Research

Nearly all of the research performed in the Rein lab is associated with algal toxins. Several projects are ongoing and involve toxin biosynthesis or metabolism, synthesis of toxin mimics, mechanism of action or isolation and identification of unknown toxins from our collection of over 75 strains of algae. Techniques that students in the Rein lab will learn and perform include organic synthesis, natural product isolation and structure elucidation, biochemistry and molecular biology applied to natural product biosynthesis, and molecular modeling.

Algal toxins are secondary metabolites produced by marine or freshwater algae or cyanobacteria. Among marine algae, the most prolific producers of algal toxins are the dinoflagellates. Dinoflagellates are members of the eukaryotic sub-group Alveolates (along with apicomplexans and ciliates).  They are biflagellate protists (unicellular eukaryotes) The picture to  the right is the dinoflagellate Prorocentrum lima stained with two fluorescent dyes.

Flagellated species of algae account for 90% of harmful algal blooms (HABs), and in this group approximately 75% are dinoflagellates.  Dinoflagellates are responsible for Red Tides, or massive blooms. Red Tides may constitute a public health threat, if the organism produces a toxin. The picture to the left is a bloom of the Florida Red Tide dinoflagellate, Karenia brevis. This was taken in Charlotte Harbor in 1998. Obviously, not all red tides are actually red.

Dinoflagellates produce secondary metabolites that are exquisitely complex. The structure of brevetoxin (K. brevis) is shown above. Brevetoxin, and a handful of structurally similar molecules called "polyether ladders" are unique to marine algae. Very little is known about the biogenic origin of these compounds. What we do know, from stable isotope incorporation experiments (done by Shimizu and Nakanishi back in the 1980s), is that they are polyketides. The carbon backbone of all polyketides is constructed by an enzyme called a polyketide synthase (PKS).

In the Rein lab, we we want to develop an understanding of the molecular genetics of dinoflagellate toxin biosynthesis. These studies are highly challenging due to the complexities of the dinoflagellate genome. We were the first to identify dinoflagellate PKS genes (Mar. Biotech. 2003, 5 (1): 1-12).  and to associate them with a dinoflagellate (Phytochemistry, 2005,  66(15),  1767-1780).

Other toxins are produced by cyanobacteria. The most frequently occurring cyanobacterial toxin is microcystin-LR, produced by several species, but primarily by Microcystis areugenosa. The photo to the left is a Microcystis bloom in the St. Lucie River (in Florida) taken in 2005. Microcystin-LR (the above figure) is a cyclic heptapeptide which contains both proteinogenic and non-proteinogenic amino acids. Microcystin-LR is a hepatotoxin and an inhibitor of serine/ threonine protein phosphatases. We are working to understand the metabolism and disposition of microcystin-LR

Like microcystin-LR, the pahayokolides are cyclic peptides containing both proteinogenic and non-proteinogenic amino acids. The pahayokolides are produced by a cyanobacteria isolated by Dr. Miroslav Gantar (FIU Biology Department) from the Florida Everglades. We published the structure of this peptide in 2007 (J. Nat. Prod., 2007, 70, 730-735).

Kainic acid (left) is a non-proteinogenic amino acid produced by the seaweed, Diginea simplex. It possesses both neurological excitatory and excitotoxic properties. As such, it has been used for decades as a tool by neuropharmacologists to understand the role of neuroreceptors. Starting in 2000, for a variety of reasons, the supply of kainic acid became severely limited, hampering neurological research. Numerous synthesis of kainic acid have been published. However, none are really practical or economically feasible due to the need for precise control over kainic acid's three contiguous stereocenters. We have been working on the synthsis of kainic acid mimics (aza analogs) by 1, 3-dipolar cycloaddition with diazoalkanes. Because 1, 3-dipolar cycloadditions are stereospecific with respect to the dipolarophile and may be stereoselective with respect to other substituents, the five-membered parent ring, can be made with control over the relative configurations of the newly formed stereocenters. Molecular docking calculations suggest that aza analogs may bind to the kainate receptor in the same fashion as kainic acid.