**3. Domoic Acid and Related Compounds**

#### *3.1. Discovery, Structure, and Some Properties*

Domoic acid (DA) **3** was first found and isolated from the red alga *Chondria armata* by Japanese scientists in the 1960s [37]. After three years of studies, its structure was determined and then refined through organic synthesis. Like kainic acid, **3** is a derivative of dicarboxylated pyrrolidine containing glutamate moiety, but in its another moiety, domoic acid bears octadienoic substituent derived from a monoterpenoid acid (Figure 1). Closely related metabolites, isodomoic acids **48**–**55**, were identified for more than 25 subsequent years in the red alga *C. armata* and contaminated mussels [38–40], as reviewed in [41,42]. All these metabolites, including isodomoic acids A-H along with domoilactones and some related compounds, also contain a glutamic acid residue and should be considered as effectors of GARs. However, as a rule, their effect is much weaker (Table 1). The number of known analogues of DA has increased, particularly in recent years [43].


**Table 1.** Metabolites related to domoic acid.

KARs, μM = displacement of kainic acid from the complex with kainate receptors.

Several compounds related to domoic acid (dainic acids) have recently been described and named as 7- -methyl-isodomoic acid A (**56**), 7- -methyl-isodomoic acid B (**57**), 7- -hydroxymethyl-isodomoic A (**58**), and 7- -hydroxymethyl-isodomoic acid B (**59**). Closely related minor metabolites **60**, **61**, which do not contain pyrrolidine ring, have also been isolated from the same alga *C. armata* (Figure 4) [43].

**Figure 4.** Dainic acids (**56**–**59**) and related metabolites (**60**,**61**) from *C. armata*.

The main turn in the fate of domoic acid and related compounds occurred after the discovery that these metabolites are produced not only by several species of red algae, but also by planktonic diatoms [43]. As a result of subsequent transfer of domoic acid (DA) (**3**) up the food chain from microalgae into edible mollusks [44–46], it may cause the so-called Amnesic Shellfish Poisoning (ASP) in humans that consume edible mollusks contaminated by DA [47–49]. The first case of ASP was described in 1987 from Canada, where 107 people, who had consumed cultivated mussel *Mytilus edulis*, showed symptoms of this disease and three of them died [45]. The diatom *Pseudo-nitzshia multiseries* was identified as a phytoplanktonic producer of DA, which also produces isodomoic acids E and F. Since 1987, it was found that this toxin and related compounds are responsible not only for human poisoning, but also for numerous cases of poisoning and mortality of birds and marine mammals [46–49].

Symptoms of ASP in humans include gastrointestinal alterations (abdominal cramp, nausea, vomiting), and/or neurological disorders (dizziness, short-term memory loss, seizure, epilepsy or coma in the acute cases) [47]. DA binds to glutamate receptors in the central nervous system (CNS) and myocardium [50] causing overexcitation and, as a consequence, neuro-excitatory behavior in humans, marine mammals, and fish [51–55]. The binding of DA to kainate or AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, subclasses of glutamate receptors, results in the intracellular accumulation of Ca2<sup>+</sup> [47,55]. In contrast to glutamic acid, DA causes long-lasting depolarization, neuronal swelling induced by intraneuronal accumulation of excess Ca2<sup>+</sup>, production of reactive oxygen species, DNA and mitochondrial damages, energy depletion, and cell death [55,56]. Along with the CNS, the heart is often also considered as a potential target site for adverse effects of DA. DA-induced cardiotoxicity has been confirmed not only in people suffering from ASP, but also in sea lions and zebrafish that died as a result of DA-poisoning [51,52].

Isodomoic acids (iso-DAs), which have an insecticidal effect against the American cockroach *Periplaneta americana* [38], were also isolated from marine diatoms including *P. australis*, *P. seriata*, and *Nitzschia navis-varingica* [57]. Some diatom species (e.g., *Nitzschia navis-varingica*) produce isodomoic acids A and B as the major toxin components [49]. However, iso-DAs are often present in the environment at lower concentration than DA [57]. The affinity of iso-DAs to the glutamate receptors is lower than that of DA (Table 1) [58]. Therefore, parent toxin is a major threat for humans and animals, in contrast to iso-DAs [59].

Thus, a series of DA isomers have been identified, while data on occurrence of DA and epidomoic acid (referred to as total DAs) and their toxicity have been used to establish the current regulation limits for DA of <sup>≤</sup>20 mg·kg−<sup>1</sup> [54]. The discovery that exposure to low levels of DA leads to long-lasting neurological effects in mammalian species [53] may suggest the need to reduce its permissible level in tissues of consumed finfish and shellfish [57].

