V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni
FULL PAPER
Figure 1. Major fragmentation pathways of the lithiated adduct of ectyoceramide (1a) as found by ESI MS/MS experiments
ganisms producing them as complex, inseparable mixtures source with MeOH/CHCl
3
(4:1) containing 1 m LiCl as the sol-
vent. Optical rotations were measured at 589 nm with a Perkin-
of homologues; in contrast, a single homologue of ectyocer-
amide is predominant in E. ferox, so compound 1a could
be obtained in pure form. The structure of the ceramide
was determined in an innovative way by use of tandem mass
spectrometry; in future work this method should allow de-
structive degradation analysis to be avoided in many cases.
The presence of ectyoceramide in E. ferox further con-
1
13
Elmer 192 polarimeter in a 10-cm microcell. H and C NMR
spectra were determined with a Bruker AMX 500 spectrometer at
5
00.13 and 125.77 MHz, respectively; chemical shifts were refer-
enced to the residual solvent signal (CDCl : δH ϭ 7.26 ppm, δC ϭ
]pyridine: δH ϭ 8.71, 7.56, and 7.19 ppm, δC ϭ
49.8, 135.3, and 123.4 ppm). For accurate measurement of the
3
7
1
7.0 ppm; [D
5
1
coupling constants, the one-dimensional H NMR spectra were
firms that marine sponges are an extremely rich source of transformed at 64 K points (digital resolution: 0.09 Hz). Homonu-
1
new glycolipid structures. Even though only a small number clear H connectivities were determined by COSY experiments.
1
of species have so far been analyzed, some general trends Through-space H connectivities were evidenced by means of an
ROESY experiment with a mixing time of 500 ms. The reverse mul-
can be outlined. First of all, all the species of sponges we
tiple-quantum heteronuclear correlation (HMQC) spectra were re-
have studied contained glycolipids, but their amounts were
corded by use of a pulse sequence with a BIRD pulse 0.5 s before
very different from species to species. In contrast, high re-
each scan to suppress the signal originating from protons not di-
producibility was observed for the kind and relative
1
3
rectly bound to C; the interpulse delays were adjusted for an aver-
amounts of glycolipids isolated from different specimens of
the same species. In addition, fatty acids found in glycolip-
ids of sponges (and sphingosines, which are biosynthesized
1
age
JC,H of 142 Hz. GLC analysis was performed with a Carlo
Erba Fractovap 4160 fitted with a 25-m SPB-1 capillary column,
with helium (5 mL/min) as carrier. High-performance liquid chro-
from fatty acids, found in spongal GSLs) are characteristic matography (HPLC) was carried out with a Varian 2510 apparatus
in that frequent occurrence of methyl-branched chains, as fitted with a Varian Star 9040 refractive index detector.
well as of odd-numbered carbon (both linear and branched)
Collection, Extraction, and Isolation: Specimens of E. ferox were
chains, is observed. However, the physiological role of such
collected in September 2000 near the coast of the island of Rum
a variety of unusual glycolipids in sponges is still unclear.
Cay (Bahamas), and identified by Prof. M. Pansini (University of
Genoa, Italy). They were frozen immediately after collection and
Recent work has demonstrated that a change in the
stereochemistry of the glycosidic linkage in monogalactopy- kept frozen until extraction. Reference specimens (ref. no. 98-55)
anosyl GSLs from the usual β to the α configuration can have been deposited at the Istituto di Zoologia, University of
significantly affect their biological activity, and α-galactosyl Genoa, Italy. The sponge (69.4 g dry weight after extraction) was
[
9]
homogenized and extracted with methanol (3 ϫ 1 L) and then with
chloroform (2 ϫ 1 L); the combined extracts were then partitioned
between H O and nBuOH. The organic layer was concentrated in
2
vacuo and afforded 11.5 g of a dark red solid, which was chromato-
graphed on a column packed with RP-18 silica gel. A fraction
GSLs are immunostimulating and antitumor compounds.
Likewise, the activity of a galactofuranosylceramide such
as ectyoceramide is certainly of interest, but could not be
examined thoroughly because of the very small amount of
ectyoceramide present in E. ferox. Preparation of synthetic
analogues of ectyoceramide in order to overcome this prob-
lem is in progress in our laboratories.
eluted with CHCl
SiO column, giving a fraction [126 mg, eluent EtOAc/MeOH (7:3)]
mainly composed of glycolipids. The main portion (110 mg) of this
fraction was peracetylated with Ac O in pyridine for 12 h. The ace-
tylated glycolipids were subjected to HPLC separation on an SiO
column [eluent: n-hexane/EtOAc (6:4)], thus affording a mixture
4.0 mg) containing ectyoceramide and other glycolipids. Further
3
(2.44 g) was further chromatographed on an
2
2
2
Experimental Section
(
General Remarks: High-resolution ESI-MS spectra were performed
with a Micromass QTOF Micro mass spectrometer, the sample be-
ing dissolved in MeCN/H O (1:1) with 0.1% TFA. ESI MS/MS
2
normal-phase HPLC purification [eluent: n-hexane/iPrOH (93:7)]
gave 1.8 mg of ectyoceramide peracetate (2b), plus minor amounts
of its homologues. Pure compound 1b (1.5 mg, 0.013% of the ex-
experiments were performed with a Finnigan LCQ ion-trap mass tract) was obtained by reversed-phase HPLC separation, with
spectrometer. The spectra were recorded by infusion into the ESI
MeOH as eluent. The natural ectyoceramide (1a, 1.1 mg) could be
Eur. J. Org. Chem. 2003, 1433Ϫ1437
1436