Amphiceramide A and B, novel glycosphingolipids from the marine sponge Amphimedon compressed

Article abstract of DOI:10.1002/ejoc.200801230

Glycolipid analysis of the Caribbean sponge Amphimedon compressa has shown it to contain two novel glycosphingoli- pids, amphiceramide A (1a) andB(2a), which possess an unusual A6-phytosphingosine. The saccharide chain of amphiceramide A is composed of a

Full text of DOI:10.1002/ejoc.200801230

FULL PAPER
DOI: 10.1002/ejoc.200801230
Amphiceramide A and B, Novel Glycosphingolipids from the Marine Sponge
Amphimedon compressa[‡]
Valeria Costantino,[a] Ernesto Fattorusso,[a] Concetta Imperatore,[a] Alfonso Mangoni,*[a]
and Roberta Teta[a]
Keywords: Amines / Carbohydrates / Glycolipids / Sponges / Structure elucidation
Glycolipid analysis of the Caribbean sponge Amphimedon
compressa has shown it to contain two novel glycosphingoli-
pids, amphiceramide A (1a) and B (2a), which possess an un-
usual ∆6-phytosphingosine. The saccharide chain of am-
phiceramide A is composed of a β-glucose residue glycosyl-
ated at the 6-position by an N-acetyl-β-glucosamine and has
never been found before in a natural product. The saccharide
chain of amphiceramide B consists of an allolactose
[Gal(1βǞ6)Glc] residue β-linked to the ceramide and is
found here for the first time in a natural glycosphingolipid.
In addition, the sponge contains a new molecular species,
acetamidoglucosyl ceramide (3a), and the known glucosyl
ceramide 4a (halicerebroside A) and melibiosyl ceramide 5a
(amphimelibioside C).
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
In addition, the sponge contains a new molecular species of
acetamidoglucosyl ceramide (3a), and the known glucosyl
ceramide (4a; halicerebroside A)[8] and melibiosyl ceramide
(5a; amphimelibioside C).[3]
Amphiceramide A and B are diglycosyl ceramides with
the same unusual ∆6-phytosphingosine moiety that is pres-
ent in amphimelibioside C. The saccharide chain of am-
phiceramide A (1a), composed of a β-glucose residue gly-
cosylated at the 6-position by an N-acetyl-β-glucosamine,
has never been found before in a natural product. The sac-
charide chain of amphiceramide B (2a) consists of an allol-
actose [Gal(1βǞ6)Glc] residue β-linked to the ceramide
and is found here for the first time in a natural glyco-
sphingolipid.
Introduction
Glycosphingolipids (GSLs) from marine sponges repre-
sent an increasingly rich family of compounds that are sig-
nificantly different from the GSLs found in mammals.[1–7]
The most distinctive structural features of sponge GSLs,
which make them unique among GSLs from animals, can
be summarized as follows. (i) The ceramide is highly hy-
droxylated with an almost constant presence of trihydroxyl-
ated sphingosines (phytosphingosines) and α-hydroxy fatty
acids. (ii) Sponge GSLs often occur as complex mixtures of
homologues, sharing the same polar part and differing in
the ceramide alkyl chains. These mixtures contain high
numbers of methyl-branched (iso and anteiso) and odd-
numbered carbon sphingosines, whereas fatty acids are usu-
ally unbranched. (iii) The oligosaccharide chains show a re-
markable chemical diversity. Unusual structural features,
like the α-anomeric configuration of the first sugar, the gly-
cosylation at the 2-position of the first sugar, or the pres-
ence of hexofuranoses, make many of these saccharide
chains unprecedented in natural glycoconjugates.
The elucidation of the stereostructures of the amphicera-
mides has been accomplished by a combination of extensive
one- and two-dimensional NMR spectroscopy, MS and
MS/MS analysis, and chemical degradation, as described
below.
As a part of our continuing study of glycolipids from
sponges, we have examined the glycolipid composition of
the Caribbean sponge Amphimedon compressa and have
shown it to contain two novel glycosphingolipids, which we
have named amphiceramide A (1a) and B (2a) (Scheme 1).
Results and Discussion
The BuOH-soluble fraction obtained from combined
chloroform and methanol extracts of A. compressa was sub-
jected first to reversed-phase and then to normal-phase sil-
ica gel column chromatography, which gave a fraction
mainly composed of glycolipids. According to our usual
procedure, the fraction was acetylated with Ac2O in pyr-
idine to give a peracetylated fraction, which is easier to pu-
rify by HPLC on an SiO2 column. HPLC separation yielded
six peracetylated glycolipids, each isolated as a mixture of
homologues. Unlike our observations with other sponges,
[‡] Glycolipids from Sponges, 21. Part 20: Ref.[1]
[a] Dipartimento di Chimica delle Sostanze Naturali, Università di
Napoli “Federico II”,
Via D. Montesano 49, 80131 Napoli, Italy
Fax: +39-081-678552
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.
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Glycosphingolipids Amphiceramide A and B
1
acetyl group is part of an amide function. Likewise, the H
NMR spectrum of compound 3d also contained one acetyl
signal, which showed that the natural compound 3a con-
tains an acetamido group. Finally, the peracetylated glyco-
lipids were deacetylated by using MeONa/MeOH (the acet-
amido groups present in compounds 1a and 3a survive
these reaction conditions) to give the corresponding natural
glycolipids 1a–5a.
Amphiceramide A (1a)
The positive-ion ESI mass spectrum of amphiceramide A
(1a) showed a sodiated pseudomolecular ion peak at m/z =
1041, in accord with the molecular formula
C72H120N2NaO24. This was confirmed by a high-resolution
measurement at m/z = 1041.7183. The ESI mass spectrum
of the peracetyl derivative 1b showed an [M + Na]+ pseudo-
molecular ion peak at m/z = 1419, which indicates the pres-
ence of nine additional acetyl groups. Compound 1b was
used for the subsequent NMR analysis.
1
The H NMR spectral features of compound 1b showed
it to be a glycosphingolipid, that is, the NH doublet at δ =
6.94 ppm of the ceramide amide function, a series of over-
lapped signals in the region between δ = 5.4 and 3.4 ppm,
nine signals at δ ≈ 2.0 ppm from the acetyl groups, an in-
tense signal at δ = 1.24 ppm indicative of a long aliphatic
chain, and a methyl (6 H) triplet at δ = 0.88 ppm as the
sole signal in the high-field region of the spectrum, which
indicates that the alkyl chains of the ceramide are both un-
branched. A second amide NH doublet at δ = 6.48 ppm
suggested the presence of an amino sugar in the molecule.
