Structures and Biogenesis of Fallaxosides D4, D5, D6 and D7, Trisulfated Non-Holostane Triterpene Glycosides from the Sea Cucumber Cucumaria fallax

Four new trisulfated triterpene glycosides, fallaxosides D4 (1), D5 (2), D6 (3) and D7 (4) have been isolated from the sea cucumber Cucumaria fallax (Cucumariidae, Dendrochirotida). The structures of the glycosides have been elucidated by 2D NMR spectroscopy and HRESIMS. All the glycosides have the lanostane aglycones of a rare non-holostane type with 7(8)-, 8(9)- or 9(11)-double bonds, one or two hydroxyl groups occupying unusual positions in the polycyclic nucleus and shortened or normal side chains. The pentasaccharide carbohydrate moieties of 1–4 have three sulfate groups. The cytotoxic activity of glycosides 1–4 against the ascite form of mouse Ehrlich carcinoma cells and mouse spleen lymphocytes and hemolytic activity against mouse erythrocytes have been studied.

Recently we have started studies on the Far-Eastern sea cucumber Cucumaria fallax [6] which contains exclusively non-holostane oligoglycosides having unusual double bond positions and uncommon sites of oxidation in their aglycone moieties. Herein we report the isolation of four new trisulfated glycosides, fallaxosides D 4 -D 7 (compounds 1-4) with earlier unknown aglycones and their structures, established by analysis of 1 H-, 13 C-NMR and 2D NMR ( 1 H´1H COSY, 1D TOCSY, HMBC, HSQC, ROESY) spectra and confirmed by HRESI mass spectrometry. The biogenesis of these unusual metabolites is also discussed.

Results and Discussion
The sea cucumbers were extracted with 70% ethanol under reflux during 5 h. The concentrated extract was sequentially submitted to the column chromatography on Polychrom-1 (powdered Teflon) in H 2 OÑ50% ethanol in order to eliminate salts and polar impurities and on Si gel using Teflon) in H2O→50% ethanol in order to eliminate salts and polar impurities and on Si gel using CHCl3/EtOH/H2O (100:125:25 and 100:150:50) as mobile phases to obtain the fraction containing polar trisulfated pentaosides (glycosides belonging to the group A7). Further separation of the fraction by HPLC on a semi-preparative reversed phase column using MeOH/H2O/NH4OAc (1 M water solution) as mobile phase in ratio 60/39/1 gave the subfractions A7I-A7V. Each of the subfractions was rechromatographed using HPLC. The HPLC of subfraction A7I with the same solvent system in ratio of 35/64/1 gave fallaxoside D4 (1) and fallaxoside D5 (2). The HPLC of subfraction A7II using the solvent system in ratio of 50/49/1 followed by 45/54/1 and 47/51/2 gave fallaxoside D7 (4). The HPLC of subfraction A7V with the same solvents in ratio of 58/41/1 gave fallaxoside D6 (3).
The presence five characteristic doublet signals at δH 4.76-5.22 (1H, d, J = 6.9-8.4 Hz), correlated by HSQC spectra with the signals of anomeric carbons at δC 102.0-105.1 in the 1 H-NMR spectra of the carbohydrate chains of fallaxosides D4 (1), D5 (2), D6 (3) and D7 (4) (Scheme 1) and known fallaxosides D1 and D2 [7] were indicative of a pentasaccharide chain and β-configurations of glycosidic bonds. The positions of all the interglycosidic linkages and the place of linkage of the carbohydrate chain to an aglycone were deduced by the analysis of the ROESY and HMBC spectra of the carbohydrate parts of the glycosides (Table 1). Indeed, the cross-peaks between H-1 of the first monosaccharide residue (xylose) and H-3 (C-3) of an aglycone; H-1 of the second mono-saccharide residue (quinovose) and H-2 (C-2) of the first monosaccaharide residue (xylose); H-1 of the third monosaccharide residue (glucose) and H-4 (C-4) of the second monosaccharide residue (quinovose); H-1 of the fourth monosaccharide residue (3-O-methylglucose) and H-3 (C-3) of the third monosaccharide unit (glucose); H-1 of the fifth monosaccharide residue (xylose) and H-2 (C-2) of the second monosaccharide residue (quinovose) were observed. The δC values characteristic of αand β-shifting effects of sulfate groups were observed for C-4 (δC 76.1) and C-5 (δC 64.0) of the first xylose residue, for C-6 (δC 67.3) and C-5 (δC 74.8) of the glucose residue and C-6 (δC 67.0) and C-5 (δC 75.5) of terminal 3-O-methylglucose residue. These data indicated the presence of a pentasaccharide carbohydrate chain with three sulfate groups. The 13 C-NMR spectra of the carbohydrate parts of compounds 1-4 (Table 1) were identical to each other and coincided with those of cucumariosides of the group A7 isolated first from Cucumaria japonica [8] and with the corresponding spectra of fallaxosides D1 and D2 having