PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of mardrugsMDPIHomeThis articleThis journalInstructions for authorsSubscribemarine drugs
 
Mar Drugs. 2017 July; 15(7): 199.
Published online 2017 June 26. doi:  10.3390/md15070199
PMCID: PMC5532641

Poecillastrosides, Steroidal Saponins from the Mediterranean Deep-Sea Sponge Poecillastra compressa (Bowerbank, 1866)

Abstract

The first chemical investigation of the Mediterranean deep-sea sponge Poecillastra compressa (Bowerbank, 1866) led to the identification of seven new steroidal saponins named poecillastrosides A–G (17). All saponins feature an oxidized methyl at C-18 into a primary alcohol or a carboxylic acid. While poecillastrosides A–D (14) all contain an exo double bond at C-24 of the side-chain and two osidic residues connected at O-2′, poecillastrosides E–G (57) are characterized by a cyclopropane on the side-chain and a connection at O-3′ between both sugar units. The chemical structures were elucidated through extensive spectroscopic analysis (High-Resolution Mass Spectrometry (HRESIMS), 1D and 2D NMR) and the absolute configurations of the sugar residues were assigned after acidic hydrolysis and cysteine derivatization followed by LC-HRMS analyses. Poecillastrosides D and E, bearing a carboxylic acid at C-18, were shown to exhibit antifungal activity against Aspergillus fumigatus.

Keywords: sponge, saponins, deep-sea, Poecillastra compressa

1. Introduction

In the marine environment, steroid and triterpenoid glycosides are widespread metabolites mainly produced by echinoderms [1,2,3], although saponins have also been isolated from other marine invertebrates such as octocorals or sponges [4,5]. To date, about 70 saponins have been reported from sponges [6] including sarasinosides from Asteropus spp. [7,8], Melophlus spp. [9,10], and Lipastrotethya sp. [11], ulososides from Ulosa sp. [12,13] and Ectoplyasia ferox [14], pandarosides and acanthifoliosides from Pandaros acanthifolium [15,16,17,18], wondosterols from the association of two sponges [19], erylosides, sokodosides, nobiloside, and formosides from Erylus spp. [20,21,22,23,24,25,26,27,28,29], ptilosaponosides from Ptilocaulis spiculifer [30], mycalosides from Mycale laxissima [31,32], feroxosides from Ectyoplasia ferox [33], and silenosides from Silene vulgaris [34]. While some sponge saponins can be oxidized on the D ring or can contain unusual side chains, the aglycone of most of them belongs to the 30-norlanostane triterpenoid family, with steroidal saponins being rather rare for sponges. Some sponge saponins were subjected to different bioassays and they usually demonstrated interesting biological activities, mostly cytotoxicity against tumor cell lines [35,36,37].

In our continuous efforts to describe the chemical diversity of marine sponges from the Mediterranean, we undertook the first chemical study of the deep-sea Tetractinellid sponge Poecillastra compressa (Bowerbank, 1866). The genus Poecillastra is known to produce a broad range of secondary metabolites such as macrolactams [38,39], nitrosohydroxyalkylamines [40], sesquiterpenes, and steroids [41,42]. We report herein the isolation and structure elucidation of seven new steroidal glycosides named poecillastrosides A–G (17) from the deep-sea sponge P. compressa (Figure 1). Their structures were deduced from spectroscopic data including 1D- and 2D-NMR experiments as well as high-resolution mass spectra (HRESIMS) analyses. Three different aglycone moieties were identified, and oxidation at the C-18 position is a common feature among all isolated saponins. Poecillastroside A (1) contains an ergostane aglycone, whereas poecillastrosides B–D (24) contain a poriferastane, and poecillastrosides E–G (57) a cholestane with a cyclopropyl ring on the side-chain.

Figure 1
Structure of poecillastrosides A–G (17).

2. Results and Discussion

The freeze-dried sponge sample (43.1 g) was macerated and repeatedly extracted with a mixture of CH2Cl2/CH3OH (1:1) under sonication. The extract (7.9 g) was fractionated by Reversed Phase C18 Vacuum Liquid Chromatography with solvent mixtures of decreasing polarity. The methanolic fraction was then purified by successive RP-Phenylhexyl and C18 HPLC yielding pure compounds 17.

