Structure and absolute stereochemistry of syphonoside, a unique macrocyclic glycoterpenoid from marine organisms

Article abstract of DOI:10.1021/jo0704917

(Figure Presented) The glycoterpenoid syphonoside (1) is the main secondary metabolite of both the marine mollusk Syphonota geographica and the sea-grass Halophila stipulacea, two Indo-Pacific species migrated to the Mediterranean Sea through the Suez Canal. The structure and the absolute stereochemistry of 1, which displays unique structural features, has been accomplished by using a combination of spectroscopic techniques, degradation reactions, and conformational analysis methods. Compound 1 was able to inhibit high density induced apoptosis in a number of human and murine carcinoma cell lines.

Full text of DOI:10.1021/jo0704917

Structure and Absolute Stereochemistry of Syphonoside, a Unique
Macrocyclic Glycoterpenoid from Marine Organisms
Margherita Gavagnin,*,† Marianna Carbone,† Pietro Amodeo,† Ernesto Mollo,†
Rosa Maria Vitale,‡ Vassilios Roussis,§ and Guido Cimino†
Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Naples), Italy, Istituto di
Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Naples, Italy, and Department of
Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, UniVersity of Athens,
Panepistimiopolis Zografou, 15771 Athens, Greece
mgaVagnin@icmib.na.cnr.it
ReceiVed March 8, 2007
The glycoterpenoid syphonoside (1) is the main secondary metabolite of both the marine mollusk Syphonota
geographica and the sea-grass Halophila stipulacea, two Indo-Pacific species migrated to the Mediterranean
Sea through the Suez Canal. The structure and the absolute stereochemistry of 1, which displays unique
structural features, has been accomplished by using a combination of spectroscopic techniques, degradation
reactions, and conformational analysis methods. Compound 1 was able to inhibit high density induced
apoptosis in a number of human and murine carcinoma cell lines.
Introduction
have a dietary origin, whereas a significantly smaller number
of compounds are biosynthesized de noVo.4-7 The “sea hares”
(family Aplysiidae) are a group of opisthobranchs frequently
found in shallow waters that generally feed on algae from which
they sequester secondary metabolites. Consequently, most of
chemicals reported from sea hares are typical algal metabolites
or derivatives of them.6,8 In the course of our investigations on
natural products from opisthobranchs,5-7 we have recently
studied the chemical constituents of the skin9 of a Mediterranean
population of the aplysiid Syphonota geographica, which is a
circumtropical species10 first recorded in the Indo-Pacific Ocean.
In this article it is suggested for the first time that there is a
Marine organisms show a great variety in the structure and
biological activity of their secondary metabolites,1 many of
which play a chemical mediation role in defense, communica-
tion, and reproduction.2 Among marine organisms, mollusks and,
in particular, gastropods of the subclass Opisthobranchia have
been recognized as a very promising sources of new bioactive
molecules.3-6 Most of the natural products from opisthobranchs
* Author to whom correspondence should be addressed. Fax + 39 0818041770;
Tel +39 0818675094.
† Istituto di Chimica Biomolecolare - CNR.
‡ Istituto di Biostrutture e Bioimmagini - CNR.
§ University of Athens.
(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep,
M. R. Nat. Prod. Rep. 2007, 24, 31-86; and earlier articles in this series.
(2) Paul, V. J. Ecological Roles of Marine Natural Products; Comstock
Publishing Associates: Ithaca, NY, 1992.
(3) Cimino, G., Gavagnin, M., Eds. Molluscs, From Chemo-ecological
Study to Biotechnological Application; Springer: Berlin, 2006; Vol. 43.
(4) Karuso, P. In Bioorganic Marine Chemistry; Scheuer P. J., Ed.;
Springer-Verlag: Berlin, 1987; Vol. 1, pp 31-60.
(5) Cimino, G.; Fontana, A.; Gavagnin, M. Curr. Org. Chem. 1999, 3,
327-372.
(6) Cimino, G.; Ciavatta, M. L.; Fontana, A.; Gavagnin, M. In BioactiVe
Natural Products: Isolation, Structure Elucidation and Biology Properties;
Tringali, C., Ed.; Taylor and Francis: London, UK, 2001; pp 577-637.
(7) Cimino, G.; Fontana, A.; Cutignano, A.; Gavagnin, M. Phytochem.
ReV. 2004, 3, 285-307.
(8) Carefoot, T. H. Oceanogr. Mar. Biol. Annu. ReV. 1987, 25, 167-
284.
(9) Gavagnin, M.; Carbone, M.; Nappo, M.; Mollo, E.; Roussis, V.;
Cimino, G. Tetrahedron 2005, 61, 617-621.
(10) Eales, N. B. Bull. Br. Mus. (Nat. Hist.) Zool. 1960, 5, 267-404.
