*2.3. Biosynthesis and Application of Kainoids*

Recently, Moore's team from the USA studied the biosynthesis of two related excitatory amino acids: domoic acid from diatoms and kainic acid from red macrophytes. After elucidating the origin of kainic acid **2**, they performed the whole genome sequencing of a kainic acid producer, *Digenea simplex*, to identify the gene clusters involved in the biosynthesis of **2** [27]. They used the new single-molecule, long-read sequencing platforms such as Oxford Nanopore Technologies. As a result, the long reads that included genes encoding the kainic acid biosynthesis were sequenced; however, due to the presence of impurity of microbial DNA, they were not assembled into the whole genome of this alga. After the analysis of *D. simpex* on a higher-throughput platform, Prometh ION, 47 Gp of sequence was generated, with the longest read being 1.2 Mb. A series of *Digenea simplex* kainic acid biosynthesis (Dskab) genes were uncovered. They included genes of annotated *N*-prenyltransferase (DsKabA), α-ketoglutarate-dependent dioxygenase (DskabC), and several retrotransposable elements such as integrase, reverse transcriptases, and RNA H domains. Both sequences, DsKabA and DskabC, were successfully expressed in *Echerichia coli* and purified. Incubation of recombinant DskabA with l-glutamate **1** and dimethylallylpyrophosphate **44** gave *N*-dimethylallyl-l-glutamic acid **45** (the so-called prekainic acid). This compound was converted into kainic acid in an aqueous extract from *D. simplex.* Another enzyme of kainic acid biosynthesis, the so-called DsKabC, was incubated with prekainic acid, α-keto-glutarate, l-ascorbate, and Fe2<sup>+</sup>, and transformed prekainic acid into kainic acid (**2**) and kainic acid lactone (**7**) (Scheme 7). The main product was confirmed to be kainic acid using isolation from the culture medium and NMR elucidation. The obtained kainic acid lactone **7** has been shown to be antagonist of iGluR in contrast to kainic acid proper.

**Scheme 7.** Biosynthesis of kainic acid.

To show the power of both chemical synthesis and biocatalysis, prekainic acid was synthesized through the reductive amination of l-glutamate with 3-methyl-2-butenal, by the method reminiscent of the cobalt-mediated strategy of Baldwin et al. [28]. For the next step, DsKabC was used without additional purification, but in a medium with E. coli expressing this enzyme. The employment of this combined approach and the purification procedure using activated carbon followed by preparative reverse phase HPLC provided 1.1 g of kainic acid with a 32% overall yield and 95% purity. It proved to be better compared to any chemical synthesis. Thus, this work has not only discovered enzymes of biosynthesis of kainic acid, but also developed a new method combining chemical and enzymatic transformations to obtain **2** for pharmacological research.

Analyses of genomes of other red algae revealed similar genes. For example, genes named as PpkabA and PpkabC were identified in the red alga Palmara palmata, the second known producer of **2**. In comparison with Digenea simplix, the kab genes in the edible P. palmata were tightly clustered, and the retrotransposable elements were absent in the intergenic region. The corresponding kab genes were also found in four other red algae species [27].

As it is well known, **2** has found a wide range of applications to model epilepsy, to determine molecular events indicating human neurodegenerative disorders, and to evaluate efficiency of various therapeutic interventions on laboratory animals with simulated diseases [29–31].

The specific interaction of kainic acid with glutamic acid receptors (GARs) in CNS was studied. Most of ionotropic kainate receptors have ligand-binding domains which form a clamshell-like structure consisting of lobes. Kainic acid, like glutamic acid, is bound between these lobes, but their influence on activities of these receptors remains to be explained [32]. As a neurotransmitter, kainic acid acts also on another class of receptors, metabotropic glutamate receptors (mGluRs), which respond slower than iGluRs, but their interaction with **2** shows an effect on the learning and memory functions.

The biological role of kainic acid in its producers was more poorly studied than distribution, biological activities, and biosynthesis. Using rabbit polyclonal antibodies for kainic acid, which did not cross-react with other amino acids including glutamate, Japanese scientists immunochemically localized this toxin in the fine cylindrical thallus of D. simpex [33]. They concluded that the presence of kainic acid on the surface of the alga can be related to its role as a protection against grazers.

Little is currently known about the functions of algal glutamate receptors-like compounds or the probable influence of kainic acid on these receptors in algae. However, the highest variability in glutamate receptors sequences was recorded from algae [34]. These receptors are associated with many plant-specific physiological functions, such as sperm signaling in moss, pollen tube growth, root meristem proliferation, and innate immune and wound responses. It should be taken into account that the main physiological roles and modes of action of plant GluRs, in contrast to those in animals, are performed in peripheral, non-neuronal tissues [35].

Further studies on some synthetic derivatives of kainic acid and analogs (Figure 3) are aimed, first, at discovering the earlier unknown events in the brain depending on kainate receptors. Second, analogous compounds can be used to create novel drugs. Some of these compounds have reached clinical trials. For example, the compound LY404039 (**46**) is a selective agonist for the metabotropic glutamate 2/3 (MGlu2/3) receptor. This preparation, when dosed as prodrug (**47**) (LY2140023), showed efficacy in schizophrenia. A series of similar compounds was studied as potential psychiatric medicines by such pharmaceutical companies as Lilly and Taisho [36].

**Figure 3.** Structures of synthetic derivatives of glutamic acid (**46**,**47**).
