Glycolipids from sponges. Part 17.1 Clathrosides and isoclathrosides, unique glycolipids from the Caribbean sponge Agelas clathrodes

Article abstract of DOI:10.1021/np050331v

Two families of unique glycolipids, clathrosides A-C (2a-4a) and isoclathrosides A-C (5a-7a) were isolated from the Caribbean sponge Agelas clathrodes. Clathrosides and isoclathrosides are glycosides of a very-long-chain alcohol derived from fatty acids, a new class of glycolipids that appears to be characteristic of marine sponges. The six compounds differ in configuration and in the branching of alkyl chains. Stereostructures of the clathrosides were determined by NMR and CD spectroscopy, mass spectrometry, and chemical degradation. Location of the methyl branch on the proper alkyl chain required an exceptional 1-D TOCSY experiment, in which coherence was transferred through as many as 13 vicinal couplings.

Full text of DOI:10.1021/np050331v

J. Nat. Prod. 2006, 69, 73-78
73
Glycolipids from Sponges. Part 17.1 Clathrosides and Isoclathrosides, Unique Glycolipids from
the Caribbean Sponge Agelas clathrodes
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
ReceiVed September 4, 2005
Two families of unique glycolipids, clathrosides A-C (2a-4a) and isoclathrosides A-C (5a-7a) were isolated from
the Caribbean sponge Agelas clathrodes. Clathrosides and isoclathrosides are glycosides of a very-long-chain alcohol
derived from fatty acids, a new class of glycolipids that appears to be characteristic of marine sponges. The six compounds
differ in configuration and in the branching of alkyl chains. Stereostructures of the clathrosides were determined by
NMR and CD spectroscopy, mass spectrometry, and chemical degradation. Location of the methyl branch on the proper
alkyl chain required an exceptional 1-D TOCSY experiment, in which coherence was transferred through as many as
13 vicinal couplings.
Glycolipids are membrane components and occur in all kingdoms
of living organisms, i.e., bacteria, plants, and animals including
man. They are usually divided into three main groups, glycogly-
cerolipids, glycosphingolipids, and isoprenoid glycosides, depending
on their lipid moiety, which can be, respectively, an acylated gly-
cerol, an acylated sphingosine (ceramide), or a terpene alcohol. A
number of glycolipids exist, however, that cannot be classified in
any of these groups. These compounds are made of a glycosyl moi-
ety (one or several saccharide units) linked to the hydroxyl group
of a fatty alcohol or a hydroxy fatty acid, or to the carboxyl group
of a fatty acid (ester linkage). They are typically found in bacteria
and yeasts and frequently possess interesting biological properties.
Porifera are another source of these “atypical” glycolipids: we
recently reported the occurrence in Plakortis simplex of simplexides
(e.g., 1), diglycosides of a very-long-chain secondary alcohol, acting
on the immune system.2 In this paper, we wish to report the isolation
and structure elucidation of the new glycolipids clathrosides A-C
(2a-4a) and their stereoisomers isoclathrosides A-C (5a-7a) from
Agelas clathrodes. Like simplexides, compounds 2a-7a are gly-
cosides of long-chain alcohols, probably derived from the conden-
sation of two fatty acid molecules. However, the aglycone is quite
different in that the glycosylated alcohol function is primary, and
an additional double bond and hydroxyl group are present.
inseparable mixture of peracetylated clathrosides B (3b) and C (4b).
Likewise, reversed-phase HPLC separation of the latter fraction
gave isoclathroside peracetate (5b) and peracetylated isoclathrosides
B (6b) and C (7b) as a mixture.
Results and Discussion
All the peracetates 2b-7b were deacetylated with MeONa/
MeOH to give the respective natural products 2a-7a. The
conditions that we usually employ for glycolipid deacetylation, i.e.,
methanolysis in MeOH/Et3N (8:2) at 50-80 °C for 12-24 h, were
not effective in this case. For example, deacetylation of compound
2b gave only small amounts of compound 2a even at 80 °C, while
the main product of the reaction was the monoacetyl derivative
2c, in which all the acetyl groups on the sugar, but not the acetyl
group on the aglycone, had been removed. This seems to be a
general trend, since in our previous work we have often observed
that deacetylation with this procedure is much faster for two or
more nearby acetoxy groups compared to an isolated one.
The 1H NMR spectrum (pyridine-d5) of clathroside A 2a showed
it to be a glycolipid. The presence of long-chain alkyl groups was
indicated by a large peak at δ l.25, and a series of signals in the
midfield region of the spectrum suggested a carbohydrate partial
structure. However, neither the usual NH doublet around δ 7 of a
Specimens of Agelas clathrodes (Porifera, class Demospongiae,
order Agelasida, family Agelasidae) were collected at Sweeting Cay
(Grand Bahamas Island) and extracted with MeOH and CHCl3.
Following our usual procedure,3 the extract was partitioned between
H2O and n-BuOH, and the organic phase was subjected to sequential
column chromatography with RP-18 and normal silica gel, giving
a crude glycolipid mixture that was acetylated with Ac2O in
pyridine.
Repeated direct-phase HPLC separation of this mixture led to a
fraction (4.9 mg) entirely composed of homologous peracetylated
clathrosides and a fraction (4.3 mg) composed of peracetylated
isoclathrosides. Reversed-phase HPLC separation of the former
fraction gave clathroside peracetate (2b) in the pure form and an
* Corresponding author. Phone: +39-081-678-532. Fax: +39-081-678-
552. E-mail: alfonso.mangoni@unina.it.
10.1021/np050331v CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/24/2005

74 Journal of Natural Products, 2006, Vol. 69, No. 1
Costantino et al.
