Nα-Fmoc-Nε-(propargyloxy)carbonyl-L-lysine

Cyanobacterial harmful algal blooms represent one of the most conspicuous waterborne microbial hazards in aquatic environments mostly due to the production of toxic secondary metabolites, mainly microcystins (MCs). Other bioactive peptides are frequently found in cyanobacterial blooms, yet their concentration and ecological relevance is still unknown. In this paper we studied the presence and concentration of cyanobacterial peptides (microcystins, anabaenopeptins, anabaenopeptilides) in 36 Greek freshwater bodies, using HPLC-DAD, ELISA, and PP1IA. Microcystins were found in more than 90% of the samples investigated, indicating that microcystin-producing strains seem to also occur in lakes without blooms. Microcystins MC-RR, MC-LR, and MC-YR were the main toxin constituents of the bloom samples. Anabaenopeptin A and B were predominant in some samples, whereas anabaenopeptolide 90A was the only peptide found in Lake Mikri Prespa. The intracellular concentrations of anabaenopeptins produced by cyanobacterial bloom populations are determined for the first time in this study; the high (>1000 µg·L(-1)) anabaenopeptin concentration found indicates there may be some impacts, at least on the ecology and the food web structure of the aquatic ecosystems. The maximum intracellular MC values measured in Lakes Kastoria and Pamvotis, exceeding 10,000 µg·L(-1), are among the highest reported.

Freshwater cyanobacteria produce lethal toxins such as microcystins and anatoxins. During the purification of microcystins in bloom samples we found that a toxic cyanobacterium produced not only microcystins but also other types-peptides in early 1990. Since then we have isolated approximately thirty peptides from freshwater cyanobacteria. In this manuscript we focused on the following topics concerning the isolated peptides: 1) how to isolate desired compounds and to determine their structures, 2) structural classification of isolated compounds, 3) isolation of similar peptides from laboratory strains and bloom materials, 4) structurally related peptides from freshwater and marine origins, 5) beta-amino acid containing peptides from cyanobacteria, 6) comprehensive analysis system for the biosynthetic study of peptides produced by cyanobacteria, 7) biological activities of isolated compounds.

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.