Triterpene Glycosides from the Far Eastern Sea Cucumber Psolus chitonoides: Chemical Structures and Cytotoxicities of Chitonoidosides E1, F, G, and H

Four new triterpene disulfated glycosides, chitonoidosides E1 (1), F (2), G (3), and H (4), were isolated from the Far-Eastern sea cucumber Psolus chitonoides and collected near Bering Island (Commander Islands) at depths of 100–150 m. Among them there are two hexaosides (1 and 3), differing from each other by the terminal (sixth) sugar residue, one pentaoside (4) and one tetraoside (2), characterized by a glycoside architecture of oligosaccharide chains with shortened bottom semi-chains, which is uncommon for sea cucumbers. Some additional distinctive structural features inherent in 1–4 were also found: the aglycone of a recently discovered new type, with 18(20)-ether bond and lacking a lactone in chitonoidoside G (3), glycoside 3-O-methylxylose residue in chitonoidoside E1 (1), which is rarely detected in sea cucumbers, and sulfated by uncommon position 4 terminal 3-O-methylglucose in chitonoidosides F (2) and H (4). The hemolytic activities of compounds 1–4 and chitonoidoside E against human erythrocytes and their cytotoxic action against the human cancer cell lines, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and monocytes THP-1, were studied. The glycoside with hexasaccharide chains (1, 3 and chitonoidoside E) were the most active against erythrocytes. A similar tendency was observed for the cytotoxicity against adenocarcinoma HeLa cells, but the demonstrated effects were moderate. The monocyte THP-1 cell line and erythrocytes were comparably sensitive to the action of the glycosides, but the activity of chitonoidosides E and E1 (1) significantly differed from that of 3 in relation to THP-1 cells. A tetraoside with a shortened bottom semi-chain, chitonoidoside F (2), displayed the weakest membranolytic effect in the series.


Introduction
Triterpene glycosides are characteristic secondary metabolites of the sea cucumbers. Extensive studies on glycosides provide significant information on the exploration of chemical diversity, properties and biological activity of a huge collection of natural products, which are a valuable and promising resource of new drugs and medicines [1][2][3][4][5][6][7][8]. The interest in these compounds is also driven by their taxonomic specificity [9][10][11] as well as the possibility of reconstructing the sequences of the biosynthetic transformations of aglycones and carbohydrate chains during biosynthesis [12,13] and of defining the peculiarities of «structure-activity relationships» based on knowledge about their structural diversity [14]. All this indicates the relevance of searching for new glycosides. The Far Eastern sea cucumber Psolus chitonoides is the fourth chemically studied representative of the genus Psolus. The animals of this species contain a complicated multicomponent mixture of glycosides. The Far Eastern sea cucumber Psolus chitonoides is the fourth chemically studied representative of the genus Psolus. The animals of this species contain a complicated multicomponent mixture of triterpene glycosides. Therefore, their separation and purification are difficult and time-consuming. Recently, we published a paper concerning the isolation, structural elucidation, and biologic activity of a series of the glycosides, named chitonoidosides A-E, isolated from P. chitonoides [15]. These compounds feature some interesting structural features, such as a new, non-holostane aglycone lacking a lactone and featuring an 18 (20)-epoxy cycle, 3-O-methylxylose residue in the carbohydrate chains of three of them, the sulfation of 3-O-methylxylose by C-4, and, finally, a rather rare architecture of tetrasaccharide carbohydrate chain branched by C-4 Xyl1. As a continuation of our research on the glycosides from this species, four new chitonoidosides, E1 (1), F (2), G (3), and H (4), are reported. The chemical structures of 1-4 were established through the analyses of the 1 H, 13 C NMR, 1D TOCSY, and 2D NMR ( 1 H, 1 H-COSY, HMBC, HSQC, and ROESY) spectra as well as HR-ESI mass spectra. All the original spectra are presented in Figures S1-S33 in the Supplementary Data section. The hemolytic activity against human erythrocytes and cytotoxic activities against human adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and monocyte THP-1 cells were examined.

