mast cells.
Erylosides also exhibit a wide spectrum of biological activities.
For example, eryloside A possesses antitumor and antifungal
properties.1 Eryloside E is an antagonist of C5a-receptor binding.3
Eryloside F was reported as a potent thrombin receptor antagonist
and activator of intacellular calcium influx.4 Erylosides G-J exhibit
moderate cytotoxicity against a human leukemia cell line.5 No-
biloside inhibits neuroaminidase from the bacterium Clostridium
perfringes.7 In addition, erylosides and related triterpene glycosides
from marine sponges appear to have multiple ecological functions.
It was shown that the top layers and swabs of the surfaces of the
sponge Erylus formosus contained sufficiently high concentrations
of erylosides to deter enemies such as reef fishes, bacterial
settlement, and fouling.13,14 It is of special interest that a mixture
of triterpene glycosides protected E. formosus from predatory reef
fishes, but when these compounds were separated into fractions,
they failed to deter feeding at natural concentrations.1
paper we described the isolation and structural elucidation of nine
new glycosides.
Results and Discussion
The ethanolic extract of the sponge E. formosus collected off
Puerto Morelos (the Caribbean Sea, Mexico) was evaporated in
Vacuo and separated by flash column chromatography on silica gel
L followed by HPLC on a Supelco Sil and Supelco C-18 columns
to give four new biosides (1-4), two new triosides (5, 6), one new
tetraoside (7), and two new hexaosides (8, 9) as white, amorphous
solids, along with the previously known bioside (10) and trioside
(11). A portion of the bioside glycoside mixture containing 1-4
and 10 was hydrolyzed with 10% hydrochloric acid with heating.
D-galactose and L-arabinose were identified as the major sugars
after their isolation by preparative TLC and comparison of optical
rotation data with those of standard monosaccharides. Taking into
consideration that one of the biosides, namely, eryloside F (10),
was previously described,4 we have designated glycosides 1-4 as
erylosides F1-F4. A preliminary inspection of NMR spectra (Tables
1, 4, 5) of 1-4 suggested the glycotriterpene nature of all of these
compounds.
Structures of 1-4 were determined by extensive application of
spectroscopic methods, especially 2D NMR techniques. Analysis
of NMR spectra established that 1-4 had identical carbohydrate
moieties and the same tetracyclic core of aglycon as in the
previously known eryloside F.4 Actually, these spectra showed
characteristic features of biosides, namely, a series of signals at δH
3.0-5.0 and two signals of anomeric carbons at δC 104.6 (CΗ)
1
and 106.7 (CH). Interpretation of H-1H COSY spectrum gave
rise to spin systems of the sugars in 1-4, which were assigned as
R-arabinopyranose (Ara) and â-galactopyranose (Gal) by analysis
of HSQC, HMBC, and NOESY data as well as by 1H-1H coupling
constant values obtained from 1D-TOCSY experiments and J(C1H1)
values17 (Table 1). The pyranose nature of the sugars in 1-4 was
determined on the basis of HMBC correlations between the
anomeric methines and the C5-Ara methylene group and C5-Gal
methine group. All sugars were connected through â-glycosidic
linkages for those belonging to the D-series and R- for L-arabinose
as followed from 13C NMR shifts (δC > 100 ppm) and spin-
decoupling constants of anomeric protons. Taking into consideration
that L-arabinose and D-galactose were identified in the hydrolysate
of the glycoside fraction, HMBC correlations H1-Ara/C-3 and H1-
Gal/C2-Ara implied the sequence â-D-galactopyranosyl-(1f2)-R-
L-arabinopyranosyl in the carbohydrate moiety as well as the
attachment of this fragment to C-3 of an aglycon.
As a rule, erylosides are present in sponges as very complicated
mixtures of closely related glycosides. Probably, the individual
glycosides differ significantly from each other in their physiological
and ecological actions. Moreover, the minor constituents appear to
be more potent than the major glycosides, as was suggested by
Pawlik and collaborators.14
In continuation of our studies on glycosides from marine
sponges,15,16 we have isolated a series of new erylosides (1-9) from
the alcoholic extract of the Caribbean sponge Erylus formosus along
with the previously known erylosides F (10)4 and H (11).5 In this
* Corresponding author. Tel: 7(4232) 311168. Fax: 7(4232) 314050.
E-mail: stonik@piboc.dvo.ru.
† Pacific Institute of Bioorganic Chemistry.
‡ Instituto di Chimica Biomoleculare (ICB-CNR).
10.1021/np060364q CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/17/2007

170 Journal of Natural Products, 2007, Vol. 70, No. 2
AntonoV et al.
H3-19 with the signal of C-9 at δC 139.8. The chemical shifts of
C-14 at 62.7-62.8 and the carboxyl carbonyl at 178.25 along with
other HMBC results, given in Table 5, indicated that 1-4 contain
a 14-carboxylanost-8(9)-ene skeleton system. This was confirmed
by the results of NOESY experiments, which gave cross-peaks H-3/
H-5, H-3/H3-28, H-1/H-11, and H-12/H-17. Cross-peaks H-20/H3-
18 and H-12/H3-21 showed the R-configuration of C-20.15,16
Erylosides F1-F4 differed from each other in side chain structures
only. Eryloside F1 (1) analyzed for C42H68O12 by combined
HRESTOFMS (+) (pseudomolecular peak at m/z 787.4464 [M +
Na]+) and 13C NMR data. The structure of its side chain followed
1
from 13C and H NMR spectra (Tables 4, 5), indicating the same
characteristic features as those of the side chain of 24-methylene-
cholesterol. Actually, the signals of an exo-methylene group were
observed at δH 4.81 d (1.4) and 4.84 s as well as at δC 106.4 s
(C-24) and 106.4 (C-31). Along with key HMBC correlations such
as H-31 (δH 4.81 d, 4.84 s)/C-23 (31.4), C-24, C-25 (33.8) and
H3-26 (δH 1.03 d, 1.04 d)/C-24, C-25, all these data confirmed the
structure of eryloside F1 (1) as 3â-O-[â-D-galactopyranosyl-(1f2)-
R-L-arabinopyranosyl]-14-carboxy-24-methylenelanost-8(9)-en-3â-
ol.
The molecular formula of eryloside F2 (2) was deduced as
C41H66O13 by HRTOFMSES (+) (pseudomolecular peak at m/z
789.4371 [M + Na]+) and 13C NMR spectra. Spectroscopic data
of 2 were very similar to those of 1 and suggested that 2 had an
additional hydroxyl group, and a double bond in the side chain
was in another position when compared with 1. The difference in
the NMR spectra was the appearance of the CH-O signals at δH
4.35 m and δC 75.4 and the 25(26) double-bond signals at δH 4.95
m, 5.25 d (H2-26) and δC 149.7 s (C-25), 109.9 t (C-26) instead of
those of the 24(28) double bond. Positions of the hydroxyl group
at C-24 and the double bond as ∆25(26) were confirmed by HMBC
cross-peaks H-26/C-24, C-27 and H3-27/C-24, C-25, C-26.
