The negative FAB-MS spectrum of PNG-1 showed a series of pseudo-molecular ion peaks [M − H]⁻ at m/z 1079–1149. The fragment ion peaks arising from cleavage of the glycosidic linkages of the major component (m/z 1121) were observed at m/z 816 [M − H − 305; A] and 654 [A − 162; ceramide] as shown in Figure 2A. These data indicated that PNG-1 contained a disaccharide (OMeNeuAc-Hexose-Ceramide). The ¹H-NMR spectrum of PNG-1 exhibited a strong broad signal due to the aliphatic methylenes at δ_H 1.22, a terminal methyl signal at δ_H 0.80 and several multiplets between δ_H 4.00 and 4.80 due to oxygenated methine and methylene protons. These results indicated that PNG-1 was a glycosphingolipid. Some typical signals of a Neu5Ac, such as δ_H 2.28 (t, J = 11.4 Hz, H_ax − 3) and 3.43 (br.d, J = 8 Hz, H_eq− 3), were also detected, suggesting PNG-1 was a ganglioside.

Furthermore, the Neu5Ac was probably methylated, since a methoxy singlet at δ_H 3.83 (3H, s) was observed. In the ¹³C-NMR spectrum, two anomeric carbon signals were clearly observed, and one of them (appearing at δ_C 100.7) was thought to be the C-2 signal of Neu5Ac.

The ceramide moiety of PNG-1 consisted of α-hydroxy fatty acids (FAs) and phytosphingosine-type long chain bases (LCBs). In the COSY spectrum, taking H-2 of the LCB as the starting point, a structural fragment from C-1 to C-6 was established. Another fragment in FA, from C-2 to C-4, was also connected by extensive analysis of COSY and TOCSY spectra. The stereochemistry of the ceramide moiety was presumed to be identical to that of PNC-1 (Figure 3a), which was isolated from the same material [7].

The structure of the sugar moiety was determined as follows. The ¹H- and ¹³C-NMR data (Table 1) showed that PNG-1 contained a disaccharide moiety consisting of galactopyranose (Galp) and mono-methylated Neu5Ac. Identification of the neutral sugar as Gal was also supported by sugar composition analysis using GC. The β-configuration of the Galp unit was determined by the J-value of the anomeric proton [δ_H 4.79 (d, J = 7.9 Hz)]. The configuration of the Neu5Ac moiety was presumed to be α-configuration, based on the chemical shifts of the anomeric carbon at δ_C 100.7 and H-3 methylene protons [8].

The position of the methoxyl group was determined to be at C-8 of Neu5Ac by HMBC correlation from the methyoxy proton (δ_H 3.83) to C-8 (δ_C 81.4). The linkage of the sugar moiety was established by HMBC and nOe correlations (Figure 4). Neu5Ac8Me was linked to C-3 of Galp by the HMBC correlation from H-3 (δ_H 4.70) of Gal to C-2 (δ_C 100.7) of Neu5Ac8Me, as well as nOe correlation between H-3 (δ_H 2.28) of Neu5Ac8Me and H-3 of Galp. In addition, Galp was attached to C-1 of LCB by the HMBC correlations of H-1 (δ_H 4.79) of Galp and C-1 (δ_C 68.9) of LCB, and of H-1 (δ_H 4.60) of LCB and C-1 (δ_C 104.0) of Galp. Accordingly, PNG-1 was characterized to be 1-O-[8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide (Figure 3b).

¹H- and ¹³C-NMR spectra of PNG-2A showed signals characteristic of gangliosides. The negative FAB-MS spectrum of PNG-2A showed a series of pseudo-molecular ion peaks [M − H]⁻ at m/z 1400–1500. The fragment ion peaks arising from cleavage of the glycosidic linkages of the major component (m/z 1431) were observed at m/z 1269 [(M − H)⁻ − 162; A], 1107 [A − 162; B], 802 [B − 305; C], and 640 [C − 162; ceramide] as shown in Figure 2B. These data indicated the sequence of the oligosaccharide of PNG-2A could be depicted as Hexose-Hexose-OMeNeuAc-Hexose-Ceramide. In the ¹H-NMR spectrum, three anomeric proton signals were clearly observed at δ_H 4.79 (d, J = 7.9 Hz), 5.50 (s), and 5.86 (s). Further, a singlet at δ_H 3.79 (3H, s) implied that the NeuAc moiety in PNG-2A was probably methylated.