Compound 1 was isolated as a yellowish amorphous solid. Its molecular formula C40H68O13 was determined by HRESIMS. The 1H NMR spectrum of 1 suggested a steroidal saponin (Table 1). First, the characteristic anomeric signals at δH 4.49 (d, J = 7.6 Hz, 1H, H-1′), 4.56 (d, J = 7.9 Hz, 1H, H-1″), and δC 101.8 (C-1′), 105.2 (C-1″) evidenced the presence of two sugar residues. The 1H NMR data of the steroid revealed one methyl singlet at δH 0.88 (s, 3H, H3-19), three methyl doublets at δH 1.02 (d, J = 6.8 Hz, 3H, H3-21) and 1.03 (d, J = 6.8 Hz, 6H, H3-26 and -27), ten methylene groups, an oxygenated methylene with the AB system at δH 3.95 and 3.59, a 1,1-disubstituted olefin at δH 4.70 and 4.71 (H2-241), seven methine groups, two oxygenated methines at δH 3.72 (m, 1H, H-3), 4.26 (td, J = 7.7, 3.7 Hz, 1H, H-16), and three quaternary carbons at C-10, C-13 and C-24. When compared to usual steroids, this aglycone lacks one characteristic methyl signal for C-18. A hydroxylation was proposed at this position based on the presence of an AB system at δH 3.59 (d, J = 11.5 Hz, 1H, H-18b) and 3.95 (d, J = 11.5 Hz, 1H, H-18a) and further key H-12b, H-14, H-17/C-18, and H2-18/C-13, C-14, C-17 HMBC correlations. Another unusual feature for the steroid moiety was evidenced in the HSQC spectrum with signals of an oxygenated methine at δH 4.26 (td, J = 7.7, 3.7 Hz, 1H, H-16) and δC 72.8 (CH, C-16). The location of this hydroxyl group at C-16 was confirmed after interpretation of key H-16/H-17 and H-16/H-15a COSY and TOCSY correlations. While most of the relative configurations were in accordance with a common steroid core, the relative configuration at C-16 was established after examination of the NOESY spectrum. Absence of clear nuclear Overhauser effect (nOe) between H-16 and H-14 but also H-18 together with some overlap between H-17 and H-22 did not allow a straightforward determination of the relative configuration at this position. However, H-16/H-15a and H-8/H-15b nOes suggested a β orientation for the hydroxyl group at C-16. As a confirmation of this orientation, the coupling constant values of H-16 were in perfect accordance with those observed for the same signal of a closely related analogue weinbergsterol B, isolated from the sponge Petrosia weinbergi [43]. NMR signals of the sugar residues were assigned by extensive COSY, TOCSY, and HSQC interpretation. HMBC experiment evidenced H-5′/C-1′, H-1″/C-2′, H-5″/C-1″ long-range correlations, thus revealing the pyranose nature of these two sugars and their connection at C-2′. Finally, the connectivity of the sugar with the aglycone at C-3 was confirmed through the key HMBC H-1′/C-3 correlation. Moving to the relative configuration of the residues, the large coupling constants between H-1′/H-2′ and H-1″/H-2″ (7.9 and 7.6 Hz, respectively) were consistent with a β configuration for both anomeric centers. This interpretation was confirmed with the one-bond coupling constant 1JCH ≈ 160 Hz for the two anomeric positions [44]. In addition, the coupling constant values of 3JH3′–H4′ 3.2 Hz and 3JH5′–H4′ close to zero suggested an axial position for the hydroxyl at C-4 and, therefore, a β-galactopyranosyl residue attached at C-3 of the aglycone [45]. For the second sugar residue, all coupling constants were measured with values between 7 and 9 Hz which implies equatorial positions for all oxygen atoms and, therefore, a β-glucopyranosyl residue connected at C-2′ of the first residue.

Table 1
NMR spectroscopic data for poecillastrosides A–D (14) in CD3OD (500 MHz for 1H NMR data and 125 MHz for 13C NMR data).

Assuming a usual absolute configuration for the aglycone, we turned towards the pyranose moieties. After hydrolysis of the acetal bonds, the resulting monosaccharides were derivatized with l-cysteine methyl ester and phenylisothiocyanate in pyridine [46]. By comparison with standards, a d absolute configuration was assigned for both glucose and galactose monosaccharides.

Compound 2 was isolated as a yellowish amorphous solid. The molecular formula of 2 was determined by HRESIMS as C41H70O13. The spectroscopic data were very similar to those of 1, thereby suggesting that both compounds were close analogues. Examination of the 1H NMR spectrum revealed the presence of an additional methyl group at δH 1.59 (d, J = 6.3 Hz, 3H, H3-242) placed on the double bond at C-241, therefore, leading to a poriferastane skeleton. The relative configuration of 2 was found to be the same as that of poecillastroside A based on nOe correlations. A key H3-242/H2-23 nOe led us to assign the configuration of the double bond as E.

Compound 3 was isolated as a pale yellowish amorphous solid with the same molecular formula C41H70O13. Both compounds 2 and 3 are, therefore, isomers. The 1H NMR spectra were almost identical except for a deshielding of the signal corresponding to H-25, from δH 2.24 in 2 to δH 2.85 for 3. We first supposed that a change in the configuration of the double had occurred. Due to the low amount of compound available, the corresponding carbons were not visible neither in the 13C NMR spectrum nor in the HSQC, HMBC spectra. We, therefore, decided to enhance the sensitivity of the HSQC spectrum using the recently developed Pure Shift HSQC experiment [47]. Gratifyingly, we were then able to observe both HSQC spots corresponding to C-241 and C-25 (Figure S24). The shielding of the C-25 signal from δC 36.0 for 2 to δC 29.8 for 3 clearly confirmed a Z configuration for the double bond of 3.

Compound 4 was isolated as a pale yellowish amorphous solid with a molecular formula C41H68O13. The 1H NMR spectrum of 4 was very similar to the one of 2 except for the absence of the signals corresponding to the AB system of H2-18 and a shielding observed for δH 2.64 (m, 1H, H-12a). The only explanation consistent with all these observations, including the molecular formula, was the replacement of the hydroxyl group at C-18 by a carboxylic acid. This interpretation was further supported by a key H-17/C-18 HMBC correlation. Based on the chemical shift of the signal H-25 the configuration of the double bond was found to be the same as in 2.

