Vesparioside from the marine sponge Spheciospongia vesparia, the first diglycosylceramide with a pentose sugar residue

Article abstract of DOI:10.1002/ejoc.200400543

The marine sponge Spheciospongia vesparia produces vesparioside (1a), a diglycosylated glycosphingolipid which is the first example of a natural diglycosylceramide with a pentose sugar residue. The structure of vesparioside was mostly determined by extensive spectroscopic analysis but determination of the nature of the alkyl chains and elucidation of the absolute stereochemistry of the sugars and of the ceramide required chemical degradation. In this respect, an improved and simplified procedure for the microscale chemical degradation of glycosphingolipids was employed here for the first time. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400543

FULL PAPER
Vesparioside from the Marine Sponge Spheciospongia vesparia, the First
Diglycosylceramide with a Pentose Sugar Residue[‡]
Valeria Costantino,[a] Ernesto Fattorusso,[a] Concetta Imperatore,[a] and Alfonso Mangoni*[a]
Keywords: Glycolipids / Natural products / Sphingolipids / Structure elucidation / Pentose
The marine sponge Spheciospongia vesparia produces ves- ceramide required chemical degradation. In this respect, an
parioside (1a), a diglycosylated glycosphingolipid which is
the first example of a natural diglycosylceramide with a pen-
tose sugar residue. The structure of vesparioside was mostly
determined by extensive spectroscopic analysis but deter-
improved and simplified procedure for the microscale chem-
ical degradation of glycosphingolipids was employed here
for the first time.
mination of the nature of the alkyl chains and elucidation of (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
the absolute stereochemistry of the sugars and of the
Germany, 2005)
Introduction
Results and Discussion
Spheciospongia vesparia was collected near Grand
Bahamas Island (Bahamas) 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 4.5 mg of pure
compound 1b. Compound 1b was deacetylated with
MeOH/MeONa yielding the natural glycolipid 1a.
Marine sponges are a rich source of novel glycosphingo-
lipids which are often characterised by unprecedented struc-
tural features. Among them, and worthy of note, are plako-
sides from Plakortis simplex[1] which are immunosuppres-
sive glycosphingolipids with a prenylated galactose as well
as α-galactoglycosphingolipids[2Ϫ4] which are a new class of
glycosphingolipids with an α-galactose as the first sugar of
the carbohydrate chain. These have been found only in
sponges of the genera Agelas and Axinella and are charac-
terised by their immunostimulating[5] and antitumor[2,6]
properties. Even though a relatively large number of papers
on this topic has appeared in the literature in recent years,
the study of glycolipids form sponges is far from being con-
cluded and the structural variety of the isolated compounds
is far from being fully explored.
In this paper, we report the isolation and structural eluci-
dation of vesparioside (1a), a new diglycosylated glyco-
sphingolipid from Spheciospongia vesparia, characterised by
the presence of a β-arabinopyranoside linked at position 6
of the first sugar residue of the chain (a β-glucopyranoside).
Vesparioside is the first example of a natural diglycosyl-
ceramide whose carbohydrate chain contains a pentose
unit.
Scheme 1
The ESI mass spectrum showed a series of sodiated
pseudomolecular ion peaks at m/z ϭ 986, 1000, 1014 and
1028, in accordance with the molecular formula
C52H101NO14 ϩ nCH2 (n ϭ 0Ϫ3). A high-resolution meas-
urement performed on the most abundant ion at m/z ϭ
1000.7265 confirmed the molecular formula C53H103NO14
for the dominant homologue.
[‡]
Glycolipids from Sponges, 14. Part 13: See ref.[4]
[a]
`
Dipartimento di Chimica delle Sostanze Naturali, Universita di
Napoli ‘‘Federico II’’,
Via D. Montesano 49, 80131 Napoli, Italy
Fax: (internat.) ϩ 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.
368
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200400543
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Diglycosylceramide with a Pentose Sugar Residue
FULL PAPER
A preliminary 1H NMR spectroscopic analysis of the
peracetyl derivative 1b in CDCl3 showed appropriate sig-
nals for a glycosphingolipid, i.e. the intense aliphatic chain
signal at δ ϭ 1.25 ppm, several signals of oxymethine and
oxymethylene groups between δ ϭ 5.4 ppm and 3.4 ppm
and a characteristic amide NH doublet at δ ϭ 6.76 ppm,
suggesting the presence of a ceramide amide function. The
1H NMR spectrum also showed, in the methyl region, a
triplet at δ ϭ 0.87 ppm (ethyl terminus) and a doublet at
δ ϭ 0.85 ppm (isopropyl terminus) the intensities of which
were not in an integral ratio with respect to those of other
signals in the spectrum. This showed that the alkyl chain
mixture differs not only in the length but also in the branch-
ing of the alkyl chains.
