J. Nat. Prod. 2007, 70, 1871–1877
1871
Isolation and Structures of Erylosides from the Carribean Sponge Erylus goffrilleri
Shamil Sh. Afiyatullov,*,† Anatoly I. Kalinovsky,† Alexandr S. Antonov,† Ludmila P. Ponomarenko,† Pavel S. Dmitrenok,†
Dmitry L. Aminin,† Vladimir B. Krasokhin,† Valentina M. Nosova,‡ and Alexandr V. Kisin‡
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100 let VladiVostoku,
159, VladiVostok 690022, Russian Federation, and State Research Institute of Chemistry and Technology of Organoelement Compounds,
Sh. EntuziastoV, 38, Moscow 111123, Russian Federation
ReceiVed July 1, 2007
Eight new triterpene glycosides, erylosides R (1), S (2), T (3), U (4), F5-F7 (5–7), and V (8), were isolated from the
sponge Erylus goffrilleri collected near Arresife-Seko Reef (Cuba). Structures of 1 and 2 were determined as the
corresponding monosides having aglycons related to penasterol with additional oxidation and methylation patterns in
their side chains. Eryloside T (3) was structurally identified as the ∆7-isomer of 1, containing an unusual (14f9)lactone ring in the tetracyclic aglycon moiety, and eryloside U (4) was shown to be the 7,8-epoxide of 3. Erylosides
F5-F7 (5–7) and V (8) contain new variants of carbohydrate chains with two (5–7) and three (8) sugar units, respectively.
Marine sponges of the genus Erylus (order Astrophorida, family
Geodidae) are a source of various saponins, erylosides, belonging
to the steroidal or triterpenoid series. Erylosides derived from 4Rmethyl-5R-cholesta-8,14-dien-3β-ol or a related nortriterpenoid were
isolated from Erylus lendenfeldi,1–3 and glycosides derived from
penasterol or its congeners were described from E. formosus,4–7
E. nobilis,8,9 E.goffrilleri,10 and Erylus sp.11 Recently, two steroid
glycosides, sokodiosides A and B, with a novel carbon skeleton
were isolated from E. placenta.12 Erylosides from Erylus spp. have
been reported to exhibit a wide spectrum of biological activities.
For example, eryloside F was discovered to possess selective
thrombin receptor antagonist activity and functional activity in a
platelet aggregation assay.5 Eryloside E exhibits immunopressive
activity,10 and nobiloside inhibits neuraminidase from the bacterium
Clostridium perfringens.9 Erylosides A, K, and L from E.
lendenfeldi1–3 and sokodiosides A and B from E. placenta12
possessed antitumor and antifungal properties.
In continuation of our studies on glycosides from marine
sponges,7,13 we have isolated a series of new erylosides (1–8) from
the ethanolic extract of the Caribbean sponge Erylus goffrilleri.
We report herein the isolation and structural elucidation of eight
new glycosides.
Results and Discussion
The ethanolic extract of the sponge was separated by lowpressure reversed-phase column chromatography on Teflon powder
Polycrome-1 followed by Si gel flash column chromatography and
by several rounds of reversed-phase HPLC to yield individual
glycosides 1–8 as colorless, amorphous solids.
The molecular formula of eryloside R (1) was determined as
C38H64O9 by a pseudomolecular ion at m/z 687.4483 [M + Na]+
in HRMALDIMS and by 13C NMR analyses. A close inspection
of the 1H and 13C NMR data (Tables 1, 4, and 5) of 1 by DEPT
and HSQC revealed the presence of nine methyls; 11 methylenes,
including one oxygen-bearing methylene; six oxygenated methines,
including one methine linked to an anomeric carbon; and three
tertiary and five saturated quaternary carbons. The remaining
functionality, corresponding to the carbon signals at δ 178.2 (C),
139.8 (C), 127.9 (C), and 75.2 (C), suggested the presence of a
carbonyl or carboxyl carbon, one tetrasubstituted double bond, and
* To whom correspondence should be addressed. Tel: 7-4232-311168.
Fax: 7(4232)314050. E-mail: afiyat@www.piboc.dvo.ru.
†
Pacific Institute of Bioorganic Chemistry.
‡
State Scientific Research Institute of Chemistry and Technology of
Organoelement Compounds.
10.1021/np070319y CCC: $37.00
Table 1. 1H and 13C NMR Data for the Carbohydrate Moieties
of Erylosides 1–4a in C5D5N
position
δC
Gal
(1fC-3)
1
107.4 CH
2
3
4
5
6
72.9 CH
75.2 CH
70.1 CH
76.5 CH
62.3 CH2
δH (J in Hz)
HMBC
NOESY
4.78 d (7.7)
C-3,C3-Gal H-3,28,
H3,5-Gal
4.44 dd (7.7, 9.5)
C1,3-Gal
4.16 dd (3.4, 9.5)
C2-Gal
H1-Gal
4.60 brd (3.4)
C2,3-Gal
4.12 brt (6.0)
C1,4,6-Gal H1-Gal
a: 4.47 dd (6.1, 10.9) C4,5-Gal
b: 4.51 dd (6.0, 10.9) C5-Gal
a
All assignment were given for 1; spectra of 2–4 had only minor
differences in chemical shift values.
one tertiary alcohol function. The IR spectrum of 1 exhibited a
characteristic band attributable to a carboxyl (1687 cm-1) group.
Interpretation of the COSY data gave rise to spin systems involving
one anomeric proton, four oxymethines, and protons of a hydroxymethyl group. On the basis of these data, a triterpene monoside
with a tetracyclic aglycon structure was suggested for 1.
