Structures and Bioactivities of Psolusosides B1, B2, J, K, L, M, N, O, P, and Q from the Sea Cucumber Psolus fabricii. The First Finding of Tetrasulfated Marine Low Molecular Weight Metabolites

Ten new di-, tri- and tetrasulfated triterpene glycosides, psolusosides B1 (1), B2 (2), J (3), K (4), L (5), M (6), N (7), O (8), P (9), and Q (10), were isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk near the Kurile Islands. Structures of these glycosides were established by two-dimensional (2D) NMR spectroscopy and HR-ESI mass-spectrometry. It is particularly interesting that highly polar compounds 9 and 10 contain four sulfate groups in their carbohydrate moieties, including two sulfates in the same terminal glucose residue. Glycoside 2 has an unusual non-holostane aglycone with 18(16)-lactone and a unique 7,8-epoxy fragment. Cytotoxic activities of compounds 1–10 against several mouse cell lines such as Ehrlich ascites carcinoma cells, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes were quite different depending both on structural peculiarities of these glycosides and the type of cells subjected to their actions. Psolusoside L (5), pentaoside, with three sulfate groups at C-6 of two glucose and one 3-O-methylglucose residue and holostane aglycone, is the most active compound in the series. The presence of a sulfate group at C-2 of the terminal glucose residue attached to C-4 of the first (xylose) residue significantly decreases activities of the corresponding glycosides. Psolusosides of group B (1, 2, and known psolusoside B) are inactive in all tests due to the presence of non-holostane aglycones and tetrasaccharide-branched sugar chains sulfated by C-2 of Glc4.


Introduction
Triterpene glycosides of sea cucumbers are well known by their structural diversity and promising biological effects [1-3], including cytotoxicity against cancer cells and antitumor activity [4][5][6]. Therefore, the search for new representatives of this class of marine natural products and studies of their biological activities seem to be relevant. Moreover, structural analysis of diverse glycosides of sea cucumbers allows us to understand the peculiarities of biosynthesis of these complicated and numerous marine metabolites.

Structural Elucidation of the Glycosides
The initial stages of isolation of compounds 1-10 were the same as for other glycosides from P. fabricii and were described earlier [10][11][12]. The individual glycosides were isolated by HPLC on reversed-phase columns to give psolusosides: B1 (1) (  The 1 H and 13 C NMR spectra corresponding to the carbohydrate chains of psolusosides B1 (1) and B2 (2) were coincident to each other and to those of known psolusoside B [12] showing the identity of their tetrasaccharide carbohydrate moieties branched by C-4 of the xylose unit and having two sulfate groups (Table S1).
The molecular formula of psolusoside B1 (1) was determined to be C55H82O31S2Na2 from the [M2Na − Na] − ion peak at m/z 1325.4164 (calc. 1325.4185) and [M2Na − 2Na] 2− ion peak at m/z 651.2157 (calc. 651.2146) in the (−)HR-ESI-MS. The signal of H-16 was observed as a broad singlet at δH 4.89 and the signal of H-17 was observed as a singlet at δH 2.97 in the 1 H NMR spectrum of 1. These data as well as corresponding signals of carbons at δC 79.9 (C-16) and δC 58.8 (C-17) (Table 1) were indicative for 18 (16)-lactone moiety (Table 1). O-acetyl group (δC 170.9 (CH3COO) and 21.6 (CH3COO) in the 13 C NMR spectrum), attached to C-20, caused the deshielding of its signal to δC 83.8 in the same manner as in the spectrum of psolusoside B [12]. The side chain of 1 was identical to that of psolusoside B due to the coincidence of those signals in the 1 H and 13 C NMR spectra. The signal at δC 199.3 corresponded The 1 H and 13 C NMR spectra corresponding to the carbohydrate chains of psolusosides B 1 (1) and B 2 (2) were coincident to each other and to those of known psolusoside B [12] showing the identity of their tetrasaccharide carbohydrate moieties branched by C-4 of the xylose unit and having two sulfate groups (Table S1).
