Synaptosides A and A1, triterpene glycosides from the sea cucumber Synapta maculata containing 3-O-methylglucuronic acid and their cytotoxic activity against tumor cells

Article abstract of DOI:10.1021/np070283+

Two novel triterpene holostane glycosides, synaptosides A (1) and A ₁ (2), have been isolated from the Vietnamese sea cucumber Synapta maculata (Synaptida, Apodida). Their structures were elucidated by spectroscopic methods (NMR and MS) and chemical transformations. Glycosides 1 and 2 have rare branched pentasaccharide carbohydrate chains featuring a 3-O-methylglucuronic acid residue not previously reported in glycosides from sea cucumbers and a 6-O-sulfated glucose. Glycoside 2 has an oxo group at C-7 and a 8(9)-double bond. All these structural features are unknown in glycosides from sea cucumbers. Glycoside 1 has moderate cytotoxic activity (IC₅₀ 8.6 μg/mL) and glycoside 2 is inactive against HeLa tumor cells.

Full text of DOI:10.1021/np070283+

J. Nat. Prod. 2008, 71, 525–531
525
Synaptosides A and A1, Triterpene Glycosides from the Sea Cucumber Synapta maculata
Containing 3-O-Methylglucuronic Acid and Their Cytotoxic Activity against Tumor Cells
Sergey A. Avilov,† Alexandra S. Silchenko,† Alexander S. Antonov,† Vladimir I. Kalinin,*,† Anatoly I. Kalinovsky,†
Alexey V. Smirnov,‡ Pavel S. Dmitrenok,† Evgeny V. Evtushenko,† Sergey N. Fedorov,† Alexandra S. Savina,†
Larisa K. Shubina,† and Valentin A. Stonik†
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, 690022, VladiVostok, Russian Federation, and
Zoological Institute of the Russian Academy of Sciences, 199164, Saint Petersburg, Russian Federation
ReceiVed June 13, 2007
Two novel triterpene holostane glycosides, synaptosides A (1) and A1 (2), have been isolated from the Vietnamese sea
cucumber Synapta maculata (Synaptida, Apodida). Their structures were elucidated by spectroscopic methods (NMR
and MS) and chemical transformations. Glycosides 1 and 2 have rare branched pentasaccharide carbohydrate chains
featuring a 3-O-methylglucuronic acid residue not previously reported in glycosides from sea cucumbers and a 6-O-
sulfated glucose. Glycoside 2 has an oxo group at C-7 and a 8(9)-double bond. All these structural features are unknown
in glycosides from sea cucumbers. Glycoside 1 has moderate cytotoxic activity (IC50 8.6 µg/mL) and glycoside 2 is
inactive against HeLa tumor cells.
Triterpene glycosides from sea cucumbers (Holothurioidea,
Echinodermata) have been the subjects of long-term systematic
investigations.1–3 Most of these glycosides have been isolated from
sea cucumbers belonging to the orders Aspidochirotida and Den-
drochirotida. Only one species belonging to the order Apodida,
Synapta maculata, has been studied. The aglycon structure of the
major glycoside was established to be holost-7-en-3ꢀ-ol-23-one,
but the full structure of the glycoside was unknown.4 Here we report
the full structures of two new triterpene glycosides, synaptosides
A (1) and A1 (2), isolated from the glycosidic fraction of S. maculata
collected in the South China Sea near the Vietnamese shore.
indicating the sequential cleavage of a carbohydrate chain of
synaptoside A (1).
13C NMR and DEPT spectra of the aglycon part of synaptoside
A (1) (Table 1) are coincident with those of cucumechinosides C
and F isolated from the sea cucumber Cucumaria echinata5 and
calcigerosides C2 and D2 from the sea cucumber Pentamera
calcigera.6,7 13C NMR and DEPT spectra of synaptoside A (1) show
signals at 180.8 (C-18), 120.0 (C-7), and 146.5 (C-8) ppm,
characteristic of holostane aglycones having a 7(8)-double bond.
The signal of a quaternary carbon at 209.18 ppm (C-23) in the 13
C
NMR spectrum along with the correlation H-24/C-23 in the HMBC
spectrum confirmed the presence of a keto group in the aglycon
side chain of synaptoside A (1). The structure of the aglycon
was also confirmed by the analysis of NOESY and COSY spectra
(Table 1).
Results and Discussion
An ethanolic extract of S. maculata was evaporated in Vacuo,
and the residue was sequentially submitted to column chromatog-
raphy on Polychrom-1 (powdered Teflon, Latvia) and silica gel to
give a glycoside fraction. This fraction was submitted to HPLC on
a Diasphere C-8 column to give pure synaptoside A (1) as a major
component and synaptoside A1 (2) as a minor component. Structures
of the isolated glycosides were elucidated on the basis of spectro-
scopic data (1H and 13C NMR, DEPT, HSQC, HMBC, COSY,
NOESY, TOCSY, and MALDI TOF MS) and chemical transforma-
tions.
The HR MALDI TOF MS (positive ion mode) of synaptoside
A (1) exhibited a pseudomolecular ion peak [M + Na]+ at m/z
1409.5147, calculated for C60H92O31SNa3 as 1409.5036 m/z, that
allowed determination of the molecular formula of synaptoside A
(1) as C60H92O31SNa2. In the MALDI TOF MS (positive ion mode)
of synaptoside A (1) the ion peaks [M + Na - SO3Na + H]+ at
m/z 1307.4 and [M + Na - 3-O-methylglucuronic acid sodium
salt + H]+ at m/z 1197.4, indicating the loss of a sulfate group and
the terminal monosaccharide residue, respectively, were observed
along with a pseudomolecular ion peak [M + Na]+ at m/z 1409.4.
The MALDI TOF MS (negative ion mode) showed a pseudomo-
lecular ion peak [M - Na]- at m/z 1363.3 along with fragmentary
ion peaks [M - Na - 3-O-methylglucuronic acid sodium salt -
glucose + H]- at m/z 989.3 and [M - Na - 3-O-methylglucuronic
acid sodium salt - glucose - quinovose + H]- at m/z 843.3,
A comparison of NMR data for the carbohydrate moiety in
synaptoside A (1) with those of known glycosides revealed that
the sugar chain of 1 was new and that some signals in the spectra
were not characteristic of typical monosaccharide units in triterpene
glycosides from sea cucumbers.