The absence of the characteristic triplet of the fatty acid
α protons at δ = 2.3 ppm suggested the presence in the cer-
amide moiety of an α-hydroxy acid, as usually found in
GSLs from marine sponges. In addition, the presence of
two methine groups resonating at δ = 134.9 and 123.7 ppm
evinced from the 13C NMR and HSQC spectra indicated a
disubstituted C=C double bond. Starting from the NH
doublet at δ = 6.94 ppm (2-H), the COSY spectrum allowed
sequential assignment of all the protons of the sphingosine
Scheme 1. Glycosphingolipids from Amphimedon compressa. The
area of each circle is proportional to the amount of the relevant
compound in the sponge.
these mixtures were relatively simple, with one single major (Table 1). In particular, the scalar coupling of the two dia-
1
homologue in each fraction, as judged by the MS and H stereotopic methylene protons with signals at δ = 2.37 and
NMR data. Therefore, the six glycolipid mixtures were sub- 2.24 ppm (5-H2) with the oxymethine proton with signal at
jected to reversed-phase HPLC separation, which yielded δ = 4.94 ppm (4-H) and the olefinic proton with signal at δ
the six pure peracetylated glycolipids 1b–5b (Scheme 1).
= 5.24 ppm (6-H) located the double bond on the sphingo-
To distinguish any acetyl group possibly present in the sine at the 6-position. In addition, the coupling constant
natural glycolipid from those introduced during the acety- between 6-H and 7-H (15.2 Hz) indicated the (E) stereo-
lation reaction, the isolation procedure was repeated by chemistry of the double bond. The HMBC correlation
using deuteriated acetic anhydride in the acetylation step, peaks for the coupling of 2-H with C-1ЈЈЈ (δ = 170.2 ppm)
which gave the pertrideuterioacetylated derivatives 1c–5c. and C-2 (δ = 48.9 ppm) confirmed the amide linkage be-
1
The H NMR spectra of compounds 2c, 4c, and 5c were tween the sphingosine and the fatty acid.
identical to those of the corresponding peracetylated deriv-
The key step in the structure elucidation of a GSL is
atives, except that no acetyl methyl singlets were present in the identification of the nature of the sugars composing the
the spectrum. This showed that all the acetyl groups came saccharide chain. This can be achieved by collecting data
1
from the acetylation reaction. The H NMR spectrum of from COSY, TOCSY, and HSQC experiments to identify
compound 1c showed one acetyl methyl singlet, indicating the spin system of each sugar unit and to assign the protons
that one acetyl group is present in the natural compound within each spin system. If the sugars are in the pyranose
1a. The subsequent structure elucidation showed that this form, the nature of each sugar can then be easily identified
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FULL PAPER
1
Table 1. H and 13C NMR spectroscopic data for 1b and 2b (CDCl3).
Position
1b
2b
δH [ppm] (mult., J [Hz])
δC [ppm] (mult.)
δH [ppm] (mult., J [Hz])
δC [ppm] (mult.)
1a
1b
2
2-NH
3
4
5a
3.80 (dd, 10.9, 4.0)
3.69 (dd, 10.9, 4.2)
4.28[a]
65.8 (CH2)
3.90[a]
66.9 (CH2)
3.61[a]
48.9 (CH)
–
72.5 (CH)
72.4 (CH)
32.7 (CH2)
4.32 (m)
48.4 (CH)
–
72.1 (CH)
72.6 (CH)
32.3 (CH2)
6.94 (d, 8.4)
5.04 (dd, 6.2, 4.9)
4.94 (ddd, 8.9, 4.9, 3.5)
2.37 (m)
6.88 (d, 8.9)
5.09 (dd, 7.3, 4.1)
4.91 (ddd, 9.3, 4.1, 3.6)
2.36 (m)
5b
2.24[a]
2.24[a]
6
7
8
5.24 (ddd, 15.2, 6.9, 6.9)
5.48 (ddd, 15.2, 6.8, 6.8)
1.96[a]
123.7 (CH)
134.9 (CH)
32.6 (CH2)
29.7 (CH2)
31.9 (CH2)
22.7 (CH2)
14.1 (CH3)
100.1 (CH)
71.2 (CH)
72.8 (CH)
68.5 (CH)
73.6 (CH)
67.6 (CH2)
5.26 (ddd, 15.2, 6.7, 6.7)
5.47 (ddd, 15.2, 6.8, 6.8)
1.95 (m)
124.1 (CH)
134.8 (CH)
32.7 (CH2)
29.8 (CH2)
32.0 (CH2)
22.8 (CH2)
14.2 (CH2)
100.6 (CH)
71.5 (CH)
72.9 (CH)
69.0 (CH)
73.4 (CH)
68.0 (CH2)
9–15, 4ЈЈЈ–19ЈЈЈ
1.24[a]
1.24[a]
16, 20ЈЈЈ
17, 21ЈЈЈ
18, 22ЈЈЈ
1Ј
2Ј
3Ј
4Ј
5Ј
6Јa
6Јb
1ЈЈ
2ЈЈ
2ЈЈ-NH
2ЈЈ-NAc
1.25[a]
1.24[a]
1.28[a]
1.28[a]
0.88 (t, 6.8)
0.88 (t, 6.8)
4.46 (d, 8.0)
4.85 (dd, 9.6, 8.0)
5.18 (t, 9.5)
4.44 (d, 8.0)
4.84 (dd, 9.7, 8.0)
5.15 (t, 9.6)
4.93 (t, 9.9, 9.5)
3.64 (m)
4.98 (dd, 10.0, 9.5)
3.65 (ddd, 10.0, 5.5, 1.7)
3.95[a]
3.88 (dd, 11.2, 1.7)
3.52 (dd, 11.9, 5.5)
4.61 (d, 8.4)
3.95[a]
3.60[a]
101.4 (CH)
54.2 (CH)
–
23.1 (CH3)
170.7 (C)
72.7 (CH)
68.7 (CH)
71.8 (CH)
62.1 (CH2)
4.52 (d, 8.0)
5.17 (t, 10.5, 8.0)
–
101.3 (CH)
68.8 (CH)
–
6.48 (d, 8.9)
1.95
–
–
–
–
–
3ЈЈ
4ЈЈ
5ЈЈ
6ЈЈa
6ЈЈb
1ЈЈЈ
2ЈЈЈ
5.24 (dd, 10.5, 9.4)
5.06 (dd, 10.0, 9.4)
3.70[a]
4.28 (dd, 12.3, 4.7)
4.10 (dd, 12.3, 2.3)
–
5.04 (dd, 10.5, 3.4)
5.38 (dd, 3.4, 1.2)
3.92 (ddd, 6.7, 6.7, 1.2)
4.20 (dd, 11.3, 6.5)
4.10 (dd, 11.3, 6.9)
–
71.0 (CH)
67.2 (CH)
70.8 (CH)
61.3 (CH2)
170.2 (C)
74.1 (CH)
31.7 (CH2)
169.8 (C)
74.1 (CH)
31.9 (CH2)
5.11 (dd, 7.5, 4.6)
1.84 (m)
5.13 (dd, 7.3, 4.8)
3ЈЈЈ
1.84[a]
OAc groups
2.22, 2.10, 2.09, 2.06, 2.04,
21.1–20.6 (CH3)
2.22, 2.15, 2.09, 2.07, 2.06,
2.04, 2.04, 2.02, 1.98, 1.98
–
21.1–20–7 (CH3)
2.03, 2.02, 2.01, 1.98
–
171.1–169.3 (C)
170.7–169.3
[a] Overlapped signal.
