Terpioside from the marine sponge Terpios sp., the first glycosphingolipid having an L-fucofuranose unit

Article abstract of DOI:10.1002/ejoc.200701220

The new diglycosylceramide terpioside (1a) has been isolated from the marine sponge Terpios sp. Terpioside is a diglycosylated glycosphingolipid which is the first example of a natural glycosphingolipid having an L-fucofuranose unit. The structure of terpioside was elucidated by extensive spectroscopic analysis, whereas chemical degradation was used to establish the nature of the alkyl chains and the absolute configuration of the sugars and of the ceramide stereogenic centers. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200701220

FULL PAPER
DOI: 10.1002/ejoc.200701220
Terpioside from the Marine Sponge Terpios sp., the First Glycosphingolipid
Having an
-Fucofuranose Unit[‡]
L
Valeria Costantino,[a] Ernesto Fattorusso,[a] Concetta Imperatore,[a] Alfonso Mangoni,*[a]
and Roberta Teta[a]
Keywords: Fucofuranose / Ceramide / Glycolipids / Sponges / Structure elucidation
The new diglycosylceramide terpioside (1a) has been iso-
lated from the marine sponge Terpios sp. Terpioside is a di-
glycosylated glycosphingolipid which is the first example of
to establish the nature of the alkyl chains and the absolute
configuration of the sugars and of the ceramide stereogenic
centers.
a natural glycosphingolipid having an L-fucofuranose unit.
The structure of terpioside was elucidated by extensive spec-
troscopic analysis, whereas chemical degradation was used
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
In this paper we report the isolation and stereostructure
elucidation of terpioside (1a), a new diglycosylated GSL
from a Caribbean Terpios sp. which has a unique sugar moi-
ety formed by a α-fucofuranoside linked to the 3-position
of a β-glucopyranoside, the sugar residue linked to the cera-
mide (Scheme 1).
Introduction
Marine sponges are being shown to produce an increas-
ingly large number of interesting biologically active glyco-
sphingolipids, such as the immunosuppressive plakosides
from Plakortis simplex[1] and the immunostimulating
α-galactoglycosphingolipids[2–4] from sponges of the genera
Agelas and Axinella.
As a part of our investigation focused on the search for
new glycosphingolipids (GSLs) from marine invertebrates,
we examined the extract of the sponge Terpios sp. (Suberiti-
dae) collected from along the Florida coast. Terpios is an
encrusting sponge of ecological importance, as revealed by
a study carried out by the Marine Laboratory of the Uni-
versity of Guam[5] which found that Terpios sponges are
efficient competitors of corals for space. They overgrow
quickly on corals and, often, kill them. Indeed, Terpios
sponges represent one of the most important causes of de-
struction of some coral reefs.
Scheme 1.
Although the chemistry of some genera of sponges from
the Caribbean area, such as Agelas and Axinella, has been
extensively studied, the Terpios genus has not yet been ex-
amined in depth. In fact, only a few papers describing the
chemistry of Terpios species from the Pacific have been pub-
lished so far, and these include reports on the isolation of
nakiterpiosin, nakiterpiosinone,[6] and terpiodiene[7] from
the Okinawan sponge Terpios hoshinata and a norcholes-
tanol[8] from the Hawaian sponge Terpios zeteki.
Results and Discussion
Samples of Terpios sp. were collected from the tropical
waters of Key Largo (Florida) and kept frozen until extrac-
tion. The specimens were extracted with chloroform and
methanol, and the combined extracts were partitioned be-
tween water and BuOH. The organic phase was dried, and
according to our standard procedure a glycolipid fraction
was obtained by subsequent reversed-phase and normal-
phase column chromatography. The glycolipid fraction was
acetylated, and the peracetylated glycolipids were subjected
to repeated HPLC on SiO2 columns to give 62.6 mg of pure
[‡] Glycolipids from Sponges, 19. Part 18: Ref.[4]
[a] Dipartimento di Chimica delle Sostanze Naturali, Università di
Napoli “Federico II”,
via D. Montesano 49, 80131 Napoli, Italy
Fax: + 39-081-678-552
E-mail: alfonso.mangoni@unina.it
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
2130
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 2130–2134

Terpioside from the Marine Sponge Terpios sp.