The correlation observed in the COSY and HSQC spectra of
the aglycon part of 1 indicated the presence of the following distinct
spin systems: -CHOH-CH2-CH2-(C-3-C-1), >CH-CH2-CH2>CH-CH2-CH2(C-5-C-7),
CH2-CH2-(C-11-C-12),
(C-17-C-15). These partial structures were further connected to
each other by HMBC correlations: H3-19/C-1, C-5, C-9, and C-10;
H3-28/C-3, C-4, C-5, and C-29; H3-29/C-3, C-4, C-5, and C-28;
H-6/C-8 and C-10; H-12/C-9, C-13, C-14, and C-18; H3-18/C-12,
C-13, C-14, and C-17; and H-15/C-8, C-13, and C-14. The position
of a double bond at ∆8,9 was evident from the long-range correlation
of H3-19 with C-9 at 139.8. The chemical shift of C-14 at δ 62.7
and HMBC correlations of H2-15 (δ 1.69, 2.48) with C-30 (δ 178.2)
indicated the attachment of a carboxyl function at C-14. The COSY
and HMBC data allowed the assignment of the signal at δ 88.3
(C-3) to a secondary oxygen-bearing carbon, adjacent to a
quaternary sp3 carbon. This information together with the observed
NOE correlations H3-19/H-2β (δ 1.90), H-6β (δ 1.55), and H-11β
(δ 2.16), H-3/H-5 (δ 1.28) and H-28 (δ 1.24), H-6R/H-28, H-6β/
H-29 (δ 1.03), and H-12R/H-17 (δ 2.07) indicated that 1 contained
a 14-carboxylanost-8(9)-ene skeleton. In addition, the long-range
COSY correlation between H3-19 and H-1R and between H3-18
and H-12R, H-17 confirmed this conclusion. The relative stereochemistry of the proton at C-3 was defined on the basis of the
1H-1H coupling constants (J ) 4.3, 11.8 Hz) observed between
H-3 and H-2R,β and assigned as axial. HMBC correlations of
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/16/2007
1872 Journal of Natural Products, 2007, Vol. 70, No. 12
1H
Table 2.
and 13C NMR Data for the Carbohydrate Moieties of Erylosides 5 and 6a in C5D5N
δC
position
δH (J in Hz)
HMBC
Gal (1fC-3)
1
2
3
4
5
6
104.8 CH
80.8 CH
75.9 CH
69.8 CH
76.2 CH
61.9 CH2
4.78 d (7.7)
4.52 dd (7.7, 9.5)
4.18 dd (3.3, 9.5)
4.57 brd (3.5)
4.07 brt (6.2)
a: 4.43 dd (5.8, 9.9)
b: 4.47 dd (6.3, 10.9)
NAc-Glc (1f2Gal)
1
2
3
4
5
6
102.8 CH
59.3 CH
78.0 CH
72.5 CH
77.4 CH
62.9 CH2
5.39 d (8.3)
4.36 m
4.13 t (9.5)
4.17 t (9.2)
3.60 m
a: 4.29 dd (5.0, 11.4)
b: 4.37 dd (3.2, 11.5)
9.12 d (6.0)
NH
Ac
a
AfiyatulloV et al.
172.5 C
23.0 CH3
NOESY
C-3
1,3-Gal, 1-NAc-Glc
H-3, H3, 5-Gal
1-NAc-Glc
H1,5-Gal
H6a-Gal
H1-Gal
H4-Gal
C2,3-Gal
C4,6-Gal
C4,5-Gal
C2-Gal
C3-NAc-Glc
C2,4-NAc-Glc
C3,6-NAc-Glc
H3,5-NAc-Glc, H2-Gal
H1,5-NAc-Glc
H1,3-Nac-Glc
C4,5-NAc-Glc
C4-NAc-Glc
2.12 s
All assignment were given for 5; spectra of 6 had only minor differences in chemical shift values.
Table 3.
1H
and 13C NMR Data for the Carbohydrate Moieties of Erylosides 7 and 8 in C5D5N
7
position
Ga1
(1fC-3)
1
δC
106.9 CH
2
71.9 CH
2
71.9 CH
3
85.0 CH
4
5
69.7 CH
76.3 CH
6
62.2 CH2
Glc
(1f3) Gal
1
106.4 CH
2
75.6 CH
3
4
5
6
78.2 CH
71.3 CH
78.4 CH
62.4 CH2
δH (J in Hz)
4.79 d (7.7)
4.62 dd
(7.7, 9.6)
4.62 dd
(7.7, 9.6)
4.26 dd
(3.6, 9.6)
4.76 d (3.7)
4.07 brt (6.0)
8
NOESY
4.04 dd
(8.0, 9.0)
4.25 m
4.25 m
3.97 m
a: 4.39 dd (
6.0, 12.0)
b: 4.51 dd (
2.5, 11.8)
position
δC
δH (J in Hz)
NOESY
HBMC
C3,5-Ara
4.74 dd (6.5, 8.4)
H-3,
H3,5a-Ara
H1-Gal
77.4 CH
4.74 dd (6.5, 8.4)
H1-Gal
3
82.2 CH
4.28 dd (2.8, 8.7)
H1,5a-Ara,
H1-Xyl
C1-Gal,
C1,3-Ara
C1-Gal,
C1,3-Ara
C2,4-Ara,
C1-Xyl
4
5
68.5 CH
65.4 CH2
4.46 t (8.6)
a: 3.77 dd (2.1, 12,4)
b: 4.28 m
H1,3-Ara
C4-Ara
H3,5-Xyl,
H3-Ara
C3-Xyl,
C3-Ara
C1,3-Xyl
H1-Xyl
C2,4-Xyl
C2,3,5-Xyl
C3,4-Xyl
C3,4-Xyl
C-3
Ara
(1fC-3)
1
105.0 CH
4.82 d (6.5)
C1,3-Gal
2
77.4 CH
C1,3-Gal
2
H1-Glc
C2-Gal
H1-Gal
C2,3Gal
C1,4,6-Gal
H-3,
H3,5-Ga1
a: 4.38 dd
(6.0, 11.0)
b: 4.42 dd
(6.2, 11.0)
5.40 d (7.8)
HBMC
C4-Gal C4,5-Gal
H3-Gal,
H3,5-Glc
H1-Glc
C3-Gal, C5-Glc
Xyl
(1f3Ara)
1
105.1 CH
5.17 d (7.5)
C1,3-Glc
2
74.8 CH
3.96 t (7.8)
C1,2,4-Glc
C2,3,5-Glc
3
4
5
78.0 CH
70.7 CH
66.9 CH2
4.10 t (8.7)
4.17 dd (5.2, 9.8)
a: 3.64 dd (9.8, 11.1)
b: 4.28 dd (5.2, 11.3)
H1-Glc
Gal
(1f2Ara)
1
2
3
4
5
6
H3-21 (δ 1.10) with C-20 (δ 37.3) and C-17 (δ 51.7) established
attachment of the side chain at C-17. COSY correlation between
H-20 (δ 1.55), H2-22 (δ 1.20, 2.06), and H2-23 (δ 1.70, 1.76) along
with HMBC correlations from H3-31 (δ 1.26) to C-23 (δ 33.4),
C-24 (δ 75.2), and C-25 (δ 38.5), from the nine proton resonances
observed for H-26, H-27, and H-32 (δ 1.14) to both C-24 and C-25,
firmly established the structure of the side chain, including the
location of the tertiary alcohol and tert-butyl group at C-24. NOESY
104.9 CH
73.3 CH
75.2 CH
69.4 CH
76.1 CH
61.1 CH2
5.35 d (7.8)
4.47 m
4.12 dd (3.7, 9.5)
4.61 brd (3.7)
3.81 brt (6.3)
4.28 m 4.48 m
H1-Xyl
H3,5-Gal,
H2-Ara
H1,5-Gal
H1,3-Gal1
C2-Ara
C1,3-Gal
C2-Gal
C2,3-Gal
C4,6-Gal
C4,5-Gal
correlations H3-18/H-20 and H-12β/H3-21 determined the
20R*configuration for C-20. Thus, eryloside R (1) was defined as
a triterpenoid monoside possessing a modified pentasterol aglycone
that was previously found in eryloside E, a bioactive constituent
of E. goffrilleri collected from the Bahama Islands.10
The 13C and 1H NMR spectra of the sugar moieties of erylosides
R, S, T, and U (1-4) showed a close similarity of all proton and
carbon chemical shifts and proton multiplicities (Table 1). The acid
Journal of Natural Products, 2007, Vol. 70, No. 12 1873
Erylosides from the Carribean Sponge Erylus goffrilleri
Table 4.