1
Table 1. H and 13C NMR spectroscopic data of vesparioside per-
acetate 1b (CDCl3)
Position
1
δH [mult., J (Hz)][a]
δC (mult.)[b]
a
b
3.84 (dd, 10.9, 2.9)
3.67[c]
66.6 (CH2)
2
4.26 (m)
48.3 (CH)
Ϫ
72.0 (CH)
73.2 (CH)
28.4 (CH2)
25.6 (CH2)
2-NH
6.76 (d, 8.7)
3
4
5
6
5.11[c]
4.85 (m)
1.59 (m)
1.32 (m)
a
b
1.19 (m)
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
4.46 (d, 8.1)
4.87 (dd, 9.6, 8.1)
5.18 (t, 9.6)
5.07 (t, 9.6)
3.63 (ddd, 9.6, 4.5, 2.5)
3.80 (dd, 11.8, 2.5)
3.48 (dd, 11.9, 4.5)
5.03 (d, 3.6)
5.14[c]
5.29 (dd, 10.8, 3.5)
5.36 (br. s)
3.94 (br. d, 13.4)
3.67[c]
100.5 (CH)
71.3 (CH)
73.0 (CH)
68.8 (CH)
73.0 (CH)
66.3 (CH2)
Structure of the Ceramide
a
b
The ceramide portion of molecule is that commonly 1ЈЈ
found in glycolipids from marine sponges, i.e. it is com-
97.1 (CH)
68.2 (CH)
67.1 (CH)
69.0 (CH)
60.5 (CH2)
2ЈЈ
3ЈЈ
posed of a trihydroxylated, saturated sphinganine and an α-
4ЈЈ
hydroxy fatty acid residue. The amide NH doublet at δ ϭ
5ЈЈ
a
b
6.76 ppm (2-NH) allowed us to assign all the protons of
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
Ac’s
Ϫ
170.0 (C)
the polar part of the sphinganine unit up to 6-H2 through
5.12[c]
74.0 (CH)
31.8 (CH2)
24.9 (CH2)
the COSY spectrum. The α-hydroxy substitution of the
1.83 (m)
1.33 (m)
1
fatty acid residue was revealed by the absence, from the H
NMR spectrum of 1b, of the characteristic triplet at δ ϭ
2.3 ppm for the fatty acid α-protons, whereas a signal at
δ ϭ 5.12 (2ЈЈЈ-H) was present in the spectrum and showed
an intense correlation peak with the amide NH doublet in
the ROESY spectrum. In addition, both 2ЈЈЈ-H and 2-NH
were shown, by the HMBC spectrum, to be coupled with
CH3 2.24, 2.14, 2.12, 2.10, 2.04, 21.0Ϫ20.5 (CH3)
2.04, 2.01, 2.00, 1.98
Ϫ
CO
171.0Ϫ169.2
[a]
Additional 1H signals: δ ϭ 1.25 (broad band, alkyl chain pro-
tons), 0.87 (t, J ϭ 7.0, n-chain Me groups), 0.85 (d, J ϭ 6.5, iso-
chain Me groups) ppm. Additional 13C signals: δ ϭ 31.9 (CH2,
[b]
the CO carbon atom at δ ϭ 170.0 (C-1ЈЈЈ) ppm. The length ω-2), 22.7 (CH2, ω-1), 22.7 (CH3, iso-chain Me groups), 14.1 (CH3,
[c]
ω) ppm. Submerged by other signals.
of the alkyl chains and the configurations of the stereogenic
centres of the ceramide were established by chemical degra-
dation (see below).
between all the ring protons (see Table 1) indicating their
axial orientations. In addition, the deshielded chemical
shifts of the oxymethine protons at positions 2Ј, 3Ј and 4Ј
indicated that the relevant hydroxyl groups were acetylated
and therefore not glycosylated. In contrast, the chemical
Structure of the Sugar Chain
The nature of the sugar unit was unambiguously deter- shifts below δ ϭ 4.0 ppm for the protons at C-6Ј suggested
mined by NMR spectroscopic analysis. The presence of two glycosylation at this position and this was confirmed by
sugar units was revealed by two doublets at δ ϭ 4.46 ppm three intense correlation peaks in the ROESY spectrum of
(J ϭ 8.1 Hz, 1Ј-H) and 5.03 ppm (J ϭ 3.6 Hz, 1ЈЈ-H) which the pentose anomeric proton, namely 1ЈЈ-H with 5Ј-H, 6Ј-
were assigned to the anomeric protons on account of the Ha and 6Ј-Hb. Evidence of the linkage of this sugar residue
chemical shift of the relevant carbon atoms (δ ϭ 100.5 ppm to the ceramide primary hydroxyl group was provided by
and 97.1 ppm, respectively) identified through the HMQC the ROESY spectrum which displayed correlation peaks of
spectrum. These protons were used in the analysis of the 1Ј-H with 1-Ha and 1-Hb, and by the HMBC spectrum
COSY spectrum as starting points for the sequential assign- which indicated a three-bond coupling between 1Ј-H and
ment of all the protons in the disaccharide unit. Four more C-1.