The presence of five monosaccharide units in the sugar chain of
the glycoside 1 was easily deduced from its 13C NMR and DEPT
spectra. These spectra showed signals of five anomeric carbons at
102.9–104.8 ppm correlated with the corresponding anomeric
protons at δ 4.79 (d, J ) 7.2 Hz), 5.12 (d, J ) 7.8 Hz), 5.00 (d, J
) 7.9 Hz), 5.31 (d, J ) 7.9 Hz), and 4.97 (d, J ) 7.9) ppm in the
HSQC spectrum (Table 2). The coupling constants of the anomeric
protons indicated a ꢀ-configuration for all of the glycosidic bonds.8
Correlations in the NOESY spectrum of 1 between the anomeric
proton of a xylose residue (monosaccharide I) and H-3 and H-31
of the aglycon moiety showed that the xylose residue was attached
to C-3 of the aglycon (Table 2). Localization of the interglycosidic
linkages of the xylose residue were established using the glycosyl-
ation shifts of C-2 and C-4 in the 13C NMR spectrum of synaptoside
A (1) to 82.3 and 77.9 ppm, corresponding with the signals of C-2
(75.1 ppm) and C-4 (71.1 ppm) in the spectrum of the model 3-O-
[ꢀ-D-xylopyranosyl]-16-oxo-holost-9(11)-en-3ꢀ-ol obtained by acid
hydrolysis of psolusoside A from Psolus fabricii.9 Moreover, the
cross-peak between H-2 of the xylose unit at 4.07 ppm and C-1 of
the second monosaccharide unit at 104.7 ppm in the HMBC
spectrum along with the analogous cross-peak between H-2 of the
xylose residue and an anomeric proton of the second sugar unit at
5.12 ppm in the NOESY spectrum confirmed the attachment of
* To whom correspondence should be addressed. Fax: +7-(4232)-
314050. E-mail: kalininv@piboc.dvo.ru.
† Pacific Institute of Bioorganic Chemistry.
‡ Zoological Institute of the Russian Academy of Sciences.
10.1021/np070283+ CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/22/2008

526 Journal of Natural Products, 2008, Vol. 71, No. 4
AViloV et al.
Table 1. 1H and 13C NMR Data and Selected HMBC and NOESY Correlations of the Aglycon Moieties of Synaptoside A (1), Its
Desulfated Derivative 3, and Desulfated Methylated Derivative 4
a
position
δC
δH mult. (J in Hz)b
HMBC
NOESY
1
2
3
4
5
6
7
8
36.1
26.9
89.2
34.9
47.9
23.2
120.0
146.5
47.3
35.4
22.7
30.0
57.7
51.3
34.1
25.5
53.5
180.8
23.9
83.2
27.1
51.8
209.2
52.2
24.5
22.4
22.3
17.2
28.6
30.7
1.46 m
1.94 m (a), 2.12 m (e)
3.31 dd (3.9, 12.0)
H-5, H-1 Xyl1
1.03 dd (6.0, 10.0)
2.04 m
5.76 m
H-3
H-19, H-30, H-31
9
3.41 m
H-19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30
31
32
1.54 m, 1.80 m
2.04 m
H-1, H-12
H-17
1.85 m, 1.63 m
2.07 m, 1.72 m
2.79 dd (3.5, 10.5)
C: 20
H-12, H-21, H-32
H-6, H-9
1.22 s
C: 1, 5, 9, 10
C: 17, 20, 22
1.66 s
H-17, H-22
3.20 d (18.0) 3.13 d (18.0)
H-21, H-24 H-21, H-22
2.43 d (6.5)
2.18 m
0.96 d (7.0)
0.96 d (7.0)
1.11 s
C: 23, 25, 26, 27
H-22
C: 24, 25, 27
C: 24, 25, 26
C: 3, 4, 5, 31
C: 3, 4, 5, 30
C: 8, 13, 14, 15
H-6
H-6, H-1 Xyl
H-17
1.27 s
1.14 s
a Recorded at 125.77 MHz in C5D5N/D2O (4:1). b Recorded at 500 MHz in C5D5N/D2O (4:1).
this sugar to C-2 of the xylose residue. The analysis of 1D TOCSY,
COSY, HSQC, HMBC, and NOESY spectra showed that the second
monosaccharide unit (monosaccharide II) in the carbohydrate moiety
of synaptoside A (1) was quinovose (Table 2).
unit were correlated with the signal of a quaternary carbon at 175.4
ppm in the HMBC spectrum of 1. Such a chemical shift (δC) is
characteristic of the carboxyl group in uronic acids. The signal at
60.4 ppm in the 13C NMR and the corresponding proton signal at
1
The presence of the cross-peak between C-4 of the xylose at
77.9 ppm and H-1 of the sulfated glucose in the HMBC spectrum
and the analogous correlation between H-4 of the xylose residue
at 4.26 ppm and H-1 of the sulfated glucose residue at 4.97 ppm
in the NOESY spectrum of 1 indicated that the fourth carbon of
the xylose residue was linked by a ꢀ-glycosidic bond with an
anomeric carbon of a sulfated glucose residue (monosaccharide V).
The presence of a sulfate group at C-6 of this glucose residue
was confirmed by comparison of the 13C NMR spectrum of the
synaptoside A (1) carbohydrate moiety with that of the known
synallactoside C from the sea cucumber Synallactes nozawae.10
Both have similar glucose residues linked to C-4 of the first xylose
unit. The C-6 and C-5 signals of the glucose residue in the 13C
NMR spectrum of 1 were shifted downfield by 5.1 ppm and shifted
upfield by 2.2 ppm, respectively, when compared with shifts of
synallactoside C, due to R- and ꢀ-shifting effects of sulfation.8
One more monosaccharide unit in the carbohydrate moiety of
synaptoside A (1) (monosaccharide III) was identified as a glucose
attached by a ꢀ-(1f4) linkage to the quinovose residue. It was
confirmed by cross-peaks H-4 Qui/C-1 Glc and H-1 Glc/C-4 Qui
in the HMBC and H-4 Qui/H-1 Glc in the NOESY spectra. The
signals H-1-H-6 and C-1-C-6 of this residue were identified by
the analysis of TOCSY, NOESY, and HSQC spectra (Table 2).
