Identification of sulfated steroidal glycosides from the starfish Heliaster helianthus by electrospray ionization mass spectrometry

The starfish Heliaster helianthus contains Thornasteroside A ( 1 ) and Helianthoside ( 2 ) and its isomer ( 3 ) as the major sulfated steroidal glycosides. Saponin containing fractions obtained by purification of extracts of H. helianthus induced escape reactions and mortality in the limpets Siphonaria lessoni and Fissurella radiosa . ESI-MS and ESI-MS/MS were successfully applied to the rapid screening of two cytotoxic fractions obtained by purification of the ethanolic extracts of arms and central disks dissected from H. helianthus . Three major compounds were identified, together with minor sulfated steroidal mono-and diglycosides of polyhydroxysteroids


Introduction
Sulfated steroidal oligoglycosides (asterosaponins) have been recognized as predominant and characteristic metabolites of starfishes. 1 Asterosaponins, which are responsible for the toxicity of these marine organisms, are accompanied by a variety of steroidal mono-and diglycosides and by free and sulfated polyhydroxysteroids in very complex mixtures of highly oxygenated compounds.Starfish extracts have drawn attention because of their wide spectrum of biological effects associated with antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory activities. 2 Recently, we have demonstrated the antifungal activity of two new sulfated hexaglycosides and two new sulfated polyhydroxylated steroidal xylosides isolated from the Patagonian starfish Anasterias minuta. 3n our continuing search for bioactive metabolites from echinoderms and aiming to elucidate the possible biological role of steroidal glycosides in starfish, we have focused our attention on the polar extracts of the limpet predatory starfish Heliaster helianthus (Lamarck, 1816), collected off Las Cruces, Chile.Previous work on this starfish led to the isolation of the common pentaglycoside Thornasteroside A (1) together with the sulfated polyhydroxylated steroidal xylosides Helianthoside (2) and its isomer (3) (Fig. 1). 4 Previous field experiments demonstrated that the limpets Lottia orbignyi and Scurria viridula display active locomotor escape response following contact with homogenates of H. helianthus. 6ecently, we observed that saponin containing fractions obtained by purification of extracts of ARKAT USA, Inc.
H. helianthus induce escape reactions and mortality in the limpets Siphonaria lessoni and Fissurella radiosa. 7To correlate the results of these field experiments with the saponin composition of the tested fractions, we used direct infusion electrospray ionization mass spectrometry as a rapid analytical tool to identify the bioactive compounds in the fractions.Mass spectrometry has played an important role in the structural analysis of natural products owing mainly to its high sensitivity, selectivity and low levels of sample consumption.With the development of soft ionization techniques such as electrospray ionization (ESI), rapid and direct analysis of polar, non-volatile and thermally labile classes of compounds has been achieved. 8ecently, ESI tandem mass spectrometry (ESI-MS/MS) has been shown to provide useful structural information on saponins in plant extracts. 9n the present work, we employed ESI mass (ESI-MS) and tandem mass spectrometries (ESI-MS/MS) for analysis of the polar steroidal glycosides responsible of the toxicity of the purified fractions of the extract of H. helianthus.

Results and Discussion
The n-BuOH extracts of the arms and central disks of H. helianthus exhibited cytotoxicity in the brine shrimp (Artemia salina L.) larvae mortality bioassay. 10Purification of both extracts by chromatography over Sephadex LH60 and further analysis by TLC and comparison with standards led to the isolation of fractions rich in polar steroidal oligoglycosides together with fractions containing predominantly steroidal monoglycosides.Two of these fractions (F4 and F8) were selected for ESI-MS studies to obtain molecular mass and structural information on the starfish steroidal glycoside mixtures.
Fraction F4 was obtained by purification of the n-BuOH extract of the arms and contained asterosaponins as the major components.The ESI-MS in the negative ion mode of this fraction (Fig. 2) displays an ion of m/z 1243 assigned to the corresponding anion [M -Na] -of Thornasteroside A (1), the only asterosaponin reported so far from H. helianthus.The ion of m/z 495 in Fig. 2 could not be assigned to fragmentation of Thornasteroside A (1). ESI-MS/MS of this ion shows SO 4 H -of m/z 97 as the only fragmentation product.The fragment ion of m/z 495 has been detected as a characteristic ion in the FAB mass spectrum in negative ion mode of asterosaponins containing (20R)-5α-cholest-9(11)-en-23-one-3β,6α-diol 3-sulfate as the aglycone. 13C-NMR analysis of fraction F4 shows the presence of signals at δ 11.9 (C-18) and 210.4 (C-23) ppm characteristic of this aglycone. 3This data is consistent with the presence of several saponin spots in the TLC of fraction F4 and the detection of ions of m/z 1431, 1407, 1391, 1257 and 1229 in addition to the desodiated anion (m/z 1243) of Thornasteroside A (1) (Fig. 2).
Fraction F8 obtained by purification of the n-BuOH extract of the central disks of H. helianthus contained sulfated polyhydroxysteroid glycosides as the major components.The ESI-MS of F8 in the negative ion mode (Fig. 4) displays an abundant ion of m/z 663 corresponding to the desodiated anions of isomeric steroidal glycosides 2 and 3, together with ions of m/z 677, 795 and 807, presumably due to desodiated anions of minor polyhydroxylated steroidal glycosides.To investigate their structures, ESI-MS/MS experiments were performed.ESI-MS/MS experiment of the ion of m/z 677 ion (Fig. 6a) shows it to dissociate mainly to HSO 4 -of m/z 97 as well as to a fragment ion of m/z 529 by the loss of a pentose unit via H transfer.This fragmentation pathway indicates that the ion of m/z 677 corresponded to a sulfated monoglycoside of a polyhydroxylated steroid 14 Da heavier than Helianthoside (2) and its isomer (3).This mass difference can be attributed to the presence of a methoxy group in one of the sugar residues.This structural feature is very common in polyhydroxysteroid glycosides isolated from starfishes.On the other hand, the ESI-MS/MS of the isolated ion of m/z 807 (Fig.