#### *3.2. Synthesis*

Synthesis of the domoic acid **3** and correction of the earlier proposed geometry of the double bonds in its side chain have been carried out [60]. The bicyclic core was constructed from the compound **62**, obtained by multistep synthesis from **1** and diene **63** using the thermic Diels-Alder reaction to give bicyclic adduct **64** with *cis*-fused ring junction. This adduct was ozonized, treated with diazomethane and 2-methyl-2-ethyl-1,3-dioxolane in the presence of p-TsOH and yielded 1,3-dioxolane **65**. The employment of borane-dimethylsulfide complex reduced the carbonyl group. Deprotection of the silyl ester and subsequent oxidation of intermediate diol with pyridinium dichromate (PDC) provided the diester **66**. Selective removal of ketal group by 60% AcOH gave the aldehyde **67**. The Wittig reaction led to **68**, the subsequent reaction with PhSeCl to the selenium-containing aldehyde **69**. The key conversions of this synthesis consisted in the obtaining of *trans*- and *cis*-enal systems from **69**. For example, the treatment with *N*-bromosuccinimide (NBS) in tetrahydrofuran and then with NaOAc led to *cis*-enal **70** as a major product. Transformation of this particular compound, but not its trans-isomer by the Wittig reaction followed by the Jones oxidation gave the product **71**. The subsequent removal of protective groups converted it into domoic acid **3**, thus confirming the *E,Z,R*-configurations in the side chain of this toxin (Scheme 8).

**Scheme 8.** Synthesis of domoic acid.

#### *3.3. Biosynthesis, Producers, Biological Action, and Environmental Role*

Several attempts were made to study the process of biosynthesis of this toxin in order to understand how and why environmental factors trigger the biosynthesis leading to high levels of DA accumulated in diatoms. Using feeding experiments, Savage et al. [61] established that DA arises as a result of condensation of geranyl diphosphate (**72**) with glutamate. Labeled by a stable isotope (deuterium), geranyl diphosphate was incorporated from culture medium into DA that was confirmed by gas

chromatography–mass spectrometry (GC-MS). It was suggested that the condensation of geranyl diphosphate with amino group glutamic acid occurred via nucleophilic substitution of the diphosphate group with the amino group of glutamic acid (**1**).

A recent publication in Science [62] provided new important data concerning the DA biosynthesis. It was shown that these genetically programmed biosynthetic pathways are encoded by a four-gene cluster which is involved in this biosynthesis and exists in genomes of toxic microalgae belonging to the genus *Pseudo-nitzschia*. In the conditions of phosphate limitation and elevated CO2 level, probably characteristic of the final stage of microalgal bloom, *Pseudo-nitzschia* spp. express a series of enzymes involved in construction of the pyrrolidine skeleton system of DA. The up-regulation of CYP450 gene, encoding an enzyme which is responsible for the formation of 7- -carboxylic acid from geranyl diphosphate **72** at its reaction with glutamic acid (**1**), was revealed. It was shown that this gene is clustered with several other genes of DA biosynthesis (the so-called dab genes). The DA biosynthetic gene cluster was established to consist of terpene cyclase (dabA), hypothetical protein Hypo (dabB), dioxygenase (dabC), CYP450 (dabD), and probably other genes. The expression of recombinant dabA without *N*-terminal transit peptide in *E. coli* and its use as a catalyst in the reaction between **72** and glutamic acid **1** confirmed this enzyme to catalyze *N*-geranylation of l-glutamic acid in Mg2+-dependent manner. This is the first and key stage in the DA biosynthesis, which yields *N*-geranyl-l-glutamic acid **61**, as was earlier suggested by Savage et al. [61]. The use of recombinant dabC in the presence of Fe3+, oxygen, and l-ascorbic acid, and α-ketoglutarate-dependent dioxygenase, or both dabC and dabD in one put, gave one of so-called dainic acids (**56**) with a non-oxidized geranyl side chain. When the primary product of this biosynthesis **61** was incubated with *Saccharamyses cerevisae* microsomes, which expressed the transmembrane dabD, *N*-geranyl-l-glutamic acid in the presence of Fe3<sup>+</sup> and oxygen was converted into small, but reproductible amounts of 7- -carboxy-*N*-geranyl-l-glutamic acid (**73**) and its 7- -hydroxy analog. Both products were identified by being compared with synthetic fractions. The product **73,** in the same conditions as were used for the transformation of **61** into **56**, was converted into an isomer of domoic acid **48**. However, no isomerase activity necessary for the conversion of **48** into the end product of this biosynthesis was found (Scheme 9). All the above results show that this biosynthesis pathway begins with the dab A-catalyzed geranylation of l-glutamic acid in chloroplasts. Therefore, the presence of dab genes is a character that allows the recognition of toxic strains of this genus. It can help identify environmental conditions, which may potentially cause neurotoxicity in microalgae and in their consumers such as mollusks and diatom-feeding invertebrates.

**Scheme 9.** Biosynthesis of domoic acid.

Thus, it has been confirmed that DA, as a potent excitatory amino acid, is produced primarily by *Pseudo-nitzschia* and *Nitzschia* diatoms distributed across the world and is naturally accumulated in filter-feeding marine organisms. Although many *Pseudo-nitzschia* species have the potential ability to produce this neurotoxin [49], *P. multiseries*, *P. seriata*, and *P. australis* were established as the most toxic species [57].

