Triterpene Glycosides from Antarctic Sea Cucumbers. 1. Structure of Liouvillosides A 1 ,A 2 ,A 3 , B 1 , and B 2 from the Sea Cucumber Staurocucumis liouWillei: New Procedure for Separation of Highly Polar Glycoside Fractions and Taxonomic Revision Alexandr S. Antonov, † Sergey A. Avilov, † Anatoly I. Kalinovsky, † Stanislav D. Anastyuk, † Pavel S. Dmitrenok, † Evgeny V. Evtushenko, † Vladimir I. Kalinin,* ,† Alexey V. Smirnov, ‡ Sergi Taboada, § Manuel Ballesteros, § Conxita Avila, § and Valentin A. Stonik † Pacific Institute of Bioorganic Chemistry of the Far East DiVision of the Russian Academy of Sciences, Pr. 100-letya VladiVostoka 159, 690022, VladiVostok, Russian Federation, Zoological Institute of the Russian Academy of Sciences, 199164, Saint Petersburg, Russian Federation, and Department of Animal Biology (InVertebrates), Faculty of Biology, UniVersity of Barcelona, AVenida Diagonal 645, 08028, Barcelona, Spain ReceiVed March 19, 2008 Five new triterpene glycosides, liouvillosides A 1 (1), A 2 (2), A 3 (3), B 1 (4), and B 2 (5), have been isolated from the Antarctic sea cucumber Staurocucumis liouViellei along with the known liouvilloside A (6), isolated earlier from the same species, and hemoiedemosides A (7) and B (8), isolated earlier from the Patagonian sea cucumber Hemioedema spectabilis. The isolation was carried out using a new chromatographic procedure including application of ion-pair reversed-phase chromatography followed by chiral chromatography on a cyclodextrin ChiraDex column. The structures of the new glycosides were elucidated using extensive NMR spectroscopy ( 1 H and 13 C NMR spectrometry, DEPT, 1 H- 1 H COSY, HMBC, HMQC, and NOESY), ESI-FTMS, and CID MS/MS, and chemical transformations. Glycosides 1-3 are disulfated tetraosides and glycosides 4 and 5 are trisulfated tetraosides. Glycosides 2 and 3 contain 3-O- methylquinovose, found for the first time as a natural monosaccharide in sea cucumber glycosides. On the basis of analyses of glycoside structures a taxonomic revision is proposed. Holothurians contain a great diversity of triterpene glycosides belonging mainly to the so-called holostane series. Their carbohy- drate chains are comprised of two to six monosaccharide units. 1-4 Holothurian triterpene glycosides are good taxonomic markers and have been used successfully to improve sea cucumber taxonomy. 2 These natural products have been found in many sea cucumber species collected from the tropical Pacific and Indian Oceans, 1 Mediterranean Sea, 5 North Atlantic, 6 and North Pacific. 7 Neverthe- less only one paper has focused on the study of triterpene glycosides from an Antarctic sea cucumber: Staurocucumis liouVillei (Vaney) (family Cucumariidae, order Dendrochirotida). 8 Two trisulfated tetraosides, liouvillosides A and B, were isolated from this holothurian. It is generally known that sea cucumbers belonging to the Cucumariidae have very complicated and often completely nonseparable mixtures of mono-, di-, and trisulfated triterpene glycosides. 2,9 This explains why it is often so difficult to isolate any glycoside constituents from glycoside fractions. As part of our search for new triterpene glycosides from sea cucumbers belonging to the Cucumariidae, 6,7,10,11 we report the reinvestigation of the glycoside fraction of S. liouVillei. A new procedure for separation of complicated polysulfated glycoside fractions was developed, and the taxonomic positions of two sea cucumber species from the Antarctic were investigated. Results and Discussion Twenty-three specimens of the sea cucumber S. liouVillei were collected near the sub-Antarctic Island of Buovet (South Atlantic Ocean) during the Antarctic expedition ANT XXI/2. Collections were made in November 2003 from the research vessel Polarstern (Alfred Wegener Institute for Polar and Marine Research) at 134 m depth using an Agassiz trawl and were extracted with ethanol. The concentrated ethanolic extract was chromatographed on a column with Teflon powder Polychrom-1 for desalting and elimination of polar substances and a Si gel column with CHCl 3 /EtOH/H 2 O (100: 100:17 and 100:125:25) to yield two glycoside subfractions, A and B. Further separation of these subfractions by HPLC using various direct and reversed-phased columns in different chromatographic systems was unsuccessful because of high polarities of the glycosides and their small structural differences, sometimes in the position of a double bond in polycyclic moieties of the aglycones only (for example, between 1 and 7 or between 4 and 8). We succeeded in separating the subfractions into eight individual glycosides by applications of direct-phase and reversed-phase ion- pair chromatography followed by ion-pair chiral chromatography on a cyclodextrin Aligrent ChiraDex column. Subfraction A was separated by HPLC on Supelco Sil [CHCl 3 /EtOH/NH 4 OAc (1 M solution), 65:45:2], Supelco C-18 [EtOH/H 2 O/NH 4 OAc (1 M solution), 55:45:2], and cyclodextrin Agilent ChiraDex [CH 3 CN/ H 2 O/NH 4 OAc (1 M solution), 60:40:1] columns, yielding liouvil- losides A 1 (1), A 2 (2), and A 3 (3) and the known glycoside identified by 13 C NMR spectrum as hemoiedemoside A (7), previously reported from Hemioedema spectabilis. 12 Subfraction B was subsequently separated by HPLC on Supelco C-18 [EtOH/H 2 O/ NH 4 OAc (1 M solution), 55:45:2] and Agilent ChiraDex BETA [CH 3 CN/H 2 O/NH 4 OAc (1 M solution), 65:35:1, 55:45:1, 50:50:1] columns to give liouvellosides B 1 (4) and B 2 (5) and the previously known liouvilloside A (6) from Staurocucumis liouVillei 8 and hemoiedemoside B (8) from H. spectabilis. 12 This is the first use of chiral chromatography to separate these types of glycoside mixtures. The 13 C NMR spectra of aglycone parts of the glycosides 1, 2, and 4 were identical and coincident with the aglycone parts of known cucumarioside A 2 -2 from Cucumaria japonica 13 (Table 1). The common aglycone of 1, 2, and 4 belongs to the holostane type [from the signals of a 18(20)-lactone at δ 179.3 (C-18) and 83.9 ppm (C-20) in the 13 C NMR spectrum] and contains a 7(8)-double bond [the signals of a tertiary carbon (C-7) at 121.8 ppm and a quaternary carbon (C-8) at 143.9 ppm in the 13 C NMR spectrum] and a 16-keto group [the signals of a quaternary carbon at 214.3 * To whom correspondence may be addressed. Fax: 7-(4232)-31-40-50. E-mail: kalininv@piboc.dvo.ru. † Pacific Institute of Bioorganic Chemistry. ‡ Zoological Institute. § University of Barcelona. J. Nat. Prod. 2008, 71, 1677–1685 1677 10.1021/np800173c CCC: $40.75  2008 American Chemical Society and American Society of Pharmacognosy Published on Web 10/08/2008