Table 1. 1H and 13C NMR Data of Clathroside Peracetate 2b and Isoclathroside Peracetate 5b
2b
5b
pos.
δH [mult., J (Hz)]
δ
C (mult.)
δ
H [mult., J (Hz)]
δC (mult.)
1
a
b
4.25 (br d, 12.5)
3.98 (br d, 12.5)
71.7 (CH2)
4.47 (br d, 12.7)
4.12 (br d, 12.7)
64.3 (CH2)
2
3
139.2 (C)
28.3 (CH2)
138.1 (C)
33.5 (CH2)
a
b
a
b
2.21 (m)
1.99 (m)
1.40 (m)
1.27 (m)
2.11 (m)
1.95 (m)
1.43 (m)
1.34 (m)
4
28.3 (CH2)
27.4 (CH2)
5-13, 5′-13′
1.32-1.21
29.9-29.4
31.9 (CH2)
22.7 (CH2)
14.1 (CH3)
127.3 (CH)
70.6 (CH)
34.9 (CH2)
1.32-1.21
29.9-29.4
31.9 (CH2)
22.7 (CH2)
14.1 (CH3)
129.2 (CH)
70.9 (CH)
35.1 (CH2)
14, 14′
15, 15′
16, 16′
1′
1.26 (submerged)
1.28 (submerged)
0.88 (t, 6.8)
1.26 (submerged)
1.28 (submerged)
0.88 (t, 6.8)
5.33 (br d, 9.3)
5.47 (ddd, 9.3, 6.8, 6.8)
1.65 (m)
5.27 (br d, 9.0)
5.37 (ddd, 9.0, 6.8, 6.8)
1.62 (m)
2′
3′
a
b
1.48 (m)
1.40 (m)
4′
1.27 (submerged)
4.47 (d, 8.1)
5.03 (dd, 9.5, 8.1)
5.20 (t, 9.5)
5.08(dd, 9.9, 9.5)
3.64 (ddd, 9.9, 4.0, 2.0)
4.25 (submerged)
4.13 (dd, 12.5, 2.0)
2.09, 2.04, 2.02, 2.02, 2.01
25.2 (CH2)
99.1 (CH)
71.4 (CH)
73.0 (CH)
68.6 (CH)
71.7 (CH)
62.1 (CH2)
1.25 (submerged)
4.53 (d, 8.1)
5.00 (dd, 9.5, 8.1)
5.20 (t, 9.5)
5.12 (dd, 9.9, 9.5)
3.89 (ddd, 9.9, 4.0, 2.3)
4.30 (dd, 12.4, 4.0)
4.18 (dd, 12.4, 2.2)
25.1 (CH2)
97.7 (CH)
71.4 (CH)
73.2 (CH)
68.2 (CH)
71.3 (CH)
61.8 (CH2)
1′′
2′′
3′′
4′′
5′′
6′′
a
b
CH3
CO
Ac’s
21.3-20.5
170.6-169.0
2.08, 2.03, 2.02, 2.02, 1.99
21.3-20.6
170.8-169.2
Scheme 1. Degradation Procedure of Clathroside A (2a)
glycosphingolid nor the characteristic quintet around δ 5.2 of H-2
of an acylated glycerol was present. The high-resolution ESI mass
spectrum of compound 2a showed a prominent pseudomolecular
ion [M + Na]+ at m/z 665.5352, corresponding to the molecular
formula C38H74O7.
The 1- and 2-D NMR experiments directed at the structural
elucidation of clathroside A were performed using the peracetate
2b, to take advantage of the better signal dispersion in the proton
spectrum of this derivative and of the possibility to distinguish
between alkoxymethine and acetoxymethine protons on the basis
of their chemical shifts.3 The 13C NMR spectrum showed the
presence of one anomeric carbon at δ 99.1 (C-1′′), and the relevant
correlation peaks of the proton at δ 1.98 (H2-3) with the carbon
atoms at δ 127.3 (C-1′) and 139.2 (C-2). The HMBC also allowed
us to locate the sugar unit at C-1 on the basis of the correlation
peak between the anomeric proton at δ 4.47 (H-1′′) and C-1 (δ
71.7). The configuration of the double bond of clathroside A was
determined as E by analysis of the ROESY spectrum of compound
2b. Strong correlation peaks were observed between H-2′ and both
protons at C-3 and between H-1′ and the protons at C-1. Finally,
compound 2b showed only a 6H triplet at δ 0.88 in the high-field
region of the 1H NMR spectrum and only one methyl signal (apart
from those of the acetyl groups) at δ 14.1 in the 13C NMR spectrum.
These data indicated that the alkyl chains linked at C-3 and C-3′
were unbranched.
1
anomeric proton at δ 4.47 (H-1′′) in the H NMR spectrum was
identified using the HSQC spectrum. Sequential assignment of the
remaining six sugar protons (four CH and one CH2) was achieved
using the COSY spectrum. This demonstrated that (a) the sugar
was a hexose and (b) it was in the pyranose form, on the basis of
the shielded chemical shift (δ 3.64) of the sole nonacetylated
oxymethine proton, i.e., H-5′′, compared to the other oxymethine
ring protons, all resonating above δ 5.0. The large coupling
constants between H-1′′ and H-2′′, H-2′′ and H-3′′, H-3′′ and H-4′′,
and H-4′′ and H-5′′ (Table 1) showed all these protons to be axial
and, therefore, that the sugar was a â-glucopyranose.