Compound 5 was isolated as a white amorphous solid with a molecular formula of C43H66O15. Despite strong differences when compared with 14, the NMR data of 5 evidenced that the molecule was a steroidal saponin (Table 2). The aglycone exhibited an unusual skeleton with the presence of a terminal methylated cyclopropyl ring on the lateral chain. This assumption was based on the shielded signals of H-25 and H-26 but also by COSY, HSQC, and HMBC data analyses with the key H-27/C-24, H-27/C-26 HMBC correlations. Further analysis of 1H NMR data revealed the E geometry of the olefinic bond (JH-22,-23 = 15.2 Hz). No clear nOe correlations were observed for assessing the relative configuration around the cyclopropane ring. Gratifyingly, comparison with literature data and synthetic analogues of sterols with an identical side-chain led us to propose a trans configuration for the substituents at C-24 and C-25 of this ring [48,49,50,51]. To confirm this configuration in our case, we decided to look further into the coupling constants of the signals corresponding to the cyclopropane protons. Only the signals of the methylene and their multiplicity were clearly identified in the 1H NMR spectrum (Figure 2). In the case of a trans configuration of the two substituents around the cyclopropane, Ha and Hb would have the same splitting pattern as they would have in the presence of a pseudo C2 axial symmetry perpendicular to the cyclopropane plane. The 3J coupling constants between protons in a cis configuration are known to be between 8 and 10 Hz while values below 7 Hz are always observed when placed in a trans configuration. The multiplicity for both signals is observed as a doublet or triplet with coupling constants around 8 and 4 Hz, respectively. This same splitting pattern for both signals is only consistent for a trans configuration. Indeed, for a cis configuration, one of the two gem protons Hb would exhibit two large 3J coupling constants of 8 Hz. We, therefore, confirm a trans configuration for the two substituents and estimate the gem 2J coupling constants between Ha and Hb to be around 4 Hz. The presence of a carboxyl group at C-18 was inferred first from the HRESIMS data and then from the deshielding of H-12a, exactly in the same manner as for compound 4. Another difference with 4 arose from the absence of the signal corresponding to the oxygenated methine at C-16. This feature was confirmed by COSY, HSQC, and HMBC correlations. Looking at the glycosidic part of the saponin, the relative configuration was similar to those of 14, therefore, confirming one galactose linked to the aglycone and one glucose linked to the galactose. HMBC showed long-range correlations between H-1″/C-3′, H-2′/CAcC 172.2), and H-6″/CAcC 172.8), thereby indicating the presence of two acetyl groups at C-2′ and C-6″. Unlike compounds 14, the glycosidic link between both sugar residues was placed at C-3′ of the galactose. Deshielding of the signal of C-3′ at δC 82.4 in the 13C NMR spectrum confirmed this new substitution pattern.

Figure 2
Assignment of the relative configuration of the disubstituted cyclopropane through 1H NMR coupling constants [52].
Table 2
NMR spectroscopic data for poecillastrosides E–G (57) in CD3OD (500 MHz for 1H NMR data and 125 MHz for 13C NMR data of 5; 600 MHz for 1H data and 150 MHz for 13C data of 6 and 7).

Compound 6 was isolated as a white amorphous solid with a molecular formula of C41H66O13. The spectroscopic data were very similar to those of 5, thereby suggesting a close aglycone moiety. However, some changes were noticed by HSQC and HMBC analyses. Indeed, in the aglycone moiety, we observed the same AB system for H2-18 as that present in compounds 13. The long-range H-17/C-18 HMBC correlation confirmed the presence of an oxygenated methylene at C-13. In the d-β-glucose residue, the chemical shifts, and the COSY data were consistent with a terminal primary alcohol at C-6″, thereby implying the loss of the acetate at this position.

Compound 7 was isolated as a white amorphous solid with a molecular formula C43H68O14. The 1H NMR spectrum evidenced the fact that 7 is a close analogue of 6. The long-range H-6″/CAcC 172.8) HMBC correlation revealed the presence of an acetate group linked at O-6″ as in compound 5. The relative configuration of 7 was the same as those of 5 and 6.

Poecillastrosides A–G were tested in a panel of antimicrobial and cytotoxicity assays, including antibacterial activity against Gram positive (methicillin resistant (MRSA) and methicillin sensitive (MSSA) Staphylococcus aureus), and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii), antifungal activity against Aspergillus fumigatus, and cytotoxicity against the hepatic tumoral cell line hep_G2. Poecillastrosides D (4) (MIC90 = 6 µg/mL) and E (5) (MIC90 = 24 µg/mL) were the only two molecules active in the assay against A. fumigatus, revealing a key role of the carboxylic acid functionality at C-18 in the antifungal activity of this structural class. On the other hand, cytotoxicity assays also revealed weak activity of some members of the family against the hep_G2 human cell line, with IC50 values of 38, 28, and 89 µg/mL for poecillastrosides B, C, and D (24), respectively. None of the compounds of this family displayed activity against the bacterial pathogens at the highest concentration tested (96 µg/mL for compound 15, and 64 µg/mL for compounds 6 and 7).

3. Material and Methods

3.1. General Experimental Procedures

Optical rotations were recorded with a PerkinElmer 343 polarimeter equipped with a 10 cm microcell and a sodium lamp. UV measurements were obtained by extraction of the Diode Array Detector (DAD) signal of the Ultra-High Pressure Liquid Chromatography (UHPLC) Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA). NMR experiments were performed on a 500 MHz (Advance, Bruker, Billerica, MA, USA) or a 600 MHz (Agilent, Santa Clara, CA, USA) spectrometer. Chemical shifts (δ in ppm) are referenced to the carbon (δC 49.0) and residual proton (δH 3.31) signals of CD3OD. High-resolution mass spectra (HRESIMS) were obtained from a mass spectrometer Agilent 6540. HPLC separation and purification were carried out on a Jasco LC-2000 series equipped with a UV detector coupled with an Evaporative Light Scattering Detector, ELSD (Sedere, Alfortville, France).