by coupling-constant analysis. Finally, the positions of the = 54.2 ppm) and 2ЈЈ-H (δ = 3.95 ppm) are indicative of an
sugar–ceramide and sugar–sugar linkages are identified on N-linked methine carbon atom, and this was confirmed by
the basis of HMBC and/or ROESY data.
the coupling of 2ЈЈ-H with the NH doublet at δ = 6.48 ppm.
According to this protocol, the two anomeric protons Coupling-constant analysis also showed for this sugar that
(signals at δ = 4.61 and 4.46 ppm) were identified from their the ring protons are all axial, and therefore the sugar resi-
HMQC correlation peaks with the corresponding anomeric due was identified as a 2-amino-2-deoxyglucopyranoside.
carbon signals (resonances at δ = 101.4 and 100.1 ppm) in Finally, the HMBC correlation peak of 1Ј-H with C-1
the 13C NMR spectrum. Then, starting from each anomeric showed that the glucose is directly linked to ceramide,
proton resonance, each sugar spin system was assigned on whereas the correlation peak of 1ЈЈ-H with C-6Ј showed
the basis of the TOCSY and COSY spectra (Table 1). Both that the glucosamine is glycosylating the glucose at the 6Ј-
sugar residues contain, in addition to the anomeric CH position. Once the gross structure of the peracetyl derivative
group, four methine groups and one methylene group, and 1b had been elucidated, NMR spectra of 1a were collected
were therefore designated hexoses. The upfield chemical and fully assigned, providing further support for the pro-
shifts of the 5Ј-H and 5ЈЈ-H signals showed that the corre- posed structure (see Exp. Sect.).
sponding oxymethine groups are not involved in an ester
The absolute configuration of the two sugars, the config-
function, which indicates that the two sugar residues are uration at C-2ЈЈЈ of the α-hydroxy fatty acid, and the config-
both in the pyranose form. Coupling-constant analysis al- urations at C-2, C-3, and C-4 of the sphingosine were deter-
lowed us to establish that all the ring protons of the sugar, mined by chemical degradation. We used a simplified ver-
whose anomeric proton resonates at δ = 4.46 ppm, are axial sion of the microscale procedure that we developed for the
and therefore that this sugar residue is a β-glucopyranoside. analysis of glycolipids isolated as mixtures of homologues
As for the second unit, the chemical shift values of C-2ЈЈ (δ (Scheme 2).[9] Compound 1a was subjected to acidic meth-
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Glycosphingolipids Amphiceramide A and B
anolysis, and the reaction products were perbenzoylated compound 1a was subjected to catalytic reduction with H2/
and subjected to HPLC to give compounds 6–9. The methyl PtO2, and the reaction product was subjected to the degra-
glycoside 6 was identified as methyl tetra-O-benzoyl-α-- dation procedure described above. HPLC separation gave a
1
1
glucopyranoside by comparison of its H NMR and CD compound whose H NMR and CD spectra were identical
spectra with those reported in the literature.[10] The methyl to those of ribo-phytosphingosine perbenzoate (10), thus
glycoside 7 was identified as methyl tri-O-benzoyl-2-benz- also defining the configuration of the perbenzoylated ∆6-
1
amido-2-deoxy-α-glucopyranoside by comparison of its H phytosphingosine 9.
NMR and CD spectra with those of an authentic sample
The structure of 1a was further supported by MS/MS
prepared from N-acetyl--glucosamine according to the data recorded by dissolving the sample in MeOH with 1 m
same procedure used to prepare compound 1a. The fatty LiCl, as previously reported,[11] and by using the [M +
ester 8 was identified as methyl (R)-2-benzoyloxydocosano- Li]+ pseudomolecular ion (m/z = 1025) as the precursor ion.
ate from the EI mass spectrum ([M+] peak at m/z = 474) The spectrum contained three peaks at m/z = 822, 660, and
1
and from the H NMR and CD spectra, which matched 687 arising from the loss of the dehydrated N-acetylglucosa-
those reported previously.[9] The stereochemistry of the per- mine, the loss of the dehydrated saccharide chain, and the
benzoylated ∆6-phytosphingosine 9 could not be deter- loss of a C22 2-hydoxyacyl group, respectively (Scheme 3).
mined directly from its spectra because reference data were
1
not available in the literature. In the H NMR spectrum of
9, the chemical shifts and coupling constants of the protons
at C-1 to C-4 were close to those of -ribo-phytosphingos-
ine perbenzoate (10), but the presence of the additional
double bond significantly affected the CD spectrum of 9,
which was remarkably different from that of the reference
compound[9] (see Supporting Information) and did not al-
low a reliable assignment of its configuration. Therefore,
Scheme 3. MS/MS fragmentation pathways of the lithiated adduct
of amphiceramides A (1a) and B (2a).
Amphiceramide B (2a)
Compound 2a showed an [M + Na]+ pseudomolecular
ion peak in the high-resolution ESI mass spectrum at m/z
= 1000.6931, in accord with the molecular formula
1
C53H101NO14. The H NMR spectrum of the peracetate 2b
was similar to that of 1b, but only one NH doublet was
present. This data suggested a diglycosyl ceramide with no
amino sugar present in the structure. The ceramide part of
the molecule appeared to be the same as that of 1b, each
proton signal showing a chemical shift close to those ob-
served in 1b and nearly identical coupling constants
(Table 1).