Extensive analysis of COSY, TOCSY, and HSQC spectra led to the establishment of two partial structures in the ceramide moiety (from C-1 of LCB to C-6 of LCB and from C-2 of FA to C-4 of FA), suggesting that PNG-2A was composed of α-hydroxy fatty acid and phytosphingosine-type LCB. The stereochemistry of the ceramide moiety of PNG-2A was presumed to be identical with that of PNC-1 isolated from the same material. The structure of the sugar moiety was characterized as follows. To determine the sugar composition, PNG-2A was subjected to methanolysis. Comparison of the GC retention times of TMS ethers of methyl glycosides obtained from PNG-2A with those from standard hexoses showed that all the neutral sugars in PNG-2A were Gals. ¹H- and ¹³C-NMR data of the sugar moiety were assigned as shown in Table 1. ¹H-¹H COSY correlations from the three anomeric proton signals revealed a galactose each for β-galactopyranose(β-Galp), α-galactopyranose(α-Galp), and β-galactofuranose(β-Galf). In the ¹³C-NMR spectrum of PNG-2A, C-1, C-2 and C-3 carbon signals were not confirmed. The other proton and carbon signals were indicative of α-Neu5Ac8Me, the same as found in PNG-1. The sugar linkage was examined by NOESY spectrum. The nOe correlations between H-1 of β-Galf and H-3 of α-Galp, H-1 of α-Galp and H-3 of α-Neu5Ac8Me, H-3 of α-Neu5Ac8Me and H-3 of β-Galp were clearly observed. Accordingly, the core structure of PNG-2A was determined to be 1-O-[β-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→4)-8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide (Figure 5).

The negative FAB-MS spectrum of PNG-2B showed a series of pseudo-molecular ion peaks [M − H]⁻ at m/z 1400–1500. The fragment ion peaks arising from cleavage of the glycosidic linkages of the major component (m/z 1417) were observed at m/z 1255, 1093, 802 and 640. These data indicated that PNG-2B contained the same oligosaccharide sequence as PNG-2A. However, the fragment loss (291 mass) from m/z 1083 to m/z 902 suggested the existence of non-substituted Neu5Ac. Hence, the oligosaccharide moiety of PNG-2B could be depicted as Hexose-Hexose-NeuAc-Hexose-Ceramide as shown in Figure 2C. In the ¹H-NMR spectrum, two anomeric proton signals were clearly observed at δ_H 5.82 (s) and 5.22 (s), and another anomeric proton signal, obscured by other resonances, was readily discernable at δ_H 4.74 by HSQC spectrum analysis. Further, the absence of methoxy methyl signals confirmed that the NeuAc moiety in PNG-2B was not methylated.

The structure of the ceramide moiety was identified as α-hydroxy FA and phytosphingosine-type LCB by comparing the ¹³C-NMR, COSY and TOCSY spectra with those of PNG-1 and PNG-2A. The structure of the tetrasaccharide moiety was characterized as follows. ¹H-¹H COSY, TOCSY and HSQC spectra revealed the correlation of β-Galp, α-Galp, α-Neu5Ac and β-Galf (Table 1). The linkage of each monosaccharide was examined by NOESY experiment. The nOe correlations between H-1 of β-Galf and H-3 of α-Galp, H-1 of α-Galp and H-9 of α-Neup5Ac, H-3 of α-Neup5Ac and H-3 of β-Galp suggested the sugar moiety was β-Galf-(1→3)-α-Galp-(1→9)-α-Neu5Ac-(2→3)-β-Galp. The structure of the oligosaccharide moiety was also confirmed by chemical methods. PNG-2B was subjected to methanolysis. Comparison of the GC retention times of TMS ethers of methyl glycosides obtained from PNG-2B with those from standard Gals showed that all the neutral sugars in PNG-2B were Gals. To verify the sugar linkages, PNG-2B was subjected to methylation analysis. Partially methylated alditol acetates prepared from the permethylated PNG-2B were characterized as the alditols derived from terminal Galf and 3-linked Galp in a ratio of approx. 1:2 by GC-MS analysis. The acetate of partially methylated Neu5Ac derived from 9-linked Neu5Ac was detected (Figure 6). These observations unambiguously confirmed the proposed structure of the tetrasaccharide moiety. Therefore, the core structure of PNG-2B was established as 1-O-[β-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→9)-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide (Figure 7).