The structure of the aglycone moiety of clathroside A was
established as follows. The 13C NMR spectrum of compound 2b
contained, in addition to the sugar carbon signals, those of one
oxymethylene carbon atom at δ 71.7, one oxymethine carbon atom
at δ 70.6, two sp2 carbon atoms (one CH at δ 127.3 and one C at
δ 139.2) accounting for one trisubstituted double bond, and several
signals of sp3 carbon atoms.
The oxymethylene protons (δ 4.25, H-1a and δ 3.98, H-1b) were
identified using the HSQC spectrum. Using these protons as a
starting point, the COSY spectrum permitted the assignment of, in
sequence, the olefinic proton at δ 5.33 (H-1′, allylically coupled to
the previous protons), the oxymethine proton at δ 5.47 (H-2′), the
aliphatic protons at δ 1.65 and 1.48 (H2-3′), and, finally, protons
resonating in the large band at δ 1.25. Another spin system belonged
to the aglycone and was composed of the allylic methylene protons
at δ 2.21 and 1.99 (H2-3), those at δ 1.40 and 1.27 (H2-4), and,
once again, protons resonating in the band at δ 1.25. The two spin
systems were connected via the HMBC spectrum, which showed
Determination of the length of the two alkyl chains of compound
2a could not be carried out by NMR analysis, but required chemical
degradation. A small amount of compound 2a (100 µg) was
subjected to Lemieux oxidation with KMnO4/NaIO4 in t-BuOH
(Scheme 1). The reaction was expected to cause extensive cleavage
of the functionalized part of the molecule, leading to two molecules
of fatty acid whose carboxyl carbon atoms originated from C-2
and C-2′, respectively. The fatty acids isolated from the reaction
mixture were methylated with CH2N2 and subjected to GC-MS
analysis, allowing us to identify methyl pentadecanoate as the sole
product of the reaction. Accordingly, the alkyl chains have the same
length, as indicated in structure 2a.
To complete the structure elucidation of the aglycone moiety of
clathroside A (2a), only the configuration at C-2′ remained to be
determined. Because of the allylic position of this carbinol, one
obvious possibility was the CD-based method developed by
Nakanishi for determination of the absolute configuration of allylic
benzoates.4 On the other hand, a selective benzoylation at C-2′

Glycolipids from Sponges
Journal of Natural Products, 2006, Vol. 69, No. 1 75
Figure 1. (a) 1H NMR spectrum of peracetylated clathrosides B (3b) and C (4b); (b) 1-D TOCSY, mixing time 600 ms, selective excitation
at δ 2.21, H-3a; the methyl triplet at δ 0.88 (H3-16) is clearly visible. (c) 1-D TOCSY, mixing time 600 ms, selective excitation at δ 1.65,
H-3′a; only the branched chain methyl protons are visible.
appeared difficult, while measurement of the CD spectrum of the
perbenzoate would have been meaningless because of the strong
interactions between the benzoate chromophores on the sugar.
However, the low propensity toward transesterification at C-2′,
described earlier, allowed a simple preparation of the desired
derivative. Compound 2a (400 µg) was treated with benzoyl
chloride in pyridine to give the perbenzoate 2d, which was subjected
to methanolysis with MeOH/Et3N (8:2) at 55 °C for 18 h. The
benzoate groups on the sugar were selectively removed, leading to
the 2′-O-benzoyl derivative 2e (Scheme 1). The CD spectrum of
compound 2e showed a positive Cotton effect at 227 nm, which,
according to Nakanishi’s model,4 implies the S configuration at
C-2′.
additional alkyl group was on the “northern” (C-3/C16) or the
“southern” (C-3′/C16′) alkyl chain, or randomly located on the two
chains.
This goal was achieved through two diagnostic 1-D TOCSY
experiments. We needed to correlate the terminal methyl groups
of the alkyl chains with the protons at C-3 or C-3′, but the presence
of a large number of overlapped methylene signals made a
sequential assignment impossible. The only way to correlate these
protons appeared to be a TOCSY sequence, and even so, coherence
had to be transferred through as many as 13 vicinal couplings for
the experiment to be successful. Therefore, we used a very long
mixing time (600 ms), which turned out to be the best compromise
between the increased coherence transfer obtained as the mixing
time increases and the overall loss of sensitivity due to relaxation
during the mixing time. In the first experiment, selective excitation
of H-3a (δ 2.21) was performed, and only the methyl triplet of the
unbranched chain (δ 0.88) appeared in the spectrum (Figure 1b);
in contrast, when H-3′a (δ 1.65) was excited, only the methyl signals
of the iso and anteiso chains were present in the spectrum (Figure
1c). Therefore, the additional methyl group was in the “southern”
chain, i.e., linked to C-13′ in clathroside B (3a) and its derivatives,
and to C-14′ in clathroside C (4a) and its derivatives.
Because clathrosides (2b-4b) could only be isolated as their
peracetyl derivatives, there was a chance that one or more acetyls
were already present in the natural glycolipids. This possibility was
excluded by subjecting a small amount of the crude glycolipid
fraction from A. clathrodes to acetylation using trideuteroacetic
anhydride instead of acetic anhydride. The isolation procedure used
for 2b-4b was repeated, except that the three homologous
clathrosides were not separated, and a fraction (0.5 mg) of a mixture
of compounds 2f-4f was obtained. The 1H spectrum of the
pertrideuteroacetylated clathrosides was very similar to that of
compounds 2b-4b, but no acetyl methyl singlet was present,
showing that all the acetyl groups had been introduced by the
acetylation reaction.