The structure of the saccharide chain of 2b was studied
by the same methods used for 1b. The sugar linked to the
ceramide is, again, a β-glucopyranoside, glycosylated at the
6Ј-position by the second sugar, which was shown to be a
β-galactopyranoside on the basis of the following evidence.
The 2ЈЈ-H proton showed a large coupling constant with
both 1ЈЈ-H and 3ЈЈ-H, and these three protons are therefore
axial. In contrast, the coupling constant between 3ЈЈ-H and
4ЈЈ-H (3.4 Hz) showed the latter to be equatorial. As for
5ЈЈ-H, its axial orientation was demonstrated by its intense
Scheme 2. Degradation scheme of amphiceramides A (1a) and B
(2a).
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V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, R. Teta
FULL PAPER
correlation peak with the axial 3ЈЈ-H in the ROESY spec-
trum.
Conclusions
The structures of the glycosphingolipids produced by the
marine sponge Amphimedon compressa, all characterized by
an unusual ∆6-phytosphingosine, have been elucidated. The
NAcGlc(1βǞ6)Glc disaccharide of amphiceramide A (1a)
has never been found before as a part of a natural com-
pound, whereas the saccharide chain of amphiceramide B
(2a) is found for the first time in a glycosphingolipid.
It is noteworthy that, in spite of the very large number
of natural glycoconjugates that have been isolated so far,
marine sponges continue to provide new examples of oligo-
saccharide chains. The biological role of this wide array of
glycoconjugates in sponges is still unknown, although the
clear species and taxon specificities of most of them suggest
that they may be involved in cell-recognition processes.
The absolute configuration of the sugar was determined
by the same degradation scheme described for 1a
(Scheme 2). In this case, however, the benzoylated α-glyco-
sides of glucose and galactose (which are the major prod-
ucts of the reaction sequence) co-eluted from the HPLC
column and could not be obtained in pure form. The corre-
sponding β-glycosides 11 and 12, although less abundant,
gave rise to distinct peaks in the chromatogram. The struc-
tures and absolute configurations of compounds 11 and 12
1
were identified by comparison of their H NMR and CD
spectra with those reported in the literature.[1]
Finally, the structure of the ceramide of 2a, and in par-
ticular the lengths of the two alkyl chains, was confirmed
by MS/MS data. The [M + Li]+ pseudomolecular ion of 2a
(m/z = 984) showed the same fragmentation pattern as 1a
(Scheme 3) with the loss of a C22 α-hydroxyacyl group lead-
ing to a fragment ion at m/z = 646.
Experimental Section
General Methods: High-resolution ESI mass spectra were recorded
with a Bruker APEX II FT-ICR mass spectrometer. ESI mass spec-
tra were recorded with an Applied Biosystem API 2000 triple-qua-
drupole mass spectrometer. All the mass spectra were recorded by
infusion into the ESI source using MeOH as the solvent. Optical
rotations were measured at 589 nm with a Perkin-Elmer 192 polari-
Compounds 3a, 4a, and 5a
1
The H and 13C NMR spectroscopic data of the perace-
tate 3b are very similar to those reported for amphicerebro-
side B heptaacetate,[8] a monoglycosyl ceramide composed
of a ∆6-unsaturated phytosphingosine and an N-acetyl-β-
glucosamine. The only difference between the two com-
pounds, that is, the unbranched C18 sphingosine of 3b in-
stead of the iso C19 sphingosine of amphicerebroside B, is
reflected in the methyl triplet (6 H) at δ = 0.88 ppm in the
1H NMR spectrum of 3b. This was confirmed by the mass
spectrum of the natural compound 3a, which showed a li-
thiated [M + Li]+ pseudomolecular ion peak at m/z = 863
indicative of a C40 ceramide. The fragment ion peak at m/z
= 525 in the MS/MS data ([M + Li – C22H42O2]+) indicated
a C22 α-hydroxy fatty acid and, consequently, a C18 sphin-
gosine.
1
meter using a 10 cm microcell. H and 13C NMR spectra were re-
corded with Varian UnityInova 700 MHz and 500 MHz NMR
spectrometers; chemical shifts are referenced to the residual solvent
signal (CDCl3: δH = 7.26 ppm, δC = 77.0 ppm; [D5]pyridine: δH
=
8.71, 7.56, and 7.19 ppm; δC = 149.9, 135.6, and 123.6 ppm). For
an accurate measurement of the coupling constants, the one-dimen-
sional 1H NMR spectra were transformed at 64K points (digital
resolution: 0.09 Hz). Homonuclear 1H connectivities were deter-
mined by COSY and TOCSY (mixing time 100 ms) experiments.
Through-space 1H connectivities were evidenced by using
a
ROESY experiment with a mixing time of 450 ms. The reverse sin-
gle-quantum heteronuclear correlation (HSQC) spectra were opti-
mized for an average 1JCH of 145 Hz. The multiple-bond heteronuc-
lear correlation (HMBC) experiments were optimized for a 3JCH of
8 Hz. GC/MS was performed with an Agilent 6850 gas chromato-
graph with an MSD HP 5975B mass-selective detector, a split/split-
less injector, and a fused-silica column (25 mϫ0.20 mm HP-5,
cross-linked 25% PhMe silicone, 0.33 mm film thickness). After a
delay of 3 min from the injection, the temperature of the column
was varied from 150 to 280 °C with a gradient of 10 °C min–1.
Quantitative determination was based on the area of the GLC
peaks. High-performance liquid chromatography (HPLC) was per-
formed with a Varian Prostar 210 apparatus equipped with a Var-
ian 350 refractive index detector or a Varian 325 UV detector.
The ESI mass spectrum of compound 4a ([M + Na]+
at m/z = 838) was consistent with the molecular formula
C46H89NO10 and indicative of a monohexosyl ceramide
with the same ceramide as compounds 1a and 2a. Examina-
tion of the 1H NMR spectrum of the peracetate 4b evi-
denced the presence of the signals of a β-glucopyranoside
(chemical shifts and coupling constants matched well those
reported for a β-glucosyl ceramide peracetate),[12] whereas
the signals of the ceramide proton were very similar to
those of 1b and 2b. Degradation analysis as described above
led to the formation of the methyl glycoside 6, the fatty Collection, Extraction, and Isolation Procedures: Specimens of Am-
phimedon compressa were collected in the summer of 2005 along
the coast of Key Largo (Florida). They were frozen immediately
after collection and kept frozen until extraction. The sponge (290 g
dry weight after extraction) was homogenized and extracted with
methanol (3ϫ5 L) and then with chloroform (3ϫ5 L). The com-
bined extracts were partitioned between H2O and nBuOH. The or-
ganic layer was concentrated in vacuo and afforded 53.3 g of a dark
oil, which was purified by chromatography on a column packed
with RP-18 silica gel. A fraction eluted with CHCl3 (4.7 g) was
further purified by chromatography on an SiO2 column, giving a
fraction [1.01 g, eluent: EtOAc/MeOH (7:3)] mainly composed of
ester 8, and the sphingosine 9, thus defining completely the
structure of 4a. Compound 4a had previously been reported
as halicerebroside A from the Red Sea sponge Halilona
sp,[8] but due to the limited spectroscopic data present in
the original paper identification by spectral comparison was
not possible.