The molecular formula of psolusoside B 1 (1) was determined to be C 55 H 82 O 31 S 2 Na 2 from the [M 2Na − Na] − ion peak at m/z 1325.4164 (calc. 1325.4185) and [M 2Na − 2Na] 2− ion peak at m/z 651.2157 (calc. 651.2146) in the (−)HR-ESI-MS. The signal of H-16 was observed as a broad singlet at δ H 4.89 and the signal of H-17 was observed as a singlet at δ H 2.97 in the 1 H NMR spectrum of 1. These data as well as corresponding signals of carbons at δ C 79.9 (C-16) and δ C 58.8 (C-17) (Table 1) were indicative for 18 (16)-lactone moiety (Table 1). O-acetyl group (δ C 170.9 (CH 3 COO) and 21.6 (CH 3 COO) in the 13 C NMR spectrum), attached to C-20, caused the deshielding of its signal to δ C 83.8 in the same manner as in the spectrum of psolusoside B [12]. The side chain of 1 was identical to that of psolusoside B due to the coincidence of those signals in the 1 H and 13 C NMR spectra. The signal at δ C 199.3 corresponded to a keto-group adjacent to a double bond (the signals of olefinic carbons at δ C 135.3 (C-8) and 169.0 (C-9)). The position of the keto-group was deduced as C-7 based on the correlations between H 2 -6 (δ H 2.42 and δ H 2.29) and C-7 (δ C 199.3) in the HMBC spectrum of 1. This was also corroborated by an isolated spin system between the doublet of doublets at δ H 1.54 (H-5) and another doublet of doublets at δ H 2.42 (H-6a) and the triplet at δ H 2.29 (H-6b) observed in the 1 H, 1 H-COSY spectrum. The 8(9)-position of double bond was confirmed by the HMBC correlations H 3 -32/C-8 and H 3 -19/C-9. So, the aglycone of psolusoside B 1 (1) is characterized by the unique combination of such structural features as 7-keto-8(9)-ene fragment and 18(16)-lactone.  [M 2Na − Na − CH 3 COOH − GlcSO 3 Na − Glc + H] − corroborating the structure of psolusoside B 1 (1).
The All these data indicate that psolusoside  (Table S2) indicating the identity of their holostane-type aglycones having 7(8)-and 25(26)-double bonds and 16-keto-group. This aglycone is common for the glycosides of sea cucumbers belonging to the orders Dendrochirotida and Aspidochirotida [2,12].
In the 1 H and 13 C NMR spectra of the carbohydrate part of psolusoside J (3) four characteristic doublets at δ H 4.60-5.12 (J = 7.3 − 8.1 Hz) and, corresponding to them, signals of anomeric carbons at δ C 101.7-105.5 were indicative of a tetrasaccharide chain and β-configurations of glycosidic bonds. The 13 C NMR spectra of tetrasaccharide carbohydrate chain of 3 and those of 1 and 2 were quite different, while the 1 H, 1 H-COSY and 1D TOCSY spectra of 3 showed the signals of four isolated spin systems assigned to one xylose and three glucose residues as in psolusosides B [12], B 1 (1), and B 2 (2). The positions of interglycosidic linkages were elucidated by the ROESY and HMBC spectra of 3 (Table 3), where the correlations between H-1 of the xylose (Xyl1) and H-3 (C-3) of the aglycone, H-1 of the second residue (glucose, Glc2) and H-2 (C-2) of the xylose (Xyl1), H-1 of the third residue (glucose, Glc3) and H-4 (C-4) of the second residue (glucose, Glc2), H-1 of the fourth residue (glucose, Glc4) and H-4 (C-4) of the first residue (xylose, Xyl1) were observed, indicating the same architecture of sugar chains in 3 and 1 and 2. The comparison of the NMR spectra of 1 and 3 showed the coincidence of the signals of three monosaccharide residues corresponding to the linear part of the carbohydrate chain (residues I-III). The signals of terminal monosaccharide unit attached to C-4 of the first (Xyl1) unit, assigned by the 1 H, 1 H-COSY and 1D TOCSY spectra of 3 were indicative of a sulfated by C-2 glucose residue due to characteristic shifting effects observed in the 13 C NMR spectrum: the signal of C-2 Glc4 was deshielded to δ C 81.2 and the signal of C-1 Glc4 was shielded to δ C 101.7 in comparison with the corresponding signals of the same sugar unit in the 13 C NMR spectrum of psolusoside I isolated by us earlier [12].