10.1021/jo0704917 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/20/2007
J. Org. Chem. 2007, 72, 5625-5630
5625

Gavagnin et al.
TABLE 1. NMR Dataa,b for Syphonoside (1)
long-range
long-range
position
δ 1H
m, J, Hz
δ
13C
m c
correlationsd
position
δ 1H
m, J, Hz
δ
13C
mc
correlationsd
1
2
3
4
5
6
1.68
2.23
6.44
-
m
m
t,3
-
19.8
27.7
138. 5
144. 6
38.7
t
t
d
s
s
t
-
-
-
1′
2′
3′
4′
5′
6′
5.51
3.37
3.43
3.37
3.59
4.27
4.54
d, 8
m
t,9
m
m
m
95.5
72.0e
78.1
71.5e
76.3
63.9
d
d
d
d
d
t
-
-
-
-
-
-
H3-19
H-3, H3-19
-
-
-
1.16
2.16
1.36
1.98
-
1.55
4.12
2.35
2.76
-
5.46
4.64
1.86
1.03
-
ddd, 3, 13, 14
bd, 13
m
m
-
m
dd, 1, 8
bd, 15
dd, 9, 15
-
bt, 7
m
s
d, 7
-
s
s
36.5
dd, 2, 12
7
8
9
10
11
12
29.5
35.8
45.7
46.6
81.1
42.4
t
H3-17
d
s
d
d
t
H3-17, H3-20
H-12a, H3-17
H3-19, H3-20
H-12a, H3-20, H-1′′′
H3-16
1′′
2′′
3′′
4′′
5′′
-
-
172.8
46.3
71.0
49.6
171.7
s
t
s
t
H2-15, H2-2′′
H2-4′′, H3-6′′
H2-2′′, H2-4′′, H3-6′′
H3-6′′
2.61
-
2.64
-
ABq, 14
-
s
-
s
H2-6′, H2-4′′
13
14
15
16
17
18
19
20
141.1
123.4
62.4
16.4
19.2
169.2
21.7
13.8
s
d
t
q
q
s
q
q
H-11, H2-12, H2-15, H3-16
H-12a, H-15, H3-16
-
H-12a, H-14
-
H-3, H-1′
H-10
6′′
1.46
s
26.9
q
H2-2′′, H2-4′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
4.30
3.20
3.40
3.40
3.28
3.71
3.91
d, 8
m
m
m
102.3
75.2
78.6
74.0
77.8
63.1
d
d
d
d
d
t
H-11, H-2′′′
-
-
-
-
-
1.33
0.99
m
H-10
dd, 2, 12
dd, 6, 12
a Bruker DPX-300, DPX-600, and AVANCE 400 MHz spectrometers, CD3OD, chemical shifts (ppm) referred to CH3OH (δ 3.34) and to CD3OD (δ
49.0). b Assignments made by 1H-1H COSY, 2D-TOCSY, HSQC, and HMBC. c By DEPT sequence. d HMBC experiments (J ) 10 Hz). e Assignments
may be reversed.
trophic relationship between the mollusk and the invasive Indo-
Pacific sea-grass Halophila stipulacea,11,12 since specimens of
the sea-grass were identified in the stomach of the mollusks.9
The presence of both species along Mediterranean coasts is due
to the phenomenon known as Lessepsian migration from the
Indo-Pacific Ocean to the Mediterranean Sea through the Suez
Canal.
Here, we report on our investigation of the chemical
constituents of the sea-grass and the digestive gland of the
mollusk, both containing a main secondary metabolite, syphono-
side (1). The structure determination and the assignment of the
absolute stereochemistry of this unique compound possessing
a novel macrocyclic glycoterpenoid skeleton were accomplished
through a combination of spectroscopic techniques, degradation
experiments, and conformational analysis methods.
a trisubstituted isolated double bond and an R,â-unsaturated
double bond, respectively. Finally, a vinyl methyl signal at δ
1.86 (H3-16) and four methyl signals at δ 0.99 (H3-20), 1.03
(H3-17), 1.33 (H3-19), and 1.46 (H3-6′′) accounted for the proton
spectrum. Detailed analysis of the 2D NMR spectra led to the
recognition of four distinct partial structures in the carbon
skeleton: a terpenoid acyl moiety, two glycopyranosyl units,
and a dicarboxylic acid residue.
The spectral data in the terpenoid part indicated the presence
of a clerodane skeleton oxidized at C-11, C-15, and C-18,
exhibiting a glycosylated secondary hydroxyl, an esterified
allylic primary hydroxyl, and an R,â-unsaturated ester function,
respectively.
Results and Discussion
Spectral Analysis. The molecular formula C38H58O17, indi-
cating 10 degrees of unsaturation, was deduced by the sodiated-
molecular ion at 809.3545 m/z in the HRESI mass spectrum.
