Euglenophycin

However, a monophyletic group of the alga is mixotrophic, namely the Rapaza viridis, meaning that it switches between photosynthesis, absorbing nutrients and engulfing other eukaryotes.

Euglenoids can contain chlorophyll and an accessory pigment, and/or astaxanthin (a carotenoid), due to which they can either be coloured either green or red.

According to recent research, euglenophycin is produced in at least six species of euglenoid algae and six of seven strains of Euglena Sanguinea.

It was found that the toxin that caused the high mortality rates is non-protein, stable when heated to 30 °C for 10 minutes and maintained activity when frozen at -80 °C for 60 days.

[5] Due to their complicated chloroplast morphology, described as a ‘peculiar chromatophore system’, the identification of E. sanguinea using microscopic techniques remains challenging.

To create the experimental standards for this analysis, euglenophycin was purified by high performance liquid chromatography (HPLC) from E. Sanguinea clonal cultures which were isolated from the mortality events in North Carolina and Texas.

In combination with mass spectrometric methods, PCR tests facilitate monitoring and risk assessment of fresh waters populated by E. Sanguinea toxic blooms.

According to NMR analysis upon extraction, the majority of the euglenophycin produced by euglenoids is in the cis-conformation with respect to the 2nd and 6th position.

Although the nitrogen and oxygen atoms are able to form hydrogen bonds, the compound is water insoluble and very stable in organic solvents, which is in accordance with its in silico predicted log(p) value of ~5.6.

Remarkably, the structure of euglenophycin, except for the butanol side chain, is extremely similar to solenopsin the major constituent of fire ant venom.

[4] Interestingly, Jeanne N. Tawara et al (1993) has investigated toxic alkaloid piperidines from Pine (Pinus) and Spruce (Picea) trees, that are structurally similar to euglenophycin and that are of polyketide origin as well (Figure 2).

[8] Due to this outstanding similarity in both structures, except for the side chain, and origin, it is probable that the synthesis pathway of these compounds is related to that of euglenophycin, taking the evolutionary relationship between algae and trees into account.

[9] The same holds for coniine, also known as ‘the killer of Socrates’, which is another compound that appears to be even more similar to euglenophycin and has been investigated by Hannu Hotti and Heiko Rischer (Figure 3).

Other enzymes may catalyze the addition of various side chains to these piperidines, yielding a range of piperidine-based compounds (Figure 4).

The investigators of the Pine and Spruce tree toxins, have proposed a structure for the polyketide intermediate and have confirmed that the side chains of the piperidine are modified after cyclization.

Research has not yet confirmed these synthesis pathways with a hundred percent confidence, however it is estimated that euglenoids employ the same mechanisms to produce euglenophycin.

Before euglenophycin was identified, researchers observed that fish exposed to cells from E. Sanguinea showed symptoms of disorientation, increased respiration and incapacity to maintain balance.

[5] Later, Zimba et al (2009) confirmed these mortalities when catfish exposed to purified euglenophycin died within 30 min of exposure.

[11] Zimba et al (2009) researched the toxicity of euglenophycin against five algae species: Oocystis polymorpha, Gonphonema parvulum, Microcystis aeruginosa, Planktothrix PCC7811 and Scenedesmus dimorphus.

In vitro research showed that solenopsin had an inhibiting effect on PI3K/AKT which are part of the mTOR pathway in mammalian cells.

Euglenophycin can inhibit VEGF (vascular endothelial growth factor) and thus prevent new veins to be constructed to provide growing tumours with oxygen and nutrients.

Figure 1. Mass spectrometric analysis of euglenophycin. A. Mass spectrum of 500 ng purified euglenophycin. B. Mass spectrum of euglenophycin extracted from a culture of E. Sanguinea. C&D. Mass spectrum of 1 ng of euglenophycin.
Figure 2. Toxic Pine and Spruce alkaloids. The figure depicts the various structures that belong to the toxic Pine (Pinus) and Spruce (Picea) toxic alkaloids of polyketide origin. They share the same piperidine nucleus, but vary in their side chains (represented with ‘R’, labelled 1-8 and 12).
Figure 3. Proposed synthesis pathway of coniine, ‘The killer of Socrates’. This figure depicts the synthesis pathway of coniine as supposed by Hannu Hotti and Heike Rischer. The carbon atoms of coniine are labeled to indicate their position in the molecule. The conjugation of acetate units by polyketide synthase yields a polyketide intermediate (between brackets) as postulated.
Figure 4. Proposed synthesis pathways of various Hemlock piperidines. Acetate units are conjugated to form a polyketide intermediate, which is reduced to a substrate for L-alanine:aminotransferase (AAT), which is converted into a keto amine. This keto amine spontaneously cyclizes into a piperidine named γ-coniceine. This product is a substrate for various enzymes that catalyze its conversion into the final Hemlock toxin.Compounds of which the structure has not yet been determined are depicted between brackets.