The third carbon of this glucose unit was linked by a glycosidic
bond with a terminal monosaccharide unit. This was confirmed by
the presence of cross-peaks between C-3 of glucose at 86.8 ppm
and H-1 of the terminal sugar unit at 5.31 ppm observed in the
HMBC spectrum of synaptoside A (1).
3.89 ppm in the H NMR spectrum indicated the presence of an
O-methyl group attached to C-3 [the cross-peak H3 (OMe)/C-3
MeGlcUa in the HMBC spectrum] of the terminal monosaccharide
unit. According to these data, 3-O-methylglucuronic acid residue
was identified in the carbohydrate moiety of synaptoside A (1) as
the terminus. Therefore, the carbohydrate moiety of 1 was a
pentasaccharide, branched at C-4 of the first xylose residue and
containing a 6-O-sulfated glucose and a 3-O-methylglucuronic acid
as terminal units.
In order to confirm the position of the sulfate group in the
carbohydrate moiety of synaptoside A (1), we obtained desulfated
derivative 3 by solvolysis of 1 in a dioxane/pyridine mixture. The
signals of the carbohydrate chain in the 13C NMR spectrum of 3
were compared with those of synaptoside A (1) (Table 3). Signals
of C-5 and C-6 of the terminal glucose residue in the 13C NMR
spectrum of 3 were shifted downfield by 1.6 ppm and upfield by
5.0 ppm, correspondingly, due to the absence of R- and ꢀ-effects
of a sulfate group, as compared with the same signals in the
spectrum of 1.8 These data confirm the presence of a sulfate group
attached to C-6 of the glucose residue in the carbohydrate moiety
of glycoside 1.
To confirm the presence of a carboxyl group in the 3-O-
methylglucuronic acid in the carbohydrate chain of 1, methylated
derivative 4 was obtained by methylation of derivative 3 with
diazomethane. Comparison of 13C NMR spectra of 3 and 4 showed
the difference in the values of chemical shifts of the fourth terminal
monosaccharide residue only (Table 3). The characteristic signal
of the carboxyl group of 3-O-methylglucuronic acid at 175.7 ppm
was absent in the 13C NMR spectrum of 4. Instead of this signal,
there was a signal of a quaternary carbon at 170.4 ppm (C-6) and
an additional signal of an O-methyl group at 52.4 ppm. These data
Analysis of the 1D TOCSY spectrum allowed assignment of the
signals of H-1-H-5 in the terminal monosaccharide unit (monosac-
charide IV) of the carbohydrate chain of 1. The signals of H-4 (at
4.07 ppm) and H-5 (at 4.13 ppm) of the terminal monosaccharide

Triterpene Glycosides from Synapta maculata
Journal of Natural Products, 2008, Vol. 71, No. 4 527
Table 2. 1H and 13C NMR Data and Selected HMBC and NOESY Correlations of the Carbohydrate Moieties of Synaptosides A (1)
and A1 (2)
a b c
δH mult. (J in Hz)d
HMBC
NOESY
,
,
position
δC
Xyl (1fC-3)
1
2
3
4
5
104.8
82.3
75.1
77.9
63.6
4.79 d (7.2)
4.07 t
4.28 t (9.0)
4.26 m
4.51 m
3.76 m
C: 3
C: 1 Qui
C: 4 Xyl
H-3,5-Xyl, H-3, H-31
H-1 Qui
H-1 Xyl
H-1 Glc2
H-1 Glc 2
H-1 Xyl
C: 3 Xyl
Qui (1f2Xyl)
1
2
3
4
5
6
104.7
75.7
75.1
86.4
71.5
17.9
5.12 d (7.8)
3.99 t (7.5)
4.10 t (8.7)
3.66 t (8,7)
3.79 m
C: 2 Xyl
C: 3 Qui
C: 4 Qui
H-5 Qui, H-2 Xyl
C: 1 Glc 1, 3 Qui
H-6 Qui, H-1 Glc 1
H-1 Qui
H-4 Qui
1.74 d (6.0)
C: 4,5-Qui
Glc 1 (1f4Qui)
1
2
3
104.1
73.8
86.9
5.00 d (7.9)
4.05 m
4.37 t (8.8)
C: 4-Qui
C: 1, 3 Glc 1
C: 1-MeGlcUa, 2,4 Glc 1
H-4 Qui, H-3,5 Glc 1
H-1
MeGlcUa, H-1 Glc 1
4
5
6
69.5
77.3
61.7
4.02 t (9.5)
4.08 m
4.46 brd (10.3)
4.14 m
C: 5 Glc 1
H-1 Glc 1
MeGlcUA (1f3Glc 1)
1
2
3
4
5
6
104.4
74.2
86.6
72.6
74.4
175.4
60.4
5.31 d (7.9)
3.97 t (8.5)
3.74 t (8.7)
4.07 t (9.5)
4.13 d (10.0)
C: 3-Glc 1
H-3 Glc 1, H-3,5 MeGlcUa
H-1 MeGlcUa
C: 1, 3 MeGlcUa
C: 2,4 MeGlcUa, OMe
C: 6 MeGlcUa
C: 1,4,6 MeGlcUa
H-1 MeGlcUa
OMe
3.89 s
C:3 -MeGlcUa
Glc 2 (1f4 Xyl)
1
2
3
4
5
6
102.9
73.8
76.8
70.7
75.8
67.4
4.97 d (7.9)
3.95 t (8.2)
4.24 t (9.0)
4.07 m
C: 4-Xyl
C: 1,3 Glc 2
C: 2,4 Glc 2
H-5 Glc 2, H-4, 5 Xyl
H-1 Glc 2
4.15 m
5.12 brd (10.7) 4.86 dd (11.2; 7.9)
C:5 Glc 2
a Recorded at 125 MHz in C5D5N/D2O (4:1). b Bold ) interglycosidic positions. c Italic ) sulfate position. d Recorded at 500 MHz in C5D5N/D2O
(4/:1).
indicated selective methylation of a carboxyl group in the 3-O-
methylglucuronic acid residue.