3.2. Biological Material

Poecillastra compressa (Bowerbank, 1866) was collected in the Mediterranean Sea, off the French coasts, on 15 October 2014 at 200 m depth using a Remotely Operated Vehicle (Super Achille, COMEX S.A., Marseille, France). The voucher specimen “CS2ACHP09_ECH04” is kept at the Marine Station of Endoume (OSU Institut Pythéas, Marseille, France).

3.3. Extraction and Isolation

The dry sponge sample (43.1 g) was ground with a mortar and extracted with a mixture of CH3OH/CH2Cl2 (1:1, v/v) at room temperature, yielding 7.9 g (18% yield from dry-weight) of extract after solvent evaporation. The crude extract was fractionated by RP-C18 vacuum liquid chromatography (elution with a decreasing polarity gradient of H2O/CH3OH from 1:0 to 0:1, then CH3OH/CH2Cl2 from 1:0 to 0:1). The CH3OH (422 mg) fraction was then subjected to RP-HPLC on a preparative phenylhexyl column, 250 mm × 19 mm, 5 µm (Xselect, Waters, Milford, CT, USA), using a mobile phase of water (A) and acetonitrile (B). The method was developed on 30 min acquisition time: isocratic 60% B for 15 min, then linear gradient to 98% B in 1 min, held at 98% B for 10 min, back to 60% B in 1 min, and held at that percentage of B for 3 min. Selected fractions from this chromatography were then purified by RP-HPLC on a semi-preparative HTec C18 column, 250 mm × 10 mm, 5 µm (Nucleodur, Macherey-Nagel, Düren, Germany), with the following methods for each subsequent purification: isocratic 47% B to afford pure 1 (4.3 mg, 9.98 × 10−3% w/w), isocratic 49% B to afford 2 (6.2 mg, 1.44 × 10−2% w/w) and 3 (1.4 mg, 3.49 × 10−3% w/w), isocratic 50% B to afford 4 (1.6 mg, 3.71 × 10−3% w/w), isocratic 51% B to afford 5 (0.9 mg, 2.09 × 10−3% w/w), and isocratic 53% B to afford 6 (0.7 mg, 1.62 × 10−3% w/w) and 7 (0.8 mg, 1.86 × 10−3% w/w).

Poecillastroside A (1): Yellow, amorphous solid; [α]D20 +12.8 (c 0.1, CH3OH); UV (DAD) λmax 195 nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS (−) m/z 755.4582 [M − H] (calcd. for C40H67O13, 755.4587, [increment] − 0.7 ppm).

Poecillastroside B (2): Yellow, amorphous solid; [α]D20 +13.2 (c 0.1, CH3OH); UV (DAD) λmax 210 nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS (−) m/z 769.4743 [M − H] (calcd. for C41H69O13, 769.4744, [increment] − 0.1 ppm).

Poecillastroside C (3): Yellow, amorphous solid; [α]D20 +13.0 (c 0.1, CH3OH); UV (DAD) λmax 212 nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS (−) m/z 769.4745 [M − H] (calcd. for C41H69O13, 769.4744, [increment] + 0.1 ppm).

Poecillastroside D (4): Yellow, amorphous solid; [α]D20 +8.9 (c 0.1, CH3OH); UV (DAD) λmax 222 nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS (+) m/z 791.4567 [M + Na]+ (calcd. for C41H68NaO13, 791.4563, [increment] + 0.5 ppm).

Poecillastroside E (5): White, amorphous solid; [α]D20 −6.2 (c 0.1, CH3OH); UV (DAD) λmax 220 nm; 1H NMR and 13C NMR data, see Table 2; HRESIMS (+) m/z 845.4307 [M + Na]+ (calcd. for C43H66NaO15, 845.4299, [increment] + 0.9 ppm).

Poecillastroside F (6): White, amorphous solid; [α]D20 −27.3 (c 0.1, CH3OH); UV (DAD) λmax 222 nm; 1H NMR and 13C NMR data, see Table 2; HRESIMS (+) m/z 789.4405 [M + Na]+ (calcd. for C41H66NaO13, 789.4401, [increment] + 0.5 ppm).

Poecillastroside G (7): White, amorphous solid; [α]D20 −14.1 (c 0.1, CH3OH); UV (DAD) λmax 225 nm; 1H NMR and 13C NMR NMR data, see Table 2; HRESIMS (+) m/z 831.4518 [M + Na]+ (calcd. for C43H68NaO14, 831.4507, [increment] + 1.3 ppm).

3.4. Determination of the Absolute Configuration of the Pyranoses

Hydrolysis of glycosides and derivatization of the subsequent monosaccharides were performed individually following previously described methodologies [46]. The monosaccharide derivatives separation was carried out by UHPLC-HRMS on Acquity BEH (Ethylene Bridged Hybrid) C18 1.7 µm, 2.1 mm × 100 mm (Waters). The column was heated at 40 °C. The eluent consisted of water with 0.1% formic acid (A) and acetonitrile/methanol/isopropanol (50:25:25, v/v/v) with 0.1% formic acid (B). The analysis was performed in isocratic mode at 13% B and at a flow rate of 360 µL/min. The injection volume was set at 3 µL. The identity of all monosaccharide derivatives was confirmed after extraction of the ion [M + H]+ at m/z 433.1098 (Figure S55).

3.5. Evaluation of the Biological Activities

Compounds 17 were tested for their ability to inhibit the growth of Gram positive bacteria (S. aureus ATCC29213 (MSSA), and S. aureus MB5393 (MRSA)) and Gram negative bacteria (E. coli ATCC25922, K. pneumoniae ATCC700603, P. aeruginosa PAO1, and A. baumannii CL5973), and fungi (A. fumigatus ATCC46645), following previously described methodologies [53,54]. Cytotoxic activity against the hepatic human tumoral cell line hep_G2 was determined as previously reported [55].

