Glycolipids from Sponges. 13. Clarhamnoside, the First Rhamnosylated α-Galactosylceramide from Agelas clathrodes. Improving Spectral Strategies for Glycoconjugate Structure Determination

Article abstract of DOI:10.1021/jo034865h

Reinvestigation of the glycosphingolipid composition of the marine sponge Agelas clathrodes revealed the presence of a new tetraglycosylated α-galactoglycosphingolipid (1a), containing an unusual L-rhamnose unit in the sugar head. The structure of the new compound was elucidated using extensive 2D NMR studies. Because of the strong overlapping of the signals of the sugar protons in the 1H spectrum, ¹³C-coupled and ¹³C-decoupled phase-sensitive HMQC spectra were used to study the multiplicity of the overlapping signals. In addition, the absolute configuration of sugars was determined using a simple and efficient, yet underutilized CD method.

Full text of DOI:10.1021/jo034865h

Glycolip id s fr om Sp on ges. 13.1 Cla r h a m n osid e, th e F ir st
Rh a m n osyla ted r-Ga la ctosylcer a m id e fr om Agela s cla th r od es.
Im p r ovin g Sp ectr a l Str a tegies for Glycocon ju ga te Str u ctu r e
Deter m in a tion
Valeria Costantino, Ernesto Fattorusso, Concetta Imperatore, and Alfonso Mangoni*
Dipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli “Federico II”,
via D. Montesano 49, 80131 Napoli, Italy
alfonso.mangoni@unina.it
Received J une 20, 2003
Reinvestigation of the glycosphingolipid composition of the marine sponge Agelas clathrodes revealed
the presence of a new tetraglycosylated R-galactoglycosphingolipid (1a ), containing an unusual
L-rhamnose unit in the sugar head. The structure of the new compound was elucidated using
extensive 2D NMR studies. Because of the strong overlapping of the signals of the sugar protons
1
in the H spectrum, 13C-coupled and 13C-decoupled phase-sensitive HMQC spectra were used to
study the multiplicity of the overlapping signals. In addition, the absolute configuration of sugars
was determined using a simple and efficient, yet underutilized CD method.
In tr od u ction
The GSL composition of the Caribbean sponge Agelas
clathrodes was investigated by our research group in
1995,4 and three immunostimulating R-Gal-GSLs (2-4)
were isolated. The availability in our laboratory of new
specimens of A. clathrodes prompted us to reexamine the
GSL content of this sponge, and this led to the identifica-
tion of a further R-Gal-GSL, clarhamnoside (1a ), a
tetraglycosylceramide which is the first R-Gal-GSL hav-
ing an L-rhamnose unit in the sugar head.
Glycosphingolipids (GSLs) are glycolipids composed of
a long-chain amino alcohol, known as a sphingoid base,
a fatty acid residue linked to its amino group (the
resulting amide is called ceramide), and a carbohydrate
chain attached to the primary hydroxyl group of the
ceramide. The scientific interest in GSLs has recently
increased on account of the role they could play as
therapeutical immunomodulating agents. Unlike com-
mon â-glucosyl- or â-galactosylceramides from higher
animals and plants, sponges of the genera Agelas and
Axinella2-6 produce R-galactoglycosphingolipids (R-Gal-
GSLs), unique glycosphingolipids with an R-galactose as
the first sugar of the carbohydrate chain. Natural R-Gal-
GSLs have been shown to possess interesting immuno-
modulating activities,3,6 and the mechanism of action of
these compounds and of their synthetic analogues has
been studied in detail. They are potent ligands of the
CD1d antigen presenting protein, and can specifically
activate natural killer T cells (NKT cells) in vivo.7 In
addition, KRN7000, the simplest R-Gal-GSL, is consid-
ered as a novel anticancer agent, acting through stimula-
tion of the immune system. The first clinical trial with
KRN7000, which included pharmacokinetic studies, has
recently been completed.8
As usual in the procedure we set up for GSLs, structure
elucidation of the new GSL 1a was achieved by extensive
2D spectroscopic analysis performed on its peracetate 1b,
supplemented by microscale degradation. One key point
of our procedure is the analysis of vicinal 1H-1H coupling
constants for sugar stereochemical assignments. How-
ever, in complex molecules such as 1b, characterized by
1
extensive signal overlapping in the H NMR spectrum,
this information can be hardly accessible using standard
NMR experiments, particularly if the overlapped signals
are coupled to each other. In this paper, we show that
1
this problem can be overcome using a J CH-resolved
spectrum obtained from a 13C-coupled HMQC experi-
ment, and apply this method to the structure elucidation
of clarhamnoside.
Resu lts a n d Discu ssion
(1) Part 12: Costantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni,
A. Eur. J . Org. Chem. 2003, 1433-1437.
A. clathrodes was extracted, in sequence, with MeOH
and CHCl3, and the extract was partitioned between
water and n-BuOH. The organic layer was subjected to
chromatography through an RP-18 column and then
through a SiO2 column. The fraction containing glycolip-
(2) Costantino, V.; Fattorusso, E.; Mangoni, A.; Aknin, M.; Gaydou,
E. M. Liebigs Ann. Chem. 1994, 1181-1185.
(3) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron
1994, 50, 2771-2784.