position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Ac
13C
NMR Data for the Aglycon Moiety of Erylosides (1–8) in C5D5N
1
35.5 CH2
27.0 CH2
88.3 CH
39.4 C
50.4 CH
18.3 CH2
27.9 CH 2
127.9 C
139.8 C
37.4 C
22.5 CH2
31.7 CH2
46.9.0 C
62.7 C
28.3 CH2
29.6 CH2
51.7 CH
18.0 CH3
19.7 CH3
37.3 CH
18.8 CH3
30.4 CH2
33.4 CH2
75.2 C
38.5 C
25.7 CH3
25.7 CH3
27.7 CH3
16.7 CH3
178.2 C
21.0 CH3
25.7 CH3
2
35.3 CH2
27.1 CH2
88.3 CH
39.4 C
50.3 CH
18.4 CH2
27.9 CH2
127.9 C
139.9 C
37.4 C
22.5 CH2
31.6 CH2
46.9 C
62.8 C
28.4 CH2
29.6 CH2
51.0 CH
17.8 CH3
19.7 CH3
36.9 CH
18.7 CH3
29.5 CH2
32.6 CH2
87.4 C
34.0 CH
17.1 CH3
16.9 CH3
27.6 CH3
16.6 CH3
178.2 C
19.8 CH3
3
4
5
30.2 CH2
26.0 CH2
88.2CH
38.8 C
47.0 CH
21.7 CH2
120.2 CH
140.2 C
88.6 C
38.1 C
25.9 CH2
34.3 CH2
45.9 C
65.1 C
22.2 CH2
28.0 CH2
53.0 CH
14.0 CH3
16.9 CH3
36.5 CH
18.5 CH3
30.1 CH2
33.1 CH2
75.1 C
38.5 C
25.7 CH3
25.7 CH3
28.8 CH3
17.5 CH3
178.5 C
21.0 CH3
25.7 CH3
30.7 CH2
25.7 CH2
88.0 CH
38.9 C
42.6 CH
20.8 CH2
54.0 CH
64.4 C
85.8 C
37.3 C
22.8 CH2
34.3 CH2
46.7 C
59.6 C
20.3 CH2
27.8 CH2
53.0 CH
14.0 CH3
16.2 CH3
35.7 CH
18.3 CH3
30.1 CH2
33.1 CH2
75.2 C
38.5 C
25.7 CH3
25.7 CH3
28.1 CH3
16.6 CH3
176.8 C
21.0 CH3
25.7 CH3
35.3 CH2
27.0 CH2
88.8 CH
39.5 C
50.3CH
18.4 CH2
27.9 CH2
127.9 C
139.9 C
37.3 C
22.5 CH2
31.8 CH2
46.9 C
62.8 C
28.3 CH2
29.5 CH2
51.6 CH
17.9 CH3
19.5 CH3
37.4 CH
18.8 CH3
30.4 CH2
33.5 CH2
75.3 C
38.5 CH
25.7 CH3
25.7 CH3
27.6 CH3
16.4 CH3
178.3 C
21.0 CH3
25.7 CH3
170.0 C 21.8 CH3
hydrolysis of the sum of these glycosides gave D-galactose as the
only sugar that was identified by GLC of the corresponding
acetylated (-)-2-octyl glycoside, using authentic samples prepared
from D- and L-galactose.14
A comparison of the 13C NMR spectra of 1–4 with published
data for R- and β-D-galactopyranosides, together with the magnitudes of 1H-1H spin coupling constants and NOE data, elucidated
the presence of a β-D-galactopyranoside unit of C1 form in 1–4.15–17
A long-range correlation H1-Gal (δ 4.78)/C-3 (δ 88.3) in the
HMBC spectra of 1–4 and downfield chemical shift of C-3 (δ 88.3)
revealed a linkage between H1-Gal and C-3 of the aglycon. This
interpretation was confirmed by a strong NOESY cross-peak from
H1-Gal to H-3. On the basis of the above data, the structure of
eryloside R was established as 3-O-(β-D-galactopyranosyl)-14carboxy-24,25-dimethyllanost-8(9)-en-3β,24-diol (1).
