Marine Bifunctional Sphingolipids from the Sponge Oceanapia ramsayi

During the course of our continuing studies on marine natural lipid products, two known sphingolipids have been isolated for the first time from a specimen of the marine sponge Oceanapia ramsayi collected at Itampolo on the west coast of Madagascar in the Indian Ocean. The structures were elucidated using NMR data and by comparison with literature data. The occurrence of these sphingolipids within other Oceanapia spp. is discussed.


Introduction
The sponge Oceanapia ramsayi study has been studied with the aim of discovering new lipid metabolites. Marine sponges belonging to the genus Oceanapia, which now includes several other generic names considered as synonyms (Phloeodictyon Carter, 1882; Rhizochalina Schmidt, 1870; Biminia Wiedenmayer, 1977; Foliolina Schmidt, 1870 [1]) present a large variety of compounds which may have interesting biological activities, including antifungal properties [2]. Sponges previously classified in Rhizochalina present as major components sugar derivatives such as rhizochalin, the first compound isolated from a marine sponge belonging to this genus [3]. Sugar derivatives such as oceanalin A were also recently isolated from Oceanapia [2]. Other compound families such as indoles [4], alkaloids [5] or ceramides [6] have also been characterized. During the course of our continuing studies on marine natural lipid products, we have now found in the sponge Oceanapia ramsayi two known compounds, rhizochalin and the corresponding aglycone, which were identified as their corresponding peracetates 1 and 2 ( Figure 1).

Results and Discussion
The structures of these compounds were determined on the basis of their 1D and 2D NMR spectra and by comparison with literature data [3]. The molecular formula of 1, isolated as pale yellow oil, was established as C 48 H 82 O 15 N 2 on the basis of NMR and EI-MS data (m/z 926, M+, calcd 926.5715). The analysis of the corresponding 1 H-and 13 C-NMR spectra (  [3,7], the sugar was identified as a galactose moiety. Moreover, the coupling constant for the anomeric proton signal H-1' (δ 4.48, J = 7.9 Hz) indicated that the galactose had a β-configuration.
The analysis of 13 C-NMR and DEPT spectra showed the presence of one methylene group bearing an oxygen (δ 61.3 ppm), two methine groups bearing a nitrogen (δ 47.8 ppm) and a carbonyl carbon (δ 211.7 ppm). HMBC correlation between H-1' and C-3 (δ 82.5 ppm) and COSY correlation between H-1' (δ 4.48 ppm) and H-3 (δ 3.48 ppm) indicated that the galactopyranosyl group was located at C-3. Moreover, the analysis of COSY and HMBC spectra showed also the presence of two NH-acetamides at δ 5.82 ppm (d, J = 8.8 Hz) and δ 5.54 ppm (d, J = 8.9 Hz), located at C-2 and C-27, respectively. Interpretation of the COSY and HMBC data revealed the presence of two aglycon terminal chains: -CH(O-)CH(NHAc)-CH 3   The structure of compound 2, obtained as a brown oil, was established by comparison of the 1-and 2-D NMR (Table 2) spectra of 2 with literature data [3,9] and those of rhizochalin peracetate (1). The analysis of the NMR spectrum showed clearly the absence of the sugar seen in compound 1. Aside from this difference, the NMR spectra are quite close. Again, the MS fragmentations at m/z 382 and m/z 284 indicated the presence of a carbonyl located at C-18. Thus, compound 2 corresponds to the peracetyl aglycone of 1, in agreement with the literature [3]. This is the first report of these compounds in the sponge Oceanapia ramsayi. These compounds have been found previously in other investigated Oceanapia spp. (or in its synonym Rhizochalina), such as O. incrustata [3], O. bartschi [4], O. philipensis [9], as well as an unidentified Oceanapia spp. [2,[5][6][7]. The common occurrence of these two compounds suggests that they might be good chemotaxonomic markers for this genus.
Sphingolipids seem to play an important role in cellular regulation. These compounds are found within the cell membranes of all living organisms. They have a protection role in the latter against infection [8]. Two-headed sphingolipids from marine sponges are striking because of their rare α,ωbifunctionalized structures and high biological activity. Since the discovery of rhizochalin, the first member of this series, only two other compounds have been isolated, which are oceanapiside [9] and the calyxoside [10]. Moreover, each one of these compounds possesses a conserved number of carbon atoms (24) in the long chains between the functionalized termini. It suggests that these compounds are derived from a similar lipid carboxyl precursor.  The absolute configurations of rhizochalin and its aglycone (threo, threo) differ from those obtained for calyxoside and oceanapiside (erythro, threo) as well as those obtained for the sphingolipids with methyl termini such as the fumonisins B1 and B2 described by Branham and Plattner [11]. These authors propose that at least two independent amino acid fatty acyl transferases, or homologous subunits in the same enzyme are operative in the biosynthesis of dimeric sphingolipids in marine sponge, one incorporating alanine with 2R,3R stereoselectivity and the second incorporating serine with 2S,3S stereoselectivity [12].

Experimental
General 1 H, 13 C, COSY, HSQC and HMBC NMR spectra were recorded on a Bruker ARX-500 instrument using standard Bruker pulse sequences.

Biological Material
A specimen of the sponge Oceanapia ramsayi (Lendelfeld, 1888; phylum Porifera, class Demospongiae, order Haplosclerida, family Phloeodictyidae) was collected at Itampolo (west coast of Madagascar Island) in 2005. It is a massive sponge, red in life, which displays numerous fistules. A voucher specimen is deposited in the Museum d'Histoire Naturelle de Marseille (MHNM, n°15830.0).