(4) Costantino, V.; Fattorusso, E.; Mangoni, A. Liebigs Ann. 1995,
1471-1475.
(5) Costantino, V.; Fattorusso, E.; Mangoni, A. Liebigs Ann. 1995,
2133-2136.
(8) Crul, M.; Mathot, R. A. A.; Giaccone, G.; Punt, C. J . A.; Rosing,
H.; Hillebrand, M. J . X.; Ando, Y.; Nishi, N.; Tanaka, H.; Schellens, J .
H. M.; Beijnen, J . H. Cancer Chemother. Pharmacol. 2002, 49, 287-
293.
(6) Costantino, V.; Fattorusso, E.; Mangoni, A.; Di Rosa, M.; Ianaro,
A.; Mafia, P. Tetrahedron 1996, 52, 1573-1578.
(7) Dutronc, N.; Porcelli, S. A. Tissue Antigens 2002, 60, 337-353.
10.1021/jo034865h CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/22/2004
1174
J . Org. Chem. 2004, 69, 1174-1179

Glycolipids from Sponges
several signals of oxymethine and oxymethylene groups
between δ 5.43 and δ 3.52, and two characteristic amide
NH doublets at δ 7.25 and at δ 6.49, suggesting the
presence of an additional amide function other than that
of ceramide, tentatively that of an aminosugar. In addi-
tion, a methyl doublet at δ 1.20 was suggestive of a
6-deoxysugar. The 1H NMR spectrum also showed in the
methyl region a triplet at δ 0.88 (ethyl terminus) and a
doublet at δ 0.86 (isopropyl terminus), whose intensities
were not in an integral ratio with respect to those of other
signals in the spectrum. This showed that the alkyl chain
mixture differed not only in the length but also in the
branching of the alkyl chains.
The ceramide part of the molecule was characterized
as composed of a 4-hydroxysphinganine and a 2-hydroxy
acid from NMR data. The amide proton of the ceramide,
which resonated as a doublet at δ 7.25 in CDCl3, was a
useful starting point for the assignment of the sphinga-
nine protons from H2-1 to H2-5 using the COSY spectrum,
whereas the proton of the hydroxy acid at δ 5.13 showed
a correlation peak in the ROESY spectrum with the same
amide doublet.
Structure elucidation of the carbohydrate moiety was
hindered by the strong overlapping of the signals of the
-CH-O protons. In particular, the chemical shifts of
several pairs of mutually coupled signals were nearly
coincident, so that evaluation of the stereochemistry of
the sugars based on coupling constant analysis was
1
impossible. Measurement of H NMR spectra in three
different deuterated solvents (CDCl3, C6D6, and acetone-
d6) was conducted in search of the best separation of
signals. Acetone-d6 was selected as the best solvent for
the subsequent experiments, although some overlapped
signals were still observed.
The presence of four sugar units was revealed by the
four anomeric carbon signals in the 13C NMR spectrum
of 1b at δ 100.4 (C-1′′′), 99.8 (C-1IV), 99.4 (C-1′), and 96.1
(C-1′′). The heteronuclear chemical shift correlation
HMQC NMR spectrum allowed the assignment of the
anomeric protons, which are not readily discernible in
1
the H NMR spectrum, as four doublets at δ 5.30 (J )
3.5 Hz, H-1′′), δ 5.15 (J ) 3.5 Hz, H-1′), δ 4.92 (J ) 8.4
Hz, H-1′′′), and δ 4.88 (J ) 1.2 Hz, H-1IV), respectively.
Combined use of the COSY, HOHAHA, and HMQC
spectra led to the identification of all the protons and
carbon atoms of the four sugars, which included one
N-acetylaminohexose, one 6-deoxyhexose, and two hex-
oses. Information on the position of the acetoxy groups
(the free hydroxyl groups in the native glycolipid) was
provided by chemical shifts, because acetoxymethine
protons are considerably more deshielded than the other
oxymethine protons (typical chemical shift ranges are δ
4.7-5.7 and 3.5-4.5, respectively). Due to overlap, the
multiplicity of some signals could only be determined
from the relevant cross-peak in the phase-sensitive
HMQC spectrum, exploiting the larger chemical shift
dispersion of the 13C nucleus. Even at a relatively low
digital resolution (0.90 Hz), the large axial-axial cou-
plings were always well visible.
ids was acetylated, and purified by repeated HPLC on
SiO2 columns, using n-hexane/EtOAc and n-hexane/
i-PrOH as eluents, and gave 4.3 mg of clarhamnoside
peracetate (1b). Native 1a was obtained by deacetylation
of compound 1b with a 0.02 M solution of MeONa in
MeOH.
As usual for GSLs from Agelas, compound 1a is
composed of a very complex mixture of homologues,
which could not be further separated. Thus, the ESI
mass spectrum showed a series of sodiated pseudomo-
lecular ion peaks at m/z 1393, 1379, 1365, 1351, 1337,
and 1323, in accordance with the molecular formula
C63H118N2O23 + nCH2 (n ) 0-5). A high-resolution
measurement performed on the most abundant ion
at m/z 1351.8489 confirmed the molecular formula
C66H124N2NaO23 for the dominant homologue.