Eryloside F3 (3) was shown to be a glycoside epimer of 2 at
C-24. Its molecular formula was also deduced as C41H66O13 by
HRTOFMSES (+) (pseudomolecular peak at m/z 789.4390 [M +
Na]+]) and 13C NMR spectroscopy. The significant difference was
only in the chemical shifts of proton and carbon atoms flanking
the hydroxyl group in the side chain. The chemical shifts of side
chain carbons of two epimeric 12-O-(â-D-glucopyranosyl)-
(20S,24R,S)-3-ketodammaran-25(26)-ene-20,24-diols, containing
similar side chains, whose structures were established by X-ray
analysis,18 were earlier reported by Malinovskaya et al.19 These
data indicated that the signals of C-24 and C-26 were low-field
shifted, while the signal of C-27 was higher field shifted in the
spectrum of the 24S-isomer, when compared with that of the 24R-
isomer. Similar, consistent patterns were found by us on comparison
of the 13C NMR spectra of 2 and 3 (Table 4). On this basis, we
assigned the configuration of C-24 as R in 2 and as S in 3. Thus,
structures of 2 and 3 were established as (24R)- and (24S)-3â-O-
Figure 1. Structures of erylosides 1-6 and 11.
Inspection of COSY-45 spectra of 1-4 allowed us to detect
several distinct spin systems belonging to the tetracyclic aglycon
moiety. The H(3)-H2(2)-H2(1) spin system and the H2(11)-H2-
(12) sequence were revealed from signals H-3 [δH 3.12 dd] and
H2-12 (δH 1.83 and 2.83 m), respectively. The latter signal gave a
cross-peak with H3-18 (δH 0.92s). The proton sequence H(17)-
H2(16)-H2(15) was indicated from the signal H-17 [δH 2.03 brq
(9.9)], which gave cross-peaks with H-20 [δH 1.56 m)] and H3-18.
Finally, the proton sequence H(5)-H2(6)-H2(7) was indicated from
the signals of H-5 [δH 1.24 dd (12.8, 2.0)]. The positioning of a
double bond at ∆8(9) was established by long-range correlation of
Table 1. 1H and 13C NMR Data for Carbohydrate Moieties of Erylosides 1-4a in C5D5N
position
δC (JC,H in Hz)
δH (J in Hz)
HMBC
NOESY
Ara (1fC-3)
1
2
3
4
5
104.6 CH (162.6)
81.3 CH
73.2 CH
68.0 CH
64.7 CH2
4.91 d (5.6)
C-3, C2,5-Ara
H-3, H3-Ara
H1-Ara
4.56 dd (5.5, 7.4)
4.33 dd (3.6, 7.6)
4.36 m
3.80 dd (2.5, 11.9)
4.31 dd (4.9, 11.7)
C1-Ara
C3,4-Ara
Gal (1f2Ara)
1
2
3
4
5
6
106.7 CH (156.9)
73.7 CH
75.0 CH
69.4 CH
76.6 CH
5.05 d (7.7)
C5-Gal, C2-Ara
C3-Gal, C1-Ara
C2-Gal
C3-Gal
C4,6-Gal
H3,5-Gal
H1-Gal
H1-Gal
4.55 dd (7.6, 9.5)
4.13 dd (3.4, 9.5)
4.67 brd (3.1)
3.98 brdd (6.0, 7.4)
4.37 dd (5.6, 10.6)
4.57 dd (8.0, 10.3)
61.1 CH2
C4,5-Gal
C4,5-Gal
a All assignment were given for 1; spectra of 2-4 had only minor differences in chemical shift values.

Erylosides from the Carribean Sponge Erylus formosus
Journal of Natural Products, 2007, Vol. 70, No. 2 171
Table 2. 1H and 13C NMR Data for Carbohydrate Moieties of Erylosides 5 and 6a in C5D5N
position
δC
δH (J in Hz)
HMBC
NOESY
Ara1 (1fC-3)
1
2
3
4
5
104.9 CH
77.0 CH
81.3 CH
68.0 CH
65.1 CH2
4.83 d (5.5)
4.72 dd (5.9, 8.4)
4.32 dd (3.2, 8.4)
4.49 m
C-3, C5-Ara1
C3-Ara1, C1-Gal
C2-Ara1
H-3, H3,5-Ara1
H1-Gal
H1-Ara1, H1-Ara2
3.75 m
H1-Ara1
4.28 m
C3,4-Ara1
Ara2 (1f3Ara1)
1
2
3
4
5
104.7 CH
72.3 CH
74.2 CH
69.0 CH
66.2 CH2
5.17 d (7.0)
4.49 brt (8.0)
4.15 dd (3.1, 8.7)
4.31 m
3.74 m
4.30 m
C3,5-Ara2, C3-Ara-1
H3,5-Ara1
H1-Ara2
H1-Ara2
C2-Ara2
C2,3-Ara2
C3-Ara2
C4-Ara2
Gal (1f2Ara1)
1
2
3
4
5
6
105.0 CH
73.2 CH
75.3 CH
69.5 CH
76.1 CH
61.3 CH2
5.30 d (7.8)
4.48 brt (8.9)
4.09 dd (3.4, 9.6)
4.61 brd (3.9)
3.75 m
C2-Ara1
C4-Gal
C2-Gal
C2,3-Gal
C1,4,6-Gal
C4,5-Gal
C4,5-Gal
H3,5-Gal, H2-Ara1
H1-Gal
H1-Gal
4.27 m
4.47 m
a All assignments were given for 5; spectra of 6 had only minor differences in chemical shift values.
[â-D-galactopyranosyl-(1f2)-R-L-arabinopyranosyl]-14-carboxyl-
anosta-8,25-diene-3â,24-diols, respectively.
Eryloside N (6) had the molecular formula C46H74O16, which
was determined by high-resolution MALDI-TOFMS (m/z 905.4870
[M + Na]+) and spectroscopic data. The general features of the 1H
and 13C NMR spectra of the aglycon part of 6 closely resembled
those of eryloside M (5) (Tables 4-6) with the exception of proton
and carbon signals belonging to the side chain. Positioning of the
double bond in the side chain as ∆24(25) followed from HMBC cross-
peaks H-24/C-26 (δC 17.4), C-27 (δC 25.5) and H3-26 (δH 1.60
brs)/C-24 (δC 125.5), C-25 (δC 130.6), and C-27. Stereochemical
peculiarities of the lanosterol-derived core of 6 were confirmed by
NOESY correlations H-5 (δH 1.25 m)/H-3 (δH 3.12 dd) and H3-
18/H-20 (δH 1.56 m) and H3-21 (δH 1.05 d)/H-12 (δH 1.82 m). On
the basis of all these data and comparison with literature7,8 the
aglycon of 6 was identified as penasterol. The attachment of the
carbohydrate chain to C-3 was confirmed by cross-peaks H-3 (δH
3.12 dd)/H1-Ara1 (δH 4.83 d) in the NOESY spectrum. Taking
into account that 5 and 6 had identical carbohydrate chains, the
structure of 6 was represented as 3â-O-{[â-D-galactopyranosyl-
(1f2)]-[R-L-arabinopyranosyl-(1f3)]-R-L-arabinopyranosyl}-14-
carboxylanosta-8,24-dien-3â-ol.