oxymethine protons and a couple of oxymethylene protons
The nature of the pentose residue was determined on the
were assigned to the first sugar (the one with the anomeric basis of the coupling constants between the ring protons.
proton at δ ϭ 4.46 ppm), suggesting a hexose. The other The 3ЈЈ-H signal displayed one large (10.8 Hz) and one
sugar, in addition to the anomeric proton at δ ϭ 5.03 ppm, small (3.5 Hz) coupling constant, showing 3ЈЈ-H to be an
only contained three oxymethine protons and a couple of axial proton flanked by one axial and one equatorial pro-
oxymethylene protons and is therefore a pentose unit.
ton. The signal of 4ЈЈ-H was a broad singlet indicating that
The hexose sugar residue could be readily recognised as a this proton is clearly equatorial and that 2ЈЈ-H is therefore
β-glucopyranoside because of the large coupling constants axial (the multiplicity of the 2ЈЈ-H signal could not be
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
369

V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni
FULL PAPER
analysed due to the overlapping of signals). Finally, the perbenzoylated with BzCl in pyridine and the reaction mix-
anomeric proton 1ЈЈ-H is equatorial based on its small ture was separated by HPLC. The chromatogram contained
(3.6 Hz) coupling constants with 2ЈЈ-H. On the basis of the two peaks which were identified as methyl tetra-O-benzoyl-
above data, the pentose residue is clearly a β-arabinopyr- β-d-glucopyranoside (2) and methyl tri-O-benzoyl-β-d-ara-
anoside. The pyranose ring closure was confirmed by the binopyranoside (3) since they each showed the same reten-
1
coupling between 5ЈЈ-Hb and C-1ЈЈ indicated by the HMBC tion times, H NMR spectra and CD spectra as those ob-
spectrum, as well as by the low-field chemical shift of 4ЈЈ- tained from authentic samples prepared with the same pro-
H indicating acetylation of the relevant OH group.
cedure from, respectively, d-glucose and d-arabinose.
Once the structure of the peracetyl derivative 1b was as-
Fraction B, containing the sphinganines and the fatty
sessed, the 1- and 2-D NMR spectra of the natural GSL 1a acid methyl esters from the methanolysis, was analysed by
were also recorded and analysed. The information provided GC-MS and was shown to contain two different un-
by the COSY and HMQC NMR spectra of 1a confirmed branched 2-hydroxy fatty acids, identified by comparison of
all the structural features determined so far and allowed the their retention times and mass spectra with those of auth-
1
assignments of all the resonances in its H and 13C NMR entic samples (Table 2). Following this, fraction B was per-
spectra (see Experimental Section).
benzoylated as described above and the reaction mixture
was separated by normal-phase HPLC. This led to a frac-
tion composed of 2-benzoyloxy fatty acid methyl esters
(fraction C) and a fraction composed of perbenzoylated 4-
hydroxysphinganines (fraction D).
Chemical Degradation
The remaining structural features of compound 1a were
determined by microscale chemical degradation. In spite of
the huge progress of NMR spectroscopy and mass spec-
trometry, chemical degradation is still an essential step in
the study of glycosphingolipids. This is particularly true for
marine glycosphingolipids which are often present in the
organisms as inseparable mixtures of homologues and are
characterised by branched and odd-carbon alkyl chains.
Many marine glycosphingolipids can only be isolated in an
amount of 1 mg or less so that even a microscale degra-
dation procedure can destroy a significant portion of the
sample. Therefore, it is very important to try to increase the
sensitivity of this analytical procedure.
Table 2. Fatty acyl composition of vesparioside 1a
The degradation procedure we used for vesparioside,
which is quite complex and somewhat different from that
used in our previous papers, is summarised in Scheme 2.