1
compound 1b. Compound 1b was deacetylated with Table 1. H and 13C NMR spectroscopic data of terpioside perace-
tate 1b (CDCl3).
MeOH/MeONa to yield the natural glycolipid 1a.
The ESI mass spectrum of the natural GSL 1a showed a
series of sodiated pseudomolecular ion peaks at m/z = 986,
1000, 1014, 1028 1042, and 1056, which suggested a series
of homologues differing in the size of the alkyl chains. A
high-resolution measurement performed on the most abun-
dant ion at m/z = 1028.7567 indicated the molecular for-
mula C55H107NO14 for the dominant homologue.
Pos.
δH [ppm] (mult., J [Hz])[a]
δC [ppm] (mult.)[b]
1a
1b
2
2-NH
3
4
5
6a
6b
1Ј
3.80 (dd, 10.4, 2.9)
3.61 (dd, 10.4, 3.9)
4.27 (m)
6.78 (d, 9.0)
5.12 (dd, 8.4, 3.2)
4.89 (ddd, 10.0, 3.2, 3.2)
1.59 (m)
1.30 (m)
1.19 (m)
4.32 (d, 8.0)
4.81 (dd, 9.5, 8.0)
3.83 (t, 9.5)
4.97 (t, 9.5)
3.53 (ddd, 9.5, 3.9, 3.9)
4.12 (m)
5.23 (d, 4.6)
5.06 (dd, 6.3, 4.6)
5.28 (t, 6.3)
3.79 (d, 6.3)
4.96 (q, 6.6)
1.21 (d, 6.6)
–
66.0 (CH2)
47.9 (CH)
–
71.7 (CH)
73.0 (CH)
28.5 (CH2)
25.6 (CH2)
The 1H NMR spectrum of the peracetyl derivative 1b
exhibited (i) an intense aliphatic chain signal at δ =
1.25 ppm, (ii) several signals due to oxymethine and oxy-
methylene groups between δ = 5.4 and 3.4 ppm, and (iii)
an amide NH doublet at δ = 6.76 ppm. These signals are
100.1 (CH)
72.0 (CH)
79.3 (CH)
69.6 (CH)
72.3 (CH)
62.3(CH2)
100.8 (CH)
75.2 (CH)
73.5 (CH)
80.4 (CH)
69.6 (CH2)
15.5 (CH3)
169.7 (C)
2Ј
3Ј
4Ј
1
indicative of a GSL structure. The H NMR spectrum also
5Ј
6Ј
showed in the methyl region a triplet at δ = 0.87 ppm (ethyl
terminus) and a doublet at δ = 0.85 ppm (isopropyl ter-
minus), the intensities of which were not in an integral ratio
relative to those of other signals in the spectrum. This
showed that the alkyl chains differ not only in the length,
but also in the branching of the alkyl chains.
1ЈЈ
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈ
6ЈЈ
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
5.17 (dd, 6.8, 4.9)
1.83 (m)
73.6 (CH)
31.8 (CH2)
24.7 (CH2)
1.32 (m)
Planar Structure
Ac CH3 2.22, 2.09, 2.08, 2.06, 2.10, 2.05, 2.03 21.0–20.6 (CH3)
CO 170.9–169.1 (C)
–
Extensive NMR analysis was used to establish the planar
structure of terpioside. The ceramide portion of the mole-
cule is that commonly found in glycolipids from marine
sponges, that is, it is composed of a trihydroxylated satu-
rated sphinganine and an α-hydroxy fatty acid residue.
Starting from the amide NH doublet at δ = 6.78 ppm (2-
NH) we were able to assign all the protons of the polar part
1
[a] Additional H signals: δ = 1.24 (br., alkyl chain protons), 0.87
(t, J = 7.0 Hz, n-chain Me groups), 0.85 (d, J = 6.5 Hz, iso-chain
Me groups) ppm. [b] Additional 13C signals: δ = 31.9 (CH2, ω-2),
22.7 (CH2, ω-1), 22.7 (CH3, iso-chain Me groups), 14.1 (CH3, ω)
ppm.
of the sphinganine up to 6-H2 by analysis of the COSY that this sugar unit is linked to the ceramide primary hy-
spectrum (see Table 1). The α-hydroxy substitution of the droxy group was provided by the HMBC correlation peak
1
fatty acid residue was revealed by the absence in the H of 1Ј-H with C-1 and the ROESY correlation peak between
NMR spectrum of 1b of the characteristic triplet at δ ≈ 1Ј-H and 1b-H. In addition, the deshielded chemical shift
2.3 ppm for the fatty acid α-protons, whereas the spectrum of the oxymethine protons at the 2Ј-, 4Ј-, and 6Ј-positions
displayed an acetoxymethine proton resonance at δ = indicated that the relevant hydroxy groups are acetylated.