Structural Elucidation of the Glycosides
The concentrated ethanolic extract of the sea cucumber Psolus chitonoides was submitted to hydrophobic chromatography on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). The glycosides were eluted after washing with water as a mobile phase to eliminate salts and inorganic impurities with 50% EtOH. The obtained glycoside fraction was separated by the chromatography on Si gel columns with the stepped gradient of eluents CHCl 3   The configurations of the monosaccharide residues in glycosides 1-4 were assigned as D based on their biogenetic analogies with all other known sea cucumber triterpene glycosides.
It was found that chitonoidosides E1 (1), F (2) and H (4) are characterized by holotoxinogenin as aglycone, which was first found in Apostichopus japonicus and is broadly distributed in sea cucumber glycosides [16]. This was deduced from the analyses of their 1 H and The configurations of the monosaccharide residues in glycosides 1-4 were assigned as D based on their biogenetic analogies with all other known sea cucumber triterpene glycosides.
It was found that chitonoidosides E 1 (1), F (2) and H (4) are characterized by holotoxinogenin as aglycone, which was first found in Apostichopus japonicus and is broadly distributed in sea cucumber glycosides [16]. This was deduced from the analyses of their 1 H and 13 C NMR spectra (Tables S1-S3, Figures S1-S6, S9-S14 and S25-S31), which coincided with each other as well as with those of the aglycones of chitonoidosides A 1 , C, and D isolated earlier from the same species-P. chitonoides [9].  Figure S8). The 1 H and 13 C NMR spectra of the carbohydrate chain of chitonoidoside E 1 (1) ( Table 1, Figures S1-S7) were coincident with those for chitonoidoside E, isolated recently [15] and demonstrated six characteristic doublets of anomeric protons at δ H 4.67-5.11 (J = 7.1-8.0 Hz) and six signals of anomeric carbons at δ C 102.3-105.2. The analysis of the 1 H, 1 H-COSY, 1D TOCSY, HSQC and ROESY spectra of 1 resulted in the assignment of the signals of two xylose residues, one quinovose, one glucose and 3-O-methylglucose, as well as 3-O-methylxylose residues. The positions of the sulfate groups were determined based on the deshielding, due to α-shifting effect, of the sulfate group's doubled signal at δ C 67.1, which is characteristic of glucopyranose units sulfated by C-6 (C-6 signals of non-sulfated glucopyranose residues are usually observed at~δ C 61.2). The signals of C-5 MeGlc4 and C-5 Glc5 were shielded due to the β-effect of the sulfate groups to δ C 75.3 and 75.5, respectively. These data indicate the presence of a hexasaccharide chain with two sulfate groups attached to C-6 MeGlc4 and to C-6 Glc5, and 3-O-methylxylose residue as the sixth sugar unit in chitonoidoside    Figure S16). The 1H NMR spectrum of the carbohydrate part of chitonoidoside F (2) exhibited four characteristic doublets at δ H 4.67-5.18 (J = 7.6-8.1 Hz), correlated by the HSQC spectrum with corresponding anomeric carbon signals at δ C 102.2-104.9. These signals were indicative of a tetrasaccharide chain with β-configurations of glycosidic bonds ( Table 2, Figures S9-S15). An isolated spin system from each monosaccharide residue was analyzed using the 1 H, 1 H-COSY and 1D TOCSY spectra. Further analysis of the HSQC, ROESY and HMBC spectra resulted in the assignment of all monosaccharide NMR signals. Using this algorithm, the monosaccharides composing the carbohydrate moiety of chitonoidoside F (2) were found to be xylose (Xyl1), quinovose (Qui2), glucose (Glc3), and 3-O-methylglucose (MeGlc4). The monosaccharide compositions of the other glycosides, reported herein, were established in the same manner. The signal of C-6 Glc3 in the 13 C NMR spectrum of 2 was deshielded to δ C 67.2, which is characteristic of sulfation at this position. The signal of C-6 MeGlc4 was observed at δ C 61.2, indicating the absence of a sulfate group at this position, although the MS data indicated the presence of two sulfate groups in 2. The signal of C-4 MeGlc4 was deshielded to δ C 76.3 when compared to the same signal of terminal 3-O-methylglucose residues of the glycosides lacking a sulfate group (δ C~7 0.0) [12]. Moreover, the signals of C-3 and C-5 MeGlc4 in the spectrum of 2 were shielded to δ C 85.3 and 76.4, respectively, due to the β-shifting effect of a sulfate group. Therefore, the 3-O-methylglucose residue in the sugar part of 2 was sulfated by C-4. This structural feature was only found once in previous research-in the glycosides of Colochirus quadrangularis [17]. The observation of 3-O-methylxylose residue sulfated by C-4 in the glycosides of P. chitonoides [15] clearly demonstrates the presence of a specific sulfatase capable of attaching a sulfate group to C-4 of monosaccharides in pyranose form.
The positions of glycosidic linkages, established by the ROESY and HMBC spectra of 2 revealed the uncommon architecture of the sugar chain with disaccharide fragment 4-O-sodium sulfate-3-O-methyl-β-D-glucopyranosyl-(1→3)-6-O-sodium sulfate-β-Dglucopyranosyl-(1→4) attached to the first (Xyl1) residue, while quinovose was a terminal unit in the reduced bottom semi-chain (Table 2). Two tetraosides whose carbohydrate part featured the same architecture were found only in the sea cucumber Thyonidium kurilensis [18].
Based on the absence of the signals of 18(20)-lactone at δ C~1 78 (C-18) and~83 (C-20) in the 13 C NMR spectrum of 3, the aglycone with 18(20)-ether bond instead of the lactone was supposed to be present. The NMR spectra of the aglycone part of chitonoidosides G (3) and A [15], where this aglycone was first found, were almost coincident with each other ( Table 3, Figures S17-S22).  These signals indicated the presence of a hexasaccharide moiety with β-glycosidic bonds. The monosaccharide composition of 3 was determined as two xyloses (Xyl1 and Xyl3), quinovose (Qui2), glucose (Glc5), and two 3-O-methylglucoses (MeGlc4 and MeGlc6). The doubled signal at δ C 67.1 indicated the presence of two sulfate groups. Using the 1H,1H-COSY and 1D TOCSY spectra their positions were deduced as C-6 MeGlc4 and C-6 Glc5. The comparison of the 13 C NMR spectra of the carbohydrate parts of the chitonoidosides G (3) and E 1 (1) demonstrated the closeness of the signals of the monosaccharide units from first to fifth. The signals of the terminal sixth monosaccharide residue in 3 were assigned as 3-O-methylglucose instead of 3-O-methylxylose in 1. The sequence of monosaccharides and the positions of the glycosidic bonds were confirmed by the correlations in the ROESY and HMBC spectra of 3 (Table 4, Figures S17-S23). Therefore, chitonoidoside G (3) is the first compound in the combinatorial library of the glycosides from P. chitonoides to feature non-sulfated 3-O-methylglucose as terminal residue in the upper semi-chain.
confirming the sequence of monosaccharides and the aglycone structure of 3.
These data indicate that chitonoidoside  (Table 5, Figures S25-S32).  The comparison of the 13 C NMR spectra of the carbohydrate parts of 4 and 2 displayed their difference only in the presence of the signals of additional xylose residue (in the bottom semi-chain of 4), as well as the glycosylation effect at C-4 Qui2, whose signal was observed at δ C 85.6, instead of δ C 76.2 (C-4 Qui2), observed in 2. Therefore, the chitonoidosides F (2) and H (4) can be considered as sequential steps in the biosynthesis of the glycosides in P. chitonoides. The