HRMALDIMS eryloside S (2) gave a quasimolecular ion at m/z
715.4351 [M + Na]+. These data, coupled with 13C NMR spectral
data, established the molecular formula of 2 as C39H64O10. The
general features of the 1H and13C NMR spectra (Tables 4, 5) of
the aglycon part of 2 closely resemble those of eryloside R (1),
with the exception of proton and carbon signals belonging to the
side chain. The difference in the NMR spectra was the appearance
of the signals of the acetoxy group (δ 2.01 s, 170.0 C, 21.8 CH3)
and downfield chemical shift of a quaternary oxygenated carbon
(C-24, δ 87.4). Furthermore, the interpretation of the COSY and
HSQC data revealed two additional isolated spin systems:
>CH-CH3 (C-25-C-26, C-27). This information and a COSY
correlation from H-20 (δ 1.52), H-22 (δ 1.05, 1.30) to H-23 (δ
1.97, 2.04) along with HMBC correlations from H3-31 (δ 1.37) to
C-23 (δ 32.6), C-24 (δ 87.4), and C-25 (δ 34.0), from the doublet
signals (J ) 7.0 Hz for each) observed for H-26 (δ 0.85) and H-27
(δ 0.92) to both C-24 and C-25, established the structure of the
side chain, including the C-24 tertiary acetoxy group. Thus, the
structure of eryloside S (2) was determined as 3-O-(β-Dgalactopyranosyl)-14-carboxy-24-acetoxy-24-methyllanost-8(9)en-3β-ol.
6
35.2 CH2
27.0 CH2
88.7 CH
39.5 C
50.2 CH
18.4 CH2
27.9 CH2
127.9 C
139.9 C
37.3 C
22.4 CH2
31.6 CH2
46.9 C
62.8 C
28.4 CH2
29.6 CH2
51.0 CH
17.9 CH3
19.5 CH3
36.8 CH
18.7 CH3
29.5 CH2
32.5 CH2
87.4 C
34.0 CH
17.1 CH3
16.9 CH3
27.5 CH3
16.4 CH3
178.2 C
19.7 CH3
7
8
35.3 CH2
27.0 CH2
88.4 CH
39.4 C
50.2 CH
18.4 CH2
27.9 CH
127.9 CH 2
139.9 C
37.3 C
22.5 CH2
31.7 CH2
46.9 C
62.7 C
28.3 CH2
29.5 CH2
51.5 CH
18.0 CH3
19.5 CH3
37.4 CH
18.8 CH3
30.4 CH2
33.5 CH2
75.2 C
38.5 C
25.7 CH3
25.7 CH3
27.5 CH3
16.6 CH3
178.3 C
21.0 CH3
25.7 CH3
35.3 CH2
27.0 CH2
88.4 CH
39.6 C
50.2 CH
18.4 CH2
27.9 CH
127.9 CH 2
139.9 C
37.3 C
22.5 CH2
31.7 CH2
46.9 C
62.8 C
28.3 CH2
29.5 CH2
51.6 CH
18.0 CH3
19.6 CH3
37.3 CH
18.8 CH3
30.3 CH2
33.4 CH2
75.3 C
38.5 C
25.7 CH3
25.7 CH3
27.4 CH3
16.4 CH3
178.3 C
20.9 CH3
25.7 CH3
169.8 C 21.9 CH3
Eryloside T (3) exhibited the molecular formula C38H62O9 as
deduced from its HRMALDIMS ([M + Na]+, m/z 685.4331) and
13C NMR spectra. A close inspection of the 1H and 13C NMR
spectral data (Tables 1, 4, and 6) of 3 revealed the presence of a
galactoside with a triterpenoid aglycon structure possessing a
secondary alcohol function at δ 88.2, two quaternary oxygenated
carbons at δ 75.1 and 88.6, and one carbonyl carbon and one
trisubstituted double bond at δ 178.5 (C), 120.2 (CH), and 140.2
(C). The IR spectrum of 3 exhibits a characteristic band at 1748
cm-1 attributable to a five-membered lactone. A major portion of
the tetracyclic backbone was assembled on the basis of COSY-45,
HSQC, and 1D-TOCSY experiments and through interpretation of
key HMBC correlations from four methyl singlets (δ 0.77 H3-18,
0.97 H3-19, 1.26 H3-28, 1.09, H3-29) and from H-7 (δ 5.58, t, J )
4.8 Hz) to C-5, C-6, C-9, and C-14 and from H-17 (δ 1.99, brq,
J ) 9.5 Hz) to C-12, C-13, C-16, C- 18, and C-20. Localization of
the trisubstituted double bond at ∆7(8) was evident from the COSY45 and 1D-TOCSY data and HMBC correlations of H-5 (δ 1.35, t,
J ) 7.5 Hz) with C-6 and C-7 and of H-11 (δ 1.71, 1.82) and
H-15 (δ 1.70) with C-8. A quaternary carbon at δ 65.1 in the 13C
NMR spectrum was assigned to C-14 on the basis of its coupling
with H-7, H-15, and H3-18. The chemical shift of this carbon and
HMBC correlations from H2-15 (δ 1.70, 1.77) to C-30 (δ 178.5)
suggested the attachment of a carbonyl function at C-14. The
positioning of a γ-lactone at C-14 and C-9 was established by longrange correlation of H3-19 with the signal of C-9 (δ 88.6).
The relative configuration of the aglycon part of 3 was assigned
on the basis of a NOESY experiment and 1H-1H coupling
constants. The magnitudes of the vicinal coupling constants (4.0,
11.6) between H-3 (δ 3.39) and H-2R,β (δ 1.88, 2.39) revealed an
equatorial configuration of the oxygen function at C-3. Observed
NOE correlations H3-19/H-29 (δ 1.09), H-1β (δ 1.56); H-2β/H29; and H-3R/H-5R (δ 1.35), H-28 (δ 1.26) indicated a trans-ring
fusion of the A and B rings, as well as the axial orientation of
H3-19. The axial orientation of H3-18 was assignable on the basis
of long-range COSY correlations H3-18/H-12R (δ 1.59), H-17 (δ
1874 Journal of Natural Products, 2007, Vol. 70, No. 12
Table 5.
1H
AfiyatulloV et al.