In the 13C NMR spectrum, 31 signals were observed between
13.8 and 102.3 ppm, corresponding to sp3 carbons along with
7 signals in the region of 123.4-172.8 ppm which were
attributed to two double bonds and three ester carbonyl groups
(Table 1). The remaining five degrees of unsaturation were thus
1
assigned to five rings. The H NMR spectrum of compound 1
Careful proton and carbon NMR analyses (Table 1) led us
to the identification of the dicarboxylic acid moiety as 3-hy-
droxy-3-methyl-glutaric acid and of both glycosyl units as
â-glucose. The complete spin sequence for the two glucosyl
residues was determined as reported in Table 1 by detailed
analysis of 1H-1H COSY and 2D-TOCSY spectra that allowed
the connection between the anomeric proton and the methylene
group in each glucose moiety. In particular, diagnostic cross-
peaks in the 2D-TOCSY experiment between the anomeric
proton H-1′ (δ 5.51) and H-5′ (δ 3.59) clearly indicated that
contained two diagnostic doublets at δ 5.51 (J ) 8 Hz) and δ
4.30 (J ) 8 Hz), which were assigned to two anomeric protons
of â-glycosyl residues, along with a series of signals in the
region of 3.20-4.64 ppm that could be attributed to protons
linked to oxygen bearing carbons. Two olefinic signals observed
at δ 5.46 (H-14) and at δ 6.44 (H-3) indicated the presence of
(11) Lipkin, Y. Isr. J. Bot. 1975, 24, 198-200.
(12) Boudouresque, C. F.; Verlaque, M. Mar. Pollut. Bull. 2002, 44,
32-38.
5626 J. Org. Chem., Vol. 72, No. 15, 2007

Syphonoside, a Unique Macrocyclic Glycoterpenoid
the same glucose unit exhibits both C-1′ and C-6′ engaged in
ester linkages (H-1′ and H2-6′ resonated at lower fields with
respect to typical values) whereas the second glucose, which is
linked to the residue through a common glycosyl linkage
(H-1′′′ resonated at δ 4.30), displays the primary hydroxyl group
at C-6′′′ free. Analysis of the HMBC experiment allowed the
connection of the four distinct parts leading to the macrocyclic
framework depicted in structure 1. Diagnostic long-range
correlations were observed between C-11 (δ 81.1) and H-1′′′
(δ 4.30), C-18 (δ 169.2) and H-1′ (δ 5.51), C-5′′ (δ 171.7) and
H2-6′ (δ 4.27 and 4.54), C-1′′ (δ 172.8) and H2-15 (δ 4.64).
These data were in agreement with the proposed structure where
the R,â-unsaturated acyl function (C-18) of the diterpene moiety
esterifies the hydroxyl group at anomeric position (C-1′) of a
â-glucose unit, the primary hydroxyl function (6′-OH) of which
is in turn linked to the carboxyl group (C-5′′) of 3-hydroxy-3-
methyl-glutaric acid, further esterified at C-1′′ with a primary
hydroxyl function (15-OH) of the same diterpene. An additional
glucose is connected by a glycosyl linkage to a secondary
hydroxyl function (11-OH) of the diterpene.
The relative stereochemistry of the clerodane diterpene part
was determined by analysis of the carbon chemical shifts and
comparison with model clerodanes. In order to accomplish such
a comparative study, compound 1 was submitted to alkaline
methanolysis leading to three fragments: a) a glucosyl terpene
2; b) the dimethyl ester of 3-hydroxy-3-methylglutaric acid, and
c) glucose. Compound 2 was acetylated to afford the â-penta-
acetyl-derivative 3, which was fully characterized with the
exception of the stereochemistry at C-11.
alkaline methanolysis of 1 and was directly treated with benzoyl
chloride to give the mixture of the corresponding R- and
â-penta-benzoyl derivatives, which were subsequently separated.
The circular dicroism profiles of both epimers were compared
with those of authentic R- and â-penta-benzoyl derivatives
prepared from D-glucose,14 indicating the D absolute stereo-
chemistry of the glucose linked at C-18 of the terpene moiety
in syphonoside (1). In order to establish the stereochemistry of
the glucose unit attached at C-11 of the clerodane moiety,
glucosyl terpene 2 was further treated with HCl/MeOH to give
a mixture of R- and â-methyl glucopyranoses, that was further
benzoylated. The CD curves of the purified benzoyl derivatives
were identical with those of the corresponding compounds
obtained from benzoylation of commercial R- and â-methyl-
D-glucopyranoses,14 thus indicating the D absolute stereochem-
istry of the sugar unit linked at C-11 of compound 1.