Eryloside O (7) analyzed for C54H86NO20 on the basis of
combined HR-MALDI-TOFMS (m/z 1092.5827 [M + Na]+) and
13C NMR analysis. NMR spectra exhibited the tetrasaccharide
nature of this molecule. In fact, there were signals of four anomeric
carbons at δC 101.6 (CH), 105.1 (CH), 106.2 (CH), and 107.6 (CH)
and those of the corresponding protons at δH 5.50, 4.67, 5.01, and
5.07 (Table 3). Identifications of sugars as three arabinose units
and one 2-N-acetylglucosamine unit were based on GC and NMR
data. The D-configuration of 2-N-acetylglucosamine and the L-
configurations of arabinoses were determined after acid hydrolysis
by preparation of acetylated (-)- and (+)-2-octylglycosides fol-
lowed by GC and comparison with those obtained from standard
sugars.20 The pyranose nature of the sugars and configurations of
glycosidic bonds were determined in the same manner as described
above. The arrangement of sugar units was established by extensive
NMR experiments. Assignments of proton and carbon signals of
the carbohydrate chain were carried out by DEPT, HSQC, 1D-
TOCSY, and COSY-45 NMR spectra. With this information in
hand, NOESY and HMBC data were used to link carbohydrate units
together. The long-range correlations of H1-2Ara with C3-Ara1
and H1-NAc-Glc with C2-Ara1 along with NOESY cross-peaks
between H2-Ara1 and H1-NAc-Glc, H3-Ara1 and H1-Ara2 (Table
3) defined the 1,3-linkage between arabinose-2 and arabinose-1 and
the 1,2-linkage between 2-N-acetyl-D-glucosamine and arabinose-
Eryloside F4 (4) was analyzed for the molecular formula
C41H64O13 by HRTOFMSES (+) (pseudomolecular peak at m/z
787.4272 [M + Na]+) and 13C NMR data. In accordance with NMR
data (Tables 4, 5) glycoside 4 differed from 2 and 3 in the presence
of a 24-ketone instead of a 24-hydroxyl group. The chemical shift
of the carbonyl carbon at δC 202.0 and HMBC correlations H-26
(δH 5.70 s, 5.97 s)/C-24 (δC 202.0), C-27 (δC 17.6) and H3-27 (δH
1.90 s)/C-24, C-25 (δC 144.4) and C-26 (δC 124.3) confirmed this
suggestion. From all these data, eryloside F4 (4) was structurally
identified as 3â-O-[â-D-galactopyranosyl-(1f2)-R-L-arabinopyra-
nosyl]-14-carboxy-3â-hydroxy-lanosta-8,25-dien-24-one.
Both new triosides designated as erylosides M (5) and N (6)
contained identical carbohydrate chains (Table 2). The trisaccharide
nature of 5 and 6 was evident from the signals of three anomeric
carbons at δC 104.7 (CH), 104.9 (CH), and 105.0 (CH) and those
of the corresponding protons at δH 5.17, 4.83, and 5.30 in the NMR
spectra. The pyranose nature of the sugars and â-glycosidic linkages
between monosaccharide units (R- for L-arabinoses) as well as the
identification of sugar units as L-arabinose and D-galactose (2:1)
were determined in the same manner as for 1-4. Their arrangement
was established by extensive NMR experiments. The long-range
correlations of H2-Ara1 with C1-Gal and H3-Ara1 with C1-Ara2
along with NOESY cross-peaks between H2-Ara1 and H1-Gal, H3-
Ara1, and H1-Ara2 defined the 1,3-linkage between the two
arabinoses and 1,2-linkage between galactose and Ara1 (Table 2).
Thus, this moiety was shown to be a branched assembly of two
units of L-arabinose and one unit of D-galactose, containing an
additional L-arabinose unit attached to C-3 of Ara1 when compared
with the carbohydrate chains of 1-4 (Table 1).
Eryloside M (5) analyzed for C47H76O16 by combined HR-
MALDI-TOFMS (m/z 919.5138 [M + Na]+) and 13C NMR
spectrometry. The spectroscopic data of the aglycon moiety of 5
were highly compatible with those obtained for 1. The positioning
of two double bonds as ∆8(9) and ∆24(31) as well as the presence of
a carboxyl group at C-14 were established in the same manner as
described above for eryloside F1 (Tables 4, 5). The attachment of
a carbohydrate chain to C-3 was confirmed by cross-peaks H-3
(δH 3.12 dd)/H1-Ara1 (δH 4.83 dd) and H1-Ara1/C-3 (δC 88.4-
88.5) in the NOESY and HMBC spectra, respectively. Therefore,
the structure of eryloside M (5) was represented as 3â-O-{[â-D-
galactopyranosyl-(1f2)]-[R-L-arabinopyranosyl-(1f3)]-R-L-ara-
binopyranosyl}-14-carboxy-24-methylenelanost-8(9)-en-3â-ol.

172 Journal of Natural Products, 2007, Vol. 70, No. 2
AntonoV et al.
Table 3. 1H and 13C NMR Data for the Carbohydrate Moiety of Eryloside 7 in C5D5N
position
δC
δH (J in Hz)
HMBC
NOESY
Ara1 (1fC-3)
1
2
3
4
5
105.1 CH
76.1 CH
84.6 CH
69.3 CH
66.4 CH2
4.67 d (8.1)
4.67 m
4.22 dd (2.6, 8.6)
4.58 brs
4.22 m
H-3, H5-Ara1
H1-NAc-Glc
H1-Ara1, H1-Ara2
C2-Ara1
C2-Ara1
C1,4-Ara1
C1,4-Ara1
3.76 brd (11.3)
H1-Ara1
Ara2 (1f3Ara1)
1
2
3
4
5
106.2 CH
73.2 CH
74.6 CH
79.8 CH
66.7 CH2
5.01 d (7.7)
4.43 brt (9.5)
4.15 brd (10.5)
4.40 brd (3.6)
4.54 dd (2.0, 12.5)
3.89 brd (11.6)
C3-Ara-1
H3,5-Ara2, H3-Ara1
C1,3-Ara2
H1-Ara2
H1-Ara-3
C2-Ara2, C1-Ara3
C1,3,4-Ara2
C1,4-Ara2
H1-Ara2
Ara3 (1f4Ara2)
1
2
3
4
5
107.6 CH
72.9 CH
74.5 CH
69.6 CH
67.4 CH2
