Investigation of the toxicity of cyanobacterial peptides by chemical biology approaches

Global warming together with the extensive agriculture favours the uncontrolled
growth of cyanobacteria, also called blue-green algae. Being one of the oldest life
forms on our planet, they have reached evolutionary perfection over time. They are
distributed at a variety of habitats, primarily in water. Cyanobacterial blooms present
immediate danger to animals and humans, as cyanobacteria are known to produce
various toxic compounds. This thesis addresses some of the open questions
regarding cyanobacterial toxins. The current project involves the isolation,
characterization, derivatization and biological and toxicological evaluation of various
toxic compounds derived from cyanobacteria.
Our attempts for labelling cyanobacterial toxins led to the development of an
optimized protocol for arginine derivatization in complex peptides. This
straightforward procedure allows modifying the arginine moiety in peptides containing
with other functional groups, such as fluorophores or biotin under mild conditions in
an operatively simple procedure. This method was applied for labelling leuprolide, a
clinical drug, for in vivo studies in Daphnia.
Microcystins are one of the most common and widely distributed
cyanobacterial toxins. Their hepatotoxicity in humans makes them an important
compound class. Microcystin-LR is the most toxic and common representative of the
microcystin family (LD50 = 50 µgkg-1), yet the precise mechanism of its action is still
under debate. To gain an insight into the uptake, distribution, accumulation and
excretion of MC-LR, biological studies are necessary. One way to investigate the
molecular mechanism of interaction of microcystin-LR is fluorescence labelling. An
efficient synthesis route for the modification of the toxin’s arginine-residue was
established, which allowed the preparation of microcystin derivatives with a variety of
markers attached (fluorescent tags, biotin, diazirine). These derivatives preserved the
parent toxicity as judged by phosphatase inhibition assays, cell viability assays, and
acute toxicity assays against Thamnocephalus platyurus.
Structure elucidation of a newly isolated toxic peptide,aeruginosin 828A, from
Planktothrix strains was conducted. 2D-NMR studies and MS-characterization of the
toxin revealed the presence of phenyllactic acid (Pla), chloroleucine (Cleu),
2-carboxy-6-(4’-sulfo-xylosyl)-octahydroindole (Choi), and 3-aminoethyl-1-N-
amidino-delta3-pyrroline (Aeap) residues. This peptide showed inhibitory activity
against thrombin and trypsin. Furthermore, for the first time within this compound
class, toxicity against Thamnocephalus platyurus was observed. This toxic peptide
could only be found in microcystin-deficient Planktothrix strains.
Another group of toxins, produced along with the microcystins via the
nonribosomal pathway, are the cyanopeptolins. A member of this group,
cyanopeptolin 1020, is a potent inhibitor of trypsin, human kallikrein and factor Xia.
Unlike the microcystins, cyanopeptolins and cyanopeptolin 1020 in particular have
not been investigated in detail so far. One way to get a better understanding of the
toxicity of this compound class are biological test with fluorescently-labelled
cyanopeptolins. A method for labelling the glutamate residue in cyanopeptolin 1020
was established. The protocol allows fast coupling on the carboxylic function of the
toxin. As the products are highly sensitive, an improvement for product stability is
under investigation.