NMR Data for the Aglycon Moieties of 1,a 5, 7, 8, 2,b and 6 in C5D5N
1
H
δH (J, Hz)
1
R: 1.30 m
β: 1.68 m
R: 2.32 m
β: 1.90 m
3.25 dd (4.3, 11.8)
2
3
5
6
7
11
12
15
16
17
18
19
20
21
22
23
25
26
27
28
29
31
32
Ac
1.28 m
R: 1.70 m
β: 1.55 m
2.35 m
2.23 m
R: 2.42 m
β: 2.16 m
R: 2.86 dt (11.6)
β: 1.86 dd (8.5, 13.0)
R: 2.48 m
β: 1.69 m
R: 2.37 m
β: 1.48 m
2.07 brq (9.3)
0.90 s
1.08
1.55
1.10
2.06
1.20
1.76
1.70
s
m
d (6.5)
m
m
m
m
2
HBMC
NOESY
C-3
C-10
C-4,28,29
C-10,19,28,29
C-5,8
H-19,29
H-5,28,
H1-Gal
H-3
H-19,29
C-8
C-8,9
C-13,18
C-9,13,14,18
C-13,14,30
C-8,14,30
C-13,16,18,20
C-12,13,14,17
C-1,5,9,10
C-17,20,22
1.14 s
1.14 s
1.24 s
C-24,25,27,32
C-24,25,26,32
C-3,4,5,29
1.03 s
1.26 s
1.14 s
C-3,4,5,28
C-23,24,25
C-24,25,26,27
H-19
H-17
H-18,21
H-18
H-18
H-12R,21
H-12β,15β,
16β,20
H-2β,6β,11β
H-18
H-12β,17
H-3,6R,29,
H1-Gal
H-2β,6β,28
a
δH (J, Hz)
R: 1.30 m
β: 1.70 m
R: 2.31 m
β: 1.88 m
3.25 dd (4.3, 11.5)
1.28 m
R: 1.72 m
β: 1.55 m
2.35 m
2.25 m
2.42 m
2.16 m
R: 2.84 m
β: 1.84 m
R: 2.55 m
β: 1.79 m
2.46 m
1.57 m
2.05 m
0.91 s
1.09
1.52
1.05
1.30
1.05
2.04
1.97
2.43
0.85
0.92
1.25
s
m
d (6.5)
m
m
m
m
m
d (7.0)
d (7.0)
s
1.03 s
1.37 s
HBMC
C-3
H-19,29
H-5,28, H1-Gal
C-4,28,29,
C1-Gal
C-4,19,29
C-8,10
H-3,6R
H-28
C-18
C-9,13,14,18
C-13,14,30
C-14,30
H-18
C-13,16,18,20
C-12,13,14,17
H-21
H-12β,20
C-1,5,9,10
H-2β
H-18
H-12β,17
C-17,20,22
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
H-31
H-31
H-26,27
H-27,31
H-26,31
H-3,6R,29,
H1-Gal
H-2β,28
H-21,26,27,Ac
2.01 s
All assignment were given for 1; spectra of 5, 7, and 8 had only minor differences in chemical shift values.
spectra of 6 had only minor differences in chemical shift values.
1.99, brq, J ) 9.5) as well as an NOE between H3-18 and H-11β
(δ 1.71). Finally, NOE cross-peaks H3-18/H-20 and H-12R/H-21
showed the R* configuration of C-20 and the β-orientation for the
side chain. The structure of the side chain was established to be
the same as that of eryloside R on the basis of COSY and HMBC
correlations. The R-orientation of a γ-lactone in 3 was assigned on
the basis of the upfield shift of the C-5 signal (47.0) when compared
with that for lanost-7-en-3β,17R-diol (50.0).15 Eryloside T (3) was
thus unambiguously determined as 3-O-(β-D-galactopyranosyl)14,9-lactone-24,25-dimethyllanost-7(8)-en-3β,24-diol. Thus, eryloside T exhibits a an unusual structural feature among known
marine sponge glycosides, a 14,9-carbolactone.
The molecular formula of eryloside U (4) was determined to be
C38H62O10 by a HRMALDIMS peak at m/z 701.4263 and was in
accordance with 13C NMR data. The IR spectrum of 4 exhibited a
characteristic band at 1748 cm-1 attributable to a five-membered
lactone. The 1H and 13C NMR spectra of the aglycon part of 4
(Tables 4 and 6) indicated the presence of a β-equatorial secondary
alcohol function at δ 88.0 (CH), a γ-lactone at 176.8 (C) and 85.8
(C), two quaternary oxygenated carbons at 64.4 (C) and 75.2 (C),
and one secondary oxygenated carbon at 54.0 (CH). The placement
of a γ-lactone at C-14 and C-9 was evident from the long-range
correlation of H3-19 (δ 1.01) with C-9 (δ 85.8) and of H3-18 (δ
1.04), H-15 (δ 1.25) with C-14 (δ 59.6). The COSY-45 and HSQC
spectra of 4 revealed the connectivity sequence of the protons in
ring B (>CH (5)-CH2 (6)-CHO (7)-). Further, the 1H and 13C
NMR spectra showed signals corresponding to a trisubstituted epoxy
methine at δH 3.13 (1H, brd, J ) 6.4 Hz) and δC 54.0. These data
NOESY
H-3
H-19
b
All assignment were given for 2;
and HMBC correlations of H-7 with C-5 (δ 42.6), C-6 (δ 20.8),
and C-8 (δ 64.4), of H-6 with C-5, C-7 (δ 54.0), and C-8, and of
H-11 with C-8 (Table 6) indicated the location of an epoxy group
at C-7, C-8. The structure of the side chain was established to be
the same as that of eryloside R on the basis of COSY and HMBC
correlations. The orientation of an epoxy group in 4 was assigned
as R, because the upfield shift of the C-5 signal (42.6) when
compared with that for 3 (47.0) may be explained by the γ-effect
of an axial oxygen function at C-7. This shift is predictable on the
basis of analysis of spectral data of lanostane derivatives.15–18 The
above data defined the structure of 4 as 3-O-(β-D-galactopyranosyl)7,8-epoxy-14,9-lactone-24,25-dimethyllanostan-3β,24-diol. This agrees
with the molecular mass difference of 16 mass units between
eryloside T and 4.
Taking into consideration that five biosides, namely, erylosides
F and F1-F4, were previously described,5,7 we have designated
glycosides 5–7 as erylosides F5–F7. The NMR spectra of erylosides
F5 (5) and F6 (6) indicated that both compounds contained identical
carbohydrate moieties (Table 2). Initial examination of the 1-D
proton and one-bond correlation NMR data suggested the presence
of two sugars (anomeric signals at δH 4.77, δC 104.8 and δH 5.42,
δC 102.8). Interpretation of the 1H-1H COSY and 1D-TOCSY
spectra gave rise to a spin system for these monosaccharides, which
were assigned as β-galactopyranose (Gal) and β-2-N-acetylglucosamine by analysis of 13C NMR, HSQC, HMBC, and NOESY
data as well as by the 3JH-H coupling constant of ring protons.19,21
The acid hydrolysis of the sum of these glycosides gave D-galactose
and D-N-acetylglucosamine, which were identified by capillary GC
Journal of Natural Products, 2007, Vol. 70, No. 12 1875
Erylosides from the Carribean Sponge Erylus goffrilleri
Table 6.
