Protease inhibitors from a Slovenian Lake Bled toxic waterbloom of the cyanobacterium Planktothrix rubescens Olga Grach-Pogrebinsky, a Bojan Sedmak b and Shmuel Carmeli a, * a School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel b National Institute of Biology, Vecna pot 111, POB 141, 1001 Ljubljana, Slovenia Received 1 July 2003; revised 11 August 2003; accepted 4 September 2003 Abstract—Three new protease inhibitors, planktopeptin BL1125, planktopeptin BL843 and planktopeptin BL1061 were isolated, along with three known compounds, anabaenopeptin A, anabaenopeptin B and anabaenopeptin F from the hydrophilic extract of Planktothrix rubescens. The planar structure of the new compounds was determined by homonuclear and inverse-heteronuclear 2D NMR techniques as well as high- resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfey’s method for HPLC and by comparison of the acid hydrolysate with authentic samples on a chiral HPLC column. The new peptides were found to be elastase and chymotrypsin inhibitors. q 2003 Elsevier Ltd. All rights reserved. Planktothrix (formerly Oscillatoria) (Oscillatoriles, Oscil- latoriaceae) is one of the genera of cyanobacteria that produce toxic freshwater blooms. Two species of this genus, Planktothrix agardhii (Gomont) Anagnostidis and Komarek and Planktothrix rubescens (DC. ex Gomont) Anagnostidis and Komarek, occasionally produce hepatotoxic blooms. The hepatotoxicity of these cyanobacteria, is associated with the toxic cyclic peptides, microcystins, 1 which are usually accompanied by a considerable amount of protease inhibitors. 2 All five classes of protease inhibitors that have been described from toxic genera of cyanobacteria are found in Planktothrix spp., namely—micropeptins, 3 aerugeno- sins, 4 microginins, 5 anabaenopeptins 6 and microveridins. 7 Serine protease inhibitors of the micropeptins type, are the most abundant protease inhibitors from cyanobacteria, with more than fifty described compounds, 8 nine of which were isolated from extracts of Planktothrix spp. 3,9a – d Twelve of the seventeen known anabaenopeptin type proteases/protein phosphatases inhibitors, were described from extracts of Planktothrix spp. 9c,10a – c Four of the nine published microviridins were isolated from Planktothrix spp. 9c,11 Only two aeruginosins 12 and two microginin 13 type protease inhibitors were found in Planktothrix spp. We hereby report the structure elucidation and biological activity of three new micropeptin-type serine protease inhibitors. As part of our ongoing research on the chemistry of cyanobacterial blooms in water bodies, a toxic strain of the cyanobacterium Planktothrix rubescens was collected, in the summer of 1999, from Lake Bled, Slovenia. The sample of the cyanobacterium was freeze-dried and extracted with 70% MeOH in H 2 O. The extract was found to inhibit several serine proteases. The active extract was flash-chromato- graphed on an ODS column. Three fractions eluted from the column, with 40–60% MeOH in H 2 O, exhibited protease inhibitory activity and were further separated on a reversed- phase HPLC column. Three new protease inhibitors, planktopeptin BL1125 (1, 380.0 mg), planktopeptin BL843 (2, 9.5 mg) and planktopeptin BL1061 ( 3, 15.6 mg) were isolated, along with three known compounds, anabaenopeptin A 10d (4, 10.7 mg), anabaenopeptin B 10d (5, 252.0 mg) and anabaenopeptin F 10a (6, 322.0 mg). Examination of the 1 H and 13 C NMR spectra, of the planktopeptins, revealed that they are structurally related to micropeptin SD944 8 and oscillapeptin G. 3 Planktopeptin BL1125 (1) was isolated as an amorphous white solid. Its molecular formula, C 54 H 79 N 9 O 17 , was deduced from high-resolution FAB MS measurements. The 1 H NMR spectrum, in DMSO-d 6 , revealed six doublet NH proton signals and two singlet proton signals between d H 6.6 and 8.5 ppm, pointing to eight amino acid residues (taking into account the NMe-aromatic amino acid and the 0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2003.09.006 Tetrahedron 59 (2003) 8329–8336 * Corresponding author. Tel.: þ972-3-6408550; fax: þ972-3-6409293; e-mail: carmeli@post.tau.ac.il Keywords: natural products; cyanobacteria; Planktothrix; protease inhibitors.