Bioactivity of the Glycosides
The cytotoxic activity of sea cucumber glycosides against different cell types and cell lines, including HeLa and THP-1, has been extensively studied [1,19]. This has led to deeper understanding of the mechanisms of the anticancer activities of glycosides [7,8,20]. Different tumor cell lines exhibit different sensitivities to the cytotoxic effects of sea cucumber glycosides, depending on their chemical structures. This can be of special interest for the development of therapy for certain types of cancer [21].
The cytotoxic activities of compounds 1-4 and chitonoidoside E against human erythrocytes and the human cancer cell lines, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, monocytes THP-1, and leukemia promyeloblast HL-60 were investigated ( Table 6). The previously tested chitonoidoside A [9] and cisplatin were used as the positive controls. All the tested compounds demonstrated strong hemolytic activity, with hexaosides 1, 3 and chitonoidoside E proving to be the most active. The human erythrocytes were more sensitive to the membranolytic action of tested compounds (Table 6) compared to the cancer cells, which is similar to previous data concerning mouse erythrocytes [12,13,15,17]. The monocyte cell line THP-1 and the erythrocytes were comparably sensitive to the action of the glycosides.
A similar tendency for 1, 3, and chitonoidoside E was observed for the cytotoxicity against the adenocarcinoma HeLa and HL-60 cells, but the demonstrated effects were moderate. Chitonoidoside E possessing a hexasaccharide chain and the aglycone with a 18(20)-ether bond was the most active; the activity of hexaosides 1 and 3, which differed through the sixth monosaccharide residue, and the activity of pentaoside 4, were close to each other against erythrocytes and HeLa and HL-60 cells, but significantly differed in relation to DLD-1 and THP-1 cells. The tetraoside with a shortened bottom semi-chain, chitonoidoside F (2), exhibited the weakest membranolytic effect in the series. The most significant difference in the activity of 2 and the other compounds of the series was observed for the DLD-1 and HL-60 cell lines, confirming the diversity in the sensitivities of the cancer cell lines. The presence of the aglycone with an 18(20)-ether bond (in 3) did not decrease the activity of the glycosides. The latter two peculiarities are in good accordance with the biologic activity of a previously analyzed series of chitonoidosides-the glycosides of P. chitonoides [15].