5.07 d (7.6)
C4-Ara2
C1-Ara3
C2-Ara3
C2-Ara3
C1,3,4-Ara3
C1,4-Ara3
H3,5-Ara3, H4-Ara2
H1-Ara3
4.51 dd (7.7, 9.1)
4.12 dd (3.5, 9.2)
4.28 brs
4.33 dd (2.2, 12.4)
3.78 brs (11.6)
H1-Ara3
NAc-Glc (1f2Ara1)
1
2
3
4
5
6
101.6 CH
57.2 CH
78.1 CH
72.9 CH
76.6 CH
63.2 CH2
5.50 d (8.5)
4.63 brq (9.0)
4.00 t (9.8)
4.07 t (9.2)
3.21 m
C2-Ara1
H3,5-NAc-Glc, H2-Ara1
H1,5-NAc-Glc
H1,3-NAc-Glc
C1,3-NAc-Glc
C2,4-NAc-Glc
C3,5,6-NAc-Glc
4.24 m
4.14 m
NH
Ac
8.66 d (9.4)
23.5
170.3
Table 4. 13C NMR Data in C5D5N for the Aglycon Moiety of Erylosides (1-9)
position
1
2
3
4
5
6
7
8
9
1
2
3
4
35.3 CH2
26.8 CH2
88.4 CH
39.4 C
35.3 CH2
26.8 CH2
88.4 CH
39.4 C
35.3 CH2
26.8 CH2
88.4 CH
39.4 C
35.3 CH2
26.8 CH2
88.4 CH
39.4 C
35.3 CH2
26.9 CH2
88.5 CH
39.6 C
35.3 CH2
26.9 CH2
88.4 CH
39.6 C
35.3 CH2
26.9 CH2
88.8 CH
39.5 C
35.3 CH2
26.9 CH2
88.6 CH
39.6 C
35.3 CH2
26.9 CH2
88.6 CH
39.6 C
5
6
7
8
50.1 CH
18.4 CH2
27.9 CH 2
128.0 C
139.8 C
37.4 C
50.2 CH
18.4 CH2
27.9 CH2
128.0 C
139.8 C
37.4 C
50.2 CH
18.4 CH2
27.9 CH2
128.0 C
139.8 C
37.4 C
50.2 CH
18.4 CH2
27.9 CH2
128.0 C
139.8 C
37.4 C
50.3CH
50.2 CH
18.4 CH2
27.9 CH2
127.9 C
139.9 C
37.4 C
50.3 CH
18.4 CH2
27.9 CH
128.0 CH 2
139.9 C
50.2 CH
18.4 CH2
27.9 CH
127.9 CH 2
139.9 C
50.3 CH
18.6 CH2
27.9 CH
127.9 CH 2
139.9 C
18.4 CH2
27.9 CH2
128.0 C
139.9 C
37.4 C
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
37.4 C
37.4 C
37.4 C
22.5 CH2
31.7 CH2
47.0 C
22.5 CH2
31.7 CH2
46.9 C
22.5 CH2
31.7 CH2
46.9 C
22.5 CH2
31.6 CH2
46.9 C
22.5 CH2
31.7 CH2
47.0 C
22.5 CH2
31.7 CH2
46.9 C
22.5 CH2
31.7 CH2
47.0 C
22.5 CH2
31.7 CH2
46.9 C
22.5 CH2
31.7 CH2
46.9 C
62.8 C
62.8 C
62.8 C
62.7 C
62.8 C
62.8 C
62.8 C
62.8 C
62.8 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.6 CH3
36.3 CH
18.6 CH3
35.0 CH2
31.4 CH2
156.4 C
33.8 CH
21.7 CH3
21.9 CH3
27.6 CH3
16.4 CH3
178.3 C
28.3 CH2
29.5 CH2
51.2 CH
17.9 CH3
19.6 CH3
36.3 CH
18.8 CH3
32.3 CH2
32.6 CH2
75.4 CH
149.7 C
109.9 CH2
17.9 CH3
27.6 CH3
16.4 CH3
178.2 C
28.3 CH2
29.5 CH2
51.1 CH
17.9 CH3
19.6 CH3
36.4 CH
18.8 CH3
32.4a CH2
32.5a CH2
75.9 CH
149.7 C
110.2 CH2
17.5 CH3
27.6 CH3
16.4 CH3
178.2 C
28.3 CH2
29.4 CH2
51.0 CH
17.9 CH3
19.5 CH3
36.0 CH
18.4 CH3
31.1 CH2
34.7 CH2
202.0 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.6 CH3
36.3 CH
18.6 CH3
35.0 CH2
31.4 CH2
156.5 C
33.8 CH
21.7 CH3
21.9 CH3
27.6 CH3
16.4 CH3
178.2 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.6 CH3
36.0 CH
18.5 CH3
36.3 CH2
25.0 CH2
125.5 C
130.6 C
17.4 CH3
25.5 CH3
27.5 CH3
16.4 CH3
178.3 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.5 CH3
36.3 CH
18.6 CH3
35.0 CH2
31.4 CH2
156.5 C
33.8 CH3
21.7 CH3
21.8 CH3
27.5 CH3
16.4 CH3
178.2 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.6 CH3
36.3 CH
18.6 CH3
35.0 CH2
31.4 CH2
156.4 C
33.8 CH3
21.7 CH3
21.9 CH3
27.5 CH3
16.4 CH3
178.2 C
28.3 CH2
29.5 CH2
51.0 CH
17.9 CH3
19.6 CH3
36.0 CH
18.6 CH3
36.3 CH2
25.0 CH2
125.5 CH
130.6 C
17.4 CH3
25.6 CH3
27.5 CH3
16.4 CH3
178.2 C
144.4 C
124.3 CH2
17.6 CH3
27.6 CH3
16.4 CH3
178.2 C
106.4 CH2
106.4 CH2
106.4 CH2
106.4 CH2
a Interchangeable signals.
1. The HMBC cross-peak H1-Ara3 (δH 5.07)/C4-Ara2 (δC 79.8)
as well as the NOESY correlation H1-Ara3/H4-Ara2 (δH 4.40)
showed that arabinose-3 was bonded with arabinose-2 by a 1,4-
linkage. On the basis of these data the carbohydrate moiety was
shown to be a branched assembly of three units of L-arabinose and
one unit of 2-N-acetyl-D-glucosamine. It contains an additional
L-arabinose unit attached to C-4 of arabinose-2 and 2-N-acetyl-D-
glucosamine instead of galactose, when compared with the carbo-
hydrate chains of 5 and 6.The spectroscopic data of the aglycon
moiety of 7 were highly compatible with those obtained for 1 and

Table 5. 1H NMR Data in C5D5N for the Aglycon Moiety of 1-5
1
2
3
4
5
δH (J, Hz)
1.28 m
1.73 m
1.88 m
2.14 m
HMBC
δH (J, Hz)
1.28 m
1.72 m
1.88 m
2.13 m
HMBC
δH (J, Hz)
1.29 m
1.73 m
1.86 m
2.14 m
HMBC
δH (J, Hz)
1.28 m
1.72 m
1.87 m
2.13 m
HMBC
δH (J, Hz)
1.71 m
1.29 m
2.09 m
1.86 m
NOESY
HMBC
1
2
H-11
3
5
6
3.12 dd (4.5, 12.0)
1.24 dd (2.0, 12.8)
1.55 m
1.70 m
2.26 m
3.11 dd (4.5, 11.9)
1.24 dd (2.1, 12.6)
1.54 m
1.68 m
2.25 m
3.11 dd (4.4, 11.9)
1.24 dd (2.0, 12.4)
1.53 m
1.68 m
2.24 m
3.12 dd (4.3, 11.7)
1.24 dd (2.0, 12.5)
1.54 m
1.68 m
2.25 m
3.12 dd (4.2,11.7) H-5, H1-Ara1 C-4,28,29
1.26 dd (2.1,12.5) H-3
1.70 m
1.54 m
2.34 m
2.24 m
7
2.33 m
2.32 m
2.32 m
2.31 m
11 2.17 m
2.41 m
12 1.83 m
2.83 m
15 1.76 m
2.52 m
16 1.52 m
2.40 m
2.16 m
2.41 m
1.82 m
2.82 m
1.75 m
2.51 m
1.57 m
2.44 m
2.17 m
2.41 m
1.82 m
2.82 m
1.74 m
2.50 m
1.56 m
2.43 m
2.15 m
2.40 m
1.80 m
2.80 m
1.73 m
2.50 m
1.57 m
2.40 m
2.42 m
2.16 m
2.83 m
1.83 m
2.53 m
1.75 m
2.40 m
1.52 m
2.03 brq
(9.7)
H-1
H-17
H3-21
C-30
C-30
C-30
C-30
C-30
C-30
17 2.03 brq
(9.9)
2.06 brq
(10.0)
2.06 brq
(9.5)
2.02 brq
(10.0)
H-12
H-20
18 0.92 s
19 1.09 s
20 1.56 m
21 1.06 d (7.0)
22 1.20 m