Conformational Analysis. Once the absolute stereochemistry
of both glucose units and the relative stereochemistry of the
bicyclic clerodane substructure were assigned, a conformational
study was undertaken to establish the absolute stereoche-
mistry at C-11, C-3′′, and of the clerodane rings of syphonoside
(1) which were not possible to be determined by direct
spectroscopic methods. The eight possible diastereomers, ob-
tained by permutation of absolute chiralities of C-11 and
C-3′′and of the overall clerodane skeleton, were labeled with a
three-letter code, based on R/S chirality of C-8, C-11, and C-3′′
atoms, respectively.
Structural determination by simulated annealing followed by
energy minimization (SA/EM),15 with restraints derived from
NOESY spectra, was performed on all eight possible diaster-
eomers, followed by a compared analysis of restraint violations.
Sixteen strong- and six medium-intensity peaks were translated
into 1.7-3.0 Å and 1.7-4.0 Å allowed distance ranges,
respectively.
The restraints involving the D-glucosyl residues either are
directly violated in some diastereomers or induce violations in
other restraints involving their partner protons, thus allowing
an unambiguous identification of the absolute stereochemistry
of syphonoside (Table 2):
(a) Systematic violations of two strong distance restraints,
corresponding to H-11/H2-1 and H-11/H3-16 NOE cross-peaks,
were observed in both RS(R/S) and SR(R/S) diastereomers
bundles, leaving us with only four out of eight possible
diastereomers: RR(R/S) and SS(R/S).
The trans-junction between the two rings of the clerodane
skeleton was indicated by the diagnostic carbon chemical shift
of the angular methyl H3-19 (δ 21.7), since the corresponding
methyl in cis-clerodanes resonates at approximately 10 ppm
lower fields.13 The equatorial orientation of H3-17 was inferred
by the chemical shift of C-6 (δ 36.5), taking into consideration
that in trans-clerodane models this carbon resonates at higher
fields, when the substituent at C-8 is axial. The relative
stereochemistry at C-9 was suggested by both biosynthetic
considerations and the carbon value of the axially oriented C-20
(δ 13.8), which was further deshielded by the presence of the
glucosylated hydroxyl group at C-11. Furthermore, a series of
NOE effects were observed between H-10 and H-8, H-11 and
H2-12, and between the two angular methyl groups H3-19 and
H3-20.
(b) Observed strong cross-peaks between methyl hydrogens
of C-6′′ and both C-2′ and C-4′ protons of the sugar moiety
helped us to assign the absolute chirality of the C-3′′ atom as
R. In fact only in this case C-6′′ methyl hydrogen atoms satisfy
their distance restraints with H-2′ and H-4′ sugar hydrogens.
(c) The SSR diastereomer can be excluded after analysis of
overall interproton distances within the final energetically
selected 10 structure bundles. In fact, several short distances in
the SSR bundle (e.g., H-3/H-2′, H-2′′′/H2-20) do not exhibit a
counterpart in NOE cross-peaks, while applied distance restraints
are violated either in their upper or lower limits (e.g., H-6′′/H-
3/H-6′′ distance in models is systematically <3 Å, but it only
corresponds to a weak NOE peak, whereas weak H-3/H-4′′ and
H-14/H-6′′ restraints are violated of 0.7 and 2 Å, respectively
in all selected structures). In addition, 6 out of 10 structures
The identity of 3-hydroxy-3-methylglutaric acid, recovered
as the dimethyl ester from the ether phase of the alkaline
methanolysis of 1, was confirmed by LC-ESIMS comparison
with an authentic sample. The glucose moiety esterified at both
C-1′ and C-6′ was recovered from the aqueous phase of the
1
(14) See Supporting Information for H NMR data and CD profiles of
benzoyl glucose derivatives.
(15) Gippert, G. P.; Yip, P. F.; Wright, P. E.; Case, D. A. Biochem.
Pharmacol. 1990, 40, 15-22.
(13) Nogueira, R. T.; Shepherd, G. J.; Laverde, A., Jr.; Marzaioli, A. J.;
Imamura, P. M. Phytochemistry 2001, 58, 1153-1157.
J. Org. Chem, Vol. 72, No. 15, 2007 5627

Gavagnin et al.