Finally, the configuration of the sugar was established as D by
subjecting compound 2e to acidic methanolysis with 1 M HCl in
92% MeOH. The resulting methyl glycosides were perbenzoylated
and subjected to HPLC. The chromatogram contained a peak that
was identified as methyl â-D-glucopyranoside (8) by comparison
of its 1H NMR and CD spectra with those of an authentic sample.5
Clathrosides B (3a) and C (4a) could not be separated, not even
as their peracetyl derivatives, and were studied as a mixture. The
high-resolution ESI mass spectrum of the mixture gave a single
[M + Na]+ pseudomolecular ion at m/z 679.5500 (molecular
formula C39H76O7), showing it to be composed of isomers, each
with one additional CH2 compared with clathroside A (2a). The
1H NMR spectrum, performed on the peracetates 3b and 4b, was
very similar to that of compound 2b, except for the methyl region,
where a complex signal was present, whose overall integration
accounted for three methyl groups. The HSQC spectrum revealed
that these signals originated from the overlapping of four methyl
signals: the triplet of an unbranched chain (δH 0.88, δC 14.1), the
doublet of an iso chain (δH 0.86, δC 22.7), and the triplet (δH 0.85,
δC 11.4) and the doublet (δH 0.84, δC 19.2) of an anteiso chain.6
The mixture of 3a and 4a was then subjected to Lemieux
degradation, followed by CH2N2 methylation as described above.
GC-MS analysis of the resulting methyl esters permitted detection
of methyl pentadecanoate (49.8%), methyl 13-methylpentadecanoate
(40.1%), and methyl 14-methylpentadecanoate (10.1%).
After structure elucidation of peracetylated clathrosides A-C
(2b-4b) was completed, the 1H and 13C NMR spectra of the natural
clathrosides (2a-4a) were assigned on the basis of its COSY and
HSQC spectra (see Experimental Section).
The above data indicated that clathrosides B (3a) and C (4a)
differed from clathroside A (2a) only by an additional methyl branch
on, respectively, the third to last or penultimate carbon atom of
one of the two alkyl chains. The ratio between 3a and 4a was
estimated as 4:1 on the basis of the integration of the GC peaks.
However, these data did not allow us to establish whether the
Isoclathroside A (5a) was isomeric with clathroside A (2a), as
shown by the high-resolution ESI mass spectrum, giving an [M +
1
Na]+ ion at m/z 665.5312. The broad features of the H and 13C
NMR spectra of the respective peracetates 5b and 2b were also
the same, with the same number of signals with similar multiplici-
ties; however, the chemical shifts of several protons and carbons

76 Journal of Natural Products, 2006, Vol. 69, No. 1
Costantino et al.
Scheme 2
and related microorganisms. It is worth noting that in the methyl
branched compounds 3a, 4a, 6a, and 7a the methyl branch is located
specifically on one of the two chains. This implies a very high
substrate selectivity of the enzyme responsible for the condensation
step or, alternatively, could suggest that a different biosynthetic
mechanism is operating for the biosynthesis of clathrosides and
isoclathrosides.
Because of the structural similarity between clathrosides and the
immunoactive simplexides (1), the activity of clathroside A (2a)
and isoclathroside A (5a) on the immune system of mammals was
assayed (MLR assay on murine cell and production of cytokines
by human basophils), but the compounds did not show any
significant activity.
Experimental Section
General Experimental Procedures. High-resolution ESIMS spectra
were obtained on a Micromass QTOF Micro mass spectrometer. ESIMS
experiments were performed on an Applied Biosystem API 2000 triple-
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 on a Perkin-Elmer 192 polarimeter using a 10 cm
microcell. CD spectra were recorded on a Jasco J-710 spectrophotometer
using a 1 cm cell. NMR spectra were determined on Varian UnityInova
700 and 500 NMR spectrometers; chemical shifts were referenced to
the residual solvent signal (CDCl3: δH 7.26, δC 77.0; pyridine-d5: δH
of the aglycone were remarkably different (Table 1). Examination
of the COSY, HSQC, and HMBC spectra showed that isoclathroside
A peracetate (5b) had the same planar structure as clathroside A
peracetate (2b) and that the sugar moiety was the same, so that the
difference must be confined in the geometry of the double bond
and/or the configuration at C-2′.
Analysis of the ROESY spectrum demonstrated that the config-
uration of the double bond was indeed different, that is to say Z.
In fact, distinct correlations between one of the protons at C-1 (δ
4.47) and H-2′ and between H-1′ and both protons at C-3 were
present in the spectrum. The configuration at C-2′ was determined
using the same procedure as for compound 2a, leading to the 2′-
O-benzoyl derivative 5e. The CD spectrum of compound 5e showed
a negative Cotton effect at 228 nm, indicating the R configuration
at C-2′; clathroside A and isoclathroside A differed also in the
configuration at C-2′.
Finally, the structures of isoclathrosides B (6a) and C (7a),
isolated as an inseparable mixture, were established in the same
way as was done for clathrosides B and C, on the basis of (a) the
high-resolution ESI mass spectrum ([M + Na]+ ion at m/z 679.5505;
(b) the 13C NMR spectrum, showing additional methyl signals at δ
22.7, 19.2, and 11.4; (c) Lemieux degradation/methylation, which
provided methyl pentadecanoate (53.4%), methyl 13-methylpen-
tadecanoate (36.9%), and methyl 14-methylpentadecanoate (9.7%);
and (d) a 1-D TOCSY experiment, showing that upon selective
excitation at δ 2.11 (H-3a) only the methyl triplet of the unbranched
chain (δ 0.88) appeared in the spectrum, and therefore that the
“northern” chain was unbranched.