1.69 m
23 1.96 m
2.20 m
24
C-12,13,14,17 0.91 s
C-12,13,14,17 0.90 s
C-12,13,14,17 0.89 s
C-12,13,14,17 0.91s
C-12,13,14,17
C-1,5,9,10
C-1,5,9,10
1.09 s
C-1,5,9,10
1.09 s
C-1,5,9,10
1.08 s
C-1,5,9,10
1.08s
1.63 m
1.08 d (6.5)
1.41 m
1.74 m
1.70 m
1.94 m
4.35 m
1.61 m
1.07 d (6.5)
1.19 m
1.88 m
1.75 m
1.93 m
4.34 m
1.55 m
1.01 d (6.5)
1.38 m
1.96 m
2.67 m
2.78 m
1.56 m
1.05 d (6.5)
1.69 m
1.20 m
2.20 m
1.96 m
H-18
H-12
C-17,20,22
C-17,20,22
C-17,20,22
C-17,20,22
C-17,20,22
25 2.24 m
26 1.04 d (6.5)
2.23 m
1.04 d (6.5)
C-23,24,26,31
C-24,25,27
4.95 m
5.25 d (2.5)
1.90 s
1.18 s
1.11 s
C-27, C-24
C-24, C-27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
4.94 m
5.20 d (2.5)
1.90 s
1.18 s
1.11 s
C-27, C-24
C-24, C-27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
5.70 s
5.97 s
1.90 s
1.18 s
1.11 s
C-24, C-27
C-27, C-24
C-24,25,26
C-3,4,5,29
C-3,4,5,28
27 1.03 d (6.5)
28 1.19 s
29 1.11 s
31 4.81 d (1.4)
4.84 s
1.03 d (6.5)
1.23 s
1.17 s
4.84 brs
4.81 brd
(1.4)
C-24,25,26
C-3,4,5,29
C-3,4,5,28
C-23,24,25
C-23,24,25
C-3,4,5,29
C-3,4,5,28
C-23,24
C-25

Table 6. 1H NMR Data in C5D5N for the Aglycon Moiety of 6-9
6
7
8
9
δH
NOESY
H-11
HMBC
δH
NOESY
HMBC
δH
1.72 m
NOESY
H3-19
HMBC
δH
1.72 m
1.28 m
2.09 m
1.87 m
3.1 dd
(4.0,11.5)
1.25 m
1.70 m
1.54 m
2.34 m
2.25 m
2.41 m
2.14 m
2.81 m
1.82 m
2.52 m
1.75 m
2.39 m
1.52 m
2.01 brq (9.4)
0.90 s
NOESY
HMBC
1
2
3
1.71 m
1.29 m
2.09 m
1.87 m
1.70 m
1.26 m
2.07 m
1.84 m
1.29 m
2.10 m
1.88 m
3.11 dd
(4.2,11.8)
1.26 m
1.70 m
1.54 m
2.33 m
2.25 m
2.41 m
2.15 m
2.82 m
1.83 m
2.52 m
1.75 m
2.40 m
1.52 m
2.03 brq (9.0)
0.90 s
H-11
3.12 dd (4.3, 11.8)
H-5,
H1-Ara1
H-3
C-4,28,29
3.07 dd (4.4, 11.9)
H-5,
H1-Ara1
H-3
C3-Ara1
C-28,29
H-5,
H1-Ara1
H-3
C-4,28,29
H-5
H1-Ara1
H-3
C-4,28,29
5
6
1.25 dd (2.0, 12.4)
1.70 m
1.24 m
1.72 m
1.54 m
2.36 m
2.26 m
2.42 m
2.15 m
2.82 m
1.83 m
2.53 m
1.76 m
2.40 m
1.51 m
2.04 m
0.90 s
1.54 m
2.34 m
2.25 m
2.42 m
2.16 m
2.82 m
1.82 m
2.53 m
H3-29
H-29
7
H-15
H-1
11
12
15
16
H-1
H-17
H3-21
H-17
H3-21
H3-21
H3-21
C-18
H-17
H3-21
C-13,18
C-13
C-13,14,18
C-13,14,30
C-8,30
C-30
C-30
C-13,30
1.74 m
2.40 m
1.52 m
H-7
17
18
19
20
21
22
2.01 brq (9.4)
0.90s
H-12
H-20
H-12
H-20
C-13,18
C-12,13,14,17
C-1,5,9,10
H-12
H-20
H-1
C-13,20,21
C-12,13,14,17
C-1,5,9,10
H-12
H-20
C-13,16,18,20
C-12,13,14,17
C-1,5,9,10
C-12,13,14,17
C-1,5,9,10
1.07s
1.06 s
1.08 s
1.08 s
1.56 m
1.05 d (6.5)
1.56 m
1.12 m
2.13 m
H-18
H-12
1.56 m
1.05 d (6.5)
1.70 m
1.22 m
2.20 m
1.97 m
H3-18
H-12
1.56 m
1.05 d (6.5)
1.71 m
1.22 m
2.20 m
1.97 m
1.56 m
1.05 d (6.5)
1.56 m
1.10 m
2.12 m
1.96 m
5.20 brt (7.0)
H3-18
H-12
C-17,20,22
C-17,20,22
C-17,20,22
23
1.95 m
5.21 brt (7.0)
C-31
24
25
26
27
28
29
31
C-22,26,27
C-23 26,27
2.24 m
C-23,24,26,31
C-24,25,27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
C-23,25
2.24 m
C-23,24,26,31
C-24,25,27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
C-23,24,25
C-23,24,25
1.60 brs
1.68 brs
1.23 s
C-24,25,27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
1.04 d (6.5)
1.03 d (6.5)
1.21 s
1.13 s
4.84 brs
4.81 brs
1.04 d (6.5)
1.03 d (6.5)
1.24 s
1.18 s
4.84 brs
4.81 brs
1.60 brs
1.68 brs
1.23 s
C-24,25,27
C-24,25,26
C-3,4,5,29
C-3,4,5,28
1.17 s
H-6
1.17 s
C-23,25

Erylosides from the Carribean Sponge Erylus formosus
Journal of Natural Products, 2007, Vol. 70, No. 2 175
Figure 2. Structures of erylosides 7, 8, and 9.
5. The identity of aglycons in these glycosides was confirmed by
HMBC and NOESY data (Table 6). The attachment of a carbo-
hydrate chain to C-3 was indicated from the H-3 (δH 3.07)/H1-
Ara1 (δH 4.67) correlation in the NOESY spectrum. Thus, the
structure of eryloside O (7) was represented as 3â-O-{[R-L-
arabinopyranosyl-(1f4)-R-L-arabinopyranosyl-(1f3)]-[â-D-2-N-
acetyl- D-glucopyranosyl-(1f2)]-R-L-arabinopyranosyl}-14-carboxy-
24-methylenelanost-8(9)-en-3â-ol.
was established by mutual HMBC correlations between two
methines at C1-Ara2 (δH 5.21) and C3-Ara1 (δC 81.9) and
confirmed by a cross-peak of H1-Ara2 (δH 5.21)/H3-Ara1 (δH 4.28)
in the NOESY spectrum. In addition, the 1,2-linkage between the
end xylose and glucose-4 was established by cross-peaks of H2-
Glc (δH 4.13)/C1-Xyl (δC 107.7) and H1-Xyl (δH 4.91)/H2-Glc in
the HMBC and NOESY spectra, respectively. The 1,4-linkage
between glucose-4 and galactose-1 was indicated by the corre-
sponding correlations H4-Gal1 (δH 4.52)/C1-Glc (δC 105.0) and
H1-Glc (δH 5.16)/H4-Gal1. Finally, the presence of a 1,3-linkage
between galactose-1 and arabinose-2 followed from the HMBC
cross-peak H1-Gal1 (δH 5.10)/C3-Ara2 (δC 83.2) and NOESY
cross-peak H1-Gal1/H3-Ara2 (Table 7). Therefore, the carbohydrate
moiety proved to be a branched assembly of two D-galactoses, two
L-arabinoses, one D-glucose, and one D-xylose, containing an
additional trisaccharide fragment attached to C-3 of arabinose-2,
when compared with the carbohydrate chains of 5 and 6.