The CD spectrum of fraction C showed a negative Cot-
Compound 1a (100 µg) was subjected to acidic meth- ton effect at λmax ϭ 232 nm (∆ε ϭ Ϫ4.4). Since the CD
anolysis with HCl in MeOH and the reaction products were spectrum of methyl (S)-2-benzoyloxybutyrate (4), prepared
partitioned between CHCl3 and H2O/MeOH (8:2) giving an from commercial (S)-2-hydroxybutyric acid, showed a posi-
aqueous layer consisting of methyl glycosides (fraction A) tive Cotton effect at λmax. ϭ 231 nm (∆ε ϭ ϩ4.7) the (R)
and an organic layer composed of sphinganines and fatty configuration of the 2-hydroxy fatty acids from compound
acid methyl esters (fraction B).
1a could be deduced.
The methyl glycoside fraction was used to determine the
The ribo relative configurations of the benzoylated sphin-
1
absolute configuration of the two sugars.4 Fraction A was ganines were determined by recording the H NMR spec-
Scheme 2. The new microscale degradation procedure for glycolipids
370
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 368Ϫ373

Diglycosylceramide with a Pentose Sugar Residue
FULL PAPER
trum of fraction D which was identical (apart from the chromatography. This is no longer necessary because sphin-
methyl region) to that of an authentic sample of d-ribo- ganines and fatty acids can be now benzoylated together
phytosphingosine perbenzoate (5).[4] Their absolute con- and, after the introduction of the benzoate chromophore,
figurations were deduced from the CD spectrum in MeCN they can be separated using an HPLC instrument equipped
solution which matched that of compound 5 recorded in with a sensitive UV detector. This can dramatically improve
the same solvent.[4]
the purity of the samples. In addition, the configurations of
After this, fraction D was subjected to acidic meth- 2-hydroxy fatty acids can now be determined using the CD
anolysis to remove the benzoyl groups and then to Lemieux spectra of their benzoylated methyl esters instead of those
oxidation with KMnO4/NaIO4 to convert the 4-hydroxy- of the underivatised methyl esters. As a consequence, the
sphinganines to carboxylic acids with three less carbon measured Cotton effect is more than three times stronger
atoms. The obtained fatty acids were methylated with (∆ε ϭ Ϫ4.4 vs. Ϫ1.3) and at a longer wavelength (232 vs.
CH2N2 and analysed by GC-MS. The results are reported 212 nm), making the measurement easier.
in Table 3, in the form of the structures of the correspond-
ing sphinganines.
The only step that still requires extensive manipulation
of the sample is the Lemieux oxidation of the sphinganines.
We could not avoid this because of the concurrent presence
of iso and anteiso alkyl chains in the sphinganines. In such
a case, conversion of the sphinganines to fatty acid methyl
esters and subsequent GC-MS analysis is mandatory for a
successful separation and quantitative analysis. In fact, fatty
acid methyl esters can be separated very effectively by GC
and their mass spectra have been very well studied allowing
location of a branching methyl at any position.
Table 3. Sphinganine composition of vesparioside 1a
In simpler cases, Lemieux oxidation could, in future
work, be replaced by reversed-phase LC-MS analysis of the
benzoylated sphinganines. This would enable a further sca-
ling-down of this analytical method making it suitable for a
detailed structural analysis of glycosphingolipids other than
those of marine origin, those from mammalian tissues for
example.
Experimental Section
General Remarks: High-Resolution ESI-MS spectra were per-
formed with a Micromass QTOF Micro mass spectrometer by dis-
solving the sample in MeCN/H2O (1:1) with 0.1% TFA. ESI MS
experiments were performed on a Applied Biosystem API 2000 tri-
ple-quadrupole mass spectrometer. The spectra were recorded by
infusion into the ESI source using MeOH as the solvent. Optical
rotations were measured at 589 nm with a PerkinϪElmer 192
polarimeter using a 10-cm microcell. 1H and 13C NMR spectra
were determined with a Varian UnityInova spectrometer at 500.13
and 125.77 MHz, respectively. Chemical shifts were referenced to
the residual solvent signal (CDCl3: δH ϭ 7.26 ppm, δC ϭ 77.0 ppm;
[D5]pyridine: δH ϭ 8.73 ppm, 7.56 ppm and 7.21 ppm; δC ϭ 149.9
ppm, 135.6 ppm and 123.6 ppm). For an accurate measurement of
the coupling constants, the 1-D 1H NMR spectra were transformed
with 64 K points (digital resolution: 0.09 Hz). Homonuclear 1H
connectivities were determined by COSY experiments. Through-
Conclusion
The novel GSL vesparioside 1a is the first example of a
natural diglycosylceramide containing a pentose unit in the
sugar head. The structure of vesparioside was determined
by the combined use of spectroscopic analysis and a new
and improved procedure for microscale chemical degra-
dation.