Conclusions
Electrospray ionization mass and tandem mass spectrometries in the negative ion mode is shown to provide a fast and suitable screening method able to characterize underivatized sulfated steroidal glycosides in purified extracts of starfish.The present study indicates that fractions F4 and F8 contain complex mixtures of asterosaponins and sulfated mono-and diglycosides of polyhydroxylated steroids, respectively.ESI-MS and ESI-MS/MS together with NMR data allowed us to confirm the presence of saponins 1-3 in and detect the presence of other minor sulfated steroidal glycosides that may contribute to the toxicity of the starfish.ESI-MS and ESI-MS/MS have great advantages for chemical screening of complex saponin mixtures.Due to the time-consuming and material-consuming isolation of pure saponins, these methodologies are important tools to obtain information on the complexity of a saponin mixture, the presence of known compounds and the chance of finding new structures.However, because the information is limited on the sugar linkage pattern, site of attachment of sulfate groups and the presence of sugar isomers, only a partial characterization of a new saponin is possible and further studies on the pure compounds using NMR spectroscopy and chemical reactions are required.

Experimental Section
General Procedures. 1 H and 13 C NMR spectra were recorded in Py-d 5 on a Bruker AM 500 spectrometer.TLC was performed on precoated Si gel F254 (n-BuOH-HOAc-H 2 O (12:3:5)) and C 18 reversed-phase plates (70% MeOH-H 2 O) and detected by spraying with p-anisaldehyde (5% EtOH).Samples were analyzed by direct infusion ESI by means of a syringe pump (Harvard Apparatus) at a flow rate of 10 µl/ min.Negative ion mode ESI-MS fingerprints and negative mode ESI-MS/MS for low energy collision-induced dissociation (CID) were acquired using a hybrid high-resolution and high-accuracy (5 ppm) Micromass Q-TOF mass spectrometer.Capillary and cone voltages were set to 3300 V and 30 V respectively, with a desolvation temperature of 100 °C.Each sample was prepared in a solution containing 50% (v/v) chromatographic grade methanol (Tedia, Fairfield, OH, USA) and 50% (v/v) deionized water basified with ammonium hydroxide (Merck, Darmstadt, Germany).Mass spectra were acquired in the range between m/z 100 and 1400.ESI-MS/MS was performed by selecting the ion of interest using the first quadrupole Q1, which was in turn subjected to 15-35 eV collisions with argon in the second rf-only collision quadrupole (Q2) while scanning the orthogonal TOF mass analyser to acquire its tandem mass spectrum.Specimens of H. helianthus were collected at Las Cruces (V Región, Chile).The organisms were identified by Dr. Claudia Muniain of the Museo de Ciencias Naturales "Bernardino Rivadavia", Buenos Aires, Argentina, where a voucher specimen is preserved (MACN-IN 36525).
Extraction and isolation of steroidal glycosides.The starfish (10 animals, 3.85 kg) were defrosted and dissected into their arms (2.7 kg) and central disks (1.14 kg).Each portion was cut into small pieces, homogenized in EtOH and filtered.The EtOH was evaporated to obtain an aqueous residue that was partitioned between H 2 O and cyclohexane and then between n-BuOH and H 2 O.The n-BuOH extracts were evaporated under reduced pressure to give glassy materials (9.5 g from the arms and 3.5 g from the central disks) toxic to the brine shrimp Artemia salina (LD 50 : 107.3 ppm and 264.3 ppm, respectively).Each n-BuOH extract was chromatographed on a Sephadex LH60 column with MeOH:H 2 O (2:1) as eluent to give fractions containing mixtures of sulfated saponins.Two of these fractions, F4 (99 mg) and F8 (139 mg) were selected for ESI mass spectrometry studies.

Figure 2 .
Figure 2. ESI-MS of fraction F4 in the negative ion mode.

Figure 3 .
Figure 3. (a) ESI-MS/MS of the ion of m/z 1243; (b) ESI-MS/MS of the ion of m/z 511.

Figure 4 .
Figure 4. ESI-MS in the negative ion mode of the mixture of polyhydroxylated glycosides in fraction F8.

Figure 5 .
Figure 5. ESI-MS/MS of the ion of m/z 663. 6b ) shows it to dissociate to an ion of m/z 657 by the loss of one pentose unit via H transfer as well as to a fragment ion of m/z 211 as observed in the ESI-MS/MS of the ion of m/z 663.This results, together with the detection of HSO 4 -as the major fragment, are consistent with a sulfated polyhydroxysteroid diglycoside detected as [M -Na] -of m/z 807.