1
8.71, 7.56, and 7.19, δC 149.8, 135.3, and 123.4). Homonuclear H
connectivities were determined by COSY experiments. Through-space
1H connectivities were evidenced using a ROESY experiment with a
mixing time of 500 ms. The reverse-detected, gradient-enhanced single-
quantum heteronuclear correlation (HSQC) spectra were optimized for
1
an average JCH of 140 Hz. The gradient-enhanced multiple-bond
heteronuclear correlation (HMBC) experiments were optimized for a
3JCH of 8 Hz. GC-MS spectra were performed on a Hewlett-Packard
5890 gas chromatograph with a mass selective detector MSD HP 5970
MS, a split/splitless injector, and a fused-silica column, 25 m × 0.20
mm HP-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 °C to 280 °C with a slope of 10 °C min-1
;
quantitative determination was based on the area of the GLC peaks.
High-performance liquid chromatographies (HPLC) were achieved on
a Varian Prostar 210 apparatus equipped with an Varian 350 refractive
index detector or a Varian 325 UV detector.
Collection. Specimens of A. clathrodes (Porifera, class Demospon-
giae, order Agelasida, family Agelasidae) were collected by scuba
(depth 23 m) during the third “Pawlik expedition” (July 2000) along
the coast of Sweeting Cay (Grand Bahamas Island) and identified by
Prof. M. Pansini, University of Genova. A reference sample (#6-00)
has been deposited at the Istituto di Zoologia, Genova, Italy. The
samples were stored at -20 °C until extraction.
Extraction and Isolation Procedure. The frozen sponge sample
of A. clathrodes #6-00 (volume of fresh material 2400 mL, dry weight
after extraction 142 g) was blended in MeOH, then extracted in
sequence with MeOH (4 × 2.5 L), MeOH/CHCl3 (2:1, 2.5 L), MeOH/
CHCl3 (1:2, 2.5 L), and CHCl3 (2 × 3 L). The MeOH and MeOH/
CHCl3 extracts were partitioned between BuOH and H2O. The BuOH
phase was concentrated in vacuo and combined with the CHCl3 extract.
The total lipophilic extract (16.2 g) was subjected to reversed-phase
chromatography on a column packed with RP-18 silica gel, by elution
with a gradient of H2O/MeOH and then CHCl3. The fraction eluted
with CHCl3 (5.88 g) was further chromatographed on a column packed
with SiO2, eluted with a gradient of n-hexane/EtOAc (9:1) to MeOH.
The fraction eluted with EtOAc/MeOH (7:3) (364 mg) was composed
only of glycolipids. This fraction was acetylated with Ac2O in pyridine
at 25 °C for 18 h. The acetylated glycolipids were subjected to HPLC
separation on an SiO2 column [eluent: n-hexane/EtOAc (6:4)], thus
Conclusion
Clathrosides and isoclathrosides are a further, structurally dif-
ferent example of a new kind of glycolipid from Porifera, namely
glycosides of alcohols with a very long chain (C32 and C33 in this
case), functionalized near the middle of the chain. An obvious
hypothesis for the biosynthesis of the C32 aglycone of clathroside
A is the coupling of two molecules of a C16 fatty acid. The first
step would be a Claisen-type condensation, followed by reduction
and dehydration (Scheme 2).
A similar biosynthetic pathway has been proposed, and to some
extent demonstrated,7 for mycolic acids (e.g., 9) from mycobacteria

Glycolipids from Sponges
Journal of Natural Products, 2006, Vol. 69, No. 1 77
affording a fraction (50.9 mg) containing peracetylated clathrosides and
isoclathrosides, together with larger amounts of other peracetylated
glycolipids. Further normal-phase HPLC purification of these fractions
[eluent: n-hexane/i-PrOH (97:3)] gave 4.9 mg of a mixture of
peracetylated clathrosides A-C (2b-4b) and 4.3 mg of a mixture of
peracetylated isoclathrosides A-C (5b-7b). Reversed-phase HPLC
separation of the former fraction on an RP-18 column [eluent: MeOH/
H2O (97:3)] gave 1.2 mg of pure clathroside A peracetate (2b) and 1.8
mg of an inseparable mixture of clathroside B peracetate (3b) and
clathroside C peracetate (4b). Likewise, reversed-phase HPLC separa-
tion of the latter fraction under the same conditions gave 0.9 mg of
pure isoclathroside A peracetate (5b) and 1.2 mg of an inseparable
mixture of isoclathroside B peracetate (6b) and isoclathroside C
peracetate (7b).
chains), 28.2 (CH2, C-4), 26.1 (CH2, C-4′), 23.0 (CH2, C-15 and C-15′),
14.3 (CH3, C-16 and C-16′); HRESIMS m/z 665.5312 ([M + Na]+,
calcd for C38H74NaO7 665.5327).
Mixture of isoclathroside B (6a) and isoclathroside C (7a):
1
colorless oil; H NMR (500 MHz, pyridine-d5), same spectrum as 5a,
except for the methyl region, δ 0.86 (6H, m); HRESIMS m/z 679.5505
([M + Na]+, calcd for C39H76NaO7 679.5483).
Oxidative Cleavage of Clathroside A (2a) and Isoclathroside A
(5a). Compound 2a (100 µg) was subjected to oxidative cleavage with
KMnO4/NaIO4 as described,8 and the resulting carboxylic acids were
methylated with CH2N2. After removal of the solvent, the residue was
analyzed by GC-MS and shown to be composed only of methyl
pentadecanoate (tR ) 15.08), identified by comparison of its retention
time and mass spectrum with those of an authentic sample. Oxidation
of isoclathroside A (5a) gave the same results.