TABLE 2. NOESY-Derived Restraints Used in Structural
Determination and Absolute Stereochemistry Assignment of (1) and
Diagnostic Violations Observed in Calculated Diastereomer Bundlesa
restraint
distance
violating
diastereomers
proton i
proton j
H2-1
H2-2
H-3
H-3
H-3
H-11
S
M
S
M
M
S
RSR, RSS, SRR, SRS
H2-15
H2-2′′
H2-4′′
H3-6′′
H-12
SSR
SSR
H-8
H-8
H-14
S
H-10
H-10
H-11
H-11
H-11
H2-12
H2-12
H2-12
H-14
H2-15
H2-16
H-2′
H-12
H-14
H2-12
H3-16
H2-20
H-14
H3-17
H-1′′′
H3-6′′
H3-6′′
H-1′′′
H3-6′′
H3-6′′
H2-4′′
H3-6′′
H-2′
S
S
S
S
S
S
S
S
M
M
S
S
S
RSR, RSS, SRR, SRS
SSR
RRS, SSS, ssr
RRS, SSS, ssr
H-4′
H2-6′
H2-4′′
H-3
S
M
X
X
SSR
SSR
FIGURE 1. Ball-and-stick and surface representation of syphonoside
(1) best conformer. Six views of the conformer are shown, obtained
by sequential rotations around either x (horizontal) or y (vertical) axis,
as indicated near each corresponding arrow. Only heavy atoms are
shown as spheres colored according to atom types. Corresponding bonds
are represented as half-sticks colored according to atom types, except
for 22-atom ring bonds, where yellow is used for “chairlike” regions
and green for extended strands. The half-transparent shaded solvent-
accessible molecular surface is also shown, painted according to atom
types.
H-20
H-2′′′
a Restrained distances are classified into S (short, 1.7-3.0 Å), M
(medium, 1.7-4.0 Å), and absent X (>4.0 Å). The latter were used for
analysis, but not imposed as restraints. Lowercase is used in the “violating
diastereomers” column when violation is only detected in 60% of the
calculated structures.
have systematic violations of 1 Å at level of H3-6′′/H-2′/H-4′
distance restraints, thus highlighting the existence of a coupling
among distance restraints involving all the chiral centers.
So, only RRR implying an ent-clerodane skeleton provided
a 10-structure bundle that converges to a single conformation
and exhibits both no appreciable restraint violation and a
counterpart of all short distances in its NOE strong peak list.
Structural features of 1 are dominated by the conformation
of its 22-membered ring, including one glucose ring and a
clerodane ring system and being responsible for 17 out of the
19 conformational degrees of freedom of the whole molecule
(excluding smaller-ring flexibility and trivial OH and CH3
rotations). The macrocycle conformation of the main (1)
conformer (Figure 1), in analogy with the “chair” conformation
of six-membered rings, can be described as “sofalike”. Here,
the “chairlike” shape derives from two mutually parallel two-
atom segments (yellow in Figure 1) while two nine-atom almost
parallel strands (green in Figure 1) in an all-trans conformation
(except for a 90° bulge in O-15-C1′′-C2′′-C3′′ torsion)
replace the single “tip” atoms of six-membered rings. Looking
at the 3D molecular surface, the macrocycle shows a rather
planar shape curved at the borders, with the external glucose
ring almost perpendicular to the macrocycle plane. So, the whole
molecule vaguely resembles a turtle in shape, with the “head”
provided by the external glucose ring, the “shell” by the
macrocycle backbone plus the clerodane unsaturated ring, and
“limbs” by the saturated clerodane ring, internal glucose ring,
C-16 methyl group, and nonbackbone atoms of 3-hydroxy-3-
methylglutaric acid (Figure 1). Polar groups are concentrated
in the “head” and “hind limbs”, the remainder of the molecule
being mostly apolar. The “lower-shell” region is concave with
a small, deep cavity in the middle, whose bottom is provided
by C-3, C-4, and O-1′ atoms (Figure 1, f).
Biological Activity of Syphonoside (1). Syphonoside was
submitted to cytotoxicity evaluation16 on a number of human
and murine cell lines (MCF7 derived from a mammary
adenocarcinoma, PC3, derived from a prostate adenocarcinoma,
A431 derived from epidermoid carcinoma, HeLa derived from
cervix adenocarcinoma, HT29 colon cancer cell line, NT2,
teratocarcinoma, P19 murine embryonic carcinoma stem cell
line). None of the concentrations of 1 (400-0.2 µM) was shown
to be cytotoxic to the cell lines used in this study, even after
long-term exposure (72 h). Surprisingly, 1 was found to decrease
the number of floating cells especially in A431 and P19 cells
with effective concentrations being 440 ( 35 nM and 360 (
42 nm, respectively. Therefore, although syphonoside (1) did
not exhibit any cytotoxic activity, it was able to inhibit high
density induced apoptosis. The suppression of apoptosis resulted
in a prolonged viability and maximum cell density.
Conclusions
The chemical analysis of the pair S. geographica/H. stipu-
lacea led to the isolation of the same macrocyclic glycoterpe-
noid, syphonoside (1), from both organisms, confirming the
trophic relationship between the mollusk and the sea-grass. This
compound displays unique structural features among marine
natural products, where analogies could be envisaged with plant
secondary metabolites, thus representing an interesting conjunc-
tion point between marine and terrestrial natural products.