Metabolic Network of Carbohydrate Chains of Chitonoidosides of the Groups A-H
The aglycones and carbohydrate chains of triterpene glycosides are biosynthesized simultaneously and independently from each other [12,22,23], leading to the tremendous structural diversity in this class of natural products from sea cucumbers. Glycosides with identical to sugar moieties that differ in their aglycone structures are considered to belong to a group of glycosides named by particular letter (some groups may consist of one compound). Thus, eight groups of chitonoidosides, A-H, were discovered in P. chitonoides, including three types of tetraosides (groups A, C, and F), differing in their architecture and monosaccharide composition, two types of pentaosides (groups D and H) with analogical differences, and three types of hexaosides (groups B, E, and G), differing in their terminal monosaccharide residues and the positions of their sulfate groups. The biosynthesis of sugar moieties occurs through the sequential connection of monosaccharides to certain positions, forming carbohydrate chains. The first branchpoint in the biosynthesis of the chitonoidosides of diverse groups is the attachment of the third sugar residue to the bioside consisting of Xyl1 and Qui2. When the third sugar (xylose) attaches to C-4 Qui2, with the subsequent attachment of 3-OMeGlc and sulfation, the growth of the sugar chain ends with the formation of tetraosides, namely the chitonoidosides of group A (such chains are considered as linear). The attachment of glucose as the third sugar unit to C-4 Xyl1, followed by the addition of glycosylation with different monosaccharide residues (3-O-methylxylose or 3-O-methylglucose) and sulfation, leads to the formation of the chitonoidosides of groups C and F ( Figure 2). The elongation of the bottom semi-chain in chitonoidoside F (by C-4 Qui2) results in chitonoidoside H formation. The further elongation of the sugar chains of the chitonoidosides belonging to the group A and additional sulfation lead to the formation of chitonoidosides of the group D, followed by the subsequent formation of chitonoidosides belonging to group G. When the elongation of the sugar chains of the chitonoidosides of group A occurs without sulfation, the chitonoidosides of the group B are biosynthesized.  Noticeably, that the sulfation of terminal monosaccharide in the upper semi-chain of tetraosides-the chitonoidosides of groups C and F -precedes the subsequent elongation of the bottom semi-chain. Therefore, these glycosides cannot be the precursors of the chitonoidosides of group E containing non-sulfated terminal 3-O-methylxylose in the upper semi-chain. Similarly, hexaosides-chitonoidosides of group B -with the only sulfate group at C-6 MeGlc4 cannot be formed from pentaosides, the chitonoidosides of group D because, of the sulfation of Glc5 in the latter. Additionally, the carbohydrate chain of the chitonoidosides of group E can be formed through the sulfation of the chain of chitonoidosides of group B or through the glycosylation of the chain of chitonoidosides of group D. All these data demonstrate that glycosylation and sulfation are competitive and parallel/simultaneous processes in carbohydrate chain biosynthesis.