4. Conclusions

Poecillastrosides A–G (17) share an unusual oxidized methyl at C-18, and they are the first saponins exhibiting this feature. The structures of poecillastrosides E–G (57) also incorporate a terminal methylated cyclopropyl ring already known in some sponge steroids and already investigated for biosynthetic studies [56]. This cyclopropanation process could lead to the cholestane skeleton, then ergostane, and finally poriferastane, all of them being present in the metabolome of this sponge. Many sterols containing a cyclopropyl ring have been isolated to date [57], but to our best knowledge, this is the first time that saponins containing a 3-membered ring on the side-chain have been reported. Poecillastrosides D (4) and E (5), bearing a carboxylic acid at C-18, were found to be the most bioactive compounds in the antimicrobial bioassays with an interesting antifungal activity against Aspergillus fumigatus.

Acknowledgments

This work was partially funded by the Swiss company Ferring. The sampling was supported by the “Agence des Aires Marines Protégées (AAMP)”, a French establishment dedicated to the protection of the marine environment. The authors are grateful to the COMEX crew who operated the MINIBEX vessel and its ROV SUPER ACHILLE. We are grateful to G. Genta-Jouve for fruitful discussions about the relative configuration of the cyclopropane. The company Cosmo International Ingredients supported the work of K. Calabro. R. Doohan (NUI Galway) is acknowledged for her help in the record of NMR spectra and H. Solanki for his help in the HRMS acquisition.

Supplementary Materials

The following are available online at www.mdpi.com/1660-3397/15/7/199/s1: HRMS and 1H, 13C, COSY, TOCSY, HSQC, HMBC, and NOESY NMR data for compounds 17 as well as procedures for absolute configuration of compound 3.

Author Contributions

Author Contributions

O.P.T. conceived and designed the experiments; E.L.K., D.R. and K.C. performed the experiments; C.D., M.d.l.C., B.C. and F.R. performed the bioassays; K.C., O.P.T., R.L. and B.S. analyzed the data; T.P. collected and identified the biomaterial; K.C., F.R. and O.P.T. wrote the paper.