PNG-1, PNG-2A and PNG-2B were each subjected to methanolysis and periodate oxidation as previously described for PNC-1 [7]. FAs and LCBs were analyzed by GC-MS as fatty acid methyl esters (FAMEs) and long chain aldehydes (LCAs), respectively. The results were summarized in Table 2 and Table 3.

Protoreaster nodosus was collected at Katsuren, the east coast of Okinawa, in June 2009. A voucher specimen was deposited in the Herbal Museum, Graduate School of Pharmaceutical Sciences, Kyushu University.

Pyloric caeca (600 g) dissected from 20 specimens of the starfish Protoreaster nodosus were extracted with CHCl₃/MeOH (1:1, v/v, 2 L) for three times at room temperature. The extract was concentrated in vacuo to give a residue, which was subjected to silica gel column chromatography eluted with CHCl₃/MeOH/H₂O (10:1:0 to 6:4:1, v/v/v) to give four fractions. Fraction 3 and Fraction 4 were each further chromatographed on a LiChroprep RP-8 (Merck, Darmstadt, Germany) column (H₂O/MeOH, 1:0 to 0:1, v/v). Fraction 3-4 containing crude, less polar ganglioside was finally purified by Sephadex LH-20 chromatography with CHCl₃/MeOH (1:1, v/v) to afford PNG-1 (70.0 mg). Fraction 4-4 containing polar gangliosides was desalted on a Sephadex LH-20 column with CHCl₃/MeOH (1:1, v/v) and subsequently subjected to repeated silica gel column chromatography to give PNG-2A (3.0 mg) and PNG-2B (7.0 mg). PNG-1, PNG-2A, and PNG-2B showed a positive reaction with resorcinol reagent on TLC plates.

For FAB-MS analysis, a JEOL SX-102 mass spectrometer (JEOL, Tokyo, Japan) was used (Xenon atom beam, 5 kV; ion source accelerating potential 10 kV; matrix, hexamethylphosphoramide + glycerol for negative ion mode).

A ShimazuQP-5050A (SHIMADZU, Kyoto, Japan) instrument was used with the following settings: EI mode, ionizing potential of 70 eV; injection temperature, 300 °C; separation temperature, 280 °C; ion source temperature, 300 °C. A fused silica capillary column INERT CAP5 (0.25 mm I.D., 30 m, GLSciences INC., Tokyo, Japan) was used for gas-chromatographic separation with Helium as a carrier at a flow rate of 30 mL/min.

Spectra were recorded on a Varian INOVA600 spectrometer. The operating conditions were as follows: ¹H: frequency, 600 MHz; sweep width, 8 kHz; sampling point, 44 k; accumulation, 64 pulses; temperature, 30 or 40 °C. ¹³C: frequency, 150 MHz; sweep width, 32 kHz; sampling point, 160 k; accumulation, 10000 pulses; temperature, 30 °C. Chemical shifts were referenced to the residual solvent signals (δ_H 7.19, δ_C 123.5) in C₅D₅N and D₂O (20/1, v/v). Conventional pulse sequences were used for DQF-COSY, NOESY, HSQC, and HMBC. The mixing time in the NOESY experiment was set to 500 ms. TOCSY spectra were acquired using the standard MLEV17 spin-locking sequence and 120 ms mixing time. All spectra were recorded using the phase-sensitive mode. The size of the acquisition data matrix was 204 × 256 words in f2 and f1, respectively. Zero filling up to 2 k in f1 was made prior to Fourier transformation. Sine-Bell or shifted sine-bell window functions, with the corresponding shift optimized for every spectrum, were used for resolution enhancement, and baseline correction was applied in both dimensions. Water suppression was carried out by selective pre-saturation placing the carrier on the solvent resonance.