Clathroside A peracetate (2b): colorless oil, [R]D25 -9 (CHCl3, c
1
0.1); H and 13C NMR, Table 1.
Oxidative Cleavage of Clathrosides B (3a) and C (4a). A small
portion of the mixture of compounds 3a and 4a (100 µg) was subjected
to oxidative cleavage with KMnO4/NaIO4 as described above. GC-MS
analysis of the resulting methyl esters mixture showed it to be composed
of methyl pentadecanoate (tR ) 15.08, 49.8%), methyl 14-methylpen-
tadecanoate (tR ) 17.10, 9.5%), and methyl 13-methylpentadecanoate
(tR ) 17.34, 40.7%). All the esters were identified by comparison of
its retention time and mass spectrum with those of an authentic sample.
Oxidative Cleavage of Isoclathrosides B (6a) and C (7a). A small
portion of the mixture of compounds 6a and 7a (100 µg) was subjected
to oxidative cleavage with KMnO4/NaIO4 as described above. GC-MS
analysis of the resulting methyl esters mixture showed it to be composed
of methyl pentadecanoate (tR ) 15.12, 53.4%), methyl 14-methylpen-
tadecanoate (tR ) 17.15, 9.7%), and methyl 13-methylpentadecanoate
(tR ) 17.39, 36.9%).
Clathroside A Perbenzoate (2d). Clathroside A (2a, 400 µg) was
benzoylated with benzoyl chloride (10 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, and the crude
reaction product was purified by HPLC (column: Luna SiO2, 5 µ;
eluent: n-hexane/i-PrOH (99:1), flow 1 mL/min, λ 280 nm), giving
pure clathroside A perbenzoate (2d): 1H NMR (CDCl3) δ 8.03 (2H, d,
J ) 7.8 Hz, benzoyl o-protons), 7.99 (4H, d, J ) 7.8 Hz, benzoyl
o-protons), 7.87 (2H, d, J ) 7.8 Hz, benzoyl o-protons), 7.82 (2H, d,
J ) 7.8 Hz, benzoyl o-protons), 7.57-7.26 (15H, m, benzoyl protons),
5.85 (1H, t, J ) 9.7 Hz, H-3′′), 5.66-5.53 (3H, m, H-2′, H-2′′, and
H-4′′), 5.39 (1H, br d, J ) 9.0 Hz, H-1′), 4.80 (1H, d, J ) 7.9 Hz,
H-1′′), 4.47 (1H, dd, J ) 12.2 and 3.0 Hz, H-6′′a), 4.35 (1H, dd, J )
12.2 and 5.6 Hz, H-6′′b), 4.29 (1H, br d, J ) 12.4 Hz, H-1a), 4.08
(1H, br d, J ) 12.4 Hz, H-1b), 3.90 (1H, m, H-5′′), 2.11 (1H, m, H-3a),
1.92 (1H, m, H-3b), 1.70 (1H, m, H-3′a), 1.49 (1H, m, H-3′b).
Isoclathroside A perbenzoate (5d) was prepared as described
above: 1H NMR (CDCl3) δ 8.04 (2H, d, J ) 7.8 Hz, benzoyl o-protons),
7.99 (2H, d, J ) 7.8 Hz, benzoyl o-protons), 7.94 (4H, d, J ) 7.8 Hz,
benzoyl o-protons), 7.90 (2H, d, J ) 7.8 Hz, benzoyl o-protons), 7.82
(2H, d, J ) 7.8 Hz, benzoyl o-protons), 7.60-7.26 (15H, benzoyl
protons), 5.91 (1H, t, J ) 9.7, H-3′′), 5.68 (1H, t, J ) 9.7 Hz, H-4′′),
5.56 (1H, dd, J ) 9.7 and 8.0 Hz, H-2′′), 5.53 (1H, m, H-2′), 5.31
(1H, br d, J ) 9.3 Hz, H-1′), 4.94 (1H, d, J ) 8.1 Hz, H-1′′), 4.72
(1H, dd, J ) 12.2 and 2.8 Hz, H-6′′a), 4.69 (1H, br d, J ) 12.9 Hz,
H-1a), 4.52 (1H, dd, J ) 12.2 and 5.2 Hz, H-6′′b), 4.45 (1H, m, H-5′′),
4.18 (1H, br d, J ) 12.9 Hz, H-1b), 1.91 (1H, m, H-3a), 1.75 (1H, m,
H-3b), 1.65 (1H, m, H-3′a), 1.50 (1H, m, H-3′b).
Mixture of clathroside B peracetate (3b) and clathroside C
peracetate (4b): colorless oil; 1H NMR (500 MHz, CDCl3), same
spectrum as 2b, except for the methyl region, δ 0.88 (3b and 4b, t, J
) 7.0 Hz, H3-16), 0.86 (4b, d, J ) 6.6 Hz, H3-16′ and H3-17′), 0.85
(3b, t, J ) 7.2 Hz, H3-16′), 0.84 (3b, d, J ) 6.5 Hz, H3-17′); 13C NMR
(CDCl3), same signals as for 2b, plus δ 39.1 (4b, CH2, C-14′), 36.6
(3b, CH2, C-13′), 34.4 (3b, CH, C-14′), 27.1 (3b, CH2, C-12′), 22.7
(4b, CH3, C-16′ and C-17′), 19.2 (3b, CH3, C-17′), 11.4 (3b, CH3,
C-16′).
Isoclathroside A peracetate (5b): colorless oil; [R]D25 -12 (CHCl3,
1
c 0.1); H and 13C NMR, Table 1.
