Chilensosides E, F, and G—New Tetrasulfated Triterpene Glycosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida): Structures, Activity, and Biogenesis

Three new tetrasulfated triterpene glycosides, chilensosides E (1), F (2), and G (3), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E (1) and F (2) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G (3) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3—a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2—excludes the possibility of this sugar chain’s further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1–3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.


Introduction
Triterpene glycosides from sea cucumbers are long-studied metabolites that still draw interest from researchers from different scientific fields. The structural studies of the compounds from holothuroids-representatives of different taxonomic groups-are predominating [1-4], but they are in close connection with biological activity research [4][5][6][7][8] and, therefore, structure-activity relationships analyses [3,9]. An additional direction of the glycosides' applied investigations is their use as chemotaxonomic markers to clarify the systematic position of the producing species of sea cucumbers [10][11][12]. This issue is also relevant for the species under investigation-Paracaudina chilensis, belonging to the family Molpadida. The systematics and phylogeny of this taxonomic group have raised questions until now [13,14]. Our recent study concerning the isolation and structural elucidation of chilensosides A-D-the glycosides from P. chilensis-demonstrated their structural similarity to the glycosides isolated from different representatives of the order Dendrochirotida, indicating the closeness of Molpadida to Dendrochirotida [15]. Chilensosides A-D contains two different aglycones and four types of carbohydrate chains, differing in the positions and quantity of sulfate groups from two (chilensosides A, A 1 , B) to three in chilensoside
Mar. Drugs 2023, 21, x FOR PEER REVIEW 2 of 14 Dendrochirotida, indicating the closeness of Molpadida to Dendrochirotida [15]. Chilensosides A-D contains two different aglycones and four types of carbohydrate chains, differing in the positions and quantity of sulfate groups from two (chilensosides A, A1, B) to three in chilensoside C and four in chilensoside D. Three out of five glycosides demonstrated relatively high hemolytic and cytotoxic activity. In continuation of the structural research on the glycosides from P. chilensis, the isolation, structure elucidation, biologic activity testing, and carbohydrate chain biogenesis of new sulfated highly polar glycosides chilensosides E (1), F (2), and G (3) are reported. Each of the three new compounds contains four sulfate groups. The first finding of two tetrasulfated glycosides in sea cucumbers has occurred fairly recently in Psolus fabricii [16]. Three years later, three other glycosides containing four sulfate groups were found in the sea cucumber Psolus chitonoides [17] and one in P. chilensis [15]. The finding of tetrasulfated glycosides in recent years could be explained by the increased ability of HPLC separation techniques, including the use of different sorbents (immobile phases) to separate earlier inseparable polar substances [18]. The expansion of knowledge concerning the structural variability of the glycosides enables the clarification of the biosynthetic pathways of these metabolites.
The chemical structures of 1-3 were elucidated by the analyses of the 1 H, 13 C NMR, 1D TOCSY, and 2D NMR ( 1 H, 1 H-COSY, HMBC, HSQC, ROESY) spectra, as well as HR-ESI mass spectra. All the original spectra are displayed in Figures S1-S24 in the Supplementary data. The hemolytic activity on human erythrocytes and cytotoxic activities on leukemia promyeloblast HL-60, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, human neuroblastoma SH-SY5Y, and monocytic THP-1 cells were tested.

