Spectroscopic Investigations of Oligopeptides from Aquatic Cyanobacteria: Characterisation of New Oligopeptides, Development of Microcystin Quantification Tools and Investigations into Microcystin Production

Cyanobacteria (blue-green algae) are a group of ancient prokaryotic organisms which have become synonymous with water quality deterioration. An array of compounds is produced by aquatic cyanobacteria, the largest group being the oligopeptides. A major class of cyanobacterial oligopeptides are the microcystins; cyclic heptapeptides which contain the unique amino acid Adda (3 amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid). Due to their ability to inhibit protein phosphatases 1 and 2A and as they are concentrated in the liver, microcystins can be highly toxic to animals. Anabaenopeptins are cyclic hexapeptides which contain a carbonyl-linked sidechain amino acid and a ring which is cyclised through the sidechain amine of the position two D-lysine. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF MS) analysis of a cyanobacterial bloom sample from Lake Wiritoa (Manawatu, New Zealand) led to the identification of a new anabaenopeptin. The putative structure for anabaenopeptin 906 was constructed using tandem MS (MS/MS) data, in conjunction with the sequences of the known anabaenopeptins. A Microcystis species (CYN06) isolated from Lake Hakanoa (Huntly, New Zealand) was investigated as it produced a large number of microcystin congeners. Liquid chromatography-MS/MS analysis was used to identify seventeen known microcystin congeners and to characterise ten new variants. Two of the new microcystins, MC-FA and MC-WA, were structurally characterised using amino acid analysis and nuclear magnetic resonance (NMR) spectroscopy. The other new congeners included MC FAba, MC WAba, MC FL, MC-WL as well as [Asp³] analogues of MC FA, MC WA, and [Asp³] analogues of two known congeners; MC-RA and MC-RAba. A review of the number of microcystin congeners produced by reported cyanobacterial strains placed CYN06 in the upper ten percentile. However, CYN06 differed from the other strains as it showed relaxed substrate specificity at position two and four and simultaneously produced microcystin congeners which contained no arginine residues, one arginine residue and two arginine residues. Microcystis CYN06 also contained an unidentified microcystin with a mass of 1014.5 Da. Structural characterisation by NMR spectroscopy indicated that it was an analogue of MC-WA which contained N-formylkynurenine at position two. As N-formylkynurenine is a known oxidation product of tryptophan it was proposed that further unidentified microcystins from CYN06 contained two other oxidation products of tryptophan; kynurenine and oxindolyalanine. This is the first report of the presence of oxidised tryptophan residues in microcystins. Analysis of a cyanobacterial hydroterrestrial mat sample collected from Miers Valley in Eastern Antarctica indicated the presence of fourteen new oligopeptides. A combination of MS/MS and amino acid analysis was used to characterise eight new microcystins which each contained a position one glycine. A recently-developed thiol derivatisation technique indicated that the position seven amino acid in each of the microcystins was very likely dehydrobutyrine. Different combinations of variable modifications at positions two, four and five resulted in eight unique structures. These included four microcystins with Adda moieties which possessed an O-acetyl group (ADMAdda) instead of the conventional C9 methoxy. As well as being the first report of microcystins containing a position one glycine, this is the first report of ADMAdda-containing microcystins in the southern hemisphere. The putative structures of six new Antarctic linear peptides were determined through a combination of mass spectrometric techniques. The compounds contained an N terminus with the molecular formula C₉H₁₄NO₂ linked to isoleucine, two aromatic amino acids and an ester linked hydroxyphenyllactic acid. The hydroxyphenyllactic acid C terminus and the unidentified N terminus suggest that these new compounds are a novel class of cyanobacterial oligopeptide. Seven different sample preparation techniques for the quantitative analysis of microcystins by MALDI-TOF MS were assessed for signal reproducibility and sensitivity using a cost-effective internal standard (angiotensin I). The sensitivity of six of the preparations was acceptable, as was the reproducibility for two thin layer preparations performed on a polished steel target. Both thin layer preparations could be used with a MALDI-TOF mass spectrometer which automatically acquires data. The thin-layer-spot preparation could also be used in an automated sample preparation work-flow. Further investigation using this preparation demonstrated that linear quantification of three different microcystin congeners ([Dha⁷] MC-LR, MC-RR and MC-YR) was possible. Use of this MALDI sample preparation will allow large numbers of microcystin-containing samples to be analysed rapidly and at low cost. A batch culture experiment using Microcystis CYN06 exhibited a decreased abundance of arginine-containing microcystins as nitrate concentrations decreased. Linear regression of the relationship between log10 microcystin content and nitrate concentration revealed slopes which were dependent upon the number of arginine residues present in the compound. During this experiment the abundance of congeners with a single arginine residue at position two did not change (p > 0.05), whilst the abundance of the congeners with a position four arginine decreased significantly (p ≤ 0.001). This suggests that there could be an element of selectivity in regards to which arginine in the microcystin structure is modulated and could explain why congener modulation in response to nitrogen concentration has not been observed previously. Whilst it was not proven that nitrogen supply was the causative factor for the congener modulation, the results from this experiment warrant further study in this area. This research has significantly expanded our knowledge of oligopeptide diversity, improved an existing method of quantifying microcystins and shed new light on the regulation of the abundance of microcystin congeners. The identification of eighteen new microcystins is a 16% increase upon the 111 presently characterised. Also reported was the identification of nine oxidised analogues of tryptophan-containing microcystins from Microcystis CYN06. The presence of oxidised Trp residues in microcystins has not been reported previously and will allow researchers working with samples of Trp-containing microcystins to now assign the oxidised analogues. Seven new cyanobacterial oligopeptides were characterised during this study, six of which may belong to a novel class of linear peptides. A sample preparation designed for the quantification of microcystins by MALDI TOF MS gave comparative performance to the previously reported method but was compatible with automated high-throughput sample preparation and data acquisition. For the first time, a culturing experiment showed a relationship between the abundance of arginine-containing microcystins and nitrogen supply