Mixture of isoclathroside B peracetate (6b) and isoclathroside
1
C peracetate (7b): colorless oil; H NMR (500 MHz, CDCl3), same
spectrum as 5b, except for the methyl region, which was the same as
in 3b-4b; 13C NMR (CDCl3), same signals as 5b, plus the same
additional signals as in 3b-4b.
Deacetylation of 2b-7b. Compound 2b (1.2 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,
then the reaction mixture was dried under nitrogen and the residue
partitioned between H2O and CHCl3. The organic layer was washed
with H2O and taken to dryness, giving 1.0 mg of clathroside A (2a).
Compounds 3b-7b were deacetylated using the same procedure.
Clathroside A (2a): colorless oil; [R]D25 -6 (CHCl3, c 0.1); 1H NMR
(500 MHz, pyridine-d5) δ 7.28 (1H, d, J ) 4.4 Hz, OH-2′′), 7.16 (1H,
d, J ) 3.0 Hz, OH-3′′), 7.14 (1H, d, J ) 4.0 Hz, OH-4′′), 6.41 (1H, t,
J ) 6.3 Hz, OH-6′′), 6.15 (1H, d, J ) 4.2 Hz, OH-2′), 6.08 (1H, br d,
J ) 8.9 Hz, H-1′), 4.98 (1H, d, J ) 7.8 Hz, H-1′′), 4.83 (1H, m, H-2′),
4.70 (1H, br d, J ) 12.3 Hz, H-1a), 4.57 (1H, m, H-6′′a), 4.40 (2H, m,
H-6′′b and H-1b), 4.25 (2H, m, H-3′′ and H-4′′), 4.11 (1H, m, H-2′′),
3.95 (1H, m, H-5′′), 2.43 (1H, m, H-3a), 2.37 (1H, m, H-3b), 1.92
(1H, m, H-3′a), 1.63 (1H, m, H-3′b), 1.59 (1H, m, H-4a), 1.49 (1H, m,
H-4b), 1.33-1.20 (alkyl chain protons), 0.86 (3H, t, J ) 6.8 Hz, H3-
16 and H3-16′); 13C NMR (pyridine-d5) δ 137.1 (C, C-2), 132.9 (CH,
C-1′), 103.7 (CH, C-1′′), 78.7 (CH, C-3′′), 78.5 (CH, C-5′′), 75.3 (CH,
C-2′′), 72.7 (CH2, C-1), 71.9 (CH, C-4′′), 67.4 (CH, C-2′), 62.9 (CH2,
C-6′′), 38.9 (CH2, C-3′), 32.2 (CH2, C-14 and C-14′), 30.2-29.7 (several
CH2, alkyl chains), 29.1 (CH2, C-4), 28.9 (CH2, C-3), 26.3 (CH2, C-4′),
23.0 (CH2, C-15 and C-15′), 14.3 (CH3, C-16 and C-16′); HRESIMS
m/z 665.5352 ([M + Na]+, calcd for C38H74NaO7 665.5327).
Mixture of clathroside B (3a) and clathroside C (3a): colorless
oil; 1H NMR (500 MHz, pyridine-d5), same spectrum as 2a, except for
the methyl region, δ 0.86 (6H, m); HRESIMS m/z 679.5500 ([M +
Na]+, calcd for C39H76NaO7 679.5483).
Clathroside A 2′-O-benzoate (2e). Compound 2d was subjected to
basic methanolysis by keeping it in 0.5 mL of MeOH/Et3N (8:2) at 55
°C for 18 h. Removal of the solvent and HPLC purification (column:
SiO2, eluent: n-hexane/i-PrOH (85:15), flow 1 mL/min, λ 280 nm)
gave pure compound 2e: CD (MeOH) λmax ) 227 nm (∆ꢀ ) +5.1);
1H NMR (CDCl3) δ 8.03 (2H, br d, J ) 7.8 Hz, benzoyl o-protons),
7.55 (1H, br t, J ) 7.5 Hz, benzoyl p-proton), 7.43 (2H, t, J ) 7.6 Hz,
benzoyl m-protons), 5.73 (1H, m, H-2′), 5.51 (1H, br d, J ) 9.2 Hz,
H-1′), 4.27-4.35 (2H, m, H-1a and H-1′′), 4.04 (1H, br d, J ) 12.9
Hz, H-1b), 3.85 (1H, dd, J ) 12.0 and 3.3 Hz, H-6′′a), 3.75 (1H, dd,
J ) 12.0 and 5.1 Hz, H-6′′b), 3.60-3.47 (2H, m, H-3′′ and H-4′′),
3.38 (1H, t, J ) 8.7 Hz, H-2′′), 3.30 (1H, m, H-5′′).