5.17 ppm (2ЈЈЈ-H) coupled with a methylene resonance at δ In contrast, the high-field chemical shift of the C-3Ј proton
= 1.83 ppm (3ЈЈЈ-H), in turn coupled with proton reso- signal (δ = 3.83 ppm) suggested glycosylation at this posi-
nances in the broad signal, relative to alkyl chain protons, tion, whereas the high-field chemical shift of the C-5Ј pro-
at δ = 1.24 ppm. In addition, the signal at δ = 5.17 ppm ton signal (δ = 3.51 ppm) confirmed that this carbon atom
showed an intense correlation peak with the amide NH is involved in the pyranose acetal function.
doublet in the ROESY spectrum.
Similarly, the second sugar spin system was recognized
The two sugar units were revealed by the presence of two from the TOCSY and COSY experiments as a 6-deox-
characteristic resonances in the anomeric region of the 13C yhexose. This was indicated by a doublet arising from three
NMR spectrum at δ = 100.1 and 100.8 ppm which were protons [δ = 1.21 ppm (6ЈЈ-H3)], which pointed to the pres-
associated through the HMQC spectrum with the corre- ence at the 6-position of a methyl group instead of the us-
sponding anomeric protons resonating as doublets at δ = ual –CH2OH group. The high-field chemical shift of the
4.32 (J = 8.0 Hz, 1Ј-H) and 5.23 ppm (J = 4.6 Hz, 1ЈЈ-H). 4ЈЈ-H signal (δ = 3.79 ppm), compared with the low-field
These protons were used in the analysis of the COSY and chemical shift of the 5ЈЈ-H signal (δ = 4.96 ppm), clearly
TOCSY spectra as starting points for the sequential assign- indicated that C-4ЈЈ links an oxygen atom involved in an
ment of all the protons in each monosaccharide unit. Data acetal function instead of an ester function; thus, the sugar
from a TOCSY experiment were particularly useful in the must be in the furanose form and not in the more common
assignment of four oxymethine protons [δ = 4.81, 3.83, 4.97, pyranose form. This second sugar unit is linked to C-3 of
and 3.53 ppm (2Ј-H, 3Ј-H, 4Ј-H, and 5Ј-H)] and a couple the first sugar as confirmed by an HMBC correlation be-
of oxymethylene protons to the sugar having the anomeric tween C-1ЈЈ and 3Ј-H and a ROESY correlation between
proton (δ = 4.32 ppm), suggesting it is a hexose. Evidence 1ЈЈ-H and 3Ј-H.
Eur. J. Org. Chem. 2008, 2130–2134
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V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, R. Teta
FULL PAPER
Stereostructure and Alkyl Chains
Table 2. Fatty acyl composition of terpioside 1a.
Composition
The study of the values of the coupling constants is a
useful method for elucidating the relative configurations of
all the chiral centers of sugars in the pyranose form and,
consequently, for establishing the nature of the sugar. In
our case, the first hexose unit was readily recognized as a
β-glucopyranoside because of the large coupling constants
between all the pairs of vicinal ring protons (see Table 1),
which pointed to their axial orientation.