Syphonoside is characterized by a novel macrocyclic skeleton
(16) Iliopoulou, D.; Mihopoulos, N.; Vagias, C.; Papazafiri, P.; Roussis,
V. J. Org. Chem. 2003, 68, 7667-7674.
5628 J. Org. Chem., Vol. 72, No. 15, 2007

Syphonoside, a Unique Macrocyclic Glycoterpenoid
) 0.13, CHCl3); IR (liquid film): 3390, 2930, 1725 cm-1; UV
(MeOH): λmax 206 (ꢀ ) 6700); selected 1H NMR values (300 MHz,
CD3OD): δ 6.57 (1H, t, J ) 3 Hz, H-3), 5.61 (1H, app. t, J ) 7
Hz, H-14), 4.36 (1H, d, J ) 8 Hz, H-1′′), 1.77 (3H, bs, H3-16),
1.33 (3H, s, H3-19), 0.99 (3H, d, J ) 6 Hz, H3-17), 0.90 (3H, s,
H3-20); HRESIMS (m/z): (M + Na)+ calcd for C27H44O9Na,
535.2883; found, 535.2859.
containing an ent-clerodane diterpenoid moiety, two D-glucose
units, and a 3-hydroxy-3-methylglutaric residue. This structure
displays an original conformation exhibiting two long nine-atom
extended strands joined by two “cyclohexane-chairlike” two-
atom junctions. The resulting three-dimensional shape of this
22-membered ring is a moderately curved turtle-shell structure
characterized by an inhomogeneous distribution of polar vs
hydrophobic regions. In fact, a prevalently polar rim (also
including the perpendicular extracyclic glucose moiety) sur-
rounds a mostly hydrophobic central region, also including a
cavity that could easily host either an all-hydrophobic group or
a small hydrophobic chain ending with a H-bond donor group
(pointing toward O1′ atom). This cavity, together with the two
long, almost parallel eight-residue strands, could represent
interaction sites with other biomolecules and, at the same time,
provide potential technological applications. In a preliminary
cytotoxicity test, syphonoside (1) did not exhibit any cytotoxic
activity against the employed cell lines but was able to inhibit
high density induced apoptosis. Therefore, this compound may
play an important role in regulating cell survival and cell death
under specific conditions but additional studies are required for
the elucidation of this role.
The aqueous phase from the workup was filtered on a Sep-Pak
and dried to afford pure D-glucose [6.0 mg, Rf ) 0.4 (70:20:10,
EtOAc:MeOH:H2O); [R]D 45.4 (c ) 0.37, H2O)]. The absolute
1
stereochemistry of D-glucose was determined by analysis of H
NMR and CD spectra of the corresponding benzoate derivatives.14
Compound 3. Glycosyl terpene 2 (2.0 mg) was dissolved in
dry pyridine (2 mL), acetic anhydride (0.5 mL) was added to the
resulting solution, and the reaction mixture was stirred overnight
at room temperature. Pyridine was removed in Vacuo, and the
residue was dissolved in diethyl ether and worked up as usual to
afford pure 3 (1.8 mg), Rf ) 0.7 (95:5, CHCl3:MeOH); [R]D -41.4
(c ) 0.1, CHCl3); IR (liquid film): 2940, 1758, 1232 cm-1; UV
1
(MeOH): λmax 206 (ꢀ ) 5250); H NMR (400 MHz, CDCl3): δ
6.52 (1H, app. t, J ) 3 Hz, H-3), 5.41 (1H, app. t, J ) 7 Hz, H-14),
5.16 (1H, app. t, J ) 9 Hz, H-3′′′), 5.00 (1H, app. t, J ) 9 Hz,
H-4′′′), 4.93 (1H, dd, J ) 8, 9 Hz, H-2′′′), 4.62 (1H, d, J ) 8 Hz,
H-1′′′), 4.75 (1H, m, H-15a), 4.46 (1H, m, H-15b), 4.21 (1H, m,
H-6′′′a), 4.13 (1H, m, H-6′′′b), 4.09 (1H, m, H-11), 3.68 (3H, s,
OMe), 3.61 (1H, m, H-5′′′), 2.40 (1H, m, H-12a), 2.22 (1H, m,
H-6a), 2.20 (1H, m, H-12b), 2.17 (1H, m, H-2a), 2.12 (3H, s, OAc),
2.10 (1H, m, H-2b), 2.07 (3H, s, OAc), 2.04 (3H, s, OAc), 2.01
(3H, s, OAc), 1.98 (3H, s, OAc), 1.75 (3H, bs, H3-16), 1.69 (1H,
m, H-1a), 1.57 (1H, m, H-1b), 1.55 (1H, m, H-8), 1.38 (2H, m,
H2-7), 1.31 (1H, m, H-10), 1.30 (3H, s, H3-19), 1.08 (1H, m, H-6b),
0.84 (3H, s, H3-20), 0.81 (3H, d, J ) 7 Hz, H3-17); 13C NMR (300
MHz, CDCl3): δ 171.4 (s, OAc), 170.3 (s, 2C, OAc), 169.4 (s,
2C, OAc), 167.8 (s, C-18), 142.8 (s, C-4), 139.4 (s, C-13), 136.1
(d, C-3), 121.6 (d, C-14), 98.8 (d, C-1′′′), 77.4 (d, C-11), 73.1 (d,
C-3′′′), 71.9 (d, C-2′′′), 71.4 (d, C-5′′′), 69.2 (d, C-4′′′), 62.4 (t,
C-6′′′), 61.3 (t, C-15), 51.2 (q, OMe), 46.6 (d, C-10), 43.5 (s, C-9),
42.5 (t, C-12), 38.0 (s, C-5), 35.7 (t, C-6), 27.9 (t, C-7), 27.2 (t,
C-2), 21.1 (2C, q, C-19 and OAc), 20.6 (4C, q, OAc), 18.8 (t, C-1),
17.5 (d, C-17), 16.2 (q, C-16), 12.5 (q, C-20); HRESIMS (m/ z):
(M + Na)+ calcd for C37H54O14Na, 745.3411; found, 745.3430.