1H
NMR Data for the Aglycon Moieties of 3 and 4 in C5D5N
3
H
1
δH (J, Hz)
HBMC
3
R: 2.00 m
β: 1.56 m
R: 2.39 m
β: 1.88 m
3.39 dd (4.0, 11.6)
5
1.35 t (7.5)
6
1.95 t (6.2)
7
11
5.58 t (4.8)
R: 1.82 m
β: 1.71 m
R: 1.59 m
β: 1.84 m
C-5,6,9,14
C-8
C-8
C-13,14,30
C-14,30
17
1.77
1.70
2.28
1.42
1.99
18
0.77 s
19
20
21
22
26
27
28
0.97
1.44
0.96
2.02
1.23
1.74
1.70
1.15
1.15
1.26
29
31
32
1.09 s
1.29 s
1.15 s
2
12
15
16
23
m
m
m
m
brq (9.5)
s
m
d (7.0)
m
m
m
m
s
s
s
4
H-19
C-4,28,29
C-4,6,7,10,
19,28
C-5,7,8,10
C-12,13,16,
18,20
C-12,13,14,17
C-1,5,9,10
H-29
H-5,28,
H1-Gal
H-3
H-19,28,29
H-15
H-18
H-21
H-18
H-7
H-21
H-11β,12β,
20
H-1β,6, H-29
H-18
C-17,20,22
C-24,25,27,32
C-24,25,26,32
C-3,4,5,29
C-3,4,5,28
C-23,24,25
C-24,25,26,27
δH (J, Hz)
NOESY
H-3,6
H1-Gal
H-6
of the corresponding acetylated (-)- and (+)-2-octyl glycosides
using authentic samples prepared from D- and L-galactose and D-Nacetylglucosamine.13 The arrangement of the sugar units was
determined by HMBC and NOESY experiments. A long-range
1H-13C correlation (H1-Gal (δ 4.78)/C-3 (δ 88.8)) (Tables 2 and
4) as well as the NOESY cross-peak between H1-Gal and H-3
revealed a linkage between the galactose and aglycone. Similary,
a long-range correlation of H-1-NAcGlc (δ 5.39)/C2-Gal (δ 80.8)
and the NOESY cross-peak between H1-NAcGlc and H2-Gal (δ
4.52) assigned the linkage between these two sugar units.
The HRMALDIMS of eryloside F5 (5) showed the quasimolecular ion at m/z 890.5214 [M + Na]+, consistent with the
molecular formula C46H77O14. The structure of the aglycon moiety
of 5 was found by extensive NMR spectroscopy (1H and 13C NMR,
COSY, HSQC, HMBC, and NOESY) (Tables 4 and 5) to be the
same as that of eryloside R. All the above data confirmed the
structure of eryloside F5 (5) as 3-O-[2-acetamido-2-deoxy-β-Dglucopyranosyl-(1f2)-β-D-galactopyranosyl]-14-carboxy-24,25dimethyllanost-8(9)-en-3β,24-diol.
The molecular formula of eryloside F6 (6) was determined as
C47H77O15 on the basis of a high-resolution MALDIMS peak at
m/z 918.5226 and was in accordance with 13C NMR data. The 1H
and 13C NMR data observed for the aglycon part of 6(Tables 4
and 5) matched those reported for eryloside S. Thus, the structure
of eryloside F6 (6) was represented as 3-O-[2-acetamido-2-deoxyβ-D-glucopyranosyl-(1f2)-β-D-galactopyranosyl]-14-carboxy-24acetoxy-24-methyllanost-8(9)-en-3β-ol.
Eryloside F7 (7) was analyzed for C44H74O14 on the basis of
HRMALDIMS and NMR data. The disaccharide nature of 7 was
R: 1.91 m
β: 1.49 m
R: 2.35 m
β: 1.83m
3.36 dd (3.9,11.8)
1.58 dd (4.0,
13.5)
R: 1.93 ddd (4.0, 6.6, 15.0)
β: 1.72 t (14.6)
3.13 brd (6.4)
2.02 m
1.87 m
R: 1.63 m
β: 1.95 m
1.64 m
1.25 m
2.23m
1.35 m
1.93 m
HBMC
C-10
C-3,5,10
C-1,3,4,10
C-4,28,29,
C1-Gal
C-4,6,9,
10,28,29
C-5,7,10
C-5,7,8,10
C-5,6,8,14
C-12
C-8,12,13
C-11,13,14,
17,18
C-9,11,14
C-30
C-8,14,30
C-13,14,15,17
NOESY
H-19
H-19
H-5,28, H1-Gal
H-3,28
H-7,28
H-7,29
H-6R,β,15
H-19
H-19
H-18,21
C-12,13,16,20
1.04 s
C-12,13,14,17
H-12β,20
1.01
1.45
0.94
1.99
1.22
1.71
C-1,5,9,10
H-1β,6β,11
H-18
H-12β
s
m
s
m
m
m
C-17,20,22
1.14 s
1.14 s
1.22 s
C-24,25,27,32
C-24,25,26,32
C-3,4,5,29
0.98 s
1.28 s
1.14 s
C-3,4,5,28
C-23,24,25
C-24,25,26,27
H-3,5,6R,29,
H1-Gal
H-6β,28
evident from its 13C and DEPT spectra (Table 3), which exhibited
two signals for anomeric carbons at δ 106.4 (CH) and 106.9
(CH) and those of the corresponding protons at δ 5.40 (d, J )
7.8 Hz) and 4.79 (d, J ) 7.7 Hz) in the 1H NMR data.
Interpretation of the 13C NMR, COSY, HSQC, and 1D-TOCSY
spectra gave rise to spin systems for these monosaccharides,
which were assigned as galacto- and gluco-β-configured residues
on the basis of chemical shifts and coupling constants of ring
protons.19,21–23 This deduction was further corroborated by
intraresidual NOE connectivity between anomeric methines and
H3,5-Gal, H3,5-Glu found in the NOESY spectrum (Table 3).