The molecular formulas of erylosides P (8) and Q (9) were
deduced as C64H104O30 and C63H102O30, respectively, by HR-
MALDI-TOFMS (m/z 1375.6443 [M + Na]+ and 1361.6402 [M
+ Na]+, respectively) and 13C NMR spectra. The NMR spectra of
8 and 9 indicated that both compounds contained identical
1
carbohydrate moieties (Table 7). Analysis of 13C and H NMR
spectra revealed the presence of six signals attributable to anomeric
carbons (δC 105.1, 104.6, 106.2, 105.0, 107.7, and 104.8) along
with six signals of the corresponding protons [δH 4.79 d (5.7), 5.21
d (6.8), 5.10 d (7.7), 5.16 d (7.8), 4.91 d (7.5), 5.32 d (7.9)],
indicating the hexasaccharide nature of this carbohydrate chain.
Hydrolysis of these compounds gave arabinose, galactose, glucose,
and xylose, 2:2:1:1, identified by GC in the form of their
corresponding peracetates of the aldononitriles. The D-configurations
of galactose and xylose and L-configuration of arabinose were
determined after acid hydrolysis by preparation of acetylated (-)-
2-octyl glycosides followed by GC and comparison with those
obtained from standard D- and L-sugars.20 All sugars were in the
pyranose forms and attached to each other by â-glycosidic bonds
beside L-arabinose units which had R-glycosidic bonds. This was
established on the basis of NMR data in the same manner as was
used for the structure elucidation of 1-7 (Table 7). The arrangement
of sugar units as well as positions of the attachment of carbohydrate
moieties to aglycons were established by a combination of the
HMBC and NOESY experiments. The mutual HMBC correlations
between the methine at C-3 (88.6) and an anomeric methine (δH
4.79) in the NMR spectra of both glycosides allowed us to place a
glycosidic linkage at this position. Two-dimensional NMR experi-
ments including COSY, DEPT, HSQC, HMQC-TOCSY, 1D-
TOCSY, HMBC, and NOESY indicated that the carbohydrate chain
includes the same trisaccharide fragment as in 5 and 6. Actually,
the 1,2-linkage between galactose-2 and arabinose-1 was confirmed
by cross-peaks H1-Gal2 (δH 5.32)/C2-Ara1 (δC 77.0) and H1-Gal2
and H2-Ara1 (δH 4.74) in the HMBC and NOESY experiments,
respectively. The 1,3-linkage between arabinose-2 and arabinose-1
The aglycon moieties of 8 and 9 were deduced by extensive
NMR spectroscopy (1H and 13C NMR, 1H-1H COSY, HMBC, and
NOESY) (Tables 2-4) to be the same as that of 1 (either 5 or 7)
and 6, respectively. Thus, the structures of erylosides P and Q were
established as 3â-O-{[â-D-xylopyranosyl-(1f2)-â-D-glucopy-
ranosyl-(1f4)-â-D-galactopyranosyl-(1f3)-R-L-arabinopyrano-
syl-(1f3)]-[â-D-galactopyranosyl-(1f2)]-R-L-arabinopyranosyl}-
14-carboxy-24-methylenelanost-8(9)-en-3â-ol (8) and 3â-O-{[â-
D-xylopyranosyl-(1f2)-â-D-glucopyranosyl-(1f4)-â-D-galactopy-
ranosyl-(1f3)-R-L-arabinopyranosyl-(1f3)]-[â-D-galactopyranosyl-
(1f2)]-R-L-arabinopyranosyl}-14-carboxylanosta-8,24-dien-3â-
ol (9).
The structures of oligoglycosides from some marine invertebrates,
such as holothurians, are used as chemotaxonomic markers on
different taxonomic levels (species, genus, subfamily). As it was
established by us earlier, holothurians belonging to the same taxon
show characteristic structural features of glycosides independent
of location of collection and season.21 It seems that similar
taxonomic specificity exists also for erylosides. Actually, all the
glycosides from E. formosus collected in different geographic areas8
demonstrate structural similarity to each other. They contain the
related 14-carboxylated aglycons of the lanostane series and
carbohydrate chains, comprising L-arabinose as the first monosac-
charide unit. Similar structural features were found for glycosides
from some other species belonging to the same genus,5 in contrast

176 Journal of Natural Products, 2007, Vol. 70, No. 2
AntonoV et al.
to another group of species including E. lendenfeldi,1,22 E. gof-
frilleri,3 and probably other related species.
In contrast with its epimer 2, compound 3 induced apoptosis of
Ehrlich carcinoma cells at a concentration of 100 µg/mL. Com-
pounds 1 and 10 at a concentration of 100 µg/mL were found to
activate Ca2+ influx into mouse spleenocytes up to 130% of the
control.
Experimental Section
General Experimental Procedures. Optical rotations were mea-
1
sured using a Perkin-Elmer 343 polarimeter. The H and 13C NMR
spectra were recorded in C5D5N on a Bruker DRX-500 spectrometer
at 500 and 125.8 MHz, respectively, using tetramethylsilane as an
internal standard. HRESIMS spectra were recorded on a Micromass
Q-TOF MICRO spectrometer coupled with an HPLC Waters Alliance
2695 equipped with a Waters 2996 photodiode and a Rheodyne 7725i
injector. The instrument was calibrated for high-resolution measure-
ments by using a PEG mixture from 200 to 1000 MW (resolution
specification 5000 fwhm, deviation <5 ppm rms in the presence of a
known lock mass). HR-MALDI-TOF mass spectra were recorded on a
Bruker Biflex III laser desorption mass spectrometer coupled with
delayed extraction using a N2 laser (337 nm) and R-cyano-4-hydroxy-
cinnamic acid as matrix. GC analyses were performed on an Agilent
6850 Series GC system equipped with a HP-5MS column at a
temperature program of 100 to 250 °C at 5 °C min-1; temperatures of
injector and detector were 150 °C and 270 °C, respectively. Low-
pressure column liquid chromatography was performed using Poly-
chrome-1 (powder Teflon, Biolar, Latvia), Sephadex LH-20 (Sigma,
Chemical Co), and silica gel L (40/100 µm, Chemapol, Praha, Czech
Republic). Glass plates, 4.5 × 6.0 cm, precoated with silica gel (5-17
µm, Sorbfil, Russia) were used for TLC. Preparative HPLC was carried
out on a Dupont-8800 chromatograph, using Silasorb-ODS (10 µm,
9.6 × 200 mm), Diasphere-110-C18 (5 µm, 10 × 250 mm), and YMC-
Pack ODS-A (5 µm, 10 × 250 mm) columns with an RIDK
refractometer detector.
Animal Material. The sponge Erylus formosus was collected off
Puerto Morelos (the Caribbean Sea, Mexico) by scuba diving from a
depth of 15 m. A voucher specimen is on deposit in the collection of
the Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia.
Extraction and Isolation. The lyophilized specimens (0.3 kg) were
macerated and extracted with EtOH (4 × 1 L). The EtOH extract
concentrated in Vacuo was separated by low-pressure reversed-phase
CC (the column 20 × 8 cm) on Teflon powder Polychrome-1 in H2O
and 50% EtOH. After elution of inorganic salts and highly polar
compounds by H2O, 50% EtOH was used to obtain the fraction of
amphiphilic compounds, including the erylosides. The obtained fraction
after evaporation in Vacuo (40 g) was subjected to silica gel flash CC
(7 × 10 cm) with a solvent gradient system of increasing polarity from
3% to 30% MeOH in CHCl3 (total volume 3 L). Fractions of 10 mL
were collected and combined by TLC examination to obtain four
subfractions. The subfraction I (140 mg) was further purified and
separated by normal-phase HPLC on a Supelco Sil column using
CHCl3-MeOH (65:7) as eluent and then by reversed-phase HPLC on
a Supelco C-18 column eluting with MeOH-H2O (72:28) and
repeatedly with MeOH-H2O (80:20) to yield erylosides F1 (1) (3 mg),
F2 (2) (2 mg), F3 (3) (3.5 mg), F4 (4) (6 mg), and F (10) (4.5 mg.).