Finally, compound 5a was identified by comparison of its
1
MS and H and 13C NMR data with those reported for am-
phimelibioside C from the Japanese sponge Amphimedon
sp.[3]
2116
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2112–2119

Glycosphingolipids Amphiceramide A and B
glycolipids. This fraction was peracetylated with Ac2O in pyridine
for 12 h. The acetylated glycolipids were subjected to HPLC sepa-
ration on an SiO2 column [eluent: n-hexane/EtOAc (6:4)]. Five
major fractions were obtained, each mainly composed of one of
the compounds 1b–5b. Axiceramide A (1b, 5.6 mg) and B (2b,
1.6 mg) were obtained in pure form from the corresponding frac-
tions after a further normal-phase HPLC purification [eluent: n-
hexane/iPrOH (85:15)] followed by reversed-phase HPLC (eluent:
MeOH). Fractions containing compounds 3b–5b were homogen-
eous as far as the polar parts of the molecules are concerned, and
3.5 Hz, 1 H, 1-Hb), 4.53 (q, J = 8.7 Hz, 1 H, 2ЈЈ-H), 4.57 (m, 1 H,
2ЈЈЈ-H), 4.75 (overlapped, 1-Ha), 4.75 (overlapped, 6Ј-Ha), 4.85 (d,
J = 7.7 Hz, 1 H, 1Ј-H), 5.25 (d, J = 8.3 Hz, 1 H, 1ЈЈ-H), 5.27
(overlapped, 1 H, 2-H), 5.70 (ddd, J = 15.2, 6.9, 6.9 Hz, 1 H, 7-H),
5.97 (ddd, J = 15.2, 6.9, 6.9 Hz, 1 H, 6-H), 8.55 (d, J = 8.9 Hz, 1
H, 2-NH), 8.92 (d, J = 7.5 Hz, 1 H, 2ЈЈ-NH) ppm. 13C NMR
(C5D5N): δ = 14.3 (CH3, C-18, C-22ЈЈЈ), 23.0 (CH2, C-17, C-21ЈЈЈ),
23.4 (CH3, acetyl Me), 25.9 (CH2, C-4ЈЈЈ), 29.7–30.3 (several CH2,
alkyl chain methylene groups), 32.1 (CH2, C-16, C-20ЈЈЈ), 33.2
(CH2, C-8), 35.5 (CH2, C-3ЈЈЈ), 37.7 (CH2, C-5), 51.6 (CH, C-2),
therefore reversed-phase HPLC (eluent: MeOH) was sufficient to 58.1 (CH, C-2ЈЈ), 62.7 (CH2, C-6ЈЈ), 70.1 (CH2, C-6Ј), 70.3 (CH2,
separate them from their minor homologues, the new molecular
species 3b (81 mg) and known compounds 4b (33 mg) and 5b
(8.8 mg).
C-1), 71.8 (CH, C-4Ј), 72.5 (CH, C-4ЈЈ), 72.5 (CH, C-2ЈЈЈ), 72.9
(CH, C-4), 75.2 (CH, C-2Ј), 75.6 (CH, C-3), 76.7 (CH, C-3ЈЈ), 77.3
(CH, C-5Ј), 78.4 (CH, C-3Ј), 78.6 (CH, C-5ЈЈ), 103.2 (CH, C-1ЈЈ),
105.3 (CH, C-1Ј), 128.4 (CH, C-6), 133.0 (CH, C-7), 171.2 (C, ace-
tyl CO), 175.7 (C, C-1ЈЈЈ) ppm.
Amphiceramide A Peracetate (1b): Amorphous solid. [α]2D5 = +2 (c
= 1.5, CHCl3). MS (ESI, positive ion mode, MeOH): m/z = 1419
1
[M + Na]+. For H and 13C NMR data, see Table 1.
Amphiceramide B (2a): Colorless solid. [α]2D5 = –7 (c = 0.5, CHCl3).
HRMS (ESI, positive ion mode, MeOH): m/z = 1000.6931 [M +
Amphiceramide B Peracetate (2b): Amorphous solid. [α]2D5 = +4 (c
= 0.5, CHCl3). MS (ESI, positive ion mode, MeOH): m/z = 1420
Na]+ (calcd. for C52H99NNaO15 1000.6907). H NMR (C5D5N):
δ = 0.85 (2 overlapping t, J = 6.7 Hz, 6 H, 18-H3 and 22ЈЈЈ-H3),
1.27 (alkyl chain methylene protons), 1.75 (m, 1 H, 4ЈЈЈ-Hb), 1.67
(m, 1 H, 4ЈЈЈ-Ha), 1.97 (m, 1 H, 3ЈЈЈ-Hb), 2.03 (m, 2-H, 8-H2), 2.18
(m, 1 H, 3ЈЈЈ-Ha), 2.69 (m, 1 H, 5-Hb), 3.02 (m, 1 H, 5-Ha), 3.91
(m, 1 H, 2Ј-H), 3.95 (m, 1 H, 5Ј-H), 4.06 (overlapped, 5ЈЈ-H), 4.08
(overlapped, 4Ј-H), 4.10 (overlapped, 3Ј-H), 4.17 (m, 1 H, 3ЈЈ-H),
4.25 (dd, J = 11.3, 6.1 Hz, 1 H, 6Ј-Hb), 4.28 (overlapped, 4-H),
4.35 (m, 1 H, 3-H), 4.41 (overlapped, 6ЈЈ-H2), 4.43 (overlapped, 1-
Hb), 4.49 (t, J = 8.3 Hz, 1 H, 2ЈЈ-H), 4.53 (br. s, 1 H, 4ЈЈ-H), 4.58
(m, 1 H, 2ЈЈЈ-H), 4.75 (dd, J = 10.7, 5.9 Hz, 1 H, 1-Ha), 4.81 (br.