Conflicts of Interest

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Stonik V.A., Kalinin V.I., Avilov S.A. Toxins from sea cucumbers (holothuroids): Chemical structures, properties, taxonomic distribution, biosynthesis and evolution. J. Nat. Toxins. 1999;8:235–248. [PubMed]
2. Makarieva T.N., Stonik V.A., Kapustina I.I., Boguslavsky V.M., Dmitrenoik A.S., Kalinin V.I., Cordeiro M.L., Djerassi C. Biosynthetic studies of marine lipids. 42. Biosynthesis of steroid and triterpenoid metabolites in the sea cucumber Eupentacta fraudatrix. Steroids. 1993;58:508–517. doi: 10.1016/0039-128X(93)90026-J. [PubMed] [Cross Ref]
3. Burnell D.J., Apsimon J.W. Chapter 6—Echinoderm Saponins. In: Scheuer P.J., editor. Marine Natural Products. Academic Press; Waltham, MA, USA: 1983. pp. 287–389.
4. Qi S., Zhang S., Huang J., Xiao Z., Wu J., Li Q. Complete 1H and 13C NMR assignments of four new steroidal glycosides from a gorgonian coral Junceella juncea. Magn. Reson. Chem. 2005;43:266–268. doi: 10.1002/mrc.1530. [PubMed] [Cross Ref]
5. Wang S.-K., Dai C.-F., Duh C.-Y. Cytotoxic Pregnane Steroids from the Formosan Soft Coral Stereonephthya crystalliana. J. Nat. Prod. 2006;69:103–106. doi: 10.1021/np050384c. [PubMed] [Cross Ref]
6. Ivanchina N.V., Kicha A.A., Stonik V.A. Steroid glycosides from marine organisms. Steroids. 2011;76:425–454. doi: 10.1016/j.steroids.2010.12.011. [PubMed] [Cross Ref]
7. Kitagawa I., Kobayashi M., Okamoto Y., Yoshikawa M., Hamamoto Y. Structures of Sarasinosides A1′, B1′, and C1′; New Norlanostane-Triterpenoid Oligoglycosides from the Palauan Marine Sponge Asteropus sarasinosum. Chem. Pharm. Bull. 1987;35:5036–5039. doi: 10.1248/cpb.35.5036. [PubMed] [Cross Ref]
8. Espada A., Jiménez C., Rodríguez J., Crews P., Riguera R. Sarasinosides D–G: Four new triterpenoid saponins from the sponge Asteropus sarasinosum. Tetrahedron. 1992;48:8685–8696. doi: 10.1016/S0040-4020(01)89444-8. [Cross Ref]
9. Lee H.-S., Seo Y., Cho K.W., Rho J.-R., Shin J., Paul V.J. New triterpenoid saponins from the sponge Melophlus isis. J. Nat. Prod. 2000;63:915–919. doi: 10.1021/np990589j. [PubMed] [Cross Ref]
10. Dai H.-F., Edrada R.A., Ebel R., Nimtz M., Wray V., Proksch P. Norlanostane triterpenoidal saponins from the marine sponge Melophlus sarassinorum. J. Nat. Prod. 2005;68:1231–1237. doi: 10.1021/np050152d. [PubMed] [Cross Ref]
11. Lee J.-H., Jeon J.-E., Lee Y.-J., Lee H.-S., Sim C.J., Oh K.-B., Shin J. Nortriterpene Glycosides of the Sarasinoside Class from the Sponge Lipastrotethya sp. J. Nat. Prod. 2012;75:1365–1372. doi: 10.1021/np300297d. [PubMed] [Cross Ref]
12. Antonov A.S., Kalinovskii A.I., Stonik V.A., Evtushenko E.V., Elyakov G.B. Structure of ulososide A, a new triterpenoid glycoside from the Ulosa sp. sponge. Russ. Chem. Bull. 1994;43:1265–1269. doi: 10.1007/BF00698257. [Cross Ref]
13. Antonov A.S., Kalinovsky A.I., Stonik V.A. Ulososide B, a new unusual norlanostane-triterpene glycoside and its genuine aglycone from the Madagascar sponge Ulosa sp. Tetrahedron Lett. 1998;39:3807–3808. doi: 10.1016/S0040-4039(98)00589-9. [Cross Ref]
14. Colorado J., Muñoz D., Marquez D., Marquez M., Lopez J., Thomas O.P., Martinez A. Ulososides and Urabosides—Triterpenoid Saponins from the Caribbean Marine Sponge Ectyoplasia ferox. Molecules. 2013;18:2598–2610. doi: 10.3390/molecules18032598. [PubMed] [Cross Ref]
15. Cachet N., Regalado E.L., Genta-Jouve G., Mehiri M., Amade P., Thomas O.P. Steroidal glycosides from the marine sponge Pandaros acanthifolium. Steroids. 2009;74:746–750. doi: 10.1016/j.steroids.2009.03.009. [PubMed] [Cross Ref]
16. Regalado E.L., Tasdemir D., Kaiser M., Cachet N., Amade P., Thomas O.P. Antiprotozoal Steroidal Saponins from the Marine Sponge Pandaros acanthifolium. J. Nat. Prod. 2010;73:1404–1410. doi: 10.1021/np100348x. [PubMed] [Cross Ref]
17. Regalado E.L., Jimenez-Romero C., Genta-Jouve G., Tasdemir D., Amade P., Nogueiras C., Thomas O.P. Acanthifoliosides, minor steroidal saponins from the Caribbean sponge Pandaros acanthifolium. Tetrahedron. 2011;67:1011–1018. doi: 10.1016/j.tet.2010.11.103. [Cross Ref]
18. Regalado E.L., Turk T., Tasdemir D., Gorjanc M., Kaiser M., Thomas O.P., Fernandez R., Amade P. Cytotoxic and haemolytic steroidal glycosides from the Caribbean sponge Pandaros acanthifolium. Steroids. 2011;76:1389–1396. doi: 10.1016/j.steroids.2011.07.010. [PubMed] [Cross Ref]
19. Ryu G., Choi B.W., Lee B.H., Hwang K.-H., Lee U.C., Jeong D.S., Lee N.H. Wondosterols A-C, three steroidal glycosides from a Korean marine two-sponge association. Tetrahedron. 1999;55:13171–13178. doi: 10.1016/S0040-4020(99)00810-8. [Cross Ref]
20. D’Auria M.V., Paloma L.G., Minale L., Riccio R., Debitus C. Structure chacterization by two-dimensional NMR spectroscopy, of two marine triterpene oligoglycosides from a pacific sponge of the genus Erylus. Tetrahedron. 1992;48:491–498. doi: 10.1016/S0040-4020(01)89011-6. [Cross Ref]