Ganglioside (ca. 1.0 mg) was heated with 5% HCl/MeOH in a sealed tube at 80 °C for 18 h. The reaction mixture was extracted with n-hexane to extract FAMEs. The remaining MeOH layer was neutralized with Ag₂CO₃ and filtered. The dried MeOH layer was reacted with 1-(trimethylsilyl) imidazole/pyridine (1:1) for 20 min at 70 °C to give the TMS ether of methyl glycosides. The TMS ethers were then analyzed by GC-MS [column temperature 100–250 °C (rate of temperature increase: 5 °C/min)].

TMS ether of methyl glycoside from D-galactose: t_R [min] = 6.7, 7.2 and 7.7, TMS ether of methyl glycoside from PNG-1: t_R [min] = 6.7, 7.2 and 7.7, TMS ether of methyl glycoside from PNG-2A: t_R [min] = 6.7, 7.2 and 7.7, TMS ether of methyl glycoside from PNG-2B: t_R [min] = 6.7, 7.2 and 7.7.

PNG-2B (ca. 0.5 mg) was added to a NaH-DMSO solution, and the mixture was stirred for 20 min. Then, CH₃I was added to the above reaction mixture, which was stirred for another 20 min to yield permethylated ganglioside. Permethylated ganglioside was hydrolyzed with 90% HCOOH/10% TFA and subsequently reduced by NaBD₄. The residue was heated with Ac₂O/pyridine, and the mixture was extacted with CHCl₃. The extract, containing partially methylated alditol acetates, was concentrated and subjected to GC-MS analysis using a constant column temperature of 175 °C. 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalacitol from 3-linked Galp, t_R [min] = 15.5: 1,4-di-O-acetyl-2,3,5,6-tetra-O-methylgalacitol from terminal Galf, t_R [min] = 12.9.

Separately, permethylated ganglioside was methanolyzed with 10% HCl in MeOH. The reaction mixture was concentrated in vacuo, and the residue was heated with Ac₂O/pyridine. The reaction mixture was subjected to GC-MS analysis of sialic acid using a constant column temperature of 175 °C. Methyl N-acetyl-9-O-acetyl-N-methyl-2,4,7,8-tetra-O-methylneuraminate from 9-linked NeuAc, t_R [min] = 25.1.

Ganglioside (ca. 0.1 mg) was heated with 5% HCl/MeOH (0.2 mL) in a sealed tube at 80 °C for 2 h. The reaction mixture was diluted with MeOH (0.3 mL) and extracted with n-hexane. The extract was concentrated in vacuo to give a mixture of FAMEs, which was subjected to GC-MS [column temperature 180–320 °C (rate of temperature increase: 4 °C/min)]. The remaining MeOH layer, which contained a mixture of lyso-cerebroside, free long chain base and methyl glycoside, was dried in vacuo. The resiude was dissolved in CH₂Cl₂ (0.1 mL), a silica gel-supported NaIO₄ reagent (ca. 0.2 mg) was added, and the mixture was vigorously stirred at room temperature. After 30 min, the reaction mixture was filtered through a short silica gel column and washed with CH₂Cl₂. Removal of solvent from the filtrate afforded a mixture of long chain aldehydes (LCAs), which was subjected to GC-MS [column temperature 180–320 °C (rate of temperature increase: 4 °C/min)]. FAMEs; methyl 2-hydroxyicosanoate, t_R [min] = 26.8: methyl 2-hydroxyhenicosanoate, t_R [min] = 27.8: methyl 2-hydroxydocosanoate, t_R [min] = 28.9: methyl 2-hydroxytricosanoate, t_R [min] = 29.9: methyl 2-hydroxytetracosanoate, t_R [min] = 30.8. LCAs; tridecanal, t_R [min] = 14.4: 12-methyl tridecanal, t_R [min] = 15.4: tetradecanal, t_R [min] = 16.0: 13-methyl tetradecanal, t_R [min] = 17.0: 12-methyltetradecanal, t_R [min] = 17.1: pentadecanal, t_R [min] = 17.5: 13-methyl pentadecanal, t_R [min] = 18.6.