d, J = 11.3 Hz, 1 H, 6Ј-Ha), 4.85 (d, J = 7.9 Hz, 1 H, 1Ј-H), 4.99
(d, J = 7.8 Hz, 1 H, 1ЈЈ-H), 5.26 (overlapped, 2-H), 5.71 (ddd, J =
15.1, 6.7, 6.7 Hz, 1 H, 7-H), 5.98 (ddd, J = 15.1, 6.9, 6.9 Hz, 1 H,
6-H), 8.57 (d, J = 9.3 Hz, 1 H, 2-NH) ppm. 13C NMR (C5D5N): δ
= 14.3 (CH3, C-18, C-22ЈЈЈ), 22.9 (CH2, C-17, C-21ЈЈЈ), 29.7–30.3
(several CH2, alkyl chain methylene groups), 32.2 (CH2, C-16, C-
20ЈЈЈ), 33.2 (CH2, C-8), 35.5 (CH2, C-3ЈЈЈ), 37.3 (CH2, C-5), 51.3
(CH, C-2), 62.4 (CH2, C-6ЈЈ), 69.8 (CH2, C-6Ј), 70.2 (CH, C-4ЈЈ),
70.5 (CH2, C-1), 71.4 (CH, C-4Ј), 72.3 (CH, C-2ЈЈЈ), 72.6 (CH, C-
2ЈЈ), 72.6 (CH, C-4), 74.9 (CH, C-2Ј), 75.1 (CH, C-3ЈЈ), 75.3 (CH,
C-3), 76.9 (CH, C-5ЈЈ), 77.1 (CH, C-5Ј), 78.2 (CH, C-3Ј), 105.3
(CH, C-1Ј), 105.8 (CH, C-1ЈЈ), 128.2 (CH, C-6), 132.7 (CH, C-7),
175.7 (C, C-1ЈЈЈ) ppm.
+
1
1
[M + Na]+. For H and 13C NMR data, see Table 1.
Compound 3b: Amorphous solid. MS (ESI, positive ion mode,
1
MeOH): m/z = 1131 [M + Na]+. H NMR (CDCl3): δ = 0.88 (t, J
= 6.7 Hz, 6 H, 18-H3 and 22ЈЈЈ-H3), 1.28 (alkyl chain methylene
protons), 1.92 (s, 3 H, N-acetyl Me group), 1.93 (overlapped, 8-
H2), 1.99–2.19 (6 s, 18 H, O-acetyl Me groups), 2.22 (overlapped,
1 H, 5-Hb), 2.34 (m, 1 H, 5-Ha), 3.46 (m, 1 H, 2Ј-H), 3.72 (over-
lapped, 1-H2), 3.72 (overlapped, 5Ј-H), 4.10 (br. d, J = 12.4 Hz, 1
H, 6Ј-Hb), 4.24 (dd, J = 12.4, 4.1 Hz, 1 H, 6Ј-Ha), 4.30 (m, 1 H,
2-H), 4.85 (d, J = 8.2 Hz, 1 H, 1Ј-H), 4.95 (overlapped, 2 H, 3-H,
4-H), 5.00 (t, J = 9.6 Hz, 1 H, 4Ј-H), 5.07 (dd, J = 4.5, 7.3 Hz, 1
H, 2ЈЈ-H), 5.23 (ddd, J = 15.2, 6.9, 6.9 Hz, 1 H, 6-H), 5.37 (t, J =
9.6 Hz, 1 H, 3Ј-H), 5.44 (ddd, J = 15.2, 6.9, 6.9 Hz, 1 H, 7-H), 6.07
(d, J = 8.6 Hz, 1 H, 2Ј-NH), 7.10 (d, J = 8.6 Hz, 1 H, 2-NH) ppm.
13C NMR (CDCl3): δ = 14.2 (CH3, C-18, C-22ЈЈ), 20.5–23.2 (sev-
eral CH3, acetyl Me groups), 22.7–31.8 (several CH2, alkyl chain
methylene groups), 32.7 (CH2, C-5), 32.7 (CH2, C-8), 47.8 (CH, C-
2), 55.5 (CH, C-2Ј), 61.9 (CH2, C-6Ј), 66.2 (CH2, C-1), 68.8 (CH,
C-4Ј), 71.9 (CH, C-5Ј), 72.2 (CH, C-3Ј), 72.6 (CH, C-4), 72.6 (CH,
C-3), 98.9 (CH, C-1Ј), 123.7 (CH, C-6), 134.7 (CH, C-7), 170.1 (C,
C-1ЈЈ), 171.2 (C, N-acetyl CO group), 169.6–177.1 (several C, O-
acetyl CO groups) ppm.
Deacetylation of Compounds 1b–4b: Compounds 1b–4b were each
dissolved in MeOH (1 mL), and a solution of MeONa in MeOH
(0.4 , 50 µL) was added. The reactions were carried out at 25 °C
for 18 h, and then the reaction mixtures were dried under nitrogen
and the residues partitioned between water and chloroform. After
removal of the solvent, the organic layers gave the corresponding
native glycosphingolipids 1a (1.5 mg), 2a (0.9 mg), 3a (11.3 mg), 4a
(2.8 mg), and 5a (5.7 mg).
Compound 3a: White solid. MS (ESI, positive ion mode, 1 m LiCl
1
in MeOH): m/z = 863 [M + Li]+. H NMR (C5D5N): δ = 0.86 (2
overlapping t, J = 6.7 Hz, 6 H, 18-H3, 22ЈЈЈ-H3), 1.27 (alkyl chain
methylene protons), 1.75 (m, 1 H, 4ЈЈЈ-Hb), 1.67 (m, 1 H, 4ЈЈЈ-Ha),
1.97 (m, 1 H, 3ЈЈ-Ha), 2.02 (m, 2 H, 8-H2), 2.17 (s, 3 H, Ac), 2.18
(overlapped, 3ЈЈ-Hb), 2.67 (m, 1 H, 5-Hb), 3.00 (m, 1 H, 5-Ha),
3.82 (m, 1 H, 5Ј-H), 4.18 (t, J = 9.2 Hz, 1 H, 4Ј-H), 4.23 (over-
lapped, 4-H), 4.24 (overlapped, 3Ј-H), 4.29 (overlapped, 6Ј-Hb),
4.29 (overlapped, 3-H), 4.44 (m, 1 H, 6Ј-Ha), 4.48 (m, 1 H, 2Ј-H),
4.54 (dd, J = 10.9, 3.9 Hz, 1 H, 1-Hb), 4.59 (overlapped, 1-Ha),
4.59 (overlapped, 2ЈЈ-H), 5.12 (d, J = 8.3 Hz, 1 H, 1Ј-H), 5.24 (m,
1 H, 2-H), 5.70 (ddd, J = 15.2, 6.6, 6.6 Hz, 1 H, 7-H), 5.94 (ddd,
J = 15.2, 6.8, 6.8 Hz, 1 H, 6-H), 8.5 (d, J = 9.3 Hz, 1 H, 2Ј-NH),
8.85 (d, J = 7.7 Hz, 1 H, 2-NH) ppm. 13C NMR (C5D5N): δ = 14.3
(CH3, C-18, C-22ЈЈЈ), 23.0 (CH2, C-17, C-21ЈЈЈ), 23.4 (CH3, acetyl
Me), 25.9 (CH2, C-4ЈЈЈ), 29.7–30.3 (several CH2, alkyl chain meth-
Amphiceramide A (1a): Colorless solid. [α]2D5 = –4 (c = 0.9, CHCl3).