Table 3. 13C NMR Data of the Carbohydrate Moieties for the
Desulfated Derivative of Synaptoside A (3), Desulfated Methylated
Derivative of Synaptoside A (4), and Synthetic Methyl (Methyl-3-
O-methyl-ꢀ-D-glucopyranoside)uronate (5)
The presense of 3-O-methylglucuronic acid as a terminal
monosaccharide residue was also confirmed by the coincidence of
the corresponding signals in the 13C NMR spectra of methylated
derivative 4 and synthetic methyl (methyl-3-O-methyl-ꢀ-D-glu-
copyranoside)uronate (5) (Table 3). Reference substance 5 was
synthesized from commercial methyl-ꢀ-D-glucopyranoside by oxi-
dation at atmospheric air on Pt/carbon catalyst followed by
etherification of the carboxyl group by methanol and methylation
of the obtained derivative by diazomethane in the presence of SbCl3.
a b
,
a b
,
δC
δC
position
3
4
position
MeGlcUA
3
4
5
Xyl (1fC-3)
1
2
3
4
5
104.9 104.8 1
82.8 82.7 2
75.2 75.3 3
77.8 77.8 4
63.7 63.6 5
6
104.3 104.6 105.1
74.1 74.1 73.7
86.4 86.2 86.3
72.5 71.9 72.1
74.4 76.0 76.2
175.7 170.4 170.6
Acid hydrolysis of synaptoside A (1) with TFA was carried out
to ascertain its monosaccharide composition. The mixture of sugars
obtained was submitted to HPLC to give individual monosaccha-
rides. Subsequent alcoholysis of each monosaccharide by (R)-(-)-
2-octanol followed by acetylation, GLC analysis, and comparison
with standard monosaccharides allowed us to determine the absolute
D-configuration of all monosaccharide residues comprising the
carbohydrate moiety of synaptoside A (1) (xylose, quinovose,
glucose, and 3-O-methylglucuronic acid). The reference sample of
3-O-methylglucuronic acid was synthesized in our laboratory.11
Hence, synaptoside A (1) is 3-O-{6-O-sodium-3-O-methyl-ꢀ-D-
glucopyranosyluronate-(1f3)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-quino-
vopyranosyl-(1f2)-[4-O-sodium sulfate-ꢀ-D-glucopyranosyl-(1f4)]-
ꢀ-D-xylopyranosyl}-23-oxo-holost-7-en-3ꢀ-ol.
Qui (1f2Xyl)
1
OMef3MeGlcUA 60.5 60.9 60.8
104.9 104.8 OMef6MeGlcUA 52.4 52.5
2
3
4
5
75.6 75.6 Glc 2 (1f4 Xyl)
75.3 75.2 1
86.5 86.5 2
71.5 71.5 3
17.9 17.9 4
5
102.8 102.8
74.1 74.2
77.2 77.3
71.0 71.0
77.4 77.4
62.4 61.9
6
Glc 1 (1f4Qui)
1
2
3
4
5
6
104.0 104.1 6
73.7 73.7
87.2 86.8
69.5 69.2
77.3 77.3
61.8 61.7
The HR MALDI TOF MS (positive ion mode) of synaptoside
A1 (2) exhibited a pseudomolecular ion peak [M + Na]+ at m/z
1423.4694, calculated for C60H90O32SNa3 as 1423.4829 m/z, that
a Recorded at 125 MHz in C5D5N/D2O (4:1). b Bold ) intergly-
cosidic positions.

528 Journal of Natural Products, 2008, Vol. 71, No. 4
AViloV et al.
Table 4. 1H and 13C NMR Data and Selected HMBC and NOESY Correlations of the Aglycon Moiety of Synaptoside A1 (2)
a
position
δC
δH mult. (J in Hz)b
HMBC
NOESY
H-11
1
34.8
26.5
87.7
39.3
50.2
36.5
200.0
135.2
167.5
39.7
24.0
26.9
58.4
46.9
34.3
26.4
49.3
178.7
17.9
83.4
27.6
52.1
209.1
52.1
24.5
22.3
22.3
16.0
26.9
25.7
1.81 m 1.29 m
2
3
4
5
6
7
8
9
1.93 m, 2.14 m
3.23 dd (3.9, 11.2)
H-5, H-31
1.68 m
2.60 m
H-3, H-31
H-19, H-30, H-31
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30
31
32
2.62 m 2.48 m
2.27 m
H-1
H-17, H-21
2.68 m, 1.83 m
2.04 m, 1.72 m
2.74 dd (2.8, 10.3)
C-18
H-12, H-21, H-32
H-6
1.39 s
C: 1, 5, 9, 10
1.70 s
C: 17, 20, 22
C: 23
H-12, H-17, H-22
H-21, H-24
3.20 d (18.0), 3.13 d (18.0)
2.42 dd (3.0, 7.2)
2.18 m
0.96 d (6.5)
0.96 d (6.5)
1.03 s
C: 23, 26, 27
C: 23
H-22
C: 24, 25, 27
C: 24, 25, 26
C: 3, 4, 5, 31
C: 3, 4, 5, 30
C: 8, 13, 14, 15
H-6
H-3, H-5, H-6
H-17
1.14 s
1.16 s
a Recorded at 125.77 MHz in C5D5N/D2O (4:1). b Recorded at 500 MHz in C5D5N/D2O (4:1).
Chart 1
allowed us to determine a molecular formula of synaptoside A1
carbon at 209.1 ppm in the 13C NMR spectrum of 2) (Table 4).
However, in the downfield region of the 13C NMR spectrum of 2,
signals at 135.2 and 167.5 ppm are present. Such signals were not
characteristic for a 7(8)- or 9(11)-double bond. The DEPT and
HSQC spectra of synaptoside A1 (2) indicated that these signals
are signals of quaternary carbons belonging to a 8(9)-double bond.
In the downfield region of the 13C NMR spectrum of 2, an additional
signal of a quaternary carbon at 200.0 ppm was found due to a
ketone group in the polycyclic system of the aglycon.