The absolute configurations of the galactose and glucose were
determined after acid hydrolysis of 7 by preparation of acetylated
(-)-2-octyl glycosides followed by GC and comparison with
corresponding authentic samples obtained from D- and Lgalactose and D- and L-glucose.14 The arrangement of the sugar
moieties in 7 was established by a combination of the HMBC
and NOESY spectra. A long-range 1H-13C correlation (H1-Gal
(δ 4.79)/C-3 (δ 88.4)) (Tables 3 and 4) as well as the NOESY
cross-peak between H1-Gal and H-3 revealed a linkage between
the galactose and aglycone. Similary, a long-range correlation
of H1-Glc (δ 5.40)/C3-Gal (δ 85.0) and the NOESY cross-peak
between H1-Glc and H3-Gal (δ 4.26) assigned the 1,3-linkage
between glucose and galactose. A close inspection of the 1H
and 13C NMR data of 7 (Tables 4 and 5) revealed that eryloside
F7 was structurally identical to eryloside R with respect to the
aglycon. All the above data confirmed the structure of eryloside
F7 (7) as 3-O-[β-D-glucopyranosyl-(1f3)-β-D-galactopyranosyl]14-carboxy-24,25-dimethyllanost-8(9)-en-3β,24-diol.
1876 Journal of Natural Products, 2007, Vol. 70, No. 12
AfiyatulloV et al.
Figure 2. Structures of erylosides 5–8.
Figure 1. Structures of erylosides 1–4.
The molecular formula of eryloside V (8) was determined to be
C48H80C17 by high-resolution MALDIMS (m/z 951.5258, [M +
Na]+) and 13C NMR analyses. Its 1H and 13C NMR spectra (Table
3) revealed three anomeric protons at δ 4.82 (d, J ) 6.5 Hz), 5.17
(d, J ) 7.5 Hz), and 5.35 (d, J ) 7.8 Hz), which correlated with
the anomeric carbon signals at δ 105.0 (CH), 105.1 (CH), and 104.9
(CH). Acid hydrolysis of 8 gave L-arabinose, D-xylose, and
D-galactose, which were identified by GC of the corresponding
acetylated (-)-2-octyl glycosides, using authentic samples prepared
from the standard monosaccharides.14 The identification of each
sugar as well as their sequence, interglycosidic linkage, and
configuration of glycosidic bonds (β for xylose and galactose and
R for arabinose) in 8 were determined by 1D and 2D NMR,
including HMBC, HMQC, NOESY, and 1H-1H coupling constant
values.19,21–23 The correlation observed in the HMBC spectrum
between H1-Ara and C-3 as well as the NOESY cross-peak H-3/
H1-Ara assigned the connectivity between arabinose and C-3 of
the aglycon. The long-range correlations of H2-Ara with C1-Gal
and H3-Ara with C1-Xyl coupled with NOESY cross-peaks
between H2-Ara and H1-Gal and between H3-Ara and H1-Xyl
defined the 1,2-linkage between galactose and arabinose and 1,3linkage between xylose and arabinose. The aglycon moiety of 8
was found by extensive NMR spectroscopy (Tables 4 and 5) to be
the same as that of eryloside R. On the basis of all the data above,
the structure of eryloside V (8) was established as 3-O-{[β-Dgalactopyranosyl-(1f2)]-[β-D-xylopyranosyl-(1f3)]-R-L-arabinopyranosyl}-14-carboxy-24,25-dimethyllanost-8(9)-en-3β,24-diol.
Erylosides R, S, T, V, F6, and F7 exhibited cytotoxic action
against tumor cells of Ehrlich carcinoma (IC50 ) 20–40 µM) in
vitro.
Experimental Section
General Experimental Procedures. Optical rotations were measured using a Perkin-Elmer 343 polarimeter. The 1H and 13C NMR
spectra were recorded in C5D5N on Bruker Avance 500 and Avance
600 spectrometers at 500 and 125.8 MHz and 600 and 150.9 MHz,
respectively, using tetramethylsilane as an internal standard. 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-hydroxycinnamic acid as matrix. GC
analyses were performed on an Agilent 6850 Series GC system
equipped with a HP-5MS column using a temperature program of 100
to 250 °C at 5 °C min-1; temperatures of injector and detector were
150 and 270 °C, respectively. Low-pressure liquid column chromatography was performed using Polychrome-1 (Teflon powder, Biolar,
Latvia) and Si gel L (40/100 µm, Chemapol, Praha, Czech Republic).
Glass plates (4.5 × 6.0 cm) precoated with Si gel (5–17 µm, Sorbfil,
Russia) were used for TLC. Preparative HPLC was carried out on a
Beckman-Altex chromatograph, using 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 was collected in February 1998 near
Arresife-Seko Reef (Cuba) by scuba diving at depths of 15–20 m. The
sponge was cut and lyophilized immediately after collection. A voucher
specimen (PIBOC 001-059) is on deposit in the collection of the Pacific
Institute of Bioorganic Chemistry, Vladivostok, Russia. The sponge
was identified as Erylus goffrilleri Wiedenmayer, 1977 (family Geodidae). The sponge was massively encrusting, up to 30 mm thick.
Texture is firm, slightly compressible; surface is smooth and gently
wrinkled. A layer of aspidasters and microoxes form a detachable crust
(up to 0.8 mm thick). The rare orthotrianes are radially arranged to
surface. The rhabdus are 650 by 10 µm, the clads 240 by 8 µm. The
choanosomal oxeas are slightly curved (1 mm by 15 µm). The thin
aspidasters are irregularly rhomb shaped 150 µm in length and 90 µm
in width. The microoxeas/microstrongyles are slightly centrotylote and
curved (30 µm by 2 µm). There are small calthrop-like oxyasters with
4–6 acanthose rays (14 µm in diameter). There is some resemblance
to Erylus formosus Sollas widely distributed in the Caribbean, but
clearly different by the morphology of the aspidasters.