Subfraction II (320 mg) was subjected to HPLC on a Supelco C-18
column with MeOH-H2O (90:10) to give erylosides M (5) (11.0 mg),
N (6) (13.0 mg), and H (11) (3.0 mg). Erylosides O (7) (9 mg), P (8)
(21 mg), and Q (9) (34 mg) were obtained from subfraction III (540
mg) by HPLC on a Supelco C-18 column using the solvent system
MeOH-H2O (95:5) and then by HPLC on a Supelco Sil using CHCl3-
MeOH (65:30) as the solvent system.
Eryloside F1 (1): amorphous solid, 3.0 mg (0.005% of dry weight);
[R]D25 -23.0 (c 0.10, MeOH); 1H and 13C NMR data, see Tables 1-3;
HR (+) ESTOFMS m/z 787.4464 [M + Na]+, calcd for C42H68O12Na
787.4603.
Eryloside F2 (2): amorphous solid, 2.0 mg (0.0033% of dry weight);
[R]D25 -32.0 (c 0.10, MeOH); 1H and 13C NMR data, see Tables 1-3;
HR (+) ESTOFMS m/z 789.4371 [M + Na]+, calcd for C41H66O13Na
789.4401.

Erylosides from the Carribean Sponge Erylus formosus
Journal of Natural Products, 2007, Vol. 70, No. 2 177
Eryloside F3 (3): amorphous solid, 3.5 mg (0.0059% of dry weight);
[R]D25 -25.0 (c 0.10, MeOH); 1H and 13C NMR data, see Tables 1-3;
HR (+) ESTOFMS m/z 789.4390 [M + Na]+, calcd for C41H66O13Na
789.4401.
Eryloside F4 (4): amorphous solid, 6.0 mg (0.01% of dry weight);
[R]D25 -12.0 (c 0.10, MeOH); 1H and 13C NMR data, see Tables 1-3;
HR (+) ES TOF MS m/z 787.4272 [M + Na]+, calcd for C41H64O13Na
787.4245.
Eryloside M (5): amorphous solid, 11.0 mg (0.018% of dry weight);
[R]D25 -10.2 (c 0.25, MeOH); 1H and 13C NMR data, see Tables 1-3;
HR-MALDI-TOF MS m/z 919.5138 [M + Na]+, 875.4 [M + Na -
CO2]+, calcd for C47H76O16Na 919.5026.
was incubated additionally 10 min at 37 °C. The intensity of
fluorescence was measured at λex ) 485 nm, λem ) 620 nm.
A suspension of Ehrlich carcinoma cells (200 µL in each well of a
96-well microplate containing 20 µL solutions of tested compound)
was incubated for 1 h at 37 °C. Then 10 µL of Hoechst 33342 water
solution was added to each well (final concentration 5 µM). After 5
min of incubation at room temperature the fluorescence of the cell
suspension was measured at λex ) 355 nm and λem ) 460 nm. The
induction of chromatin condensation (early apoptosis) was determined
by comparison of fluorescence intensity of Hoechst 33342 (apoptotic
cells) and propidium iodide (necrotic cells) in the cell suspension.
Mouse lymphocytes (spleenocytes) were obtained from a mouse
spleen. For this purpose a spleen was isolated and cut with scissors
into small-sized slices in PBS (pH 7.4) and then pressed through nylon
gauze (280 mesh). The obtained suspension was washed twice in PBS
by centrifugation (2000 rpm, 10 min). The final concentration of cells
in the incubation medium was (2-5) × 106 cells/mL. A solution of
calcium green-1/AM (final concentration 10 µM) was added to the
suspension, and the cells were incubated with the fluorescent probe
during 60-90 min at 37 °C. Then the suspension of spleenocytes was
washed in PBS by a centrifugation at 1500 rpm, and 100 µL of the
suspension was placed in wells of a 96-well microplate including 10
µL solutions of tested compounds. The incubation was conducted during
5 min at 37 °C, and the intensity of the fluorescence was measured at
λex ) 485 nm, λem ) 518 nm. The intensity of the fluorescence in all
experiments was measured with a Fluoroscan Ascent (ThermoLab-
systems, Finland) fluorescent plate reader.23
Eryloside N (6): amorphous solid, 13.0 mg (0.022% of dry weight);
[R]D25 -14.0 (c 0.25, MeOH); 1H and 13C NMR data, see Tables 4, 5;
HR-MALDI-TOF MS m/z 905.4740 [M + Na]+, calcd for C46H74O16-
Na 905.4870, 861.5 [M + Na - CO2]+.
Eryloside O (7): amorphous solid, 21.0 mg (0.035% of dry weight);
[R]D -7.0 (c 0.25, MeOH); H and 13C NMR data, see Tables 4, 5;
HR-MALDI-TOF MS m/z 1092.5827 [M + Na]+, calcd for C54H76O87O20-
NNa 1092.5714.
25
1
Eryloside P (8): amorphous solid, 9.0 mg (0.015% of dry weight);
[R]D25 -10.2 (c 0.25, MeOH); 1H and 13C NMR data, see Tables 4, 5;
HR-MALDI-TOF MS m/z 1375.6443 [M + Na]+, calcd for C64H104O30-
Na 1375.6506.
Eryloside Q (9): amorphous solid, 34 mg (0.057% of dry weight);
[R]D25 -11.0 (c 0.25, MeOH); 1H and 13C NMR data, see Tables 4, 5;
HR-MALDI-TOF MS m/z 1361.6402 [M + Na]+, calcd for C63H102O30-
Na 1361.6349, 1317.5 [M + Na - CO2]+, 1185.5 [M + Na - CO2 -
C5H8O4]+.
Acknowledgment. This study was carried out in the framework of
a bilateral project between Pacific Institute of Bioorganic Chemistry
of the Russian Academy of Sciences (Russia) and Instituto di Chimica
Biomoleculare (ICB-CNR), Italy. The research described in this
publication was made possible in part by the Program of Presidium of
RAS “Molecular and Cell Biology” and a grant supporting leading
scientific schools.
Eryloside F (10): amorphous solid, 4.5 mg (0.0075% of dry weight);
[R]20D -41.0 (c 0.1, MeOH); HR-MALDI-TOF MS m/z 1361.6502 [M
+ Na]+, calcd for C63H102O30Na 1361.6349. 1H and 13C NMR spectra
were identical with those reported in the literature.4
Eryloside H (11): amorphous solid, 3.0 mg; [R]20 -11.2 (c 0.1,
D
MeOH); HR-MALDI-TOF MS m/z 1361.6502 [M + Na]+, calcd for
C63H102O30Na 1361.6349. 1H and 13C NMR spectra were identical with
those reported in the literature.5
Supporting Information Available: 2D NMR spectra of com-
pounds 1-9. This material is available free of charge via the Internet
at htpp://pubs.acs.org.