(2) as C60H90O32SNa2. The MALDI TOF MS (positive ion mode)
peaks of synaptoside A1 (2) were analogous to the peaks of
synaptoside A (1) [M + Na - SO3Na + H]+ at m/z 1321.4 and
[M + Na - 3-O-methylglucuronic acid sodium salt + H]+ at m/z
1211.3. The MALDI TOF MS (negative ion mode) of synaptoside
A1 (2) showed a pseudomolecular ion peak [M - Na]- at m/z
1377.4 and fragmentary ion peaks [M - Na - 3-O-methylglucu-
ronic acid sodium salt + H]- at m/z 1165.3, [M - Na - 3-O-
methylglucuronic acid sodium salt - glucose + H]- at m/z 1003.3,
and [M - Na - 3-O-methylglucuronic acid sodium salt - glucose
- quinovose + H]- at m/z 857.3, demonstrating the sequential loss
of monosaccharide residues in the carbohydrate chain of synaptoside
A1 (2).
The typical position of a ketone group in the sea cucumber
saponin aglycons is C-16.2 However, the presence of a keto group
at C-16 is impossible in 2 because of the signal of a secondary
carbon, CH2-16, at 26.4 ppm in the 13C NMR and DEPT spectra.
Moreover, in the COSY spectrum protons of two methylene groups
[H2-15 at 2.68 (m) and 1.83 (m) ppm; H2-16 at 2.04 (m) and 1.72
(m) ppm)] and a methine group [H-17 at 2.74 (dd, J ) 2.8, 10.3
The analysis of 13C NMR spectral data of the aglycon moiety of
synaptoside A1 (2) showed the presence of the signal of C-18 at
178.7 ppm characteristic for the 18(20)-lactone. The signals of
carbons C-22-C-27 were coincident with the corresponding signals
in the 13C NMR spectrum of synaptoside A (1). This indicated their
side chains have a ketone group at C-23 (the signal of the quaternary
Hz)] of the ring
D formed an isolated spin system
CH2(15)-CH2(16)-CH(17).
Comparison of the 13C NMR spectrum of synaptoside A1 (2)
with that of the 7-keto derivative of lanosterol12 reveals considerable

Triterpene Glycosides from Synapta maculata
Journal of Natural Products, 2008, Vol. 71, No. 4 529
coincidence of the carbon signals of rings A, B, and C of polycyclic
systems of these compounds. This allows us to suggest an additional
keto group occurs at C-7 of the aglycon of synaptoside A1 (2).
The aglycon structure proposed for synaptoside A1 (2) containing
an 8(9)-double bond and a 7-ketone group is confirmed by COSY,
NOESY, and HMBC spectral data (Table 4).
Comparison of the NMR spectra of carbohydrate moieties of
synaptosides A (1) and A1 (2) (Table 2) shows coincidence of all
monosaccharide residue signals. This indicates the identity of the
sugar parts of the glycosides.
(reversibly) calcium transportation, and to inhibit oocyte maturation
before the spawning season in order to synchronize reproductive
processes in a population.27 When oxidation of the terminal glucose
residue into glucuronic acid was lost during further evolution of
sea cucumbers, the 3-O-methyl groups of terminal monosaccharide
residues were conserved because they increased membranolytic
activity of glycosides that was useful for protection against predatory
fish.27 Glycosides having no 3-O-methyl at the terminal monosacca-
haride unit are less active as membranolytic toxins compared to
similar substances having a 3-O-methyl group.28
Therefore, synaptoside A1 (2) is 3-O-{6-O-sodium-3-O-methyl-
ꢀ-D-glucopyranosyluronate-(1f3)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-
quinovopyranosyl-(1f2)-[4-O-sodium sulfate-ꢀ-D-glucopyranosyl-
(1f4)]-ꢀ-D-xylopyranosyl}-7,23-dioxo-holost-8-en-3ꢀ-ol.
Synaptosides A (1) and A1 (2) were studied as potential cytotoxic
agents using the HeLa human tumor cell line. Glycoside 1 showed
moderate cytoxicity, with an IC50 of 8.6 µg/mL. Glycoside 2 was
not active against these tumor cells in concentrations of 14.1 µg/
mL. The absence of activity of glycoside 2 may be explained by
the different position of the double bond in the aglycon nucleus,
8(9), but not 7(8). Hence the cytotoxic activity depends on the
configuration of the aglycon nucleus.
Sugar moieties of sea cucumber glycosides are comprised of two
to six monosaccharide residues. The first is always xylose and the
rest of the monosaccharides are usually glucose, quinovose, 3-O-
methylglucose, and rarely 3-O-methylxylose.1–3 Aglycons of the
majority part of the glycosides are referred to a holostane type,
having an 18(20)-lactone along with 7(8)- or 9(11)-double bonds.
Synaptosides A (1) and A1 (2) are characterized by uncommon
chemical features in the carbohydrate moieties. These glycosides
are the first representatives of triterpene glycosides from sea
cucumbers containing methylated glucuronic acid as one of
monosaccharide residues in carbohydrate chains. 3-O-Methylglu-
curonic acid was found in capsule polysaccarides of bacteria,13,14
but has never been identified in sea cucumber triterpene glycosides
or any other triterpene glycosides. Moreover, synaptoside A1 (2)
contains an unusual polycyclic nucleus having a 7-oxo-8(9)-en
system that is unique in sea cucumber triterpene glycosides.
Taxonomists consider the order Apodida as evolutionarily distant
from other orders in the class based on morphological15–18,20 and
paleontological21,22 data and partially on analysis of 18S rRNA.19,23
The presence of unique 3-O-methylglucuronic acid residues as
terminal monosaccharides in both 1 and 2 correlates well with this
point of view.
Lanosterol is a triterpenoid characteristic of the animal kingdom
that is a precursor of sterols. It has also been suggested to be
biosynthetic24,25 and most probably a phylogenetic precursor of
aglycons from sea cucumber triterpene glycosides. The presence
of the 8(9)-double bond in the aglycon moiety of synaptoside A1
(2), like in lanosterol, suggests the Apodida (including the family
Synaptidae) is the most primitive order in the class Holothurioidea.