The δ C of the signals of C-2 and C-1 of the fourth monosaccharide unit (Glc4) in the 13 C NMR spectrum of psolusoside J (3) were very close to those in the 13 C NMR spectrum of 1, corroborating the presence of a sulfate group at C-2 of this residue (Glc4). The correlations between H-2/H-3/H-4 in this monosaccharide residue, deduced by the 1 H, 1 H-COSY spectrum of 3, indicated the signal of H-4 Glc4 at δ H 4.90. The signal of the corresponding carbon (C-4 Glc4), deduced by the HSQC spectrum, was downshifted to δ C 77.3 as compared with the same signal (C-4 Glc4) at δ C 70.7 in the 13 C NMR spectrum of 1. Actually, the signals at δ C~7 0.4-70.8 were absent and the signals of C-3 Glc4 and C-5 Glc4 were upshifted to δ C 75.6 and 76.6, correspondingly, in the 13 C NMR spectrum of 3 due to β-shifting effect of sulfate group, when compared with the corresponding signals in the 13 C NMR spectrum of 1. Considering that (−)HR-ESI-MS indicated the presence of three sulfate groups as well as the NMR data, the attachment of the third sulfate group to C-4 of Glc4 was supposed. The signal at δ C 62.4 (C-6 Glc4) was characteristic for carbons of non-sulfated hydroxy-methylene groups of glucopyranose residues and excluded the positioning of the third sulfate group at C-6 Glc4 that confirmed our supposition. Hence psolusoside J (3) is a trisulfated tetraoside with two sulfate groups attached to the same glucose residue. To the best our knowledge, this structural feature is first found in the glycosides. Table 3. 13 The 13 C NMR spectra of the aglycone moieties of the glycosides 4-10 were identical to each other (Table S3) and to those of psolusosides E, F, and G containing 16-ketoholosta-9(11),25-dien-3β-ol as an aglycone, known earlier and frequently occurring in the glycosides of sea cucumbers [12].
The molecular formula of psolusoside K (4) was determined to be C  (Table 4) as described above indicating the presence of a tetrasaccharide carbohydrate chain branched by C-4 of the xylose residue (Xyl1). The monosaccharide composition of 4, deduced from the 1 H, 1 H-COSY and 1D TOCSY spectra, was the same as in glycosides 1-3. The comparison of the 13 C NMR spectra of trisulfated compounds 3 and 4 showed the coincidence of the signals corresponding to three monosaccharide residues (residues I-III in the formula) forming the linear part of the sugar chain. The signals of C-2 Glc4 at δ C 80.3 and C-1 Glc4 at δ C 101.4 in the 13 C NMR spectrum of 4 were very close to those in the spectrum of 3 that indicated the attachment of a sulfate group to C-2 Glc4 in psolusoside K (4). All of the signals of this monosaccharide residue were assigned using the 1 H, 1 H-COSY and 1D TOCSY spectra. The doublet at δ H 5.00 and the doublet of doublets at δ H 4.63 corresponded to the protons of the hydroxy-methylene group of the terminal glucose unit (H 2 -6 Glc4) and were deshielded as compared with the corresponding signals in the 1 H NMR spectrum of 3. The signal at δ C 67.4 (C-6 Glc4) also indicated the presence of a sulfate group at C-6 of Glc4 in addition to another sulfate group at C-2 of