The sugar linked to the ceramide is an R-galactopy-
ranose, as shown by the following evidence: the upfield
chemical shifts of H-5′ (δ 4.32) indicated a pyranose
sugar, while the upfield chemical shift of H-2′ (δ 4.15)
showed that this pyranose is glycosylated at C-2′. The
1
A preliminary H analysis of peracetyl derivative 1b
in CDCl3 showed appropriate signals of a glycosphin-
golipid: the intense aliphatic chain signal at δ 1.24,
J . Org. Chem, Vol. 69, No. 4, 2004 1175

Costantino et al.
galacto configuration was evinced by the mutual large
axial-axial couplings (J ) 12 Hz) for H-2′ and H-3′ (δ
5.25), while the small coupling constants of H-4′ (δ 5.42,
dd, J ) 3.3 and 1.1 Hz) showed its equatorial orientation.
To define the orientation of H-5′, we used an ROESY 2D
NMR experiment, in which a correlation peak between
H-5′ and H-3′ pointed to a 1-3 diaxial relationship
between the two protons. Finally, the R-stereochemistry
of the glycosidic linkage was revealed by the small
coupling constant of H-1′ (δ 5.17, d, J ) 3.7 Hz). The
linkage of this sugar directly to the ceramide was shown
by the prominent cross-peak of H-1′ with H-1a and H-1b
in the ROESY spectrum and by the correlation peak of
H-1′ with C-1 in the HMBC spectrum.
F IGURE 1. Partial plot of the 13C-coupled HMQC spectrum
of 1b. The top trace is the section at δ 69.7 of the spectrum,
IV
which was used to measure coupling constants of H-3
.
The structure of the second sugar, also an R-galacto-
pyranoside, was determined in a similar way. This sugar
is linked to the first one, as shown by an ROESY
correlation between H-1′′ (δ 5.30) and H-2′, and is
glycosylated at C-6′′, as shown by the upfield chemical
shift of the protons at C-6′′, both below δ 4.0, as well as
by the correlation peak between H-1′′′ (δ 4.92) and C-6′′
(δ 65.3) in the HMBC spectrum.
The third sugar of the chain is a 2-acetamido-2-deoxy-
â-galactopyranoside. The 8.3 Hz coupling constant be-
tween H-1′′′ (δ 4.92) and H-2′′′ (δ 3.66) indicated the axial
nature of these protons. The presence of an acetamido
rather than an acetoxy group at position 2′′′ was sug-
gested by the upfield chemical shifts of H-2′′′ and C-2′′′
(δ 54.5), and by the presence of a D2O exchangeable
doublet at δ 7.34, coupled with H-2′′′. The large mutual
coupling between H-2′′′ and H-3′′′ (11.1 Hz) showed their
axial orientation, while the ROESY correlation of H-3′′′
with H-5′′′ (δ 4.00) demonstrated the axial nature of
H-5′′′. Finally, the equatorial orientation of H-4′′′ (δ 5.40)
was evident from its multiplicity and coupling constants
(br d, J ) 3.3 Hz). The upfield chemical shift of H-3′′′
(δ 4.45) and the HMBC correlation between H-1IV (δ 4.88)
and C-3′′′ (δ 75.5) evidenced the linkage of this sugar with
the last one of the chain.
TABLE 1. 1H a n d 13C NMR Da ta of 1b (Aceton e-d 6)
position
1
δH (mult, J (Hz))
δC (mult)
a
b
3.92 (dd, 11.4, 6.2)
3.80 (dd, 11.4, 3.9)
4.41 (m)
70.5 (CH2)
2
49.9 (CH)
2-NH
3
4
5
6
1′
2′
3′
4′
5′
6′
7.66 (d, 9.1)
5.22 (br d, 9)d
5.02c
73.2 (CH)
73.5 (CH)
32.9 (CH2)
26.0 (CH2)
99.4 (CH)
72.5 (CH)
69.4 (CH)
69.4 (CH)
68.1 (CH)
62.5 (CH2)
1.83 (m)
1.41c
5.15 (d, 3.5)
4.15 (dd, 12, 4)d
5.25 (br d, 12)d
5.42 (dd, 3.3, 1.2)
4.32 (br t, 6.8)
4.15c
a
b
4.06 (dd, 11.4, 6.8)
5.30 (d, 3.5)
5.15 (dd, 12, 4)d
5.24c
1′′
2′′
3′′
4′′
5′′
6′′
96.1 (CH)
68.8 (CH)
68.8 (CH)
68.9 (CH)
68.5 (CH)
65.5 (CH2)
5.47 (dd, 3.0, 1.1)
4.38c
a
b
3.87 (t, 8.7)
3.65c
1′′′
2′′′
4.92 (d, 8.4)
100.4 (CH)
54.5 (CH)
3.66c
2′′′-NH
3′′′
4′′′
5′′′
6′′′
7.34 (d, 8.1)
4.45 (dd, 11.1, 3.3)
5.40 (br d, 3.3)
4.00c
75.5 (CH)
70.2 (CH)
72.3 (CH)
63.3 (CH2)
The remaining spin system belonged to a rhamnose.