The new degradation procedure described here allowed
us: (a) to confirm the nature of the sugars in the carbo-
hydrate chain and determine their absolute configurations, space 1H connectivities were highlighted using a ROESY experi-
ment with a mixing time of 500 ms. The reverse multiple-quantum
heteronuclear correlation (HMQC) spectra were recorded using a
pulse sequence with a BIRD pulse of 0.5 s before each scan to
suppress the signal originating from protons not directly bound to
13C nuclei. The interpulse delays were adjusted for an average 1JC,H
of 142 Hz. The gradient-enhanced multiple-bond heteronuclear
(b) to determine the relative and absolute configuration of
the sphinganines as well as the nature of their alkyl chains
and (c) to determine the configuration at C-2 of the 2-
hydroxy fatty acids and the nature of their alkyl chains. The
modifications we made to the method, in comparison with
those previously reported, were aimed at improving sensi-
tivity and reducing manipulation of the sample and thus
the consequent risk of contamination.
3
correlation (HMBC) experiment was optimised for a JC,H of
8.3 Hz. GC-MS spectra were performed with a HewlettϪPackard
5890 gas chromatograph with a mass selective detector MSD HP
5970 MS, a split/splitless injector and an HP-5 fused-silica column
In previous methods,[4,7] the sphinganine fraction was
separated from the fatty acid fraction using SiO2 column of dimensions 25 m ϫ 0.20 mm (cross-linked 25% Ph Me silicone,
Eur. J. Org. Chem. 2005, 368Ϫ373
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V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni
FULL PAPER
0.33-mm film thickness). The temperature of the column was va-
ried, after a delay of 3 min from the injection, from 150 °C to 280
°C with a rate of 10 °C·minϪ1. Quantitative determination was
based on the areas of the GLC peaks. High performance liquid
chromatography (HPLC) was performed with a Varian Prostar 210
apparatus equipped with a Varian 350 refractive index detector or
a Varian 325 UV detector.
3ЈЈЈ), 51.2 (CH, C-2), 64.3 (CH2, C- 5ЈЈ), 69.0 (CH2, C-6Ј), 70.0
(CH, C-4ЈЈ), 70.6 (CH2, C-1), 70.7 (CH, C-2ЈЈ), 70.7 (CH, C-3ЈЈ),
71.7 (CH, C-4Ј), 72.2 (CH, C-4), 72.3 (CH, C-2ЈЈЈ), 75.0 (CH, C-
2Ј), 75.7 (CH, C-3), 76.8 (CH, C-5Ј), 78.3 (CH, C-3Ј), 101.7 (CH,
C-1ЈЈ), 105.3 (CH, C-1Ј), 175.3 (C, C-1ЈЈЈ) ppm; Composition in
fatty acids: Table 2. Composition in sphinganines: Table 3.
Methanolysis of 1a: Compound 1a (100 µg) was dissolved in 1 n
HCl in 91% MeOH (500 µL) and the resultant solution was kept
for about 12 h at 80 °C in a sealed tube. The reaction mixture was
dried under nitrogen and partitioned between CHCl3 and H2O/
MeOH (8:2). The aqueous layer was concentrated to give a mixture
of methyl glycosides (fraction A), whereas the organic layer con-
tained a mixture of α-hydroxy acid methyl esters and sphinganines
(fraction B).
Collection, Extraction and Isolation: Specimens of Spheciospongia
vesparia were collected in the summer of 2000 along the coast of
Grand Bahamas Island (Bahamas) and identified by Prof. M. Pan-
sini (University of Genoa). They were frozen immediately after col-
lection and kept frozen until extraction. The sponge (100 g dry
weight after extraction) was homogenised and extracted with meth-
anol (3 ϫ 1 L) and then chloroform (3 ϫ 1 L). The combined ex-
tracts were partitioned between H2O and nBuOH. The organic
layer was concentrated in vacuo to afford 12.1 g of a dark green
oil which was chromatographed on a column packed with RP-18
silica gel. A fraction eluted with CHCl3 (3.6 g) was further chroma-
tographed on an SiO2 column giving a fraction [285 mg, eluent
EtOAc/MeOH (7:3)] mainly composed of glycolipids. This fraction
was peracetylated with Ac2O in pyridine at 25 °C for 12 h. The
acetylated glycolipids were subjected to HPLC separation on an
SiO2 column [eluent: n-hexane/EtOAc (6:4)], thus affording a mix-
ture (11.7 mg) containing 1b and other glycolipids. Further normal-
phase HPLC purification [eluent: n-hexane/iPrOH (95: 5)] gave
4.5 mg of vesparioside peracetate (1b).