Glc4. So, psolusoside K (4) is an isomer of psolusoside J (3) by the sulfate position and is the second glycoside from sea cucumbers that contains two sulfate groups bonded to the same monosaccharide residue. The The  Table 5). Analysis of the 1 H, 1 H-COSY and 1D TOCSY spectra of psolusoside L (5) showed the presence of one xylose, one quinovose, two glucose, and one 3-O-methylglucose residues. The presence of a quinovose residue was confirmed by the 1 H and 13 C NMR spectra demonstrating the characteristic doublet at δ H 1.59 (H-6 Qui2) and the signal at δ C 17.7 (C-6 Qui2). The positions of interglycosidic linkages and the consequence of monosaccharides in the chain of 5 were established by analysis of the ROESY and HMBC spectra (Table 5) indicating the presence of branched pentasaccharide moiety with glucose, attached to C-4 Xyl1, and 3-O-methylglucose, attached to C-3 Glc3, as terminal residues. The 13 C NMR spectrum of 5 demonstrated three signals at δ C 67.0, 67.5, and 67.6, corresponding to sulfated hydroxy-methylene groups of glucopyranose residues that indicated the sulfation of two glucose and 3-O-methylglucose units in the carbohydrate chain of 5.  The comparison of the 13 C NMR spectrum of the sugar part of psolusoside L (5) with those of known achlioniceosides A 1 , A 2 , and A 3 , with identical carbohydrate chains, isolated earlier from the sea cucumber Rhipidothuria racowitzai [14] showed the coincidence of the signals of four monosaccharide residues in their spectra. The signals of terminal 3-O-methylglucose residues of the novel and known compounds were different due to the absence of a sulfate group in this residue of known compounds. All these data indicated that psolusoside L (5) Table 6). Analysis of the 1 H, 1 H-COSY and 1D TOCSY, ROESY, and HMBC spectra of psolusoside M (6) showed the same monosaccharide composition and architecture of the carbohydrate chain as in 5. Actually, the comparison of their 13 C NMR spectra showed the closeness of the signals corresponding to the monosaccharides from the first to the fourth. The differences of the 13 C NMR spectra of compounds 6 and 5 were concerned with the terminal glucose residue (Glc5) connected to C-4 Xyl1. The characteristic signals at δ C 100.9 (C-1 Glc5) and at δ C 80.6 (C-2 Glc5) in the 13 C NMR spectrum of 6 were very close to the corresponding signals in the spectra of the compounds 1-4 indicating the presence of a sulfate group at C-2 Glc5 in the psolusoside M (6). At the same time, the hydroxy-methylene group of this sugar was free from sulfation, since the signal of C-6 Glc5 was observed at δ C 61.8. Two signals of sulfated hydroxy-methylene groups of the glucose (Glc3) and 3-O-methylglucose (MeGlc4) residues were observed at δ C 67.5 and 67.0 in the 13 C NMR spectrum of 6. Therefore, psolusoside M (6)

All these data indicate that psolusoside M (6) is 3β-O-{6-O-sodium-sulfate-3-O-methyl-β-dglucopyranosyl-(1→3)-6-O-sodium-sulfate-β-d-glucopyranosyl-(1→4)-β-d-quinovopyranosyl-(1→2)-[2-O-sodium-sulfate-β-d-glucopyranosyl-(1→4)]-β-d-xylopyranosyl}-16-ketoholosta-9(11),25-diene.