Such a point of view was supported earlier by morphological,15–18,20
paleontological,21,22 and 18S rRNA gene sequence19,23 data. This
point of view correlates well with the data on the structures of
triterpene glycosides of Synapta maculata. The absence of cytotoxic
activity of synaptoside A1 (2) having an 8(9)-double bond in the
aglycon moiety also reveals the primitive position of the order
Apodida because membranolytic activity (including cytotoxicity)
of triterpene glycosides ought to be increased during sea cucumber
evolution as an adaptive response against evolving predatory fish.27
Glucuronic acid in water solution may easily form glucurone,
6(3)-lactone.26 The presence of a 3-O-methyl group in the glu-
coronic acid residue completely prevents the formation of this
lactone and correspondingly decreases the hydrophilicity of gly-
coside. It may be adaptive because maintaining the optimal
hydrophilicity may contribute to the ability of glycosides to increase
microviscosity of sea cucumber oocyte membranes, to block
Experimental Section
General Experimental Procedures. All melting points were
determined with a Kofler-Thermogenerate apparatus. Specific rotation
was measured on a Perkin-Elmer 343 polarimeter. NMR spectra were
recorded on a AMX Bruker 500 spectrometer at 500.12/125.67 MHz
(1H/13C) in C5D5N with TMS as an internal reference (δ ) 0). GLC
analysis was carried out on an Aligent 6850 Series apparatus, carrier
gas He (1.7 mL/min) at 100 °C (0.5 min) f 250 °C (5 °C/min, 10
min), capillary column HP-5 MS (30 m × 0.25 mm); temperatures of
injector and detector were 150 and 280 °C, respectively. The MALDI
TOF MS (positive and negative ion modes) were recorded using a
Bruker mass spectrometer, model BIFLEX III, with impulse extraction
of ions, on an R-cyano-4-hydroxycinnamic acid matrix. HPLC was
performed using an Agilent 1100 chromatograph equipped with a
differential refractometer on a Diasphere C8 (4.6 × 250) column.
Animal Material. Specimens of the sea cucumber Synapta maculata
(family Synaptidae; order Apodida) were collected in January 2005
during the 30th scientific cruise of the research vessel Akademik Oparin
in Van Phong Bay (Vietnam), South China Sea, using scuba at a depth
of 2–12 m. The sea cucumber was identified by Dr. V. S. Levin (Pacific
Institue of Bioorganic Chemistry), and a voucher specimens is on
deposit in the collection of the Zoological Institute, the Russian
Academy of Sciences, Saint Petersburg.
Extraction and Isolation. The sea cucumbers were minced and
extracted twice with refluxing 70% EtOH. The dry wt of the residue
after extraction was 34.5 g. The combined extracts were concentrated
to dryness in Vacuo, dissolved in H2O, and chromatographed on a
Polychrom-1 column (powdered Teflon, Biolar, Latvia), eluting first
inorganic salts and polar impurities with H2O and then the glycosides
with 60% acetone. The latter fraction was submitted to sequential
chromatography on Si gel columns eluting with CHCl3/EtOH/H2O (100:
150:50 and 100:125:25) solvent systems to give 563 mg of glycoside
fraction A, as an individual spot on TLC. The glycosides were separated
by HPLC on a Diasphere C8 (4 × 250) column, with 50% MeOH as
mobile phase, to give 150 mg of synaptoside A (1) as a major
compound. A minor fraction was rechrogmatographed in the same
conditions with 55% MeOH as mobile phase to give 7 mg of
synaptoside A1 (2).
20
Synaptoside A (1): mp 285–287 °C; [R]D –16 (c 0.1, pyridine);
for 1H and 13C NMR data, see Tables 1 and 2; MALDI TOF MS
(positive ion mode) m/z (rel int) 1409.4 (C60H92O31SNa3 [M + Na]+,
1), 1307.4 ([M + Na - SO3Na + H]+, 0.3), 1197.4 ([M + Na - 3-O-
methylglucuronic acid sodium salt + H]+, 0.2); MALDI TOF MS
(negative ion mode) m/z (rel int) 1363.3 (C60H92O31SNa [M - Na]-,
1), 989.3 ([M - Na - 3-O-methylglucuronic acid - glucose + H]-,
0.3), 843.3 ([M - Na - 3-O-methylglucuronic acid sodium salt -
glucose - quinovose + H]-, 0.2); HR MALDI TOF MS (positive ion
mode) [M + Na]+ at m/z 1409.5147, calculated for C60H92O31SNa3 as
1409.5036 m/z.
20
Synaptoside A1 (2): mp 268–270 °C; [R]D –3 (c 0.1, pyridine);
for 1H and 13C NMR data, see Tables 1 and 4; MALDI TOF MS
(positive ion mode) m/z (rel int) 1423.4 (C60H90O32SNa3 [M + Na]+,
1), 1321.4 ([M + Na - SO3Na + H]+, 0.3), 1211.3 ([M + Na - 3-O-
methylglucuronic acid sodium salt + H]+, 0.2), 1109.3 ([M + Na -
SO3Na - 3-O-methylglucuronic acid + H]+, 0.2); MALDI TOF MS
(negative ion mode) m/z 1377.4 (C60H90O32SNa [M - Na]-, 1), 1165.3
([M - Na - 3-O-methylglucuronic acid sodium salt + H]-, 0.3), 1003.3
([M - Na - 3-O-methylglucuronic acid sodium salt - glucose + H]-,
0.2), 857.3 ([M - Na - 3-O-methylglucuronic acid - glucose sodium
salt - quinovose + H], 0.2); HR MALDI TOF MS (positive ion mode)
[M + Na]+ at m/z 1423.4694, calculated for C60H90O32SNa3 as
1423.4829 m/z.

530 Journal of Natural Products, 2008, Vol. 71, No. 4
AViloV et al.
Desulfation of Synaptoside A (1). A sample of synaptoside A (1,
5 mg) was dissolved in a mixture of pyridine/dioxane (1:1) and refluxed
for 1 h. The obtained mixture was concentrated in Vacuo. The residue
was chromatographed on a Si gel column with CHCl3/EtOH/H2O (100:
50:4) to give 4 mg of the desulfated derivative 3; see Tables 1 and 3
for NMR data.
Methylation of Desulfated Derivative 3 with Diazomethane. A
sample of derivative 3 (4 mg) was dissolved in 2 mL of MeOH and
placed on a magnetic stirrer. A solution of CH2N2 (2 mL) in diethyl
ether was added to the solution, and the mixture was concentrated in
Vacuo. The residue was chromatographed on a Si gel column with
CHCl3/EtOH/H2O (100:75:10) to give 3.5 mg of the desulfated
methylated derivative 4; see Tables 1 and 3 for NMR data.