Fatty acid methyl ester
[%]
Methyl 2-hydroxy-20-methylhenicosanoate (iso-C22
Methyl 2-hydroxydocosanoate (n-C22
Methyl 2-hydroxy-21-methyldocosanoate (iso-C23
Methyl 2-hydroxy-20-methyldocosanoate (ante-iso-C23
Methyl 2-hydroxytricosanoate (n-C23
Methyl 2-hydroxy-22-methyltricosanoate (iso-C24
Methyl 2-hydroxy-21-methyltricosanoate (ante-iso-C24
Methyl 2-hydroxytetracosanoate (n-C24
Methyl 2-hydroxy-23-methyltetracosanoate (iso-C25
Methyl 2-hydroxy-22-methyltetracosanoate (ante-iso-C25
)
3.6
1.7
17.0
4.2
4.4
2.2
)
)
)
)
)
)
5.3
)
22.3
15.5
5.0
As for the furanose sugar unit, the ROESY spectrum
showed correlation peaks between 1ЈЈ-H/4ЈЈ-H, 2ЈЈ-H/4ЈЈ-H,
)
)
and 3ЈЈ-H/5ЈЈ-H, which indicated that 1ЈЈ-H, 2ЈЈ-H, and 4ЈЈ- Methyl 2-hydroxypentacosanoate (n-C25
)
11.7
3.2
1.8
Methyl 2-hydroxy-24-methylpentacosanoate (iso-C26
Methyl 2-hydroxy-23-methylpentacosanoate (ante-iso-C26
Methyl 2-hydroxyhexacosanoate (n-C26
)
H lie on the same side of the five-membered ring, whereas
3ЈЈ-H and the CHOHCH3 group at C-4ЈЈ are on the other
side. The configuration at C-5 could not be established on
the basis of the ROESY data, so the sugar could be either
an α-fucofuranoside (6-deoxy-α-galactofuranoside) or a 6-
)
)
2.1
In particular, analysis of the perbenzoylated methyl gly-
deoxy-β-altrofuranoside. Degradation analysis (see below) coside fraction led to the isolation of the 6-deoxyglycoside
showed the former to be the case. 3, which was tentatively identified as tri-O-benzoyl-α-fuco-
1
The remaining structural features of terpioside were es- pyranoside on the basis of its H NMR spectrum. An au-
tablished by chemical degradation. The natural glycolipid thentic sample of tri-O-benzoyl-α-fucopyranoside was pre-
1a (300 µg) was subjected to acidic methanolysis, and the pared from -fucose by acidic methanolysis and subsequent
reaction products were separated, perbenzoylated, and ana- perbenzoylation, and its 1H NMR and CD spectra were
lyzed by CD and GC-MS (Scheme 2). This procedure, set recorded. The spectra were identical to those of compound
up in our laboratory and described in detail in a previous 3, thus confirming that the outer sugar of terpioside is a
paper,[4] allowed us to establish the absolute configuration fucose and showing that its absolute configuration is .
of each sugar and of the hydroxy acid, and the relative and
Once the structure of the peracetyl derivative 1b had
absolute configurations of the phytosphingosine, as well as been elucidated, 30 mg of the compound were deacetylated
to identify the alkyl chains of fatty acids (Table 2) and using MeOH in MeONa to give the natural glycolipid 1a
sphingosines (Table 3).
in quantitative yield. The one- and two-dimensional NMR
Scheme 2. Microscale degradation analysis of terpioside (1a).
Table 3. Sphinganine composition of terpioside 1a.
Sphinganine
Composition [%]
(2S,3S,4R)-2-Amino-16-methyl-1,3,4-heptadecanetriol (iso-C18)
(2S,3S,4R)-2-Amino-15-methyl-1,3,4-heptadecanetriol (ante-iso-C18)
(2S,3S,4R)-2-Amino-1,3,4-octadecanetriol (n-C18)
(2S,3S,4R)-2-Amino-16-methyl-1,3,4-octadecanetriol (ante-iso-C19)
(2S,3S,4R)-2-Amino-1,3,4-nonadecanetriol (n-C19)
4.5
15.5
4.1
70.1
5.8
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Eur. J. Org. Chem. 2008, 2130–2134

Terpioside from the Marine Sponge Terpios sp.
a mixing time of 500 ms. The reverse multiple-quantum heteronu-
clear correlation (HMQC) spectra were recorded by using a pulse
sequence with a BIRD pulse 0.5 s before each scan to suppress
the signal originating from protons not directly bound to 13C; the
interpulse delays were adjusted for an average 1JCH of 142 Hz. The
gradient-enhanced multiple-bond heteronuclear correlation
spectra of the natural GSL 1a were recorded and analyzed.
The information provided by the COSY and HMQC NMR
spectra of 1a confirmed all the structural features deter-
mined for its peracetylated derivative and allowed the as-
signment of all the resonances in its 1H and 13C NMR spec-
tra (see Exp. Sect.).