Methyl Tetra-O-benzoyl-β-D-glucopyranoside (2): d-Glucose
(2.0 mg) was subjected to acidic methanolysis as described above.
The resultant 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 and after 30 min was dried under
nitrogen. Methyl benzoate was removed by keeping the residue un-
der vacuum for 24 h with an oil pump. The residue was purified by
HPLC (column: Luna SiO2, 5 µ; eluent: n-hexane/iPrOH, 99:1,
flow 1 mL/min, UV dedector) affording the glycoside 2 (tR
ϭ
1
10.2 min). H NMR (CDCl3, ppm): δ ϭ 3.53 (s, 3 H, OMe), 4.14
(m, 1 H, 5-H), 4.49 (dd, J ϭ 12.0, 5.4 Hz, 1 H, 6-Hb), 4.63 (dd,
J ϭ 12.0, 3.1 Hz, 1 H, 6-Ha), 4.75 (d, J ϭ 7.8, 1 H, 1-H), 5.50 (dd,
J ϭ 9.8, 7.8 Hz, 1 H, 2-H), 5.66 (t, J ϭ 9.8 Hz, 1 H, 4-H), 5.89 (t,
J ϭ 9.8, 1 H, 3-H), 7.55Ϫ7.24 (12 H, overlapping signals, benzoyl
protons), 7.80 (t, J ϭ 8.1 Hz, 2 H, benzoyl ortho protons), 7.94 (d,
J ϭ 8.1 Hz, 2 H, benzoyl ortho protons), 7.88 (d, J ϭ 8.1 Hz, 2 H,
benzoyl ortho protons), 8.00 (d, J ϭ 8.1 Hz, 2 H, benzoyl ortho
protons). CD (MeCN): λmax. (∆ε) ϭ 235 nm ( ϩ15), 220 nm ( Ϫ2).
Vesparioside Peracetate (1b): Colourless oil, [α]2D5 ϭ Ϫ18 (CHCl3,
1
c ϭ 0.3). H and 13C NMR: Table 1. Composition in fatty acids:
Table 2. Composition in sphinganines: Table 3.
Deacetylation of 1b: Compound 1b (3.0 mg) was dissolved in
MeOH (950 µL) and a 0.4 m solution of MeONa in MeOH was
added (50 µL). The reaction was allowed to proceed for 18 h at 25
°C and the mixture was then dried under nitrogen and the residue
partitioned between water and chloroform. After removal of the
solvent, the organic layer gave 2.1 mg of the native glycosphingoli-
pid 1a.
Methyl Tri-O-benzoyl-α-D-arabinopyranoside (3): d-Arabinose
(2.0 mg) was subjected to acidic methanolysis followed by ben-
zoylation as described above. HPLC purification under the same
conditions as above afforded the glycoside 3 (tR ϭ 12.5 min). 1H
NMR (CDCl3, ppm): δ ϭ 3.54 (s, 3 H, OMe), 3.89 (br. d, J ϭ
12.9 Hz, 1 H, 5-Hb), 4.31 (dd, J ϭ 12.9 and J ϭ 3.7 Hz, 1 H, 5-
Ha), 4.64 (d, J ϭ 6.3 Hz, 1 H, 1-H), 5.58 (dd, J ϭ 9.0, 3.5 Hz, 1
H, 3-H), 5.73Ϫ5.65 (overlapping signals, 2 H, 2-H and 4-H),
7.58Ϫ7.28 (overlapping signals, 9 H, benzoyl protons), 7.89 (d, J ϭ
7.9 Hz, 2 H, benzoyl ortho protons), 8.00 (d, J ϭ 7.9 Hz, 2 H,
benzoyl ortho protons), 8.03 (d, J ϭ 7.9 Hz, 2 H, benzoyl ortho
protons). CD (MeCN): λmax. (∆ε) ϭ 237 nm ( Ϫ52), 221 nm ( ϩ10).