The doublet at δ 1.12 (H3-6IV) was coupled with the signal
of H-5IV (δ 4.03), which was in turn coupled with that of
H-4IV (δ 5.00), which appeared as a well-resolved triplet
(J ) 11.0 Hz). Therefore, H-3IV (δ 5.09) and H-5IV are
both axially oriented. Unfortunately, the nearly coinci-
dent chemical shifts of H-2IV (δ 5.10) and H-3IV prevented
us from measuring their coupling constants. Even the
HMQC spectrum was not useful in this case, because the
two strongly coupled protons gave rise to distorted
multiplets. This left the orientation of H-2IV, as well as
that of H-1IV, undetermined.
a
b
4.14c
4.00c
1IV
2IV
3IV
4IV
5IV
6IV
1V
4.88 (d, 1.2)
5.10 (br d, 3)e
5.09 (dd, 11, 3)e
5.00 (t, 11)d
4.03c
99.8 (CH)
71.0 (CH)
69.7 (CH)
71.5 (CH)
68.1 (CH)
17.9 (CH3)
170.8 (C)
75.1 (CH)
29.3 (CH2)
1.12 (d, 6.2)
2V
4.98c
1.73 (m)
1.62 (m)
3V
a
b
a
1
Additional H signals: δ 2.152 (3H, Ac), 2.143 (3H, Ac), 2.132
(3H, Ac), 2.129 (3H, Ac), 2.106 (3H, Ac), 2.088 (3H, Ac), 2.081 (3H,
Ac), 2.041 (3H, Ac), 2.023(6H, 2 Ac), 2.017 (3H, Ac), 1.996 (3H,
Ac), 1.927 (3H, Ac), 1.922 (3H, Ac), 1.916 (3H, Ac), 1.25 (broad
band, alkyl chain protons), 0.87 (t, J ) 7.0 Hz, n-chain Me groups),
To solve this problem, a 13C-coupled HMQC spectrum
was obtained (Figure 1). In this experiment, each cor-
relation peak is split into two correlation peaks along the
1
1H dimension by the large J CH coupling constant (about
b
0.86 (d, J ) 6.5 Hz, isochain Me groups). Additional 13C signals:
150 Hz for the oxymethine protons). In particular, the
cross-peak of H-3IV was split into two peaks centered at
δ 5.22 and 4.91 on our 500 MHz spectrometer, which
were completely resolved from all other signals of the
sugar. Consequently, the signal of H-3IV appeared as a
well-defined first-order multiplet in this spectrum, with
only one large axial-axial coupling constant. The orien-
tation of H-2IV was therefore determined to be equatorial,
and the 6-deoxysugar was established to be a rhamnose.
δ 171.6-170.4 (several C, Ac carbonyl groups), 32.9 (CH2, ω-2),
26.5 (CH2, C-4V), 23.5 (CH3, NAcGal Ac methyl group), 23.5 (CH2,
ω-1), 23.1 (CH3, isochain Me groups), 21.8-21.5 (several CH3, Ac
methyl groups), 14.5 (CH3, ω). c Submerged by other signals.
d
Coupling constants were measured from the HMQC NMR
experiment. e Coupling constants were measured from the 13C-
coupled HMQC NMR experiment.
Finally, the anomeric configuration of the rhamnose
was determined on the basis of the coupling constant
1176 J . Org. Chem., Vol. 69, No. 4, 2004

Glycolipids from Sponges
TABLE 2. F a tty Acyl Com p osition of 1a
TABLE 3. Sp h in ga n in e Com p osition of 1a
between H-1IV and C-1IV, which was also provided by the
13C-coupled HMQC experiment. It is known that the 1J CH
coupling constant of an axial anomeric proton is ap-
proximately 160 Hz, while that of an equatorial anomeric
proton is ∼170 Hz.7 The measured coupling constant
between H-1IV and C-1IV (J ) 172 Hz) clearly pointed to
the R anomeric configuration of the rhamnose.
Once the structure of the peracetyl derivative 1b was
1
assessed, the assignment of the H and 13C NMR reso-
nances of the natural GSL 1a became a feasible task.
This was performed on the basis of the COSY, HOHAHA,
and HMQC NMR spectra, and is reported in Table 1. The
13C assignment was particularly useful to confirm the R
anomeric configuration of the rhamnose, because reso-
nances of oxymethine carbon atoms of this sugar were
all within 1 ppm from those reported for known R-rham-
nopyranosides, while they were significantly different (∆δ
up to 3.2 ppm) from those of â-rhamnopyranosides.9
The composition in fatty acids and sphingoid bases of
the ceramide part of compound 1a was established by
degradation of a small amount of sample as previously
reported.4 The sample was subjected to acidic metha-
nolysis, and the products, consisting of methyl glycosides,
sphinganines, and fatty acid methyl esters were sepa-
rated chromatographically. Fatty acid methyl esters were
directly analyzed by GC-MS, and were identified as
unbranched 2-hydroxy fatty acid by comparison of their
retention times and mass spectra with those of authentic
samples (Table 2). The absolute configuration at C-2 of
the methyl esters was determined as R on the basis of
the CD spectrum, showing a negative Cotton effect at
λmax ) 212 nm (∆ꢀ ) -1.3), which is a general feature of
the 2(R)-hydroxy fatty acid derivatives.10
The fraction composed of 4-hydroxysphinganines was
divided into two portions. One portion was treated with
KMnO4/NaIO4 to convert 4-hydroxysphinganines to car-
boxylic acids with three less carbon atoms, which were
methylated with CH2N2 and analyzed by GC-MS. The
results are reported in Table 3, in the form of structures
of the corresponding sphinganines. The other portion was
perbenzoylated with benzoyl chloride in pyridine, and
purified by HPLC. The ribo relative configuration of the
benzoylated sphinganines was determined by recording
1
their H NMR spectrum, which was identical (apart from
the methyl region) to that of an authentic sample of
D-ribo-phytosphingosine perbenzoate (5). Their absolute
configuration was deduced from the CD spectrum in
MeCN solution, which matched that of compound 5
recorded in the same solvent.
