dCyanobacteria produce numerous volatile organic compounds (VOCs), such as ␤-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only ␤-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as ␤-cyclocitral, ␤-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including ␤-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of ␤-cyclocitral. ␤-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments.

LC/MS/MS under ion trap conditions was used to analyze microcystins produced by cyanobacteria. Tandem mass spectrometry using MS 2 was quite effective since ions arising from cleavage at a peptide bond provide useful sequence information. The fragmentation was confirmed by a shifting technique using structurally-related microcystins and the resulting fragmentation pattern was different from those determined by triple stage MS/MS and four sector MS/MS. Analysis of a mixture of microcystins in a bloom sample was successfully performed and two new microcystins were identified by LC/MS/MS under ion trap conditions. Thus, LC/MS/MS under ion trap conditions is effective for the structural characterization of microcystins.

Volatile compounds, such as beta-cyclocitral, geosmin, and 2-methylisoborneol, from cyanobacteria showed a lytic activity against cyanobacteria. Particularly, beta-cyclocitral caused an interesting color change in the culture broth from green to blue during the lysis process. In the present study, the lytic behavior of various cyanobacteria with beta-cyclocitral was investigated, and a mechanism for the blue color formation was developed. beta-Cyclocitral lysed both the laboratory strains of any genera and bloom samples including many species of cyanobacteria, and caused the characteristic color change from green to blue. beta-Cyclocitral provided a characteristic behavior, such that the absorption maxima of chlorophyll-a and beta-carotene disappeared, but that of phycocyanin still remained after 12 h, which indicated that beta-cyclocitral decomposed chlorophyll-a and beta-carotene rapidly, so that the inherent colors from the tolerant water-soluble pigments became observable in the cultured broth. This phenomenon was confirmed by another experiment using Phormidium (NIES-611), which showed a pink color derived from phycoerythrin. beta-Cyclocitral was more easily oxidized when compared with similar aldehyde compounds, so that the pH of the solution quickly decreased to 4.5. An oxidation product of beta-cyclocitral in water solution was isolated and identified as 2,6,6-trimethylcyclohexene-1-carboxylic acid. This study provides support that beta-cyclocitral derived from cyanobacteria plays an important role in the lysis of cyanobacteria and participates in the blue color formation under natural conditions.

Melophlins, tetramic acid derivatives possessing a long alkyl chain, have been isolated from the marine sponge Melophlus sarasinorum collected in Indonesia.2-4) Tetramic acid derivatives were also reported from other species of marine sponges. [5][6][7][8][9][10][11] During our continuing study on biologically active metabolites from marine organisms, we found four new melophlins, named melophlins P, Q, R, and S (1-4, Chart 1), from the ethanol extract of a marine sponge Melophlus cf. sarasinorum collected in Palau, together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11). Three of the new melophlins Q-S (2-4) and seven known compounds (5-11) were also detected in the ethanol extract of a Palauan Melophlus sp., which had a different appearance from that of the above species.We describe herein the isolation, structures, and growth inhibitory activity against the murine leukemia cell line L1210 of four new melophlins.Ethanol extract of M. cf. sarasinorum inhibited growth of L1210 cells. Bioassay-guided isolation from the extract by repeated column chromatographies and HPLC gave four new melophlins P-S (1-4, Chart 1) and five previously known melophlins D (6), G (8), H (9), I (10), and O (11). Melophlins A (5) and E (7) were also detected in a fraction obtained by silica gel column chromatography. Ethanol extract of the other Melophlus sp. showed cytotoxicity against L1210 cells, and seven known compounds 5-11 were isolated from the extract. Three new compounds 2-4 were contained in a fraction separated by a silica gel column, but melophlin P (1) was not detected in any fractions. The structures of 5-11 were assigned on the basis of their spectral data and confirmed by comparing the data with reported values. 2,3)The 1 H- (Table 1) and 13 C-NMR signals (Table 2) of four new compounds 1-4 were assigned by 2D NMR ( 1 H-1 H COSY, HMQC, and HMBC) experiments. The UV spectra of 1-4 suggested that these compounds were also tetramic acid derivatives. 2,3,8,12) The 1 H-and 13 C-NMR spectra of 1-4 showed the presence of an N-CH 3 group (d H 2.95, d C 26.3 or 26.4) and a methyl group (d H 1.33, d C 14.9 or 15.0) ascribed to 5-CH 3 . HMBC correlations were observed from N-CH 3 to C-2 and C-5, 5-CH 3 to C-4 and C-5, H-5 to C-4 and 5-CH 3 , and from H 2 -7 to C-3 and C-6 in the HMBC spectra of 1-4. These spectral data revealed that 1-4 have the same tetramic acid moiety possessing the 5-CH 3 group as that of melophlins B, 2) C, 3) J, 3) and L-O (11), 3) which was also confirmed by comparison of the NMR data for 1-4 with those of the reported values for B, C, J, and L-O. The 1 H-and 13 C-NMR spectra of 1-4 showed that these compounds were mixtures of two tautomers (exo A and exo B forms) [13][14][15] in the ratio of 9 : 1 at the tetramic acid moiety as similar to the other melophlins. 2,3)Melophlin P (1) was isolated as a yellowish oil. The ESI-MS of 1 showed an (MϩH) ϩ ion at m/z 366. The molecular formula (C 22 H 39 NO 3 ) was determined from high-resolution (HR) FAB-MS and NMR data. In th...

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