Vesparioside (1a): Colourless amorphous solid, [α]2D5 ϭ Ϫ12 (c ϭ
0.1 in MeOH). ESI MS (positive ion mode, MeOH): m/z ϭ 986,
1000, 1014, and 1028 ([M ϩ Na]ϩ series). HRESI MS (positive ion
ϩ
mode, MeOH): m/z ϭ 1000.7265 ([M ϩ Na]ϩ, C53H103NNaO14
gives 1000.7276). 1H NMR ([D5]pyridine): δ ϭ 0.86 (n- and iso-
chain Me groups), 1.25 (large band, alkyl chains), 1.67 (m, 2 H,
4ЈЈЈ-H2), 1.71 (m, 2 H, 6-H2), 1.90 (m, 1 H, 5-Hb), 1.99 (m, 1 H,
3ЈЈЈ-Hb), 2.21 (m, 1 H, 3ЈЈЈ-Ha), 2.23 (m, 1 H, 5-Ha), 4.00 (ddd,
J ϭ 7.9, 7.9, and 3.2 Hz, 1 H, 2Ј-H), 4.04 (overlapped, 5Ј-H), 4.07
(overlapped, 5ЈЈ-Hb), 4.11 (overlapped, 4Ј-H), 4.18 (overlapped, 3Ј-
H), 4.18 (overlapped, 6Ј-Hb), 4.25 (m, 1 H, 4-H), 4.30 (overlapped,
5ЈЈ-Ha), 4.32 (overlapped, 3-H), 4.38 (br. s, 1 H, 4ЈЈ-H), 4.55 (over-
lapped, 3ЈЈ-H), 4.56 (overlapped, 1-Hb), 4.58 (overlapped, 2ЈЈЈ-H),
4.63 (overlapped, 2ЈЈ-H), 4.64 (br. d, J ϭ 10.5 Hz, 1 H, 6Ј-Ha), 4.73
(dd, J ϭ 10.7, 6.0 Hz, 1 H, 1-Ha), 4.97 (d, J ϭ 7.9 Hz, 1 H, 1Ј-H),
5.28 (m, 1 H, 2-H), 5.55 (d, J ϭ 3.1 Hz, 1 H, 1ЈЈ-H), 6.01 (d, J ϭ
6.5 Hz, 1 H, 4-OH), 6.30 (d, J ϭ 2.9 Hz, 1 H, 4ЈЈ-OH), 6.50 (d,
J ϭ 7.3 Hz, 1 H, 2ЈЈ-OH), 6.61 (d, J ϭ 5.4 Hz, 1 H, 3ЈЈ-OH), 6.79
(d, J ϭ 6.3 Hz, 1 H, 3-OH), 7.31 (2 H, overlapped, 2Ј-OH and 4Ј-
Methyl (S)-2-Benzolyoxybutanoate (4): (S)-2-Hydroxybutanoic acid
(Fluka, 10 mg) was treated with an excess of an ethereal solution
of CH2N2 until the yellow colour persisted and then benzoylated
as described above. The crude reaction mixture was purified under
the same conditions as above to give 7.5 mg of the ester 4 (tR
ϭ
5.5 min). 1H NMR (CDCl3, ppm): δ ϭ 1.09 (t, J ϭ 7.4, 3 H, 4-
H3). 2.03 (m, 2 H, 3-H2), 3.77 (s, 3 H, OMe), 5.21 (t, J ϭ 6.1 Hz,
1 H, 2-H), 7.46 (t, J ϭ 7.6 Hz, 2 H, benzoyl meta protons), 7.59
(t, J ϭ 7.5 Hz, 1 H, benzoyl para proton), 8.09 (d, J ϭ 8.0 Hz, 2 H,
benzoyl ortho protons). CD (MeCN): λmax. (∆ε) ϭ 231 nm (ϩ4.7).
Absolute Stereochemistry of Methyl Glycosides from Compound 1a:
OH), 7.38 (d, J ϭ 4.0 Hz, 1 H, 3Ј-OH), 7.66 (d, J ϭ 5.1 Hz, 1 H, Fraction A from methanolysis of compound 1a was benzoylated
2ЈЈЈ-OH), 8.58 (d, J ϭ 9.2 Hz, 1 H, 2-NH). 13C NMR ([D5]pyridine, with benzoyl chloride (20 µL) in pyridine (200 µL) at 25 °C for
ppm): δ ϭ 14.2 (CH3, n-chain Me groups), 22.8 (CH3, iso-chain
16 h. The reaction was then quenched with MeOH and after 30 min
Me groups), 23.0 (CH2, n-chain ω-1 CH2 groups), 25.9 (CH2, C- was dried under nitrogen. Methyl benzoate was removed by keeping
6), 26.5 (CH2, C-4ЈЈЈ), 30.5Ϫ29.5 (several CH2, alkyl chains), 32.1 the residue under vacuum for 24 h with an oil pump. The residue
(CH2, n-chain ω-2 CH2 groups), 33.9 (CH2, C-5), 35.6 (CH2, C-
was purified by HPLC (column: Luna SiO2, 5 µ; eluent: n-hexane/
372
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 368Ϫ373

Diglycosylceramide with a Pentose Sugar Residue
iPrOH, 99:1, flow 1 mL/min). The chromatogram contained two The esters obtained were analysed by GC-MS and the results are
FULL PAPER
peaks which were identified as compounds 2 and 3 by a comparison
of their retention times, 1H NMR spectra and CD spectra with
those of the authentic samples prepared from d-glucose and d-ar-
abinose.
compiled in Table 3, expressed in terms of the original sphingan-
ines.