Experimental Section
General experimental procedures were performed as previously
described.9
Collection and Extraction. The collection and the extraction
of S. geographica individuals (seven animals) has already been
reported.9 A sample of H. stipulacea was collected from the same
site as the mollusk in November 2003 and was frozen at -20 °C.
This material (dry residue 103.0 g) was extracted according to the
standard procedures.9 The n-BuOH layer from the acetone extract
was evaporated under reduced pressure to afford 1.31 g of crude
extract. Analogously, the n-BuOH layer of the digestive gland
extract of the mollusk was evaporated to dryness, yielding 618.0
mg of residue. Both extracts were analyzed by TLC (chloroform/
methanol in variable ratios) and compared.
Isolation of 1. The n-BuOH extract (618 mg) of the mollusk
was subjected to Sephadex LH-20 chromatography using a mixture
of chloroform/methanol in 1:1 ratio as eluent, to yield a fraction
(115 mg) which was further purified on preparative TLC (SiO2,
7:3, CHCl3:MeOH) to give 49.2 mg of pure metabolite 1 (ca. 8%
of the extract). An aliquot (500 mg) of the n-BuOH soluble part of
the extract of the sea-grass was chromatographed under the above-
described conditions to give18.8 mg of pure 1 (ca. 4% of the
extract).
Acid Methanolysis of 2. Compound 2 (10 mg) was dissolved
in a 1 N HCl solution in MeOH (2 mL), and the obtained solution
was stirred for 12 h at 40 °C. After the usual workup, the reaction
mixture was dried and partitioned between CHCl3 and H2O/MeOH,
8:2. The aqueous layer was concentrated, filtered on a Sep-Pak
cartridge, and dried. The 1H NMR spectrum of this fraction
indicated that it was a mixture of R-and â-methyl glucopyranose,
the absolute stereochemistry of which was determined by analysis
Syphonoside (1). Rf ) 0.50 (7:3, CHCl3:MeOH); [R]D -7.0 (c
) 0.54, MeOH); CD (EtOH) [θ]209 2754, [θ]237 -8574; IR (liquid
film): 3500, 2887, 1714 cm-1; UV (MeOH): λmax 206 (ꢀ )
1
11 660); H and 13C NMR in Table 1; HRESIMS (m/z): (M +
1
Na)+ calcd for C38H58O17Na, 809.3572; found, 809.3545.
of H NMR and CD spectra of the corresponding tetra-benzoate
derivatives.14
Alkaline Methanolysis of 1. A 25 mg amount of 1 was dissolved
in a 0.5 M solution of MeONa in MeOH (2.5 mL), and the resulting
mixture was stirred at room temperature for 1.5 h. The reaction
mixture was first chromatographed on a DOWEX-50W column
eluted with methanol (100 mL) and then extracted with Et2O to
afford 15.0 mg and 4.5 mg of residue from the ethereal and aqueous
phases, respectively. An aliquot of the ethereal phase was analyzed
by HPLC (Phenomenex, Kromasil 5 µm 100A C18; eluent CH3-
CN/H2O 4:6; flow 1 mL/min). The dimethyl ester of 3-hydroxy-
3-methylglutaric acid was identified by comparison of its retention
time (tR 4 min) with that of an authentic sample. An additional
LC-ESIMS analysis was conducted confirming the identity of
3-hydroxy-3-methylglutaric dimethyl ester. The remaining ether
soluble residue was purified on preparative TLC (SiO2, 9:1, CHCl3:
MeOH) to give 12.0 mg of compound 2 (Rf ) 0.4); [R]D -4.8 (c
Conformational Analysis of 1. Syphonoside structures were
obtained by restrained SA/EM.15 Experimental NOE intensities were
converted into 22 proton-proton distance restraints classified into
two ranges: 1.7-3.0 Å and 1.7-4.0 Å, corresponding to strong/
strong-medium and medium/medium-weak NOE peaks, respec-
tively. Weak and very weak interactions, as well as peaks
corresponding to fixed distances in glucose rings, were not included.