The molecular formula of psolusoside N (7) was determined to be C 60 H 91 O 37 Table 7). Analysis of the 1 H, 1 H-COSY and 1D TOCSY spectra of psolusoside N (7) showed the presence of one xylose, three glucose, and one 3-O-methylglucose residues. The positions of interglycosidic linkages and the consequence of monosaccharides in the carbohydrate chain of 7 were established in the same manner as for 1-6 ( Table 7) indicating the presence of branched pentasaccharide moiety having the same architecture as in compounds 5 and 6. The comparison of the 13 C NMR spectra of 7 and 5 showed the closeness of the signals of all the monosaccharide residues except for the signals assigned to the second sugar units in their chains. Actually, in the 1 H and 13 C NMR spectra of 7, the signals characteristic of quinovose residue were absent but two doublets of doublets at δ H 4.95 (H-6a Glc2) and at δ H 4.75 (H-6b Glc2) and the signal at δ C 61.0 (C-6 Glc2), assigned to hydroxy-methylene group of glucopyranose moiety, were detected. These data indicated the replacement of quinovose by the glucose residue in the second position of a carbohydrate chain in psolusoside N (7) as compared with psolusoside L (5). Three sulfate groups were supposed to attach the C-6 of two glucose and 3-O-methylglucose residues due to the signals at δ C 67.4, 67.5, and 66.9 observed in the spectrum of 7. The carbohydrate chain of psolusoside N (7) is the first found in the glycosides from holothurians.
The In the 1 H and 13 C NMR spectra of the carbohydrate part of psolusoside O (8), five characteristic doublets at δ H 4.60-5.12 (J = 7.0-8.6 Hz) and, corresponding to them, signals of anomeric carbons at δ C 101.0-104.8 indicated a pentasaccharide carbohydrate chain and β-configurations of glycosidic bonds (Table 8).  Analysis of the 1 H, 1 H-COSY and 1D TOCSY spectra of psolusoside O (8) showed the same monosaccharide composition and positions of interglycosidic linkages as in the carbohydrate chain of compound 7 (Table 8). The coincidence of the molecular formulae of 8 and 7 and the presence of three-charged ions in the (−)HR-ESI-MS of 8 indicated their difference in the position of a sulfate group. Really, the signals of monosaccharide residues from the first to the fourth were almost coincident in their 13 C NMR spectra. The characteristic signals at δ C 101.0 and δ C 80.6 indicated the bonding of a sulfate group to C-2 of a terminal residue which glycosylates C-4 Xyl1. Analysis of the 1 H, 1 H-COSY and 1D TOCSY spectra of 8 showed this unit is a glucose (Glc5). Indeed, the comparison of the 13  All these data indicate that psolusoside O (8) The molecular formula of psolusoside P (9) was determined to be C  Table 9). Analysis of the 1 H, 1 H-COSY and 1D TOCSY spectra of psolusoside P (9) showed the presence of one xylose, one quinovose, two glucose, and one 3-O-methylglucose residues. The positions of interglycosidic linkages and the consequence of monosaccharides in the chain of 9 established by the ROESY and HMBC spectra were the same as in the glycosides 5 and 6 ( Table 9). The comparison of the 13 C NMR spectra of the compounds 9 and 5 showed the coincidence of the signals corresponding to the monosaccharides from the first to the fourth indicating their identity in these glycosides. The signals of the fifth terminal sugar residue assigned by the 1 H, 1 H-COSY and 1D TOCSY spectra corresponded to the glucose residue sulfated by C-6 (the signal at δ C 67.9 (C-6 Glc5)). Thus, three sulfate groups were positioned at C-6 of 3-O-methylglucose (MeGlc4) and C-6 of two glucose residues (Glc3 and Glc5) in the carbohydrate chain of psolusoside P (9). The position of the fourth sulfate