21. Gulavita N.K., Wright A.E., Kelly-Borges M., Longley R.E., Yarwood D., Sills M.A. Eryloside E from an Atlantic sponge Erylus goffrilleri. Tetrahedron Lett. 1994;35:4299–4302. doi: 10.1016/S0040-4039(00)73338-7. [Cross Ref]
22. Stead P., Hiscox S., Robinson P.S., Pike N.B., Sidebottom P.J., Roberts A.D., Taylor N.L., Wright A.E., Pomponi S.A., Langley D. Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity. Bioorg. Med. Chem. Lett. 2000;10:661–664. doi: 10.1016/S0960-894X(00)00063-9. [PubMed] [Cross Ref]
23. Shin J., Lee H.-S., Woo L., Rho J.-R., Seo Y., Cho K.W., Sim C.J. New triterpenoid saponins from the sponge Erylus nobilis. J. Nat. Prod. 2001;64:767–771. doi: 10.1021/np010047d. [PubMed] [Cross Ref]
24. Antonov A.S., Kalinovsky A.I., Stonik V.A., Afiyatullov S.S., Aminin D.L., Dmitrenok P.S., Mollo E., Cimino G. Isolation and Structures of Erylosides from the Carribean Sponge Erylus formosus. J. Nat. Prod. 2007;70:169–178. doi: 10.1021/np060364q. [PubMed] [Cross Ref]
25. Jaspars M., Crews P. A triterpene tetrasaccharide, formoside, from the Caribbean Choristida sponge Erylus formosus. Tetrahedron Lett. 1994;35:7501–7504. doi: 10.1016/S0040-4039(00)78328-6. [Cross Ref]
26. Takada K., Nakao Y., Matsunaga S., van Soest R.W.M., Fusetani N. Nobiloside, a New Neuraminidase Inhibitory Triterpenoidal Saponin from the Marine Sponge Erylus nobilis. J. Nat. Prod. 2002;65:411–413. doi: 10.1021/np010480n. [PubMed] [Cross Ref]
27. Fouad M., Al-Trabeen K., Badran M., Wray V., Edrada R., Proksch P., Ebel R. New steroidal saponins from the sponge Erylus lendenfeldi. ARKIVOC. 2004;37:17–27.
28. Sandler J.S., Forsburg S.L., Faulkner D.J. Bioactive steroidal glycosides from the marine sponge Erylus lendenfeldi. Tetrahedron. 2005;61:1199–1206. doi: 10.1016/j.tet.2004.11.039. [Cross Ref]
29. Okada Y., Matsunaga S., van Soest R.W.M., Fusetani N. Sokodosides, Steroid Glycosides with an Isopropyl Side Chain, from the Marine Sponge Erylus placenta. J. Org. Chem. 2006;71:4884–4888. doi: 10.1021/jo060653j. [PubMed] [Cross Ref]
30. Gabant M., Schmitz-Afonso I., Gallard J.-F., Menou J.-L., Laurent D., Debitus C., Al-Mourabit A. Sulfated Steroids: Ptilosteroids A–C and Ptilosaponosides A and B from the Solomon Islands Marine Sponge Ptilocaulis spiculifer. J. Nat. Prod. 2009;72:760–763. doi: 10.1021/np800758c. [PubMed] [Cross Ref]
31. Kalinovsky A.I., Antonov A.S., Afiyatullov S.S., Dmitrenok P.S., Evtuschenko E.V., Stonik V.A. Mycaloside A, a new steroid oligoglycoside with an unprecedented structure from the Caribbean sponge Mycale laxissima. Tetrahedron Lett. 2002;43:523–525. doi: 10.1016/S0040-4039(01)02184-0. [Cross Ref]
32. Antonov A.S., Afiyatullov S.S., Kalinovsky A.I., Ponomarenko L.P., Dmitrenok P.S., Aminin D.L., Agafonova I.G., Stonik V.A. Mycalosides B–I, Eight New Spermostatic Steroid Oligoglycosides from the Sponge Mycale laxissima. J. Nat. Prod. 2003;66:1082–1088. doi: 10.1021/np0300030. [PubMed] [Cross Ref]
33. Campagnuolo C., Fattorusso E., Taglialatela-Scafati O. Feroxosides A–B, two norlanostane tetraglycosides from the Caribbean sponge Ectyoplasia ferox. Tetrahedron. 2001;57:4049–4055. doi: 10.1016/S0040-4020(01)00287-3. [Cross Ref]
34. Glensk M., Wray V., Nimtz M., Schöpke T. Silenosides A–C, Triterpenoid Saponins from Silene vulgaris. J. Nat. Prod. 1999;62:717–721. doi: 10.1021/np980505r. [PubMed] [Cross Ref]
35. Wang W., Hong J., Lee C.-O., Im K.S., Choi J.S., Jung J.H. Cytotoxic Sterols and Saponins from the Starfish Certonardoa semiregularis. J. Nat. Prod. 2004;67:584–591. doi: 10.1021/np030427u. [PubMed] [Cross Ref]
36. Wang W., Jang H., Hong J., Lee C.-O., Bae S.-J., Shin S., Jung J.H. New cytotoxic sulfated saponins from the starfish Certonardoa semiregularis. Arch. Pharm. Res. 2005;28:285–289. doi: 10.1007/BF02977793. [PubMed] [Cross Ref]
37. Kicha A.A., Ivanchina N.V., Huong T.T.T., Kalinovsky A.I., Dmitrenok P.S., Fedorov S.N., Dyshlovoy S.A., Long P.Q., Stonik V.A. Two new asterosaponins, archasterosides A and B, from the Vietnamese starfish Archaster typicus and their anticancer properties. Bioorg. Med. Chem. Lett. 2010;20:3826–3830. doi: 10.1016/j.bmcl.2010.04.005. [PubMed] [Cross Ref]
38. Rashid M.A., Gustafson K.R., Crouch R.C., Groweiss A., Pannell L.K., Van Q.N., Boyd M.R. Application of High-Field NMR and Cryogenic Probe Technologies in the Structural Elucidation of Poecillastrin A, a New Antitumor Macrolide Lactam from the Sponge Poecillastra Species. Org. Lett. 2002;4:3293–3296. doi: 10.1021/ol026564k. [PubMed] [Cross Ref]
39. Takada K., Choi B.W., Rashid M.A., Gamble W.R., Cardellina J.H., Van Q.N., Lloyd J.R., McMahon J.B., Gustafson K.R. Structural Assignment of Poecillastrins B and C, Macrolide Lactams from the Deep-Water Caribbean Sponge Poecillastra Species. J. Nat. Prod. 2007;70:428–431. doi: 10.1021/np0604984. [PubMed] [Cross Ref]