Methylene protons in the NMR spectra of syphonoside were not
resolved, but they were described using an “ambiguous restraint”
approach in which, for each restraint, all the possible distances
between the two methylene protons and their NOE counterpart
proton(s) were calculated and weighted by the inverse of the sixth
power of their values. Calculations were performed with Sander
J. Org. Chem, Vol. 72, No. 15, 2007 5629

Gavagnin et al.
module of AMBER8,17 using AMBER GAFF parametrization18 and
semiempirical AM1-BCC charges.19 Starting structures for all eight
sampled syphonoside diastereomers were built with Ghemical
1.01.20 Conformational sampling was obtained by restrained
simulated annealing (SA), including an implicit solvation model
(RIWSA), based on a generalized Born21 with surface area
contribution22 approach (GBSA). The starting structure of each
syphonoside diastereomer underwent 50 SA cycles of 100 000 MD
steps, where system temperature was linearly raised from 10 to
1200 K (step 1 to 5000) and then kept constant at 1200 K (step
5001 to 50 000), and finally linearly decreased down to 10 K (step
50 001 to 100 000). A time step of 1 fs, with no constraints or
restraints on bond lengths, a nonbonded cutoff of 16 Å and a 0.05
fs time constant for heat bath coupling were used, with all other
parameters set at their default values. A semiparabolic penalty
function, with a force constant of 20 kcal mol-1 Å-2, was applied
to interprotonic distances larger than upper-limit values. trans-
Alkene bonds were forced into a trans orientation (ω ) 180°) by
torsional constraints with a force constant of 50 kcal mol-1 operating
for deviations higher than 20° from the trans form. Final structures
were energy minimized (EM) with 100 steps of steepest descent
followed by a conjugate gradient method, down to a gradient norm
value less than 10-3 kcal mol-1 Å-1 (thus leading to the final SA/
EM structure set), both with the same penalty function used for
SA (REM) and under totally unrestrained conditions (UEM).
MOLMOL23 program was used for structural analysis.
Cytotoxicity Assays. Cytoxicity of 1 was evaluated according
to MTT dye reduction assay.24-26
Acknowledgment. We are grateful to Mr. F. Castelluccio
for his valuable technical assistance. Thanks are also due to
Mr. R. Turco for drawings and to Mr. C. Iodice for spectro-
photometric measurements. The NMR spectra were recorded
at the ICB NMR Service, the staff of which is acknowledged.
Particular thanks are due to Mrs. Dominique Melck for NMR
spectra processing. The authors thank also Dr. Panagiota
Papazafiri (University of Athens, Dept. of Biology) for her
assistance with cytotoxicity evaluation.
Supporting Information Available: MS and 1D- and 2D-NMR
spectra of syphonoside (1), H NMR spectra of compounds 2 and
3, CD spectra of benzoyl glucose derivatives and additional
experimental procedures. This material is available free of charge
via the Internet at http:// pubs.acs.org.
(17) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C.
L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D.
A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak,
V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.; Ross, W. S.;
Kollman, P. A. AMBER 8, University of California, San Francisco, 2004.
(18) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D.
A. J. Comput. Chem. 2004, 25, 1157-1174.
1
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2000, 21, 132-146.
(23) Koradi, R.; Billeter, M.; Wu¨thrich, K. J. Mol. Graph. 1996, 14,
51-55.
(20) Rowley, C.; Hassinen, T. Ghemical, 2001-2002; licensed un GNU
GPL, Copyright 2006 Tommi Hassinen, Kuopio, Finland (http://
www.bioinformatics.org/ghemical/ghemical/index.html).
(21) Tsui, V.; Case, D. A. Biopolymers 2001, 56, 275-291.
(22) Weiser, J.; Shenkin, P. S.; Still, W. C. J. Comput. Chem. 1999, 20,
217-230.
(24) Mossmann, T. J. Immunol. Methods 1983, 65, 55-63.
(25) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R.
H.; Boyd, M. R. Cancer Res. 1988, 48, 589-601.
(26) Experimental details are reported in Supporting Information.
5630 J. Org. Chem., Vol. 72, No. 15, 2007