Penasulfate A, a New r-Glucosidase Inhibitor from a Marine Sponge Penares sp. † Yoichi Nakao, ‡ Takashi Maki, ‡ Shigeki Matsunaga, ‡ Rob W. M. van Soest, § and Nobuhiro Fusetani* ,‡ Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, and Institute for Systematics and Ecology, The University of Amsterdam, P.O. Box 94766, 1090 GT, Amsterdam, The Netherlands Received February 3, 2004 A new R-glucosidase inhibitor, penasulfate A, has been isolated from a marine sponge Penares sp. 1 Its structure was elucidated by spectral and chemical methods to be a scalemic mixture of methyl pipecolates acylated with a novel sulfated fatty acid. Significant roles of glycosidases in various biological functions, including immune response, oncogenesis, me- tastasis of tumors, viral and bacterial infections, and differentiation of neural cells, 2 have become obvious through the recent advances in glycobiology. 2 R-Glucosidases are known to be involved in glycogenolysis and glycoprotein processing in the endoplasmic reticulum (ER). In addition to the application for treatment of diabetes, R-glucosidase inhibitors, which cause misfolding of viral glycoproteins and interfere with the viral life cycle, are expected to be antiviral drugs against HIV or HBV infection. 3 Previously, we have reported penarolide sulfates A 1 (1) and A 2 (2), whose structures were assigned as proline- containing macrolide trisulfates, from a marine sponge Penares sp. 4 Further investigation of this sponge led to the isolation of a new inhibitor, penasulfate A (3). 5 In this paper we describe the isolation, structure elucidation, and bio- logical activities of this compound. Results and Discussion The MeOH extract of the frozen sponge (150 g, wet weight) was partitioned between CH 2 Cl 2 and H 2 O, and the aqueous layer was further extracted with n-BuOH. The n-BuOH layer was separated by flash chromatography on ODS with aqueous MeOH containing 0.1 M NaClO 4 . The fraction eluted with 90% MeOH, which showed potent inhibition against R-glucosidase, was separated by repeti- tive reversed-phase HPLC on ODS to afford a peak and was further purified by two steps of recycling HPLC on ODS to yield an apparently homogeneous fraction (fr. 1). Because NMR data of fr. 1 did not show any hint that it is a mixture, 6 we started structure elucidation using this sample. The IR spectrum (1240 cm -1 ), the negative ion mode FABMS [at m/z 97 (HSO 4 - ) and 80 (radical anion of SO 3 - )], and characteristic MS fragmentation pattern ob- served at m/z 776 (M - Na) - , 754 (M - 2Na + H) - , and 674 (M - Na - NaO 3 S + H) - of fr. 1 indicated the presence of two sulfate groups. The composition of the ion peak at m/z 776 was determined as [C 36 H 67 NO 11 S 2 Na] - on the basis of HRFABMS [m/z 776.4019 (M - Na) - , Δ -2.2 mmu]. The 1 H NMR, 13 C NMR, COSY, and HMQC 7 spectra exhibited the presence of a terminal (CH 3 -28: δ C 14.4; δ H 0.89 t, J ) 6.9 Hz), a branched (CH 3 -29: δ C 20.1; δ H 0.85 d, J ) 6.5), and an oxygenated (OCH 3 -1′: δ C 52.7; δ H 3.72 s) methyl, a large methylene envelope (δ H 1.3), lower field methylenes (δ H 1.35-2.4), a nitrogenous methylene (CH 2 - 6′: δ C 44.9; δ H 3.87 and 3.21) and methine (CH-2′: δ C 53.4; δ H 5.25), and two oxygenated methines (CH-14 and 15: δ C 81.3; δ H 4.46, 2H), together with two carbonyl carbons (C-1 and 1′: δ C 175.9 and 173.1, respectively). Interpretation of HOHAHA 8 and HMBC 9 data led to partial structures a-d (Figure 1). The connectivities from the methine at δ H 5.25 (H-2′) to the methylene protons at δ H 3.87 and 3.21 (H 2 -6′) via three consecutive methylenes (H 2 -3′,4′, and 5′) were readily deduced from the HOHAHA spectrum. An HMBC correlation from H-2′ to C-6′ con- nected C-2′ and C-6′ via a nitrogen atom to form a piperidine unit. Further HMBC cross-peaks, C-1/H-2′ and H-6′, and C-1′/H-2′ and MeO-1′, secured the partial struc- ture a as a methyl pipecolate in which the nitrogen atom was acylated. Two sulfated methines showed identical resonances at δ H 4.46/δ C 81.3. HMBC correlations observed between the protons (δ H 4.46) and carbons (δ C 81.3) led to partial † Dedicated to the late Dr. D. John Faulkner (Scripps) and the late Dr. Paul J. Scheuer (Hawaii) for their pioneering work on bioactive marine natural products. * To whom correspondence should be addressed. Tel: +81-3-5841-5299. Fax: +81-3-5841-8166. E-mail: anobu@mail.ecc.u-tokyo.ac.jp. ‡ The University of Tokyo. § The University of Amsterdam. Figure 1. HOHAHA and HMBC correlations for partial structures a-d. Figure 2. Recycling HPLC charts: (a) the chromatogram of the active fraction to afford fraction 1 [Inertsil ODS-3, n-PrOH/MeCN/H2O (4:2: 4) containing 0.2 M NaClO4] and (b) the chromatogram of fr. 1 to afford penasulfate A (3) [Develosil C30-UG-5, with n-PrOH/MeCN/H2O (35: 25:40) containing 0.2 M NaClO4]. 1346 J. Nat. Prod. 2004, 67, 1346-1350 10.1021/np049939e CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 07/02/2004