group at C-4 Glc5 was established by the comparison of the 13 C NMR spectra of psolusosides P (9) and L (5). The signal of C-4 Glc5, deduced by the 1 H, 1 H-COSY spectrum of 9, was deshielded to δ C 77.1 due to α-shifting effect of the sulfate group, as compared with the corresponding signal in the 13 C NMR spectrum of 5 observed at δ C 70.7. Oppositely, the signal of C-5 Glc5 was shielded to δ C 73.7 in the spectrum of 9 due to the β-shifting effect of the sulfate group as compared with the spectrum of 5 (δ C 75.65 (C-5 Glc5)). So, psolusoside P (9) is the first case of triterpene glycoside having four sulfate groups, in that two of them were connected to one monosaccharide residue. The (−)ESI-MS/MS of 9 demonstrated the fragmentation of [M 4Na − Na] − ion at m/z 1631.4. The peaks of fragment ions were observed at m/z: 1265.4 [M 4Na − Na − C 6 H 8 O 11 S 2 Na 2 (Glc(SO 3 Na) 2 )] − , 1233.4 [M 4Na − Na − C 7 H 12 O 9 SNa (MeGlcSO 3 Na) − NaSO 3 ] − , 1145.5 [M 4Na − Na − C 6 H 9 O 11 S 2 Na 2 (Glc(SO 3 Na) 2 − NaSO 4 ] − , 1089.4 [M 4Na − Na − C 7 H 12 O 9 SNa (MeGlcSO 3 Na) − C 6 H 8 O 7 SNa (GlcSO 3 Na)] − , 969.4 [M 4Na − Na − C 7 H 12 O 9 SNa (MeGlcSO 3 Na) − C 6 H 8 O 7 SNa (GlcSO 3 Na) − NaHSO 4 ] − , and 943.3 [M 4Na − Na − C 7 H 12 O 9 SNa (MeGlcSO 3 Na) − C 6 H 8 O 7 SNa (GlcSO 3 Na) − C 6 H 10 O 4 (Qui)] − corroborating the structure of carbohydrate chain of psolusoside P (9).
All these data indicate that psolusoside P (9)  In the 1 H and 13 C NMR spectra of the carbohydrate part of psolusoside Q (10), five characteristic doublets at δ H 4.61-5.12 (J = 6.7-8.4 Hz) and, corresponding to them, signals of anomeric carbons at δ C 101.5-104.8, were indicative of a pentasaccharide chain and β-configurations of glycosidic bonds ( Table 10). The molecular weights of tetrasulfated psolusosides P (9) and Q (10) differed by 16 amu in HR-ESI-MS that along with the absence of the signals corresponding to the quinovose residue in the NMR spectra of 10 indicated the presence of a glucose residue in the second position of its carbohydrate chain. Actually, the coincidence of the signals of monosaccharide residues from the first to the fourth the 13 C NMR spectra of psolusosides Q (10), N (7), and O (8) confirmed this supposition. Analysis of the 1 H, 1 H-COSY, 1D TOCSY, ROESY, and HMBC spectra of psolusoside Q (10) showed the same monosaccharide composition and the consequence of monosaccharides in the chain of 10 as in psolusosides N (7) and O (8) ( Table 10). The characteristic signals at δ C 101.5 (C-1 Glc5) and δ C 80.3 (C-2 Glc5) indicated attachment of a sulfate group to C-2 of the fifth residue (Glc5) in the sugar part of 10. The signal of C-6 Glc5 was assigned by the HSQC spectrum of 10, demonstrating the correlation of the both doublet at δ H 5.02 (H-6a Glc5) and doublet of doublets at δ H 4.64 (H-6b Glc5) with the corresponding resonance at δ C 67.5 that indicated the presence of an additional sulfate group at C-6 Glc5 in psolusoside Q (10). All these data show that psolusoside Q (10) has a new carbohydrate chain with four sulfate groups, in that two of them are attached to C-2 and C-6 of the same (Glc5) residue.
The ( All these data indicate that psolusoside Q (10) is 3β Thus, highly polar tetrasulfated glycosides are first discovered in sea cucumbers. Although polysulfated polysaccharides are common biopolymers of marine macrophytes and invertebrates, low molecular weight metabolites, containing several sulfate groups are extremely rare. So far, trisulfated natural compounds such as steroid glycosides were found only in sponges [15][16][17] and trisulfated triterpene glycosides, in some representatives of the class Holothuroidea [18,19].