HRMS (ESI, positive ion mode, MeOH): m/z = 1041.7183 [M +
Na]+ (calcd. for C54H102N2NaO15+ 1041.7172). 1H NMR (C5D5N):
δ = 0.86 (2 overlapping t, J = 6.7 Hz, 6 H, 18-H3, 22ЈЈЈ-H3), 1.27
(alkyl chain methylene protons), 1.75 (m, 1 H, 4ЈЈЈ-Hb), 1.67 (m, 1
H, 4ЈЈЈ-Ha), 1.94 (m, 1 H, 3ЈЈЈ-Hb), 2.03 (m, 2 H, 8-H2), 2.17 (s, 3
H, Ac), 2.18 (overlapped, 3ЈЈЈ-Ha), 2.68 (m, 1 H, 5-Hb), 3.02 (m,
1 H, 5-Ha), 3.91 (overlapped, 5ЈЈ-H), 3.93 (overlapped, 4Ј-H), 3.94
(overlapped, 2Ј-H), 3.95 (overlapped, 5Ј-H), 4.11 (m, 1 H, 3Ј-H), ylene groups), 32.1 (CH2, C-16, C-20ЈЈЈ), 33.2 (CH2, C-8), 35.5
4.14 (dd, J = 11.2, 6.3 Hz, 1 H, 6Ј-Hb), 4.21 (m, 1 H, 4ЈЈ-H), 4.28 (CH2, C-3ЈЈЈ), 37.7 (CH2, C-5), 50.6 (CH, C-2), 57.8 (CH, C-2Ј),
(m, 1 H, 4-H), 4.32 (overlapped, 6ЈЈ-Hb), 4.33 (overlapped, 3-H),
4.40 (m, 1 H, 3ЈЈ-H), 4.47 (m, 1 H, 6ЈЈ-Ha), 4.50 (dd, J = 10.3,
62.4 (CH2, C-6Ј), 69.0 (CH2, C-1), 72.3 (CH, C-4Ј), 72.6 (CH, C-
2ЈЈ), 72.8 (CH, C-3Ј), 75.4 (CH, C-3), 77.1 (CH, C-4), 78.5 (CH,
Eur. J. Org. Chem. 2009, 2112–2119
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2117

V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, R. Teta
FULL PAPER
C-5Ј), 102.0 (CH, C-1Ј), 128.1 (CH, C-6), 132.9 (CH, C-7), 172.2
(C, acetyl CO), 175.7 (C, C-1ЈЈ) ppm.
MeOH (1 mL), and the solution obtained was kept at 80 °C for
12 h. The resulting mixture was dried under nitrogen, benzoylated
with benzoyl chloride (100 µL) and pyridine (1 mL), and purified
by HPLC [column: Luna SiO2, 5 µm; eluent: n-hexane/iPrOH
(99:1); flow: 1 mLmin–1].
Methyl Tri-O-benzoyl-2-benzamido-2-deoxy-α-glucopyranoside (7):
N-Acetyl--glucosamine (11.2 mg) was subjected to acidic meth-
anolysis as described below. The resulting methyl glycosides were
benzoylated with benzoyl chloride (200 µL) in pyridine (2 mL) at
25 °C for 16 h. The reaction was quenched with MeOH, and, after
30 min, the mixture was dried under nitrogen. Methyl benzoate was
removed by keeping the residue under vacuum with an oil pump
for 24 h. The residue was purified by HPLC [column: Luna SiO2,
5 µm; eluent: n-hexane/iPrOH (97:3); flow: 1 mLmin–1; UV detec-
(2S,3S,4R)-1,3,4-O-Benzoyl-2-benzoylaminooctadecane-1,3,4-triol
(10): CD (MeCN): λmax (∆ε) = 233 (–8), 221 (+2) nm. The 1H
NMR spectrum was identical to that of an authentic sample of -
ribo-phytosphingosine perbenzoate.[13]
Methanolysis of Amphiceramide B and Analysis of Methyl Glyco-
sides from 2a: Amphiceramide B (2a; 0.7 mg) was subjected to
acidic methanolysis as described above for compound 1a. The reac-
tion mixture was dried under nitrogen and benzoylated with ben-
zoyl chloride (100 µL) and pyridine (1 mL) at 25 °C for 16 h. The
reaction was quenched with MeOH and dried under nitrogen. The
residue was then purified by HPLC [column: SiO2, 4.6ϫ250 mm,
5 µm; eluent: n-hexane/iPrOH (99.5:0.5); flow: 1 mLmin–1; UV de-
tector: 260 nm], and the two peaks observed were identified as
methyl tetra-O-benzoyl-β--glucopyranoside (11; tR = 28.8 min)
1
tor: 280 nm] and afforded the methyl glycoside 7 (tR = 15 min). H
NMR (CDCl3): δ = 3.50 (s, 3 H, OMe), 4.37 (ddd, J = 9.9, 4.9,
2.8 Hz, 1 H, 5-H), 4.48 (dd, J = 12.1, 4.9 Hz, 1 H, 6-Hb), 4.62 (dd,
J = 12.1, 2.8 Hz, 1 H, 6-Ha), 4.76 (ddd, J = 10.6, 9.1, 3.5 Hz, 1 H,
2-H), 5.02 (d, J = 3.5 Hz, 1 H, 1-H), 5.76 (t, J = 9.7 Hz, 1 H, 4-
H), 5.83 (dd, J = 10.6, 9.5 Hz, 1 H, 3-H), 6.62 (d, J = 9.1 Hz, 1 H,
2-NH), 7.28–7.59 (overlapping signals, 12 H, benzoyl protons), 7.68
(d, J = 7.8 Hz, 2 H, benzoyl ortho protons), 7.90 (d, J = 7.9 Hz, 2
H, benzoyl ortho protons), 7.93 (d, J = 7.9 Hz, 2 H, benzoyl ortho
protons), 8.06 (d, J = 8.0 Hz, 2 H, benzoyl ortho protons) ppm.