25
1
Isoclathroside A (5a): colorless oil; [R]D -2 (CHCl3, c 0.1); H
NMR (700 MHz, pyridine-d5) δ 7.26 (1H, d, J ) 3.6 Hz, OH-2′′),
7.18 (1H, br s, OH-3′′ or OH-4′′), 7.11 (1H, br s, OH-4′′ or OH-3′′),
6.53 (1H, t, J ) 5.4 Hz, OH-6′′), 6.06 (1H, d, J ) 4.2 Hz, OH-2′),
5.87 (1H, br d, J ) 8.6 Hz, H-1′), 4.99 (1H, d, J ) 7.8 Hz, H-1′′), 4.97
(1H, m, H-2′), 4.85 (1H, br d, J ) 11.8 Hz, H-1a), 4.62 (br d, J ) 11.8
Hz, H-1b), 4.60 (overlapped, H-6′′a), 4.37 (1H, ddd, J ) 11.6, 5.9,
and 5.9 Hz, H-6′′b), 4.20 (2H, overlapped, H-3′′ and H-4′′), 4.10 (1H,
m, H-2′′), 4.01 (1H, m, H-5′′), 2.50 (1H, m, H-3a), 2.30 (1H, m, H-3b),
1.91 (1H, m, H-3′a), 1.71 (1H, m, H-3′b), 1.55 (2H, m, H2-4), 1.33-
1.20 (alkyl chain protons), 0.86 (3H, t, J ) 6.8 Hz, H3-16 and H3-16′);
13C NMR (pyridine-d5) δ 135.5 (CH, C-1′), 102.5 (CH, C-1′′), 78.7
(CH, C-3′′), 78.4 (CH, C-5′′), 75.2 (CH, C-2′′), 71.9 (CH, C-4′′), 67.4
(CH, C-2′), 65.6 (CH2, C-1), 62.9 (CH2, C-6′′), 39.1 (CH2, C-3′), 35.1
(CH2, C-3), 32.2 (CH2, C-14 and C-14′), 30.2-29.7 (several CH2, alkyl
Isoclathroside A 2′-O-benzoate (5e) was prepared as described
1
above: CD (MeOH) λmax ) 228 nm (∆ꢀ ) -5.5); H NMR (CDCl3)
δ 8.02 (2H, br d, J ) 7.8 Hz, benzoyl o-protons), 7.55 (1H, br t, J )
7.6 Hz, benzoyl p-proton), 7.43 (2H, br t, J ) 7.8 Hz, benzoyl

78 Journal of Natural Products, 2006, Vol. 69, No. 1
Costantino et al.
m-protons), 5.90 (1H, m, H-2′), 5.43 (1H, br d, J ) 9.5 Hz, H-1′),
4.40 (1H, d, J ) 7.8, H-1′′), 4.35 (2H, br s, H-1a and H-1b), 3.95 (1H,
m, sugar proton), 3.82 (1H, m, sugar proton), 3.59 (2H, m, sugar
protons), 3.48 (1H, m, sugar proton), 3.35 (1H, m, H-2′′).
edged. This research project is funded by the Italian Government, MIUR
PRIN (Italy).
Supporting Information Available: 1-D and 2-D NMR spectra
of clathroside A peracetate (2b) and isoclathroside A peracetate (5b),
1H and 13C NMR spectra of clathroside A (2a) and isoclathroside A
(5a), 1H NMR spectra of compounds 2d, 2e, 3b/4b, 5d, 5e, and 6b/7b,
and CD spectra of compounds 2e, 5e, and 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
Absolute Configuration of Glucose. Clathroside A 2′-O-benzoate
(2e) was subjected to acidic methanolysis with 1 M HCl/MeOH. The
reaction mixture was kept for 12 h at 80 °C in a sealed tube, then
dried under nitrogen and partitioned between CHCl3 and H2O/MeOH
(8:2). The aqueous layer, containing methyl glycosides, was dried and
subsequently benzoylated with benzoyl chloride (10 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: SiO2; eluent:
n-hexane/i-PrOH (99:1), flow 1 mL/min). The chromatogram contained
one peak (tR ) 11.8 min), which was identified as methyl 2,3,4,6-
tetra-O-benzoyl-â-D-glucopyranoside (8) by comparison of its 1H NMR
and CD spectra with those of an authentic sample.5 The procedure was
repeated using isoclathroside A 2′-O-benzoate (5e) as substrate, with
the same results.
References and Notes
(1) Part 16: Barbieri, L.; Costantino, V.; Fattorusso, E.; Mangoni, A. J.
Nat. Prod. 2005, 68, 1527-1530.
(2) Costantino, V.; Fattorusso, E.; Mangoni, A.; M. Di Rosa, M.; Ianaro,
A. Bioorg. Med. Chem. Lett. 1999, 9, 271-276.
(3) Fattorusso, E.; Mangoni A. In Progress in the Chemistry of Organic
Natural Products; W. Hertz, W.; Kirby, G. W.; Moore, R. E.;
Steiglich, W.; Tamm, Ch. Eds.; Springer-Verlag: Wien, 1997; Vol.
72, pp 215-301.
(4) Gonnella, N. C.; Nakanishi, K.; Martin, V. S.; Sharpless, K. B. J.
Am. Chem. Soc. 1982, 104, 3775-3776.
(5) Costantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni, A. Eur. J.
Org. Chem. 2005, 368-373.
Acknowledgment. The authors are grateful to Prof. J. R. Pawlik,
UNCW, for inviting them to participate in the third “Pawlik Expedition”
under the NSF project grant entitled “Assessing the Chemical Defenses
of Caribbean Invertebrates”, during which the material was collected,
and to Prof. M. Pansini, University of Genova, for the sponge taxonomy
determination. Mass and NMR spectra were recorded at the “Centro
di Servizi Interdipartimentale di Analisi Strumentale”, Universita` di
Napoli “Federico II”. The assistance of the staff is gratefully acknowl-
(6) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy, 3rd ed.;
VCH: Weinheim, 1987; p 183.
(7) Portevin, D.; de Sousa-d’Auria, C.; Houssin, C.; Grimaldi, C.; Chami,
M.; Daffe, M.; Guilhot, C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
314-319.
(8) Costantino, V.; Fattorusso, E.; Mangoni, A.; M. Di Rosa, M.; Ianaro,
A.; Maffia, A. Tetrahedron 1996, 52, 1573-1578.
NP050331V