Synthesis of Methyl (Methyl-3-O-methyl-ꢀ-D-glucopyranoside)-
uronate (5). To a solution of 0.49 g of commercial methyl-ꢀ-D-
glucopyranoside in 10 mL of H2O was added 15 mg Pt/carbon catalyst,
and the reaction mixture was stirred during 5 h at 60 °C as atmospheric
air bubbled through the reaction mixture. To the reaction mixture was
added 2.5 mL of 1 N H2O solution of NaHCO3 in several portions,
maintaining a pH of 7.0–8.5. The catalyst was removed by filtration,
the solution was deionized by cation exchange resin KU-2 (H+), and
the solution was evaporated to dryness in Vacuo. The residue was
dissolved in absolute MeOH, refluxed 1 h, and evaporated to dryness.
The residue was chromatographed on a Si gel column (MeOH/CHCl3,
1:9) to give 0.16 g of methyl (methyl-ꢀ-D-glucopyranoside)uronate,
mp 149–150 °C, [R]D20 –25.2 (c 0.8, MeOH). To the solution of 0.12 g
of methyl (methyl-ꢀ-D-glucopyranoside)uronate in 1.5 mL of MeOH
were added 0.01 mM SbCl3 and 5 mL of a 0.5 N solution of CH2N2 in
CH2Cl2. The solution was kept for 2 h at room temperature and
evaporated in Vacuo. The residue was chromatographed on a Si gel
column (MeOH/CHCl3, 0.5:9.5) to give 0.07 g of methyl (methyl-3-
O-methyl-ꢀ-D-glucopyranoside)uronate (5), syrup, [R]D20 ) –22.4 (c
0.5, MeOH); for 13C NMR see Table 3.
28.32, 29.03, 29.24, and 29.46 min), and 3-O-methyl-D-glucuronic acid
(retention times 35.24, 35.52, and 36.14 min).
Tumor Cells Viability Assay. The effect of compounds 1 and 2 on
cell viability was evaluated using MTS reduction into its formazan
product.30 The HeLa cells were cultured for 12 h in 96-well plates
(6000 cells/well) in RPMI media (100 µL/well) containing 10% FBS.
The media was replaced with 5% FBS-RPMI containing known
concentrations of the compounds, and the cells were incubated for 22 h.
A 20 µL amount of MTS reagent was added into each well, and MTS
reduction was measured 2 h later spectrophotometrically at 492 and
690 nm as background using a µQuant microplate reader (Bio-Tek
Instruments, Inc.). The means ( SD from six samples of two
independent experiments were calculated. The statistical computer
program Statistica 6.0 for Windows (StatSoft, Inc., 2001) was used to
compute SD and IC50 in corresponding experiments. The IC50 value
for 1 is 8.6 µg/mL. Glycoside 2 was not active in concentrations of
14.1 µg/mL.
Acknowledgment. The authors thank the Institute of Oceanography
of the Vietnamese Academy of Sciences and Technology for providing
the opportunity to collect the animals. The authors acknowledge
financial support by grants from the Presidium of the Russian Academy
of Sciences “Molecular and Cell Biology” and the President of the
Russian Federation No. NSH-6491.2006.4, RFBR Grant No. 06-04-
96016, FEB RAS Grants No. 06-III-B-05-128, 06-III-A-05-122, and
FEB-UrB RAS Grant No. 06-2U-0-05-009. The authors are very
appreciative of Prof. J. Lawrence from the University of South Florida
(Tampa, FL) for useful discussions of the manuscript and correction
of the English.
References and Notes
(1) Stonik, V. A.; Kalinin, V. I.; Avilov, S. A. J. Nat. Toxins 1999, 8,
235–248.
Acid Hydrolysis and Isolation of Individual Monosaccharides
from Synaptoside A (1). The acid hydrolysis of synaptoside A (1) (7
mg) was conducted in a solution of 0.2 M trifluoroacetic acid (TFA)
(0.3 mL) in a stoppered vial on a H2O bath at 100 °C for 30 min. The
H2O layer was extracted with CHCl3 (3 × 0.5 mL) and concentrated
in Vacuo. The mixture of resulting sugars was submitted to HPLC on
a NH2 9.4 column (4 × 250) with CH3CN/H2O/TFA (65:35:0.1) as
the mobile phase to give only individual 3-O-methylglucuronic acid
and a mixture of the remaining sugars. This mixture was rechromato-
graphed on the same column with CH3CN/H2O (90:10) as the mobile
phase to isolate xylose, quinovose, and glucose, identified as described
below.
(2) Kalinin, V. I.; Silchenko, A. S.; Avilov, S. A.; Stonik, V. A.; Smirnov,
A. V. Phytochem. ReV. 2005, 4, 221–236.
(3) Kobayashi, M.; Hori, M.; Kan, K.; Yasuzawa, T.; Matsui, M.; Suzuki,
Sh.; Kitagawa, I. Chem. Pharm. Bull 1991, 39, 2282–2287.
(4) Kuznetsova, T. A.; Kalinovskaya, N. I.; Kalinovsky, A. I.; Elyakov,
G. B. Khim. Prirod. Soedin. 1985, 5, 667–669.
(5) Miyamoto, T.; Togawa, K.; Higuchi, R.; Komori, T.; Sasaki, T. Liebigs
Ann. Chem. 1990, 453–460.
(6) Avilov, S. A.; Antonov, A. S.; Drozdova, O. A.; Kalinin, V. I.;
Kalinovsky, A. I.; Stonik, V. A.; Riguera, R.; Lenis, L. A.; Jimenez,
C. J. Nat. Prod. 2000, 63, 65–71.
(7) Avilov, S. A.; Antonov, A. S.; Drozdova, O. A.; Kalinin, V. I.;
Kalinovsky, A. I.; Stonik, V. A.; Riguera, R.; Lenis, L. A.; Jimenez,
C. J. Nat. Prod. 2000, 63, 1349–1355.
(8) Shashkov, F. S.; Chizhov, O. S. Bioorg. Khim. 1976, 2, 437–497.
(9) Kalinin, V. I.; Kalinovsky, A. I.; Stonik, V. A. Khim. Prirod. Soedin.
1985, 2, 212–217.