The methyl glycosides were used to determine the
absolute configuration of the four sugars, which resulted
to be D for galactose and N-acetylgalactosamine and L
(9) Agrawal, P. K.; J ain, D. C.; Gupta, R. K.; Thakur, R. S.
Phytochemistry 1985, 24, 2479-2496.
(10) Cymerman., C. J .; Pereira, W. E., J r. Tetrahedron 1970, 26,
3457-3460.
J . Org. Chem, Vol. 69, No. 4, 2004 1177

Costantino et al.
chiral columns, have been preferred. In our hand, this
method appears to be effective and sensitive, and fits well
with the microscale degradation procedure needed for
analysis of the alkyl chains of ceramide.
Exp er im en ta l Section
Collection , Extr a ction , a n d Isola tion . Specimens of A.
clathrodes were collected in the summer of 2000 along the coast
of Grand Bahamas Island (Sweetings Cay) and identified by
Prof. M. Pansini (University of Genoa). They were frozen
immediately after collection and kept frozen until extraction.
Reference specimens were deposited at the Istituto di Zoologia,
University of Genoa, Italy. The sponge (160 g of dry weight
after extraction) was homogenized and extracted with metha-
nol (3 × 1 L) and then with chloroform (3 × 1 L); the combined
extracts were partitioned between H2O and n-BuOH. The
organic layer was concentrated in vacuo and afforded 34.9 g
of a dark brown oil, which was chromatographed on a column
packed with RP-18 silica gel. A fraction eluted with CHCl3
(7.8 g) was further chromatographed on a SiO2 column, giving
a fraction [857 mg, eluent EtOAc/MeOH (7:3)] mainly com-
posed of glycolipids. This fraction was peracetylated with Ac2O
in pyridine for 12 h. The acetylated glycolipids were subjected
to HPLC separation on a SiO2 column [eluent n-hexane/EtOAc
(2:8)], thus affording a mixture (51 mg) containing 1b and
other glycolipids. Further normal-phase HPLC purification
[eluent n-hexane/i-PrOH (85:15)] gave 4.3 mg of clarhamnoside
1b.
for rhamnose, as follows. Authentic samples of D-galac-
tose, N-acetyl-D-galactosamine, and L-rhamnose were
subjected to acidic methanolysis in the same conditions
as compound 1a , followed by perbenzoylation with ben-
zoyl chloride in pyridine. After HPLC purification, the
major reaction products, methyl tri-O-benzoyl-R-L-rham-
nopyranoside (6), methyl tetra-O-benzoyl-R-D-galactopy-
ranoside (7), and methyl tri-O-benzoyl-2-deoxy-2-benza-
mido-R-D-galactopyranoside (8), were identified on the
1
basis of their H NMR spectra.
The methyl glycoside mixture from methanolysis of
compound 1a was also subjected to benzoylation, and the
reaction mixture was separated on HPLC. The chromato-
gram contained three peaks, which were collected and
identified (apart from their absolute configuration) as
compounds 6-8 on the basis of their retention times.
Because sugar benzoates contain several exciton-coupled
chromophores, CD spectroscopy was used to elucidate the
absolute configuration of compounds 6-8. The CD spec-
trum of each glycoside from clarhamnoside 1b was
recorded, and matched that of the corresponding syn-
thetic glycoside, thus showing the absolute stereochem-
istry of glycosides from 1b and reference glycosides to
be the same.
Cla r h a m n osid e P er a ceta te (1b). The peracetylated de-
rivative 1b, [R]D25 ) +36 (c ) 0.43 in CHCl3), was obtained as
a colorless oil. 1H and 13C NMR: Table 1. Composition in fatty
acids: Table 2. Composition in sphinganines: Table 2.
Dea cetyla tion of 1b. Compound 1b (3.0 mg) was dissolved
in 950 µL of MeOH, and 50 µL of a 0.4 M solution of MeONa
in MeOH was added. The reaction was allowed to proceed for
18 h at 25 °C, 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
2.1 mg of the native glycosphingolipid 1a (quantitative yield).
25
Cla r h a m n osid e (1a ). White solid. [R]D ) +25 (c ) 0.20
in MeOH). HRESIMS (positive ion mode, MeOH): m/z
1351.8489 ([M + Na]+, C66H124N2NaO23 gives 1351.8442.