Structure Elucidation of the Glycosides
The crude glycosidic mixture of Paracaudina chilensis was isolated by hydrophobic chromatography of the concentrated ethanolic extract on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). Its further separation using chromatography on Si gel columns with a stepped gradient of the system of eluents CHCl 3 /EtOH/H 2 O in the ratios of 100:100:17, 100:125:25, and 100:150:50 was followed by the additional purification of the obtained fractions, yielding the subfractions I.0, I.1, II, III.1, and III.2. The individual glycosides 1-3 ( Figure 1) have been isolated using HPLC of subfractions II and III.2 on silicabased columns Supelcosil LC-Si (4.6 × 150 mm), and reversed-phase semipreparative columns Supelco Ascentis RP-Amide (10 × 250 mm) and Diasfer 110 C-8 (4.6 × 250 mm). The sugar configurations in the glycosides 1-3 were assigned as D, along with the biogenetic analogies with all other known triterpene glycosides from the sea cucumber. The sugar configurations in the glycosides 1-3 were assigned as D, along with the biogenetic analogies with all other known triterpene glycosides from the sea cucumber.
The holostane aglycones of chilensosides E (1), F (2), and G (3) ( Table 1, Tables S1 and S2, Figures S1-S7, S9-S15 and S17-S23) have 9(11)-and 24(25)-double bonds as well as a 16-oxo-group, and are identical to each other and those of chilensosides A 1 , B, C, and D, isolated earlier [15]. The identity of the aglycones of compounds 1-3 was evidenced by the coincidence of their NMR spectroscopic data. The structure of the aglycone moiety of 1 was established based on 2D NMR spectra analyses, where the characteristic features were found: 18 (20)-lactone signals at δ C 176.9 (C-18) and δ C 83.1 (C-20), 9(11)-double bond signals at δ C 151.1 (C-9), δ C 111.3 (C-11) and δ H 5.37 (brs, H-11), and the downfield signal of quaternary carbon at δ C 214.6 that corresponded to a carbonyl group at C-16.         4 Glc3)). The HSQC spectrum correlated them with two carbon signals at δ C 83.8 (C-3 Glc3) and 67.4 (C-6 Glc3). These data indicated that two sulfate groups were linked to the Glc3 residue at C-3 and C-6. The ROESY and HMBC correlations of H-3 Glc3 with any protons or carbons of neighboring monosaccharide residues were absent. These data indicated that Glc3 is a terminal residue of the bottom semi-chain. The attachment of the third sulfate group to C-6 Glc4 was deduced from the characteristic signal at δ C 67.7 assigned in the same manner as for Glc3. The position of the last sulfate group was established to be at C-4 MeGlc5 due to the deshielding of its signal to δ C 76.1 when compared with corresponding signals in the spectra of chilensosides A and C observed at δ C 70.0 [15]. Moreover, all the signals of this monosaccharide unit in the 13 (Table 3, Figures  S9-S15). Two glucose residues (Glc3 and Glc4) were sulfated by C-6, which was deduced from the presence of characteristic signals of the sulfated hydroxy methylene groups at δ C 67.4 and 68.5, while the 3-O-methylglucose residue did not bear any sulfates (δс 70.0 (C-4 MeGlc5) and δс 62.0 (C-6 MeGlc5)). An additional two sulfates were attached to C-3 Glc3 and C-4 Glc4, deduced from the downfield shifting of their signals to 83.9 and 75.5, respectively. The comparison of the signals corresponding to the Glc3 residue in the 13 C NMR spectra of chilensosides E (1) and F (2) showed their coincidence corroborating the presence of sulfate groups at C-3 Glc3 and C-6 Glc3 in 2. The same procedure was conducted for the signals of the Glc4 residue in the 13 C NMR spectra of chilensosides A, A 1 , C [15], and F (2) and confirmed the sulfate groups attachment to C-4 Glc4 and C-6 Glc4 in 2. The deshielding of the signal of C-4 Glc4 to δ C 75.5 due to the α-shifting effect of the sulfate group in the spectrum of 2 compared with the same signal in the spectrum of 1 (δ C 69.5) and corroborated the position of the sulfate group. Therefore, chilensoside F (2) has a tetrasulfated sugar chain with two disulfated glucose residues.
The ( (Table 4, Figures S17-S23).  The positions of sulfate groups were established as a result of NMR spectra analyses. The typical values of chemical shifts observed due to the sulfate groups shifting effects were found. The signal of C-6 Glc3 was deshielded to δ C 67.4, and the signal of C-5 Glc3shielded to δ C 74.7, indicating the sulfation of the hydroxymethylene group of this residue. At the same time, the signal of C-3 Glc3 was observed at δ C 86.1 due to the glycosylation effect appearing because of the attachment of the terminal (Glc4) unit to this position. The ROESY correlation H-1 Glc4/H-3 Glc3 confirmed this supposition. As a result of the analysis of the isolated spin system corresponding to the Glc5 residue, two deshielded signals (in comparison with the signals of non-sulfated carbons) were assigned to sulfated carbons, C-4 Glc5 (δ C 75.3) and C-6 Glc5 (δ C 68.2). Another downfield shifted signal at δ C 80.1 was attributed to position 3 of Glc5 glycosylated by the sixth monosaccharide unit. The last sulfate group was attached to C-6 of the terminal residue in the upper semi-chain-MeGlc6. A corresponding signal (C-6 MeGlc6) was observed at δ C 66.8. The presence of a terminal non-methylated glucose unit (Glc4) was established by the shielded signal of C-3 Glc4 at δ C 77.2, which was observed instead of the signal at δ C~8 6.5 in 3-O-methylated derivatives. The absence of the signal of the second OMe-group at δ C~6 0.5 in the 13 C NMR spectrum of 3 was an additional confirmation. Hence, chilensoside G (3) is a tetrasulfated hexaoside that expanded the list of the most polar glycosides of sea cucumbers found so far.