Extraction and Isolation. The lyophilized specimens (0.1 kg) were
macerated and extracted with EtOH (4 × 500 mL) and 70% EtOH (2
× 500 mL). The combined extracts were concentrated to dryness and
separated by low-pressure RP CC (20 × 8 cm column) on Polychrome-1
Teflon powder 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. After
evaporation of the solvent, half of the residual material (4.5 g) was
subjected to Si gel flash CC (7 × 13 cm) with a solvent gradient system
of increasing polarity from 5% to 30% EtOH in CHCl3 (total volume
3 L). Fractions of 10 mL were collected and combined by TLC
examination to obtain two subfractions. Subfraction I (540 mg) was
further purified and separated by RP HPLC on a Diasphere-110-C18
column eluting with MeOH-H2O (90:10) and repeatedly chromatographed on a YMC-Pack ODS-A column in the same system to yield
erylosides R (1) (180 mg), S (2) (5.5 mg), T (3) (3.0 mg), and U (4)
(3 mg). Subfraction II (240 mg) was subjected to HPLC on a Diasphere110-C18 column with MeOH-H2O (85:15) and then on a YMC-Pack
ODS-A column using MeOH-H2O-CHCl3(75:25:5) to give erylosides
A5 (5) (3.5 mg), A6 (6) (12.0 mg), A7 (7) (10.0 mg), and V (8) (8 mg).
Eryloside R (1): colorless, amorphous solid; 180 mg; [R]D20 -38.0
(c 0.1, MeOH); IR (CD3OD) 1687 cm-1; 1H and 13C NMR data, see
Tables 1, 4, 5; HR MALDI TOF MS m/z 687.4483 [M + Na]+, calcd
for C38H64O9Na 687.4448.
Eryloside S (2): colorless, amorphous solid; 5.5 mg; [R]D20 -24.5
(c 0.2, MeOH); IR (CD3OD) 1685 cm-1; 1H and 13C NMR data, see
Tables 1, 4, 5; HR MALDI TOF MS m/z 715.4351 [M + Na]+, calcd
for C39H64O10Na 715.4397.
Eryloside T (3): colorless, amorphous solid; 3.0 mg; [R]D20 -14.0
(c 0.1, MeOH); IR (CD3OD) 1748 cm-1; 1H and 13C NMR data, see
Erylosides from the Carribean Sponge Erylus goffrilleri
Tables 1, 4, 6; HR MALDI TOF MS m/z 685.4331 [M + Na]+, calcd
for C38H62O9Na 685.4292.
Eryloside U (4): colorless, amorphous solid; 3.0 mg; [R]D20 -55.0
(c 0.1, MeOH); IR (CD3OD) 1748 cm-1; 1H and 13C NMR data, see
Tables 1, 4, 6; HR MALDI TOF MS m/z 701.4263 [M + Na]+, calcd
for C38H62O10Na 701.4241.
Eryloside F5 (5): colorless, amorphous solid; 4.0 mg; [R]D20 -41.0
(c 0.1, MeOH); 1H and 13C NMR data, see Tables 1, 3; HR MALDI
TOF MS m/z 890.5214 [M + Na]+, calcd for C46H77O14NNa 890.5242.
Eryloside F6 (6): colorless, amorphous solid; 12.0 mg; [R]D20 -35.0
(c 0.25, MeOH); 1H and 13C NMR data, see Tables 4, 5; HR MALDI
TOF MS m/z 918.5226 [M + Na]+, calcd for C47H77O15NNa 918.5191.
Eryloside F7 (7): colorless, amorphous solid; 10.0 mg; [R]D20 -29.5
(c 0.2, MeOH); 1H and 13C NMR data, see Tables 4, 5; HR MALDI
TOF MS m/z 849.4898 [M + Na]+, calcd for C44H74O14Na 849.4976.
Eryloside V (8): colorless, amorphous solid; 8.0 mg; [R]D -24.52
(c 0.2, MeOH); 1H and 13C NMR data, see Tables 4, 5; HR MALDI
TOF MS m/z 951.5258 [M + Na]+, calcd for C48H80O17Na 951.5293.
Acidic Hydrolysis of Erylosides R-U (1–4). A solution of a
mixture of compounds 1–4 (each 1.5 mg) in 0.2 M TFA (0.5 mL) was
heated in a stoppered reaction vial at 100 °C for 1 h. The H2O layer
was extracted with CHCl3 and then neutralized with Dowex (HCO3-).
The residue obtained after evaporation of the H2O layer was purified
on a Zorbax NH2 column (5 µm, 4.6 × 150 mm) eluting with
CH3CN-H2O (90:10) to yield 1.1 mg of galactose. The monosaccharide
was treated with (-)-2-octanol (0.2 mL) in the presence of trifluoroacetic acid (1 drop) in a stoppered reaction vial at 130 °C overnight.13
The mixture was evaporated to dryness and acetylated with Ac2O in
pyridine. The acetylated (-)-2-octyl glycoside was analyzed by GC
using the corresponding authentic samples prepared from D- and
L-galactose.
Acidic Hydrolysis of Erylosides F5 and F6 (5, 6). A solution of a
mixture of compounds 5 and 6 (each 4.0 mg) in 2 N HCl (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 a Zorbax
NH2 column (5 µm, 4.6 × 250 mm) eluting with CH3CN-H2O (90:
10) to yield 0.8 mg of galactose and 0.7 mg of 2-N-acetylglucosamine.
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. Retention time
for the L-GlcNAc derivative was determined for (+)-2-octyl glycoside
of the corresponding D-sugar according to Leontein.13
Acidic Hydrolysis of Eryloside F7 (7). Compound 7 (6.0 mg) was
hydrolyzed as described above for erylosides F5 and F6. 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 and D- and L-glucose.14
Acidic Hydrolysis of Eryloside V (8). Compound 7 (6.0 mg) was
hydrolyzed as described above for erylosides F5 and F6. 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 Dand L-xylose.14
Bioassay. Ehrlich carcinoma cellls were grown intraperitoneally in
albino mice, 18–20 g in weight. Cells were harvested on the seventh
to tenth day after inoculation and washed twice by centrifugation (450
g, 10 min) in cold phosphate-buffered saline (PBS). Then 100 µL of
Journal of Natural Products, 2007, Vol. 70, No. 12 1877
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
was incubated additionally for 10 min at 37 °C. The fluorescence
intensity was measured at λex ) 485 nm, λem ) 620 nm.
Acknowledgment. This study was supported by the program grants
RFBR N 06-04-48578 and 05-04-48246, the grant of Supporting of
the Leading Science Schools 6491.2006.4, and Program of Presidium
of RAS “Molecular and Cell Biology”.
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