ESIMS (positive ion mode, MeOH): m/z 1393, 1379, 1365,
1351, 1337, 1323 ([M + Na]+ series). 1H NMR (pyridine-d5):
δ 9.04 (1H, d, J ) 8.5 Hz, NH-2′′′), 8.60 (1H, d, J ) 9.8 Hz,
NH-2), 5.78 (1H, br s, H-1IV), 5.63 (1H, d, J ) 3.0 Hz, H-1′),
5.55 (1H, d, J ) 3.3 Hz, H-1′′), 5.27 (1H, d, J ) 7.8 Hz, H-1′′′),
5.16 (1H, m, H-2), 5.07 (1H, m, H-5′′), 4.92 (1H, m, H-2′′′), 4.73
(overlapped, H-2′), 4.72 (overlapped, H-4′′′), 4.66 (overlapped,
H-2IV), 4.64 (overlapped, H-2V), 4.63 (overlapped, H-3′′′), 4.61
(overlapped, H-5IV), 4.60 (overlapped, H-2′′), 4.58 (overlapped,
H-1a), 4.57 (overlapped, H-3′), 4.49 (overlapped, H-4′), 4.48
(overlapped, H-5′), 4.48 (overlapped, H-3′′), 4.48 (overlapped,
H-3IV), 4.47 (overlapped, H-6′′a), 4.42 (overlapped, H-1b), 4.40
(overlapped, H-3), 4.35 (overlapped, H2-6′′′), 4.34 (overlapped,
H-6′′b), 4.32 (overlapped, H2-6′), 4.27 (overlapped, H-4), 4.27
(overlapped, H-4′′), 4.25 (partially overlapped, t, J ) 9.4 Hz,
H-4IV), 3.95 (1H, m, H-5′′′), 2.19 (1H, m, H-3Va), 2.08 (1H, m,
H-5a), 2.00 (1H, m, H-3Vb), 1.90 (1H, m, H-5b), 1.56 (3H, d,
J ) 6.2 Hz, H-6IV), 1.25 (large band, alkyl chains), 0.86 (n-
chain and isochain Me groups). 13C NMR (pyridine-d5): δ 175.3
(C, C-1V), 104.2 (CH, C-1IV), 102.6 (CH, C-1′′′), 99.0 (CH, C-1′′),
98.1 (CH, C-1′), 79.8 (CH, C-3′′′), 76.8 (CH, C-5′′′), 76.0 (CH,
C-3), 75.6 (CH, C-2′), 74.0 (CH, C-4IV), 73.0 (CH, C-5′), 72.7
(CH, C-4), 72.7 (CH, C-3IV), 72.5 (CH, C-2IV), 72.5 (CH, C-2V),
71.2 (CH, C-4′), 71.2 (CH, C-5′′), 71.0 (CH, C-4′′), 70.4 (CH,
C-2′′), 70.4 (CH, C-3′′), 70.4 (CH2, C-6′′), 70.4 (CH, C-5IV), 70.1
(CH, C-3′), 69.6 (CH, C-4′′′), 68.6 (CH2, C-1), 62.4 (CH2, C-6′),
62.1 (CH2, C-6′′′), 53.6 (CH, C-2′′′), 51.1 (CH, C-2), 35.8 (CH2,
C-3V), 33.8 (CH2, C-5), 30.5-29.5 (several CH2 groups, alkyl
chains), 23.4 (CH3, Ac methyl group), 22.8 (CH3, isochain Me
Con clu sion
The novel R-Gal-GSL 1a is the first example of an
R-Gal-GSL containing the unusual rhamnose unit in the
sugar head. It is a further proof of the ability of sponges
of the genus Agelas to synthesize a variety of unique
GSLs, characterized by an R-galactose as sugar directly
linked to the ceramide.
Structure elucidation of clarhamnoside was made
possible by two NMR methods, which improve the
standard strategy for the NMR analysis of a carbohydrate
chain: 13C-coupled HMQC for J CH-resolved interpreta-
1
tion of the coupling constant of overlapping protons, and
correlation of the one-bond C-H coupling constant of the
anomeric proton to the anomeric configuration at the
glycosidic linkage of a pyranose sugar with an equatorial
proton at C-2.
The absolute stereochemistry of the sugar components
was determined by matching CD spectra of the respective
benzoates with authentic standards. A similar method
was proposed over 10 years ago,11 but since then it has
been used only occasionally, while more sophisticated
derivatizations, as well as HPLC or GC separation on
(11) Kaluarachchi, K.; Bush, C. A. Anal. Biochem. 1989, 179, 209-
215.
1178 J . Org. Chem., Vol. 69, No. 4, 2004

Glycolipids from Sponges
groups), 18.6 (CH3, C-6IV), 14.3 (CH3, n-chain Me groups).
