Chem. Pharm. Bull. 50(11) 1467—1470 (2002)

ingolipids (GSLs) from echinoderms, a series of studies on the isolation and structural elucidation of the GSLs from sea cucumber species have been performed in our laboratory. In the study of the GSLs of the sea cucumber Holothuria pervicax (Torafunamako in Japanese), we reported the isolation and structure of four new ganglioside molecular species. Continuing the preceding studies, the isolation and characterization of cerebrosides from H. pervicax was conducted. In this paper, we report the isolation and characterization of glucocerebrosides from the whole bodies of H. pervicax. The less polar lipid fraction, which was obtained from the chloroform/methanol extract of the whole bodies of H. pervicax, was subjected to repeated silica gel column chromatography to give three cerebroside molecular species, HPC-1, HPC-2, and HPC-3, each showing a single spot on silica gel thin-layer chromatography (TLC). HPC-1, HPC-2, and HPC-3 exhibit strong hydroxy and amide absorptions in their IR spectra, and a series of molecular ion peaks in their positive FAB mass spectra, respectively. In their C-NMR spectra (Fig. 1, Table 1), they reveal characteristic signals of a sphingosine-type b-glucocerebroside possessing an unsubstituted fatty acid (HPC-1), a sphingosine-type b-glucocerebroside possessing a 2-hydroxy fatty acid (HPC-2), and a phytosphingosine-type b-glucocerebroside possessing a 2-hydroxy fatty acid (HPC-3), respectively. Therefore, they are suggested to be the molecular species of three typical types of glucocerebrosides. Their structures shown in Fig. 1 were characterized by comparison of their C-NMR spectral data with those of known glucocerebrosides hitherto obtained, and by means of the results of their chemical degradations, namely methanolysis followed by the GC-MS analysis of the methanolysis products, fatty acid methyl ester (FAM) and long-chain base (LCB), as shown in Fig. 2 and the Experimental section. The absolute configuration of their glucose moiety (D-form) was determined by the Hara method (Experimental section). Based on the considerable interest in and importance of determining the molecular species composition of GSLs, the isolation and structural elucidation of glucocerebroside components in the most polar molecular species, HPC-3, was conducted at this time. HPC-3 could be separated by reversed-phase HPLC into eighteen peaks (Fig. 3a), and could be recovered to give six fractions, 5, 8, 9, 11, 14 and 16. They behaved as pure compounds in HPLC. However, fractions 11, 14 and 16 were still regarded as heterogeneous compounds, respectively, since they afforded plural FAMs upon methanolysis. Each fraction, 11, 14 and 16, was successively separated into plural peaks by using recycling reversed-phase (C30) HPLC, and seven fractions could be isolated as shown in Fig. 3b and c. Thus, ten components, designated as HPC-3-A—HPC-3-J, were obtained from the parent glucocerebroside mixture, HPC-3. In the negative FAB mass spectrum, they reveal single quasi-molecular ion peaks [M Na] at m/z: 838 (HPC-3-A), 852 (HPC-3-B), 826 (HPC-3-C), 840 (HPC-3-D and HPC-3-E), 854 (HPC-3-F, HPC-3-G and HPC-3-H), 868 (HPC-3-I and HPC-3-J). These were confirmed as being the glucocerebroside component of HPC-3, since their C-NMR spectra are identical to that of HPC-3 (Table 1). Upon methanolysis, they yielded methyl 2-hydroxytricosenoate (HPC-3-A), methyl 2-hydroxytetracosenoate (HPC-3-B), methyl 2-hydroxydocosanoate (HPC-3-C, HPC3-D and HPC-3-F ), methyl 2-hydroxytricosanoate (HPC-3E, HPC-3-G and HPC-3-I), methyl 2-hydroxytetracosanoate (HPC-3-H and HPC-3-J) and 2-amino-1,3,4-trihydroxy-heptadecane (HPC-3-A, HPC-3-B, HPC-3-C, HPC-3-E and HPC-3-H), 2-amino-1,3,4-trihydroxy-octadecane (HPC-3-D, HPC-3-G and HPC-3-J), 2-amino-1,3,4-trihydroxy-nonadecane (HPC-3-F and HPC-3-I) as their fatty acid and LCB components, respectively. The location and geometry of the double bond in the fatty acyl moiety of HPC-3-A and HPC3-B were determined as follows. The mass spectra of the dimethyl disulfide (DMDS) derivatives of the FAM from HPC-3-A [(a) in Fig. 4] and HPC-3-B [(b) in Fig. 4] show remarkable fragment-ion peaks at m/z: 173 and 303 for (a) and m/z: 173 and 317 for (b) due to cleavage of the bonds between the carbons bearing the methylthio groups (Fig. 4). These data indicate that the double bonds in the fatty acyl moieties of HPC-3-A and HPC-3B are located at C-14 and C-15, respectively. Furthermore, the geometry (Z) of the double bonds of both compounds were determined from the d value (27.6) of the allylic carbon November 2002 Chem. Pharm. Bull. 50(11) 1467—1470 (2002) 1467