Bioactivity of the Glycosides
The cytotoxic activities of the compounds 1-10 as well as known earlier psolusosides G (used as a positive control) and B [12] against mouse erythrocytes (hemolytic activity), the ascite form of mouse Ehrlich ascites carcinoma cells, neuroblastoma Neuro 2A cells, and normal epithelial JB-6 cells are presented in Table 11. The biological effects of the investigated substances were quite different due to the diverse structures of their aglycones and carbohydrate chains. Moreover, hemolytic effects of these compounds were higher than their cytotoxicity against other cells, especially against the Ehrlich ascites carcinoma cells. For instance, psolusoside P (9) demonstrated high hemolytic action, but moderate cytotoxicity against Neuro 2A and JB-6 cells and was not active against mouse Ehrlich carcinoma cells (ascite form). The analogic dependency was observed for psolusosides M (6) and O (8), which were not cytotoxic against all the cell lines except erythrocytes. Psolusoside L (5) was shown to be the most active substance in the series. It has a holostane-type aglycone and pentasaccharide chain with three sulfate groups at C-6 of two glucose and 3-O-methylglucose residues. It is very unusual for a glycoside with three sulfate groups to demonstrate high cytotoxic properties, because it is known that sulfate groups attached to the C-6 position of the terminal glucose and 3-O-methylglucose residues greatly decrease the activity of pentaosides branched by the second monosaccharide unit (quinovose) sugar chains [3]. Probably, the peculiarities of architecture of a carbohydrate chain of 5 (the branching at C-4 Xyl1) compensate the negative influence of the three sulfate groups.
The activity of psolusoside N (7) was slightly lower than that of 5, due to the presence of a glucose residue as the second unit in the sugar chain instead of the quinovose (in 5) that is in good accordance with the earlier observations of the glycoside's SAR [3]. The alteration of the sulfate position attached to the terminal (glucose) residue from C-6 Glc5 to C-2 Glc5 caused the extreme decrease in the activity. This was illustrated by the effects of psolusoside M (6) differing from the compound 5 in this character only and demonstrating much lower hemolytic action than 5 and the absence of the activity against other tested cells. The same relationship was observed for psolusosides N (7) and O (8) differing from each other in the position of the sulfate group in the fifth (Glc5) residue.
The tetrasulfated (at C-6 Glc3, C-6 MeGlc4, C-6 Glc5, and C-4 Glc5) psolusoside P (9) demonstrated high hemolytic and moderate cytotoxic action against Neuro-2A and JB-6 cells and was not active against ascites of Ehrlich carcinoma. However, it was much more active than trisulfated psolusoside M (6) containing sulfate group at C-2 Glc5. The activity of tetrasulfated psolusoside Q (10) was also strongly reduced by the sulfate group attached to C-2 Glc5 as well as by the presence of glucose in the second position of its carbohydrate chain.
Psolusosides B [12], B 1 (1), and B 2 (2) were not active in all the tests due to the presence of non-holostane aglycones in combination with the tetrasaccharide-branched carbohydrate chain sulfated by C-2 of terminal residue (Glc4) attached to C-4 Xyl1. Moreover, psolusosides J (3) and K (4) with carbohydrate chains with the same architecture and sulfate group at C-2 of the terminal residue (Glc4) were also inactivated despite the presence of holostane aglycones.

Animals and Cells
Specimens of the sea cucumber Psolus fabricii (family Psolidae; order Dendrochirotida) were collected in the Sea of Okhotsk near Onekotan Island (Kurile Islands). Sampling was performed with a scallop dredge in August-September 1982 at a depth of 100 m during expedition works on fishing seiners "Mekhanik Zhukov" and "Dalarik". Sea cucumbers were identified by V.S. Levin. Voucher specimens were preserved in the A.V. Zhirmunsky National Scientific Center of Marine Biology, Vladivostok, Russia.
CD-1 mice weighing 18-20 g were purchased from RAMS 'Stolbovaya' nursery (Russia) and kept at the animal facility in standard conditions. All experiments were conducted in compliance with all of the rules and international recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental Studies.