the same carbohydrate chain [6]. The structure of such a carbohydrate chain was previously elucidated by desulfation whereby a known desulfated derivative was obtained. This derivative was obtained The 13 C-NMR spectra of the carbohydrate parts of compounds 1-4 (Table 1) were identical to each other and coincided with those of cucumariosides of the group A 7 isolated first from Cucumaria japonica [8] and with the corresponding spectra of fallaxosides D 1 and D 2 having the same carbohydrate chain [6]. The structure of such a carbohydrate chain was previously elucidated by desulfation whereby a known desulfated derivative was obtained. This derivative was obtained from a monosulfated glycoside where very detailed chemical evidence of the sugar sequence was obtained by a variety of methods including specific enzymatic hydrolysis, periodate oxidation and Smith degradation, etc. All the obtained progenins were characterized by 13 C-NMR [8]  The NMR spectra of the aglycone part of fallaxoside D 4 (1) revealed the presence of 24 carbon atoms (Table 2), including in six methylenes, five methines and six methyl groups as well as seven quaternary carbon signals, that corresponded to the 22,23,24,25,26,27-hexanorlanostane type of aglycones previously found in glycosides of C. fallax [6]. The resonances of two olefinic carbons with δ C 139.9 (C-8) and 142.9 (C-9) were assigned to the tetrasubstituted double bond. The HMBC correlations between H-32 (δ H 1.00, 3H, s) and C-8 and between H-11 (δ H 4.81, 1H, brd, J = 6.8 Hz) and C-9 as well as between H-19 (δ H 1.57, 3H, s) and C-9 confirmed the 8(9)-double bond position. The signal at δ C 211.8 indicated the presence of a keto group. The signals at δ H 2.83 (1H, t, J = 8.8 Hz, H-17) and δ C 58.9 (C-17) were assigned to a methine group adjacent to a ketone group. The HMBC correlations from H-17 and H-21 (δ H 2.14, 3H, s) to the carbon at δ C 211.8 (C-20) allowed the positioning of the group at C-20. There were two downshifted resonances in the 13 C-NMR spectrum of 1 at δ C 67.7 (C-7) and δ C 65.2 (C-11) corresponding to the oxygen bearing allylic type methines suggesting the presence of two hydroxyls. Their positions at C-7 and C-11 were corroborated by the correlations from H-6b (δ H 1.99, 1H, m) to C-7 and from H-12b (δ H 2.27, 1H, d, J = 14.2 Hz) to C-11 in the HMBC spectrum as well as ROESY correlations between H-1(2.47 m) and H-11 and between H-5 (1.07 dd, 2.7, 15.6 Hz) and H-7. Seeing that absolute configuration at C-5 with α-orientation of hydrogen, as well as configurations of C-10, C-13, C-14, and C-17 stereocenters in the sea cucumber triterpene glycosides were established earlier [9], the H-5-H-7 H-15α-H7 and H-32-H-7 NOESY correlations are indicative of β-orientation of hydroxyl group at C-7. The configuration of the C-11 stereocenter in 1 was proposed by analysis of the ROESY spectrum and MM2 optimized models of 1. The β-orientation of hydroxyl group at C-11 was proposed, based on the comparison of MM2 optimized models of aglycones with αand β-oriented hydroxyls. The observed in the ROESY spectrum of 1 correlations from H-11 to H-1 and vice versa would be realized for both 11-α and 11β-hydroxyls. However, the correlations from H-11 with methyl groups H-19 and H-18 should be the realized in the ROESY spectrum at 11α-hydroxy orientation. Nevertheless, these correlations were absent in the ROESY spectrum of 1. Hence the β-orientation of the hydroxyl group at C-11 is the most probable. However, the ions observed in the mass spectra corresponded to the loss of water upon ionization of fallaxoside D 4 (1), presumably due to the lability of the dihydroxy-ene-fragment in the rings B and C of its aglycone. The structure of the carbohydrate chain of 1 was also confirmed by fragment ion peaks at m/z 929, corresponding to a monodesulfated carbohydrate chain [MeGlcOSO 3 Na + GlcOSO 3 Na + The structure of the aglycone moiety of fallaxoside D 5 (2) was proved to be similar and NMR spectra also confirmed the 22,23,24,25,26,27-hexanorlanostane skeleton system of its aglycone. The difference in the spectra (Table 3) was in the presence of an additional signal of a ketone group at δ C 201.4 instead of one of hydroxyl group signals in the 13 C-NMR spectrum. The signals of the quaternary olefinic carbons indicated the presence of 8(9)-double bond and were downshifted (δ C 140.0 and 163.1) when compared with those in the 13 C-NMR spectrum of 1, suggesting that the ketone group adjoins the double bond. Additionally, the signals at δ H 2.56 (1H, m, H-6a) and δ H 2.50 (1H, m, H-6b) demonstrated the formation of a spin coupled system in the COSY spectrum with the signal of H-5 (1H, dd, J = 3.9, 13.5 Hz) only and were also noticeably downshifted. These data indicated the closeness of these atoms to the ketone group that was positioned at C-7. The HMBC correlation from both H 2 -6 to C-7 (δ C 201.4) and C-10 (δ C 40.6) confirmed the ketone position. Hence, the hydroxyl group at C-7 of the aglycone of 1 was substituted with a ketone group in the aglycone of 2. The signal at δ C 64.1 (C-11) in the 13 C-NMR spectrum of 2, correlated by the HSQC with the resonance at δ The orientation of hydroxyl group was proposed as α based on the ROESY spectrum. Clear NOE correlations from H-11 to both β-oriented methyl groups Me-18 and Me-19 were observed. Hence the hydroxyl groups at C-11 are opposite oriented in fallaxosides D 4 (1) and D 5 (2) The 13 C-NMR spectrum of the aglycone part of fallaxoside D 6 (3) revealed the presence of 30 carbons indicating the presence of an aglycone with normal non-shortened side chain ( Table 4). The absence of a γ-lactone was deduced from the absence of the signal at « δ C 176 and contemporary presence of the resonances of Me-18 (δ C 24.8, C-18, δ H 1.28, 3H, s, H-18) in the 13 C-and 1 H-NMR spectra, respectively, indicating the aglycone of fallaxoside D 6 (3) to be of the lanostane type without a lactone, in contrast with aglycones of the majority of sea cucumber glycosides. The resonances of an olefinic methine group at δ C 122.2 (C-7) and δ H 5.59 (1H, m, H-7) in NMR spectra as well as of an olefinic quaternary carbon at δ C 149.7 (C-8) in the 13 C-NMR spectrum were assigned to the 7(8)-double bond typical of many sea cucumber glycosides [1,9].  (Table 4) corroborated the configurations of stereocenters C-3, C-5, C-10, C-13, C-14, C-17 as well as (20R) configuration (NOE between H-16 and H-23 and vice versa) established earlier for similar aglycone of frondoside C [10]. A ROESY correlations of a signal at 2.28 brd, J = 12.8 Hz, H-9 with signals of methyl group at C-10 and C-14 established the rare 9β-H configuration in 3 characteristic of 7(8)-unsaturated sea cucumber glycosides [1,9,11]. Thus, these data allowed us to determine all the structural and stereochemical features of this unusual aglycone. Table 4. NMR spectrosopic data (700 MHz, C 5 D 5 N/D 2 O (4/1 v/v)) of the aglycone moiety of fallaxoside D 6 (3).  The structure of the aglycone moiety of fallaxoside D 7 (4) was found by extensive NMR spectroscopy (Table 5) to be similar to that of 1-2, indicating the presence of a 22,23,24,25,26,27-hexanorlanostane aglycone with the keto group at C-20 (δ C 212.5). The double bond was positioned as 9 (11) according to the signal of the quaternary olefinic carbon at δ C 147.6 (C-9) and olefinic methine resonances at δ C 116.0 (C-11) in the 13 C-NMR spectrum and δ H 5.27 (1H, m, H-11) in the 1 H-NMR spectrum. The long range COSY correlation H-8/H-11 as well as the characteristic correlation from Me-19 (δ H 1.00, 3H, s, H-19) to C-9 confirmed the double bond position. The signal at δ C 71.6 (C-7) in the 13 C-NMR spectrum of 4, correlated by the HSQC with the resonance at δ H 3.83 (1H, m, H-7) indicated the attachment of hydroxyl group to this carbon. Its position as C-7 was confirmed by the COSY spectrum, where the signals of spin coupled system H-5/H 2 -6/H-7/H-8 were observed. On the base of all the above discussed data, the structure of fallaxoside D 7 (4)  The cytotoxic activities of fallaxosides D 4 (1), D 5 (2), D 6 (3) and D 7 (4) against mouse spleen lymphocytes and ascite form of mouse Ehrlich carcinoma cells along with hemolytic activity against mouse erythrocytes were studied. None of these glycosides were active in these tests at the dosage studied (IC 50 > 100 µM/mL). This could be explained by the absence of the 18(20)-lactone moiety that is essential for the membranolytic action of sea cucumber glycosides and by the presence of three sulfate groups that decreased the membranolytic activity of the glycosides [12].
It is most probable that holostane type glycosides play a role in chemical defense against predators because of their strong membranolytic activities and not only as regulators of oocyte maturation. Non-holostane glycosides may play only a regulatory role and are evolutionary precursors of holostane glycosides [12].

Animal Material
The samples of Cucumaria fallax (Cucumariidae, Dendrochirotida) were collected during the 41-st scientific cruise of the research vessel Akademik Oparin in the Pacific Ocean near Black Brothers Islands,