Composition in fatty acids: Table 2. Composition in sphinga-
nines: Table 2.
as above afforded 8 (tR ) 47.2 min) as the major reaction
1
product. H NMR (CDCl3): δ 8.16 (2H, d, J ) 7.8 Hz, benzoyl
ortho protons), 8.03 (2H, d, J ) 7.8 Hz, benzoyl ortho protons),
7.92 (2H, d, J ) 7.8 Hz, benzoyl ortho protons), 7.68 (2H, d,
J ) 7.8 Hz, benzoyl ortho protons), 7.66-7.35 (12 H, overlap-
ping signals, benzoyl protons), 6.51 (1H, d, J ) 9.5 Hz, NH-2),
5.96 (1H, br d, J ) 3.1 Hz, H-4), 5.77 (1H, dd, J ) 10.8 and
3.2 Hz, H-3), 5.16-5.08 (2H, m, H-1 and H-2), 4.60 (1H, dd,
J ) 10.8 and 6.9 Hz, H-6a), 4.54 (1H, br dd, J ) 6.9 and
5.4 Hz, H-5), 4.43 (1H, dd, J ) 10.8 and 5.4 Hz, H-6b), 3.49
(3H, s, OMe). CD (MeCN): λmax ) 238 nm (∆ꢀ ) +46), 221 nm
(∆ꢀ ) -19).
Absolu te Ster eoch em istr y of Meth yl Glycosid es fr om
Com p ou n d 1a . Fraction A from methanolysis of compound
1a was benzoylated with benzoyl chloride (20 µL) in pyridine
(200 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 under
vacuum for 24 h with an oil pump. The residue was purified
by HPLC (column Luna SiO2, 5 µm; eluent n-hexane/i-PrOH,
99:1; flow 1 mL/min). The chromatogram contained three
peaks, which were identified as compounds 6-8 by a com-
parison of their retention times and CD spectra with those of
the respective synthetic glycosides (see the Results and
Discussion for details).
An a lysis of F a tty Acid Meth yl Ester s. Fraction B from
methanolysis of compound 1a was analyzed by GC-MS, and
its components were identified by a comparison of their
retention times and mass spectra with those of authentic
samples. The results are compiled in Table 2. In addition, the
CD spectrum (EtOH) of the mixture was recorded: λmax ) 212
nm (∆ꢀ ) -1.3).
Sph in gan in es: Oxidative Cleavage an d GC-MS An aly-
sis. Fraction C from methanolysis of compound 1a was divided
into two portions. One portion was subjected to oxidative
cleavage with KMnO4/NaIO4 as described,6 and the resulting
carboxylic acids were methylated with diazomethane. The
obtained esters were analyzed by GC-MS, and the results are
compiled in Table 3, expressed in terms of original sphinga-
nines.
Meth a n olysis of 1a . A small amount (300 µg) of 1a was
dissolved in 1 mL of 1 N HCl in 91% MeOH, and the obtained
solution was kept for about 12 h at 80 °C in a sealed tube.
The reaction mixture was dried under nitrogen, and parti-
tioned between CHCl3 and H2O/MeOH (8:2). The aqueous layer
was concentrated to give a mixture of methyl glycosides
(fraction A). The organic layer was concentrated and dissolved
in a small quantity of CHCl3, and the solution was passed
through a SiO2 (70-230 mesh) column. Elution with 15 mL of
1% pyridine in CHCl3 gave a mixture of R-hydroxy acid methyl
esters (fraction B), and subsequent elution with 1% pyridine
in MeOH afforded a mixture of sphinganines (fraction C).
D-r ibo-P h ytop h in gosin e P er ben zoa te (5). (2S,3S,4R)-2-
Amino-1,3,4-octadecanetriol (D-ribo-phytosphingosine, Sigma,
1.0 mg) was benzoylated with benzoyl chloride (50 µL) in
pyridine (500 µL) at 25 °C for 16 h. After quenching with
MeOH, the reaction mixture was dried and purified by HPLC,
giving 1.1 mg of compound 5. 1H NMR (CDCl3): δ 8.04 (2H, d,
J ) 7.9 Hz, benzoyl ortho protons), 8.00 (2H, d, J ) 7.9 Hz,
benzoyl ortho protons), 7.93 (2H, d, J ) 7.9 Hz, benzoyl ortho
protons), 7.83 (2H, d, J ) 7.9 Hz, benzoyl ortho protons), 7.60-
7.34 (12 H, overlapping signals, benzoyl protons), 7.07 (1H, d,
J ) 8.9 Hz, NH-2), 5.76 (1H, dd, J ) 6.7 and 3.7 Hz, H-3),
5.55 (1H, ddd, J ) 8.6, 4.4, and 3.7 Hz, H-4), 5.12 (1H, m,
H-2), 4.69 (1H, dd, J ) 11.8 and 3.5 Hz, H-1a), 4.58 (1H, dd,
J ) 11.8 and 5.8 Hz, H-1b), 1.99 (2H, m, H2-5), 1.42 (2H, m,
H2-6), 1.34-1.16 (large band, alkyl chain), 0.87 (3H, d, J )
6.9 Hz, H3-18). CD (MeCN): λmax ) 234 nm (∆ꢀ ) -7.3), 222
nm (∆ꢀ ) +1.8).