The museum tetraploid strain of murine ascite Ehrlich carcinoma (EAC) cells from the All-Russian Oncology Center (Moscow, Russia) was used. EAC cells were injected into the peritoneal cavity of CD-1 mice. Cells for experimentation were collected 7 days after inoculation. For this purpose, mice were killed by cervical dislocation, and the ascitic fluid containing tumor cells was collected with a syringe. The cells were washed triply by centrifugation at 2000 rpm (450 g) for 10 min in PBS (pH 7.4) followed by resuspension in RPMI-1640 medium containing 8 µg/mL gentamicin (BioloT, Saint Peterburg, Russia). Neuroblastoma Neuro 2A cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS; BioloT, Saint Petersburg, Russia), normal epithelial JB-6 cells were cultured in DMEM medium containing 5% fetal bovine serum (BioloT, Saint Petersburg, Russia), and 1% penicillin/streptomycine (Termo Fisher Scientific (Invitrogen), Waltham, Massachusetts, USA).

Extraction and Isolation
The sea cucumbers (about 800 specimens, average weight of one specimen is about 100 g) were minced and extracted twice with refluxing 60% EtOH. The extract was evaporated to water residuum and lyophilized followed by extraction with CHCl 3 /MeOH (1:1). The obtained extract was evaporated and submitted to the subsequent extraction by EtOAc/H 2 O to remove the lipid fraction. those by the second monosaccharide residue, which is a glucose instead of a quinovose. Psolusosides N (7) and O (8) are the structural analogs of psolusosides L (5) and M (6), correspondingly, having identical sulfate groups positions. Tetrasulfated psolusoside Q (10) differs from psolusoside P (9) by the positions of sulfation-at C-2 and C-6 of terminal (Glc5) residue. Tetrasulfated glycosides have not ever been found in any natural objects.
The present investigation is conclusive in a series of research concerning the glycosides of the sea cucumber Psolus fabricii. Generally, 27 new and 5 known earlier triterpene glycosides have been isolated from this animal. These compounds contain six previously unknown aglycones and 13 novel carbohydrate chains.
The sulfated oligosaccharide moieties predominate in the glycosides of P. fabricii. Monosulfated trisaccharide (psolusosides H and H 1 ) and linear tetrasaccharide (psolusosides E and F) moieties, disulfated branched tetrasaccharide (psolusosides B, B 1 (1), B 2 (2), and I) or linear tetrasaccharide (psolusosides A and G) carbohydrate chains, trisulfated branched tetrasaccharide (psolusosides J (3) and K (4)) carbohydrate chains, and finally pentasaccharide trisulfated (psolusosides L (5), M (6), N (7), O (8)) and tetrasulfated (psolusosides P (9) and Q (10)) carbohydrate chains were found in glycosides of P. fabricii. The sugar chains also differ from each other by the second monosaccharide unit (quinovose, glucose, or xylose). The most variable structural feature of the carbohydrate chains of the glycosides 1-10 is the quantity (one or two) and positions of the sulfate groups in terminal glucose unit, attached to C-4 Xyl1. There are three combinations of such positions of sulfate groups in these residues: C-2 and C-4, C-2 and C-6, or C-4 and C-6. Whereas the single sulfate group bonds only to C-2 or C-6 of terminal glucose unit.
It is interesting to note that diverse groups of psolusosides (A-Q) (a certain group of glycosides consists of substances with the same carbohydrate chain and diverse aglycones) characterized by different structural variability of aglycones. All psolusosides belonging to groups C and D (both containing hexasaccharide non-sulfated sugar chains) have the holostane-type aglycones with 9(11)-double bond, 16-keto-group, and different side chains (5 variants). Psolusosides of the group B contain exclusively non-holostane aglycones with 18(16)-lactone and 7(8)-double bond, completely different from the aglycones of the other groups of psolusosides. These could be explained by their special biological functions in the organism-producer. Four holostane aglycones with 7(8)-, or 9(11)-double bond were found in five glycosides having trisaccharide (psolusosides H and H 1 ) or tetrasaccharide-branched carbohydrate chains (psolusosides I, J, K).
Hence, the biogenetic analysis of the structures of glycosides found in P. fabricii showed that carbohydrate chains and aglycones biosynthesis possesses a mosaic (combinatoric) character, which also has some trends.