Acidic Hydrolysis of Erylosides F1-F4 (1-4). A solution of
compounds 1-4 (each 1.5 mg) in 2 N HCl (0.5 mL) was heated in a
stoppered reaction vial at 100 °C for 2 h. The water layer was extracted
with CHCl3 and then neutralized with Dowex (HCO3-). The residue
obtained after evaporation of the H2O layer was separated on an Agilent
ZORBAX carbohydrate analysis column (5 µm, 4.6 × 250 mm) eluting
with CH3CN-H2O (75:25) to yield 1.0 mg of D-galactose, [R]20D +79.0
References and Notes
(1) Carmely, Y.; Roll, M.; Kashman, Y. J. Nat. Prod. 1989, 52, 167-
170.
(2) D’Auria, M. V.; Paloma, L. G.; Riccio, R.; Debitus, C. Tetrahedron
1992, 48, 491-498.
(3) Gulavita, N. K.; Wright, A. E.; Kelly-Borges, M.; Longley, R. E.
Tetrahedron Lett. 1994, 35, 4299-4302.
(c 0.1, H2O) and 0.8 mg of L-arabinose, [R]20 +101.0 (c 0.08, H2O).
D
Acidic Hydrolysis Followed by GC Monosaccharide Analysis of
Eryloside O (7). A solution of compound 7 (3.5 mg) in 1 N TFA (1
mL) was heated in a stoppered reaction vial at 100 °C for 2 h. The
residue obtained after evaporation of the H2O layer was separated on
an Agilent ZORBAX carbohydrate analysis column (5 µm, 4.6 × 250
mm) eluting with CH3CN-H2O (75:25) to yield 1.0 mg of galactose,
0.7 mg of arabinose, and 0.8 mg of 2-N-acetylglucosamine. The
monosaccharides were treated with (-)-2-octanol (0.2 mL) in the
presence of trifluoroacetic acid (1 drop) in a stoppered reaction vial at
130 °C overnight. The mixtures were evaporated to dryness and
acetylated with Ac2O in pyridine. The acetylated (-)-2-octyl glycosides
were analyzed by GC using the corresponding authentic samples
prepared from D- and L-galactose, and D- and L-arabinose. Retention
time for the L-GlcNAc derivative was determined for (+)-2-octyl
glycoside of the corresponding D-sugar according to Leontein.20
Acidic Hydrolysis and GC Analysis of Monosaccharide Com-
position of Eryloside P (8). Compound 8 (6.5 mg) was hydrolyzed as
described above for erylosides F1-F4. The absolute configurations of
the monosaccharides were determined by GC of the acetylated (-)-
2-octyl glycosides using the corresponding authentic samples prepared
from D- and L-galactose, D- and L-arabinose, and D- and L-xylose.20
Bioassays. Ehrlich carcinoma cells were grown intraperitoneally in
albino mice, 18-20 g of weight. Cells were harvested on the seventh
to tenth day after inoculation and washed twice by centrifugation (450g,
10 min) in cold phosphate-buffered saline (PBS). Then 100 µL of the
cell suspension (final cell concentration (2-5) × 106 cells/mL) was
placed into wells of a 96-well microplate containing 10 µL solutions
of tested compounds. The incubation was conducted within 1 h at 37
°C. Then 10 µL of an aqueous solution of propidium iodide (final
concentration 2.5 µg/mL) was added to each well, and the microplate
(4) Stead, P.; Hiscox, S.; Robinson, P. S.; Pike, N. B.; Sidebottom, P.
J.; Roberts, A. D.; Taylor, N. L.; Wright, A. E.; Pomponi, S. A.;
Langley, D. Biorg. Med. Cem. Lett. 2000, 10, 661-664.
(5) Shin, J.; Lee, H. S.; Woo, L.; Rho, J. R.; Seo, Y.; Cho, K. W.; Sim,
C. J. J. Nat. Prod. 2001, 64, 767-771.
(6) Sandler, J. S.; Forsburg, S. L.; Faulkner, D. J. Tetrahedron 2005,
61, 1199-1206.
(7) Takada, K.; Matsunaga, S.; van Soest, R. W. V.; Fusetani, N. J. Nat.
Prod. 2002, 65, 411-413.
(8) Jaspars, M.; Crews, P. Tetrahedron Lett. 1994, 35, 7501-7504.
(9) Campagnuolo, C.; Fattorusso, E.; Taglialatela-Scafati, O. Tetrahedron
2001, 57, 4049-4055.
(10) Cheng, J. F.; Kobayashi, J.; Nakamura, H.; Ohizumi, Y.; Hirata, Y.;
Sasaki, T. J. Chem. Soc., Perkin Trans. 1988, 8, 2403-2406.
(11) Shoji, N.; Umeyama, A.; Motoki, S.; Arihara, S.; Ishida, T.; Nomoto,
K.; Kobayashi, J.; Takei, M. J. Nat. Prod. 1992, 55, 1682-1685.
(12) Takei, M.; Umeyama, A.; Shoji, N.; Arihara, S.; Endo, R. J. Pharm.
Sci. 1995, 84, 228-230.
(13) Kubanek, J.; Whalen, K. E.; Engel, S.; Kelly, S. R.; Henkel, T. P.;
Fenical, W.; Pawlik, J. R. Oecologia 2002, 131, 125-136.
(14) Kubanek, J.; Pawlik, J. R.; Eve, T. M.; Fenical, W. Mar. Ecol. Prog.
Ser. 2000, 207, 69-77.
(15) Kalinovsky, A. I.; Antonov, A. S.; Afiyatullov, S. S.; Dmitrenok, P
.S.; Evtuschenko, E. V.; Stonik, V. A. Tetrahedron Lett. 2002, 43,
523-525.
(16) Antonov, A. S.; Afiyatullov, S. S.; Kalinovsky, A. I.; Ponomasrenko,
L. P.; Dmitrenok, P. S.; Aminin, D. L.; Agafonova, I. G.; Stonik, V.
A. J. Nat. Prod. 2003, 66, 1082-1088.
(17) Fujioka, T.; Iwamoto, M.; Iwase, Y.; Okabe, H.; Mihashi, K.;
Yamachi, T. Chem. Pharm. Bull. 1988, 36, 2772-2777.

178 Journal of Natural Products, 2007, Vol. 70, No. 2
AntonoV et al.
(18) Ilyin, S. G.; Malinovskaya, G. V.; Antipin, M. Ju.; Struchkov, Ju.
G. Khim. Prirodn. Soedin. 1991, 667-672 (in Russian).
(19) Malinovskaya, G. V.; Novikov, V. L.; Denisenko, V. A.; Uvarova,
N. I. Khim. Prirodn. Soedin. 1980, 346-351 (in Russian).
(20) Leontein, K.; Lindberg, B.; Lonngren, J. Carbohydr. Res. 1978, 62,
359-362.
(21) Elyakov, G. B; Stonik, V. A.; Levina, E. V.; Slanke, V. P.;
Kuznetsova, T. A.; Levin, V. S. Comp. Biochem. Physiol. 1973, 44B,
325-336. Stonik, V. A.; Elyakov, G. B. Bioorganic Marine
Chemistry; Scheuer, P. J., Ed.; Springer-Verlag, New York, 1988;
Vol. 2, pp 43-86. Stonik, V. A.; Kalinin, V. I.; Avilov, S. A. J.
Nat. Toxins 1999, 8, 235-248, and other articles in this series.
(22) Fouad, M.; Al-Trabeen, K.; Badran, M.; Wray, V.; Edrada, R.;
Proksch, P.; Ebel, R. ArkiVoc. 2004, 17-27.
(23) Levina, E. V.; Kalinovsky, A. I.; Andriyashenko, P. V.; Dmitrenok,
P. S.; Aminin, D. L.; Stonik, V. A. J. Nat. Prod. 2005, 68,
1541-1544.
NP060364Q