There have been numerous bloom events caused by highly toxic DA-producing microalgae along the USA and Canadian coasts over the past 15 years. In many cases, it led to mass poisoning of marine mammals and birds [48]. For instance, in the spring of 2015, an unusually intense and long-lasting toxigenic bloom of *Pseudo-nitzschia* microalgae associated with an abnormal warming of water, was observed in a vast area from California to Alaska. Besides the mass poisoning of marine animals, it caused significant economic losses due to the closure of farms that cultivated bivalves and crabs [63]. Thus, DA exposure has become more widespread due to the higher intensity of toxigenic *Pseudo-nitzschia* blooms and related consumption of DA-contaminated mollusks.

A suggestion can be made that the isomerase activity necessary for the transformation of less active biosynthetic precursors into DA may be present not only in these microalgae but also in consumers, or in symbionts and/or epiphytic microorganisms. It is possible that bacteria play an important role in accumulation of DA in microalgae, but the details of this are still elusive [47]. It was established that axenic diatom cultures produce less DA than xenic ones and that bacteria can enhance DA production [64]. It was shown, for example, that after the addition of the gamma-proteobacterium *Alteromonas macleodii*, isolated from the Russian clone of *P. multiseries*, to an axenic culture of another clone of this species, the amount of DA produced by the latter significantly increased [65]. The stimulating effect of bacteria on the DA production has also been found for another known DA-producer, *Nitzschia* sp. [66]. However, mechanisms of bacteria's influence on intracellular DA levels in microalgae remain insufficiently studied [49].

There are a few other hypotheses that discuss environmental factors influencing toxicity of diatoms. It was suggested that DA production may arise as a defense against grazing. However, the effect of predatory copepods on toxicity of microalgae has not been definitively confirmed [67]. The hypothesis that DA may be involved in a high-affinity iron uptake system seems likely [68]. Trick et al. [69] demonstrated that the addition of iron stimulated the growth of toxigenic *Pseudo-nitzschia* spp., providing a competitive advantage over other phytoplankton. Each of these hypotheses requires further validation.

#### **4. Dysiherbaine and Related Compounds**

#### *4.1. Discovery and Structures*

In 1997, Japanese scientists described a new potent marine excitatory amino acid **74** [70], isolated through homogenization of the sponge *Dyidea herbacea* in water, centrifugation of the extract, and precipitation of high-molecular-weight compounds with 2-propanol. Further purification of water-soluble materials using several types of column chromatography yielded this toxin known as disyherbaine (Figure 5). Its structure was identified as a novel diamino dicarboxylic acid using NMR, FABMS, ISIMS, and other methods of molecular structure analysis. Structurally, the acid contains the bicyclic core consisting of *cis*-fused tetrahydropyran and tetrahydrofuran rings with a glutamic acid fragment attached to the tetrahydrofuran moiety. Dysiherbaine core contains four contiguous stereogenic centers with an additional quaternary stereocenter in the tetrahydrofuran moiety at the site of amino acid section attachment. This excitatory acid stimulates binding of kainic acid and 1-amino-3-hydroxy-5-methyl-4-isooxazolepropionic acid, but not *N*-methyl-D-aspartic acid to the rat brain synaptic membranes, suggesting that **74** is an agonist of KARs in CNS.

Radioligand binding assay showed that dysiherbaine binds kainic acid receptors at significantly lesser concentrations than other agonists of KARs. Intraperitoneal injection into mice (20 μg/kg) caused the same behavior as that observed after injection of domoic acid. In general, dysiherbaine exhibits the most potent epileptogenic activity among so far known amino acids. ED50 for substitution of kainic acid, labeled by tritium in kainate receptors by dysiherbaine is 0.00074 μM. This excitatory amino acid is useful for evaluating the physiological roles of KARs in the central nervous system [71].

The above-mentioned findings explained the increased attention to synthesis **74** and related compounds. Another excitatory amino acid, neodysiherbaine A **75** (Figure 5), was isolated by a bioassay-guided chromatographic procedure as a minor constituent of the aqueous extract from the same sponge species. Its structure was deduced by NMR and MS methods and unambiguously confirmed by the total synthesis. After HPLC purification, Japanese scientists obtained as small amount of **75** as 0.26 mg [72]. Neodysiherbaine A is also potent agonist of KARs which induces characteristic epilepsy-like seizures in mice. Like dysiherbaine, it activates neuronal glutamate receptors with a considerable preference toward kainic acid receptors. However, neodysiherbaine is less active in comparison with dysiherbaine: the same activity associated with crowding out kainic acid from kainate receptors is induced by 0.052 μM of neodysiherbaine [72].

**Figure 5.** Structures of dysiherbaine (**74**) and neodysiherbaine (**75**).

After the discovery of dysiherbaine, it was established that various fragments of its structure play different roles in interaction with receptors. The glutamate section of this molecule is responsible for binding to a receptor, while structural and stereochemical features in the tetrahydropyran ring determine the profile of its activity, in particular, agonistic or antagonistic properties [70,71].