To check that no acetyl groups were present in the natu-
ral terpioside 1a before the peracetylation reaction used for
its isolation, we performed the acetylation reaction on the
3
(HMBC) experiment was optimized for a JCH of 8.3 Hz. GC-MS
spectra were performed with a Hewlett-Packard 5890 gas chroma-
tograph with an MSD HP 5970 MS mass-selective detector, a split/
splitness injector, and a fused-silica column (25 mϫ0.20 mm HP-
glycolipid fraction using trideuterioacetic anhydride and 5; cross-linked 25% Ph Me silicone, 0.33-mm film thickness). The
temperature of the column was varied after a delay of 3 min from
the injection from 150 to 280 °C with a gradient of 10 °Cmin–1;
quantitative determination was based on the area of the GLC
peaks. High-performance liquid chromatography (HPLC) was
achieved with a Varian Prostar 210 apparatus equipped with a Var-
ian 350 refractive index detector or a Varian 325 UV detector.
then purified the mixture to give the deuterio derivative 1c.
The H NMR spectrum of 1c was identical to that of 1b,
except that no acetyl methyl singlet was present.
1
Conclusions
Collection, Extraction, and Isolation: Specimens of Terpios sp. were
collected in December 2005 from along Key Largo coast (Florida)
and identified by Prof. S. Zea (University of Colombia). They were
frozen immediately after collection and kept frozen until extraction.
The sponge (220 g of dry weight after extraction) was homogenized
and extracted with methanol (3ϫ1 L), and then with chloroform
(3ϫ1 L); the combined extracts were partitioned between H2O and
nBuOH. The organic layer was concentrated in vacuo and afforded
20 g of a dark green oil, which was purified by chromatography on
a column packed with RP-18 silica gel. A fraction eluted with
CHCl3 (5.9 g) was further purified by chromatography on an SiO2
column, giving a fraction [820 mg; eluent: EtOAc/MeOH (7:3)]
mainly composed of glycolipids. This fraction was peracetylated
with Ac2O in pyridine for 12 h. The acetylated glycolipids were
subjected to HPLC separation on an SiO2 column [eluent: n-hex-
ane/EtOAc (6:4)], thus affording 62 mg of terpioside peracetate 1b.
The lipophilic extract of Caribbean Terpios sp. was ana-
lyzed and a new GSL, terpioside, was isolated. The struc-
ture of terpioside was determined by the combined use of
spectroscopic analysis and microscale chemical degrada-
tion.
Although glycolipids containing -fucose in the pyranose
form (including the important Lewis GSLs expressed by
some human cancer cells) are relatively common, terpioside
is the first natural GSL containing an -fucose in the fu-
ranose form in the sugar part of the molecule. Terpioside
is also the first GSL reported from sponges of the genus
Terpios.
The alkyl chains of terpioside ceramide are also worthy
of note. Methyl-branched alkyl chains are commonly found
in glycosphingolipids obtained from sponges, but they are
usually part of the sphinganines, whereas the fatty acid resi-
dues are generally unbranched. In contrast, in terpioside iso
and ante-iso fatty acids comprise more than 50 mol-% of
the total fatty acid residues.
Terpioside Peracetate 1b: Colorless oil, [α]2D5 = –8 (c = 0.15, CHCl3).
ESI-MS (positive ion mode, MeOH): m/z = 1434, 1420, 1406, 1392,
1378, 1364 ([M + Na]+ series). 1H and 13C NMR: see Table 1.
Composition of the fatty acids: see Table 2. Composition of the
sphinganines: see Table 3.
Deacetylation of 1b: Compound 1b (30 mg) was dissolved in MeOH
(950 µL) and a 0.4  solution of MeONa in MeOH (50 µL) was
added. The reaction was allowed to proceed at 25 °C for 18 h and
then the reaction mixture was dried under nitrogen and the residue
partitioned between water and chloroform. After removal of the
solvent, the organic layer gave 22 mg of the native glycosphingoli-
pid 1a.
These findings extend the variety of GSLs isolated from
marine sponges and represent an additional proof of the
chemical diversity of marine sponge chemistry.