Supporting Information Available: (See also footnote on the first
1
page of this article). H NMR and ESI MS spectra of compound
1a. 1-D and 2-D NMR spectra of compound 1b. 1H NMR and
CD spectra of the degradation products 2Ϫ5.
Analysis of Fatty Acid Methyl Esters: Fraction B from methanolysis
of compound 1a was analysed by GLC-MS and its components
identified by a comparison of their retention times and mass spec-
tra with those of authentic samples. The results are compiled in
Table 2.
Acknowledgments
This work is the result of a project sponsored by MIUR PRIN
(Italy). We wish to thank Prof. J. R. Pawlik (University of North
Carolina) for giving us the opportunity to join in an expedition to
the Caribbean Sea during which the sponge S. vesparia was col-
lected and Prof. M. Pansini (Istituto di Zoologia, University of
Genoa, Italy) for identifying the sponge. Mass and NMR spectra
were recorded at the ‘‘Centro di Servizi Interdipartimentale di
Analysis of Fraction B: Fraction B from methanolysis of com-
pounds 1a was benzoylated as described above and the crude reac-
tion mixture was purified by HPLC (column: Luna SiO2, 5 µ; elu-
ent: n-hexane/iPrOH, 99:1, flow 1 mL/min). The chromatogram
contained two peaks which were identified as being due to a mix-
ture of homologous benzoylated fatty acid methyl esters (fraction
C, tR ϭ 4.0 min) and a mixture of perbenzoylated sphinganines
(fraction D, tR ϭ 7.2 min) on the basis of their respective 1H
NMR spectra.
`
Analisi Strumentale’’, Universita di Napoli ‘‘Federico II’’. The
assistance of the staff is gratefully acknowledged.
[1]
V. Costantino, E. Fattorusso, A. Mangoni, M. Di Rosa,
Methyl (R)-2-Benzoyloxyalkanoate: (Fraction C, mixture of homol-
ogues) 1H NMR (CDCl3, ppm): δ ϭ 0.87 (ω Me group), 1.25 (large
band, alkyl chain), 1.99 (m, 2 H, 3-H2), 3.78 (s, 3 H, OMe), 5.23
(t, J ϭ 6.0 Hz, 1 H, 2-H), 7.44 (t, J ϭ 7.6 Hz, 2 H, benzoyl meta
protons), 7.58 (t, J ϭ 7.5 Hz, 1 H, benzoyl para proton), 8.10 (d,
J ϭ 8.0 Hz, 2 H, benzoyl ortho protons). CD (MeCN): λmax. (∆ε) ϭ
231 nm (Ϫ4.4).
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Ianaro, B. Pisano, Eur. J. Org. Chem. 2001, 4651Ϫ4656.
V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, J.
Org. Chem. 2004, 69, 1174Ϫ1179.
T. Natori, M. Morita, K. Akimoto, Y. Koezuka, Tetrahedron
1994, 50, 2771Ϫ84.
M. Crul, R. A. A. Mathot, G. Giaccone, C. J. A. Punt, H.
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V. Costantino, E. Fattorusso, A. Mangoni, M. Di Rosa,
[3]
[4]
[5]
(2S,3S,4R)-1,3,4-O-Benzoyl-2-benzamidoamino-1,3,4-alkanetriol:
(Fraction D, mixture of homologues): CD (MeCN): λmax. (∆ε) ϭ
[6]
1
233 nm ( Ϫ7), 220 nm (ϩ1); the H NMR spectrum was identical
(apart from the methyl region) to that of an authentic sample of
d-ribo-phytosphingosine perbenzoate (5).[4]
[7]
Oxidative Cleavage and GC-MS Analysis of Sphinganines: Fraction
D was debenzoylated by acidic methanolysis as described above,
subjected to oxidative cleavage with KMnO4/NaIO4 as described[8]
and the resultant carboxylic acids were methylated with CH2N2.
[8]
A. Ianaro, P. Maffia, Tetrahedron 1996, 52, 1573Ϫ1578
Received July 30, 2004
Eur. J. Org. Chem. 2005, 368Ϫ373
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
373