(10) Silchenko, A. S.; Avilov, S. A.; Antonov, A. S.; Kalinin, V. I.;
Kalinovsky, A. I.; Stonik, V. A.; Smirnov, A. V.; Riguera, R.; Jimenez,
C. J. Nat. Prod. 2002, 65, 1802–1808.
(11) Marsh, C. A. J. Chem. Soc. 1952, 1578–1582.
(12) Emmons, G.; Wilson, W.; Schroefer, G. Magn. Reson. Chem. 1989,
27, 1012–1024.
(13) Rodriguez-Carvajal, M. A.; Tejero-Mateo, P.; Espartero, J. L.; Ruiz-
Sainz, J. E.; Buendia-Claveria, A. M.; Oliero, F. J.; Yang, S. S.; Gil-
Serrano, A. M. Biochem. J. 2001, 357, 505–511.
Synthesis of 3-O-Methyl-D-glucuronic Acid. 3-O-Methyl-1,2-
isopropiliden-R-D-glucuronic acid (0.2 g) was obtained from commercial
1(2),5(6)-diisopropilidene-R-D-glucofuranoside by Haworth metalation29
with (CH3)2SO4 in NaOH solution followed by hydrolysis with
CH3COOH, and oxidation by atmospheric air expulsion through the
reaction mixture in the presence of Pt/carbon catalyst as was described
by Marsh11 It was dissolved in 20% HCO2H (6 mL), hydrolyzed for
1.5 h at 60 °C, and evaporated in Vacuo to give 3-O-methyl-D-
glucuronic acid: 0.16 g, syrup; [R]D +39.1 (c 1.2, H2O); 13C NMR
20
(D2O) δ 173.8, 172.9, 96.7, 93.0, 85.3, 82.8, 75.1, 73.7, 71.5, 71.3,
71.2, 71.2, 60.8, 60.5.
Determination of the Absolute Configuration of Monosaccharides.
One drop of concentrated TFA and 0.2 mL of (-)-2-octanol (Aldrich)
were added to the dry residue of each sugar, and the ampules were
sealed and then heated on a glycerol bath at 130 °C for 6 h. The
derivatives obtained from each monosaccharide were evaporated in
Vacuo and treated with a mixture of pyridine/acetic anhydride (1:1, V
) 0.6 mL) for 24 h at room temperature. The acetylated (-)-2-
octylglycosides were analyzed by GLC using the corresponding
authentic samples: D-xylose, D-quinovose, D-glucose, and 3-O-methyl-
D-glucuronic acid treated by the same procedure. The following peaks
were detected: D-xylose (retention times 24.51, 24.70, and 25.02 min),
D-quinovose (retention times 24.03, 24.24, 24.66, and 24.86 min),
D-glucose (retention times 28.32, 29.01, 29.24, and 29.44 min), and
3-O-methyl-D-glucuronic acid (retention times 35.24, 35.52, and 36.14
min). Retention times of authentic samples were as follows: D-xylose
(retention times 24.51, 24.71, and 25.00 min), D-quinovose (retention
times 24.02, 24.24, 24.64, and 24.86 min), D-glucose (retention times
(14) Rodriguez-Carvajal, M. A.; Rodrigues, J. A.; Soria-Diaz, M. E.; Tejero-
Mateo, P.; Buendia-Claveria, A; Gutierrez, R.; Ruiz-Sainz, J. E.;
Thomas-Oates, J.; Gil-Serrano, A. M. Biomacromolecules 2005, 6,
1448–1456.
(15) Ostergren, H. In Zoologiska Studier Tillagnade Professor T. Tullberg
pa hans 65-ars dag; Almqvist & Wiksells Boktryckeri-A.-B.: Uppsala,
1907; pp 191–215..
(16) Becher, S. Ergebnis. Fortschrit. Zool. 1909, 1, 403–490.
(17) Cuénot, L. In Traité de Zoologie. Anatomie, Systématique, Biologie;
De Grasse, P. P., Ed.; Masson: Paris, 1948; Vol. 11, pp 3–272..
(18) Smirnov, A. V. Zool. Zhurn. 1984, 63, 547–553.
(19) Kerr, A. M.; Kim, J. Zool. J. Linn. Soc. 2001, 133, 63–81.
(20) Smirnov, A. V. Bull. Mosk. Obshch. Ispyt. Prirody. Otd. Geol. 2007,
82, 87–88.
(21) Gilliland, P. M. Spec. Pap. Paleont. 1993, 47, 1–147.
(22) Smirnov, A. V. In Echinoderm Research 1998. Proceedings of the
fifth European Conference of Echinoderms. Milan (Italy), 7–12
September, 1998; A. A. Balkema: Rotterdam, Brookfield, 1999; pp
393–395..

Triterpene Glycosides from Synapta maculata
Journal of Natural Products, 2008, Vol. 71, No. 4 531
(23) Lacey, K. M. J.; McCormack, G. P.; Keegan, B. F.; Powell, R. Mar.
Biol. 2005, 147, 1149–1154.
(24) Sheikh, Y. M.; Djerassi, C. Chem. Commun. 1976, 1057–1058.
(25) Makarieva, T. N.; Stonik, V. A.; Kapustina, I. I.; Boguslavsky, V. M.;
Dmitrenok, A. S.; Kalinin, V. I.; Cordeiro, M. I.; Djerassi, C. Steroids
1993, 58, 508–517.
(26) Kochetkov, N. K.; Bochkov, A. F.; Dmitriev, B. A.; Usov, A. I.;
Chizhov, O. S.; Shybaev, V. N. Carbohydrate Chemistry; Khimiya:
Moscow, 1967.
Products); Atta-ur-Rachman, Ed.; Elsevier Science Publisher: The
Netherlands, 2007; Vol. 35, pp 135–196..
(28) Kalinin, V. I.; Prokofieva, N. G.; Likhatskaya, G. N.; Schentsova, E. B.;
Agafonova, I. G.; Avilov, S. A.; Drozdova, O. A. Toxicon 1996, 34,
475–483.
(29) Haworth, W. N. J. Chem. Soc. 1915, 107, 8–16.
(30) Baltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G. Bioorg. Med.
Chem. Lett. 1991, 1, 611–614.
(27) Kalinin, V. I.; Aminin, D. L.; Avilov, S. A.; Silchenko, A. S.; Stonik,
V. A. In Studies in Natural Product Chemistry (BioactiVe Natural
NP070283+