Experimental Section
General Remarks: High-resolution ESI-MS data were recorded
with a Micromass QTOF Micro mass spectrometer; sample were
dissolved in MeCN/H2O (1:1) with 0.1% TFA. ESI-MS data were
recorded with an Applied Biosystem API 2000 triple-quadrupole
mass spectrometer. The spectra were recorded by infusion of the
samples into the ESI source using MeOH as the solvent. Optical
rotations were measured at 589 nm with a Perkin-Elmer 192 polari-
Terpioside (1a): Colorless amorphous solid, [α]2D5 = –25 (c = 0.15,
MeOH). HR-MS (ESI, positive ion mode, MeOH): calcd. for
[C55H107NNaO14]+ 1028.7589; found 1028.7567 [M + Na]+. 1H
NMR ([D5]pyridine): δ = 0.86 (n- and iso-chain Me groups), 1.28
(large band, alkyl chains), 1.54 (d, J = 6.5 Hz, 3 H, 6ЈЈ-H), 1.65
(m, 1 H, 6-Hb), 1.69 (m, 1 H, 4ЈЈЈ-Hb), 1.75 (m, 1 H, 4ЈЈЈ-Ha), 1.88
(m, 1 H, 5-Hb), 1.90 (m, 1 H, 5-Ha), 1.99 (m, 1 H, 3ЈЈЈ-Hb), 2.22
(m, 1 H, 5-Ha), 3.75 (m, 1 H, 5Ј-H), 3.95 (ddd, J = 7.8, 7.8, 3.7 Hz,
1 H, 2Ј-H), 4.08 (ddd, J = 9.2, 9.2, 3.5 Hz, 1 H, 4Ј-H), 4.15 (m, 1
H, 4-H), 4.16 (overlapped, 4ЈЈ-H), 4.23 (overlapped, 3Ј-H), 4.24
(overlapped, 6Ј-Hb), 4.26 (m, 1 H, 3-H), 4.36 (overlapped, 5ЈЈ-H),
4.56 (m, 1 H, 2ЈЈЈ-H), 4.47 (dd, J = 10.8 and 7.0 Hz, 1 H, 1-Ha),
4.65 (dd, J = 10.8, 3.9 Hz, 1 H, 1-Hb), 4.85 (d, J = 7.9 Hz, 1 H,
1Ј-H), 5.24 (m, 2-H), 6.08 (br. s, 1 H, 4-OH), 6.14 (d, J = 4.5 Hz,
1 H, 1ЈЈ-H), 6.25 (br. s, 1 H, 2ЈЈ-OH), 6.43 (br. s, 1 H, 6Ј-OH), 6.53
(br. s, 1 H, 5ЈЈ-OH), 6.86 (br. s, 1 H, 3-OH), 7.10 (br. s, 1 H, 4Ј-
OH), 7.36 (br. s, 1 H, 2Ј-OH), 7.45 (br. s, 1 H, 3ЈЈ-OH), 7.73 (br.
1
meter using a 10-cm microcell. H and 13C NMR spectra were de-
termined with a Varian Unity Inova spectrometer at 500.13 and
125.77 MHz, respectively; chemical shifts are referenced to the re-
sidual solvent signal (CDCl3: δH = 7.26 ppm, δC = 77.0 ppm; [D5]-
pyridine: δH = 8.73, 7.56, and 7.21 ppm; δC = 149.9, 135.6, and
123.6 ppm). For an accurate measurement of the coupling con-
1
stants, the one-dimensional H NMR spectra were transformed at
1
64K points (digital resolution: 0.09 Hz). Homonuclear H connec-
1
tivities were determined by COSY experiments. Through-space H
connectivities were evidenced through a ROESY experiment with
Eur. J. Org. Chem. 2008, 2130–2134
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2133

V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, R. Teta
FULL PAPER
s, 1 H, 2ЈЈЈ-OH), 8.53 (d, J = 9.5 Hz, 1 H, 2-NH) ppm. 13C NMR
([D5]pyridine): δ = 14.3 (CH3, n-chain Me groups), 20.6 (CH3, C-
6ЈЈ), 22.8 (CH3, iso-chain Me groups), 22.9 (CH2, n-chain ω-1 CH2
Analysis of Fraction B: Fraction B from the methanolysis of com-
pounds 1a was benzoylated as described above and the crude prod-
uct of the reaction was purified by HPLC [column: Luna SiO2, 5 µ;
groups), 25.6 (CH2, C-4ЈЈЈ), 26.3 (CH2, C-6), 30.5–29.5 (several eluent: n-hexane/iPrOH (99:1); flow: 1 mLmin–1]. The chromato-
CH2, alkyl chains), 32.1 (CH2, n-chain ω-2 CH2 groups), 33.8
(CH2, C-5), 35.3 (CH2, C-3ЈЈЈ), 51.4 (CH, C-2), 61.9 (CH2, C-6Ј), logues of benzoylated fatty acid methyl esters (fraction C, tR
65.9 (CH, C-5ЈЈ), 61.9 (CH2, C-6Ј), 69.2 (CH, C-4Ј), 72.1 (CH, C- 4.0 min) and a mixture of perbenzoylated sphinganines (fraction
gram shows two peaks, which were identified as a mixture of homo-
=
1
4; CH, C-2ЈЈЈ), 74.9 (CH, C-3ЈЈ), 77.9 (CH, C-5Ј), 79.2 (CH, C- D, tR = 6.9 min) on the basis of their respective H NMR spectra.