Meth yl Tr i-O-ben zoyl-r-L-r h a m n op yr a n osid e (6). L-
Rhamnose (2.5 mg) was subjected to acidic methanolysis as
described above. The resulting methyl glycosides were ben-
zoylated 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 under vacuum for 24 h
with an oil pump. The residue was purified by HPLC (column
Luna SiO2, 5 µm; eluent n-hexane/i-PrOH, 99:1; flow 1 mL/
min), affording 6 (tR ) 6.0 min) as the major reaction product.
1H NMR (CDCl3): δ 8.10 (2H, d, J ) 7.8 Hz, benzoyl ortho
protons), 7.97 (2H, d, J ) 7.8 Hz, benzoyl ortho protons), 7.83
(2H, d, J ) 7.8 Hz, benzoyl ortho protons), 7.62 (1H, t, J ) 7.5
Hz, benzoyl para proton), 7.52 (1H, t, J ) 7.5 Hz, benzoyl para
proton), 7.49 (2H, d, J ) 7.7 Hz, benzoyl meta protons), 7.42
(1H, t, J ) 7.5 Hz, benzoyl para proton), 7.39 (2H, d, J ) 7.7
Hz, benzoyl meta protons), 7.26 (2H, d, J ) 7.7 Hz, benzoyl
meta protons), 5.82 (1H, dd, J ) 10.1 and 3.4 Hz, H-3), 5.67
(1H, t, J ) 10.0 Hz, H-4), 5.65 (1H, br s, H-1), 4.91 (1H, br s,
H-1), 4.18 (1H, d quartet, J ) 9.9 and 6.3 Hz, H-5), 3.51 (3H,
Sp h in ga n in es: P er ben zoyla tion . The remaining portion
of fraction C was benzoylated as described above and purified
by HPLC (column Luna SiO2, 5 µm; eluent n-hexane/i-PrOH,
99:1; flow 1 mL/min), giving a fraction composed of perben-
1
zoylated sphinganines. H NMR (CDCl3): same spectrum as
for compound 5, except for the methyl region, where signals
at δ 0.87 (t, J ) 7.0, n-chain Me groups) and 0.86 (d, J ) 6.5,
isochain Me groups) were present. CD (MeCN): λmax ) 233
nm (∆ꢀ ) -8.0), 221 nm (∆ꢀ ) +1.5).
Ack n ow led gm en t. This work was a result of a
project sponsored by MIUR PRIN (Italy). We thank Prof.
J . R. Pawlik (University of North Carolina) for giving
us the opportunity to join in the expedition to the
Caribbean Sea, during which the sponge A. clathrodes
was collected, and Prof. M. Pansini (Istituto di Zoologia,
University of Genoa, Italy) for identifying the sponge.
Mass and NMR spectra were recorded at the Centro
Interdipartimentale di Analisi Strumentale, Universita`
di Napoli “Federico II”. The assistance of the staff is
gratefully acknowledged.
s, OMe), 1.38 (3H, d, J ) 6.3 Hz, H3-6). CD (MeCN): λmax
237 nm (∆ꢀ ) +91), 222 nm (∆ꢀ ) -26).
)
Meth yl Tetr a -O-ben zoyl-r-D-ga la ctop yr a n osid e (7). D-
Galactose (2.0 mg) was subjected to acidic methanolysis
followed by benzoylation as described above. HPLC purification
in the same conditions as above afforded 7 (tR ) 7.2 min) as
1
the major reaction product. H NMR (CDCl3): δ 8.07 (2H, d,
J ) 7.8 Hz, benzoyl ortho protons), 8.00 (2H, d, J ) 7.8 Hz,
benzoyl ortho protons), 7.96 (2H, d, J ) 7.8 Hz, benzoyl ortho
protons), 7.77 (2H, d, J ) 7.8 Hz, benzoyl ortho protons), 7.62-
7.36 (12 H, overlapping signals, benzoyl protons), 6.00 (1H,
br d, J ) 3.2 Hz, H-4), 5.97 (1H, dd, J ) 10.8 and 3.2 Hz, H-3),
5.66 (1H, dd, J ) 10.8 and 3.5 Hz, H-2), 5.31 (1H, br d, J )
3.5 Hz, H-1), 4.60 (2H, m, H-5 and H-6a), 4.37 (1H, m, J ) 7.1
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal procedures, 1H NMR and ESI MS spectra of 1a , 1H and
13C NMR spectra and COSY, TOCSY, ROESY, HMQC, 13C-
coupled HMQC, and HMBC 2D NMR spectra of compound 1b,
1H NMR and CD spectra of phytosphingosine benzoate 5 and
of benzoylated methyl glycosides 6-8, and CD spectrum of
2-hydroxy fatty acid methyl esters from 1b. This material is
available free of charge via the Internet at http://pubs.acs.org.
and 5.1 Hz, H-6b), 3.47 (3H, s, OMe). CD (MeCN): λmax
238 nm (∆ꢀ ) +85), 223 nm (∆ꢀ ) -32).
)
Meth yl Tr i-O-ben zoyl-2-ben za m id o-2-d eoxy-r-D-ga la c-
top yr a n osid e (8). 2-Acetamido-2-deoxy-D-galactose (3.0 mg)
was subjected to acidic methanolysis followed by benzoylation
as described above. HPLC purification in the same conditions
J O034865H
J . Org. Chem, Vol. 69, No. 4, 2004 1179