CD (MeCN): λmax (∆ε) = 231 (+4) nm.
and methyl tetra-O-benzoyl-β--galactopyranoside (12; tR
=
1
30.7 min) by a comparison of their retention times and H NMR
and CD spectra with those reported previously.[1]
Methanolysis of Amphiceramide A (1a): Compound 1a (1.2 mg) was
dissolved in 1  HCl in 91% MeOH (500 µL), and the obtained
solution was kept at 80 °C for about 12 h. The reaction mixture
was dried under nitrogen and benzoylated with benzoyl chloride
(50 µL) in pyridine (500 µL) at 25 °C for 16 h. The reaction was
then quenched with MeOH. After 30 min, the reaction mixture was
dried under nitrogen. Most of the methyl benzoate was removed
by keeping the residue under vacuum with an oil pump for 24 h.
The residue was then purified by HPLC [column: Luna SiO2, 5 µm;
eluent: n-hexane/iPrOH (99:1); flow: 1 mLmin–1]. The chromato-
gram showed four peaks: methyl glycoside 6, identified as methyl
tetra-O-benzoyl-α--glucopyranoside (tR = 10.8 min) by compari-
son of its 1H NMR and CD spectra with those reported in the
literature,[9] methyl tri-O-benzoyl-2-benzamido-2-deoxy-α-glucopy-
ranoside (7; tR = 43.3 min), identified by comparison of its 1H
NMR and CD spectra with those of an authentic sample of 7, the
benzoylated fatty acid methyl ester 8 (tR = 4.0 min), recognized as
methyl (R)-2-benzoyloxydocosanoate because of its EI mass, 1H
NMR, and CD spectra, which matched those reported pre-
Supporting Information (see footnote on the first page of this arti-
cle): ESI mass spectra and 1H NMR spectra of compounds 1a and
1
2a; H, 13C, COSY, ROESY, HSQC, and HMBC NMR spectra of
1
1
compound 1b; H and HSQC NMR spectra of compound 2b; H
NMR and CD spectra of compounds 7 and 9.
Acknowledgments
This work was supported by the Italian MIUR PRIN 2007 (grant
no. 2007KZ9W5J). We thank Prof. J. R. Pawlik (University of
North Carolina) for giving us the opportunity to join the expedi-
tion to the Caribbean Sea during which the sponge A. compressa
was collected and Professor S. Zea (Departamento de Biología y
Centro de Estudios en Ciencias del Mar – CECIMAR, Universidad
National de Colombia) for identifying the sponge. NMR and mass
spectra were recorded at the Centro Interdipartimentale di Analisi
Strumentale, Università di Napoli “Federico II”; the assistance of
the staff is gratefully acknowledged.
viously,[9] and the perbenzoylated ∆6-phytosphingosine 9 (tR
=
10.5 min).
(2S,3S,4R)-1,3,4-O-Benzoyl-2-benzoylaminooctadec-6-ene-1,3,4-
triol (9): H NMR (CDCl3): δ = 0.87 (d, J = 6.9 Hz, 3 H, 18-H3),
1.34–1.16 (br., alkyl chain), 1.88 (m, 2 H, 8-H2), 2.62 (m, 1 H, 5-
Hb), 2.74 (m, 1 H, 5-Ha), 4.59 (dd, J = 12.0, 6.1 Hz, 1 H, 1-Hb),
4.67 (dd, J = 12.0, 6.0 Hz, 1 H, 1-Ha), 5.15 (m, 1 H, 2-H), 5.41
(ddd, J = 15.2, 7.2, 7.2 Hz, 1 H, 6-H or 7-H), 5.52 (ddd, J = 15.2,
[1] V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, J.
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[2] E. Fattorusso, A. Mangoni in Progress in the Chemistry of Or-
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Moore, W. Steiglich, Ch. Tamm), Springer, Vienna, 1997, pp.
215–301.
1
7.0, 7.0 Hz, 1 H, 7-H or 6-H), 5.56 (m, 1 H, 4-H), 5.72 (t, J = [3] C. Emura, R. Higuchi, T. Miyamoto, R. W. M. VanSoest, J.
Org. Chem. 2005, 70, 3031–3038.
5.2 Hz, 1 H, 3-H), 7.14 (d, J = 9.1 Hz, 1 H, 2-NH), 7.33–7.63 (over-
lapping signals, 12 H, benzoyl protons), 7.83 (d, J = 7.7 Hz, 2 H,
benzoyl ortho protons), 7.93 (d, J = 7.7 Hz, 2 H, benzoyl ortho
protons), 8.01 (d, J = 7.7 Hz, 2 H, benzoyl ortho protons), 8.04 (d,
J = 7.7 Hz, 2 H, benzoyl ortho protons) ppm. CD (MeCN): λmax
(∆ε) = 248 (–1), 222 (+8) nm.
[4] N. Borbone, S. De Marino, M. Iorizzi, F. Zollo, C. Debitus, A.
Ianaro, B. Pisano, Eur. J. Org. Chem. 2001, 4651–4656.
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Catalytic Reduction and Methanolysis of Compound 1a: Compound
1a (0.5 mg) was dissolved in EtOH (1 mL), and a small amount of
PtO2 was added. The suspension was kept under H2 at 1 atm for
12 h, then filtered through a Titan PTFE membrane disc filter
(0.2 µm), and dried. The residue was dissolved in 1  HCl in 92%
[7] T. Natori, Y. Koezuka, T. Higa, Tetrahedron Lett. 1993, 34,
5591–5592.
[8] S. Hirsch, Y. Kashman, Tetrahedron 1989, 45, 3897–3906.
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2112–2119

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Freigang, L. Teyton, Bioorg. Med. Chem. 2008, 16, 2077–2085.
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[13]
V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni, J.
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Received: December 11, 2008
Published Online: March 18, 2009
Eur. J. Org. Chem. 2009, 2112–2119
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2119