2ЈЈ), 84.1 (CH, C-3Ј), 86.4 (CH, C-4ЈЈ), 105.1 (CH, C-1Ј), 103.1
Methyl (R)-2-Benzoyloxyalkanoate: Fraction C from the mixture of
(CH, C-1ЈЈ), 105.3 (CH, C-1Ј), 175.3 (C, C-1ЈЈЈ) ppm. Composition
homologues. CD (MeCN): λmax = 229 (∆ε = –3.4) nm. The 1H
of the fatty acids: see Table 2. Composition of the sphinganines:
NMR spectrum was identical to that reported in the literature.[4]
see Table 3.
(2S,3S,4R)-1,3,4-O-Benzoyl-2-benzoylamino-1,3,4-alkanetriol: Frac-
Methanolysis of 1a: Compound 1a (100 µg) was dissolved in 1 
tion D from the mixture of homologues. CD (MeCN): λmax = 233
HCl in 91% MeOH (500 µL), and the solution obtained was kept
(∆ε = –1), 222 (∆ε = +2) nm. The 1H NMR spectrum was identical
in a sealed tube at 80 °C for about 12 h. The reaction mixture was
(apart from the methyl region) to that of an authentic sample of
dried under nitrogen and partitioned between CHCl3 and H2O/
MeOH (8:2). The aqueous layer was concentrated to give a mixture
-ribo-phytosphingosine perbenzoate.[9]
Oxidative Cleavage and GC-MS Analysis of Sphinganines: Fraction
D was debenzoylated by acidic methanolysis as described above
and subjected to oxidative cleavage with KMnO4/NaIO4 as de-
scribed in the literature.[2] The resulting carboxylic acids were meth-
ylated with CH2N2. The esters obtained were analyzed by GC-MS,
and the results are compiled in Table 3, expressed in terms of origi-
nal sphinganines.
of methyl glycosides (fraction A), whereas the organic layer con-
tained a mixture of α-hydroxy acid methyl esters and sphinganines
(fraction B).
Methyl Tri-O-benzoyl-α-L-fucopyranoside (3): -Fucose (2.0 mg)
was subjected to acidic methanolysis as described above. The re-
sulting methyl glycosides were benzoylated with benzoyl chloride
(50 µL) in pyridine (500 µL) at 25 °C for 16 h. The reaction was
then quenched with MeOH; after 30 min, the mixture was dried
under nitrogen. Methyl benzoate was removed by keeping the resi-
due under vacuum with an oil pump for 24 h. The residue was
purified by HPLC [column: Luna SiO2, 5 µ; eluent: n-hexane/
iPrOH (99:1); flow: 1 mLmin–1; UV detector: 280 nm] affording
Supporting Information (see footnote on the first page of this arti-
1
cle): H, 13C, COSY, ROESY, TOCSY, HSQC, and HMBC NMR
spectra of compound 1b; 1H, 13C, and COSY NMR spectra of
1
compound 1a; H NMR and CD spectra of compound 3.