Bioactivity of the Glycosides
The cytotoxic activities of chilensosides E-G (1-3) against human cell lines, erythrocytes, and cancer cells, including neuroblastoma SH-SY5Y, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, leukemia promyeloblast HL-60, and monocytic THP-1 have been studied. The earlier tested chitonoidoside L [3] was used as the positive control in all the tests ( Table 5). The activity of the glycosides against SH-SY5Y, HeLa, and DLD-1 cells was examined using an MTT assay and against HL-60 and THP-1 cells using an MTS assay.  Erythrocytes, in agreement with earlier published data [15,19], exhibited an increased sensitivity to the membranolytic action of sea cucumber glycosides compared to cancer cells. The erythrocytes are a traditional and convenient model for investigating the membranolytic action of the glycosides. All compounds 1-3 showed moderate hemolytic activity, allowing us to suppose the cytotoxic doses of the investigated compounds will be relatively high. Chilensosides E-G (1-3) were not cytotoxic against cancer cell lines even at the maximal tested concentration (100 µM). As previously observed, the presence of four sulfate groups alone did not deplete the activity [17]. However, the decreasing membranolytic activity caused by the increasing number of sulfates was reported earlier for some of the glycosides [20]. Generally, the influence of sulfate groups on the activity of the glycosides significantly depends on the structural characteristics of their carbohydrate chains [9]. So, in the case of chilensosides E-G (1-3), the combination of sulfates quantity and positions presumably negatively affected their activity. This was in good agreement with the previous tests of cytotoxicity of tetrasulfated chilensoside D, which has been inactive in relation to three of five cancer cell lines [15]. The analysis of the structure-activity relationships of the glycosides isolated from P. chilensis and the comparison between the effects of trisulfated chilensoside C, tetrasulfated chilensosides D [15], and E-G (1-3) have allowed the finding of the common feature of tetrasulfated glycosides that presumably extremely decreased their activity against cancer cells-the presence of a sulfate group at C-6 Glc3 (on the bottom semi-chain). However, the much more complicated influence of the glycosides' different structural features, including aglycones' structures, carbohydrate chains, architecture and composition, and their combinations on the glycoside/membrane interactions is obvious.

Biogenesis of Chilensosides A-G
The majority of chilensosides share the same overall structures, including the same aglycones and monosaccharide composition. Consequently, biosynthesis of the glycosides of P. chilensis looks somewhat strictly directed. However, the combinatorial features of biosynthesis [18,21] have become evident when the character of sulfation of sugar chains of these glycosides is analyzed. The biogenetic analysis of this series of glycosides has revealed that they form a network instead of biogenetic rows due to the enzymatic introduction of sulfate groups in almost all possible positions (Figure 3). The trend has been derived from the biogenetic analysis that the upper semi-chains are sulfated before the bottom ones, and C-6 of the glucose residues attached to C-4 Xyl1 take precedence in the sulfation. Noticeably, the presence of a sulfate group at C-3 Glc3-the terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2-excludes the possibility of further sugar chain elongation. Thus, chilensosides C [15] and F (2) could not be the biosynthetic precursors of hexaoside chilensoside G (3). More likely, chilensoside G (3) is biosynthesized through the chilensosides A and D (Figure 2), having a free hydroxyl group available for glycosylation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, a characteristic feature of the mosaic type of biosynthesis [18,21].

Animals and Cells
The sea cucumber Paracaudina chilensis (family Caudinidae; order Molpadida) (36 specimens) was harvested in Troitsa Bay, Sea of Japan  Noticeably, the presence of a sulfate group at C-3 Glc3-the terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2-excludes the possibility of further sugar chain elongation. Thus, chilensosides C [15] and F (2) could not be the biosynthetic precursors of hexaoside chilensoside G (3). More likely, chilensoside G (3) is biosynthesized through the chilensosides A and D (Figure 2), having a free hydroxyl group available for glycosylation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, a characteristic feature of the mosaic type of biosynthesis [18,21].

Animals and Cells
The sea cucumber Paracaudina chilensis (family Caudinidae; order Molpadida) (36 specimens) was harvested in Troitsa Bay, Sea of Japan The study was carried out in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of G.B > Elyakov Pacific Institute of Bioorganic Chemistry (Protocol No. 0037.12.03.2021).