40. Natori T., Kataoka Y., Kato S., Kawai H., Fusetani N. Poecillanosine, a new free radical scavenger from the marine sponge Poecillastra spec. aff. tenuilaminaris. Tetrahedron Lett. 1997;38:8349–8350. doi: 10.1016/S0040-4039(97)10260-X. [Cross Ref]
41. Killday K.B., Longley R., McCarthy P.J., Pomponi S.A., Wright A.E., Neale R.F., Sills M.A. Sesquiterpene-Derived Metabolites from the Deep Water Marine Sponge Poecillastra sollasi. J. Nat. Prod. 1993;56:500–507. doi: 10.1021/np50094a008. [PubMed] [Cross Ref]
42. Makarieva T.N., Stonik V.A., D’Yachuk O.G., Dmitrenok A.S. Annasterol sulfate, a novel marine sulfated steroid, inhibitor of glucanase activity from the deep water sponge Poecillastra laminaris. Tetrahedron Lett. 1995;36:129–132. doi: 10.1016/0040-4039(94)02192-E. [Cross Ref]
43. Sun H.H., Gross S.S., Gunasekera M., Koehn F.E. Weinbersterol disulfates A and B, antiviral steroid sulfates from the sponge Petrosia weinbergi. Tetrahedron. 1991;47:1185–1190. doi: 10.1016/S0040-4020(01)86375-4. [Cross Ref]
44. Tvaroska I., Taravel F.R. Carbon-Proton Coupling Constants In The Conformational Analysis of Sugar Molecules. In: Derek H., editor. Advances in Carbohydrate Chemistry and Biochemistry. Volume 51. Academic Press; Waltham, MA, USA: 1995. pp. 15–61. [PubMed]
45. Stenutz R. Coupling Constants of Pyranoses. [(accessed on 19 February 2017)]; Available online: http://www.stenutz.eu/sop/a704.html.
46. Wang Y.-H., Avula B., Fu X., Wang M., Khan I.A. Simultaneous Determination of the Absolute Configuration of Twelve Monosaccharide Enantiomers from Natural Products in a Single Injection by a UPLC-UV/MS Method. Planta Med. 2012;78:834–837. doi: 10.1055/s-0031-1298432. [PubMed] [Cross Ref]
47. Paudel L., Adams R.W., Király P., Aguilar J.A., Foroozandeh M., Cliff M.J., Nilsson M., Sándor P., Waltho J.P., Morris G.A. Simultaneously Enhancing Spectral Resolution and Sensitivity in Heteronuclear Correlation NMR Spectroscopy. Angew. Chem. Int. Ed. 2013;52:11616–11619. doi: 10.1002/anie.201305709. [PMC free article] [PubMed] [Cross Ref]
48. Bonini C., Kinnel R.B., Li M., Scheuer P.J., Djerassi C. Minor and trace sterols in marine invertebrates. 38. Isolation, structure elucidation, and partial synthesis of papakusterol, a new biosynthetically unusual marine sterol with a cyclopropyl-containing side chain. Tetrahedron Lett. 1983;24:277–280. doi: 10.1016/S0040-4039(00)81384-2. [Cross Ref]
49. Catalan C.A.N., Lakshmi V., Schmitz F.J., Djerassi C. Minor and trace sterols in marine invertebrates. 39. 24ξ,25ξ-24,26-Cyclocholest-5-en-3β-ol, a novel cyclopropyl sterol. Steroids. 1982;40:455–463. doi: 10.1016/0039-128X(82)90022-8. [PubMed] [Cross Ref]
50. Fujimoto Y., Kimura M., Terasawa T., Khalifa F.A.M., Ikekawa N. Stereocontrolled synthesis and determination of the C-24 and C-25 stereochemistry of glaucasterol. Tetrahedron Lett. 1984;25:1805–1808. doi: 10.1016/S0040-4039(01)90046-2. [Cross Ref]
51. Kobayashi M., Mitsuhashi H. Marine sterols. XII. Glaucasterol, a novel C27 sterol with a unique side chain, from the soft coral Sarcophyton glaucum. Steroids. 1982;40:665–672. doi: 10.1016/0039-128X(82)90007-1. [PubMed] [Cross Ref]
52. Wiberg K.B., Nist B.J. The Nuclear Magnetic Resonance Spectra of Cyclopropane Derivatives. J. Am. Chem. Soc. 1963;85:2788–2790. doi: 10.1021/ja00901a026. [Cross Ref]
53. Audoin C., Bonhomme D., Ivanisevic J., Cruz M., Cautain B., Monteiro M., Reyes F., Rios L., Perez T., Thomas O.P. Balibalosides, an Original Family of Glucosylated Sesterterpenes Produced by the Mediterranean Sponge Oscarella balibaloi. Mar. Drugs. 2013;11:1477–1489. doi: 10.3390/md11051477. [PMC free article] [PubMed] [Cross Ref]
54. Braña A.F., Sarmiento-Vizcaíno A., Pérez-Victoria I., Otero L., Fernández J., Palacios J.J., Martín J., de la Cruz M., Díaz C., Vicente F., et al. Branimycins B and C, Antibiotics Produced by the Abyssal Actinobacterium Pseudonocardia carboxydivorans M-227. J. Nat. Prod. 2017;80:569–573. doi: 10.1021/acs.jnatprod.6b01107. [PubMed] [Cross Ref]
55. Cautain B., de Pedro N., Schulz C., Pascual J., Sousa T.d.S., Martin J., Pérez-Victoria I., Asensio F., González I., Bills G.F., et al. Identification of the Lipodepsipeptide MDN-0066, a Novel Inhibitor of VHL/HIF Pathway Produced by a New Pseudomonas Species. PLoS ONE. 2015;10:e0125221 doi: 10.1371/journal.pone.0125221. [PMC free article] [PubMed] [Cross Ref]
56. Wessjohann L.A., Brandt W., Thiemann T. Biosynthesis and Metabolism of Cyclopropane Rings in Natural Compounds. Chem. Rev. 2003;103:1625–1648. doi: 10.1021/cr0100188. [PubMed] [Cross Ref]
57. Gunasekera S.P., Cranick S., Pomponi S.A. New Sterol Ester from a Deep Water Marine Sponge, Xestospongia sp. J. Nat. Prod. 1991;54:1119–1122. doi: 10.1021/np50076a035. [Cross Ref]

Articles from Marine Drugs are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)