1
the glycoside 3 (tR = 6.6 min). H NMR (CDCl3): δ = 1.29 (d, J =
Acknowledgments
6.5 Hz, 3 H, 6-H), 3.47 (s, 3 H, OMe), 4.39 (br. q, J = 6.5 Hz, 1
H, 5-H), 5.24 (d, J = 3.6 Hz, 1 H, 1-H), 5.64 (dd, J = 10.7, 3.6 Hz,
1 H, 2-H), 5.76 (br. d, J = 3.4 Hz, 1 H, 4-H), 5.95 (dd, J = 10.7,
3.4 Hz, 1 H, 3-H), 7.24 (t, J = 7.7 Hz, 2 H, benzoyl meta protons),
7.37 (t, J = 7.4 Hz, 2 H, benzoyl meta protons), 7.42 (t, J = 7.6 Hz,
1 H, benzoyl para proton), 7.53–7.45 (overlapping signals, 3 H,
benzoyl protons), 7.61 (t, J = 7.5 Hz, 1 H, benzoyl para proton),
7.79 (d, J = 7.9 Hz, 2 H, benzoyl ortho protons), 7.98 (d, J =
7.9 Hz, 2 H, benzoyl ortho protons), 8.11 (d, J = 8.0 Hz, 2 H, ben-
zoyl ortho protons) ppm. CD (MeCN): λmax = 237 (∆ε = –42), 222
(∆ε = +14) nm.
This work is a result of a project sponsored by MIUR PRIN (Italy)
. We wish to thank Prof. J. R. Pawlik (University of North Caro-
lina) for collecting the sponge and Professor S. Zea (Departamento
de Biología y Centro de Estudios en Ciencias del Mar-CECIMAR,
Universidad National de Colombia) for identifying the sponge.
Mass and NMR spectra were recorded at the Centro di Servizi
Interdipartimentale di Analisi Strumentale, Università di Napoli
“Federico II”. The assistance of the staff is gratefully acknowl-
edged.
Absolute Stereochemistry of Methyl Glycosides from Compound 1a:
Fraction A from the methanolysis of compound 1a was benzoyl-
ated with benzoyl chloride (20 µL) in pyridine (200 µL) at 25 °C
for 16 h. The reaction was then quenched with MeOH; after
30 min, the mixture was dried under nitrogen. Methyl benzoate was
removed by keeping the residue under vacuum with an oil pump
for 24 h. The residue was purified by HPLC [column: Luna SiO2,
5 µ; eluent: n-hexane/iPrOH (99:1); flow: 1 mL min–1]. The chroma-
togram showed two peaks: methyl tetra-O-benzoyl-α--glucopyr-
anoside (2), identified by comparison of its 1H NMR and CD spec-
tra with those reported in the literature,[4] and methyl tri-O-ben-
zoyl-α--fucopyranoside (3), identified by comparison of its 1H
NMR and CD spectra with those of the authentic samples pre-
pared from -fucose.
[1] V. Costantino, E. Fattorusso, A. Mangoni, M. Di Rosa, A. Ian-
aro, J. Am. Chem. Soc. 1997, 119, 12465–12470.
[2] V. Costantino, E. Fattorusso, A. Mangoni, M. Di Rosa, A. Ian-
aro, P. Maffia, Tetrahedron 1996, 52, 1573–1578.
[3] V. Costantino, M. D’Esposito, E. Fattorusso, A. Mangoni, N.
Basilico, S. Parapini, D. Taramelli, J. Med. Chem. 2005, 48,
7411–7417.
[4] V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, S.
Freigang, L. Teyton, Bioorg. Med. Chem.; DOI:10.1016/
j.bmc.2007.10.098.
[5] G. Plucer-Rosario, Coral Reefs 1987, 5, 197–200.
[6] T. Teruya, S. Nakagawa, T. Koyama, H. Arimoto, M. Kita, D.
Uemura, Tetrahedron 2004, 33, 6989–6993.
[7] T. Teruya, S. Nakagawa, T. Koyama, K. Suenaga, D. Uemura,
Chem. Lett. 2002, 31, 38–39.
[8] C. Delseth, L. Tolela, C. Djerassi, P. J. Scheuer, R. J. Wells,
Helv. Chim. Acta 2004, 62, 101–109.
[9] V. Costantino, E. Fattorusso, E. C. Imperatore, A. Mangoni,
J. Org. Chem. 2004, 69, 1174–1179.
Analysis of Fatty Acid Methyl Esters: Fraction B from the meth-
anolysis of compound 1a was analyzed by GLC-MS and its compo-
nents identified by comparison of their retention times and mass
spectra with those of authentic samples. The results are compiled
in Table 2.
Received: December 21, 2007
Published Online: March 10, 2008
2134
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 2130–2134