General Experimental Procedures
Specific rotation, PerkinElmer 343 Polarimeter (PerkinElmer, Waltham, MA, USA); NMR, Bruker AMX 500 (Bruker BioSpin GmbH, Rheinstetten, Germany) (500.12/125.67 MHz ( 1 H/ 13 C) spectrometer; ESI MS (positive and negative ion modes), Agilent 6510 Q-TOF apparatus (Agilent Technology, Santa Clara, CA, USA), sample concentration 0.01 mg/mL; HPLC, Agilent 1260 Infinity II with a differential refractometer (Agilent Technology, Santa Clara, CA, USA); columns Supelcosil LC-Si (4.6 × 150 mm, 5 µm) and Ascentis RP- Noticeably, that the sulfation of terminal monosaccharide in the upper semi-chain of tetraosides-the chitonoidosides of groups C and F-precedes the subsequent elongation of the bottom semi-chain. Therefore, these glycosides cannot be the precursors of the chitonoidosides of group E containing non-sulfated terminal 3-O-methylxylose in the upper semi-chain. Similarly, hexaosides-chitonoidosides of group B-with the only sulfate group at C-6 MeGlc4 cannot be formed from pentaosides, the chitonoidosides of group D because, of the sulfation of Glc5 in the latter. Additionally, the carbohydrate chain of the chitonoidosides of group E can be formed through the sulfation of the chain of chitonoidosides of group B or through the glycosylation of the chain of chitonoidosides of group D. All these data demonstrate that glycosylation and sulfation are competitive and parallel/simultaneous processes in carbohydrate chain biosynthesis.

Animals and Cells
The specimens of the holothurian Psolus chitonoides (family Psolidae; order Dendrochirotida) were harvested in the Bering Sea during the 14th expedition cruise on board The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Pacific Institute of Bioorganic Chemistry (Protocol No. 0037.12.03.2021).

Extraction and Isolation
The sea cucumbers were minced and kept in EtOH at +10 • C. Next, they were extracted twice with refluxing 60% EtOH. The combined extracts were concentrated to dryness in a vacuum, dissolved in H2O, and chromatographed on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). Eluting first the inorganic salts and impurities with H2O and then the glycosides with 50% EtOH produced 3200 mg of crude glycoside fraction, which was submitted to stepwise column chromatography on Si gel using CHCl3/EtOH/H2O  Table S1 and Table 1 Table S2 and Table 2

Cytotoxic Activity (MTT Assay Applied for HeLa Cells)
All the studied substances (including the chitonoidoside A and cisplatin, used as positive controls) were tested in concentrations from 0.1 µM to 100 µM using twofold dilution in d-H2O. The cell suspension (180 µL) and solutions (20 µL) of the tested compounds in different concentrations were injected in wells of 96 well plates (1 × 104 cells/well) and incubated at 37 • C for 24 h in 5% CO 2 . After incubation, the tested substances with medium were replaced by 100 µL of fresh medium. Next, 10 µL of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) stock solution (5 mg/mL) was added to each well, followed by the incubation of the microplate for 4 h. Subsequently, 100 µL of SDS-HCl solution (1 g SDS/10 mL d-H2O/17 µL 6 N HCl) was added to each well and incubated for 18 h. The absorbance of the converted dye, formazan, was measured with a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. The cytotoxic activity of the tested compounds was calculated as the concentration that caused 50% cell metabolic activity inhibition (IC50). The experiments were carried out in triplicate, p < 0.05.

Cytotoxic Activity (MTS Assay Applied for DLD-1, THP-1 and HL-60 Cells)
The cells of the HL-60 line (6 × 10 3 /200 µL) were placed in 96 well plates at 37 • C for 24 h in a 5% CO 2 incubator. The cells were treated with tested substances and chitonoidoside A and cisplatin were used as positive controls at concentrations from 0 to 100 µM for an additional 24 h of incubation. Next, the cells were incubated with 10 µL MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) for 4 h, and the absorbance in each well was measured at 490/630 nm with plate reader PHERA star FS (BMG Labtech, Ortenberg, Germany). The experiments were carried out in triplicate and the mean absorbance values were calculated. The results were presented as the percentage of inhibition that produced a reduction in absorbance after the tested compound treatment compared to the non-treated cells (negative control), p < 0.01.

Hemolytic Activity
The erythrocytes were isolated from human blood through centrifugation with phosphatebuffered saline (PBS) (pH 7.4) at 4 • C for 5 min by 450× g on centrifuge LABOFUGE 400R (Heraeus, Hanau, Germany) three times. Next, the residue of the erythrocytes was resuspended in an ice-cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm, and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution (including chitonoidoside A used as positive control) in V-bottom 96 well plates. After 1 h of incubation at 37 • C, the plates were exposed to centrifugation for 10 min at 900× g on laboratory centrifuge LMC-3000 (Biosan, Riga, Latvia). Next, 100 µL of supernatant was carefully selected and transferred into new flat-plates, respectively. The lysis of the erythrocytes was determined by measuring of the concentration of hemoglobin in the supernatant with microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA), λ = 570 nm. The effective dose causing 50% hemolysis of erythrocytes (ED50) was calculated using the computer program SigmaPlot 10.0. All the experiments were performed in triplicate, p < 0.01.

Conclusions
The continuation of the research into triterpene glycosides from the sea cucumber Psolus chitonoides resulted in the isolation of four previously unknown chitonoidosides E 1 (1), F (2), G (3) and H (4). The compounds characterized by two types of the aglycones (holotoxinogenin and its structural analog with 18(20)-epoxy-cycle instead of a lactone) were also found earlier in the series of chitonoidosides A-E. The compounds 1-4 differed in the number of monosaccharides in their sugar chains (from four to six), the architecture of these chains (for tetra-and pentaosides), their monosaccharide composition, and the positions of their sulfate groups. Terminal 3-O-methylglucose unit was sulfated by C-4 in chitonoidosides F (2) and H (4), which is a rare position for a sulfate group from sea cucumber glycosides. Notably, in the first series of the studied glycosides from P. chitonoides [15] 3-O-methylxylose residue sulfated by C-4 was found. This indicates the presence of specific sulfatase in this species of the sea cucumber, capable of attaching the sulfate group to C-4 of monosaccharides in pyranose form. The observed "structureactivity relationships" were as follows: tetraosides with a shortened bottom semi-chain displayed the weakest membranolytic effect; and the activity of the glycosides with the new-type aglycone with a 18(20)-ether bond instead of 18(20)-lactone was comparable with that of the substances with holostane-type aglycones. Hexaosides and tetraosides with linear carbohydrate chains (having bottom semi-chain) were the most active in the series of the glycosides from P. chitonoides. The pathways of the biosynthetic transformations were analyzed based on the structures of eight types of carbohydrate chains found in the glycosides of P. chitonoides. The analysis revealed and confirmed that glycosylation and sulfation are parallel and competitive processes. All the discussed data broaden knowledge about structural diversity of triterpene glycosides from the sea cucumbers and help us to the understand the complicated biosynthesis process of this class of metabolites, which is currently a large gap in knowledge for scientists.